This paper presents a biosensor implementation for the detection of protein molecules using specific antibodies. Affinity sensors allow the detection and quantification of target molecules in complex mixtures by affinity-based interactions. Immobilized antibody molecules are the probes that bind to

specific protein molecules (targets) in biological fluids. In this study, inter-digitated electrodes in the form of capacitance on glass slide were designed, fabricated and used to measure the changes in the dielectric properties of the inter-digitated capacitances. Our results in this study present that

with a careful design of micro-interdigitated capacitors, a wider dynamic range and higher sensitivity can be achieved for the detection and quantification of C-Reeactive Protein

Başağa, Hüveyda

Gül, Özgür

Gürbüz, Yaşar

Kallempudi, Sreenivasa Saravan

Sezerman, Uğur

English

Sabanci University Research Database

RESEARCHARTICLECopyright © 2008 American Scientific PublishersAll rights reservedPrinted in the United States of AmericaSENSOR LETTERSVol. 6, 1–5, 2008Label-Free Biosensors for the Detection andQuantification of Cardiovascular Risk MarkersKallempudi S. Saravan, Ozgur Gul, Huveyda Basaga, Ugur Sezerman, and Yasar Gurbuz∗Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul 34956, Turkey(Received: xx Xxxx xxxx. Revised/Accepted: xx Xxxx xxxx)This paper presents a biosensor implementation for the detection of protein molecules using specificantibodies. Affinity sensors allow the detection and quantification of target molecules in complexmixtures by affinity-based interactions. Immobilized antibody molecules are the probes that bind tospecific protein molecules (targets) in biological fluids. In this study, inter-digitated electrodes in theform of capacitance on glass slide were designed, fabricated and used to measure the changesin the dielectric properties of the inter-digitated capacitances. Our results in this study present thatwith a careful design of micro-interdigitated capacitors, a wider dynamic range and higher sensitivitycan be achieved for the detection and quantification of C-Reeactive Protein.Keywords: Biosensor, Antibody, Capacitive, Cardiovascular, C-Reactive Protein.1. INTRODUCTIONBiosensors are fast, direct, and with ability to performlabel-free measurements are attractive for different appli-cations. The affinity based interactions/sensing approachesby name mean use of specific antigen/antibody, allow thedetection and quantification of target molecules in complexmixtures. So, with this approach we can detect the specificproteins without labeling (Label-free) them. Immobilizedantibody molecules are the probes that bind to specific pro-tein molecules (targets) in biological fluids. Inter-digitatedelectrodes in the form of capacitance on glass slide canbe used to measure the changes in the dielectric prop-erties of the inter-digitated capacitances upon antigenbinding.1Besides the inexpensive production and low sample con-sumption, main advantages of inter-digitated, capacitiveimmuno-sensors are (1) the label-free detection mecha-nisms and (2) the fast and reliable measurements. Unlikeother detection mechanisms used to quantify protein con-centrations, capacitive method does not need labelingof samples prior to analysis. Upon binding of proteinmolecules to the immobilized antibody molecules on thesurface of electrodes, dielectric properties between theinter-digitated electrodes changes and measurement canbe taken immediately after hybridization.23 The detectionmechanism of these sensors is based on the change in∗Corresponding author; E-mail: yasar@sabanciuniv.eduthe dielectric constant of the inter-digitated capacitance.This change arises, at the simplest form, from the equationC = oA/d, where C is the capacitance between theinter-digitated electrodes,  is the dielectric constant ofthe medium between the plates, o is the dielectric con-stant of free space, A is the area of the plates and d isthe distance. When a change in dielectric constant occurs,a counter change occurs in capacitance value in betweenthe electrodes.1This change in the dielectric constant, hence in thecapacitance, is correlated to the bound antigen moleculesto