9/14/2015 Patent WO2009001220A2 ­ Functionalization of microscopy probe tips ­ Google Patents

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Patents

Publication number WO2009001220 A2Publication type ApplicationApplication number PCT/IB2008/002466Publication date Dec 31, 2008Filing date Jun 25, 2008Priority date Jun 26, 2007

Also published as WO2009001220A3, WO2009001220A4,WO2009001220A8

Inventors Ola Nilsen, 5 More »

Applicant Uni I Oslo, 6 More »

Export Citation BiBTeX, EndNote, RefMan

Patent Citations (6), Referenced by (2), Classifications (21),Legal Events (3)

External Links: Patentscope, Espacenet

CLAIMS (1)

1. WHAT IS CLAIMED IS:

1. A functionalized AFM probe comprising an AFM tipcoated with at least one material chosen from biological,organic, organic­inorganic hybrid, bone­ like, and an implant­like material.

2. A functionalized AFM probe of claim 1 , wherein the probecomprises at least one material chosen from Si, Si3N4, and

SiN.

3. A functionalized AFM probe of claims 1 ­2, wherein theprobe further comprises gold, aluminum, or platinum.

4. A functionalized AFM tip of claims 1­3, wherein thebiological material comprises a hybrid film comprising one ormore biomolecules chosen from neurotransmitters, ligands,amino acids, antibodies, sugars, peptides, proteins, fattyacids, spingolipids, and lipids.

5. A functionalized AFM tip of claims 1­4, wherein thebiological material comprises a hybrid film comprising one ormore biomolecules chosen from bioadhesives, cellattachment factors, biopolymers, blood proteins, enzymes,extracellular matrix proteins and biomolecules, growthfactors and hormones, 2'­ deoxy­nucleic acids, ribo­nucleicacids, receptors, synthetic biomolecules, synthetic DNA,synthetic RNA, synthetic biopolymers, synthetic peptides,recombinant proteins, synthetic enzyme inhibitors, syntheticand extracted vitamins, pharmaceuticals, and biologicallyactive ions.

6. A functionalized AFM tip of claim 5, wherein one or morebioadhesive is chosen from marine mussel adhesiveproteins, fibrin­like proteins, spider­web proteins, plant­derived adhesives, adhesives extracted from marine animals,insect­ derived adhesives, fibrin, fibroin, Mytilus edulis footprotein (mefpi , "mussel adhesive protein"), other musseladhesive proteins, proteins and peptides with glycine­richblocks, proteins and peptides with poly­alanine blocks, andsilks.

7. A functionalized AFM tip of claims 5­6, wherein one ormore cell attachment factor is chosen from the groupcomprising ankyrins, cadherins, connexins, dermatan

Functionalization of microscopy probe tips WO 2009001220 A2

ABSTRACT

The invention comprises a method of functionalizing scanning probe microscope(SPM) tips to image and/or measure interactions between surfaces, including thesurfaces of inorganic, organic­inorganic hybrid, organic, magnetic/conductive,hard coatings and biological materials. The invention further comprises the use ofatomic layer deposition (ALD) to functionalize SPM tips.

DESCRIPTION

FUNCTIONALIZATION OF MICROSCOPY PROBE TIPS

[001] This application claims priority to U.S. Patent Application Nos. 60/929,399,filed June 26, 2007; 60/935,008, filed July 23, 2007; and 61/039,890, filed March27, 2008, the contents of which are incorporated herein by reference.BACKGROUND AND SUMMARY AT THE INVENTION

[002] The invention relates to the functionalization of scanning probe microscope(SPM) probe tips to study interactions between inorganic, organic­ inorganichybrid, organic, and/or biological materials. The invention also relates to thefunctionalization of atomic force microscope (AFM) and magnetic forcemicroscope (MFM) probe tips. The invention further relates to the use of atomiclayer deposition (ALD) as the method to functionalize these tips.

[003] The invention also relates to the use of ALD to deposit organic and/orbiomolecules on probe tips, and the use of these tips to image sample surfacesand study the interactions between the material deposited on the tip and asample surface.

[004] Molecular biologists today do not have effective tools to image realtimecellular and sub­cellular "topo­dynamics." Instead, researches often image cellsthat are fixed and prepared for microscopy. Advanced molecular imagingtechniques that utilize fluorochromes, antibodies, and probes to analyzemolecular interactions are almost always restricted to imaging of biologicalprocesses that have been arrested by fixation. Therefore, our biological knowledgeis based in part on distorted still images of the dynamic situations that make uplife. The study of real­time topo­dynamics of molecular interactions that takeplace in and on cells will dramatically increase our understanding of cellular andsub­cellular functions, clearing the road for new advances in biological,biomedical, and biomaterial sciences.

[005] The invention thus further relates to a method for investigating, in real­time,the topo­dynamics of a biological surface using an SPM equipped with a probecomprising a cantilever and a tip coated via ALD with an organic and/or inorganicand/or organic­ inorganic hybrid and/or magnetic/conductive and/or hard coatedand/or biomolecular film. Scanning Probe Microscopy

[006] SPM is the branch of microscopy used to image and characterize samplesurfaces at the nanometer scale with a physical probe. The probe scans andinteracts with the surface to measure some property of that surface. SPM is abroad term used to describe various imaging applications. The type of interactionmeasured between the probe tip and the sample surface determines the type ofSPM technique required.

Atomic Force Microscopy

[007] AFM images sample surfaces. It can reveal structures precisely up to sub­nanometer resolution in three dimensions. For example, in one embodiment, theAFM raster­scans a surface using a probe, as shown in Figure 1. As the probe

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sulphate, entactin, fibrin, fibronectin, glycolipids, glycophorin,glycoproteins, heparan sulphate, heparin sulphate,hyaluronic acid, immunglobulins, keratan sulphate, integrins,laminins, N­CAMs (Calcium independent AdhesiveMolecules), proteoglycans, spektrin, vinculin, and vitronectin.

8. A functionalized AFM tip of claims 5­7, wherein one ormore biopolymer is chosen from the group comprisingalginates, Amelogenins, cellulose, chitosan, collagen,gelatins, oligosaccharides, and pectin.

9. A functionalized AFM tip of claims 5­8, wherein one ormore blood protein is chosen from the group comprisingalbumin, albumen, cytokines, factor IX, factor V, factor VII,factor VIII, factor X, factor Xl, factor XII, factor XIII,hemoglobins (with or without iron), immunglobulins(antibodies), fibrin, platelet derived growth factors (PDGFs),plasminogen, thrombospondin, and transferrin.

10. A functionalized AFM tip of claims 5­9, wherein one ormore enzyme is chosen from the group comprisingabzymes, adenylate cyclase, alkaline phosphatase,carboxylases, collagenases, cyclooxygenase, hydrolases,isomerases, ligases, lyases, metallo­matrix proteases,nucleases, oxidoreductases, peptidases, peptide hydrolase,peptidyl transferase, phospholipase, proteases, sucrase­isomaltase, TIMPs, and transferases.

11. A functionalized AFM tip of claims 5­10, wherein one ormore extracellular matrix protein or biomolecule is chosenfrom the group comprising ameloblastic amelogenins,collagens (I to XII), dentin­sialo­protein (DSP), dentin­ sialo­phospho­protein (DSPP), elastins, enamelin, fibrins,fibronectins, keratins (1 to 20), laminins, tuftelin,carbohydrates, chondroitin sulphate, heparan sulphate,heparin sulphate, hyaluronic acid, lipids and fatty acids, andlipopolysaccarides.

12. A functionalized AFM tip of claims 5­11 , wherein one ormore growth factor or hormone is chosen from the groupcomprising activins; Amphiregulin (AR); Angiopoietins (Ang 1to 4); Apo3 (a weak apoptosis inducer also known asTWEAK, DR3, WSL­1 , TRAMP or LARD); Betacellulin(BTC); Basic Fibroblast Growth Factor (bFGF, FGF­b);Acidic Fibroblast Growth Factor (aFGF, FGF­a); 4­1 BBLigand; Brain­derived Neurotrophic Factor (BDNF); Breastand Kidney derived Bolokine (BRAK); Bone MorphogenicProteins (BMPs); B­Lymphocyte Chemoattractant/B cellAttracting Chemokine 1 (BLC/BCA­1 ); CD27L (CD27 ligand);CD30L (CD30 ligand); CD40L (CD40 ligand); A Proliferation­inducing Ligand (APRIL); Cardiotrophin­1 (CT­1); CiliaryNeurotrophic Factor (CNTF); Connective Tissue GrowthFactor (CTGF); Cytokines; 6­cysteine Chemokine (ΘCkine);Epidermal Growth Factors (EGFs); Eotaxin (Eot); EpithelialCell­derived Neutrophil Activating Protein 78 (ENA­78);Erythropoietin (Epo); Fibroblast Growth Factors (FGF 3 to19); Fractalkine; Glial­derived Neurotrophic Factors (GDNFs);Glucocorticoid­ induced TNF Receptor Ligand (GITRL);Granulocyte Colony Stimulating Factor (G­ CSF);Granulocyte Macrophage Colony Stimulating Factor (GM­CSF); Granulocyte Chemotactic Proteins (GCPs); GrowthHormone (GH); I­309; Growth Related Oncogene (GRO);lnhibins (Inh); Interferon­inducible T­cell AlphaChemoattractant (I­TAC); Fas Ligand (FasL); Heregulins(HRGs); Heparin­Binding Epidermal Growth Factor­LikeGrowth Factor (HB­EGF); fms­like Tyrosine Kinase 3 Ligand(Flt­3L); Hemofiltrate CC Chemokines (HCC­1 to 4);Hepatocyte Growth Factor (HGF); Insulin; Insulin­like GrowthFactors (IGF 1 and 2); Interferon­gamma Inducible Protein 10

traverses the sample surface, small changes in the height of the cantilever tipmay be detected by a laser. Raster­scanning the probe across a sample surfacesgenerates a topological map of the surface.

[008] Atomic force microscopy has revolutionized the way in which researchersexplore biological structures at the single­molecule level. See B. P. Jena and J.K. Hόrber, "Atomic Force Microscopy in Cell Biology" in Methods in Cell Biology,San Diego: Academic Press, 2002. It can provide three­dimensional views ofsamples with minimal sample preparation. See A. Engel and D.J. Muller, NatureStructural Biology, 7(9): 715­18 (Sept. 2000). Compared to the conventionalinstruments for studying sample surfaces, such as the profilometer, the AFM hasa much sharper tip and small loading force. This may result in improved lateralresolution of surface images.

[009] In one embodiment, atomic force microscopy may allow researchers toimage samples under more natural aqueous conditions. In contrast, microscopictechniques relying on electron scanning (SEM) or electron transmission (TEM)image samples under high vacuum.

[010] The AFM not only maps surface topography, but may also be used to mapsurface forces. To probe surface forces, the AFM may "tap" a sample surfacewith the probe tip, as illustrated in Figure 2. By monitoring the cantileverdeflection and by knowing the spring constant of the cantilever, one may obtainadditional information about the sample, including its hardness and adhesiveness.Alternatively, the AFM may be operated with the tip in contact with the samplesurface.

[011] This molecular force probing (MFP) has emerged as a powerful tool forexploring intermolecular forces and dynamics. In some embodiments, MFP's

allow measurement of pico­newton (10"12 N) forces associated with singlemolecules. T.E. Fisher et al., Nature Structural Biology, 7(9): 719­24 (Sept.2000). This sensitivity allows the AFM to probe the molecular basis of biologicalphenomena and properties as diverse as molecular recognition [see B. Samori,Chemistry (Weinheim an der Bergstrasse, Germany) 6(23): 4249­55 (Dec. 1 ,2000); E. L. Florin et al., Science, 264(5157): 415­17 (Apr. 15, 1994)]; proteinfolding and unfolding [see A.F. Oberhauser et al., Nature, 393(6681 ): 181­85(May 14, 1988)]; DNA mechanics [see M. Rief et al., Nature Structural Biology,6(4): 346­49 (Apr. 1999)]; and focal cell adhesion [see M. Benoit et al., NatureCell Biology, 2(6): 313­ 17 (Jun. 2000)]. In one embodiment, AFM/MFP may beused to reveal the interaction between individual ligands and receptors, either onisolated molecules or on cellular surfaces.

[012] Other techniques are available for probing the interaction forces at biologicalsurfaces and may be used in conjunction with AFM. These include the use ofshear flow detachment [see J. L. Mege et al., Ce// Biophysics, 8(2): 141­60 (Apr.1986)], surface force apparatus [see D. E. Leckband et al., Science, 255(5050):1419­21 (Mar. 13, 1992)], biomembrane force probe [see R. Merkel et al., R,Nature, 397(6714): 50­53 (Jan. 7, 1999)], and optical tweezers [see A. Ashkin andJ. M. Dziedzic, Proc. Nat'l Acad. ScL U.S.A., 86(20): 7914­18 (Oct. 1989)].

[013] AFM/MFP is a force­measuring technique that can be used to map thenanoscale lateral distribution of single molecular recognition sites on biosurfaces.Procedures to probe the forces, dynamics, and localization of molecularrecognition interactions are now well established.

[014] AFM­Magnetic force microscopy (MFM) is a straightforward special mode ofoperation of non­contact scanning force microscope. Detection of magneticinteractions on a local scale is possible by equipping the force microscope with amagnetic probe, which then can be raster­scanned across any magnetic sample.MFM is applicable under various environmental conditions, in most cases evenwithout requiring any special sample preparation procedure. MFM is an importantanalytical tool whenever the near­surface stray­field variation of a magneticsample is of interest. See Koblischka and Hartmann, Ultramicroscopy, 97: 103­112 (2003).

[015] The recording industry became an important field of industrial application.See Rugar, Mamin et al., Journal of Applied Physics, 68: 1169­1183 (1990). MFMalso exhibits some serious shortcomings and they have not been overcome sofar: In the general situation, the method yields only qualitative information aboutthe magnetic object and it is difficult to improve the resolution to values below 100

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(IP­10); lnterleukins (IL 1 to 18); Interferon­gamma (IFN­gamma); Keratinocyte Growth Factor (KGF); KeratinocyteGrowth Factor­2 (FGF­10); Leptin (OB); Leukemia InhibitoryFactor (LIF); Lymphotoxin Beta (LT­B); Lymphotactin (LTN);Macrophage­Colony Stimulating Factor (M­CSF);Macrophage­derived Chemokine (MDC); MacrophageStimulating Protein (MSP); Macrophage InflammatoryProteins (MIPs); Midkine (MK); Monocyte ChemoattractantProteins (MCP­1 to 4); Monokine Induced by IFN­gamma(MIG); MSX 1 ; MSX 2; Mullerian Inhibiting Substance (MIS);Myeloid Progenitor Inhibitory Factor 1 (MPIF­1 ); NerveGrowth Factor (NGF); Neurotrophins (NTs); NeutrophilActivating Peptide 2 (NAP­ 2); Oncostatin M (OSM);Osteocalcin; OP­1 ; Osteopontin; OX40 Ligand; Plateletderived Growth Factors (PDGF aa, ab and bb); PlateletFactor 4 (PF4); Pleiotrophin (PTN); Pulmonary andActivation­regulated Chemokine (PARC); Regulated onActivation, Normal T­cell Expressed and Secreted(RANTES); Sensory and Motor Neuron­derived Factor(SMDF); Small Inducible Cytokine Subfamily A Member 26(SCYA26); Stem Cell Factor (SCF); Stromal Cell DerivedFactor 1 (SDF­1 ); Thymus and Activation­regulatedChemokine (TARC); Thymus Expressed Chemokine (TECK);TNF and ApoL­related Leukocyte­expressed Ligand­1 (TALL­1 ); TNF­related Apoptosis Inducing Ligand (TRAIL); TNF­related Activation Induced Cytokine (TRANCE); LymphotoxinInducible Expression and Competes with HSV GlycoproteinD for HVEM T­lymphocyte receptor (LIGHT); PlacentaGrowth Factor (PIGF); Thrombopoietin (Tpo); TransformingGrowth Factors (TGF alpha, TGF beta 1 , TGF beta 2);Tumor Necrosis Factors (TNF alpha and beta); VascularEndothelial Growth Factors (VEGF­A1B1C and D).

13. A functionalized AFM tip of claims 5­12, wherein one ormore 2'­ deoxy nucleic acid is chosen from the groupcomprising A­DNA; B­DNA; artificial chromosomes carryingmammalian DNA (YACs); chromosomal DNA; circular DNA;cosmids carrying mammalian DNA; DNA; Double­strandedDNA (dsDNA); genomic DNA; hemi­methylated DNA; linearDNA; mammalian cDNA (complimentary DNA; DNA copy ofRNA); mammalian DNA; methylated DNA; mitochondrialDNA; phages carrying mammalian DNA; phagemids carryingmammalian DNA; plasmids carrying mammalian DNA;plastids carrying mammalian DNA; recombinant DNA;restriction fragments of mammalian DNA; retroposonscarrying mammalian DNA; single­ stranded DNA (ssDNA);transposons carrying mammalian DNA; T­DNA; and virusescarrying mammalian DNA; Z­DNA.

14. A functionalized AFM tip of claims 5­13, wherein one ormore ribonucleic acid is chosen from the group comprisingacetylated transfer RNA (activated tRNA, charged tRNA);circular RNA; linear RNA; mammalian heterogeneousnuclear RNA (hnRNA), mammalian messenger RNA(mRNA); mammalian RNA; mammalian ribosomal RNA(rRNA); mammalian transport RNA (tRNA); mRNA; poly­adenylated RNA; ribosomal RNA (rRNA); recombinant RNA;retroposons carrying mammalian RNA ; ribozymes; transportRNA (tRNA); and viruses carrying mammalian RNA.

15. A functionalized AFM tip of claims 5,­14 wherein one ormore receptor is chosen from the group comprising the CDclass of receptors CD; EGF receptors; FGF receptors;Fibronectin receptor (VLA­5); Growth Factor receptor, IGFBinding Proteins (IGFBP 1 to 4); lntegrins (including VLA 1­4); Laminin receptor; PDGF receptors; Transforming GrowthFactor alpha and beta receptors; BMP receptors; Fas;Vascular Endothelial Growth Factor receptor (Flt­1 ); and

nm See Hartmann, Annual Review of Materials Science, 29: 53­ 87 (1999);Wickramasinghe, Acta Materialia, 48: 347­358 (2000).

[016] Commercially available MFM tips comprise magnetic or conductive coatingswith radius > 30 nm. However, reducing this radius, in order to improve theresolution of the image, usually does not provide adequate conductivity due to thechanging geometry of the tip.

[017] The spatial resolution obtained by MFM may be related to both themagnetized part of the probe, which is actually exposed to the sample stray field,and to the probe­sample distance. Thus, in order to improve the lateral resolution,one may decrease the coated layer thickness. By coating tips by an atomic layerand/or several layers of conductive and/or magnetic material (Fe, Co, Ni, Nd2Fe­

I4B, Samarium­cobalt magnets (SmCo5), ferrites which are various mixtures of

iron oxides such as Hematite (Fe2O3) or Magnetite (Fe3O4) and the oxides of

other metals, Alnico which is an alloy composed primarily of aluminium, nickeland cobalt, PANiCNQ, which is a combination of emeraldine­based polyaniline(PANi) and tetracyanoquinodimethane (TCNQ)) with the described ALD techniqueone will increase and achieve higher resolution.

[018] MFM coated tips may be used as a tool to characterize the magneticproperties of magnetic recording media such as harddrive, memory cards, andmagnetic strips, to map the surfaces of semiconductor chips for criticaldimension control, or to map Dynalbeads or other magnetic spheres withantibodies attached onto cellmembranes.

