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Biosensors and Bioelectronics 24 (2008) 893–899

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

urface plasmon resonance protein sensor using Vroman effect

eokheun Choi ∗, Yongmo Yang, Junseok Chaelectrical Engineering Department, Arizona State University, Tempe, AZ 85287, USA

r t i c l e i n f o

rticle history:eceived 15 April 2008eceived in revised form 27 June 2008ccepted 16 July 2008vailable online 31 July 2008

eywords:

a b s t r a c t

We report a new surface plasmon resonance (SPR) protein sensor using the Vroman effect for real-time,sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorp-tion onto the surface, directly depending upon protein’s molecular weight. The sensor uses SPR for highlysensitive biomolecular interactions detection and the Vroman effect for highly selective detection. By usingthe Vroman effect we bypass having to rely on bio-receptors and their attachment to transducers, a processknown to be complex and time-consuming. The protein sensor is microfabricated to perform real-timeprotein detection using four different proteins including aprotinin (0.65 kDa), lysozyme (14.7 kDa), strep-

roman effecturface plasmon resonance (SPR)tomic force microscope (AFM)rotein sensorelf-assembled monolayer (SAM)icrofluidics

tavidine (53 kDa), and isolectin (114 kDa) on three different surfaces, namely a bare-gold surface and twoothers modified by OH- and COOH-terminated self-assembled monolayer (SAM). The real-time adsorptionand displacement of the proteins are observed by SPR and evaluated using an atomic force microscope(AFM). The sensor can distinguish proteins of at least 14.05 kDa in molecular weight and demonstrate avery low false positive rate. The protein detector can be integrated with microfluidic systems to provide

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. Introduction

Scientists have reported potentially fast, simple and inexpensiveest techniques to detect disease-associated biomarkers (Rifai et al.,006). Biomarker is an important parameter to assess the progressf disease and monitor the effects of treatment. For instance, molec-lar biomarkers allow early diagnosis of bladder cancer (Oehr,005). Many protein biomarkers have been discovered for aidf early diagnosis in the past (Sahab et al., 2007). Nevertheless,xisting diagnostic approaches based on proteomics are still lim-ted partially due to (i) low sensitivity at ultra low concentrationiomarkers, (ii) complex three-dimensional protein conformation,nd (iii) diverse forms of proteins.

The most popular technique for the complex description of aroteome is protein microarray technology. The microarray allowsnalysis of thousands of molecules of interest simultaneouslyGlokler and Angenendt, 2003). Although DNA microarray chips areeing actively used currently (Han et al., 2006), research on proteinicroarray is still in its infancy. This is in part due to its difficulty in

etecting the very low abundance of biomarker proteins and find-ng appropriate bio-receptors which have high enough affinity tonalyze and immobilize them (Daniels and Pourmand, 2007).

∗ Corresponding author.E-mail address: shchoi2@asu.edu (S. Choi).

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956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2008.07.036

e analytical capability.© 2008 Elsevier B.V. All rights reserved.

Extensive research activities have been focused on both highelectivity bio-receptors and high sensitivity transducers to detectroteins. A protein sensor consists of a bio-receptor to selec-ively recognize target entities and a transducer to monitor theecognition event. A variety of transduction techniques have beenntroduced in laboratory-based analytical methods and in-fieldnalytical techniques. These techniques include electrochemical,ptical, piezoelectric, magnetic or thermometric transductions. Thextensive research on transducers leads extremely high sensitivityng–pg mL−1) (Daniels and Pourmand, 2007).

Transducers can largely be categorized as either labeled orabel-free system. Labeling techniques have been broadly employedor biochemical analysis including proteins detection. However,he chemical labeling may modify the target proteins charac-eristics, altering their behavior (Haab, 2003). Moreover, theabeling procedure is time-consuming and labor-intensive, andt is often difficult to achieve accurate quantification due toariable labeling efficiency for different proteins. The label-freeechnique, on the other hand, can eliminate the labeling pro-ess and is becoming a rather attractive alternative for biologicalnalysis (Yu et al., 2006). Among various label-free mechanisms,urface plasmon resonance (SPR) has been one of the leading

echniques due to its extremely high sensitivity, offering detec-ion limits up to few ppt (pg mL−1) (Ince and Narayanaswamy,006; Li et al., 2007). SPR is a surface-sensitive analyticalool responding to slight changes in refractive index occur-ing adjacent to the metal film. Thus, binding of proteins on

