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Sensors and Actuators B 114 (2006) 10711082

Review

Preparation of ormosil and its applications in theimmobilizing biomolecules

Vijay Shyam Tripathi, Vivek Babu Kandimalla, Huangxian Ju

Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry, Nanjing University, Nanjing 210093, PR China

Received 8 April 2005; received in revised form 6 July 2005; accepted 6 July 2005Available online 10 August 2005

Abstract

Biomolecules such as enzymes, antibodies, etc., are highly sensitive and specic in catalysis and recognition. These characteristics makethem as potential recognition and catalytic agents in different elds. Attempts have been made to utilize their harness by immobilizing them insuitable matrices/supports. In recent years solgel technology has appeared as a greatly promising tool in entrapment of active biomolecules.The introduction of various organic functional groups, such as amino, glycidoxy, epoxy, hydroxyl, etc., into alkoxide monomers leads toorganically modied solgel glasses (ormosil). The preparation of such organic/inorganic composites provides a means to produce silicatematerials with continuously tunable chemical and physical properties by simply changing the precursors employed, their molar ratio, or both.Recently ormosils have been employed in multifarious applications in industrial and medical elds and show promising results in preservingnative activity of biomolecules. This review article discusses about the basic chemistry, characterization, advances and biosensor applicationsof ormosil. The attractive features of ferrocene linked/entrapped ormosil are also incorporated. 2005 Elsevier B.V. All rights reserved.

Keywords: Ormosil; Biosensors; Ferrocene; Encapsulation; Mediated biosensors; Non-mediated biosensors

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10712. Chemistry of solgel process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10723. Characterization of ormosil lms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10734. Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10755. Ormosil based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075

5.1. Non-mediated biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10765.2. Mediated biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076

6. Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10787. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079

1. Introduction

The developments in solgel technology have permittedthe formation of ceramic materials in desired shapes at low

Corresponding author. Tel.: +86 25 83593593; fax: +86 25 83593593. E-mail address: [email protected] (H. Ju).

temperatureand triggeredseveraldomestic and technologicalapplications [1,2] . The preparation of silica gel via hydroly-sisof tetraethylorthosilicate, [Si(OC 2H5)4] in acidicmediumresulted in the productions of glass-like materials in variousforms like bers, monolithic optical lenses and compositeglass [3]. Major limitation in preparation of conventionalceramic materials is the need of high temperature during

0925-4005/$ see front matter 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2005.07.037


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processing andthe difculty in forming desired complex geo-metrical congurations of the materials. During 1970s Royand his co-workers [4] recognized the potential of solgelprocess for achieving very high level of chemical homo-geneity. They synthesized a large number of ceramic oxidecompositionsinvolvingAl,Si, Ti, Zn,etc.,whichcouldnot be

produced by traditional ceramic technology. The pioneeringwork of Ilers group [5] in silica chemistry led to commer-cialdevelopment of colloidal silica powders [6]. Thisconcepthas led to the production of a wide variety of composites withcontrolled morphologies and particle size [6,7] .

The most widely used starting precursors for the fabrica-tion of silica-based materials (solgels) are tetramethoxysi-lane (TMOS) and tetraethoxysilnae (TEOS). The introduc-tion of various organic functional groups into inorganicalkoxide has led to organically modied solgel glasses,known as ormosils. Ormosils have several attractive featurescompared to inorganic solgels. Firstly, they allow specicbinding of an enzyme to the silica network, for example onsilica grafted with aminosilane by Michael coupling throughglutaraldehyde to an amine bearing enzyme. Secondly, theyallow the encapsulation of catalysts with effective reten-tion properties in case of strong interaction with the organicbranch, or better, the covalent bonding of a charge transfercofactor to the composite material via a chemical reactionwith thepreviously grafted groups. Andthey make it possibleto tune the wettability of composite material by a judiciouschoice of the ratio of hydrophilic to hydrophobic monomers[8,9]. With respect to analytical applications, ormosil derivedmaterials can be designed to a controlled active thicknessof the sensing device and controlled porosity and provide

a versatile way to prepare modied electrodes [1012] . Themajor advantages of theormosil technology are that themate-rials can be prepared at a relatively low temperature andtheir compositions can be easily changed according to theapplications. For example, it is possible to prepare the elec-trodes using different conducting species, organosilanes orpolymer additives, redox mediators and several enzymes,each of which can be used to ne tune the properties of the electrode. The conducting species contain graphite andpalladium and the incorporation of gold colloids that wasreported in recent work [13]. Ormosils and polymers aregood to modulate enzyme activity. For example, some of theresearchers demonstrated that the use of polycationic poly-mersinto ormosil materialscouldimprovethe performance of avoproteins [14,15] , while some studies have demonstratedthat the incorporation of copolymers into silica-basedglassescan improve the activity of entrapped glucose oxidase foramperometric detection of glucose [16]. The biomoleculessuch as atrazine chlorohydrolase [17], lipase [18], lipaseand human serum albumin [19] entrapped in ormosils showimproved performances including storage stability, excellentactivity retention, etc. By taking these advantages and utiliz-ing the advances in ormosil technology in last two decadesseveral enzymes have been successfully encapsulated intoormosil and employed in design of biosensors [9]. In recent

years several reviews were also appeared on solgel encapsu-lated biomolecules [2027] , and few on ormosil [9,28] . Thisreview tries to describe all important aspects and reports of the ormosil with emphasis on active biomolecules.

