CuxS platform as advanced signal amplification under anodic bias

Journal Pre-proof Ultrasensitive photoelectrochemical immunosensor for procalcitonin detection with porous nanoarray BiVO4 /Cux S platform as advanced signal amplification under anodic bias Jinhui Feng, Faying Li, Lei Liu, Xuejing Liu, Yanrong Qian, Xiang Ren, Xueying Wang, Qin Wei

PII:

S0925-4005(20)30032-0

DOI:

https://doi.org/10.1016/j.snb.2020.127685

Reference:

SNB 127685

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

13 October 2019

Revised Date:

4 January 2020

Accepted Date:

6 January 2020

Please cite this article as: Feng J, Li F, Liu L, Liu X, Qian Y, Ren X, Wang X, Wei Q, Ultrasensitive photoelectrochemical immunosensor for procalcitonin detection with porous nanoarray BiVO4 /Cux S platform as advanced signal amplification under anodic bias, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127685

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Ultrasensitive photoelectrochemical immunosensor for procalcitonin detection with porous nanoarray BiVO4/CuxS platform as advanced signal amplification under anodic bias

Jinhui Fenga,1, Faying Li a,1, Lei Liua, Xuejing Liua, Yanrong Qiana, Xiang

. Collaborative Innovation Center for Green Chemical Manufacturing

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a

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Rena, Xueying Wanga, Qin Weia,* [email protected]

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and Accurate Detection, Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and

Corresponding author: (Qin Wei);

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*

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Chemical Engineering, University of Jinan, Jinan 250022, PR China

Fax: + 86 531 82767367;

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Tel: + 86 531 82767872;

Highlights  An ECASE-based PEC sandwich-type immunosensor for PCT detection was designed. Page 1 of 35

 The porous nanoarray BiVO4/CuS was prepared as matrix material.  The photocurrent could be significantly enhanced by the ECASEbased PEC strategy.  PS@Ab2 bioconjugate act as labels for enhancing the sensitivity of PEC immunosensor.  The PEC immunosensor exhibited a low detection limit of 17.3 fg

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mL-1.

Abstract

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A new, porous nanoarray BiVO4/CuxS (PN-BiVO4/CuxS) platform as

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an advanced signal amplification strategy under anodic bias on the process of electrochemical catalysis assisted self-enhancing based

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photoelectrochemical (ECASE-based PEC) without electron donors was established for ultrasensitive procalcitonin (PCT) detection. The system

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operated upon that the PN-BiVO4/CuxS electrode could enhance separation of e-/h+ pairs under illumination of visible-light because the energy levels between BiVO4 and CuxS matched well. Afterwards, the photoexcited electrons could shift to working electrode as output signal, while the photoexcited holes could oxidize water under anodic bias to Page 2 of 35

produce hydrogen peroxide (H2O2) that was subsequently reduced by CuxS, which resulted in significantly enhanced photocurrent intensity by electrochemical process. To further enhance the sensitivity of PEC immunosensor, amino-modified polystyrene (PS) nanoparticles act as labels for immobilizing secondary antibodies (Ab2) to form PS@Ab2 conjugates for construction of sandwich-type immunosensor on the basis

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of ECASE-based PEC strategy. The designed PEC immunosensor

exhibited a linear concentration range from 50 fg mL-1 to 100 ng mL-1,

with a low detection limit of 17.3 fg/mL (S/N=3) for PCT, and achieved

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good selectivity, acceptable stability, and high sensitivity. This work first

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conducted the ECASE-based PEC system, and we believed it will provide a new perspective for developing innovative signal amplification toward

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detection of other biomarker in the future.

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Keywords: porous nanoarray BiVO4, ECASE-based PEC, procalcitonin,

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signal amplification, photoelectrochemical immunosensor

1. Introduction The septicemia, as an acute systemic infection, caused by a pathogenic bacteria infection.[1] Accurate detection of disease-related biomarkers is essential in fields of biomedical and diagnostic research. Page 3 of 35

The procalcitonin (PCT), a typical biomarker, has been explored as a reliable diagnosis and prognosis of septicemia.[2,3] Moreover, it has been demonstrated that septicemia can rapidly result in tissue damage, organ dysfunction and death without treatment in time.[4] Therefore, accurately and early detection of PCT plays an important role to the health of human beings.