894 S. Choi et al. / Biosensors and Bioelectronics 24 (2008) 893–899

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he surface and their subsequent affinity interactions could beonitored in real-time without labeling (Bally et al., 2006).Selectivity has always been an enduring problem in protein sen-

or because of difficulty in finding appropriate bio-receptors withigh affinity and high specificity. The bio-receptors have a specificffinity site that captures the target analyte, thereby determiningelectivity. Many bio-receptors have been used, including antibod-es, protein lysates, lectins, peptides, and aptamers (Chaerkady andandey, 2008). However, a weak binding affinity with analytes,on-specific adsorption, and low reproducibility remain significant

imitations. Besides these limitations on bio-receptors, integrat-ng them on to the transducer is another significant challenge. Inrder to integrate highly ordered bio-receptors on to the surfacef the transducers, linker molecules such as self-assembled mono-ayer (SAM) have often been used. For instance, thiol-ended SAM

s attached on a gold surface as a linker molecule for subsequentntibody bio-receptor to capture antigen (Shankaran and Miura,007). Forming linker molecules are a time-consuming and labor-

ntensive process and often become the bottle neck of high yieldensors. Obviously, not all bio-receptors have appropriate linker

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obic and (2) the hydrophilic surfaces when the LMW protein adsorbs first and thenW protein comes later and (3) no angle change occurs.

olecules to the transducer surface, which limits practical appli-ations significantly.

In this paper, we present a new proof-of-concept SPR pro-ein sensor offering high sensitivity and selectivity simultaneouslyithout using a bio-receptor. The selectivity results from the

ompetitive nature of protein adsorption onto the surface, whichirectly relies upon protein’s molecular weights, namely theroman effect (Vroman and Adams, 1969). The Vroman effectas been studied extensively in the biochemistry community

or protein adsorption competition to materials having differ-nt surface chemistries and surface energies (Noh and Vogler,007). Fig. 1 briefly describes the Vroman effect. A low-moleculareight (LMW) protein initially covering the surface is dis-laced by a high-molecular weight (HMW) protein (Vroman anddams, 1969; Horbett and Brash, 1995; Green et al., 1999). This

ffect occurs, as it is more thermodynamically stable in naturehen HMW proteins replace LMW ones. However, the reverse

equence does not occur. When HMW proteins cover the sur-ace first, LMW ones arriving later do not displace the formerFig. 1(3)).

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We utilize the molecular weight differences for the selectivity ofhe protein sensor. When a mixture of proteins goes through sam-le preparation stages including mixer, concentrator, and separator,he proteins are grouped as their properties depending upon sam-le preparation mechanisms such as hydrophobicity, ion strength,nd size. One of the mechanisms is size exclusion chromatogra-hy (SEC), which separates a mixture of proteins depending uponheir sizes (Yang and Chae, 2007). The eluted proteins from thereparation stages are grouped based upon their sizes and then gohrough the protein sensor. The protein sensor forms an array ofensing surfaces which are covered by different size of proteins.sing the Vroman effect, we identify target proteins and monitor

he concentration in real-time using SPR.In Section 2, the surface preparation and device fabrication are

iscussed. Experimental and evaluation methods are presented inection 3. In Section 4, we demonstrate SPR measurements resultssing four different proteins. Finally, concluding remarks follow inection 5.

. Sensing surface preparation and microfluidic enclosure

.1. Sensing surface formation

Three different surfaces are prepared to observe protein adsorp-ion: bare-gold, OH-terminated SAM, and COOH-terminated SAM.his is to characterize Vroman effect on three different hydropho-icity surfaces. The Vroman effect occurs due to the energyreference of hydrophobicity/hydrophilicity surfaces; thus the sur-

ace modification greatly affects biosensor performance (Horbettnd Brash, 1995). The adsorption performance of the three surfacess presented in Section 4. Glass substrates (BK7, n = 1.517) are firstleaned in piranha solution (a 3:1 ratio of H2SO4 and H2O2) for0 min. The cleaned glass is coated with Cr/Au (3/48 nm) by ther-

al evaporation. Then the substrates are cleaned by oxygen plasma

Harrick Plasma Inc.) and then immersed in an ethanol solution ofifferent alkane-thiols at 1 mM for 24 h at room temperature toorm SAMs. COOH- and OH-terminated SAMs are formed on theubstrate with 11-mercaptoundecanoic acid and 11-mercapto-1-

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ctronics 24 (2008) 893–899 895

ndecanol. Finally the substrates are rinsed with ethanol and water,nd thoroughly dried using nitrogen.