2. Chemistry of solgel process

The solgel chemistry paves a versatile path for the lowtemperature synthesis of silica matrices. In typical solgelprocess alkoxide monomers (TMOS or TEOS) undergohydrolysis to form silanols,silanols then link together to formsiloxanes, nally through condensation silanols react withsiloxanes to form porous solgel matrices after aging anddryingprocesses underambient atmospheres [28]. Thechem-istry of such a process can be expressed in Fig. 1. Sol is thedispersion of colloidal particles with diameters of 1100 nmin a liquid. Gel is an interconnected, rigid network with poresof submicrometer dimensions and polymeric chains whoseaverage length is greater than a micrometer. The term gelembraces a diversity of combinations of substances that canbe classied into four categories: (a) well-ordered lamel-lar structures, (b) covalent polymeric networks, completelydisordered, (c) polymer networks formed through physicalaggregation, predominantly disordered, and (d) particulardisordered structures [28]. When the pore of the liquid isremoved at or near ambient pressure by thermal evapora-tion, drying, shrinkage occurs and the monolith is termed asxerogel. If the pore liquid is primarily alcohol, the monolithis often termed as alcogel. The generic term of gel usuallyapplies to either xerogels or alcogels. A gel is dened as

dried when the physically adsorbed water is evacuated; dry-ing process substantially reduces thepore size. As mentionedabove, the introduction of organic functional groups such asamino-, glycidoxy- and epoxy-hydroxyl, etc. into the alkox-idemonomersleads to theormosil [28]. TEOSandsomeotherormosil monomer structures and ormosil formation are givenin Fig. 2. The organic modication is mainly employed toreduce the degree of cross-linking, improve lm adhesion toits support, reduce theconcentration of surface silanol groupsand the ion exchange capacity, alter partition coefcientsor introduce reactive functional groups that can be subse-quently used for anchoring molecular recognition specieson pre-prepared xerogels. While selecting ormosil matri-ces for biosensor applications the characteristics, such ashydrophobicity, hydrophilicity, porosity, optical properties,lm thickness, hard ness and cracking, should be taken intoconsideration. When hydrophobic silica-forming monomersare used such as Epoxy, the resulting matrices will reject

Fig. 1. Reaction scheme of solgel lm formation.


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Fig. 2. TEOS and commonly used ormosil monomers and reaction schemefor ormosil formation.

water, leaving only segregated islands of carbon at the outer-most surface in contact with electrolyte [1]. Hence, the ratioof hydrophilic andhydrophobic monomers should be in opti-mal ratio for sensor design or retaining the better activityof the biomolecule. The variation of the number and typeof organic moieties included on the silicon monomer resultsin a variety of pore sizes that can be created in the ormosilnetwork. The pore size corresponds to the physical size of the organic subsistent ( Fig. 2). The mechanical and opti-cal properties of glass, prepared by the solgel process, canbe improved using modied alkoxide precursors RSi(OEt) 3to form organicinorganic hybrid matrices. Where R is anorganic group such as methyl, vinyl or amyltriethoxysilane.

The covalently bound organic groups decrease the mechani-cal tension during the drying process [29]. In order to avoidphase separation, functionalized alkoxides F-R -Si(OEt) 3,where F is a functional group such as amino or isocyanateand R is an alkyl spacer, are usually used to graft cova-lently dopants onto the matrix. After drying, optically clearand dense inorganicorganic hybrid xerogels (30 mm diam-eter and 15 mm thick) can be obtained [30,31] . The reactivearea of ormosil can be determined by its wettability, whichcan be increased by incorporating readily leachable/water-soluble components such as polyethyleneglycol in the matri-ces and dissolving them out by immersing the electrodesin an electrolyte solution [32]. This increases the porosityfor the penetrating electrolyte, thereby increasing the wettedsection inside the solgel matrices [1]. Changing the ratioof tetralkoxysilane to organotrialkoxysilane can control thecation exchange capacity and polarity of porous surface [1].The homogeneous sized nano particles can also be obtainedvia aqueous dispersion process, which is difcult thoroughnormal solgel drying and grinding processes [33].