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Photoelectrochemical (PEC) bioanalysis, have attracted increasing attention because of their outstanding merits with high selectivity and

sensitivity thanks to the reduced background signals by total separation of

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the excitation source and detection signal. Besides, excellent signal

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amplification is essential for fast and ultrasensitive detection in PEC immunoassay which have good stability, easy miniaturization, and low

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manufacturing cost.[5-10] To amplify the response signal, previous

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strategies mainly including photoelectrochemical-chemical-chemical redox cycling route,[11,12] enzyme-assisted generation of signaling

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species, [13,14] and co-sensitization strategy with cascade energy level [15,16] have been adopted. Although these methods have good

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sensitivity, while they often should be subjected to laborious combinations of materials, heavy consumption of electron donor or biomolecules, and unstable sensor signal. Therefore, building a simple, effective, and stable signal amplification strategy is still highly attractive among ultrasensitive PEC immunoassay. Page 4 of 35

Recently, we first demonstrated that porous nanoarray BiVO4/CuxS (PN-BiVO4/CuxS) platform as an advanced signal amplification strategy under anodic bias on the process of electrochemical catalysis assisted self-enhancing based photoelectrochemical (ECASE-based PEC) without electron donors was constructed PEC bioanalysis. Unfortunately, such a process has not been conducted for amplifying the response signal of

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PEC immunoassay.

The BiVO4, an attractive n-type semiconductor with superior intrinsic properties included strong absorption of visible-light and

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moderate band-gap has found wide application in photocatalysis and

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biosensor.[17-19] Importantly, the separation of electron-hole (e-/h+) pairs was enhanced because the lighter of effective masses electrons and holes

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in BiVO4.[20] And, porous nanoarray of BiVO4 with large specific

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surface area and more reactive sites can accelerate electron transfer rate, and further improve signal stability and reproducibility of sensor.[21-23]

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In addition, metals sulfides have been widely used in biosensor field.[24,25] Particularly, CuxS with a band-gap value about 2.0 eV was

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not only used in PEC biosensor due to effectively absorption of visiblelight, but also applied in electrochemical biosensor due to its good reduction property towards H2O2 and significant electron transfer ability.[26-28] Therefore, the CuxS combined with PN-BiVO4 synthetic nano hetero-structures was used in this work. The nano hetero-structures Page 5 of 35

with excellent charge transfer rate, effective cost, good chemical stability have been adopted in electrocatalytic, photocatalytic, biosensor, and solar cell widely. [29-34] Specifically, based on PN-BiVO4/CuxS platform, the signal amplification derived from ECASE-based PEC system operated upon two process: (1) the separation rate of e-/h+ pairs was enhanced by PN-BiVO4/CuxS electrode under illumination of visible-light, due to the

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energy levels between BiVO4 and CuxS matching well;[35] (2)

photoexcited electrons as output signal could shift to working electrode

while photoexcited holes could oxidize water, producing reactive species

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(H2O2) under an anodic bias. Then, the hole-drived H2O2 was reduced by

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CuxS to achieve signal amplification by effectively inhibiting recombination of e-/h+ pairs and excellent performance of electrochemical

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properties.

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To further enhance the sensitivity of PEC immunosensor, the sandwich-type PEC immunosensor was employed in this work. The

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mono-dispersed polystyrene (PS) nanoparticles with uniform size, good stability, and high surface reaction capability have been widely applied in

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biosensor.[36] In this work, amino-modified PS nanoparticles act as labels for immobilizing secondary antibodies (Ab2) to form PS@Ab2 conjugates. It is demonstrated that PS@Ab2 conjugates could facilitate the decrease of photocurrent intensity, due to its insulation property and steric hindrance, which contributed to an excellent sensitivity for PEC Page 6 of 35

immunosensor. In this work, the designed ECASE-based PEC sandwichtype immunosensor achieved excellent signal amplification and further increased the sensitivity of PCT detection. This work revealed the novel strategy of ECASE-based PEC, which offered a new perspective for developing innovative signal amplification toward ultrasensitive PEC

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biosensors in the future.

2. Experimental

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2.1 Materials and Apparatus

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Supporting Information (SI).