.2. Surface characterization

The three different surfaces are characterized by contactngle measurement (Easy Drop FM 40, Kruss GmbH Inc.) andourier transform infrared spectroscopy (FTIR, Nicolet Contin-um, Thermo Electron Corp.). The contact angles for bare-gold,OOH-, and OH-terminated SAMs are 82.6 ± 0.77◦, 41.4 ± 0.63◦, and8.2 ± 0.32◦, respectively. As expected, the bare-gold surface is veryydrophobic and OH- and COOH-SAM surfaces hydrophilic. Theifference between OH- and COOH-SAM surfaces is minimal, yethis small difference makes a significant impact on the proteindsorption/desorption. The compositions of the SAM surfaces arevaluated by FTIR. All spectra are obtained using p-polarized lightncident on the substrate at an angle of 78◦ with respect to theormal surface. FTIR spectra show that the methylene stretchingf the alkyl chains for both COOH- and OH-terminal SAMs has twobsorption bands at 2917 and 2846 cm−1. While the spectrum ofhe COOH-SAM indicates the C O absorption band at 1706 cm−1,he OH-SAM spectrum is visible with the C–O stretching band at040 cm−1, thus validating the formation of SAMs on the gold sub-trate.

.3. Microfluidic device

In order to facilitate potential integration with microfluidic sys-ems, the sensor is enclosed by microfluidic channels/chambersFig. 2). First, Cr/Au are deposited (3/48 nm) on a glass substrate500 �m, Pyrex 7740, Corning Inc.). A cover glass, 1-mm thick,s patterned and etched to have 1-mm wide and 50 �m deep

uidic channels using hydrofluoric acid (HF) for 25 min. Ports

or inlets/outlets, 9.7 mm in height and 6.4 mm in diameter, areechanically drilled. The glass substrate and the cover glass are

onded by epoxy films (Upchurch Scientific) and cured at 120 ◦Cor 20 min.

) microfluidic enclosure, and (d) the bio-sensor chip on a SPR.

896 S. Choi et al. / Biosensors and Bioele

Fig. 3. (a) Dependence of the SPR angle shift obtained with single protein exper-iment on the molecular weight. Angle shift of each protein on bare-gold; blacksquares, on COOH-SAM; red circles, and on OH-SAM; blue triangles. Dotted plotsare the empirical SPR data and solid lines correspond to the fittings. AFM images offour proteins (b) aprotinin (c) lysozyme (d) streptavidine (e) isolectin on bare-goldsurface and isolectin on (f) COOH-SAM and (g) OH-SAM surfaces.

Fig. 4. Real-time SPR displacement profiles of aprotinin by lysozyme (1) on the bare-gold, (2) COOH-SAM and (3) OH-SAM surface. Group A is selected as LMW proteinsand Group B as HWM proteins. After LMW protein is injected and stabilized angleshift is obtained, HMW protein is injected. In this figure, aprotinin is Group A andlysozyme is Group B.

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. Experimental methods and evaluation

.1. SPR setup

The fabricated microfluidic chip is mounted on the SPR ana-ytical system (Biosensing Instrument Inc.). We monitor the anglehift in real-time as protein solution flows through the microfluidichannels driven by an external syringe pump. Initially, phosphate-uffered saline (PBS) is circulated for 20 min until the angle shifttabilizes. Once the angle shift stabilizes, the protein sample flowshrough the microfluidic channels at 10 �l/min, which generates anngle shift proportional to molecular interactions on the surface.hen protein adsorption completes, we let PBS wash the surface

o remove excess weakly bound proteins. As shown in Fig. 1, inrder to observe the Vroman effect, small proteins such as aprotinin0.65 kDa) and lysozyme (14.7 kDa) form molecular bonds on theurface first. The bond strength depends upon its surface hydropho-icity. The more hydrophobic the surface is the stronger the bond

s (Wertz and Santore, 2001). Then, large proteins such as strepta-idine (53 kDa) and isolectin (114 kDa) flow through the channelsnd replace existing bonds formed by the LMW proteins. Finally, weerform the aforementioned procedure in reverse, adding HMW toMW proteins, to confirm the Vroman effect (Fig. 1(3)).