SPM Tips

[019] SPM tips are probe tips comprising, for example, SiN, Si3N4, or SiN. The

tip may be coated, for example, with gold, aluminum, or platinum. These tips mayaslo be coated with other materials, for example MFM tips may be coated with aferromagnetic film. Some probe tips are coated using imprecise techniques thatmay deposit multilayered coatings, which can severely affect the spring constantof the cantilevers in unpredictable ways dramatically decreasing the resolution ofSPM images and maps. Moreover, these coating techniques could in many caseinduce a bending of the cantilever that prevent the laser from reflecting onto thedetector with a correct angle. In this case, these cantilevers are useless,impossible to calibrate. Care should be taken to avoid these potential problems.

[020] Various research efforts have focused on coating the available probe tipswith other materials. For example, a Langmuir­Blodgett trough may be used todeposit a monolayer of ampiphillic molecules. However, this technique exerts noatomic control of the deposited material and has limited application to inorganicmaterials.

[021] Some industry­specific SPM applications require a physical contact tip­hardsurface, for example for quality control in the production process for siliconwafers, magnetic hard disks and tapes. When tip­hard surfaces are needed,prolonging the integrity of the probe tip is desired for long­term use, specifically forhigh resolution.

[022] A probe tip with a longer lifetime can reduce the cost to the companyrequiring tip­hard surfaces, for example in labor costs (e.g. manually changing thecantilever) and production costs (e.g. delay in the quality control process).

ALD

[023] ALD deposits films one monolayer at a time using alternating self­terminating gas­to­surface reactions. H.S. Nalwa (ed.), Handbook of Thin FilmMaterials, Vol. 1 , Academic Press, San Diego, CA, 2001. ALD may also bereferred to as atomic layer epitaxy ("ALE") or as atomic layer chemical vapordeposition ("ALCVD").

[024] The deposition of alumina (AI2O3) by ALD, which is known in the art, is one

non­limiting example of how the technique works. See Figure 3. First, thealuminum atoms are placed on the surface using the precursor materialtrimethylaluminum (AI(CH3)3, TMA). Most materials have a native monolayer of

hydroxyl groups on the terminating surface. These hydroxyl groups serve asactive sites for the reaction with TMA. When TMA is pulsed into the reactionchamber, it reacts with all sterically available hydroxyl groups and forms a new

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Vitronectin receptor.

16. A functionalized AFM tip of claims 5­15, wherein one ormore synthetic DNA is chosen from the group comprising A­DNA; antisense DNA; B­ DNA; complimentary DNA (cDNA);chemically modified DNA; chemically stabilized DNA; DNA ;DNA analogues ; DNA oligomers; DNA polymers; DNA­RNAhybrids; double­stranded DNA (dsDNA); hemi­methylatedDNA; methylated DNA; single­ stranded DNA (ssDNA);recombinant DNA; triplex DNA; T­DNA; and Z­DNA.

17. A functionalized AFM tip of claims 5­16, wherein one ormore synthetic RNA is chosen from the group comprisingantisense RNA; chemically modified RNA; chemicallystabilized RNA; heterogeneous nuclear RNA (hnRNA);messenger RNA (mRNA); ribozymes; RNA; RNA analogues;RNA­DNA hybrids; RNA oligomers; RNA polymers;ribosomal RNA (rRNA); and transport RNA (tRNA).

18. A functionalized AFM tip of claims 5­17, wherein one ormore synthetic biopolymer is chosen from the groupcomprising cationic and anionic liposomes; celluloseacetate; hyaluronic acid; polylactic acid; polyglycol alginate;polyglycolic acid; poly­prolines; polysaccharides.

19. A functionalized AFM tip of claims 5­18, wherein one ormore synthetic biopolymer is chosen from the groupcomprising decapeptides containing DOPA and/or diDOPA;peptides with sequence "Ala Lys Pro Ser Tyr Pro Pro Thr TyrLys"; peptides where Pro is substituted with hydroxyproline;peptides where one or more Pro is substituted with DOPA;peptides where one or more Pro is substituted with diDOPA;peptides where one or more Tyr is substituted with DOPA;peptide hormones; peptide sequences based on the abovelisted extracted proteins; peptides containing an RGD (ArgGIy Asp) motif.

20. A functionalized AFM tip of claims 5­19, wherein one ormore synthetic enzyme inhibitor is chosen from the groupcomprising pepstatin, poly­ prolines, D­sugars, D­aminocaids, cyanide, diisopropyl fluorophosphates (DFP),metal ions, N­tosyl­l­phenylalaninechloromethyl ketone(TPCK), Physostigmine, Parathion, and penicillin.

21. A functionalized AFM tip of claims 5­20, wherein one ormore synthetic or extracted vitamin is chosen from the groupcomprising biotin; calciferol (vitamin D's; vital for bonemineralisation); citrin; folic acid; niacin; nicotinamide;nicotinamide adenine dinucleotide (NAD, NAD+);nicotinamide adenine dinucleotide phosphate (NADP,NADPH); NAD+retinoic acid (vitamin A); riboflavin; vitaminB's; vitamin C; vitamin E; and vitamin K's.

22. A functionalized AFM tip of claims 5­21 , wherein one ormore pharmaceuticals is chosen from the group comprisingantibiotics, cyclooxygenase inhibitors, hormones,inflammation inhibitors, NSAI D's, painkillers, prostaglandinsynthesis inhibitors, steroids, and tetracycline.

23. A functionalized AFM tip of claims 5­22, wherein one ormore biomolecules is chosen from the group comprisingadenosine di­phosphate (ADP); adenosine mono­phosphate(AMP); adenosine tri­phosphate (ATP); amino acids; cyclicAMP (cAMP); 3,4­dihydroxyphenylalanine (DOPA); 5'­di(dihydroxyphenyl­l_­ alanine (diDOPA); diDOPA quinone;DOPA­like o­diphenols; fatty acids; glucose; hydroxyproline;nucleosides; nucleotides (RNA and DNA bases);prostaglandin; sugars; sphingosine 1 ­phosphate; andrapamycin.

24. A functionalized AFM tip of any of the preceding claims,

terminating layer of AI(CH3)X fragments. In addition, some methane (CH4) is

formed as a byproduct. This process is self­limiting in that the reaction stopswhen no more active surface sites are available for reaction. Excess TMA andmethane may be removed from the reaction chamber under vacuum and perhapsby purging with inert gas.

[025] In one embodiment, the next step in the process is to introduce water in thegas phase. Water will react with all available methyl (CH3) groups, forming a new

hydroxyl­terminated surface and more methane. This step is also self­limiting.Excess water and methane may be removed from the system under vacuum andperhaps by purging with inert gas.

[026] The surface now once again has a monolayer of hydroxyl groups as itsterminating layer. Thus, the procedure may be repeated until the desired filmthickness is achieved, depositing exactly one monolayer each cycle.

[027] Different precursors may be used to produce inorganic films. Appropriateprecursors include, but are not limited to, those shown in the table below.

[028] Additional precursors capable of producing inorganic films are well­ known inthe art. See, e.g., R. L. Puurunen, J. Appl. Phys., 97: 121301­52 (2005).Inorganic precursors include, but are not limited to BCI3, BBr3, B(OMe)3, AICI3,

AIBr3, AIMe2CI, AIMe2O',Pr, AIMe2H1 AI(OEt)3, AI(O0Pr)3, a trialkyl aluminum,

GaCI3, GaMe3, GaCI, GaBr, GaI, GaMe3, GaEt3, Ga(acac)3, Ga, GaEt2CI,

GaEt2Me, InCI3, InMe3, InEt3, ln(acac)3, In1, InEtMe2, InCI, InCIMe2, CF3, SiCI4,

SiCI3H1 SiCI2H2, SiH4, Si2H6, SiCI3H, Si(OEt)4, Si(O"Bu)4, ('BuO)3SiOH, GeCI4,

GeMe2H2, GeEt2H2, GeH4, Ge2H6, SnCI4, SnEt4, SnMe4, SnI4, SnCI4, PbBr2,

PbI2, Pb(OAc)2, Pb(O1Bu)2, Pb(thd)2, Pb(detdc)2, YCp3, Y(CpMe)3, Y(thd)3, Cd,

CdMe2, CdCI2, PCI3, POCI3, SbCI5, Bi(Ph)3, Bi[N(SiMe3)2]3 TiCI4, TiI4,

Ti(NMe2)4, Ti(NEt2)4, Ti(NMeEt)4, Ti(O'Pr)4, Ti(OEt)4, ZrCI4, ZrI4, ZrCp2CI2,

ZrCp2Me2, Zr(O'Pr)2(dmae)2, Zr(O^Bu)4, Zr(dmae)4, Zr(thd)4, Zr(NMe2)4,

Zr(NEtMe)4, Zr(NEt2J4, HfCI4, HfCI2[N(SiMe3)2]2, HfI4, Hf(O^Bu)4,

Hf(OfBu)2(mmp)2, Hf(mmp)4, Hf(ONEt2)4, Hf(NEt2J4, Hf(NEtMe)4,

Hf[N(SiMe3)2]2CI2, Hf(NOs)4, VOCI3, VO(C1Pr)3, VO(acac)2, Nb(OEt)5, NbCI5,

TaF5, TaCI5, TaBr5, TaI5, Ta(OEt)5, Ta(NMe2)5, Ta(NEt2),, Ta(NEt)(NEt2)3,

Ta(NfBu)(NEt2)3, Ta(NfBu)(NEtMe)3, CrO2CI2, Cr(acac)3, MoCI5, WF6, WOCI4,

WFxOy, W(N'Bu)2(NMe2)2, Mn(thd)3, MnCI2, Mn, Fe(acac)3, FeCI3, Fe(thd)3,

Fe(^BuAMD)2, Ru(CpEt)2, RuCp2, Ru(Od)3, Ru(thd)3, Co('PrAMD)2, Co(acac)3,

Co(thd)2, lr(acac)Ca(thd)3, NiCp2, Ni(acac)2, Ni(apo)2, Ni(dmg)2, Ni('PrAMD)2,

Ni(thd)2, Pd(thd)2, Pd(hfac)2, Pt(CpMe)Me3, Pt(acac)2) Cu(acac)2, Cu(thd)2,

Cu(hfac)2, CuCI, Cu ('PrAM D), ZnCI2, ZnMe2, ZnEt2, Zn(OAc)2, Zn,

Zn[N(SiMe3)2], HgMe2, Mg, Mg(Cp)2, Mg(thd)2, Ca(thd)2, CaCp2, Sr(Cp'Pr3)2,

Sr(thd)2, Sr(methd)2, Sr(CpMe5)2, Ba(CpMe5)2, Ba(thd)2, ScCp3, Sc(thd)3,

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wherein the biological material comprises a serotonin hybridfilm.

25. A functionalized AFM tip of any of the preceding claims,wherein the organic material comprises a film containingpeptide bonds.

26. A functionalized AFM tip of any of the preceding claims,wherein the organic material is chosen from a peptide filmand a polyamide film.

27. A functionalized AFM tip of any of the preceding claims,wherein the organic­inorganic hybrid material is chosen froma glycine hybrid film, a 4­ aminobenzoic acid hybrid film, anda 4­aminobenzophenone hybrid film.

28. A functionalized AFM tip of claims any of the precedingclaims, wherein the bone­like material is chosen fromcalcium phosphate and Ca2(PO4)F.

29. A functionalized AFM tip of claims any of the precedingclaims, wherein the implant­like material comprises one ormore sodium oxides, silicate, calcium oxides, calciumsulfates, calcium phosphates, calcium carbonates,hydroxyapatite, hydrides, vanadium, platinum, hafnium, goldhydroxide, fluorides and oxides of titanium, ferro­titaniumalloys, and tantalum metals.

30. A method of coating an AFM tip comprising using ALD tocoat said tip with a biological, organic, organic­inorganichybrid, bone­like, or implant­like material.

31. The method of claim 30, comprising exposing said AFMtip to a precursor comprising a reactive inorganic species inan inert environment, optionally purging said environmentwith an inert gas, exposing said tip to a biological material inan inert environment, and optionally purging said environmentwith an inert solvent.

32. The method of claims 30­31 , wherein the biologicalmaterial is chosen from neurotransmitters, ligands, aminoacids, nucleic acids, antibodies, sugars, peptides, proteins,fatty acids, spingolipids, and lipids.

33. The method of claims 30­32, wherein the biologicalmaterial is chosen from bioadhesives, cell attachmentfactors, biopolymers, blood proteins, enzymes, extracellularmatrix proteins and biomolecules, growth factors andhormones, 2'­ deoxy­nucleic acids, ribo­nucleic acids,receptors, synthetic biomolecules, synthetic DNA, syntheticRNA, synthetic biopolymers, synthetic peptides,recombinant proteins, synthetic enzyme inhibitors, syntheticand extracted vitamins, pharmaceuticals, and biologicallyactive ions.

34. The method of claim 33, wherein the bioadhesive ischosen from marine mussel adhesive proteins, fibrin­likeproteins, spider­web proteins, plant­ derived adhesives,adhesives extracted from marine animals, insect­derivedadhesives, fibrin, fibroin, Mytilus edulis foot protein (rnefpi ,"mussel adhesive protein"), other mussel adhesive proteins,proteins and peptides with glycine­rich blocks, proteins andpeptides with poly­alanine blocks, and silks.

35. The method of claims 33­34, wherein the cell attachmentfactor is chosen from ankyrins, cadherins, connexins,dermatan sulphate, entactin, fibrin, fibronectin, glycolipids,glycophorin, glycoproteins, heparan sulphate, heparinsulphate, hyaluronic acid, immunglobulins, keratan sulphate,integrins, laminins, N­ CAMs (Calcium independent AdhesiveMolecules), proteoglycans, spektrin, vinculin, and vitronectin.

La[N(SiMe3)2]3, La(1PrAMD)3, La(thd)3, Ce(thd)4, Ce(thd)3phen, Pr[N(SiMe3)2]3,

Nd(thd)3, Sm(thd)3, Eu(thd)3, Gd(thd)3, Dy(thd)3, Ho(thd)3, Er(thd)3, Tm(thd)3>and Lu[Co(SiMe3)]2CI.

[029] ALD's unique deposition mechanism distinguishes it from other depositionor crystal growth techniques. First, ALD exhibits different growth dynamics. Thisis because the precursors attach only at the available surface sites, and not ontop of the same precursor molecules that have already attached. The packingdensity of the precursors on the surface controls the growth rate. Therefore, unlikemost other deposition and crystal growth techniques, ALD monolayer growth maynot depend on the distribution of the precursor or rate of formation of growth stepson the crystallites forming the film.

[030] The deposition mechanism may also make ALD unique in its ability tosequentially deposit substantially uniform monolayers on all exposed surfaces.For example, this may result in conformal coverage of cantilevers, and mayprovide a thin film that may not significantly influence the spring constant.

BRIEF DESCRIPTION OF THE DRAWINGS

[031] Figure 1 shows the function of an AFM probe. As the probe scans asurface, small changes in the height of the cantilever tip are detected by a laser.

[032] Figure 2 shows the AFM in the force­probing "tapping" mode. The AFMprobe taps the sample surface.

[033] Figure 3 demonstrates the ALD growth of alumina.

[034] Figure 4 shows the growth rate of TiO2 from TiCI4 and H2O and AI2O3 from

TMA and H2O as a function of temperature using a pulsing sequence of 2 s of 2 s

metal precursor, 1 s purge, 2 s H2O, and 1 s purge.

[035] Figure 5 shows a force curve generated from a human osteoblast cellprobed with an AFM tip that was coated with TiO2 by ALD.

[036] Figure 6 shows a force curve generated from a sample area with little or noadhesion to an AFM tip that was coated with TiO2 by ALD. This force curve likely

reveals the interaction of the TiO2 tip and the SiO2 sample substrate. [037] Figure

7 is a map of an area of human osteoblast. Darker areas have less adhesion,whereas lighter area show strong adhesion.

[038] Figure 8 shows the temperature dependence of the growth rate of CaCO3 by

ALD.

[039] Figure 9 shows the quartz crystal microbalance (QCM) (also known asquarts crystal monitor (QCM)) results from a deposition using TMA and glycine ina pulsing pattern of 1 s TMA, 1 s purge, 2 s glycine, 1 s purge.

[040] Figure 10 shows the QCM results from the deposition using TiCI4 and

glycine in a pulsing pattern of 1 s TiCI4, 1 s purge, 2 s glycine, 1 s purge.

[041] Figure 11 shows the QCM results from the deposition using TiCI4 and 4­

aminobenzoic acid in a pulsing sequence of 4 s TMA, 3 s purge, 7 s 4­aminobenzoic acid, 3 s purge.

[042] Figure 12 shows the QCM results from the deposition using TiCI4 and 4­

aminobenzophenone in a pulsing pattern of 1 s TiCI4, 2 s purge, 1 s 4­

aminobenzophenone, 1 s purge.

[043] Figure 13 shows an AFM probe tip coated with an organic functional group(A), and molecular force probing of the surface of a bone cell. B is the adhesiveforce between the peptide and its receptor, C. D represents another receptor thatis not specific to the peptide coating.

[044] Figure 14 shows an uncoated probe tip, a conventional magnetic coatedprobe tip, and a probe tip coated according to the methods described herein. Thegeometry of the conventional coated tip is changed in comparison with the tipcoated using the disclosed method.

[045] Figure 15 shows the change in appearance of an uncoated probe tipcompared with a probe tip prepared according to the disclosed coating method.

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36. The method of claims 33­35, wherein the biopolymer ischosen from alginates, Amelogenins, cellulose, chitosan,collagen, gelatins, oligosaccharides, and pectin.

37. The method of claims 33­36, wherein the blood protein ischosen from albumin, albumen, cytokines, factor IX, factor V,factor VII, factor VIII, factor X, factor Xl, factor XII, factor XIII,hemoglobins (with or without iron), immunglobulins(antibodies), fibrin, platelet derived growth factors (PDGFs),plasminogen, thrombospondin, and transferrin.

38. The method of claims 33­37, wherein the enzyme ischosen from abzymes, adenylate cyclase, alkalinephosphatase, carboxylases, collagenases, cyclooxygenase,hydrolases, isomerases, ligases, lyases, metallo­matrixproteases, nucleases, oxidoreductases, peptidases, peptidehydrolase, peptidyl transferase, phospholipase, proteases,sucrase­isomaltase, TIMPs, and transferases.

39. The method of claims 33­38, wherein the extracellularmatrix protein or biomolecule is chosen from ameloblastin,amelogenins, collagens (I to XII), dentin­sialo­protein (DSP),dentin­sialo­phospho­protein (DSPP), elastins, enamelin,fibrins, fibronectins, keratins (1 to 20), laminins, tuftelin,carbohydrates, chondroitin sulphate, heparan sulphate,heparin sulphate, hyaluronic acid, lipids and fatty acids, andlipopolysaccarides.