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All the materials and apparatus used were provided in the

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2.2 Synthesis of BiVO4/CuxS

The BiVO4 was synthesized by electrodeposition procedure to form

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precursor of BiOI then annealed.[37] The detailed offered in SI. CuxS was deposited on PN-BiVO4 via a SILAR process. To acquire this, the Cu

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precursor was gained by mixed solution of CuSO4 (1 mmol, 8 mL) and Na2S2O3 (6 mmol, 40 mL) in ultrapure water. Then, the ITO/PN-BiVO4 electrode was immersed into above mixed solution for 20 s, followed by immersion in Na2S·9H2O (0.1 mol/mL) for 40 s. Finally, the PNBiVO4/CuxS electrode was obtained after washing with ultrapure water. Page 7 of 35

2.3 Preparation of PS@Ab2 bioconjugate Amino-modified PS nanospheres 360 μL (d = 250 nm) was thoroughly washed with ultrapure water by centrifugation. Then, 600 μL glutaraldehyde was added and oscillated at room temperature. After washing, 1 mL Ab2 (10 μg/mL) was added and incubated for overnight at

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4 °C under continuous shaking. Then, 500 uL of BSA solution (1%) was added and incubated for 60 min. After centrifugation and washing, the

obtained PS@Ab2 bioconjugate was added into 3 mL PBS of pH 7.4 for

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dispersion and then stored at 4 °C for further using.

2.4 Fabrication PEC sandwich-type immunosensor

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Scheme 1

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The PEC sandwich-type immunosensor was showed in Scheme 1. In the first, the ITO/PN-BiVO4/CuxS electrode formed by deposition of

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CuxS on the PN-BiVO4 via SILAR process. Then, 5 μL of chitosan and 5 μL glutaraldehyde 2.5% (v/v) were added on the ITO/PN-BiVO4/CuxS

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electrode. After cleaning, 10 μL of Ab1 was dropped to achieve amino crosslinking.[38,39] After that, 4 μL of 1% BSA was dropped on ITO/PNBiVO4/CuxS/Ab1 electrode to block nonspecific binding sites. Then, 10 μL of PCT with various concentrations were added above electrode incubating at 4 °C for 60 min. Finally, 10 μL of PS@Ab2 bioconjugate Page 8 of 35

was coated on the ITO/PN-BiVO4/CuxS/Ab1/BSA/PCT electrode for 60 min at 4 °C. Above process of each step were washed with buffer solution. Then, the designed PEC sandwich-type immunosensor was fabricated successfully.

2.5 PEC detection

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The PEC tests were performed in 0.1 mol L-1 PBS (pH 7.4). The

current-time plots recorded in a home-built PEC system based traditional three-electrode system: ITO electrode as the working electrode, a

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3. Results and discussion

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electrode as the reference electrode.

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platinum wire as the auxiliary electrode, and a saturated calomel

3.1 Characterization of PN-BiVO4/CuxS photoelectrode and PS

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Figure 1

BiOI, PN-BiVO4, CuxS and PN-BiVO4/CuxS were characterized by

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SEM and HR-TEM images in Fig. 1. The BiOI exhibited a 2D thin plate structure with a thickness about 1.224 µm (Fig. 1A). And, PN-BiVO4 with irregular clavate exhibited a thickness about 1.346 µm on the surface of ITO in Fig. 1B. As shown in Fig. 1C, the morphology of CuxS was irregular spherical. Fig. 1D revealed that a mass of CuxS nanoparticles Page 9 of 35

were dispersed on the surface of PN-BiVO4 electrode by deposition. To make it clear, HR-TEM image in Fig. 1E also exhibited the successful coupling of PN-BiVO4 with CuxS. And inset showed two different lattice spacing of 0.310 nm and 0.322 nm, assigning to the (1 2 1) facet of PNBiVO4 and (1 1 0) facet of CuxS, respectively.[40] Elemental mapping image in Fig. 1G presented that elements of V, O, Bi, Cu, and S were

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uniform distribution, further suggesting the successful formation of PNBiVO4/CuxS.

The XRD patterns included pure BiOI, PN-BiVO4, CuxS, and PN-

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BiVO4/CuxS were shown in Fig. 1F. The main diffraction peaks of pure

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BiOI can be indexed to the tetragonal phase of BiOI (JCPDS No. 100445).[41] For bare PN-BiVO4, the peaks at 29.3°, 31.4° and 35.2° can

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respond to diffractions from (1 2 1), (0 4 0) and (0 0 2) planes of the scheelite-monoclinic BiVO4 (JCPDS No. 14-0688).[37] The major

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diffraction peaks at 2ө values around 32.1° and 48.3° can be attributed to

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the (1 0 3) and (1 1 0) plane of the hexagonal CuxS (JCPDS No. 060464).[40] The peaks in the blue curve proved that PN-BiVO4/CuxS

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prepared successfully.

