Ratiometric ultrasensitive electrochemical immunosensor based on redox substrate and immunoprobe

In this work, we presented a ratiometric electrochemical immunosensor based on redox substrate and immunoprobe. Carboxymethyl cellulose-Au-Pb2+ (CMC-Au-Pb2+) and carbon-Au-Cu2+ (C-Au-Cu2+) nanocomposites were firstly synthesized and implemented as redox substrate and immunoprobe with strong current signals at −0.45 V and 0.15 V, respectively. Human immunoglobulin G (IgG) was used as a model analyte to examine the analytical performance of the proposed method. The current signals of CMC-Au-Pb2+ (Isubstrate) and C-Au-Cu2+ (Iprobe) were monitored. The effect of redox substrate and immunoprobe behaved as a better linear relationship between Iprobe/Isubstrate and Lg CIgG (ng mL−1). By measuring the signal ratio Iprobe/Isubstrate, the sandwich immunosensor for IgG exhibited a wide linear range from 1 fg mL−1 to 100 ng mL−1, which was two orders of magnitude higher than other previous works. The limit of detection reached 0.26 fg mL−1. Furthermore, for human serum samples, the results from this method were consistent with those of the enzyme linked immunosorbent assay (ELISA), demonstrating that the proposed immunoassay was of great potential in clinical diagnosis.

Scientific RepoRts | 6:35440 | DOI: 10.1038/srep35440 incubation of IgG, indicating that IgG immune reacted with anti-IgG (curve e). The resistance further increased when the probes were coated, demonstrating that the probe was fixed (curve f). These resistance increases were derived from that the proteins hindered the electron transport 30,31 .
The pH of the detection solution can significantly affect the sensitivity of the immunosensor. The effect of pH on the current response was investigated as shown in Fig. S6. After immunoassay, the I substrate generally increased from pH 4.0 to 5.5 and then slightly decreased from pH 5.5 to 6.0. The change trend of I probe caused by pH was similar with that of I substrate . This indicated that pH 5.5 was optimal for detecting IgG.
Under the optimized pH, the analytical property of the proposed immunosensors for IgG was investigated by detecting a series of standard IgG samples (Fig. 4A). I substrate was irregularly changed with adding IgG, which was consistent with that of I probe . Obviously, no satisfactory calibration curves would be obtained between single current signal (I substrate or I probe ) and logarithm of IgG concentration. Unexpectedly, a good linear relation between I probe /I substrate and logarithm of IgG concentration existed (Fig. 4B). The linear regression equation was I probe /I substrate = 0.0346Lg C (ng mL −1 ) + 0.6416 with a correlation coefficient of 0.998. The proposed immunosensor for IgG exhibited a wide linear detection range (LDR) from 1 fg mL −1 to 100 ng mL −1 with an ultralow limit of detection (LOD) of 0.26 fg mL −1 (Fig. 4B). The results indicated that the two-channel ratiometric method was effective for ultrasensitive electrochemical immunoassay. The comparison of this method with some previous works was illustrated in the Table 1. It can be seen that present method possessed a wider LDR and lower LOD.  To investigate the repeatability, 10 ng mL −1 IgG was determined by the proposed immunosensor for three times. The results showed small deviation of ± 2.35%, proving its fantastic repeatability. In order to testify the specificity, the 10 ng mL −1 IgG samples containing 100 ng mL −1 interfering substances, such as glucose (GC), dopamine (DA), uric acid (UA), ascorbic acid (AA), glutamic acid (Glu), lactic acid (LA) and BSA, were analyzed. As shown in Fig. S7, the obtained current signals were not affected by the interference substances, demonstrating the admirable specificity of this immunosensor. The stability of the immunosensor was examined by determining the IgG. After 14 days, the capacity of the immunosensor remained 85%, suggesting the good stability of the immunosensor. Compared with some recently reports, results indicated that the proposed immunosensor was more sensitive (Table 1).
Generally, for a normal adult, the concentration of IgG is about 7-16 mg mL −1 in serum. The human serum was firstly determined by ELISA method. After being diluted 1:10 6 , the received human serum samples were investigated by the proposed immunosensor. The results were listed in the Table S1, and relative error (RE) was less than 10%. The results of this immunoassay were consistent with the ELISA method, indicating its great potentials in clinic diagnosis.