40. The method of claims 33,­39 wherein the growth factor orhormone is chosen from activins; Amphiregulin (AR);Angiopoietins (Ang 1 to 4); Apo3 (a weak apoptosis induceralso known as TWEAK, DR3, WSL­1 , TRAMP or LARD);Betacellulin (BTC); Basic Fibroblast Growth Factor (bFGF,FGF­b); Acidic Fibroblast Growth Factor (aFGF, FGF­a); 4­1BB Ligand; Brain­derived Neurotrophic Factor (BDNF);Breast and Kidney derived Bolokine (BRAK); BoneMorphogenic Proteins (BMPs); B­LymphocyteChemoattractant/B cell Attracting Chemokine 1 (BLC/BCA­1); CD27L (CD27 ligand); CD30L (CD30 ligand); CD40L (CD40ligand); A Proliferation­inducing Ligand (APRIL);Cardiotrophin­1 (CT­1 ); Ciliary Neurotrophic Factor (CNTF);Connective Tissue Growth Factor (CTGF); Cytokines; 6­cysteine Chemokine (6Ckine); Epidermal Growth Factors(EGFs); Eotaxin (Eot); Epithelial Cell­derived NeutrophilActivating Protein 78 (ENA­78); Erythropoietin (Epo);Fibroblast Growth Factors (FGF 3 to 19); Fractalkine; GMaI­derived Neurotrophic Factors (GDNFs); Glucocorticoid­induced TNF Receptor Ligand (GITRL); Granulocyte ColonyStimulating Factor (G­CSF); Granulocyte MacrophageColony Stimulating Factor (GM­CSF); GranulocyteChemotactic Proteins (GCPs); Growth Hormone (GH); I­309;Growth Related Oncogene (GRO); lnhibins (Inh); Interferon­inducible T­cell Alpha Chemoattractant (I­TAC); Fas Ligand(FasL); Heregulins (HRGs); Heparin­Binding EpidermalGrowth Factor­Like Growth Factor (HB­EGF); fms­likeTyrosine Kinase 3 Ligand (Flt­3L); Hemofiltrate CCChemokines (HCC­1 to 4); Hepatocyte Growth Factor (HGF);Insulin; Insulin­ like Growth Factors (IGF 1 and 2); Interferon­gamma Inducible Protein 10 (IP­10); lnterleukins (IL 1 to 18);Interferon­gamma (IFN­gamma); Keratinocyte Growth Factor(KGF); Keratinocyte Growth Factor­2 (FGF­10); Leptin (OB);Leukemia Inhibitory Factor (LIF); Lymphotoxin Beta (LT­B);Lymphotactin (LTN); Macrophage­ Colony Stimulating Factor(M­CSF); Macrophage­derived Chemokine (MDC);Macrophage Stimulating Protein (MSP); MacrophageInflammatory Proteins (MIPs); Midkine (MK); MonocyteChemoattractant Proteins (MCP­1 to 4); Monokine Inducedby IFN­gamma (MIG); MSX 1 ; MSX 2; Mullerian InhibitingSubstance (MIS); Myeloid Progenitor Inhibitory Factor 1

[046] Figure 16 shows results from X­ray diffraction analysis used to measureTiO2 thickness.

[047] Figure 17 shows SEM images of cantilevers demonstrating themeasurement of the width of cantilevers at the pyramidal base of the tip, beforeand after TiO2 deposition.

[048] Figure 18 shows SEM images of cantilever tip morphology before and afterTiO2 deposition (A= control, B= 1 nm, C=5.3nm, D=10.9nm, E=98.4nm,

F=114nm of Tiθ2). The change in tip morphology is more pronounced with tips

coated with 98.4 and 114nm Of TiO2.

[049] Figure 19 shows spring constant measurements before and after TiO2coating.

[050] Figure 20 shows adhesion force results between non­coated and TiO2coated (6nm) tips versus Nunclon surface in liquid. The results show a significantdifference in adhesive force for TiO2 coated cantilevers versus non­ coated on

Nunclon (p<0.001 ).

[051] Figure 21 shows adhesion force results between uncoated and TiO2 coated

(6nm) tips versus collagen I surface in liquid. The results show a significantdifference in adhesive force for TiO2 coated cantilevers versus collagen I (p<0.001

).

[052] Figure 22A shows SEM images of uncoated cantilever tips before and after18 scans with AFM against ZrO2. Figure 22B shows the before and after images

superposed.

[053] Figure 23 shows AFM images of uncoated cantilever tip surface morphologybefore and after 18 scans with AFM.

[054] Figure 24A shows SEM images of TiO2 coated cantilever tips before and

after 18 scans with AFM against ZrO2. Figure 24B shows the before and after

images superposed.

[055] Figure 25 shows AFM images of TiO2 coated cantilever tip morphology

before and after 18 scans with AFM.

[056] Figure 26A shows SEM images of ZrO2 coated cantilever tips before and

after 18 scans with AFM against ZrO2. Figure 26B shows the before and after

images superposed.

[057] Figure 27 shows AFM images of ZrO2 coated cantilever tip morphology

before and after 18 scans with AFM.

[058] Figure 28A shows SEM images of AI2O3 coated cantilever tips before and

after 18 scans with AFM against ZrO2. Figure 28B shows the before and after

images superposed.

[059] Figure 29 shows AFM images of AI2O3 coated cantilever tip morphology

before and after 18 scans with AFM. [060] Figure 30 shows AFM images of afloppy disk scanned with an AC240 cantilever (Si, uncoated) demonstrating thatthe AC240 uncoated cantilever could not detect the magnetic informationcontained in the floppy disk.

[061] Figure 31 shows AFM images of a floppy disk scanned with a standardMFM cantilever which did demostrate that the MFM cantilever could detectinformation contained in a floppy disk.

[062] Figure 32 shows AFM images of a floppy disk scanned with Fe2CoO4coated cantilever. The results indicate that the Fe2CoO4 coated cantilever had

higher sensitivity to the magnetic field.

[063] Figure 33 shows AFM images of a floppy disk scanned with a RC800 PSA(SiN) uncoated cantilever demonstrating that the RC800 cantilever could notdetect the magnetic information contained in the floppy disk.

[064] Figure 34 shows AFM images of a floppy disk scanned with an RC800 PSA

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(MPIF­1 ); Nerve Growth Factor (NGF); Neurotrophins (NTs);Neutrophil Activating Peptide 2 (NAP­2); Oncostatin M(OSM); Osteocalcin; OP­1 ; Osteopontin; OX40 Ligand;Platelet derived Growth Factors (PDGF aa, ab and bb);Platelet Factor 4 (PF4); Pleiotrophin (PTN); Pulmonary andActivation­regulated Chemokine (PARC); Regulated onActivation, Normal T­cell Expressed and Secreted(RANTES); Sensory and Motor Neuron­ derived Factor(SMDF); Small Inducible Cytokine Subfamily A Member 26(SCYA26); Stem Cell Factor (SCF); Stromal Cell DerivedFactor 1 (SDF­1 ); Thymus and Activation­regulatedChemokine (TARC); Thymus Expressed Chemokine (TECK);TNF and ApoL­related Leukocyte­expressed Ligand­1 (TALL­1 ); TNF­related Apoptosis Inducing Ligand (TRAIL); TNF­related Activation Induced Cytokine (TRANCE); LymphotoxinInducible Expression and Competes with HSV GlycoproteinD for HVEM T­lymphocyte receptor (LIGHT); PlacentaGrowth Factor (PIGF); Thrombopoietin (Tpo); TransformingGrowth Factors (TGF alpha, TGF beta 1 , TGF beta 2);Tumor Necrosis Factors (TNF alpha and beta); VascularEndothelial Growth Factors (VEGF­A1B1C and D).

41. The method of claims 33­40, wherein the 2'­deoxynucleic acid is chosen from A­DNA; B­DNA; artificialchromosomes carrying mammalian DNA (YACs);chromosomal DNA; circular DNA; cosmids carryingmammalian DNA; DNA; Double­stranded DNA (dsDNA);genomic DNA; hemi­methylated DNA; linear DNA;mammalian cDNA (complimentary DNA; DNA copy of RNA);mammalian DNA; methylated DNA; mitochondrial DNA;phages carrying mammalian DNA; phagemids carryingmammalian DNA; plasmids carrying mammalian DNA;plastids carrying mammalian DNA; recombinant DNA;restriction fragments of mammalian DNA; retroposonscarrying mammalian DNA; single­stranded DNA (ssDNA);transposons carrying mammalian DNA; T­DNA; and virusescarrying mammalian DNA; Z­DNA.

42. The method of claims 33­41 , wherein the ribo­nucleicacid is chosen from acetylated transfer RNA (activatedtRNA, charged tRNA); circular RNA; linear RNA; mammalianheterogeneous nuclear RNA (hnRNA), mammalianmessenger RNA (mRNA); mammalian RNA; mammalianribosomal RNA (rRNA); mammalian transport RNA (tRNA);mRNA; poly­adenylated RNA; ribosomal RNA (rRNA);recombinant RNA; retroposons carrying mammalian RNA ;ribozymes; transport RNA (tRNA); and viruses carryingmammalian RNA.

43. The method of claims 33,­42 wherein the receptor ischosen from the group comprising the CD class of receptorsCD; EGF receptors; FGF receptors; Fibronectin receptor(VLA­5); Growth Factor receptor, IGF Binding Proteins(IGFBP 1 to 4); lnteghns (including VLA 1­4); Lamininreceptor; PDGF receptors; Transforming Growth Factor alphaand beta receptors; BMP receptors; Fas; VascularEndothelial Growth Factor receptor (Flt­1 ); and Vitronectinreceptor.

44. The method of claims 33­43, wherein the synthetic DNAis chosen from A­DNA; antisense DNA; B­DNA;complimentary DNA (cDNA); chemically modified DNA;chemically stabilized DNA; DNA ; DNA analogues ; DNAoligomers; DNA polymers; DNA­RNA hybrids; double­stranded DNA (dsDNA); hemi­ methylated DNA; methylatedDNA; single­stranded DNA (ssDNA); recombinant DNA;triplex DNA; T­DNA; and Z­DNA.

45. The method of claims 33,­44 wherein the synthetic RNA

(SiN) cantilever coated woth Fe2O3 which did demonstrate that the Fe2O3 coated

cantilever had higher sensitivity to the magnetic field than the uncoated cantilever.

[065] Figure 35 shows AFM images of a floppy disk scanned with an RC800 PSA(SiN) cantilever coated woth CoFe3O4 which did demonstrate that the Fe2O3coated cantilever had higher sensitivity to the magnetic field than the uncoatedcantilever.

DETAILED DESCRIPTION

[066] Surprisingly, ALD may be used to deposit a myriad of coatings on probetips, expanding the range of surface imaging and force­probing capabilities. Theinvention comprises functionalizing probe tips to image sample surfaces and toprobe interactions between the tips and sample surfaces, for example for AFMand MFM applications. The invention further comprises the use of ALD as themethod of functionalization. The invention also comprises the actual use of thefunctionalized tips to image sample surfaces and/or to probe interactions betweenthe tips and sample surfaces. The material coating the tip and the sample surfacemay be the same or different, and may be inorganic, organic­inorganic hybrid,organic, magnetic/conductive, biological materials, and/or hard coatings. [067]The invention further comprises the use of ALD to produce organic and/orbiomolecular films. These films may be deposited on any appropriate surface, forexample on an AFM tip. A tip coated with an organic and/or biomolecular filmmay be used to image sample surfaces and probe intermolecular forces betweenthe surface and the organic and/or biomolecular tip coating. Appropriate samplesurfaces include any surface that may be imaged by SPM, however, biologicalsurfaces are of particular interest.

[068] In one embodiment, ALD deposits substantially uniform films of pinhole­freematerials on substrates having complex geometrical shapes. This technique ofmodifying probe tips may provide several advantages. For example, in oneembodiment, ALD conserves the low spring constant of the probe. Therefore, itmay modify tips while retaining the high resolution of the SPM.

[069] As used herein, "substantially uniform" means that there is a detectableamount of material on all exposed surfaces. "Substantially uniform" films mayinclude, but are not limited to, films resulting from the sequential deposition of oneor more monolayers of material, wherein each monolayer may be the same ordifferent as the previous layer.

[070] The term "monolayer" a film or layer of material approximately one moleculeof precursor thick. The term therefore may vary depending on the film beingdeposited.

[071] Examples of other embodiments of using ALD may include at least one ofthe following advantages. The technique is well­suited to the deposition ofinorganic, organic, organic­inorganic hybrid, and biological materials. At relativelylow temperatures, it can produce surfaces terminated by a desired functionality.Tailoring the surfaces of probe tips may allow one to construct and study amyriad of tip­specimen surface interactions on a molecular level.

[072] In one embodiment, a precursor molecule should not undergo reactions withitself.

[073] In order to improve and/or ensure the saturation density of the terminatinghydroxyl groups on the surface of the SPM tip, the surface may be pulsed withwater before depositing the initial precursor molecule. Any excess water may bepurged or evacuated from the reaction chamber before pulsing the surface with thefirst precursor. [074] In one embodiment, the functionalization of the probe tipscan be performed by deposition of one or more monolayers of different materialsthat either mimic bone, simple extracellular matrices, implant surfaces, orbiological signal molecules (ligands).

Bone­Like Materials

[075] In one embodiment, ALD may be used to produce a film that mimics thetermination of bone material by terminating in, for example, a calcium phosphatelayer. Bone­ or implant­like materials are those that either induce or conductformation of new bone. Such material often have chemical structures involvingsodium oxides, silicate, calcium oxides, calcium sulphates, calcium phosphates,calcium carbonates, hydroxyapatite, hydrides, vanadium, platinum, hafnium, gold

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is chosen from antisense RNA; chemically modified RNA;chemically stabilized RNA; heterogeneous nuclear RNA(hnRNA); messenger RNA (mRNA); ribozymes; RNA; RNAanalogues; RNA­DNA hybrids; RNA oligomers; RNApolymers; ribosomal RNA (rRNA); and transport RNA(tRNA).

46. The method of claims 33­45, wherein the syntheticbiopolymer is chosen from cationic and anionic liposomes;cellulose acetate; hyaluronic acid; polylactic acid; polyglycolalginate; polyglycolic acid; poly­prolines; polysaccharides.

47. The method of claims 33­46, wherein the syntheticenzyme inhibitor is chosen from pepstatin, poly­prolines, D­sugars, D­aminocaids, cyanide, diisopropyl fluorophosphates(DFP), metal ions, N­tosyl­l­phenylalaninechloromethylketone (TPCK), Physostigmine, Parathion, and penicillin.

48. The method of claims 33­47, wherein the synthetic orextracted vitamin is chosen from biotin; calciferol (vitaminD's; vital for bone mineralisation); citrin; folic acid; niacin;nicotinamide; nicotinamide adenine dinucleotide (NAD,NAD+); nicotinamide adenine dinucleotide phosphate(NADP, NADPH); NAD+retinoic acid (vitamin A); riboflavin;

vitamin B's; vitamin C; vitamin E; and vitamin K1S.

49. The method of claims 33­48, wherein the pharmaceuticalis chosen from antibiotics, cyclooxygenase inhibitors,hormones, inflammation inhibitors, NSAID's, painkillers,prostaglandin synthesis inhibitors, steroids, and tetracycline.

50. The method of claims 33­49, wherein the biologicallyactive ions is chosen from adenosine di­phosphate (ADP);adenosine mono­phosphate (AMP); adenosine tri­phosphate(ATP); amino acids; cyclic AMP (cAMP); 3,4­dihydroxyphenylalanine (DOPA); 5'­di(dihydroxyphenyl­L­alanine (diDOPA); diDOPA quinone; DOPA­like o­diphenols;fatty acids; glucose; hydroxyproline; nucleosides;nucleotides (RNA and DNA bases); prostaglandin; sugars;sphingosine 1 ­phosphate; and rapamycin.

51. The method of claims 30­50, wherein the biologicalmaterial is serotonin.

52. The method of claims 31­52, wherein the precursor ischosen from BCI3, BBr3, B(OMe)3, AICI3, AIBr3, AIMe2CI,

AIMe2C1Pr, AIMe2H, AI(OEt)3, AI(CPr)3, a trialkyl aluminum,

GaCI3, GaMe3, GaCI, GaBr, GaI, GaMe3, GaEt3, Ga(acac)3,

Ga, GaEt2CI, GaEt2Me, InCI3, InMe3, InEt3, ln(acac)3, In,,

InEtMe2, InCI, InCIMe2, CF3, SiCI4, SiCI3H, SiCI2H2, SiH4,

Si2H6, SiCI3H, Si(OEt)4, Si(O"Bu)4, ('BuO)3SiOH, GeCI4,

GeMe2H2, GeEt2H2, GeH4, Ge2H6, SnCI4, SnEt4, SnMe4,

SnI4, SnCI4, PbBr2, PbI2, Pb(OAc)2, Pb(O4Bu)2, Pb(thd)2,

Pb(detdc)2, YCp3, Y(CpMe)3, Y(thd)3, Cd, CdMe2, CdCI2,

PCI3, POCI3, SbCI5, Bi(Ph)3, Bi[N(SiMe3)2]3 TiCI4, TiI4,

Ti(NMe2J4, Ti(NEt2J4, Ti(NMeEt)4, Ti(CPr)4, Ti(OEt)4, ZrCI4,

ZrI4, ZrCp2CI2, ZrCp2Me2, Zr(O'Pr)2(dmae)2, Zr(OfBu)4,

Zr(dmae)4, Zr(thd)4, Zr(NMe2J4, Zr(NEtMe)4, Zr(NEt2J4,

HfCI4, HfCI2[N(SiMe3)2]2, HfI4, Hf(OfBu)4, Hf(θ'Bu)2(mmp)2,

Hf(mmp)4, Hf(ONEt2J4, Hf(NEt2)* Hf(NEtMe)4,

Hf[N(SiMe3)2]2CI2, Hf(NOa)4, VOCI3, VO(O',Pr)3, VO(acac)2,

Nb(OEt)5, NbCI5, TaF5, TaCI5, TaBr5, TaI5, Ta(OEt)5,

Ta(NMe2)5, Ta(NEt2J5, Ta(NEt)(NEt2J3, Ta(N'Bu)(NEt2)3,

Ta(N^Bu)(NEtMe)3, CrO2CI2, Cr(acac)3, MoCI5, WF6,

WOCI4, WFxOy, W(NfBu)2(NMe2)2, Mn(thd)3, MnCI2, Mn,

hydroxide, fluorides and oxides of titanium, ferro­titanium alloys, and tantalummetals, or combination of above mention materials.

[076] For example, Ca(Cp)2 may serve as one precursor reacting with available

surface sites. Triphenyl phosphine, P(Ph)3, may then be pulsed into the reaction

chamber, forming calcium­phosphorous bonds at the surface. An oxygen sourcesuch as ozone or water may then be pulsed in to the reaction chamber to createthe terminal phosphate groups.

[077] Alternatively, calcium phosphate materials may be deposited using Ca(Cp)2as a first precursor, followed by H3PO4, POCI3, or other suitable phosphate

precursors.

[078] In anther embodiment, Ca2(PO4)F may be deposited using the following

sequence:

Step 1 : Ca(Cp)2 + 2H2O = Ca(OH)2 + 2H­Cp Step 2a: 3Ca(OH)2 + 2P(Ph)3 =

Ca3(PO3J2 + 6H­Ph Step 3a: Ca3(PO3)2 + O3 = Ca3(PO4J2 Step 2b: 3Ca(OH)2 +

2POCI3 = Ca3(PO4)2 + 6HCI Step 2c: 2Ca(OH)2 + POCI3 = Ca2(PO4)(OH) +

3HCI Step 3c: Ca2(PO4)(OH) + NH4F = Ca2(PO4)F + NH3 + H2O [079] Said bone

material may be deposited on a probe tip. These tips may optionally be used toimage sample surfaces and to study the interactions between the bone­likematerial and sample surfaces.

Implant­Like Materials

[080] In another embodiment, implant­like surfaces can be deposited by usingmaterials including, but not limited to, titanium oxide (TΪO2). TΪO2 may be

deposited by using TiCI4 and water as alternating precursors. Implant­like

materials often have chemical structures involving sodium oxides, silicate,calcium oxides, calcium sulphates, calcium phosphates, calcium carbonates,hydroxyapatite, hydrides, vanadium, platinum, hafnium, gold hydroxide, fluoridesand oxides of titanium, ferro­titanium alloys and tantalum metals, or combinationof these materials.

[081] In another embodiment, said implant­like material may be deposited on aprobe tip. These tips may then be used to image sample surfaces and to studythe interactions between the implant­like material and sample surfaces.