X-ray photoelectron spectroscopy (XPS) was used to identify the

chemical states of the PN-BiVO4/CuxS. As shown in Fig. S1A, the elements of V, O, Bi, S and Cu exhibited PN-BiVO4/CuxS spectrum. The peaks of V 2p at 524.3 and 516.6 eV can be ascribed to the typical V 2p Page 10 of 35

1/2

and V 2P 3/2, referring to V5+ ions in the crystal lattice (Fig.

S1B).[42,43] The peaks located at 535.3 and 531.2 eV for O 1s in Fig. S1C.[44] As shown in Fig. S1D, the peaks of Bi 4f 5/2 and Bi 4f 7/2 are located at 167.5 and 158.3 eV, respectively, which revealed to Bi3+ ions in the crystal lattice. And the S 2p band consists of two peaks, 163.2 eV for S22- (Cu2S and CuS) and 161.6 eV for S2-.[37] The Cu 2p regions peaks at

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Cu 2p 1/2 and Cu 2p 3/2 are situated at 951.3 and 931.7 eV in Fig. S1E,

respectively. In addition, there are several weak satellite peaks, which are

the evidence of divalent Cu-species.[40] The result demonstrated that PN-

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BiVO4/CuxS was synthesized successfully.

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Fig. S2A showed that the morphology of PS is micro-sphere with uniform size about 200 nm. The FT-IR spectrum of amino-modified PS

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nanospheres was presented in Fig. S2B, the peaks at 698.2 cm-1, 755.1

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cm-1 are characteristic bending-vibration absorption of C-H bond and peaks at 1452.3 cm-1 and 1492.8 cm-1 attributed to stretching modes C - C

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bond on benzene ring.[45] The peak at 1541.0 cm-1 and 1730.1 cm-1 corresponded to the phenyl ring scaffold containing C = C and C = O

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stretching modes, respectively. The absorption peaks at 3026.2 cm-1 and 3455.0 cm-1 related to C-H stretching vibration in olefins and - NH2 antistretching vibration, respectively.[46] Therefore, the FT-IR spectrum confirmed the success amination of PS. Figure 2 Page 11 of 35

Fig. 2A compared the PEC performance of PN-BiVO4, and PNBiVO4/CuxS with no bias and anodic bias (0.8 V). The photocurrent intensity of PN-BiVO4 with anodic bias (curve a', 0.8 V) increased evident comparing without bias (curve a, 0 V) under visible-light illumination. The anodic bias could increase the efficient separation rate of photoexcited e-/h+ pairs because holes could oxidize water to produce

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H2O2. The photocurrent intensity of PN-BiVO4/CuxS with anodic bias

(curve b', 0.8 V) increased to about 7.2 times higher comparing without bias (curve b, 0 V) under visible-light illumination, owing to good

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reduction of CuxS toward hole-drived H2O2. Thus, the anodic bias is an

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essential factor for the process of ECASE-based PEC. Fig. 2B compared CV performance of PN-BiVO4, and PN-BiVO4/CuxS under dark and

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visible-light. There was no redox peak of PN-BiVO4 under dark (curve a)

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and a little peak under visible-light illumination (curve a'). However, after deposition of CuxS, a significant reduction peak of PN-BiVO4/CuxS

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(curve b) was obtained under dark, however further increased reduction peak of PN-BiVO4/CuxS (curve b') was acquired under visible-light.

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Hence, the light also is an essential factor for the process of ECASEbased PEC biosensor. The PEC performance of the PN-BiVO4/CuxS electrode in PBS and in PBS with additional H2O2 (0.1 mol/L) were evaluated in Fig. 2C under 0.8 V anodic bias and visible-light illumination. The photocurrent Page 12 of 35

intensity of PN-BiVO4 in PBS (curve a') was higher comparing in PBS + H2O2 (curve a), which because additional H2O2 may hinder the separation of e-/h+ pairs. The photocurrent intensity of PN-BiVO4/CuxS in PBS (curve b') increased significantly comparing in PBS + H2O2 (curve b) because the additional H2O2 would impede the generation of hole-drived H2O2 based on chemical equilibrium. Moreover, the baseline of curve b

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was not zero when turn off the light and the photocurrent was higher than baseline at the beginning (Fig. S3), which confirmed that CuxS has good reduction toward additional H2O2. Therefore, we choose the PBS as

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electrolyte solution in this work to reduce interference from additional

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H2O2.