Processes such as ageing, drying, stabilization and densi-cation inuence the solgel structure since they are relatedto the rates of hydrolysis andcondensation that determine thestructure of the gel. It is essential to understand the kineticsof the hydrolysis and condensation reactions. Additionally,

many other factors including many species that present in thesolution inuence the kinetics of hydrolysis and condensa-tion [34]. Thevariables of major importance are temperature,condensation electrolyte (acid/base), nature of the solventand the type of alkoxide precursor. In fact, many studies havereported the variation of gelation time, viscosity or textural

characteristics (specic surface area) of the gel as a func-tion of experimental conditions [3539] , without alternationin hydrolysis and condensation. The inuence of electrolyteconcentration on the hydrolysis of TEOS in different sol-vents shows that the hydrolysis rate increases linearly withthe increasing concentration of H + or H3O+ in acidic mediaor OH in basic medium. The nature of alkoxy group onthe silicon atom also inuences the rate constant. In general,the long and bulky alkoxy group leads to low rate constantof hydrolysis [36]. The nuclear magnetic resonance spectra(NMR) of silicon were reported to investigate the conden-sation of aqueous silicates at high pH [40]. Hydrolysis andcondensation reactions initiate numerous reactive sites whenmixing TMOS with water. The size of the sol particles andthe cross-linking within the particles (i.e. density) dependupon the pH and the ratio of [H 2O]/[Si(OR) 4]. The time of gelation ( t g) also changes the chemistry of solgel signi-cantly [41]. Yamane et al. [42] reported that the curve of t gversus pH was bell shape, in other words, gelation could benearly instantaneous for very acidic or basic solution of sili-con alkoxides. This behavior is very different from the gelsprepared by destabilization of a silica sol where the curvehas a S shape, with maximum t g around the isoelectric pointof silica (pH 2) and a minimum t g near pH 56 [43]. Theanion and solvent also play an important role in the kinetics

of gelation, which can be either acidically or basically cat-alyzed. The amount of water for hydrolysis of alkoxysilanehas a dramatic inuence on gelation time [43]. For low watercontent, generally an increase in the amount of hydrolysiswater decreases the gelation time, though there is a dilutioneffect as well. It can be predicted that for higher water con-tent the gelation time increases with the increasing quantityof water. Furthermore viscosity of solgel precursors signif-icantly inuences the physical form, e.g. ber coating andmonolith of casted gel [4448] .

3. Characterization of ormosil lms

Although many reports indicated silica net works areable to retain the structure and activity of a wide variety of enzymes [49,50] , some proteins maycompletely unfolduponencapsulation as reported for apomyoglobin by Eggers andValentine [51]. Biomolecules are highly sensitive and fragilein nature; hence their vicinity should be mild and closer tothe native environment (inside the cell) after immobilization.Commonly the factors such as polarity, local microviscosityandinteractionswith pore walls, andpreferential partitioninginto a given phase have an impact on the dynamics, stabilityand accessibility of the dopant. Hence there is a great need to


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understand the nature of the local microenvironments withinnano composites. It requires suitable methods to characterizethe solgel materials. Following characterization proceduresof solgel structure have been adopted so far.

Firstly, thenature of the local microenvironment(s) withina solgel-derived nanocomposite is an important factor in

designing materials for sensing applications. The widelyemployed methods to characterize the solgel matricesinclude small-angle X-ray scattering (SAXS), neutron scat-tering (SANS) and light scattering (SALS), NMR [52], anduorescence spectroscopy [5355] . SAXS allows the deter-mination of a characteristic length of a particle (Guiniersradius of gyration or electronic radius of gyration) and a frac-tal dimension, which give some information on the structureof the polymer (branched versus linear) and on the growthmechanism. The application of SAXS to a number of gelsystems hasbeen reported by various authors [5355] . SANShas been applied to the study of silica sols [56,57] , as it yieldsstructural information on complex biological systems in realtime without damaging the structures involved [58]. Morerecently lypase enzyme was entrapped into ormosil preparedusing tetramethylorthosilicate and methyltrimethoxysilaneusing NaF as a catalyst. The relationship between gel struc-ture and catalyst was evaluated using SANS. Scattering stud-ies were conducted on both immobilized lipase and lipasein free solution. Scattering studies on free enzyme providedevidence for multiple populations of enzyme aggregates andshowed that choice of solvent affected the degree of aggre-gation. The presence of the enzyme during the gel formationconferred structural changes in the gel matrix [58]. SALShave received very little attention in the solgel literature.

However, its characteristic dimension probed by visible lightscattering is 710nm, and therefore, it cannot be used tocharacterize the early stage of the gelation process. Recentdevelopment of short wavelengthUV lasersmaymake it pos-sible to extend light-scattering studies to 3 nm, and therebyto follow most of the gelation process.