Inorganic­Organic Hybrid Materials

[082] In another embodiment of the invention, ALD may also be used to depositan organic­inorganic hybrid films by alternating an inorganic precursor with anorganic precursor having at least one functional group. For sequential depositionsof inorganic/organic precursors, the organic precursor must have at least twofunctional groups. An organic precursor with only one functional group may beused as the terminating layer. See WO 2006/071126A1.

[083] In one embodiment, the initial precursor used to deposit an inorganic­hybridfilm may be an inorganic precursor selected from a group consisting of metalalkyls, metal cycloalkyls, metal aryls, metal amine, metal silylamine, metalhalogenides, metal carbonyls and metal chelates, where the metal is selectedfrom the group comprising Al, Si, Ge, Sn, In, Pb, alkali metals, alkaline earthmetals, 3d­insertion metals, 4d­insertion metals, 5d­insertion metals, lanthanides,and actinides. [084] This precursor may be pulsed into the reaction chamberunder conditions such that it reacts with the available surface sites. Any excessinorganic precursor molecules may optionally be removed from the reactionchamber by purging with inert gas or evacuating the chamber.

[085] In one embodiment, the inorganic precursor bound to the surface may thenbe reacted with an organic precursor by pulsing said organic precursor into thereaction chamber. The organic precursor may be an organic compound with twoor more reactive substituents selected from the group comprising ­OH, ­ OR, =O,­COOH, ­SH, ­SO4H, ­SO3H, ­PH2, ­PO4H, ­PO3H, ­PRH1 ­NH2, ­NH3I, ­ SeH, ­

SeO3H, ­SeO4H, ­TeH, ­AsH2, ­AsRH, ­SiH3, ­SiRH2, ­SiRR1H, ­GeH3, ­ GeRH2,

­GeRR'H, amine, alkyl amine, silated amine, halogenated amine, aceticanhydride, imide, azide and nitroxyl; where R and R' may be a C­MO aryl, alkyl,

cycloalkyl, alkenyl or alkynyl group. Pulsing conditions should be used such thatthe organic precursor reacts with the inorganic­functionalized surface forming an

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Fe(acac)3, FeCI3, Fe(thd)3, Fe(^BuAMD)2, Ru(CpEt)2,

RuCp2, Ru(Od)3, Ru(thd)3, Co('PrAMD)2, Co(acac)3,

Co(thd)2, lr(acac)Ca(thd)3, NiCp2, Ni(acac)2, Ni(apo)2,

Ni(dmg)2, Ni('PrAMD)2, Ni(thd)2, Pd(thd)2, Pd(hfac)2,

Pt(CpMe)Me3, Pt(acac)2, Cu(acac)2, Cu(thd)2, Cu(hfac)2,

CuCI, Cu('PrAMD), ZnCI2, ZnMe2, ZnEt2, Zn(OAc)2, Zn,

Zn[N(SiMe3)J, HgMe2, Mg, Mg(Cp)2, Mg(thd)2, Ca(thd)2,

CaCp2, Sr(Cp1Pr3J2, Sr(thd)2, Sr(methd)2) Sr(CpMe5J2,

Ba(CpMe5J2, Ba(thd)2, ScCp3, Sc(thd)3, La[N(SiMe3J2J3,

La(PrAMD)3, La(thd)3, Ce(thd)4, Ce(thd)3phen,

Pr[N(SiMe3)2]3, Nd(thd)3, Sm(thd)3, Eu(thd)3, Gd(thd)3,

Dy(thd)3, Ho(thd)3, Er(thd)3, Tm(thd)3) and Lu[Co(SiMe3)]2CI.

53. The method of claims 30­52, comprising exposing saidAFM tip to a precursor gas comprising molecules having twoanhydride moieties or having two acyl chloride moieties,optionally purging with an inert gas, exposing said tip to aprecursor gas comprising molecules having two aminogroups, and optionally purging with an inert solvent.

54. The method of claims 30­52, comprising exposing saidAFM tip to a first precursor gas comprising molecules havingtwo amino groups, optionally purging with an inert gas,exposing said tip to precursor gas comprising molecules twoanhydride moieties and/or molecules having two acylchloride moieties, and optionally purging with an inertsolvent.

55. The method of claims 54 and 55 wherein the precursor ischosen from 1 ,2,4,5­benzenetetracarboxylic anhydride andnonanedioyl chloride.

56. The method of claims 54, 55, and 56 wherein saidprecursor is chosen from ethylenediamine, 1 ,6,­diaminohexame, 1 ,4­phenylenediamine, and 4,4'­oxydianiline.

57. The method of claims 30­52, comprising exposing saidAFM tip to a precursor gas comprising molecules of the typeH2N­R­C≡C­R' in an inert environment, optionally purging

said environment with an inert gas, exposing said tip to aprecursor gas comprising ozone in an inert environment, andoptionally purging said environment with an inert gas.

58. The method of claims 30­52, comprising exposing saidAFM tip to a precursor gas comprising molecules of the typeH2N­R­C=C­R' in an inert environment, optionally purging

said environment with an inert gas, exposing said tip to aprecursor gas comprising ozone in an inert environment, andoptionally purging said environment with an inert gas.

59. The method of claim 58 wherein the AFM tip surface isfirst terminated with carboxylic anhydride moieties.

60. The method of claims 30­52, comprising exposing saidAFM tip to an inorganic precursor in an inert environment,optionally purging said environment with an inert gas,exposing said tip to an organic precursor in an inertenvironment, and optionally purging said environment with aninert gas.

61. The method of claim 60, wherein the organic precursor ischosen from glycine, 4­aminobenzoic acid, and 4­aminobenzophenone.

62. The method of claim 60 and 61 , wherein the inorganicprecursor is chosen from BCI3, BBr3, B(OMe)3, AICI3, AIBr3,

AIMe2CI, AIMe2O',Pr, AIMe2H, AI(OEt)3, AI(CPr)3, a trialkyl

inorganic­organic hybrid layer. Any excess organic precursor may optionally beremoved by either purging the reaction chamber with inert gas and/or evacuatingthe chamber.

[086] In one embodiment, the sequential deposition of inorganic and organicprecursors may optionally be repeated using the same or different inorganic andorganic precursors until the desired film thickness and surface termination isachieved. For sequential deposition, the organic precursor must have more thanone reactive substituent.

[087] In one embodiment, an organic precursor having only one or more reactivesubstituent may be used to deposit the terminating layer of the hybrid film. Thereactive substituent of said organic molecule may be chosen from the groupcomprising ­OH, ­OR, =O, ­COOH, ­SH, ­SO4H, ­SO3H, ­PH2, ­PO4H, ­PO3H, ­

PRH, ­NH2, ­NH3I, ­SeH, ­SeO3H, ­SeO4H, ­TeH, ­AsH2, ­AsRH, ­SiH3, ­SiRH2, ­

SiRR1H, ­GeH3, ­GeRH2, ­GeRR'H, amine, alkyl amine, silated amine, acetic

anhydride, halogenated amine, imide, azide, and nitroxyl; where R and R' may bea C­ι­10 aryl, alkyl, cycloalkyl, alkenyl, or alkynyl group.

[088] In another embodiment, said inorganic­organic hybrid material may bedeposited on a probe tip. These tips may optionally be used to image samplesurfaces and to study the interactions between the bone­like material and samplesurfaces.

[089] ALD has been used to produce functionalized inorganic­organic hybrid filmsusing numerous combinations of precursors. The following examples of suitableprecursors exemplify the types of precursors suitable for this technique, but arenot meant to limit the scope of this invention: TMA and hydroquinone (Hq), TMAand malonic acid; TMA and terephtalic acid; ZrCI4 and Hq; TiCI4 and Hq; TiCI4and ethylenediamine. Additional inorganic precursors include, but are not limitedto, BCI3, BBr3, B(OMe)3, AICI3, AIBr3, AIMe2CI, AIMe2CPr, AIMe2H, AI(OEt)3,

AI(O"Pr)3, a trialkyl aluminum, GaCI3, GaMe3, GaCI, GaBr, GaI, GaMe3, GaEt3,

Ga(acac)3, Ga, GaEt2CI, GaEt2Me, InCI3, InMe3, InEt3, ln(acac)3, In1, InEtMe2,

InCI, InCIMe2, CF3, SiCI4, SiCI3H, SiCI2H2, SiH4, Si2H6, SiCI3H, Si(OEt)4,

Si(O0Bu)4, ('BuO)3SiOH, GeCI4, GeMe2H2, GeEt2H2, GeH4, Ge2H6, SnCI4,

SnEt4, SnMe4, SnI4, SnCI4, PbBr2, PbI2, Pb(OAc)2, Pb(O1Bu)2, Pb(thd)2,

Pb(detdc)2, YCp3, Y(CpMe)3, Y(thd)3, Cd, CdMe2, CdCI2, PCI3, POCI3, SbCI5,

Bi(Ph)3, Bi[N(SiMe3)2]3 TiCI4, TiI4, Ti(NMe2)4, Ti(NEt2)4, Ti(NMeEt)4, Ti(O7Pr)4,

Ti(OEt)4, ZrCI4, ZrI4, ZrCp2CI2, ZrCp2Me2, Zr(O'Pr)2(dmae)2, Zr(OfBu)4, Zr(dmae)4,

Zr(thd)4, Zr(NMe2)4, Zr(NEtMe)4, Zr(NEt2)4, HfCI4, HfCI2[N(SiMe3)2]2, HfI4,

Hf(O^Bu)4, Hf(O'Bu)2(mmp)2, Hf(mmp)4, Hf(ONEt2)4, Hf(NEt2)4, Hf(NEtMe)4,

Hf[N(SiMe3)2]2CI2, Hf(NO3)4, VOCI3, VO(C1Pr)3, V0(acac)2, Nb(OEt)5, NbCI5,

TaF5, TaCl5, TaBr5, TaI5, Ta(OEt)5, Ta(NMe2J5, Ta(NEt2)5, Ta(NEt)(NEt2)3,

Ta(N'Bu)(NEt2)3, Ta(N^Bu)(NEtMe)3, CrO2CI2, Cr(acac)3, MoCI5, WF6, WOCI4,

WFxOy, W(N'Bu)2(NMe2)2, Mn(thd)3, MnCI2, Mn, Fe(acac)3, FeCI3, Fe(thd)3,

Fe(^BuAMD)2, Ru(CpEt)2, RuCp2, Ru(Od)3, Ru(thd)3, Co(PrAMD)2, Co(acac)3,

Co(thd)2, lr(acac)Ca(thd)3, NiCp2, Ni(acac)2, Ni(apo)2, Ni(dmg)2, Ni('PrAMD)2,Ni(thd)2, Pd(thd)2, Pd(hfac)2, Pt(CpMe)Me3, Pt(acac)2, Cu(acac)2, Cu(thd)2,

Cu(hfac)2l CuCI, Cu('PrAMD), ZnCI2, ZnMe2, ZnEt2, Zn(OAc)2, Zn, Zn[N(SiMe3J2],

HgMe2, Mg, Mg(Cp)2, Mg(thd)2, Ca(thd)2, CaCp2, Sr(Cp'Pr3)2, Sr(thd)2,

Sr(methd)2, Sr(CpMe5)2, Ba(CpMe5J2, Ba(thd)2, ScCp3, Sc(thd)3,

La[N(SiMe3)2]3, La(1PrAMD)3, La(thd)3, Ce(thd)4, Ce(thd)3phen, Pr[N(SiMe3)2]3,

Nd(thd)3, Sm(thd)3, Eu(thd)3, Gd(thd)3, Dy(thd)3, Ho(thd)3, Er(thd)3, Tm(thd)3,

and Lu[Co(SiMe3)]2CI.

[090] Some of the functional groups imagined to undergo reactions by the ALDprinciple and thus suitable as functional groups to attach active molecules to thesurface are described in the following. For all the proposed reaction mechanisms,it is likely that the reaction scheme in reality is somewhat shifted or different.Thus, the reaction schemes should not be interpreted limiting the scope of theinvention. The main principal is that the reaction results in film formation.Therefore, the following reaction schemes serve only to show the potential of thefollowing functional groups to form a component of a film.

9/14/2015 Patent WO2009001220A2 ­ Functionalization of microscopy probe tips ­ Google Patents

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aluminum, GaCI3, GaMe3, GaCI, GaBr, GaI, GaMe3, GaEt3,

Ga(acac)3, Ga, GaEt2CI, GaEt2Me, InCI3, InMe3, InEt3,

ln(acac)3, In1, InEtMe2, InCI, InCIMe2, CF3, SiCI4, SiCI3H,

SiCI2H2, SiH4, Si2H6, SiCI3H, Si(OEt)4, Si(O0Bu)4,

('BuO)3SiOH, GeCI4, GeMe2H2, GeEt2H2, GeH4, Ge2H6,

SnCI4, SnEt4, SnMe4, SnI4, SnCI4, PbBr2, PbI2, Pb(OAc)2,

Pb(O1Bu)2, Pb(thd)2, Pb(detdc)2, YCp3, Y(CpMe)3, Y(thd)3,

Cd, CdMe2, CdCI2, PCI3, POCI3, SbCI5, Bi(Ph)3,

Bi[N(SiMe3)2]3 TiCI4, TiI4, Ti(NMe2J4, Ti(NEt2J4, Ti(NMeEt)4,

Ti(CPr)4, Ti(OEt)4, ZrCI4, ZrI4, ZrCp2CI2, ZrCp2Me2,

Zr(O'Pr)2(dmae)2, Zr(OfBu)4, Zr(dmae)4, Zr(thd)4, Zr(NMe2)4,

Zr(NEtMe)4, Zr(NEt2J4, HfCI4, HfCI2[N(SiMe3)2]2, HfI4,

Hf(OfBu)4, Hf(OfBu)2(mmp)2, Hf(mmp)4, Hf(ONEt2J4,

Hf(NEt2J4, Hf(NEtMe)4, Hf[N(SiMe3)2]2CI2, Hf(NO3J4, VOCI3,

VO(O',Pr)3, VO(acac)2, Nb(OEt)5, NbCI5, TaF5, TaCI5,

TaBr5, TaI5, Ta(OEt)5, Ta(NMe2)S, Ta(NEt2J5, Ta(NEt)

(NEt2J3, Ta(N'Bu)(NEt2)3, Ta(N^Bu)(NEtMe)3, CrO2CI2,

Cr(acac)3, MoCI5, WF6, WOCI4, WFxOy, W(N'Bu)2(NMe2)2,

Mn(thd)3, MnCI2, Mn, Fe(acac)3, FeCI3, Fe(thd)3,

Fe(fBuAMD)2, Ru(CpEt)2, RuCp2, Ru(Od)3, Ru(thd)3,

Co('PrAMD)2, Co(acac)3, Co(thd)2) lr(acac)Ca(thd)3, NiCp2,

Ni(acac)2, Ni(apo)2, Ni(dmg)2, Ni( rAMD)2, Ni(thd)2, Pd(thd)2,

Pd(hfac)2, Pt(CpMe)Me3, Pt(acac)2, Cu(acac)2, Cu(thd)2,

Cu(hfac)2, CuCI, Cu('PrAMD), ZnCI2, ZnMe2, ZnEt2,

Zn(OAc)2, Zn, Zn[N(SiMe3)2], HgMe2, Mg, Mg(Cp)2,

Mg(thd)2, Ca(thd)2, CaCp2, Sr(Cp'Pr3)2, Sr(thd)2, Sr(methd)2)Sr(CpMe5J2, Ba(CpMe5J2, Ba(thd)2, ScCp3, Sc(thd)3,

La[N(SiMe3Js]3, La(1PrAMD)3, La(thd)3, Ce(thd)4,

Ce(thd)3phen, Pr[N(SiMe3)2]3, Nd(thd)3, Sm(thd)3, Eu(thd)3,

Gd(thd)3, Dy(thd)3, Ho(thd)3, Er(thd)3, Tm(thd)3, and

Lu[Co(SiMe3)J2CI.

63. The method of claims 30­52, comprising exposing saidAFM tip to a calcium precursor, optionally purging saidenvironment with an inert gas, exposing said tip to aphosphorous precursor in an inert environment, optionallypurging said environment with an inert solvent, and exposingsaid environment to a pulse of either water or NH4F.

64. The method of claim 63, wherein the calcium precursor ischosen from Ca(Cp)2 and Ca(thd)2.

65. The method of claims 64 and 65, wherein thephosphorous precursor is chosen from POCI3, P(Ph)3, and

(CH3O)3PO.

66. The method of claims 30­52, wherein the implant­likematerial deposited comprises one or more sodium oxides,silicate, calcium oxides, calcium sulfates, calciumphosphates, calcium carbonates, hydroxyapatite, hydrides,vanadium, platinum, hafnium, gold hydroxide, fluorides andoxides of titanium, ferro­titanium alloys, or tantalum metals.

67. The method of claims 30­52 wherein the AFM tip iscoated using liquid­phase ALD in an inert atmosphere,comprising exposing said AFM tip to a first precursorcontaining a reactive inorganic species in an inert solvent,optionally rinsing said tip with an inert solvent, exposing saidtip to a biological material dissolved or suspended in an inertsolvent, and optionally rinsing said tip with an inert solvent.

68. The method of claims 66 and 67, wherein the inorganic

[091] Concerning possible reactions between metal containing precursors andorganic molecules with functional groups, organic molecules with functionalgroups that have some degree of acidity, i.e., that can donate a proton may bepreferred. This proton will be used to complete the alkane molecule or halogenacid molecule from the inorganic precursor and let the reaction proceed. Hvdroxylgroups

[092] Hydroxyl groups (R­OH) provide an oxygen and hydrogen for a possiblereaction. These may react readily with electropositive metals, including but notlimited to metal alkyls and metal halides, whereby metal alkoxides and alkyl orhydrohalogen acids may be produced, respectively.

[093] Two partial reactions that may take place between a diol (HO­R­OH) andTMA ((CH3)3AI) are given below: HO­R­OH(g) + CH3­AI­I → CH4(g) + HO­R­O­AI­|

2HO­R­I + (CH3J3AKg) → 2CH4(g) + CH3­AI­(O­R­I)2

[094] Electropositive metals known to readily undergo such reactions are: Al, Mg,Si, Ti, V, and several other metals including, but not limited to, Zn, Mn, Fe, Co,and/or Cr. Ether groups

[095] Ether groups (­OR) may react and form adducts to metals in the film. Thesebonds may be rather weak, but may still form the basis of structure formation forfilms produced at low temperatures. An example of such a reaction scheme is:

R'O­R(g) + CH3­Mn­I → R1O­R­Mn­I 2RO­R­I + (CHs)3AKg) → 2R'­CH3(g) + CH3­

AI­(O­R­I)2 [096] Only one half reaction is presented, because the film formed via

this reaction path may use another type of functional group in its structure to forma film in the next step. Ketone groups

[097] Ketones (R=O) may interact with metal atoms, and molecules with morethan one ketone moiety may chelate metal atoms. One such example is theformation of compounds with β­ketones. An example of such visualized reactionscheme is: O=R'­R=O (g) + Mn­| → R,R'(=O)2 ­Mn­|

[098] Only one half reaction is presented, because the film formed via thisreaction path may use another type of functional group in its structure to form thefilm in the next step. Carboxyl groups

[099] Carboxyl groups (­COOH) have the same building units as for hydroxyls (­OH) and ketones (=O).

[0100] Two partial reactions that take place between a dicarboxylic acid (HOOC­R­COOH) and TMA ((CH3)3AI) are given below. HOOC­R­COOH(g) + CH3­AI­| →

CH4(g) + HOOC­R­COO­AI­I 2HOOC­R­I + (CH3)3AI(g) → 2CH4(g) + CH3­AI­

(COO­R­I)2

[0101] TMA may also react with both the =O and the ­OH of a carboxyl acid.