Based on the above analysis, the possible electron-transfer

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mechanism of ECASE-based PEC was on the following process details in

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Fig. 2D. Under visible-light, the photo-generated electrons jumped to conduction band (CB) of CuxS and the electrons in CB of CuxS also

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shifted to the BiVO4, then electrons transferred to the ITO electrode. While the photo-generated holes could oxidize water, producing H2O2

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under an anodic bias. The consumption of holes would facilitate the transfer of electrons from CB of CuxS to BiVO4 then to ITO. Importantly, signal amplification was achieved by the good reduction towards holedrived H2O2 of CuxS, which further increased photocurrent by excellent performance of electrochemical properties. Therefore, the photocurrent Page 13 of 35

response of the immunosensor enhanced evidently.

3.2 Characterization of PEC sandwich-type immunosensor Figure 3 Electrochemical impedance spectroscopy (EIS) was used to characterize the modified electrodes. The electron-transfer resistance (Ret)

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equals the semicircle diameter, which represents redox probe at the

electrode interface by electron transfer kinetics. Inset in Fig. 3A illustrates the equivalent circuit, which includes four elements. The data in Table S1

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was that the fitted value was simulated by ZSimpWin software. The

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experimental curves in the Nyquist plots in a solution containing 0.1 mol/L KCl and 2.5 mmol/L Fe(CN)63-/4 from 0.1 to 105 Hz were shown in

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Fig. 3A, the bare ITO electrode presented a little Ret (curve a). After

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adding PN-BiVO4 and CuxS on the bare ITO electrode, the Ret (curve b, c) increased evidently. When the Ab1, BSA, PCT modified on ITO/PN-

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BiVO4/CuxS electrode, the Ret (curve d, e, f) increased obviously. Finally, when PS@Ab2 was added on the above modified electrode, the Ret (curve

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g) further increased. The results testified that the sandwich-type immunosensor was fabricated successfully. Cyclic voltammogram (CV) was also applied to study stepwise of prepared immunosensor. As shown in Fig. 3B, the scanning potential in 5 mmol/L K3[Fe(CN)6] for CV was monitored from -0.2 V to 0.6 V. The Page 14 of 35

bare ITO has good redox peaks (curve a), while decorating PN-BiVO4 (curve b) and CuxS (curve c) the redox peak current decreased. Subsequently, the redox peaks current further decreased, when the electrode were incubated Ab1, BSA, PCT, PS@Ab2 (curve d, e, f, g), respectively, which electron transfer is likely to blocking by biological substances. These results also confirmed that the ECASE-based PEC

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sandwich-type immunosensor were successfully constructed as expected.

3.3 Optimization of conditions for sandwich-type immunosensor

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To obtain best sensing performances, experiment conditions of

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reaction time of Na2S·9H2O for growth CuxS and bias for ECASE-based PEC process were optimized in Fig. S4. The maximum photocurrent

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intensity was 40 s in Fig. S4A, because the more reaction time the thicker

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CuxS will inhibit electron transfer. Therefore, the optimal time for growth CuxS was 40 s. The maximum photocurrent intensity was at 0.8 V anodic

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bias (Fig. S4B), due to appropriate bias could increase e-/h+ pairs separation. Hence, 0.8 V anodic bias was chosen as the ideal bias in this

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study.

3.4 Analysis of PEC sandwich-type immunosensor for PCT Figure 4 Under the optimal conditions, various concentrations of PCT were Page 15 of 35

assayed by the fabricated ECASE-based PEC sandwich-type immunosensor based on ITO/PN-BiVO4/CuxS electrode. As shown in Fig. 4A, the photocurrent was linearly related to the logarithm of PCT concentration. The linear regression equation is: I (μA) = 62.918 – 3.531 log (cPCT, ng mL-1), with correlation coefficient of 0.996. Corresponding PEC intensity curves were presented in Fig. 4B, with increasing of PCT

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concentration from 50 fg mL-1 to 100 ng mL-1, more PS@Ab2

bioconjugate increased steric hindrances, which resulted in decreasing

photocurrent intensity. The detection limit was 17.3 fg mL-1 that lowered

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than other previous works in Table S2, which attributed to the effect of

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ECASE-based PEC.