.2. Evaluation using AFM

The SPR angle shift only indicates the size and rate of molecularnteractions on the surface. This does not verify protein adsorptionctually occurs. We use an AFM (Digital Instruments Nanoscope IIIsultimode AFM) to evaluate the protein adsorption. The surfaceorphology changes as protein adsorption occurs. Obviously, theFM does not support real-time molecular interaction measure-ents, yet it offers a powerful laboratory tool to observe protein

dsorption. The measurements are performed at air-ambient (25 ◦Cnd 35% relative humidity). We use 42 Nm−1 silicon AFM tips withhe resonance frequency of 250–390 kHz, and scanning rate of 2 Hzlines per second).

. Results and discussion

.1. Single protein adsorption

We use four different proteins to characterize the protein sensor:protinin (0.65 kDa), lysozyme (14.7 kDa), streptavidine (53 kDa),nd isolectin (114 kDa) (Sigma–Aldrich and Invitrogen). The pro-eins are diluted using PBS 1× (1.15 g/L Na2HPO4, 0.20 g/L KCl,.20 g/L KH2PO4, 8.0 g/L NaCl, pH 7.4).

Fig. 3(a) presents the SPR response analytical curve plottedlong with the molecular weight of each protein. The SPR anglehifts at a given surface are proportional to the molecular weightsf the proteins. For instance, hydrophobic surface (bare-gold)roduces 135, 168, 347, and 547 mDeg angle shifts for apro-inin (0.65 kDa), lysozyme (14.7 kDa), streptavidine (53 kDa), andsolectin (114 kDa), respectively. The trend remains regardless ofydrophobic/hydrophilic (COOH- and OH-SAM covered) surfaceroperties. The fitted lines show very linear responses for all sur-

aces; r-squared of the three lines are 0.9933, 0.9742 and 0.8994or bare-gold, COOH-SAM, and OH-SAM covered surfaces, respec-ively. Hydrophilic surfaces show much smaller angle shifts than

ydrophobic surface. For isolectin, hydrophobic (bare-gold) surfacehows 547 mDeg whereas COOH- and OH-SAM covered surfaceshow 91 and 22 mDeg, respectively. This is because proteins ineneral adsorb more on hydrophobic than hydrophilic surfacesLatour, 2005). The SPR angle shifts suggest the following: (i) higher

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S. Choi et al. / Biosensors and

olecular weight proteins produce a thicker monolayer than lowerolecular weight proteins and (ii) proteins adsorb more on the

ydrophobic than hydrophilic surface, resulting in a more denselyacked layer on the hydrophobic surface (Fig. 3(e–g)).

These phenomena are due to the characteristic structure ofroteins. Generally, proteins have hydrophobic residues buriedithin the core of the proteins and their hydrophilic residues fac-

ng outside. The adsorption of any protein onto a solid surfaces highly related to hydrophobic attraction. Hydrophilic residuesorm the orientation of the adsorbed protein and do not takeart in the adsorption process itself (Latour, 2005). Therefore,roteins rapidly adsorb onto a hydrophobic surface, unfold andpread their hydrophobic core over the surface. On the other hand,ydrophilic surfaces have weaker protein adsorption, making themasily detachable by subsequent PBS flow. Fig. 3(b) shows AFMmages of adsorbed proteins on three different surfaces. The imageslearly support the SPR measurements; proteins adsorb more onydrophobic than hydrophilic surfaces. For instance, the bare-goldurface is covered 44% more than COOH- and OH-SAM surfaces bysolectin.

.2. Protein displacement

LMW proteins are initially attached on the three different sur-aces and HMW proteins are subsequently flown to observe proteinisplacement. The surfaces need to be saturated; otherwise HMWroteins tend to adsorb the space between LMW proteins if theyo not form a fully packed protein monolayer (Green et al., 1999).he surfaces are saturated using 0.05% (w/v) proteins to form aully packed monolayer. The angle shift of SPR profiles increases atach protein adsorption and stabilizes after washing weakly boundroteins from the surface. The final angle shift represents a fullydsorbed monolayer on the surfaces. The proteins form monolayer,ot multi-layered structures at steady-state. We verify the mono-

ayer formation by reversing the injection sequence later in theection.