Thiol groups

[0102] Thiol groups (­SH) may form participate in similar types of reactions astheir isoelectronic hydroxyl relatives (­OH). However, metal affinity towardssulphur differs from that towards oxygen. Elements including, but not limited to,Pb, Au, Pt, Ag, Hg, and others react and form stable bonds towards sulphur.

[0103] Two partial reactions that may take place between a di­thiol (HS­R­ SH)and Pt(thd)2 are given below: HS­R­SH(g) + thd­Pt­| → Hthd(g) + HS­R­S­Pt­|

2HS­R­I + Pt(thd)2(g) → Hthd(g) + thd­Pt­S­R­|

Sulphate groups

[0104] Sulphate groups (­SO4H) may react with electropositive metals in ways

similar to hydroxyls or ketones.

[0105] Two partial reactions that may take place between a disulphate (HSO4­R­

SO4H) and TMA are given below: HSO4­R­SO4H (g) + CH3­AI­I → CH4(g) +

HSO4­R­SO4­AI­I 2HSO4­R­I + (CHa)3AKg) → 2CH4(g) + CH3­AI­(SO4­R­I)2

Sulphite groups

[0106] Sulphite groups (­SO3H) may react in ways similar to sulphate groups.

9/14/2015 Patent WO2009001220A2 ­ Functionalization of microscopy probe tips ­ Google Patents

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precursor is chosen from BCI3, BBr3, B(OMe)3, AICI3, AIBr3,

AIMe2CI, AIMe2C1Pr, AIMe2H, AI(OEt)3, AI(O"Pr)3, a trialkyl

aluminum, GaCI3, GaMe3, GaCI, GaBr, GaI, GaMe3, GaEt3,

Ga(acac)3, Ga, GaEt2CI, GaEt2Me, InCI3, InMe3, InEt3,

ln(acac)3, In,, InEtMe2, InCI, InCIMe2, CF3, SiCI4, SiCI3H,

SiCI2H2, SiH4, Si2H6, SiCI3H, Si(OEt)4, Si(O0Bu)4,

('BuO)3SiOH, GeCI4, GeMe2H2, GeEt2H2, GeH4, Ge2H6,

SnCl4, SnEt4, SnMe4, SnI4, SnCI4, PbBr2, PbI2, Pb(OAc)2,

Pb(O1Bu)2, Pb(thd)2, Pb(detdc)2, YCp3, Y(CpMe)3, Y(thd)3,

Cd, CdMe2, CdCI2, PCI3, POCI3, SbCI5, Bi(Ph)3,

Bi[N(SiMe3)2]3 TiCI4, TiI4, Ti(NMe2)4, Ti(NEt2)4, Ti(NMeEt)4,

Ti(CPr)4, Ti(OEt)4, ZrCI4, ZrI4, ZrCp2CI2, ZrCp2Me2,

Zr(O'Pr)2(dmae)2, Zr(O^Bu)4, Zr(dmae)4) Zr(thd)4, Zr(NMe2)4,

Zr(NEtMe)4, Zr(NEt2J4, HfCI4, HfCI2[N(SiMe3)2]2, HfI4,

Hf(OfBu)4, Hf(O'Bu)2(mmp)2, Hf(mmp)4> Hf(ONEt2)4,

Hf(NEt2)4, Hf(NEtMe)4, Hf[N(SiMe3)2]2CI2, Hf(NO3J4, VOCI3,

VO(O',Pr)3, VO(acac)2, Nb(OEt)5, NbCI5, TaF5, TaCI5,

TaBr5, TaI5, Ta(OEt)5, Ta(NMe2)S, Ta(NEt2)5, Ta(NEt)

(NEt2)3, Ta(NfBu)(NEt2)3, Ta(NfBu)(NEtMe)3, CrO2CI2,

Cr(acac)3, MoCI5, WF6, WOCI4, WFxOy, W(NfBu)2(NMe2)2,

Mn(thd)3, MnCI2, Mn, Fe(acac)3, FeCI3, Fe(thd)3,

Fe(^BuAMD)2, Ru(CpEt)2, RuCp2, Ru(Od)3, Ru(thd)3,

Co('PrAMD)2, Co(acac)3, Co(thd)2, lr(acac)Ca(thd)3> NiCp2,

Ni(acac)2, Ni(apo)2, Ni(dmg)2, Ni(1PrAMD)2, Ni(thd)2,

Pd(thd)2, Pd(hfac)2, Pt(CpMe)Me3, Pt(acac)2, Cu(acac)2,

Cu(thd)2, Cu(hfac)2) CuCI, Cu('PrAMD), ZnCI2, ZnMe2,

ZnEt2, Zn(OAc)2, Zn, Zn[N(SiMe3J2], HgMe2, Mg, Mg(Cp)2,

Mg(thd)2, Ca(thd)2, CaCp2, Sr(Cp'Pr3)2, Sr(thd)2, Sr(methd)2,

Sr(CpMe5J2, Ba(CpMe5J2, Ba(thd)2, ScCp3, Sc(thd)3,

La[N(SiMe3)2]3, La('PrAMD)3, La(thd)3, Ce(thd)4,

Ce(thd)3phen, Pr[N(SiMe3)2]3, Nd(thd)3) Sm(thd)3, Eu(thd)3,

Gd(thd)3, Dy(thd)3, Ho(thd)3, Er(thd)3, Tm(thd)3, and

Lu[Co(SiMe3)]2CI.

69. The method of claims 67 and 68, wherein the inertsolvents are each chosen from toluene, hexane, andheptane.

70. The method of claims 30 ­ 69, wherein said method isrepeated 1 ­ 2000 times.

71. A functionalized AFM tip prepared using the method ofclaims 30 ­ 70.

72. A method of studying a surface comprising using anAFM tip as claimed in claims 1 ­ 29 in an AFM to imageand/or probe a sample surface.

73. The method of claim 72, wherein the sample surfacecomprises a biological, organic, inorganic­hybrid, bone, orimplant­like material.

74. The method of claim 72, wherein the sample surfacecontains a cell, protein, enzyme, antibody, or peptide.

75. The method of claim 72, wherein the sample surface is abone cell.

76. A method of studying a surface comprising using anAFM tip as claimed in claim 1 ­ 29 in an AFM to study theintermolecular forces between said tip and a sample surface.

77. The method of claim 76, wherein the sample surface

[0107] Two partial reactions that take place between a disulphite (HSO3­R­ SO3H)

and TMA ((CH3)3AI) are given below: HSO3­R­SO3H (g) + CH3­AI­I → CH4(g) +

HSO3­R­SO3­AI­I 2HSO3­R­I + (CH3)3AI(g) → 2CH4(g) + CH3­AI­(SO3­R­I)2

Phosphide groups

[0108] Two partial reactions that may take place between a di­phospide (H2P­R­

PH2) and Ni(thd)2 are given below: H2P­R­PH2(g) + thd­Ni­| → Hthd(g) + H2P­R­

PH­Ni­I 2H2P­R­I + Ni(thd)2(g) → Hthd(g) + thd­Ni­PH­R­|

Phosphate groups

[0109] Two partial reactions that may take place between a diphosphate (HPO4­

R­PO4H) and TMA ((CH3J3AI) are shown below: HPO4­R­PO4H (g) + CH3­AI­I →

CH4(g) + HPO4­R­PO4­AI­I 2HPO4­R­I + (CHs)3AKg) → 2CH4(g) + CH3­AI­(PO4­

R­I)2 Amine groups

[0110] Amine groups, alkyl amines, or silated amines, or halogenated amines,may react with compounds including but not limited to SnI2, SnI4, PbI2, PbI4,

CuI2, CuI4 or similar compounds to form perovskite­related hybrid materials as

described by D. B. Mitzi (D. B. Mitzi, Progress in Inorganic Chemistry, 48: 1­121(1999); D. B. Mitzi, Chem. of Materials, 13: 3282­98 (2001 )).

[0111] One proposed reaction mechanism is: Snl4(g) + NH4I­R­I → SnI4­NH4I­R­I

SnI4­I + NH4l­R­H4NI(g) → NH4I­R­H4NI­SnI4­I

[0112] A redox­reaction with Sn(IV)­Sn(II) and formation of I2(g) might be involved

here. Alternatively, by using divalent halogenides, the reaction might be visualizedas:

Snl2(g) + NH4I­R­I → SnI3­NH4­R­I SnI3­I + NH4l­R­H4NI(g) → NH4I­R­H4N­SnI4­I

[0113] In addition or in the alternative, amines may react similar to hydroxylgroups. Two potential partial reactions that may take place between a diamine(H2N­R­NH2) and TMA ((CH3)3AI) are given below: H2N­R­NH2(g) + CH3­AI­I →

CH4(g) + H2N­R­NH­AI­I 2H2N­R­I + (CHa)3AKg) → 2CH4(g) + CH3­AI­(NH­R­I)2

[0114] Both of the H­atoms on one of the amines may react with TMA.

[0115] The following functional groups will react in a similar way: ­OH, ­SH, ­SeH1­TeH, ­NH2, ­PH2, ­AsH2, ­SiH3, ­GeH3. ­SO4H, ­SO3H, ­PO4H, ­PO3H, SeO3H,

SeO4H. In all cases where more than one H is present, the other H may be

substituted by another organic group R, where R is straight and branched chainalkane, cycloalkane, an aryl group, a heteroaryl group, or a functional groups.

[0116] For precursors with more than one functional group, the functional groupsneed not be of the same type. Different functional groups with different reactivitymay form a monolayer of organic molecules with a degree of ordering. In addition,different inorganic precursors may have different affinities for the different groups.Different organic and inorganic precursors may be used to produce variousterminating surfaces.

[0117] The organic compound carrying the functional groups is not particularlylimited but can be any organic molecule that can be brought into the gas phase. Itis preferred that the organic precursor molecule with more than one functionalgroup will have some form of structural or steric hindrance to prevent all of itsfunctional groups from reacting with the same surface. For organic precursorswith more than one reactive site, it is preferred that at least one reactive site doesnot react with the active surface sites and remains for use in a subsequentreaction. Otherwise, there are no limitations on the structure of the organicprecursors. The organic molecule may influence the acidity of the protons on thefunctional groups. The organic compound may be a non­branched alkane,branched alkane, cyclo alkane, alkene, a monocyclic or polycyclic aromaticgroup, a heterocyclic aromatic group, where these compounds, in addition to thefunctional groups, may be substituted or not substituted with other organic groupslike alkyl.

[0118] Some of the inorganic precursors that may be used to make inorganic­organic hybrid films are described below. All the suggested reactions are only

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comprises a biological, organic, inorganic­hybrid, bone, orimplant­like material.

78. The method of claim 76, wherein the sample surfacecontains a cell, protein, enzyme, antibody, or peptide.

79. The method of claim 76, wherein the sample surface is abone cell.

80. A functionalized SPM tip comprising a SPM tip coatedwith at least one material chosen from biological, organic,inorganic, organic­inorganic hybrid, magnetic/conductive andhard coatings.

81. A functionalized SPM tip of claim 80, wherein the SPMtip is a MFM tip coated in a magnetic/conductive film.

82. The MFM tip of claim 81 , wherein the film is chosenfrom ferromagnetic, ferrimagnetic and paramagnetic films.

83. The MFM tip of claim 81­82, wherein the magnetic film is(Fe1Co)3O4.

84. The MFM tip of claim 81­83, wherein the (Fe1Co)3O4 film

is 5­200 nm thick.

85. A functional ized SPM tip of claim 81 , wherein the SPMtip comprises a hard coating chosen from Tiθ2, AI2O3, ZrO2,

Ti­Nitride, multi­layer and hybrid hard coatings.

86. A SPM probe coated with TiO2.

illustrations of possible reactions, and are not to be interpreted as limitations.Metal alkyls

[0119] Metal alkyls and metal cycloalkyls may be rather reactive and hence mayreact with most organic functional groups. Examples of possible metal alkyls are:AI(CH3)3, Zn(Et)2, Zn(Me)2, and MgCp2I. Metal halogenides

[0120] Some electropositive metal halogenides may be rather reactive andtherefore may undergo reaction with many organic functional groups. Someexamples are AICI3, TiCI4, SiCI4, SnCI4, Si(CH3)2CI2. Metal carbonyls

[0121] Metal carbonyls may also be reactive, including but not limited to:Fe2(CO)9, Mn(CO)x. Metal chelates

[0122] Reactive metal chelates may include, but are not limited to: VO(thd)2,

Mn(HMDS)2, Fe(HMDS)2, TiO(thd)2, Pt(thd)2, where HMDS stands for

hexamethyl­disilazane.

[0123] Other possible chelates are beta­ketones such as acetylacetonates,fluorinated thd­compounds and ethylenediaminetetra acetic acid (EDTA).

[0124] The metal for the inorganic precursor is selected from the group consistingof Al, Si, Ge, Sn, In, Pb, alkali metals, alkaline earth metals, 3d­insertion metals,4d­insertion metals, 5d­insertion metals, lanthanides, and actinides. Metals ofparticular interest may include, but are not limited to, Cu, Ni, Co, Fe, Mn, and V.

[0125] In one embodiment of the invention, hybrid films may be fabricated via ALDusing TMA as the inorganic precursor and Hq and/or PhI as organic precursor(s).This may result in films of aluminium benzene oxides. Films are produced byusage of TMA­PhI, TMA­Hq, and a controlled mixture of the type TMA­ PhI­TMA­Hq. The growth kinetic may be investigated using quartz crystal monitor (QCM)measurements. The films may optionally be analyzed by Fourier transformedinfrared spectroscopy (FT­IR). Active surfaces terminating in hydroxy! groups onaromatics or metal alkyls are obtained by using Hq/Phl or TMA respectively as the last type of precursor.

[0126] In another embodiment of the invention, hybrid films may be deposited from TMA and one or more organic precursorsthat will react with the methyl­aluminum surface, including but not limited to 1 ,4­benzenedicarboxylic acid, 1 ,3­benzenedicarboxylic acid, 1 ,3,5­benzenetricarboxylic acid, and/or 1 ,2,4,5­ benzenetetracarboxylic acid.

Organic Films

[0127] In one embodiment of the invention, ALD may be used to deposit organic films. As described in M. Putkonen et al., J.Mater. Chem., 17: 664 (2007) polyimide films may be grown using anhydrides and diamines. Suitable anhydrides includethose with two anhydride moieties, for example:

1 ,2,4,5­Benzenetetracarboxylic anhydride (Pyromellitic dianhydride, PMDA)

[0128] Similarly, suitable diamines include those with two amino groups. For example:

Ethylenediamine, EDA 1,6­Diaminohexane, DAH

1,4­Phenylenediamine, PDA 4,4'­oxydianiline, ODA

[0129] In one embodiment of the invention, 1 ,2,4,5­Benzenetetracarboxylic anhydride may react with a diamine to form a filmusing ALD.

[0130] In addition, polyamide films may be deposited using an acyl chloride and a diamine. See A. Kubono et al., Thin SolidFilms, 289: 107 (1996). Suitable acyl chlorides include those with two or more acyl chloride groups. For example,nonanedioyl chloride (azelaoyl dichloride (ADC)) may be used with diaminoheptane to deposit organic films.

[0131] Alternatively, polyamide films may be deposited by alternating dicarboxylic acids and diamines.

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[0132] In another embodiment, ALD may be used to deposit organic films on an AFM tip. These tips may optionally be usedto image sample surfaces and to study the interactions between the bone­like material and sample surfaces.

[0133] Where a precursor contains two or more functional groups, the functional groups may be the same or different. Theprecursor should leave a reactive site suitable for succeeding growth with another type of precursor, unless no furtherdeposition steps are desired. For production of the final terminating surface, the requirement of two or more types offunctional groups does not apply. Rather the precursor should have at least one functional group that will undergo reactionswith the previous surface, and also contain the groups that should finally terminate the surface.

Biomolecular Films

[0134] In another embodiment, ALD may be used to produce biomolecular films. Biological surfaces may be constructed byusing highly reactive metal precursors to link the native hydroxyl group terminating layer to the desired biological molecules.

[0135] The term "biomolecules" is intended to cover and comprise within its meaning a very wide variety of biologically activemolecules in the widest sense of the word, be they natural biomolecules (i.e. naturally occurring molecules derived fromnatural sources), synthetic biomolecules (i.e. naturally occurring molecules prepared synthetically as well as non­naturallyoccurring molecules or forms of molecules prepared synthetically) or recombinant biomolecules (i.e. prepared through the useof recombinant techniques).

[0136] A non­limiting list of main groups of and species biomolecules that are contemplated as being suitable for coatingprobe tips in accordance with the invention is given below. Extracted biomolecules Bioadhesives:

[0137] Bioadhesives include biomolecules that mediate attachment of cells, tissue, organs or organisms onto non­biologicalsurfaces like glass, rock etc. This group of bio­molecules includes the marine mussel adhesive proteins, fibrin­like proteins,spider­web proteins, plant­derived adhesives (resins), adhesives extracted from marine animals, and insect­derived adhesives(like resilins).

[0138] Some specific non­limiting examples of adhesives include, but are not limited to: fibrin; fibroin; Mytilus edulis footprotein (mefpi , "mussel adhesive protein"); other mussel's adhesive proteins; proteins and peptides with glycine­rich blocks;proteins and peptides with poly­alanine blocks; and silks. Cell attachment factors: [0139] Cell attachment factors includebiomolecules that mediate attachment and spreading of cells onto biological surfaces or other cells and tissues. This group ofmolecules typically contains molecules participating in cell­ matrix and cell­cell interaction during vertebrate development,neogenesis, regeneration, and repair. Typical biomolecules in this class are molecules on the outer surface of cells like theCD class of receptors on white blood cells, immuneglobulins, and haemagglutinating proteins, and extracellular matrixmolecules/ligands that adhere to such cellular molecules.

[0140] Typical examples of cell attachment factors with potential for use as bioactive coating on metal hybrid ­coated probetips include, but are not limited to: ankyrins; cadherins (Calcium dependent adhesion molecules); connexins; dermatansulphate; entactin; fibrin; fibronectin; glycolipids; glycophorin; glycoproteins; heparan sulphate; heparin sulphate; hyaluronicacid; immunglobulins; keratan sulphate; integrins; laminins; N­CAMs (Calcium independent Adhesive Molecules);proteoglycans; spektrin; vinculin; vitronectin.

Biopolvmers:

[0141] Biopolymers are any biologically prepared molecules that, under the right conditions, may be assembled intopolymeric, macromolecular structures. Such molecules constitute important parts of the extracellular matrices where theyparticipate in providing tissue resilience, strength, rigidity, integrity etc. Some important biopolymers with potential for use asbioactive coating on metal hybrid­ coated cantilever include, but are not limited to: alginates; Amelogenins; cellulose;chitosan; collagen; gelatins; oligosaccharides; pectin.

Blood proteins:

[0142] Blood proteins typically contain any dissolved or aggregated protein that normally is present whole blood. Suchproteins can participate in a wide range of biological processes like inflammation, homing of cells, clotting, cell signaling,defense, immune reactions, metabolism etc. Typical examples with potential for use as bioactive coating on metal hybrid ­coated cantilever include, but are not limited to: albumin; albumen; cytokines; factor IX; factor V; factor VII; factor VIII; factorX; factor Xl; factor XII; factor XIII; hemoglobins (with or without iron); immunglobulins (antibodies); fibrin; platelet derived growthfactors (PDGFs); plasminogen; thrombospondin; transferrin.

Enzymes:

[0143] Enzymes are any protein or peptides that have a specific catalytic effect on one ore more biological substrates whichcan be virtually anything from simple sugars to complex macromolecules like DNA. Enzymes are potentially useful fortriggering biological responses in the tissue by degradation of matrix molecules, or they could be used to activate or releaseother bioactive compounds in the implant coating. Some important examples with potential for use as bioactive coating onmetal hybrid ­coated cantilever include, but are not limited to: abzymes (antibodies with enzymatic capacity); adenylatecyclase; alkaline phosphatase; carboxylases; collagenases; cyclooxygenase; hydrolases; isomerases; ligases; lyases;metallo­matrix proteases (MMPs); nucleases; oxidoreductases; peptidases; peptide hydrolase; peptidyl transferase;phospholipase; proteases; sucrase­ isomaltase; TIMPs; transferases.

