Verification of the Operating Principle
The first step in verifying the silver enhancement principle is to measure the size of the silver-enhanced gold nanoparticles in the silver enhancer solution (Ted Pella, Inc., CA) with respect to the silver enhancing time. For this experiment, Zetasizer Nano (Malvern Instruments Ltd, UK) was used to characterize the particle size. Figure shows the linear relationship between the silver enhancing time and the size of gold particles. We have observed that the 40-nm gold nanoparticles will reach average size of 1.2 µm when exposed to the silver enhancer for approximately 10 min. This interesting property makes the silver enhancement principle suitable for signal amplification in conductimetric biosensors.
The relationship between the silver-enhanced gold particle size and the silver enhancing time
To evaluate the detection capabilities of the biochip, we dispense 2.5 mM/mL gold anti-IgG conjugate onto the biochip surface for 1 h and then treat the biochip with silver enhancer solution (Ted Pella, Inc., CA). To stop silver enhancement, the biochip was rinsed with distilled water and was dried with N2. Figure shows the microscopic observations before and after 35 min silver enhancing time. As gold nanoparticles are 40 nm size and they can not be observed by in Fig , where as they can be clearly observed after silver enhancement (see Fig. ). It can also be seen that the silver-enhanced gold particles form a bridge between the interdigitated electrodes. Figure shows the SEM image of the bridge formed by silver-enhanced gold particles and then verifies the operating principle.
Microphotographs for the biochip active surface. a before silver enhancement b 35 min after silver enhancement
SEM image of the bridge formed by enlarged gold particles
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).
Figure shows the conductance between the electrodes increases with the increasing exposure to the silver enhancer solution where the insert figure shows the conductance measurement in the logarithmic domain. The conductance increases when the biochip is exposed more in the silver enhancer solution as expected. It also clear shows the response has three distinct operation region (A, B, and C), which verifies the operating principle of the silver-enhanced gold nanoparticle-based biochip. During the sub-threshold region (labeled as B) of the operation, gold nanoparticles grow in the presence of silver enhancer solution thus leading to a shorter path for electron transport. But during this stage, enhanced gold particles have not formed the bridge to short the electrodes. With the increase in enhancement time, the consistent growth of silver-enhanced particles completely bridges the area between the electrodes, and there is immediate transition from state B to C when it happened (shown a step from B to C in the inserted figure in Fig. ). In the stage C, more bridges formed by gold nanoparticles are building up in parallel, thus leading to more conductance increase until it become to a more stable state. Bovine IgG biochips were used as negative control experiments, and we have observed that the conductance of “control” biochip start to increase at 10 min. It means that the non-specific binding actually occurred, but the number of such events is much smaller than the number of specific binding events. Thus, the biochip is able to detect target IgG in the presence of background noise. We have seen that the measurement results are stable several days after the experiments have been conducted. Also, the results in Fig. show that the detection range that can be achieved by proposed biosensor is 40 dB with respect to the control conductance.
Figure 8 Conductance of the biochips measured as a function of silver enhancing time. (Inset) Conductance measurement in logarithmic domain, which can clearly show that the biomolecular transistor exhibits three different types of responses (labeled as A, B, and (more ...)
We also conducted rabbit IgG detection using different IgG concentrations, and Fig. shows the conductance measurements at 45 min with three IgG concentrations and the control experiment. For each concentration, the experiment is repeated three times, and the standard deviation is also shown in the graph. It clearly shows that pathogenic and non-pathogenic cases can be easily distinguished even with low concentration of IgG. As seen in the graph, the conductance of biochip decrease with the decrease of the IgG concentration level, implying a decreasing of the signal-to-noise ratio. Similar experiments have been performed using anti-mouse IgG biochip with the mouse IgG detection, and the results are shown in the Fig. . These repeated and controlled experimental results verify the functionality of proposed biochip to detect biomolecules.
Steady state conductance measurement of rabbit IgG detection
Steady state conductance measurement of mouse IgG detection
Some researchers have argued that the conductivity-based silver-enhanced detection is not applicable to quantitative concentration assays, because the electrodes are short circuited above a certain density of the silver-enhanced gold particles [4
]. In the next experiment, we will show that the quantitative analysis can be achieved by adjusting silver enhancing time. Figure shows the relation of the silver enhancing time required to reach a conductance range of 3.8–5 mS as a function of rabbit IgG concentration. It is interesting to note that 240 pg/mL rabbit IgG can be detected when the conductance increases to 3.8 mS at the silver enhancing time of 42 min.
Quantitative analysis: the silver enhancing time required to reach a conductance range of 3.8–5 mS as a function of IgG concentrations
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 [14