the capture antibodies on the surface, between the elec-trodes. The dielectric constant of the antibodies changesthe dielectric constant between the electrodes/fingers andthis result in a change in capacitance. Inter-digitated elec-trode arrays are used in order to increase the active areafor higher sensitivity to binding events. Atomic ForceMicroscopy (AFM) and electrochemical methods are usedto measure this change, but impedance analysis gives fasterresults.4Biosensor applications for various human serum pro-teins are available. C-reactive protein (CRP) is one of theinflammation markers in human serum.5 Recent articleshave shown significance of inflammatory markers in earlydetection of cardiovascular disease.67 CRP is emergingas a new marker for acute phase inflammation and hasshowed a lot of potential for earlier detection of cardio-vascular diseases.89Sensor Lett. 2008, Vol. 6, No. 5 1546-198X/2008/6/001/005 doi:10.1166/sl.2008.521 1RESEARCHARTICLELabel-Free Biosensors for the Detection and Quantification of Cardiovascular Risk Markers Saravan et al.2. STRUCTURAL MODELLING ANDEXPERIMENTAL2.1. Structural Design and ModelingMulti-finger types of capacitors are widely used inmicrostrip technology. The capacitor itself is definedbetween the two ports as shown in Figure 1(a). The inter-digital capacitor couples the two coplanar waveguidesby the electromagnetic field in its region. In a nutshell,the variation of medium between the fingers of inter-digitalcapacitor, will lead to variation in effective dielectric con-stant of the area. This directly changes the total capaci-tance of inter-digital capacitance. Figure 1(b) depicts theequivalent circuit of the inter-digital capacitor. Magneticcoupling between the fingers, a transformer with the self-inductances L1 and L2 and the mutual inductance M isused. In order to decrease these inductances, long fingersare not being used. The ohmic losses that occur due to thecurrent flow through the fingers can be descried by two fre-quency dependent resistors Rf1 and Rf2. There resistancesmust be decreased for instance using thicker metal layerto get rid of losses. The capacitances Cp1 and Cp2 repre-sent the stray fields from the fingers to the ground plane.Therefore in our design the distance between the groundlayer and inter-digital electrodes is increased.The simulation of this type of structures at high fre-quencies is not easy and electromagnetic simulators likeMOMENTUM/HFSS should be used for high accuracy.In this work, modeling and simulation of inter-digitalcapacitors are performed using ADS (Advance DesignSystem) MOMENTUM® and HFSS® tools.The structure/model, shown in Figure 1, is simulatedin the frequency range of 1 GHz to 5 GHz. One of theports is grounded during the simulations. The optimum fre-quency range, selected the one having a maximum capac-itance change, is between 2–3 GHz. This frequency rangeis also known as industrial, scientific and medical (ISM)radio bands, originally reserved internationally for non-commercial use of RF electromagnetic fields for industrial,scientific and medical purposes. Figure 2 presents thefabricated/realized inter-digital capacitor structure.(a) (b)Fig. 1. Two-port, inter-digitated capacitors (a) and its electricalmodel (b).Fig. 2. Nickel IDC with SU8 wells.The S-parameters of the structure in Figure 1 areextracted from the simulation results and actual valuesare measured using a Network Analyzer. The resultingcapacitance values from simulations and measurements forthe mentioned frequency range are presented in Figure 3,respectively. The Figure 3 envisages that the capacitancevalue by simulation at 2.354 GHz is found as 1.829 pF,Figure 3(a) that is very close to measured value of 1.8 pF,Figure 3(b).2.2. Sensor FabricationThe fabrication flow for the structure shown in Figure 2 ispresented in Figure 4. Very thin tungsten layer of 30 nmis sputter deposited on the glass microscope slide, whichis used to improve the adhesion of gold on substrate.Then 300 nm of gold is deposited using sputter deposi-tion, as seen in step (A). Following this step, the goldlayer is patterned by wet etching with the mask seen in thestep (B). I2/KI/H2O solution is used to etch gold layer