Ormosil can be characterized by scanning electronmicroscopy, BET adsorption experiments, differential ther-mogravimetric analysis, and impedance spectroscopy to getinformation on the surface characters (smoothness, cracks,thick ness, etc.), porosity, structure and properties of thematerials [57]. X-ray photoelectron spectroscopy has beenemployed to evaluate the chemical stability of solgel lmscontaining covalently-bonded ferrocene moieties, coated onglassycarbon, and to monitor thechemical oxidationof tetra-sulfur groups immobilized by covalent grafting to a silicatelm coated on indiumtin oxide electrode [59]. The distribu-tionofZrO 2 particleswithin a solgel silicamaybestudied byvarioustechniquesbefore incorporating theresultingmaterialinto carbon paste, including gas adsorption, scanning elec-tron microscopy, X-ray diffraction, and thermogravimetry[60]. Composite lms made of hybrids of naon and sil-ica have been analyzed by transmission electron microscopy,energy-dispersive analysis of X-ray, modulated differentialscanning calorimetry, and electrochemical impedance spec-

troscopy using two stainless steel electrodes, which revealthat thecomposite displays additional properties with respectto those of the isolated Naon and silica materials [61].The thermogravimetry/mass spectra and Fourier transforminfrared (FTIR) spectra indicate the entrapment of urease ina solgel silicalm canforma silicanetwork [62]. FTIRspec-

troscopyandUVvisabsorptionspectrometryareextensivelyused methods for characterizing organically modied or pureSiO2 modied electrodes containing encapsulated organic ororganometallic species [59,6368] . In uorescence charac-terization of the solgel-derived material the selection of auorescent probe is the most critical factor. A wide variety of uorescent probesexist and typically fall into various classessuch as pH sensitive probes, solvatochromic probes, rigi-dochromic probes, anisotropy probes, and so on [69]. These(probes) tend to be incorporated into the materials duringthe early stages of the solgel process when the hydrolyzedprecursor and entrapped molecule(s) are mixed and allowedto gel. During the subsequent condensation reactions theprobe molecules are entrapped within the pores as the mate-rial forms and may become part of the matrix network [69].Bottini et al. [70] studied the conformation and stability of myoglobin in organically net works through absorption anduorescence spectra. Marino et al. [71] used Raman spec-troscopy to investigate the protonation of doped moleculessuch as disperse red dye molecules in ormosil matrices.

Among the electrochemical methods cyclic voltamme-try has been often used to characterize the electrochemicalbehaviors of ormosil modied electrodes comprising elec-troactive centers or encapsulating electroactive components.Theelectrochemistry of Cu II in ammonicalmedium ata silica

gelmodied carbon paste electrode [72,73] shows thatcopperspecies are electrochemically accessible only because theyretain enough mobility to diffuse out of the silica material onthe time scale of the voltammetric measurement. This agreeswith a subsequent work by Borgo et al. [67] who reporteda redox behavior for Cu II initially loaded on aminopropyl-grafted silica similar to that usually observed for solutionspecies. On the contrary, Bond et al. [74] suggested an intra-silica charge transfer mechanism when studying the voltam-metric reduction of several metal ions (Hg II, AgI, CuII, PbII)adsorbed onto a new thick-walled form of mesoporous sil-ica. The electrochemical characterization of ceramic-carboncomposite electrodes was mainly performed by the Levsgroup [75,76] . They studied theinuenceof thecarbon source(graphite, acetylene black or Ketjen black) and compared theresults to conventional carbon electrodes by using variousorganic and inorganic redox couples.

Electrochemical techniques are also exploited to evaluatethe porosity of ormosil-based materials as well as the asso-ciated mass transfer reactions. For example, Collinson et al.[77] demonstrated that ultra microelectrodes could be effec-tively used to probe molecular transport in microstructure gelmaterial and provided an effective mean to get accurate val-uesof diffusion coefcients of target moleculesencapsulatedin solgel-derived silica monoliths. Voltammetry can also be


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directly used to characterize mass transport in SiO 2 modiedelectrodes, as well as to select the optimal experimental con-ditions togeta sensordevicedisplaying thehighest sensitivityas possible [78,79] . Besides the mass transfer limitation, theoverall electrochemical reaction might also be limited bythe heterogeneous rate of electron transfer, as exemplied

by Markovich and Mandler [80], via the electrochemistry of hexacyanoferrate at an indium tin oxide electrode coveredwith an octadecylsilane monolayer.

4. Advances

Inorganic solgel matrices are not highly biocompatibleand bristle in nature. Organic modication in solgel precur-sormay provide betterway of controllingnanoporous geome-tryof ormosil suitable for sensordesign [81,82] . The additionof syntheticor natural polymers such aspoly(ethylene glycol)(PEG) to TEOS and organosilane-derived solgel materialoffers enhanced material properties such as optical clarityand dehydration/rehydration stability, results in signicantimprovement in the medium term stability of entrappedlipase as compared to entrapment in absence of polymeradditives [83]. A graft copolymer of poly(vinylalcohol) with4-vinylpyridine has been hybridized into solgel net, and theformed organicinorganic composite lm displays an excel-lent adhesion to electrodes andincreases the sensitivityof theenzyme electrode [16]. The ormosil methyltrimethoxysilane(MTMOS) doped with chitosan shows good biocompati-bility for immobilization of glucose oxidase (GOD) [84].Pandey et al. [8,32] added an appropriate organic polymer

PEG in the starting sol solution to prevent the formation of crack, thus increasing the lm stability. The ormosilcarbonelectrodes modied by the incorporation of Meldola1sblue into the MTMOS (hydrophobic) greatly reducedthe potential value to 0.2 in sensing the NADH [85].The surface renewability of the electrodes by mechanicalpolishing showed relative standard deviation less than8% for successive surface renewals of mediator-modiedelectrodes.

