Principle of the assay
The electrochemical detection of OA was demonstrated in Fig. 1. As the recognition element, the aptamer OA34 was immobilized on the electrode surface by self-assembling with AuNPs, and its directional arrangement was achieved following treatment with MCH, making adaptive folding and binding with targets easier. After binding with OA, the alteration of the aptamer conformation from loose to tight caused a substantial increase in the electron-transfer current as measured by CV, and the quantitative detection of OA can be completed by establishing the relationship between them.
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The optimization of modified condition and the characterization of AuNPs/CS/SPCE
To improve the electrochemical response as much as possible, the relevant conditions of modification with CS and AuNPs were optimized in this study.
The increase in peak currents can be attributed to the conductivity of CS obviously, but a possible explanation for the decrease might be that the film formed by excess CS layers was so thick that the charge transfer was inhibited (Fig. 2 A).
The reduction peak currents increased obviously with the increase in scan cycles (equivalent to deposition time) at the beginning and tended to stabilize after 5 cycles because of the limited electrodes surface area. To bind as much aptamers as possible on electrodes, 6 cycles were adopted to deposit AuNPs (Fig. 2 B).
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After the modification with CS and AuNPs, SEM was used to characterize the different morphologies of SPCE, CS/SPCE and AuNPs/CS/SPCE. The SPCE working surface consisted of lots of irregular and blocky structure (Fig. 3 A), and there was little significant difference with drip-coated CS (Fig. 3 B). Then the surface was obviously attached with uniform and dense particles additionally after deposition (Fig. 3 C), which could be inferred that AuNPs had been modified successfully on the surface of electrode with an average particle size less than 100 nm. Furthermore, the CV curves recorded in 0.2 M sulfuric acid (Fig. 3 D) performed a reduction peak current corresponded to the potential about 0.4 V, which ment the characteristic reduction peak of Au.
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Feasibility of the method and the optimization of experimental condition
The electrochemical behaviors of different modification processes were characterized by CV. Fig. 4 (A) showed that the SPCE without any modification exhibited low redox peak currents (curve a). After modification with drip-coated CS and AuNPs by electrodeposition on the surface, the currents increased about 7 μA because of the good conductivity of CS and AuNPs (curve b). While the currents decreased evidently with incubation in aptamers solution overnight (curve c), which proved that the aptamers had been immobilized on the electrode successfully. Then the currents decreased even further after the blocking of the nonspecific sites with MCH (curve d). Finally, the incubation of OA increased the currents due to the specific binding of aptamers and targets (curve e).
In this work, considering the sensitivity and nonspecific adsorption of the sensor, we optimized the aptamer concentration, MCH blocking time and target incubation time respectively to achieve the best detection conditions, which were selected according to the reduction peak currents differences before and after operation. Fig. 3 (B, C and D) showed the signal differences of reduction peak current (Δip), and the optimal setting values of these three parameters were finally determined as 10 μM, 60 min and 60 min.
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Analytical Performance, stability and selectivity of the aptasensor
The sensor response to OA concentrations ranging from 0.001 ng/mL to 100 ng/mL was measured with the above-mentioned optimal conditions (Fig. 5 A). The change of the reduction peak current as per the aptamer conformation upon binding with OA was observed clearly by the reduction percentage (Δip/ ip,0) (where the ip,0 was the reduction peak current before the binding of the aptasensor with OA ) of signal here. The data points in the calibration curve represented three independent measurements. In order to intuitively show aptasensor response to OA concentrations, the curve was displayed with the reduction percentage of reduction peak currents to the logarithmic values of the OA concentrations. And there was a linear increase of the signal with the concentrations changing from 0.01 ng/mL to 100 ng/mL (y= 6.70303 +1.13752*x, R2 = 0.99877, where x was the logarithmic value of the OA concentration, and y was the reduction percentage of the reduction peak current before and after binding with OA). The limit of detection (LOD) of the biosensor was calculated to be about 6.7 pg/mL (S/N=3), which was better than or comparable to some of the previously reported electrochemical and other aptasensors.
In this work, the stability of the sensor was confirmed by inter-day trials for five continuous days. The peak currents after combining the sensor with 10 ng/mL OA were measured by CV (Fig. 5 B). During the scanning process, the reduction peak currents (ip,r) were always slightly higher than the oxidation peak currents (ip,o). And there was no significant difference both of them for five consecutive days, indicating the good stability of this method.
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Analysis of Real Samples
In this work, the relevant extracts were obtained from fresh mussel and scallop samples by the sample processing steps described in the methods section. The extracts spiked with different known concentrations including 1, 50, 100 ng/mL of OA was tested using the constructed aptasensor. And the recovery statistics for each of the samples examined was shown in Table.1, ranging from 92.30 % to 115.98 % for an average of n = 3 replicates with an RSD from 8.64 % to 17.28 %, which represented a good performance in real samples.