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3.5 Stability and selectivity of PEC sandwich-type immunosensor The performance of fabricated immunosensor could be affected by

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stability of the photocurrent intensity. As exhibited in Fig. 4C, there was no significant change of photocurrent in the repeating 12 cycles for 280 s

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(incubating of PCT with 1.0 ng mL-1), declaring a remarkable

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photocurrent stability of prepared immunosensor. Moreover, the longtime stability also influenced the performance of fabricated immunosensor. The photocurrent of prepared immunosensor (Fig. S6) maintained 95.8%, 93.6% and 91.7% of its initial photocurrent after storing in a refrigerator at 4 °C for 2 weeks, 4 weeks and 6 weeks, Page 16 of 35

respectively, proving good long-time stability. The selectivity was conducted by using interfering substances of brain natriuretic peptides (BNP), prostate-specific antigen (PSA), squamous cell carcinoma antigen (SCCA) in Fig. 4D. No distinct interferential photocurrent signals were acquired when interfering substances were added on blank electrode or PCT, indicating the selectivity of the fabricated ECASE-based PEC

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sandwich-type immunosensor was satisfactory for PCT detection.

3.6 Sample analysis in human serum

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Table 1

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The accuracy and feasibility of the PEC sandwich-type immunosensor for real sample analysis in human serum was investigated

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by standard addition methods. The human serum obtained from local

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hospital was diluted 50 times and detected to be 0.75 ng mL-1 using the proposed immunosensor. The recoveries with 97.4 - 102.2% and the RSD

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with 1.2 - 3.5% were exhibited in Table 1, indicating that the fabricated ECASE-based PEC sandwich-type immunosensor has enough accurate

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and sensitive for detecting PCT in human serum.

4 Conclusion

In summary, we first presented a new ECASE-based PEC system on Page 17 of 35

the PN-BiVO4/CuxS photoelectrode as an advanced signal amplification to achieve ultrasensitive sandwich-type PEC immunoassay for PCT detection. The effective separation of e-/h+ pairs was acquired on the PNBiVO4 photoelectrode under illumination of visible-light. And then signal amplification was obtained by good reduction toward hole-drived H2O2 of CuxS, which increased photocurrent intensity by excellent performance of

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electrochemical property. Moreover, the PN-BiVO4 with porous

nanoarray provided a high surface area and stable sensing platform to gain the stability of the signal. The PS@Ab2 conjugates as label

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constructed sandwich-type immunosensor for ultrasensitive detection of

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PCT. More importantly, such a concept of ECASE-based PEC has not been reported, and good performance of fabricated immunosensor

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provides a new strategy for development of detection other biomarker in

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the future PEC biosensors.

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Declaration of interests

The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

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Acknowledgments This work was supported by the National Key Scientific Instrument and Equipment Development Project of China (No.21627809); National Natural Science Foundation of China (Nos.21575050, 21777056,

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21505051), Jinan Scientific Research Leader Workshop Project (2018

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GXRC024).

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Page 27 of 35

Author Biographies Jinhui Feng studies in school of chemistry and chemical engineering, University of Jinan as doctoral student.

Faying Li studies in school of chemistry and chemical engineering,

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Université du Québec as doctoral student.

Lei Liu received Ph.D. degree from China university of Geosciences

(Beijing). Now, she is a post-doctoral at University of Jinan. Her main

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catalysis of functional nanomaterials.

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research interests are electrocatalysis, photocatalysis and heterogeneous

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Xuejing Liu received Ph.D. degree from University of Chinese Academy

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of Sciences. Now, she is a post-doctoral at University of Jinan. Her main

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research interest is stoichiometric calculation.

Yanrong Qian studies in school of chemistry and chemical engineering,

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University of Jinan as postgraduate student.

Xiang Ren Ph.D. degree from University of Jinan. He is an associate professor at University of Jinan. His main research interests are the determination of electrochemical immunosensor. Page 28 of 35

Xueying Wang a professor and received the Ph.D. degree from Wuhan University. She is a lecturer University of Jinan. She dedicates to the surfactant and biological macromolecules interaction.