Conclusion
In summary, CMC-Au-Pb 2+ and C-Au-Cu 2+ firstly synthesized as redox species, with parallel current signals. A novel ratiometric electrochemical immunosensor was successfully developed using CMC-Au-Pb 2+ and C-Au-Cu 2+ to construct the redox substrate and probe, respectively. The linear range of the proposed immunoassay (1 fg mL −1 to 100 ng mL −1 ), two orders of magnitude higher than other sandwich electrochemical immunoassay. This immunosensor exhibited excellent sensitivity, reproducibility, specificity, stability and practicability. This ratiometric method could be easily extended to the detection of other targets.  Apparatus. TEM was performed with a JEOL-100CX electron microscope under 80 kV accelerating voltage (H7650, Hitachi, Japan). SEM was performed with a Hitachi SU8010 SEM. X-ray photoelectron spectroscopy (XPS) analysis was conducted on an ESCALAB 250 X-ray Photoelectron Spectroscope (Thermofisher, American). The UV-vis spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were recorded by a UV-2550 spectrophotometer (Shimadzu, Japan) and a FT-IR spectrophotometer, respectively. EIS measurements were performed using Princeton PARSTAT 2273 (America). SWV was performed on a CHI832 electrochemical work station (Chenhua Instruments Co., Shanghai, China) with a three-electrode system which was consisted of a platinum wire as the auxiliary electrode, an Ag/AgCl electrode (saturated KCl) as the reference electrode and a GCE as the working electrode.

Materials
Synthesis of C-Au-Cu 2+ immunoprobe. The stepwise fabrication procedures of immunoprobe were shown in Fig. 2. CNP was synthesized according to the literature 32 . In brief, a homogeneous solution was produced by 1% glucose mixed with 10% sodium citrate. Further, the prepared solution was heated to 170 °C for 30 min in microwave reaction instrument (250 W). CNP was centrifuged, dispersed. After mixing CNP with 100 μ L 4% HAuCl 4 , a microwave reaction (100 °C, 10 min) was carried out. The CNP-Au nanocomposite was centrifuged and dispersed to 5 mL water. For adsorbing Cu 2+ , 1 mL CNP-Au was mixed with 1 mL100 mM Cu 2+ for 3 h. Then the obtained C-Au-Cu 2+ was centrifuged, washed and dispersed to 1 mL water. 5 mL C-Au-Cu 2+ was mixed with 500 μ L anti-IgG with stirring gently for 8 h. After that, the obtained C-Au-Cu 2+ /anti-IgG was blocked by 1% BSA for 1 h, and CNP-Au-Cu 2+ /anti-IgG/BSA was centrifuged, washed for several times, and dispersed to 5 mL water.
Synthesis of CMC-Au. After a homogeneous solution was prepared by 1 mL 1% CMC and 10 μ L 4% HAuCl 4 , 40 μ L 1.5% NaBH 4 was quickly injected with vortex mixing for 1 min. The solution turned from light yellow to red. Then, the CMC-Au was obtained and diluted to 16 mL prior to use.

Fabrication of immunosensor.
The schematic illustration of the fabrication procedure of the proposed immunosensor was shown in Fig. 2. GCE (Φ = 4 mm) was prepared by polishing with the alumina powders and successively sonication washing with water. After 20 μ L CMC-Au was dropped and dried at 37 °C, the obtained GCE was dipped in 10 mM Pb 2+ for 10 min. After that, a thin CMC-Au-Pb 2+ film was formed and washed with plenty of deionized water. 80 μ L 100 μ g mL −1 anti-IgG was incubated on CMC-Au-Pb 2+ /GCE for 12 h at 4 °C. The obtained electrode was incubated with 1% BSA at 37 °C for 1 h to block the remaining active points. Therefore, the immunosensor was accomplished and stored at 4 °C.
Electrochemical measurement. The immunosensor was incubated with 80 μ L IgG for 1 h at 37 °C. Next, 20 μ L C-Au-Cu 2+ /anti-IgG/BSA was incubated for 1 h at 37 °C. The modified GCE was carefully washed with water after each step. Subsequently, SWV was performed from − 1.3 V to 0.6 V in acetate buffer with pulse amplitude of 25 mV and an increase E of 4 mV s −1 .