By taking the advantages of ormosils, Lev and Tsion-sky [86] prepared macroporous chromatographic mediaby incorporation of cluster-forming materials along withthe precursors of lms. Nakanishi et al. [87] introducedsolgel derived monoliths for high-pressure liquid chro-matography. Lev and co-workers [88] reported that theincorporation of H 2O2 into solgel precursors of methyl-ormosil favored in synthesis of macroporous monoliths,whichwould be useful in chromatographicapplications.Dur-ing the solgel process the decomposition of the H 2O2 fromminiature bubbles, which formed templates for the poly-condensation, yielded fractured or powdery materials. Theencapsulation of myoglobin in the organically modied sil-ica bulks prepared using three organic functionalities (3-aminopropyl)-trimethoxysilane, 3-(trimethoxysilyl)-propylmethacrylate and (3-glycidyloxypropyl)-trimethoxysilane

retained its activity and structure even after treated withdenaturing agent guanidinium hydrochloride (GdHCl). Theorganic functionalities acted as surfactants that interacteddirectly with the hydrophobic residues of myoglobin thenformed micelles around the protein to prevent the forma-tion of unfavorable water structure promoted by GdHCl

[89].

5. Ormosil based biosensors

The use of solgel glass for the development of elec-trochemical biosensors has received great attention becauseof its sturdiness and possible commercial applications. Anumber of publications are available on the applications of solgel glass for the development of electrochemical sensors[90]. Four approaches dependent on the convenience, stabil-ity and response can be used for immobilizing the enzymeon electrode surface: entrapment of enzyme in ormosil lm,attachment of enzyme on the surface of ormosil lm, immo-bilization of enzyme in a sandwich conguration and abilayer conguration ( Fig. 3). Wang and Pamidi [91] devel-oped biogel-based carbon inks that displayed compatibilitywith the screen-printing device for microband electrodes. Inrecent years Pandeys group [9294] successfully immobi-lized glucose oxidase (GOD), horseradish peroxidase (HRP)and acetylcholinesterase in ormosils. These immobilizedenzymes were evenly distributed in the solgel matrices andgavegood responsewhen employed forbiosensing.Theaddi-tion of pore forming agents and conductive materials, PEG,poly vinylalcohol (PVA) and graphite, palladium, ruthenium,

respectively can improve the electrochemical signals consid-erably. Diffusional penetration of a mediator into the proteinyields a sufciently short electron transfer distance for theelectrical activation of the biocatalyst. Penetration of themediator close to the enzyme active center inside the pro-tein matrix canbe controlled by thehydrophobic/hydrophilicproperties of the mediator and the enzyme, the size andshape of the mediator and the electrostatic charge interac-tion between the mediator and the enzyme. The mediatorscan diffusionally shuttle electrons between the electrode and

Fig. 3. Different approaches for the immobilization of biomolecules andmediator molecules in ormosil matrices.


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enzymes in several congurations: soluble, immobilized asmonolayers (or multilayers), or incorporated into porousmatrices.

5.1. Non-mediated biosensors

The amperometric biosensor that does not require theparticipation of redox molecules having reversible electro-chemistry is referred to as non-mediated biosensor, whereastheparticipationof redoxmediatorin signal transduction gen-eratesa categoryof mediatedbiosensors [9597] . Usually thecharge transfer and sensitivity of non-mediated biosensorsmust be improved because the most of redox enzyme activesitesare deep inside, whichmakesthe electronshuttle becomeslower or poor. The porosity and wettablility of the ormosillm play key roles in biosensor performance. The ormosillm developed using 3-aminopropyl-triethoxysilane (rela-tively hydrophilic precursor) and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane (relatively hydrophobic precursor) isvery smooth without cracking and has a good performancewhen doping GOD [32]. Furthermore, the preparation of this ormosil lm is very simple with one-step gelation pro-cess as compared to those prepared by following com-plex protocol of gelation, which requires sonication forthe homogenization of monomers, and additive suspension[75,98,99] . Theporosity and wettability can also be modiedby employing the additives such as PEG along with bioac-tive compounds [8,32] . To evaluate the effects of PEG andgraphite on GODentrappedormosil that were prepared using3-aminopropyltriethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyl-trimethoxysilane as precursors, different compositions