As discussed in the previous section, surface properties are aey to protein adsorption. In this section, we demonstrate that sur-ace properties are critical to proteins displacement, which cane used for the protein sensor. Fig. 4 shows an example of thedsorption/displacement profiles of sequentially injected two pro-eins with different molecular weights on the bare-gold, COOH-nd OH-SAM surface. First, four proteins are grouped as “A” andB”, representing LMW and HMW proteins, respectively. “A” grouprotein is adsorbed on the surface and subsequently “B” group pro-ein reaches to the surface to replace initially adsorbed protein,roman effect. For instance, when aprotinin is firstly injected anddsorbed on the surface, lysozyme, streptavidine, and isolectin cane used to displace aprotinin. Therefore, six cases occur from fourroteins. Among them, Fig. 4 indicates that aprotinin is displacedy lysozyme on different surfaces. Other cases follow similar pat-erns, and the SPR responses are summarized in Table 1. First, “A”roup proteins are injected along with PBS. When the SPR profileseach steady-state plateau, “B” group proteins flow to the chan-el. For reliable displacement rate, “B” group proteins reach theurface that is covered by “A” group proteins after the SPR pro-le stabilizes. When “B” group proteins reach the surface the SPRrofile changes from the steady-state. This means that some por-ion of LMW proteins adsorbed on the surface are displaced byMW proteins. On bare-gold surface in Fig. 4(1), the angle shift is

arge at the steady-state while hydrophilic surfaces provide smallngle changes. Table 1 summarizes all SPR angle shifts and theisplacement percentage on the bare-gold, COOH- and OH-SAMurfaces, respectively. The angle shift indicates how many LMWroteins are replaced by HMW ones. The bigger the difference in

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ctronics 24 (2008) 893–899 897

olecular weights between LMW and HMW proteins, the biggerhe angle shifts. For example, the displacement of aprotinin byysozyme, which is the smallest difference in molecular weights,hows the smallest angle change whereas the largest angle changeccurs when aprotinin is displaced by isolectin. This is becausehe angle shift is proportional to the MW of proteins. The dis-lacement percentage, on the other hand, is not proportional tohe MW differences. For instance, on a bare-gold surface whenprotinin (0.65 kDa) is replaced by lysozyme (14.7 kDa), 17% of apro-inin is displaced by lysozyme. When aprotinin is displaced by aarger protein, streptavidine (53 kDa), only 11% of aprotinin is dis-laced.

The angle shift depends upon the types of surface. Theydrophilic surface has a higher displacement percentage thanhe hydrophobic surface. On the bare-gold surface, LMW proteinsdsorb first, strongly binding to the surface and undergoing con-ormational spreading (Wertz and Santore, 2001). Subsequentlyrriving HMW proteins do not replace all the initially adsorbed pro-eins from the surface. However, hydrophilic surfaces show a higherisplacement percentage because their weak bonds between theydrophilic residues of the proteins and the surface do not inducehe conformational spreading of proteins. The weak bonds allow theigh percentage displacement. From our measurements, the dis-lacement rate of aprotinin by HMW proteins on the hydrophobicurface is below 20% whereas the rate increases to minimum 30%nd sometimes up to 100% on the hydrophilic surfaces. The higherisplacement rate represents higher selectivity. On the other hand,he hydrophobic surface produces much higher SPR angle shiftshan hydrophilic surfaces. The SPR angle shifts for all four proteinsre more than 100 mDeg on the bare-gold surface whereas OH-AM covered surface produces only 7–12 mDeg. The larger anglehifts indicate higher sensitivity. Therefore, the bare-gold surfacehows the highest sensitivity yet limited selectivity while the OH-AM covered surface has the best selectivity yet low sensitivity. Aecessary trade-off between sensitivity and selectivity exists andhe protein sensor should optimize surface properties for the targetrotein.

In the case of the hydrophilic surfaces, OH- and COOH-SAM,he difference of hydrophilicity is minimal, 38.2◦ and 41.4◦; yetheir adsorption and displacement behaviors are different. Almostll cases of proteins displacement show that OH-SAM has higherisplacement rate than COOH-SAM. A minute change of surfaceroperties deliver significant differences in selectivity and sensi-ivity characteristics of the protein sensor.