Extracellular Matrix proteins and biomolecules:

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[0144] Specialized cells, e.g. fibroblasts and osteoblasts, produce the extracellular matrix. This matrix participates in severalimportant processes. The matrix is crucial for i.a. wound healing, tissue homeostasis, development and repair, tissuestrength, and tissue integrity. The matrix also decides the extracellular milieu like pH, ionic strength, osmolarity etc.Furthermore extracellular matrix molecules are crucial for induction and control of biomineral formation (bone, cartilage,teeth). Important extracellular proteins and biomolecules with potential for use as bioactive coating on metal hybrid ­coatedcantilever include: ameloblastic amelogenins; collagens (I to XII); dentin­sialo­protein (DSP); dentin­sialo­phospho­ protein(DSPP); elastins; enamelin; fibrins; fibronectins; keratins (1 to 20); laminins; tuftelin; carbohydrates; chondroitin sulphate;heparan sulphate; heparin sulphate; hyaluronic acid; lipids and fatty acids; lipopolysaccarides.

Growth factors and hormones:

[0145] Growth factors and hormones are molecules that bind to cellular surface structures (receptors) and generate a signalin the target cell to start a specific biological process. Examples of such processes are growth, programmed cell death,release of other molecules (e.g. extracellular matrix molecules or sugar), cell differentiation and maturation, regulation ofmetabolic rate etc. Typical examples of such biomolecules with potential for use as bioactive coating on metal hybrid ­coatedprobe tips include, but are not limited to: activins (Act); Amphiregulin (AR); Angiopoietins (Ang 1 to 4); Apo3 (a weakapoptosis inducer also known as TWEAK, DR3, WSL­1 , TRAMP or LARD); Betacellulin (BTC); Basic Fibroblast GrowthFactor (bFGF, FGF­b); Acidic Fibroblast Growth Factor (aFGF, FGF­a); 4­ 1BB Ligand; Brain­derived Neurotrophic Factor(BDNF); Breast and Kidney derived Bolokine (BRAK); Bone Morphogenic Proteins (BMPs); B­LymphocyteChemoattractant/B cell Attracting Chemokine 1 (BLC/BCA­1 ); CD27L (CD27 ligand); CD30L (CD30 ligand); CD40L (CD40ligand); A Proliferation­inducing Ligand (APRIL); Cardiotrophin­1 (CT­1 ); Ciliary Neurotrophic Factor (CNTF); ConnectiveTissue Growth Factor (CTGF); Cytokines; 6­cysteine Chemokine (ΘCkine); Epidermal Growth Factors (EGFs); Eotaxin (Eot);Epithelial Cell­derived Neutrophil Activating Protein 78 (ENA­78); Erythropoietin (Epo); Fibroblast Growth Factors (FGF 3 to19); Fractalkine; Glial­derived Neurotrophic Factors (GDNFs); Glucocorticoid­induced TNF Receptor Ligand (GITRL);Granulocyte Colony Stimulating Factor (G­CSF); Granulocyte Macrophage Colony Stimulating Factor (GM­CSF);Granulocyte Chemotactic Proteins (GCPs); Growth Hormone (GH); I­ 309; Growth Related Oncogene (GRO); lnhibins (Inh);Interferon­inducible T­cell Alpha Chemoattractant (I­TAC); Fas Ligand (FasL); Heregulins (HRGs); Heparin­ Binding EpidermalGrowth Factor­Like Growth Factor (HB­EGF); fms­like Tyrosine Kinase 3 Ligand (Flt­3L); Hemofiltrate CC Chemokines (HCC­1 to 4); Hepatocyte Growth Factor (HGF); Insulin; Insulin­like Growth Factors (IGF 1 and 2); Interferon­ gamma InducibleProtein 10 (IP­10); lnterleukins (IL 1 to 18); Interferon­gamma (IFN­gamma); Keratinocyte Growth Factor (KGF); KeratinocyteGrowth Factor­2 (FGF­10); Leptin (OB); Leukemia Inhibitory Factor (LIF); Lymphotoxin Beta (LT­B); Lymphotactin (LTN);Macrophage­Colony Stimulating Factor (M­CSF); Macrophage­derived Chemokine (MDC); Macrophage Stimulating Protein(MSP); Macrophage Inflammatory Proteins (MIPs); Midkine (MK); Monocyte Chemoattractant Proteins (MCP­1 to 4);Monokine Induced by IFN­gamma (MIG); MSX 1 ; MSX 2; Mullerian Inhibiting Substance (MIS); Myeloid Progenitor InhibitoryFactor 1 (MPIF­1 ); Nerve Growth Factor (NGF); Neurotrophins (NTs); Neutrophil Activating Peptide 2 (NAP­2); Oncostatin M(OSM); Osteocalcin; OP­1 ; Osteopontin; OX40 Ligand; Platelet derived Growth Factors (PDGF aa, ab and bb); PlateletFactor 4 (PF4); Pleiotrophin (PTN); Pulmonary and Activation­regulated Chemokine (PARC); Regulated on Activation, NormalT­cell Expressed and Secreted (RANTES); Sensory and Motor Neuron­derived Factor (SMDF); Small Inducible CytokineSubfamily A Member 26 (SCYA26); Stem Cell Factor (SCF); Stromal Cell Derived Factor 1 (SDF­1 ); Thymus and Activation­regulated Chemokine (TARC); Thymus Expressed Chemokine (TECK); TNF and ApoL­ related Leukocyte­expressed Ligand­1(TALL­1); TNF­related Apoptosis Inducing Ligand (TRAIL); TNF­related Activation Induced Cytokine (TRANCE); LymphotoxinInducible Expression and Competes with HSV Glycoprotein D for HVEM T­ lymphocyte receptor (LIGHT); Placenta GrowthFactor (PIGF); Thrombopoietin (Tpo); Transforming Growth Factors (TGF alpha, TGF beta 1 , TGF beta 2); Tumor NecrosisFactors (TNF alpha and beta); Vascular Endothelial Growth Factors (VEGF­A1B1C and D).

2'­Deoxy­Nucleic acids (DNA):

[0146] DNA encodes the genes for proteins and peptides. Also, DNA contains a wide array of sequences that regulate theexpression of the contained genes. Several types of DNA exist, depending on source, function, origin, and structure. Typicalexamples for DNA based molecules that can be utilized as bioactive, slow release coatings on probe tips (local gene­therapy)include, but are not limited to: A­DNA; B­DNA; artificial chromosomes carrying mammalian DNA (YACs); chromosomal DNA;circular DNA; cosmids carrying mammalian DNA; DNA; Double­stranded DNA (dsDNA); genomic DNA; hemi­methylatedDNA; linear DNA; mammalian cDNA (complimentary DNA; DNA copy of RNA); mammalian DNA; methylated DNA;mitochondrial DNA; phages carrying mammalian DNA; phagemids carrying mammalian DNA; plasmids carrying mammalianDNA; plastids carrying mammalian DNA; recombinant DNA; restriction fragments of mammalian DNA; retroposons carryingmammalian DNA; single­stranded DNA (ssDNA); transposons carrying mammalian DNA; T­DNA; viruses carryingmammalian DNA; Z­DNA.

Ribo­Nucleic Acids (RNAs):

[0147] RNA is a transcription of DNA­encoded information. In some viruses, RNA is the essential information­encoding unit.Besides being an intermediate for expression of genes, RNAs have been shown to have several biological functions.Ribozymes are simple RNA molecules with a catalytic action. These RNAs can catalyze DNA and RNA cleavage andligation, hydrolyze peptides, and are the core of the translation of RNA into peptides (the ribosome is a ribozyme).

[0148] Typical examples of RNA molecules with potential for use as bioactive coating on metal hybrid coated probe tipsinclude, but are not limited to: acetylated transfer RNA (activated tRNA, charged tRNA); circular RNA; linear RNA;mammalian heterogeneous nuclear RNA (hnRNA), mammalian messenger RNA (mRNA); mammalian RNA; mammalianribosomal RNA (rRNA); mammalian transport RNA (tRNA); mRNA; poly­adenylated RNA; ribosomal RNA (rRNA);recombinant RNA; retroposons carrying mammalian RNA ; ribozymes; transport RNA (tRNA); viruses carrying mammalian

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RNA.

Receptors:

[0149] Receptors are cell surface biomolecules that bind signals (e.g. hormone ligands and growth factors) and transmit thesignal over the cell membrane and into the internal machinery of cells. Different receptors are differently "wired" imposingdifferent intracellular responses even to the same ligand. This makes it possible for the cells to react differentially to externalsignals by varying the pattern of receptors on their surface.

[0150] Receptors typically bind their ligand in a reversible manner, making them suitable as carriers of growth factors that areto be released into the tissue. Thus by coating cantilever with growth factor receptors, and then load these receptors withtheir principal ligands, a bioactive surface is achieved that can be used for controlled release of growth factors to thesurrounding tissues following implantation. Examples of suitable receptors with potential for use as bioactive coating on metalhybrid coated cantilever includes: The CD class of receptors CD; EGF receptors; FGF receptors; Fibronectin receptor (VLA­5); Growth Factor receptor, IGF Binding Proteins (IGFBP 1 to 4); lntegrins (including VLA 1­4); Laminin receptor; PDGFreceptors; Transforming Growth Factor alpha and beta receptors; BMP receptors; Fas; Vascular Endothelial Growth Factorreceptor (Flt­1 ); Vitronectin receptor.

Synthetic Biomolecules

[0151] Synthetic biomolecules are molecules that are based on and/or mimic naturally occurring biomolecules. Bysynthesizing such molecules a wide array of chemical and structural modification can be introduced that can stabilize themolecule or make it more bioactive or specific. Thus if a molecule is either too unstable or unspecific to be used fromextracts it is possible to engineer them and synthesize them for use as implant surface coatings.

[0152] Furthermore, many biomolecules are so low abundant that extraction in industrial scales is impossible. Such rarebiomolecules have to be prepared synthetically, e.g. by recombinant technology or by (bio­) chemistry. Below is listed severalclasses of synthetic molecules that can be potentially useful for implant coatings:

Synthetic DNA: [0153] Synthetic DNA molecules are biomolecules. These include, but are not limited to: A­DNA; antisenseDNA; B­DNA; complimentary DNA (cDNA); chemically modified DNA; chemically stabilized DNA; DNA ; DNA analogues ;DNA oligomers; DNA polymers; DNA­RNA hybrids; double­stranded DNA (dsDNA); hemi­methylated DNA; methylated DNA;single­stranded DNA (ssDNA); recombinant DNA; triplex DNA; T­DNA; Z­DNA.

Synthetic RNA:

[0154] Synthetic RNA molecules are biomolecules. These include, but are not limited to: antisense RNA; chemically modifiedRNA; chemically stabilized RNA; heterogeneous nuclear RNA (hnRNA); messenger RNA (mRNA); ribozymes; RNA; RNAanalogues; RNA­DNA hybrids; RNA oligomers; RNA polymers; ribosomal RNA (rRNA); and transport RNA (tRNA).

Synthetic Biopolymers:

[0155] The term biomolecules encompasses synthetic biopolymers, including but not limited to cationic and anionicliposomes; cellulose acetate; hyaluronic acid; polylactic acid; polyglycol alginate; polyglycolic acid; poly­prolines;polysaccharides.

Synthetic peptides:

[0156] Synthetic peptides are also encompassed by the term biomolecules. These peptides include, but are in no way limitedto: decapeptides containing DOPA and/or diDOPA; peptides with sequence "Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys";peptides where Pro is substituted with hydroxyproline; peptides where one or more Pro is substituted with DOPA; peptideswhere one or more Pro is substituted with diDOPA; peptides where one or more Tyr is substituted with DOPA; peptidehormones; peptide sequences based on the above listed extracted proteins; peptides containing an RGD (Arg GIy Asp) motif.Recombinant proteins:

All recombinantly prepared peptides and proteins

Synthetic Enzyme inhibitors:

[0157] Synthetic enzyme inhibitors range from simple molecules, like certain metal ions, that block enzyme activity bybinding directly to the enzyme, to synthetic molecules that mimic the natural substrate of an enzyme and thus compete withthe principle substrate. An implant coating including enzyme inhibitors could help stabilizing and counteract breakdown ofother biomolecules present in the coating, so that more reaction time and/or higher concentration of the bioactive compoundis achieved. Examples of enzyme inhibitors include, but are not limited to: pepstatin; poly­prolines; D­sugars; D­aminocaids;Cyanide; Diisopropyl fluorophosphates (DFP); metal ions; N­tosyl­l­phenylalaninechloromethyl ketone (TPCK);Physostigmine; Parathion; Penicillin.

Vitamins (synthetic or extracted):

[0158] The term biomolecules also encompasses synthetic and extracted vitamins, including but not limited to: biotin;calciferol (Vitamin D's; vital for bone mineralisation); citrin; folic acid; niacin; nicotinamide; nicotinamide adenine dinucleotide(NAD, NAD+); nicotinamide adenine dinucleotide phosphate (NADP1 NADPH); NAD+retinoic acid (vitamin A); riboflavin;

vitamin B's; vitamin C (vital for collagen synthesis); vitamin E; and vitamin K's.

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Other bioactive molecules for AFM probe tip coatings:

[0159] Other suitable molecules for coating include, but are not limited to: adenosine di­phosphate (ADP); adenosine mono­phosphate (AMP); adenosine triphosphate (ATP); amino acids; cyclic AMP (cAMP); 3,4­dihydroxyphenylalanine (DOPA); 5'­di(dihydroxyphenyl­L­alanine (diDOPA); diDOPA quinone; DOPA­like o­ diphenols; fatty acids; glucose; hydroxyproline;nucleosides; nucleotides (RNA and DNA bases); prostaglandin; sugars; sphingosine 1 ­phosphate; and rapamycin.

Drugs for AFM probe tip coatings:

[0160] Pharmaceuticals incorporated in a hybrid layer of the probe tip coating may be utilized for local effects like improvinglocal resistance against invading microbes, local pain control, local inhibition of prostaglandin synthesis; local inflammationregulation, local induction of biomineralisation and local stimulation of tissue growth. Examples of pharmaceuticals suitablefor incorporation into metal hydride layers include, but are not limited to: Antibiotics; cyclooxygenase inhibitors; hormones;inflammation inhibitors; NSAID's; painkillers; prostaglandin synthesis inhibitors; steroids, tetracycline (also as biomineralizingagent).

Biologically active ions for AFM probe tip coatings:

[0161] Ions are important in a diversity of biological mechanisms. By incorporating biologically active ions in metal hybridcoated layers on cantilever it is possible to locally stimulate biological processes like enzyme function, enzyme blocking,cellular uptake of biomolecules, homing of specific cells, biomineralization, apoptosis, cellular secretion of biomolecules,cellular metabolism and cellular defense. Examples of bioactive ions for incorporation into metal hybrid coated include, butare not limited to: calcium; chromium; copper, fluoride; gold; iodide; iron; potassium; magnesium; manganese; selenium;silver; sodium; zinc.

[0162] In one embodiment of the invention, ALD may be used to form films containing biomolecules. In another embodiment,TMA (AI(CH3)3) molecules or similar precursors are pulsed into the chamber to form an initial reactive layer on the surface. A

biological precursor is then pulsed into the chamber, forming bonds between the biomolecules and the aluminum atoms onthe surface.

[0163] In another embodiment, the biological precursor may be serotonin. TMA (AI(CH3)3) molecules or similar precursors are

pulsed into the chamber to form an initial reactive layer on the surface. Either the free amine group or the hydroxyl group willreact with the aluminum atoms at the surface to form a film.

serotonin

[0164] Serotonin is a monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system (CNS)and enterochromaffin cells in the gastrointestinal tract of animals including humans. In the central nervous system, serotoninis believed to play an important role in the regulation of anger, aggression, body temperature, mood, sleep, vomiting,sexuality, and appetite. The bioamine serotonin (5­hydroxytryptamine; 5­HT), is a well­known neurotransmitter in the centralnervous system. In addition, serotonin plays important roles in normal embryogenesis and cell growth, as well as being aregulator of physiological functions such as peristalsis in the gastrointestinal tract and blood pressure regulation. SeeFrishman et al., J Clin. Pharmacol., 35: 541­ 572 (1995); Gershon et al., Aliment Pharmacol. Then, 13(Suppl 2): 15­30 (1999).In 2001 , a relationship between serotonin and bone was suggested by the demonstration of functional receptors for serotoninin osteoblastic cells. Westbroek et al., J. Cell. Biochem., 101 (2): 360­8 (2001 ). Serotonin may also regulate bone cellproliferation, differentiation, and activation in vitro. Bliziotes et al., Bone 29: 477­486 (2001 ); Westbroek et al., J. Cell.Biochem.,101 (2): 360­8 (2001 ); Gustafsson et al., J. Cell. Biochem. 97: 1283­1291 (2006). Moreover, long­term serotoninadministration may result in increased bone mineral density (BMD), stiffer bones, and altered bone architecture in rats.Gustafsson et al., J. Cell. Biochem. 97: 1283­1291 (2006). These in vivo serotonergic effects on bone may be direct viaserotonin receptors, but may also be indirect via interaction with other bone regulating substances such as leptin andadiponectin. J. Yamada et al., Eur. J. Pharmacol., 383: 49­51 (1999); J. Yamakawa et al., Diabetes Care, 26: 2477­2478(2003). [0165] Serotonin and its transporter may also play a role in bone metabolism. The expression of the rate­limitingenzyme in serotonin synthesis, tryptophan hydroxylase, in osteoblasts and osteoclasts, has been demonstrated, indicatingthat bone cells may be capable of synthesizing serotonin. Gustafsson et al., J. Cell. Biochem., 98: 139­151 (2006). Themembrane­bound serotonin transporter (5­HTT) expression has also been demonstrated in both osteoblasts and osteoclasts.Bliziotes et al., Bone 29: 477­486; Gustafsson et al., J. Cell. Biochem., 98: 139­151 (2006), and is responsible for the cellularinternalization of serotonin, and is thus a key protein in serotonergic signaling and serotonin metabolism. The serotoninreceptor­bearing bone cells may not only be able to respond to serotonin, but may also be able to regulate serotoninavailability themselves, via its transporters as well as via synthesis.

[0166] Modulation of the serotonergic system may influence bone metabolism. In addition, long­term use of fluoxetine mayeffect on bone health. The exact mechanisms of serotonin action on bone metabolism are still unclear. Serotonin may actdirectly on the receptors on the bone cells, or may also act indirectly via other factors important in bone metabolism, like

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leptin and adiponectin. See Ducy et al., Cell 100: 197­207 (2000); Takeda et al., Cell, 111 : 305­317 (2002); Gordeladze andReseland, J. Cell. Biochem., 88: 706­712 (2003); Luo et al., Exp Cell Res 309: 99­109 (2005); Oshima et al., Biochem.Biophys. Res. Commun., 331 : 520­526 (2005). In one embodiment of the invention, the use of serotonin coated probe tipsmay enhance the understanding of the relationship between serotonin and bone cells.

[0167] In another embodiment, ALD may be used to deposit biomolecular films on an AFM tip. These tips may optionally beused to image sample surfaces and to study the interactions between the bone­like material and sample surfaces.

[0168] Where a precursor contains two or more functional groups, the functional groups may be the same or different. Theprecursor should leave a reactive site suitable for succeeding growth with another type of precursor, unless no furtherdeposition steps are desired. For production of the final terminating surface, the requirement of two or more types offunctional groups does not apply. See WO 2006/071126A1. Rather the precursor should have at least one functional groupthat will undergo reactions with the previous surface, and also contain the groups that should finally terminate the surface.