suchasGOD,GOD along with PEG,GOD and graphite pow-der (12 (m), and GOD along with PEG and graphite pow-der were designed. Composition 4 exhibited relatively goodresponse in the presence of glucose in 0.1 M pH 7.0 phos-phate buffer at 25 C. Upon addition of glucose the anodiccurrent corresponding to the oxidation of hydrogen peroxideincreased greatly. The magnitude of anodic current in com-position 4 was greater than those in other compositions. Thiswas mainly due to relatively less concentration of oxygenat the site of enzymatic reaction required for the formationof hydrogen peroxide. It was also been reported that neitherhighly hydrophobic nor totally hydrophilic solgel matriceswere desirable for sensing application [1,100] . When chem-ical modiers such as metal dispersion, water-soluble poly-mers and proteins were added to the materials, the resultingelectrodes became more hydrophilic. It has been reported [1]that a blank solgel electrode without any hydrophilic mod-ier shows the highest water content angle (80 ) and in turnthe lowest wettability, whereas the solgel electrodes withall hydrophilic modiers (carbon, PEG and Pd-GOD) showthe lowest water contact angle (42 ) and the highest wetta-bility (42m 2 /g). An increase in the wetted area increases thewetted conductive surface accessible to the solution and alsothe corresponding electrochemically active area and capac-itive current. On the other hand, the non-wetted area does

not contribute to the capacitive or faradic currents. Ingersolland Bright [99] reported on the effect of addition time of dopant and used oxygen as the analyte to study sensor per-formance. The ormosil entrapped GOD also exhibited goodperformance by the addition of hydorphilic modiers such asgraphite and PEG into ormosils mainly due to the increase in

wettability, which increased the oxygen diffusion [92].The non-mediated potentiometric biosensor has alsobeen reported. Gulcev et al. [101] co-entrapped carboxy-seminaphtharhodauor-1-dextran conjugate and hydrolyticenzyme (urease/lipase) in ormosil to develop a reagentlesspH based biosensor. The doping of PVA improved enzymeactivity and enhanced the accessibility of different lms.The response was 25-fold higher than that obtained fromTEOS alone.

5.2. Mediated biosensors

Ferrocene derivatives, organic dyes, ferricyanide, Ru-complexes and other electrochemically active substanceshave been employed as mediators to improve the elec-tron transfer of biomolecules with the conductive support[102] . Among these ferrocene derivatives are particularlywell studied for electrochemical biosensing owing to theirgood stability, high degree of characterization andtheir appli-cation potential bioanalysis. Soluble redox-enzymes electri-cally contacted by the use of diffusional electron transfermediators have been extensively reviewed [102]. The reac-tion of GOD has been extensively studied with a number of articial electron acceptors including organic dyes such asphenazine methosulfate, 2,6-dichlorophenolindophenol, and

N , N , N , N -tetramethyl-4-phenylenediamine. However, thesemediators have a number of limitations such as poor stabil-ity and the pH dependence of their redox potentials [102] .Other simple inorganic redox species such as hexacyanofer-rate,hexacyanoruthenate andpentaaminepyridineruthenium[103] do not suffer from these problems. These inorganiccompounds have almost ideal electrochemistry and are morestable than the organic dyes. The application of inorganicmediators has been exemplied with other oxidases suchas sarcosine oxidase and lactate oxidase [104] . Inorganicmediators are difcult to tune for solubility and electro-chemicalproperties, as theycannot bemodied or derivatizednearly as easily as their organic counterparts. The major-ity of these problems have been overcome by the use of ferrocene derivatives as electron acceptors for soluble oxi-dases (e.g. GOD). The published values for the second-orderrate constant ( k et) for the reaction of the reduced active cen-ter of GOD (FADH 2) and an oxidized ferrocene derivativerange from 2.6 104 to 5.25 105 M 1 s 1 [102]. Thereis no simple correlation between k et and formal potential E 0 , the positively charged ferrocene derivatives are favoredfor the mediated electron transfer from GOD. This effectoriginates from the electrostatic attraction of the positivelycharged oxidized mediator and the negatively charged GOD.Comparison of the mediating efciency of charged electron


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relays and charged enzymes always takes electrostatic inter-actions into account [105] . The size of the mediator is alsoan important factor. It has been shown that ferrocene deriva-tives includedin cyclodextrin cavitiesdo notmediate electrontransport fromenzyme [106] . To improve thecontact betweenelectron relay molecules and redox enzymes, micellar sys-

tems composed of ferrocene-functionalized surfactants havebeenapplied [107] . The incorporation of graphite powder andPEG into the ormosil as precursors exhibits excellent bio-electrochemical properties [32]. The voltammograms of thesoluble ferrocenemonocarboxylicacidshowsreversible elec-trochemistry of ferrocene, and the incorporation of graphiteparticles increases the wetted surface area of the electrodeas well as facilitates the electron transfer within the solgelmatrix as a result of increases electronic conductivity of theelectrode. Chen et al. [84] reported a glucose biosensor usingferrocene as a mediator, in which enzyme was immobilizedin ormosilchitosan composite. The adsorbed ferrocene pro-vided good shuttle of electrons between the enzyme and theelectrode and the presence of chitosan provided stabilizingmicroenvironmentaround the enzyme. In some of the reportsferrocene was rstly adsorbed on electrode surface, and thenthe ormosil composite containing GOD was covered withormosil in construction of glucose biosensor [84].

