Photoelectrochemical-Chemical-Chemical Redox Cycling for

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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Photoelectrochemical-Chemical-Chemical Redox Cycling for Advanced Signal Amplification: Proof-of-Concept Toward Ultrasensitive Photoelectrochemical Bioanalysis Bing Wang,†,‡,⊥ Li-Ping Mei,‡,⊥ Yan Ma,† Yi-Tong Xu,‡ Shu-Wei Ren,§ Jun-Tao Cao,*,† Yan-Ming Liu,† and Wei-Wei Zhao*,‡

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College of Chemistry and Chemical Engineering, Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, China ‡ State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China § Xinyang Central Hospital, Xinyang 464000, China S Supporting Information *

ABSTRACT: Signal amplification is essential for ultrasensitive photoelectrochemical (PEC) bioanalysis. Exploration of the facile and efficient route for multiple signal amplification is highly appealing. Herein, we present the concept of photoelectrochemical-chemical-chemical (PECCC) redox cycling as an advanced signal amplification route and a proof-of-concept toward ultrasensitive PEC bioanalysis. The system operated upon the bridging between the enzymatic generation of signaling species ascorbic acid (AA) from a sandwich immunoassay and the PECCC redox cycling among the ferrocenecarboxylic acid as redox mediator, the AA, and the tris(2-carboxyethyl)phosphine as reducing agent at the Bi2S3/Bi2WO6 photoelectrode. Exemplified by myoglobin (Myo) as target, the proposed system achieved efficient regeneration of AA and thus signal amplification toward the ultrasensitive split-type PEC immunoassay. This work first exploited the PECCC redox cycling, and we believe it will attract more interest in the research of PEC bioassays on the basis of advanced redox cycling.

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oxidizing) agents and an electrode, resulting in the much faster redox reactions and regeneration of these signaling species.27 We hypothesize that such a process also possesses great potential in improving sensitivity of PEC bioanalysis. Unfortunately, such a possibility has not been found. To verify the hypothesis, on the basis of a Bi2S3/Bi2WO6 heterojunction photoelectrode, we designed the PECCC RCA system consisting of a redox mediator, a proper reductant, as well as the signaling indicator produced from an enzymeassisted immunoassay (see Supporting Information for detailed Experimental Section). Specifically, as shown in Scheme 1, the system operated upon the initial oxidation of ferrocenecarboxylic acid (FcA) as redox mediator by the holes in the Bi2S3/ Bi2WO6 photoelectrode, triggering the RCA processes in which the FcA would be regenerated from the oxidized product FcA+ by AA and the subsequent recovery via reaction between dehydroascorbic acid (DHA) and the TCEP. On other hand, the signal indicator AA was sourced from the enzymatic conversion of ascorbic acid 2-phosphate (AAP) by alkaline phosphatase (ALP) label of a sandwich immuno-

his work presents the concept of redox cycling as an advanced signal amplification route and proof-of-concept toward ultrasensitive photoelectrochemical (PEC) bioanalysis. Unique signal amplification has long been highly pursued in the field due to the need for fast and ultrasensitive bioassays and the trend toward miniaturized assays.1−7 For amplification to be achieved, previous strategies mainly resorted to the use of various functional nanoprobes,8−11 DNA-based amplification techniques,12−17 and enzyme-assisted generation of signaling species.18−22 Although these methods have good sensitivity, they often suffer from laborious procedures, heavy consumption of biomolecules, and thus high economic costs. Therefore, facile, selective, and reproducible signal amplification is still highly appealing toward ultrasensitive PEC bioanalysis. On the basis of repeatedly coupled reduction and oxidation reactions, redox cycling amplification (RCA) represents a useful method for regenerating signal reporters in electrochemical detection.23−25 Recently, we first revealed the feasibility of RCA-based PEC bioanalysis, and a single-cycling chemical RCA scheme was operated upon the oxidation of ascorbic acid (AA) by the photoelectrode and its regeneration by the tris(2-carboxyethyl)phosphine (TCEP).26 Comparatively, electrochemical−chemical−chemical RCA refers to a signaling species redox-cycled by two different reducing (or © XXXX American Chemical Society

