Silver-enhanced labeling method has been employed in immunochromatographic assays for improving the sensitivity of detecting pathogens. In this paper, we apply the silver enhancement technique for biomolecular signal amplification in a gold nanoparticle-based conductimetric 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 to electron hopping between silver islands and the electrolyte and (b) an above-threshold region where the conduction is due to a direct flow of electrons. These two regions are characterized by different conduction slopes, and we show that combining the information from both these regions can improve the sensitivity of the biochip. Results from fabricated prototypes show a dynamic range of more than 40 dB and with a detection limit less than 240 pg/mL. The fabrication of the biochip is compatible with standard complementary metal–oxide–semiconductor (CMOS) processes making it ideal for integration in next-generation CMOS biosensors.
KeywordsGold nanoparticle Silver enhancement Biomolecules Biochip Biosensor
Biosensors have emerged as important analytical tools for detecting and controlling disease outbreaks, which according to the United States Department of Agriculture (USDA) cause $2.9–6.7 billion worth of losses every year . Biosensors typically consist of a biological recognition layer (e.g. enzymes, antibodies, DNA etc.) integrated in proximity to a transducer which converts the binding event between the target and its specific probes into a measurable signal. For instance, in the most widely used enzyme-linked immunosorbent assay (ELISA) technique, the hybridization event between antibodies and antigen is reported using a colorimetric signal and with detection limits approaching picomolar range. Out of all detection methods used in biosensors, optical-based technique is the most popular one because of its high-sensitivity and its ability to remotely interrogate the information on the biosensor using light or laser. However, biosensors with electrical readouts offer several advantages over their optical counterparts due to their reduced cost, reduced form factor, and the ease of signal acquisition [2, 3]. One of the major challenges in the electrical or impedance based detection is low signal-to-noise ratio when compared to optical detection, which is attributed to the large magnitude of the background signal . In this regard, a biomolecular amplification technique called “silver enhancement” could be ideal to boost the signal-to-noise ratio (SNR) of conductimetric biosensors to be comparable to that of its optical counterparts. In fact, silver enhancement has been previously proposed and used for improving the detection range in optical biosensors. In [4–7], silver enhancement has been used in conjunction with labeling with gold nanoparticles for optical detection in immunoassays. In , it was reported that the conjugation significantly increased the detection limit of ricin to 100 pg/mL. We show in this paper that for conductimetric biosensors, silver enhancement significantly improves the SNR and in the process can achieve detection sensitivity comparable or better than an optical based system. Also, performing signal enhancement at the biomolecular level before performing electrical read-out would reduce the effects of background interference [8–10].
The model conductimetric biochip used for this study has been constructed using functionalized gold nanoparticles on the high-density interdigitated microelectrode array. The interdigitated electrodes provide a large active area to facilitate binding between the analyte and the detection probe and hence have several advantages over non-interdigitated electrode arrays [11, 12]. The salient features of this study include: (a) a simple and robust electrical detection method using a combination of gold nanoparticle labels with silver amplification technique; (b) characterization of the extent to which the nanoparticle adsorption can be quantified using silver enhancement; (c) characterization of two distinct biomolecular transistor responses that are the sub-threshold and the above-threshold regions of the operation, and (d) characterization of the biochip sensitivity and the detection limit using repeated and controlled experiments. This paper is organized as follows: Sect. 2 describes the operating principle of the silver enhancement technique when applied to gold nanoparticles and the high-density microelectrode biochip. Section 3 describes the fabrication method of biochips and surface functionalization of biochips. Section 4 presents experimental results of detecting biomolecules using rabbit and mouse IgG as model antigen, which verify the principle of silver enhancement and the functionality of the biochip. Section 5 concludes with a brief discussion and the future work.
Biochip Architecture and Principle of Operation
Biochip Fabrication and Surface Functionalization
Results and Discussions
Verification of the Operating Principle
Based on the principle of silver enhancement, we conduct IgG detection by first applying rabbit IgG onto the active area of the anti-rabbit IgG biochip allowing incubation for 30 min. Goat anti-rabbit IgG and gold conjugates were then applied and were incubated for 30 min. Excess gold conjugates were washed with PBS solution. Electrical measurements are taken after each treatment of the biochip with the silver enhancer solution, and the conductance between the electrodes was measured using a BK multimeter Model AK-2880A (Worchester, MA).
We have shown the experiments to verify the principle of silver-enhanced electrical detection of biomolecules using rabbit IgG as model antigen. One issue that other researchers have not addressed in the silver enhancement method is the accuracy and possible false positive errors. Due to the sensitivity of the presence of gold nanoparticles when exposing to silver, it might have a high level of false positive results. The typical method of prevention is to extensively wash the biochips to alleviate non-specific binding. However, the method is time consuming, and it is not always effective. Another alterative solution is to embed the biochip with error-correction function by employing encode-decoding scheme similar to the approach that we have previously reported .
In this paper, we have designed and characterized a silver enhancement technique for amplifying the signal for gold nanoparticle label at biomolecular level. The gold nanoparticles serve as nucleation sites about which a reduction reaction deposits silver and hence enlarges the size of the gold nanoparticle. Using silver enhancer solution, the gold antiparticles can grow into a micro size particle and ultimately can bridge the gap between electrodes, leading a measurable change in conductance. Comprehensive experiments have verified the effectiveness of surface functionalization and the functionality of biochip. The proposed biochip in conjunction with silver enhancement provides a simple, effective, and sensitive way of detecting trace quantity of biomolecules.
This work is supported in part by a research grant from the National Science Foundation: NSF ECCS-0622056. Authors also would like to thank Lurie Nanofabrication Facility at University of Michigan for the fabrication of biochips.
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