[0169] Of particular interest is the study of interactions between biological materials and different types of surfaces. In oneembodiment, probe tips may be coated with specific peptides using a sequence of amino acid analogs as ALD precursors.Peptides are formed by combining amino acids through amide bonds. For example, di­acyl chlorides of amino acids andamino acids with two amino groups may be used to deposit peptide films. Alternatively, dicarboxylic acids and diamines maybe used to form peptide films.

[0170] These tips may then be used to probe biological surfaces. In one embodiment, depending on the sequence of thepeptide, it will be possible to map the distribution, affinity, and dynamics of different receptors, for example, integrin receptorsin a bone cell membrane. Magnetic/Conductive Coatings

[0171] Magnetic films may be constructed from magnetic materials that include, by way of non­limiting example,ferromagnetic, ferrimagnetic and paramagnetic materials. Examples of conductive films include, but are not limtied toruthenium, palladium, molybdenum, TiN, LaNiθ3, ZnO:AI, iridium, platinum and copper. Currently available MFM tips usually

have a film thickness >30nm. One way to improve resolution of MFM is to reduce the film thickness, although this may leadto a reduction in the conductivity of the probe.

[0172] In at least one embodiment of the present disclosure, ALD may be used to deposit magnetic/conductive films on probetips, for example Fe­Co with a thickness of 25±5 nm while maintaining stiffness of the probe equal to currently available MFMtips, thereby reducing film thickness without altering stiffness.

[0173] In one embodiment, a film is constructed by the ALD technique using suitable precursors for forming magneticmaterials, according to the procedure described in Dalton Transactions, 2008, pgs. 253­259 by Lie et al., where Co(thd)2 and

Fe(thd)3 in combination with ozone are used as the precursors to form (Fe1Co)3O4 materials. The magnetic properties of the

film may be further improved by annealing in a controlled oxygen and temperature environment.

[0174] In yet another embodiment, ALD may be used to deposit magnetic films by depositing an oxide or mixture of oxidesthat become ferro/ferri­magnetic when being subjected to suitable reduction or oxidation processes. A non­limiting list ofexamples are: Films of Fe2O3 may become ferrimagnetic Fe3O4 (magnetite) upon reduction, Cr2O3 may become

ferromagnetic CrO2 upon oxidation and the magnetic properties of manganite perovskites may be tuned depending on the

oxidation potensial. In addition films containtn mixtures of oxides may be reduced to magnetic metallic states by suitablereduction processes such as annealing in pure H2 at elevated temperatures, such as the process described in International

Application No. WO 2002/045167. Hard Coatings

[0175] Hard coatings may be useful for maintaining sharp tips over time, for example when a physical contact tip­hard surfaceis used. In at least one embodiment, ALD may be used to deposit coatings on probe tips, for example to prolong the lifetimeof the tip and the recording time of the probe. Thin layers of hard coatings enable conservation of the low spring constant ofthe probe and the geometry of the cantilever to remain unchanged over time.

[0176] Non­limiting examples of hard coatings include TiO2, AI2O3, ZrO2, Ti­ Nitride, multi­layered materials and hybrid hard

coatings.

[0177] In one embodiment, ALD may be used to coat SPM tips with hard coatings, for example by pulsing TiCI4 and

ammonia (NH3) precursors into the reaction chamber onto the surface according to the ALD principle, using a carrier gas at

elevated temperature and repeating until desired thickness is obtained for Ti­ Nitride films, as described for example, inInternational Application No. WO 2007/013924 and Journal of Applied Physics, 2005, 97, 121301 , by Puurunen.

TiO? Coated SPM Probes

[0178] The stiffness of the cantilever of the probe and the radius of the probe tip in contact with the substrate are twoparameters that may affect the resolution and the quality of the data.

[0179] The material composing the outer surface of the current commercially available probes include: SiN, Si3N4, and SiN

coated with Au, Al or Pt, which may exhibit short life­times under certain conditions. Accordingly, the need to replace wornprobes over short periods of time may be necessary, particularly for industries that use SPM applications requiring physicalcontact with the surface. [0180] Disclosed herein is a probe coated with Tiθ2, that may further strengthen the probe ascompared to currently available silicon­based probes, and thus prolong the life­time of the SPM probes.

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Example 1 Titanium Oxide AFM Tips

[0181] TiCI4 and H2O were used to coat AFM tips with TiO2. Films were grown in a commercial F­120 Sat reactor (ASM

Microchemistry) by using TiCl4 (Fluka; 98%) and H2O (distilled) as precursors. Both precursors were kept at room

temperature in vesels outside the reactor during the deposition. The reactor pressure was maintained at ca. 1.8 mbar by

employing an N2 carrier­gas flow of 300 cm3 min~1 supplied from a Nitrox 3001 nitrogen purifier with a purity of 99.9995%

inert gas (N2 + Ar) according to specifications.

[0182] The films were grown using a pulsing scheme of 2 s pulse of TiCI4 followed by a purge of 1 s. Water was then admitted

using a pulse of 2 s followed by a purge of 1 s. This complete pulsing scheme makes up one pulsing cycle and the films weremade using different numbers of such cycles (typically from 20­2000 cycles). Films can be formed in a relatively large

temperature interval as shown in Figure 4. Using a deposition temperature of 150 0C we obtained a growth rate of 0.054nm/cycle.

[0183] The deposition may be expressed accordingly: Step 1 : TiCU(g) + I­OH → 1­0­TiCI3 + HCI(g)

Step 2:

1­0­TiCI3 + H2O(g) → |­O­Ti­(OH)3 + 3HCI(g)

[0184] The reactions may be shifted so that the liberation of HCI(g) is more in step 1 and less in step 2 depending on thereaction conditions. See R.L. Puurunen, J. Appl. Phys. 97 (2005) 121301.

[0185] By performing the deposition at a reactor temperature at or below 165 0C, the resulting layer may be practicallyamorphous. The amorphous film may optionally be converted into the TiO2 forms rutile or anatase by post annealing.

Alternatively, the structure may be controlled in situ as described in J. Aarik et al., J. Cryst. Growth 148: 268 (1995) where

anatase is deposited in the range 165 ­ 350 0C and rutile is obtained at temperatures above 350 0C.

[0186] Alternatively, polycrystalline films of pure rutile, pure Tiθ2­ll, or a mixture of them both may be formed. See J. Aarik, A.

Aidla, V. Sammelselg, H. Siimon, T. Uustare, J. Cryst. Crowth 169 (1996) 496. Polycrystalline films of pure rutile, pure TiO2­

II, or a mixture of them both can be produced by varying the water pressure during deposition at 400 0C for otherwise similarconditions using TiCI4 and H2O as precursors. The TiO2­ll structure is an orthorhombic phase isomorphous to D­PbO2.

[0187] Molecular force probing (MFP/AFM Asylum research, Santa Barbara U.S.) with titanium oxide coated AFM cantilevers(AC160 Olympus SilicaNitril, Tokyo, Japan) with tip radii 10 nm and height 10 μm was used to scan mouse pre­ osteoblasts

(donated) in DMEM (Dulbecco/Vogt Modified Eagle's Minimal Essential Medium, Cambrex Biosciences, UK), at 22 0C inaqueous contact mode.

[0188] Mouse pre­osteoblasts (Type, MC3T3 E1 , CRL­2593, ATCC, Manassas, USA) were placed with DMEM in anenvironmental chamber (Bioheater, Asylum Research, USA) on a polymer slide (NUNC™ Brand Pocket, SlideFlask,Roskilde, Denmark) without any fixation chemicals.

[0189] 400 force curves were generated. Representative curves are shown in Figures 5 and 6. Figure 5 reveals strongadhesion between the TiO2­coated AFM tip and the osteoblast.

[0190] These force curves were then used to generate a map of adhesion strengths across the cell surface, shown in Figure7.

Example 2 Titanium Oxide Coated SPM Cantilevers

[0191] SPM cantilevers (RC­800PSA, Olympus, Tokyo, Japan) were coated in a commercial F­120 Sat reactor (ASMMicrochemistry) by using TiCI4 (Aldrich; 99%) and H2O (distilled) as precursors, such as the process described in J. Aarik,

A. Aidla, E. Uustare, V. Sammelselg, Journal of Crystal Growth, 148(1995) 268. Both precursors were kept at roomtemperature in vessels outside the reactor during the deposition and pulsed into the reactor without an external carrier gas.[0192] To provide saturated growth, the deposition process followed a pulsing scheme of 0.6 s pulse of TiCI4 followed by a 1 s

purge. Water was then admitted using a 2 s pulse followed by a 1 s purge. Deposition was performed at three different reactor

temperatures: 1500C, 3000C, and 4000C conducive to providing amorphous, anatase and rutile phases respectively. Usingdeposition temperatures of 150°C and 300°C, we obtained a growth rate of 0.046 and 0.040 nm/cycle respectively.

[0193] A silicon substrate was subjected to the deposition process described above and compared with cantilevers coatedwith titanium oxide to measure and compare film thickness and crystallinity.

[0194] The crystallinity of the films was analyzed by x­ray diffraction (Siemens D5000 diffractometer) in Θ­2Θ mode usingCuKa radiation. The results from the x­ray diffraction analysis are shown in Figure 16. SEM was used to measure TiO2 film

thickness using a TM­1000 (Hitachi tabletope microscope, Tokyo, Japan) and a Philips XL 30 ESEM (FEI Electronics Optics,Eindhoven, Netherlands). The results for these measurements are shown in Figures 17 and 18.

[0195] Cantilever stiffness, according to coating thickness, was also analyzed by AFM. AFM (MFP­3D, Asylum research,Santa Barbera, U.S.) was performed on uncoated cantilevers and TiO2­coated cantilevers with seven different thicknesses: 1 ,

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5.4, 6, 10.9, 12.5, 30, 50, 75, 98.4, 114, and >120nm. Spring constant (i.e. stiffness of the cantilevers) was measured byforce curve analysis. The results are shown in Figure 19.

[0196] MFP was used to analyzed adhesion forces between coated and non­coated cantilevers versus cell culture plates. 900force­curves were performed using MFP to test the physical/chemical activation of the tip surface by a 6nm TiO2 coating

compared with SiN non­coated tip. The substrates used for analysis were the Nunclon™Δ Surface (NUNC A/S, Roskilde,Denmark), and collagen I coated plates (NUNC A/S, Roskilde, Denmark). The tests were performed in deionized water atroom temperature. Results are shown in Figures 20 and 21. Example 3 Ti­O­N Surfaces

[0197] TiOxNy surfaces may be produced by varying the usage of H2O or NH3 as precursor in the reaction scheme described

for growth of TiO2. The reaction scheme may be as follows: Step 1 : TiCU(g) + I­OH → 1­0­TiCI3 + HCI(g)

Step 2a:

1­0­TiCI3 + 3H2O(g) → |­O­Ti­(OH)3 + 3HCI(g)

Step 2b:

1­0­TiCI3 + 3NH3(g) → |­O­Ti­(NH2)3 + 3HCI(g)

Example 4 Calcium Carbonate

[0198] Calcium carbonate films were grown in a commercial F­120 Sat reactor (ASM Microchemsitry) by using Ca(thd)2(Strem Chemicals; 99.9%; Hthd = 2,2,6,6­tetramethylheptan­3,5­dione) and ozone as precursors, with CO2 (AGA; 99.99 %)

as controlling atmosphere in selected runs. Ozone gas with a flow of 500 cm3 min"1 was produced by feeding pure O2 (AGA

99.999%) into an ozone generator (OT­020, OzoneTechnology; giving an ozone concentration of 15 vol.% according to

specifications). The applied sublimation temperature for Ca(thd)2 was 195 0C. The reactor pressure was maintained at ca. 1.8

mbar by employing an N2 carrier­gas flow of 300 cm3 min"1, supplied from a Nitrox 3001 nitrogen purifier with a purity of

99.9995% inert gas (N2 + Ar) according to specifications. Additional CO2 (AGA 99%) was introduced to produce well

crystalline carbonate. The CO2 was introduced at a flow of approximately 400 cm3 min"1.

[0199] The films were made using a pulsing scheme of 1 s Ca(thd)2 followed by a purge of 0.5 s and then a 2 s pulse of O3followed by a purge of 0.8 s. In addition to this sequence, an additional pulse of 3 s CO2 was introduced followed by a purge

of 1 s. This sequence forms a cycle may be repeated until the desired film thickness is achieved. Films using a total of 250 ­5000 cycles have been produced. [0200] Under these conditions, the growth rate varied relatively little with depositiontemperature, and was at ca. 0.045 nm/ cycle, as shown in Figure 8.

Example 5 Calcium Phosphates

[0201] Calcium phosphate films may be grown in a commercial F­120 Sat reactor (ASM Microchemsitry) by using Ca(Cp)2(Cp = cyclopentadienyl), H2O (distilled), and POCI3 (99% Fluka) as precursors. Ca(Cp)2 may be sublimed at ca. 85 °C, and

H2O and POCI3 may be kept at room temperature in vessels outside the reactor during the deposition. The reactor pressure

may be maintained at ca. 1.8 mbar by employing an N2 carrier­gas flow of 300 cm3 min~1, supplied from a Nitrox 3001

nitrogen purifier with a purity of 99.9995% inert gas (N2 + Ar) according to specifications.

[0202] The films may be grown using a pulsing scheme of 2 s pulse of Ca(Cp)2 followed by a purge of 1 s. Water may then be

admitted using a pulse of 2 s followed by a purge of 1 s. POCI3 may then be pulsed for 2 s followed by a purge of 1 s. Water

may then be admitted using a pulse of 2 s followed by a purge of 1 s. This pulsing scheme comprises one pulsing cycle.Films may be made using different numbers of these cycles (typically from 20­2000 cycles). Films may be formed at suitabledeposition temperatures. The films may be made by repeating the pulsing cycles until the desired thickness was achieved.

[0203] The deposition may be expressed as follows: Step 1 : I­OH + Ca(Cp)2(g) = |­O­Ca­Cp + H­Cp(g)

Step 2:

I­Ca­Cp + H2O(g) = |­Ca­OH + H­Cp(g)

Step 3:

2I­OH + POCI3(g) = |­PO3CI + 2HCI(g)

Step 4:

1­PO3CI + H2O(g) = 1­PO4H + HCI(g) [0204] Alternatively, films may be grown as follows. Films may be grown using Ca(Cp)2

(Cp = cyclopentadienyl), H2O (distilled), and PPh3 (Ph = phenyl) (98.5% Fluka) as precursors. Ca(Cp)2 may be sublimed at

ca. 85 0C, while H2O and PPh3 may be kept at room temperature in vessels outside the reactor during the deposition. The

reactor pressure may be maintained at ca. 1.8 mbar by employing an N2 carrier­gas flow of 300 cm3 min~1, supplied from a

Nitrox 3001 nitrogen purifier with a purity of 99.9995% inert gas (N2 + Ar) according to specifications.

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[0205] The films may grown using a pulsing scheme of 2 s pulse of Ca(Cp)2 followed by a purge of 1 s. Water may then be

admitted using a pulse of 2 s followed by a purge of 1 s. PPh3 may then be pulsed for 2 s followed by a purge of 1 s. Water

may then be admitted using a pulse of 2 s followed by a purge of 1 s. Ozone may was be then admitted for 3 s followed by apurge of 1 s. This complete pulsing scheme makes up one pulsing cycle and the films were made using different numbers ofsuch cycles (typically from 20­2000 cycles). Films may be formed at suitable deposition temperatures. The films may bemade repeating the pulsing cycles until the desired thickness is achieved.

[0206] This deposition sequence may be expressed as follows: Step 1 : I­OH + Ca(Cp)2(g) = |­O­Ca­Cp + H­Cp(g)

Step 2:

I­Ca­Cp + H2O(g) = |­Ca­OH + H­Cp(g)

Step 3:

I­OH + P(Ph)3(g) = |­O­P(Ph)2 + H­Ph(g)

Step 4:

1­0­P(Ph)2 + 2H2O(g) = |­O­P(OH)2 + 2H­Ph(g)

Step 5: 1­0­P(OH)2 + O3(g) = 1­0­PO(OH)2 + O2(g)

[0207] Alternatively, ozone may be pulsed in place of H2O in step 4. Step 5 may then be expressed as: Step 4b: |­O­P(Ph)2+ xO3 = |­O­PO(OH)2 + CO2(g) + H2O(g)

[0208] Alternatively, the process may be modified to produce fluorapatite by pulsing NH4F in place of water in one or more

pulses according to the reaction:

Ca2(PO4)(OH) + NH4F = Ca2(PO4)F + NH3 + H2O

[0209] An alternative approach to obtain Ca­P­O films is to use the precursor pairs Ca(thd)2 + O3 and (CH3O)3PO + H2O as

precursors in a mixed fashion. The film is formed by first producing a monolayer of CaCO3 using the Ca(thd)2 + O3 precursor

pair as described in Example 3 and theafter transforming this into Ca3(PO4)2 by using the (CH3O)3PO + H2O precursor pair.

The Ca3(PO4J3 film can be transformed into hydroxyapatite by subsequent treatment of the film in moist N2 at temperatures

above 500 0C.

Example 6 Calcium Fluoride

[0210] CaF2 films may be produced using Ca(thd)2 and HF as precursors. See M. Ylilammi & T. Ranta­aho, J. Electrochem.

Soc, 141 : 1278 (1994).

[0211] The reactions may proceed as follows. Overall: Ca(thd)2 + 2HF = CaF2 + 2Hthd Step 1 : |­Ca­F2 + Ca(thd)2(g) = |­Ca­

F2­Ca(thd)2

Step 2: |­Ca­thd2 + 2HF(g) = |­Ca­F2 + 2Hthd(g) [0212] Alternatively, the fluoride may be introduced using TiF4 or TaF5 as

precursors. The films may be formed according to the reaction scheme:

2Ca(thd)2(g) + TiF4(g) = 2CaF2(S) + Ti(thd)4(g)

5Ca(thd)2(g) + 2TaF5(g) = 5CaF2(S) + 2Ta(thd)5(g)

[0213] The TiF4 precursor may be sublimed in the reactor at 140­145 0C. The TaF5 precursor may be sublimed in the reactor

at 45­50 0C. The Ca(thd)2 precursor may be sublimed in the reactor at 195 °C. The pulse times for Ca(thd)2 may be 2 s or

longer followed by a purge of 1 s. The pulse time for TiF4 or TaF5 may be 1 s followed by a purge of 1 s.

Example 7 Glycine Hybrid Films

[0214] Glycine hybrid films were grown in a commercial F­120 Sat reactor (ASM Microchemsitry) by using either TiCI4 (Fluka;

98%) or TMA (trimethylaluminium; Withco, 98%) and glycine (Aldrich, 99%). The metal precursors were kept at room

temperature in vessels outside the reactor during the deposition. Glycine was sublimed in the reactor at 200 0C. The reactor

pressure was maintained at ca. 2 mbar by employing an N2 carrier­gas flow of 300 cm3 min~1 supplied from a Nitrox 3001

nitrogen purifier with a purity of 99.9995% inert gas (N2 + Ar) according to specifications.