In order to evaluate the selectivity of the protein sensor, weharacterize the proteins adsorption in the reverse sequence. HMWroteins are injected first and then LMW ones later (Fig. 5). On allurfaces, the angle shifts are almost negligible, less than 1 mDeg,ndicating that the reverse sequence does not allow the replace-

ent (SPR profiles on COOH- and OH-SAM surfaces are not shown).his supports the selectivity of the SPR protein sensor in Table 1.ig. 5 also demonstrates that proteins form monolayer. If the pro-eins form multi-layered structures, then the SPR angle shouldhange when subsequent proteins are injected on top of existingound proteins. A few techniques to study the multi-layer for-ation of proteins have been introdueced, such as total internal

eflection fluorescence (TIRF microscopy), ellipsometry, and AFMSchmidt et al., 1990; Gunning et al., 1998). These techniques have

onitored the thickness changes of proteins on a surface, leadingo a better understanding of the protein layers as a result. These

echniques have monitored the thickness changes of proteins on aurface, leading to a better understanding of the protein layers as aesult. However, none has been able to fully verify the monolayerormation. SPR can detect molecular interactions of up to 200 nmbove the metal surface; thus the measurement strongly suggests

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Table 1Summary of the SPR angle shifts on the bare-gold, OH-, and COOH-SAM surfaces

Angle shifts represent sensitivity and the sequential angle shifts indicates selectivity. Bare-gold shows the highest sensitivity yet limited selectivity. On the other hand OH-SAM offers the best selectivity yet low sensitivity.

S. Choi et al. / Biosensors and Bioele

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hat the reverse sequence of protein displacement does not occurnd the protein layer remains monolayer.

This proof-of-concept protein sensing technique using Vromanffect offers both sensitivity and selectivity without using bio-eceptors. By eliminating the use of bio-receptors it is possible toeduce manufacturing cost and to encourage an uptake of reliableiniaturized protein sensors in processing. No perfect separator

xists to date. Thus the eluted sample from the separator has mul-iple proteins. A mixture of different molecular weight proteins canlso be detected using the protein sensor. When multiple proteinsre in a solution, mass transfer rate of each protein influences theirdsorption. The mass transfer rate is a strong function of its solutiononcentration and inversely proportional to its molecular weightFournier, 1999). Therefore, the smaller proteins tend to arrive firstt a surface, and then be displaced from the surface in an exchangeeaction by larger proteins. However, the Vroman effect cannot betilized for a mixture of similar molecular weight proteins. Sup-ose two surfaces are prepared to capture a target protein; one isovered by a little smaller MW than the target protein and the others covered by a little bigger MW protein. The sensor only looks at

W of target proteins and if it falls between the two initially cov-red proteins, then the Vroman effect allows one to be displacednd not the other. If the mixture of protein sample contains similarW proteins to the target protein, it is virtually impossible to detect

he target protein. There are options to mitigate the challenge. Onef them is to use isoelectric point; proteins are charged depend-

ng upon the pH of the surrounded buffer solution. The isoelectricoint has been used to distinguish similar MW proteins in 2D elec-rophoresis. One can combine different options together with theroman effect, it may be possible to detect the target protein in aixture of similar MW proteins.

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ctronics 24 (2008) 893–899 899

. Conclusion

Conventional protein sensors often fail to satisfy both high selec-ivity and sensitivity requirements because of complex structurend multiple forms of proteins. Existing protein sensors utilize aio-receptor to capture target molecules for selectivity, yet manyhallenges exist on having appropriate bio-receptors and inte-rating the bio-receptors into the transducer. In this paper, weresent a high sensitivity and high selectivity protein sensor with-ut using bio-receptors. Instead we use the Vroman effect whichakes advantage of the competitive nature of proteins adsorptionsn the surfaces. The sensor uses SPR, which measures real-timeolecular interactions with extremely high sensitivity. We demon-

trate that LMW proteins are displaced by HMW proteins using fourifferent proteins, aprotinin, lysozyme, streptavidine, and isolectin,et the reverse sequence does not occur. The protein sensor can dis-inguish at least 14.05 kDa in molecular weight and demonstrate aery low false positive rate.

We have also demonstrated the effects of surface proper-ies on protein adsorption/displacement using well-defined SAMsf alkane-thiols. Strong interaction of protein with hydrophobicurface induces conformational spreading and transition to an irre-ersibly adsorbed state. The strong binding makes the proteinisplacement very difficult. On the other hand, COOH- and OH-SAModified hydrophilic surfaces allow weak binding with proteins,

esulting in a high chance of displacement, and thus high selectiv-ty.

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