In the absence of ferrocene an ormosil made using palla-dium-linked glycidoxypropyltrimethoxysilane precursor,trimethoxysilane, HCl and tetrathifulvalenetetracyanoquin-odimethane (TTF-TCNQ) powder at 25 C was used toprepare an electrocatalytic biosensor for glucose [108]. Alarge electrocatalytic current to the order of 8000 A/cm 2

was observed on the addition of 300 mM glucose. Pyrorolo-

quinoline quinone (PQQ) is a redox cofactor present in anumber of dehydrogenases. It has been shown to catalyzenon-enzymatic reactions, including the oxidation of thiols todisulde, and successfully immobilized in ormosil preparedfrom 3-aminopropyltrimethoxysilane (APTES) and 2-(3,4-epoxycyclohexyl) ethyl-trimethoxysilane. The entrappedPQQexhibitsgoodelectrochemicalperformance against cys-teine and glutathione oxidation and reproducibility [109] .

The encapsulation/linking (with precursors) of redoxmaterials within solgel glass has gained a signicant shareof attention in sensor designing because such a compos-ite system provides very close contact of biomoleculeswith transducer surface. Pankratov and Lev [110] reportedferrocene-mediated carbon ceramic electrodes with lim-ited storage and in-use stability. Several other reports onferrocene-encapsulated solgel glasses, including those of Lev et al. [11,74,98] , are available. Audebert et al. [111]reported several electrodes modied with organicinorganichybrid gels containing a ferrocene unit covalently linkedin silica network and organicinorganic hybrid gels formedby hydrolysispolycondensation of some trimethoxysilyl-ferrocenes [112] . Collinson et al. [113] reported the elec-troactivity of redox probes encapsulated in solgel-derivedsilicate lm based on anionic and cationic gel-doped probes,i.e., [Fe(CN) 63

/4 ], [IrCl62 /3 ] and ferrocenemethanol

Fig. 4. Ormosil monomers conjugated with ferrocene derivatives.

[FcCH 2OH0/+]. Fig. 4 shows typically mediator molecules(ferrocene derivatives) incorporated into solgel matricesby physically doping or by using organosilicon precursors.Other reports on ferrocene-based solgel sensors are alsoavailable [114] . A biocompatible Pd-linked ormosil mate-rial with encapsulation of ferrocene has also been reported,which shows redox electrochemistry similar to that of solubleferrocene in solution and good electrocatalytic behaviors toredox proteins (alcohol dehydrogenase) and cofactors suchas NADH [115] .

Two ormosils prepared using ferrocene carboxaldehydeand a mixture of 3-aminopropyltrimethoxy silane with2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane and ferro-cene monocarboxylic acid and a mixture of 3-glycidoxy-propyltrimethoxysilane with trimethoxysilane have beenreported [116] . The encapsulated ferrocene derivatives inormosils show quasi-reversible electrochemistry comparedto ferrocene-linked to ormosil [28,116] . Another ferrocene-encapsulated palladium-linked ormosil shows a reversibleelectrochemistry with a peak separation of 58 mV at the scanrates less than 50 mV/s [117] . The good reversibility is dueto the presence of palladium, which plays a crucial role inelectron transfer [118] . This ferrocene-encapsulated ormosilhas been employed in development of glucose and dopaminebiosensors [118120] . Thesilicate-basedelectrodespreparedwith ferrocene-linked ormosil precursorsshow good stabilitywithout any leaching of the mediator and the enzyme [121].Recently we conjugated the ferrocene monocarboxylic acidwith an inert protein bovine serum albumin (BSAFMC)through carbodiimide linkage. The conjugate was success-fully entrapped in an ormosil prepared using APTES and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers.The entrapped BSAFMC exhibited reversible redox peakswithout any leaching of mediator [122] . To evaluate theapplicability of the BSAFMC doped ormosil in biosensors,


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enzymes and multiwall carbon nanotubes were also dopedinto theormosil composite. Theglassy carbonelectrode mod-ied with enzymes and nanoparticles doped ormosil com-posite lm exhibited good sensitivity, reproducibility andstability towards the determination of glucose and H 2O2[122,123] .

6. Other applications

Ormosils have also been used in other different elds suchas manufacture of new contact lenses and fresnel lenses,preparation of laser components for opticsm, second-ordernon-linear optically active nanocomposites and bone repair-ingmaterials, synthesisof photochromiccoatings andporoussolvent absorbers, andso on [29,124129] . The biomoleculesor dyes entrapped in ormosil nanoparticles can be efcientlyemployed in drug delivery and other pharmaceutical andmedical applications [130]. The organically modied andanticancer drug doped nanoparticles (diameter 30nm)obtained through aqueous dispersion have been used inphotodynamic cancer treatment [131]. Water-insoluble pho-tosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide, was entrapped in non-polar core of micelles by hydrolysis of triethoxyvinylsilane, the resultednanoparticles were spherical, highly monodispersed, andstable in aqueous system. Irradiation of the photosensitizingdrug entrapped in the nanoparticles with light of 650nmwavelength resulted in efcient generation of singlet oxygen,which could inactivate the cells. The drug-doped nanoparti-cles were up taken into the cytosol of tumor cells and caused

the damage to the impregnated tumor cells upon irradiation.DNA molecules bound through electrostatic interactiononto ormosil nanoparticles could be employed in non-viralvector gene therapy [132] . The bound DNA moleculeswere highly resistant from nucleases degradation. Bypaying more affords, it would be possible to use efcientlythe manipulation of surface reactive groups on ormosilnanoparticles in optical tracking, drug delivery and druginteractions.