[0215] The films were grown using a pulsing scheme of 1 s pulse of TiCI4 or TMA followed by a purge of 2 s. Glycine was

then admitted using a pulse of 2 s followed by a purge of 2 s. This complete pulsing scheme makes up one pulsing cycle andthe films were made using different numbers of such cycles (typically from 2­2000 cycles). Quarts crystal microbalanceresults from such measurements performed at 250 °C are shown in Figures 9 and 10. Typical growth rates of these films at

250 0C are 0.47 nm/cycle for Ti­Glycine and 0.53 nm/cycle for Al­Glycine. Example 8 4­Aminobenzoic Acid Hybrid Films

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[0216] 4­Aminobenzoic acid (Aldrich; 99%) films were grown in a commercial F­120 Sat reactor (ASM Microchemsitry) byusing TiCI4 (Fluka; 98%). The metal precursors were kept at room temperature in vessels outside the reactor during the

deposition. 4­aminobenzoic acid was sublimed in the reactor at 160 °C. The reactor pressure was maintained at ca. 2 mbar

by employing an N2 carrier­gas flow of 300 cm3 min~1, supplied from a Nitrox 3001 nitrogen purifier with a purity of 99.9995%

inert gas (N2 + Ar) according to specifications.

[0217] The films were grown using a pulsing scheme of 1 s pulse of TiCI4 followed by a purge of 1 s. 4­aminobenzoic acid

was then admitted using a pulse of 2.5 s followed by a purge of 1 s. This complete pulsing scheme makes up one pulsingcycle and the films were made using different numbers of such cycles (typically from 2­2000 cycles). Quarts crystal

microbalance results from such measurements performed at 200 0C are shown in Figure 11. Typical growth rates of thecompound at 200 °C are: 1.0 nm/cycle for Ti­4­aminobenzoic acid.

Example 9 4­Aminobenzophenone Hybrid Films

[0218] 4­Aminobenzophenone hybrid films were grown in a commercial F­ 120 Sat reactor (ASM Microchemsitry) by usingTiCI4 (Fluka; 98%). The metal precursors were kept at room temperature in vessels outside the reactor during the deposition.

4­aminobenzophenone (Fluka; 97%) was sublimed in the reactor at 126 °C. The reactor pressure was maintained at ca. 2

mbar by employing an N2 carrier­ gas flow of 300 cm3 min"1, supplied from a Nitrox 3001 nitrogen purifier with a purity of

99.9995% inert gas (N2 + Ar) according to specifications.

[0219] The films were grown using a pulsing scheme of 1 s pulse of TiCI4 followed by a purge of 1 s. 4­aminobenzophenone

was then admitted using a pulse of 2.5 s followed by a purge of 1 s. This pulsing scheme makes up one pulsing cycle, andthe films were made using different numbers of such cycles (typically from 2­2000 cycles). Quarts crystal microbalance

results from such measurements performed at 200 0C are shown in the Figure 12. Example 10 Sertonin Hybrid Films

[0220] Sertonin films may be grown in a commercial F­120 Sat reactor (ASM Microchemsitry) by using TiCI4 (Fluka; 98%).

The metal precursors may be kept at room temperature in vessels outside the reactor during the deposition. Serotoninhydrochloride (Sigma) may be sublimed in the reactor at a suitable sublimation temperature, which may be approximately

200 °C. The reactor pressure may be maintained at ca. 2 mbar by employing an N2 carrier­gas flow of 300 cm3 min~1, which

may be supplied from a Nitrox 3001 nitrogen purifier with a purity of 99.9995% inert gas (N2 + Ar) according to specifications.

[0221] A suitable pulsing scheme may employ a 1 s pulse of TiCI4 followed by a purge of 1 s. Serotonin may then be

admitted using a 2.5 s pulse followed by a purge of 1 s. This example pulsing scheme makes up one pulsing cycle. Ti­sertonin films may be made using different numbers of such cycles (typically from 2­2000 cycles).

[0222] Serotonin has both a hydroxyl and two amines as functional groups and should react according to the reactionmechanisms proposed above for hybrid films.

Example 11 Amino acids and Peptide Films

[0223] Films of amino acids may be deposited by forming peptide bonds according to the reaction scheme shown below.

[0224] The carboxylic acids may be reacted with amines, forming water as a byproduct. See M. Putkonen et al., J. Mater.Chem., 17: 664 (2007).

[0225] Alternatively, the chloride salts of the carboxylic acids may be reacted with amines, forming HCI as a byproduct, asshown below. The iodine or bromine salts may also be used. See A. Kubono et al., Thin Solid Films 289: 107 (1996). Overallreaction:

CI­OC­R­CO­CI + H2N­R'­NH2 = [CI­OC­R­CO­NH­R'­NH2] + HCI(g)

Step 1 :

I­R­CO­CI + H2N­R'­NH2(g) = |­R­CO­NH­R'­NH2 + HCI(g)

Step 2:

|­R­CO­NH­R'­NH2 + CI­OC­R­CO­CI(g) = I­R­CO­NH­R'­HN­OC­R­CO­CI + HCI(g)

[0226] Alternatively, molecules of the type: Y1­NH­R­CO­CI + H2N­R'­CO­ Y2 may be used to deposit films where each Y

group may be a blocking agent that prevents self­polymerization. In one embodiment, Yi and/or Y2 may be a substituted or

unsubstituted alkyl group. Step 1 : I­R­CO­CI + H2N­R^NH­Y1Cg) = I­R­CO­NH­R'­NHΛ + HCI(g)

Step 2: l­R­CO­NH­R'­NH­Yi + H2O(g) = |­R­CO­NH­R'­NH2 + YrO­H(g)

Step 3:

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|­R­CO­NH­R'­NH2 + CI­OC­R­CO­Y2(g) = |­R­CO­NH­R'­H N­OC­R­CO­Y2 + HCI(g)

Step 4:

|­R­CO­NH­R'­HN­OC­R­CO­Y2 + H2O(g) = I­R­CO­NH­R'­HN­OC­R­CO­OH +

Y2H(g)

[0227] Alternatively, molecules of the type: H2N­R­C=C­R' or preferrably H2N­R­C≡C­R' may be used together with ozone to

form peptide bonds. The reaction mechanism may utlilize ozonolysis of the unsaturated bonds (preferrably the alkyne bond)to transform these into carboxylic acid or carboxylic anhydride groups. The reaction mechanism for formation of carboxylicanhydride groups from alkyne bonds is given in the figure below: R ≡Ξ≡Ξ R'

In,

[0228] Molecules of the type H2N­R­C≡C­R' may be pulsed into a reaction chamber. The amine group may then react with the

previous surface to form a peptide bond. This reaction may occur, but is not limited to, through an anhydride on that surface.Excess precursor may then optionally be removed from the reaction chamber. Ozone may then be admitted. The ozone mayreact with the alkyne bond to form a carboxylic anhydride. Excess ozone may then be removed from the reaction chamber.The resulting surface may then consist of carboxylic anhydrides that may function as reactive goups for formation of newpeptide bonds by admission of further amines and liberation of a carboxylic acid containing the R' group. The process maythus be repeted with the same type or different types of amines. When molecules of the type H2N­R­C=C­R' are used as

precursors, the ozonolysis may result in carboxylic acid groups that may be used for further reaction with amines and/oramine salts to produce peptide bonds.

Example 12 Large Biomolecules

[0229] Biomolecular materials may be attached to surfaces as follows. A surface (such as an AFM tip) may be treated withor exposed to water vapour or liquid water to form a terminating hydroxyl layer.

[0230] In an inert atmosphere, this surface may then be dipped into a solution of TMA or other highly reactive metalcomplexes such as, but not limited to, TiCU, diethyl zinc, magnesium cyclopendadienyl, in an inert organic solvent, forexample but not limited to, heptane, hexane, or toluene. The concentration of TMA (metal complex) may be in the range of,but not limited to, 0.0001 ­ 10 M in the inert solvent. This surface may then be rinsed gently with an inert organic solvent toremove excess TMA or other metal complex. [0231] The surface may then be dipped into a separate solution containing anorganic biomolecule. This biomolecule will then attach to the surface by reaction with the reactive metal complexes. Theorganic biomolecules may be in an inert organic solvent. The identity of the biomolecule may be any biomolecule containing afunctional group that will react with the methyl­aluminum (or other metal complex) surface. Suitable biomolecules include butare in no way limited to RGD peptide and RNA and/or DNA molecules.

Example 13

[0232] AFM tips coated with peptides can be used to map and probe receptors on cell membrane surfaces. This is illustratedin Figure 13. An AFM cantilever tip may be coated with peptide ("A"). Molecular force probing can then be used to map thesurface of a bone cell. If A contains the motif Arg­Gly­Asp (RGD) tri­peptides, there will be an adhesive force ("B") betweenthe peptide and the integrin receptor ("C") on the cell membrane. This adhesive force will be specific to the interactionbetween peptide A and the integrin (αvβ3/ αvβ5 and α5βi). The peptide will not interact as strongly with other receptors,

represented by "D" in Figure 13. Therefore, a map of force curves can reveal the location, distribution, and abundance of thereceptor specific to the peptide on the AFM tip.

[0233] Depending on the peptide sequence, one can obtain topo­dynamic three­dimensional maps of different receptors onthe cell membrane. By switching between tips coated with different materials, the distribution in 3D of several competing orinteracting receptors on the same cell can be mapped and the binding forces between ligands and receptors measured.

Example 14

[0234] An AFM cantilever coated with serotonin according to the invention described herein (such as in Example 9) may beused in an atomic force microscope equipped for observation in an incubation chamber that is designed to facilitate thesupport of living human bone cells (such as the MFP­3D, Asylum research Santa Barbara, US). The cantilever may bebrought in contact with the surface of a cell in the incubation chamber, and may be used to scan the cell surface while themicroscope is in MFP mode (molecular force probing). The MFP mode may allow for recoding of adhesive forces between thecantilever and the cell surface. An affinity map representing the distribution of serotonin receptors may be constructed of thecell surface. The distribution, concentration, modulation, and dynamics of the cell's serotonin receptors may be analyzedwhile they are growing on different substrates, in different media, and at different time points in differentiation. In addition,pharmaceuticals (like SSRIs) and biological signal molecules (i.e. hormones, cytokines, fluoride, calcium, growth factors etc)may be introduced to the system while the cell is being observed to investigate the influence of such additives. In addition,

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the serotonin­coated AFM cantilever may be used to "load" (mechanically stress) single cells and at the same time may beused to monitor the serotonin receptor activity, thus analyzing the activity and function of the serotonin system in real time ina living loaded cell. To detect and measure adhesion force at around pico newton level, the cantilever used in the MFP modemay be elastic (with a spring constant less than 0.07 nN/nm). The adhesion forces is applied on living cells (e.g. humanosteoblasts, or any other cell line, or human cell culture) placed in appropiate growth media (e.g. Osteoblast Basal Medium,OBM TM, Clonetics, Cambrex, Walkersville, US). The living cells and medium may be placed in an environmental chamber

(Bioheater ®, Asylum Research, Santa Barbara, US) in order to keep temperature constant at 37 0C or alter the temperatureto any desired value. The mapping of the osteoblast's adhesion forces was conducted in an area of 80 μm x 80 μm, with 400adhesion points within this area. The cantilever was moved down toward the cell at a speed of 500 nm/sec and retracted atthe same speed. The deflection's trigger point for the cantilever when the serotonin coated cantilever tip touches the cell'smembrane was 15 nm. The adhesion forces between the serotonin coated cantilever and the specific serotonin receptor onthe osteoblast cell membrane, was higher regions without receptors. Example 15

[0235] The wear resistance of A) uncoated, commercially available (SiN) AFM cantilevers (control) were compared with AFMcantilevers coated with a thin layer (=10nm) of B) titanium dioxide (TiO2), C) zirconium dioxide (ZrO2) and D) aluminum oxide

(AI2O3). [0236] AFM cantilevers coated with «10nm of TiO2 were prepared according to the present invention. A layer of TiO2was deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry). The

deposition was performed using TiCI4 (Fluka) and H2O (distilled) as precursors at a deposition temperature of 150 0C. Both

precursors were used at room temperature. A thickness of 10 nm was reached after 148 deposition cycles.

[0237] AFM cantilevers coated with =10nm of ZrO2 were prepared according to the present invention. A layer of ZrO2 was

deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry). The deposition

was performed using ZrCI4 (Aldrich) and H2O (distilled) as precursors at a deposition temperature of 250 0C. Both precursors

were used at room temperature. A thickness of 10 nm was reached after 67 deposition cycles.

[0238] AFM cantilevers coated with =10nm of AI2O3 were prepared according to the present invention. A layer of AI2O3 was

deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry). The depositionwas performed using AI(CH3)3 (trimethylaluminium, TMA) (Witco) and O3 as precursors at a deposition temperature of 300

0C. The TMA precursor was used at room temperature while the O3 precursor was delivered from an OT­ 020 ozone generator

provided with 99.999% O2 (AGA) at a rate of 500 seem. A thickness of 10 nm was reached after 91 deposition cycles.

[0239] All wear resistance tests were performed at room temperature (230C), alternative contact mode (AC mode), using aMFP­3D AFM (Asylum Research, Santa Barbara, USA). The hardness and wear resistance of the cantilevers were testedagainst ZrO2 surfaces. The Q was set at 110, in mixed mode (attractive and repulsive imaging). Q is a parameter that

improves the phase contrast imaging (the shift from repulsion to attraction gets more sensitive with a high Q). Keeping aconstant Q gives comparatives measurements possible between cantilevers. The free amplitude in AC mode was set around60 nm, and the set point during scanning fixed at 40 nm. 18 scans were performed with the same cantilever, the sameparameters, over the same area.

[0240] SEM (Philips XL 30 ESEM, FEI Electron Optics, Eindhoven, Netherlands) was also used to compare images of thecantilever tips tbefore and after AFM scanning. The before and after images were also superposed to reflect differences inwear damage of the tip due to scanning friction against the ZrO2 surfaces between A) uncoated cantilevers, B) cantilevers

coated with TiO2, C) cantilevers coated with ZrO2, and D) cantilevers coated with AI2O3. Results are shown in Figures 22­29.

Example 16

[0241] Commercially available cantilevers (Olympus Si AC240, uncoated; ASYMFM, coated with » 50nm cobalt chrome; andOlympus RC800, uncoated) were compared with cantilevers coated with magnetic coatings according to the invention todetermine magnetic field sensitivity.

Olympus Si AC240 and ASYMFM cantilevers

[0242] A commercially available Olympus Si AC240 cantilever and a commercially available MFM cantilever (ASYMFM)served as controls (i.e., no surface modification was performed).

[0243] An Olympus Si AC240 cantilever was coated with Fe2CoO4 (thickness « 10nm) according to the invention. A layer of

(Co1Fe)3O4 was deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry).

The deposition was performed using Co(thd)2 and Fe(thd)3 (Hthd = 2,2,6, 6­tetramethyl­3,5­heptadione) and O3 as precursors

at a deposition temperature of 200 0C. The Fe(thd)3 precursor was sublimed at 115 0C, the Co(thd)2 precursor was sublimed

at 118 0C while the O3 precursor was delivered from an OT­020 ozone generator provided with 99.999% O2 (AGA) at a rate of

500 seem. A thickness of ca. 10 nm was reached after 1030 deposition cycles of metal.

[0244] The magnetic experiments for all of the Olympus Si AC240 and ASYMFM cantilevers were performed at room

temperature (23 0C), in alternative contact mode (AC mode), using a MFP­3D atomic force microscope (AFM) (AsylumResearch, Santa Barbara, USA) equipped with a variable field module (VFM, Asylum Research, Santa Barbara, USA). The

ability of the cantilevers in detecting any magnetic field on a 31/4­inch floppy disk full with data was tested and used as areference for the other tested surface coatings.

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[0245] The Q was set at 200, in mixed mode (attractive and repulsive imaging). Q is a parameter that improves the phasecontrast imaging (the shift from repulsion to attraction gets more sensitive with a high Q). Keeping a constant Q givescomparatives measurements possible between cantilevers. The free amplitude in AC mode was set around 60 nm, and theset point during scanning fixed at 45 nm. The magnetic scanning was performed at a distance of 100 nm above the analysedsurface (floppy disk). The scan size was chosen at 30 μm x 30 μm and the scan speed at 75.12 μm/s. The target field of theVFM stage was kept close to 0 Gauss in order to keep the data on the floppy disk. (Increasing this Field to almost 1000Gauss erases the magnetic storage of the disk). Results are shown in Figures 30­32.

Olympus RC800 cantilevers

[0246] A commerically available Olympus RC800 PSA cantilever, SiN (uncoated) served as control (i.e., no surfacemodification).

[0247] An Olympus RC800 PSA cantilever was coated with Fe2O3 (thickness « 10nm) according to the invention. A layer of

Fβ2θ3 was deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry). The

deposition was performed using Fe(thd)3 (Hthd = 2,2,6,6­ tetramethyl­3,5­heptadione) and O3 as precursors at a deposition

temperature of 200 0C. The Fe(thd)3 precursor was sublimed at 115 0C while the O3 precursor was delivered from an OT­020

ozone generator provided with 99.999% O2 (AGA) at a rate of 500 seem. A thickness of 10 nm was reached after ca. 834

deposition cycles.

[0248] An Olympus RC800 PSA cantilever was coated with Fe2CoO4 (thickness « 10nm) according to the invention. A layer

of Fe2CoO4 was deposited using the ALD (atomic layer deposition) technique in a F­120 Sat reactor (ASM Microchemistry).

The deposition was performed using Co(thd)2 and Fe(thd)3 (Hthd = 2,2,6, 6­tetramethyl­3,5­heptadione) and O3 as precursors

at a deposition temperature of 200 0C. The Fe(thd)3 precursor was sublimed at 115 0C, the Co(thd)2 precursor was sublimed

at 118 0C while the O3 precursor was delivered from an OT­020 ozone generator provided with 99.999% O2 (AGA) at a rate of

500 seem. A thickness of ca. 10 nm was reached after 1030 deposition cycles of metal.

[0249] The magnetic experiments with the Olympus RC800 cantilevers were performed at room temperature (23 0C), incontact mode, using a MFP­3D atomic force microscope (AFM) (Asylum Research, Santa Barbara, USA) equipped with avariable field module (VFM, Asylum Research, Santa Barbara, USA). The ability of the cantilevers in detecting any magnetic

field on a 31/2­inch floppy disk full with data was tested and used as a reference for the other tested surface coatings. [0250]The magnetic scanning was performed at a distance of 300 nm above the analysed surface (floppy disk). The scan size waschosen at 30 μm x 30 μm and the scan speed at 75.12 μm/s. The target field of the VFM stage was kept close to 0 Gauss inorder to keep the data on the floppy disk. (Increasing this Field to almost 1000 Gauss erases the magnetic storage of thedisk). Results are shown in Figures 33­35.

PATENT CITATIONS

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DE19636582C1 * Sep 9, 1996 Nov 27, 1997 ForschungszentrumJuelich Gmbh Sensor for measuring ion concentrations

EP0511662A1 * Apr 29, 1992 Nov 4, 1992 Matsushita ElectricIndustrial Co., Ltd.

Scanning probe microscope, molecular processing method using thescanning probe microscope and DNA base arrangement detectingmethod

EP0540839A1 * Aug 18, 1992 May 12, 1993 Matsushita ElectricIndustrial Co., Ltd.

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* Cited by examiner

REFERENCED BY

Citing Patent Filing date Publication date Applicant Title

CN101975854A * Oct 21, 2010 Feb 16, 2011 中国科学院化学研究所

Single molecule force spectroscopy­based anti­cancer medicamentidentification method

CN101975854B Oct 21, 2010 Jul 17, 2013 中国科学院化学研究所

Single molecule force spectroscopy­based anti­cancer medicamentidentification method

* Cited by examiner

CLASSIFICATIONS

International Classification G01Q60/42, G01Q60/56, G01Q70/18

Cooperative Classification B82Y35/00, C23C16/45555, C23C16/405, G01Q60/56, G01Q60/42, G01Q70/18, C23C16/45525, C23C16/406, C23C16/34

European Classification B82Y35/00, C23C16/455F2K, G01Q60/42, G01Q60/56, G01Q70/18, C23C16/40H, C23C16/455F2, C23C16/34, C23C16/40J

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