Received: August 20, 2018 Accepted: October 9, 2018 Published: October 9, 2018 A

DOI: 10.1021/acs.analchem.8b03798 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

the (200) plane of Bi2WO6,28 and the fringes of d = 0.380 nm agreed with the (101) plane of Bi2S3.29 Elemental mapping results of Figure 1f show that elements of Bi, W, and S were well-distributed, further confirming the formation of the heterojunction. X-ray photoelectron spectroscopy (XPS) analysis of the sample was also performed as shown in Figure S1. Figure 1g displays the X-ray powder diffraction (XRD) results of Bi2WO6, Bi2S3, and Bi2S3/Bi2WO6 samples. The XRD patterns of Bi2WO6 and Bi2S3 could be indexed to the orthorhombic Bi2WO6 (JCPDS No. 39-0256)30 and orthorhombic Bi2S3 (JCPDS No. 17-0320),31 respectively, and the sharp diffraction peaks identified that two crystals were wellcrystallized. The peaks in the pattern of Bi2S3/Bi2WO6 heterojunction stated clearly that the heterojunction consisted of Bi2WO6 and Bi2S3. The Raman spectra of the samples are shown in Figure 1h. The peaks in the range of 600−1000 cm−1 of Bi2WO6 could be assigned to the stretching of the W−O bands.32 Specifically, the band around 700 cm−1 was due to an antisymmetric bridging mode associated with the tungstate chain. The bands at 790 and 820 cm−1 were interpreted as the antisymmetric and symmetric Ag modes of terminal O−W−O. Moreover, the band located at 310 cm−1 was associated with translational modes involving simultaneous motions of Bi3+ and WO66−. Regarding Bi2S3, the peaks at 142, 183, 236, and 256 cm−1 were identical with those of the literature data.33,34 As for the spectrum of the Bi2S3/Bi2WO6 heterojunction, its peaks included all the characteristic bands of Bi2WO6 and Bi2S3, indicating the formation of the heterojunction. The Fourier transform infrared (FT-IR) spectra and UV−vis diffuse reflectance spectra (DRS) of the samples were also recorded as shown in Figure S2. Figure 2a compares the PEC performance of Bi2WO6, Bi2S3, and Bi2S3/Bi2WO6 heterojunctions on the indium tin oxide (ITO) substrates. As seen from curve a, Bi2WO6/ITO had hardly any response due to the fast recombination of electron− hole pairs on the Bi2WO6 surface,35,36 whereas the response of Bi2S3/ITO was more evident (curve b). Significantly, the Bi2S3/Bi2WO6/ITO demonstrated much stronger signal due to the band matching in the heterojunction (curve c). This phenomenon could be explained visually by Scheme 1b. The electron−hole (e−-h+) pairs initially generated by light illumination within both components. Then, the electrons on

Scheme 1. (a) Sandwich Immunorecognition and ALPCatalyzed AA Formation and (b) PECCC RCA on Bi2S3/ Bi2WO6 Photoelectrode

reaction (anti-Myo-SiO2-ALP) in a 96-well plate, and then, the generated AA was transferred into the detection cell containing the FcA and TCEP. Exemplified by myoglobin (Myo) as the analyte, the designed system realized the efficient regeneration of AA and thus signal amplification toward an ultrasensitive split-type PEC immunoassay. This work presented the new concept of PECCC RCA, and we believe it offered a different perspective for the investigation and implementation of innovative signal amplification routes toward ultrasensitive PEC bioanalysis.

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RESULTS AND DISCUSSION Bi2WO6, Bi2S3, and Bi2S3/Bi2WO6 heterojunctions were first prepared and subjected to scanning electron microscopy (SEM) and transmission electron microscope (TEM) characterization. Panels a and b in Figure 1 show that Bi2WO6 possessed nanosheet (NS)-assembled microsphere structure with diameters of ∼3.8 μm and thickness of ∼30 nm, and the Bi2S3 has a nanorod (NR) structure with widths and lengths of 10−37 nm and 20−140 nm, respectively. Figure 1c shows that plenty of Bi2S3 NRs were deposited on the surface of Bi2WO6 NSs. The TEM image in Figure 1d also clearly revealed the successful coupling of Bi2WO6 and Bi2S3. In Figure 1e, the lattice spacing of 0.273 nm could be assigned to