A ber-optic microbial sensor for determination of biochemical oxygen demand (BOD) was reported usingan oxygen-sensitive uorescent material and two dif-ferent kinds of seawater microorganisms immobilizedin ormosil. This report used Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) perchlorateas the oxygen uo-rescent quenching indicator [133] . Nano-sized (20100nm)photonic explorers for bioanalysis with biologically local-ized embedding have been developed for specically usein biological environments to monitor small species likeH+, Ca2+ , Na+, Mg2+ and glucose, etc. [134,135] . Ana-lytes can diffuse through the matrix and interact with thesensing dye. These sensors typically contain a referencedye. Koo et al. [136] reported an oxygen sensor based onentrapment of platinum porphyrins in ormosil nanoparti-cles. The ormosil nanoparticles ( 120nm) were prepared

via a solgel-based process, which included the formationof core particles with phenyltrimethoxysilane as a precur-sor followed by the coating layer with methyltrimethoxysi-lane as a precursor. The highly permeable structure andthe hydrophobic nature of the ormosil nanoparticles, aswell as their small size made the sensor show good sensi-

tivity for detection of dissolved oxygen. The hydrophobicormosil matrices such as methyltriethoxysilane (MTEOS)and ethyltriethoxysilane (ETEOS) show enhanced perfor-mance of dissolved oxygen sensor [137]. Chen et al. [138]reported anoxygensensor usinga rutheniumcomplex asoxy-gen sensitive indicator and TMOS, dimethyldimethoxysilane(DiMe-DMOS) as ormosil precursors. The enhanced sen-sitivity to dissolved oxygen was reported to be due to thehigh hydrophobicity of the ormosil lm. Another potentialapplication of ormosils is design of molecularly imprintedpolymer against diverse analytes [139] . Marx et al. [140,141]reported molecularly imprinted polymers using ormosilsagainst to parathion and paroxon and employed them forquantication.

7. Conclusions

This review article discussed the ormosil basic chem-istry, characterization, advances and biosensor/biologicalapplications. Ormosil matrices offer tailorable hydrophilic,hydrophobic, ionic, and H-bonding capacities as well aselectrochemical activities and controllable porosity andare highly stable, inert and non-biodegradable. By dopingconductive materials and natural polymers into ormosil

matrices their conductivity and biocompatibility can befurther improved. By creating more reactive groups onormosil surfaces, these matrices can be exploited foranchoring molecular-recognizing receptor and attachingsensing elements on optrode, electrode and on several othertransduction surfaces especially for mimicking biologicalprocesses. Another challenge is, despite widely utilized, thesolgel process is inherently complicated, and its mecha-nism, the gel microstructure, the effects of different factorson the stability and activity of immobilized biocatalystsneed to be understood. The applications of ormosil matricescan further be extended and utilized in optical trackingof cell metabolisms, nano medicine, gene therapy, chro-matography and biosensors by paying more affords in nearfuture.

Acknowledgements

We gratefully acknowledge the nancial support of the Distinguished Young Scholar Fund to H.X. Ju(20325518), the National Natural Science Foundation of China (20275017). V.S. Tripathi and V.B. Kandimalla arehighly thankful to Nanjing University for providing Post-doctoral fellowships.


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Biographies

Vijay Shyam Tripathi received his MSc degree in chemistry and PhDdegree in analytical chemistry from Banaras Hindu University, Varanasi,India in 1997 and 2003, respectively. He is currently working as aPost-doctoral fellow in the Department of Chemistry, Nanjing University,China. His research interest includes electroanalytical chemistry, solgelchemistry, biosensor and bioelectronics.

Vivek Babu Kandimalla received his BSc degree (1993) in biology andMSc degree (1996) in biotechnology from Nagarjuna University, Nagar-

juna Nagar, India and PhD in biotechnology in 2002 form Andhra Univer-sity, Visakhapatnam, India. He is a Post-doctoral fellow in the Departmentof Chemistry, Nanjing University, Nanjing China. His research interestsinclude the development of electrochemical biosensors, antibodies pro-duction, quantum dots (QDs) conjugation with antibodies and aptamers,cellular imaging and in situ imaging tools design.

Huangxian Ju received his BSc (1986), MSc (1989) and PhD (1992)degrees in chemistry from Nanjing University, Nanjing, China. He wasappointed as a Research Scientist at this University in 1992 and becamea Full Professor in 1999. During January 1996 to July 1997 he was aPost-doctoral fellow in the Department of Chemistry, Montreal University,Canada. His research interests include analytical biochemistry, biosensors,electroanalysis and clinical chemistry.

Documents

199 SAB Tripathi