and30% hydrogen peroxide (H2O2) is used to etch the under-lying tungsten layer. After that, an oxide layer of 50 nmis deposited on the electrodes, step (C), and the top of thecontact pads are opened using HF solution, step (D). Theoxide layer is deposited to improve the coating of epoxysilane layer, which will be used to immobilize antibodiesand to insulate the electrodes. Length of each electrode is750 m and width is 25 m. The distance between twoelectrodes is 25 m. To ease the process of fabricationgold metal is replaced with Nickel. Image Reversal Tech-nique with AZ 5214 E is used to Pattern Nickel Metal,step (D) in the above process is omitted as Nickel read-ily forms oxide. A 40 m deep SU8 wells were patternedover the inter-digitated structure for easing the antibodyimmobilization on the sensor structure. Figure 2 shows thefabricated Nickel IDC with SU8 wells.2.3. Materials and ReagentsMonoclonal antibody and purified antigens for C-ReactiveProtein were purchased from Fitzgerald Industries Inter-national (Concord, MA, USA). Alexa-488 conjugated2 Sensor Letters 6, 1–5, 2008RESEARCHARTICLESaravan et al. Label-Free Biosensors for the Detection and Quantification of Cardiovascular Risk Markers(a)(b)Fig. 3. Simulated (a) and measured (b) values of the inter-digitalcapacitors.rabbit anti-mouse antibody was purchased from Invitro-gen (Carlsbad, CA, USA). (3-glycidoxypropyl)-trimethoxysilane (GPTS), BSA, PBS and Tween 20 were purchasedfrom Sigma (USA). Fluorescent scanning was carried outusing an ArrayWoRx(R) Biochip Reader (Applied Preci-sion, Marlborough, UK) and analyzed by using accom-panying software. PBS-T (1X PBS, 0.5% Tween 20) andDiluent buffer (2% BSA in BPS-T) were used for all wash-ing and dilution steps, otherwise noted.2.4. Surface Activation and Antibody Immobilization2% of (3-glycidoxypropyl)-trimethoxy silane (GPTS) solu-tion was used to coat SiO2 surface. After one hour silaniza-tion reaction, sensors were washed several times withethanol and dried using centrifuge. 2 l of C-reactive pro-tein antigen at 0.5 mg/ml concentration was added ontosurface and incubated for two hours at room temperature.After immobilization step, biosensor surface was blockedusing 2% BSA for nonspecific binding of antigen. AfterFig. 4. Inter-digital capacitor fabrication/process.several times washing with PBS-T, sensors were stored at4 C until use.2.5. MeasurementsPurified antigens were diluted in diluent buffer and incu-bated with sensors for one hour. After several washingsteps, sensors were dried using centrifuge. Karl-Suss PM-5RF Probe Station and Agilent-8720ES S-Parameter Net-work Analyzer were used for all capacitance measure-ments. Network Analyzer were calibrated using SOLT(short-open-load-through) method and S-Parameter data ofthe capacitor were measured. Finally, capacitance values(C) were extracted from the measurements at certain fre-quencies (f ). Only 1 to 5 GHz range was scanned.3. RESULTS AND DISCUSSION3.1. Antibody ImmobilizationImmobilization of C-reactive protein specific antibodiesonto epoxy coated biosensor surface was checked usingAlexa-488 labeled antibodies. Fluorescent conjugated anti-mouse antibodies were hybridized with capture antibodieson the surface. Fluorescent scanner was used to scan thesensors fabricated on the microscope slide by followingthe method mentioned in experimental section.Fluorescent image can be seen in Figure 5. We havefound that 0.5 mg/ml antibody concentration is optimumfor immobilization procedure. 2% BSA blocking is shownto be efficient, as seen in Figure 5, where no fluorescentsignal was detected when only BSA blocked sensor wasincubated with fluorophore labeled anti-mouse antibody.Sensor Letters 6, 1–5, 2008 3RESEARCHARTICLELabel-Free Biosensors for the Detection and Quantification of Cardiovascular Risk Markers Saravan et al.Fig. 5. Antibody immobilization confirmed by hybridization withAlexa-488 labeled anti-mouse antibodies.3.2. Antigen DetectionC-reactive protein antigen were diluted to different con-centration using diluent buffer and hybridized with sen-sor for 1 hour. After several washing steps and drying,capacitance measurements were carried out by followingthe protocol in experimental section. Capacitance