Figure 1. SEM of (a) Bi2WO6, (b) Bi2S3, and (c) Bi2S3/Bi2WO6; (d) TEM, (e) high-resolution TEM, and (f) elemental mapping of Bi2S3/Bi2WO6; (g) XRD patterns and (h) Raman spectra of Bi2S3/Bi2WO6. B

DOI: 10.1021/acs.analchem.8b03798 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 2. (a) PEC tests of Bi2WO6/ITO (black curve), Bi2S3/ITO (red curve), and Bi2S3/Bi2WO6/ITO (blue curve) in PBS solution containing 0.1 M AA, (b) CV on bare ITO electrode, and (c) photocurrents obtained on Bi2S3/Bi2WO6/ITO electrode in different tris buffer solutions (curve a, TCEP; curve b, AA; curve c, FcA; curve d, AA/TCEP; curve e, FcA/AA; curve f, FcA/TECP; curve g, FcA/AA/TCEP). The concentrations of FcA, AA, and TCEP are 0.2, 0.2, and 0.5 mM, respectively. (inset in b) Amplified CV curve of AA measured on the ITO electrode.

higher than that of the single-cycling RCA, which had great potential for improving the sensitivity of the bioanalysis. Incidentally, the influence of the mass ratio of Bi2WO6 to Bi2S3 in the heterojunctions as well as the concentration of Bi2S3/ Bi2WO6 heterojunction on the PEC performance and the effects of the concentrations of FcA and TCEP to PECCC redox cycling were evaluated as shown in Figure S3. Under the optimal conditions, variable concentrations of Myo were assayed by the as-developed PECCC RCA-based sensor. Figure 3a shows that the enhanced signal along with

the conduction band (CB) of Bi2S3 were rapidly transferred to the CB of Bi2WO6 and collected by ITO as current signal, whereas the holes on the valence band (VB) of Bi2WO6 injected to the VB of Bi2S3. Obviously, as compared to the pure Bi2WO6 and Bi2S3, the formation of the heterojunction could accelerate the transfer of photoexcited charge carriers and thus enhance the photocurrent intensity. As shown in Figure 2b, the feasibility of the proposed PECCC RCA strategy was first investigated by the cyclic voltammogram (CV) behaviors of the ITO electrode in different tris buffer solutions. There was no redox peak of TCEP (curve a), and no obvious oxidation peak of AA was observed (curve b and Figure 2b inset), which agreed with the previous report.37 The FcA oxidation occurred at ∼0.540 V (curve c), which clearly indicated that the FcA was more easily oxidized than AA. Moreover, because of the TCEP-enabled AA regeneration from DHA, the presence of TCEP caused the oxidation currents of AA to increase over a broad potential range (curve d). In the FcA/AA system, an obvious oxidation peak occurred at the potential of the FcA oxidation. This is because FcA could mediate the electron transfer between AA and the electrode. In the FcA/TCEP system, the addition of TCEP to the FcA solution did not induce an apparent change in the potential of FcA (curve f), indicating that the oxidized form of FcA was not reduced by TCEP. Significantly, in the FcA/AA/TCEP system (curve g), the anodic peak current further increased compared to that of FcA/AA, suggesting that the presence of TCEP could accelerate the regeneration of AA and thus promote the reaction between FcA and AA. The PEC performance of the heterojunction electrode in different Tris buffer solutions was then evaluated as depicted in Figure 2c. It can be seen that TCEP has no effect on increasing the photocurrent (curve a), whereas both AA (curve b) and FcA (curve c) could obviously enhance the signals, which could be attributed to the easy oxidization of the two substances by the photogenerated holes of the photoelectrode and thus depression of the electron−hole pair recombination. For AA/TCEP system, its response was much higher than that of AA because of the AA regeneration from the DHA (curve d). Similarly, signal enhancement of FcA/AA was caused by the regeneration of FcA from the reaction between FcA+ and AA (curve e). In addition, for the FcA/TCEP system, it was revealed that the FcA could not be regenerated by TCEP (curve f). However, in the FcA/AA/TCEP system, the highest photocurrent was observed, indicating that the coexistence of FcA, AA, and TCEP enabled running of the PECCC RCA (curve h). These results clearly demonstrated that the amplification effect of the double-cycling RCA strategy was

