Biomolecules Detection Using a Silver-Enhanced Gold Nanoparticle-Based BiochipAbstractIntroductionBiochip Architecture and Principle of OperationBiochip Fabrication and Surface FunctionalizationResults and DiscussionsVerification of the Operating PrincipleIgG DetectionConclusionsAcknowledgmentsReferencesSPECIAL ISSUE ARTICLEBiomolecules Detection Using a Silver-Enhanced GoldNanoparticle-Based BiochipYang Liu•Deng Zhang•Evangelyn C. Alocilja•Shantanu ChakrabarttyReceived: 30 September 2009 / Accepted: 16 January 2010 / Published online: 2 February 2010Ó The Author(s) 2010. This article is published with open access at Springerlink.comAbstract Silver-enhanced labeling method has beenemployed in immunochromatographic assays for improv-ing the sensitivity of detecting pathogens. In this paper, weapply the silver enhancement technique for biomolecularsignal amplification in a gold nanoparticle-based conduc-timetric biochip. We show that the response of the silver-enhanced biochip comprises two distinct regions namely:(a) a sub-threshold region where conduction occurs due toelectron hopping between silver islands and the electrolyteand (b) an above-threshold region where the conduction isdue to a direct flow of electrons. These two regions arecharacterized by different conduction slopes, and we showthat combining the information from both these regions canimprove the sensitivity of the biochip. Results from fabri-cated prototypes show a dynamic range of more than 40 dBand with a detection limit less than 240 pg/mL. Thefabrication of the biochip is compatible with standardcomplementary metal–oxide–semiconductor (CMOS) pro-cesses making it ideal for integration in next-generationCMOS biosensors.Keywords Gold nanoparticle  Silver enhancement Biomolecules  Biochip  BiosensorIntroductionBiosensors have emerged as important analytical tools fordetecting and controlling disease outbreaks, whichaccording to the United States Department of Agriculture(USDA) cause $2.9–6.7 billion worth of losses every year[1]. Biosensors typically consist of a biological recognitionlayer (e.g. enzymes, antibodies, DNA etc.) integrated inproximity to a transducer which converts the binding eventbetween the target and its specific probes into a measurablesignal. For instance, in the most widely used enzyme-linked immunosorbent assay (ELISA) technique, thehybridization event between antibodies and antigen isreported using a colorimetric signal and with detectionlimits approaching picomolar range. Out of all detectionmethods used in biosensors, optical-based technique is themost popular one because of its high-sensitivity and itsability to remotely interrogate the information on the bio-sensor using light or laser. However, biosensors withelectrical readouts offer several advantages over theiroptical counterparts due to their reduced cost, reduced formfactor, and the ease of signal acquisition [2, 3]. One of themajor challenges in the electrical or impedance baseddetection is low signal-to-noise ratio when compared tooptical detection, which is attributed to the large magnitudeof the background signal [3]. In this regard, a biomolecularamplification technique called ‘‘silver enhancement’’ couldbe ideal to boost the signal-to-noise ratio (SNR) of con-ductimetric biosensors to be comparable to that of itsoptical counterparts. In fact, silver enhancement has beenpreviously proposed and used for improving the detectionrange in optical biosensors. In [4–7], silver enhancementhas been used in conjunction with labeling with goldnanoparticles for optical detection in immunoassays. In [5],it was reported that the conjugation significantly increasedY. Liu (&)  S. ChakrabarttyElectrical and Computer Engineering, Michigan StateUniversity, East Lansing, MI 48824, USAe-mail: [email protected]. Zhang  E. C. AlociljaBiosystems and Agricultural Engineering, Michigan StateUniversity, East Lansing, MI 48824, USA123Nanoscale Res Lett (2010) 5:533–538DOI 10.1007/s11671-010-9542-0the detection limit of ricin to 100 pg/mL. We show in thispaper that for conductimetric biosensors, silver enhance-ment significantly improves the SNR and in the process canachieve detection sensitivity comparable or better than anoptical based system. Also, performing signal enhancementat the biomolecular level before performing electrical read-out would reduce the effects of background interference[8–10].The model conductimetric biochip used for this studyhas been constructed using functionalized gold nanoparti-cles on the high-density interdigitated microelectrodearray. The interdigitated electrodes provide a large activearea to facilitate binding between the analyte and thedetection probe and hence have several advantages overnon-interdigitated electrode arrays [11, 12]. The salientfeatures of this study include: (a) a simple and robustelectrical detection method using a combination of goldnanoparticle labels with silver amplification technique; (b)characterization of the extent to which the nanoparticleadsorption can be quantified using silver enhancement; (c)characterization of two distinct biomolecular transistorresponses that are the sub-threshold and the above-thresh-old regions of the operation, and (d) characterization of thebiochip sensitivity and the detection limit using repeatedand controlled experiments. This paper is organized asfollows: Sect. 2 describes the operating principle of thesilver enhancement technique when applied to gold nano-particles and the high-density microelectrode biochip.Section 3 describes the fabrication method of biochips andsurface functionalization of biochips. Section 4 presentsexperimental results of detecting biomolecules using rabbitand mouse IgG as model antigen, which verify the prin-ciple of silver enhancement and the functionality of thebiochip. Section 5 concludes with a brief discussion andthe future work.Biochip Architecture and Principle of OperationThe principle of conductimetric biochip detection is shownin Fig. 1 where initially probes specific to the target mol-ecules are immobilized in the regions between two elec-trodes. When the analyte is applied, the target biomoleculesinteract with the specific probes. The secondary antibodiesconjugated with gold (Au) nanoparticle are then applied tothe biochip, which leads to the formation of a sandwicharray as shown in Fig. 1a. This configuration is denoted asthe ‘‘cutoff’’ region, since the current