valueswere extracted from measurements for each different anti-gen concentration. We have observed an inductive behav-ior after 2.7 GHz frequency, therefore capacitance valuesfor frequencies higher than 2.7 GHz were not considered.Capacitance change versus frequency graph can be seen inFigure 6.Concentrations versus capacitance changes were cal-culated for some of the frequency points, as shown inFigure 7(a) and changes in capacitance at various stagesblank(0), epoxy coated(1), antibody coated(2), blocked(3),and antigen incubation(4), were presented in Figures 7(b)and (c). These frequencies can be used for further exper-iments. We have found that concentration versus capac-itance change at 2.62 GHz is linear over 100 ng/ml to800 ng/ml antigen concentration range, with R2 equals to0.97, as presented in Figure 8.We have showed that capacitance change upon bindingof antigen to the immobilized capture antibodies on thesurface is correlated to the antigen concentration in thebuffer. Although the detection range is not low comparingFig. 6. Frequency versus capacitance change for different antigenconcentration.(a)(b)(c)Fig. 7. Concentration versus capacitance change at different frequen-cies (1–25 means Capacitance measured after epoxy coating that will beincubated with 25 ng/ml antigens).Fig. 8. Capacitance change versus antigen concentration at 2.62 GHz.4 Sensor Letters 6, 1–5, 2008RESEARCHARTICLESaravan et al. Label-Free Biosensors for the Detection and Quantification of Cardiovascular Risk Markersto other available systems, optimization of capacitorgeometries can further enhance the sensor detection limits.4. CONCLUSIONSIn this study, we have analyzed the reliability of inter-digitated electrodes for biosensor applications. Biosensorsbased on antibody–antigen interactions can be used forquantification of biomarkers in human serum. High sen-sitivity and improved detection range can be obtainedthrough optimization of topology/geometry of capacitors.Furthermore, integration with microfluidics systems forsample delivery can further improve the overall stabil-ity and reproducibility of inter-digitated electrodes basedimmuno-biosensor.Although we have used C-reactive protein as a modelsystem, this antibody based, label-free detection systemcan be applied to other areas, such as, microbiologicaldetection, DNA detection or biomarker detection.Inter-digitated electrodes can be further designed inan array format and integrated into other Micro-Electro-Mechanical devices/systems for further improvements ofperformance, cost, reliability, etc. Using arrays of capac-itors may allow us to quantify more than one marker ata time and enhance the diagnostic power of biosensor.Besides high throughput capability of these biosensors,further integration of other electronics parts can improvethe signal-to-noise ratio as well.Acknowledgment: We thank Bulent Koroglu for hisvaluable contribution to the processing of devices. We alsothank the Scientific and Technological Research Coun-cil of Turkey (TÜBI˙TAK) for the financial support forthis project under the contract number 107E014 andtitle “RF Transmitter–Based Transducer for BiosensorApplications.”References and Notes1. C. Bergren, Electroanalysis 13, 173 (2001).2. M. DeSilva, Y. Zhang, P. Hesketh, G. Maclay, S. Gendel, andJ. Stetter, Biosensors Bioelectron. 10, 675 (1995).3. A. R. Varlan, J. Suls, W. Sansen, D. Veelaert, and De. Loof, Sens.Actuators, B 44, 334 (1997).4. G. Zeng and Z. Zheng, Proteomics 5, 4347 (2005).5. M. P. De Maat and A. Trion, Curr. Opin. Lipidol. 15, 651 (2004).6. P. M. Ridker, Circulation 108, e81-5 (2003).7. V. B. Patel, M. A. Robbins, and E. J. Topol, Cleve Clin. J. Med. 68,521 (2001).8. M. B. Clearfield, J. Am. Osteopath. Assoc. 105, 409 (2005).9. F. Lombardi, F. Tundo, P. Terranova, P. M. Battezzati, M. Ramella,A. Bestetti, and L. Tagliabue, Int. J. Cardiology 98, 313, (2005).Sensor Letters 6, 1–5, 2008 5

Label-free biosensors for the detection and quantification of cardiovascular risk markers

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Label-free biosensors for the detection and quantification of cardiovascular risk markers

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