Figure 3. (a) PEC response of the sensor to different target concentrations, (b) as-derived linear curve, (c) selectivity test with HAS, hIgG, cTNI, and Myo concentrations corresponding to 4.0 × 10−2, 1.0 × 10−2, 3.0 × 10−11, and 1.0 × 10−10 g/mL, respectively, and (d) stability test of the electrode in 0.1 M AA electrolyte.

the higher target concentration, which could be attributed to more target causing enhanced generation of AA and thus accelerated regeneration of FcA, promoting PECCC RCA for photocurrent production. Figure 3b shows the corresponding linear curve, which could be described in the equation I = 33.38 + 1.78log C (R2 = 0.998) within the range of 1.0 × 10−13 to 1.0 × 10−7 g/mL with an experimental detection limit of 3.0 × 10−14 g/mL, which was comparable to those of some recent immunosensors as shown in Table S3.38−41 The selectivity was studied by using the interfering proteins, such as human serum albumin (HSA), human IgG (hIgG), cardiac troponin I (cTnI), and their mixture plus the target. Figure 3c shows that the target Myo and the mixture samples yielded much higher C

DOI: 10.1021/acs.analchem.8b03798 Anal. Chem. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS We gratefully appreciate the support from the National Natural Science Foundation of China (21675136, 21675080, and 21874115), Plan for Scientific Innovation Talent of Henan Province (2017JR0016), the Natural Science Foundation of Jiangsu Province (BK20170073), Science & Technology Innovation Talents in Universities of Henan Province (18HASTIT003), Funding Scheme for the Young Backbone Teachers of Higher Education Institutions in Henan Province (2016GGJS-097), and the Nanhu Young Scholar Supporting Program of XYNU.

signals than those of HSA, hIgG, and cTnI, whereas the results of HSA, hIgG, and cTnI were similar to the blank test, indicating good selectivity for Myo measurements. The stability of the electrode was tested in 0.1 M AA. Figure 3d shows no obvious changes in the signal intensity in the repeated measurements within 300 s, demonstrating the steady signal output. The practical application of the sensor was studied by analysis of seven human serum samples with the results in Table S1. The results were consistent with the standard values provided by ROCHE ECL analyzer from the hospital. The relative errors between the two methods were no more than 6.7%. The relative standard deviations (RSDs) of the proposed method were within 5.4%. The recovery of Myo was determined by the standard addition method in real serums. From Table S2, the recoveries were in the range of 80.0− 103.1% with RSDs of less than 6.0%, indicating acceptable accuracy for the detection in real samples.

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CONCLUSIONS This work first investigated the PECCC redox cycling on the Bi2S3/Bi2WO6 photoelectrode as an advanced signal amplification route and thus realized the ultrasensitive split-type PEC immunoassay of Myo. In such a system, the signal indicator AA was generated from the enzyme-assisted immunoassay and then steered into the PECCC redox cycling; there, the photogenerated hole would oxidize the FcA, and the FcA would be regenerated from FcA+ by AA and AA would be regenerated by TCEP. The PECCC RCA, combined with the enzymatic amplification, achieved obvious signal amplification and thus ultrasensitive detection of Myo. In brief, such a concept of PECCC RCA has not been reported, and the resulting immunoanalysis also exhibited good performance. More importantly, the mechanism revealed in this proof-ofconcept offers a new perspective for the future development of ultrasensitive PEC bioanalysis. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03798. Experimental section, XPS spectra, FT-IR spectra, and UV−vis DRS of the samples, CV of AA measured on the glassy carbon electrode, experimental optimization, practical application, and performance comparison (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yan-Ming Liu: 0000-0003-3381-2307 Wei-Wei Zhao: 0000-0002-8179-4775 Author Contributions ⊥

B.W. and L.-P.M contributed equally to this work.

Notes

The authors declare no competing financial interest. D

DOI: 10.1021/acs.analchem.8b03798 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.8b03798 Anal. Chem. XXXX, XXX, XXX−XXX