Qin Wei, a professor and DSc, has devoted herself to analytical teaching and scientific research. Her main research interests are the determination

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of protein and nucleic acid by photometry and the electrochemical

immunosensor preparation. She has published over two hundred articles on analysis, immunosensor and applied successfully for many research

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projects, such as Biomaterials, Advanced Functional Materials.,

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Biosensors & Bioelectronics, Sensors and Actuators B-Chemical and

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na

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ACS Applied Materials & Interfaces.

Page 29 of 35

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Figure Captions

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Scheme 1. Schematic of construction proposed ECASE-based PEC

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ur

na

lP

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sandwich-type immunosensor.

Page 30 of 35

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Fig. 1. SEM images of (A) BiOI electrode (inset: side-view of the BiOI

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electrode), (B) PN-BiVO4 electrode (inset: side-view of the PN-BiVO4 electrode), (C) CuxS nanoparticles, (D) PN-BiVO4/CuxS electrode (inset: SEM magnification of the PN-BiVO4/CuxS), (E) HR-TEM image of PNBiVO4/CuxS, (F) XRD dates of BiOI, PN-BiVO4, CuxS and PNBiVO4/CuxS. (G) Elemental mapping image of PN-BiVO4/CuxS. Page 31 of 35

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Fig. 2. (A) Photocurrent response under light illumination with no bias (curve a, b) and 0.8 V anodic bias (curve a', b'), (B) CV response under

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dark (curve a, b) and light illumination (curve a', b'), (C) Photocurrent

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response under light illumination and 0.8 V anodic bias in PBS + H2O2 electrolyte solution (curve a, b) and PBS electrolyte solution (curve a', b'),

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a and a': ITO/PN-BiVO4 electrode, b and b': ITO/PN- BiVO4/CuxS electrode, PBS (0.1 mol L-1 KH2PO4 and 0.1 mol L-1 Na2HPO4, pH 7.4),

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(D) Electron-transfer mechanism of ECASE-based PEC immunosensor in PBS.

Page 32 of 35

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Fig. 3. (A) EIS plots and (B) CV curves of (a) bare ITO electrode, (b) ITO/PN-BiVO4 electrode, (c) ITO/PN-BiVO4/CuxS electrode, (d)

ITO/PN-BiVO4/CuxS/Ab1 electrode, (e) ITO/PN-BiVO4/CuxS/Ab1/BSA

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electrode, (f) ITO/PN-BiVO4/CuxS/Ab1/BSA/PCT electrode, (g) ITO/PNBiVO4/CuxS/Ab1/BSA/PCT/PS@Ab2 electrode (the EIS plots was

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recorded in a solution containing 0.1 mol/L KCl and 2.5 mmol/L

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Fe(CN)63-/4 from 0.1 to 105 Hz and CV curves was monitored by the

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scanning potential from -0.2 V to 0.6 V in 5 mmol/L K3[Fe(CN)6]).

Page 33 of 35

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Fig. 4. (A) The curve of I corresponding to the concentrations of PCT, (Y

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= I (μA), x = cPCT), (B) Photocurrent response to different concentrations

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of PCT based on PEC sandwich-type immunosensor (from a to j: 50 fg mL-1, 100 fg mL-1, 500 fg mL-1, 1 pg mL-1, 10 pg mL-1, 100 pg mL-1, 500

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pg mL-1, 1 ng mL-1, 10 ng mL-1, 100 ng mL-1), (C) Stability evaluation of the PEC sandwich-type immunosensor, (D) Selectivity of PEC sandwich-

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type immunosensor: blank, BNP, PSA, SCCA, PCT, PCT + 100 ng mL-1 BNP, PCT + 100 ng mL-1 PSA, PCT + 100 ng mL-1 SCCA, (cPCT = 1.0 ng mL-1, error bars=SD (n=5)).

Page 34 of 35

Table 1. Analytical application of PCT in serum sample based on ECASE-based PEC sandwich-type immunosensor Spiked

concentration -1

(ng mL )

Found

concentration -1

RSD

Recovery

(n=5, %)

(%)

2.3

97.4

3.5

101.2

1.4

99.4

1.2

102.2

concentration -1

(ng mL )

(ng mL )

0.01

0.74

0.1

0.86

1

1.74

10

10.98

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Samples

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lP

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0.75

Page 35 of 35