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A Potentiometric Addressable Photoelectrochemical Biosensor for Sensitive Detection of Two Biomarkers Shupei Zhang, Hong Dai, Zhensheng Hong, and Yanyu Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02101 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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Analytical Chemistry

A Potentiometric Addressable Photoelectrochemical Biosensor for Sensitive Detection of Two Biomarkers Hong Dai a* , Shupei Zhang a, Zhensheng Hong b, Yanyu Lin c 1

a College of Chemistry and Chemical Engineering, Fujian Normal University, Fuzhou 350108, P. R. China b College of Physics and Energy, Fujian Normal University, Fuzhou 350108, P. R. China c Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, and Department of Chemistry, Fuzhou University, Fuzhou 350002, P. R. China ABSTRACT: It is a great challenge to fabricate multiplex and convenient photoelectrochemical biosensors for ultrasensitive determination of biomarkers. Herein, a fascinating potentiometric addressable photoelectrochemical biosensor was reported for double biomarkers detection by varying the applied bias in the detection process. In this biosensor, the nanocomposite of cube anatase TiO2 mesocrystals and polyamidoamine dendrimers modified dual disk electrode as excellent photoelectrochemical sensing matrix. Subsequently, two important biomarkers in serum for prostate cancer, prostate specific antigen and human interleukin-6, were immobilized onto the different disks of modified electrode via glutaraldehyde bridges. Then another two photosensitizers, graphitic carbon nitride and CS-AgI labeled different antibodies were self-assembled onto electrode surface by corresponding competitive immune recognition reaction. The photocurrents changed with target antigen concentration at different critical voltages enables us to selectively and quantitatively determine targets. The results demonstrated that this potentiometric addressable photoelectrochemical biosensing strategy not only owned great promise as a new point-of-care diagnostic tool for early detection of prostate cancer but also can be conveniently expanded to multiplex biosensing by simply change biomarkers. More importantly, this work provides an unambiguous operating guideline of multiplex photoelectrochemical immunoassay.

KEYWORDS： potentiometric addressable, photoelectrochemical biosensor, double biomarkers

1

Corresponding author, Phone/fax: (+86)-591-22866135. E-mail: [email protected]

ACS Paragon Plus Environment

(H. Dai);

Analytical Chemistry INTRODUCTION

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The immunoassay of tumor markers has been demonstrating its significance in the application of cancer detection, prevention, and therapy.1-2 However, the detection of single biomarker is often not efficient for the diagnostic purpose because of its limited specificity.2 Therefore, multiplex immunoassay with noteworthy advantages including high sample throughput, low sample consumption, short assay time, improved assay efficiency, and low cost, has recently gained considerable attention to meet the increasing demand for diagnostic application.2-3 So far, many different methods, such as electrochemical,4 electrochemiluminescent,2 fluorescent5, chemiluminescent6 and photoelectrochemical (PEC)7 technique, have been developed for the multiplex detection of biomarkers. Among these strategies, PEC analysis with low undesired background noise and high sensitivity holds significant promise for multiplex detection because that the photocurrent heavily depends on the photoactive material,8 wavelength of excitation light,9 the applied potential in the detection process.10 However, most PEC biosensors were fabricated based on different photoactive species for the determination of signal target.11-14 Recently, Yu’s group prepared the multiplex PEC immunosensor for three tumor markers determination by using electrode arrays.15 Later, Hu’s group established a lightaddressable PEC biosensor for the multiplexed detection of DNA sequences.7 Heretofore, there are few reports about the multiplex PEC detection. Therefore, it is significance to develop PEC biosensor of multianalyte detection. In the PEC process, photocurrent was produced when the photogenerated electrons or holes in semiconductor transferred to electrode and then to the external electronic circuit, which the photocurrent value and polarity can be changed by mediating the applied bias.10 Inspired by this, it can be deduced that there must be a certain bias voltage in the external electronic circuit which make semiconductor produce a zero photocurrent, which we named this certain bias voltage as critical voltage because that the photocurrent polarity will change when the critical voltage was exceeded. Namely, different photoactive materials possessed different critical voltages. Therefore, when two different photoactive materials were modified onto the same working electrode with different sections, the photocurrent of one photoactive material at its critical voltage will not influenced the photocurrent of the other photoactive material and the reverse is also true. The above inference provides a possibility for us to discriminate the source of photocurrent between different photoactive materials based on the applied bias, which we named this method potentiometric addressable technology. Obviously, photoactive material was the important element in the potentiometric addressable technology. However, when the two photocurrents of photoactive materials possess same photocurrent polarity at 0 V, the photocurrent value of one photoactive material will be reduced at the critical voltages of the other photoactive material. To obtain the high sensitivity of biosensor, two different photoactive materials with different photocurrent polarities at 0 V applied potential is necessary. Herein, graphitic carbon nitrides (g-C3N4) with anodic photocurrent and CS-AgI with cathodic photocurrent were selected by experiment investigations. In the two photoactive materials, g-C3N4 has received many attentions as a metal-free photocatalyst in PEC

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sensing application due to its good chemical stability, low cost, easily-synthesized, appealing electronic structure with a medium-bandgap.16-17 And CS-AgI as a visible light active material demonstrated good phtocatalytic property in the photodegradation of organic pollutants.18 In order to further improve the photocurrent of this biosensor, cube anatase TiO2 mesocrystals (CAM) with proper band structure was introduced. Due to the well match of energy level between CAM and g-C3N4, CS-AgI, the photocurrents of CAM/g-C3N4 and CAM/CS-AgI were enhanced comparing to the bare g-C3N4 and CS-AgI. More importantly, the photocurrent polarities of g-C3N4 and CS-AgI were not changed after the introduction of CAM. Hereinto, the high crystallinity and subunit alignment of TiO2 mesocrystals significantly reduce crystal defects and grain boundaries, and the periodically hierarchical structures enhance the lightmatter interaction.19 Additionally, the high porosity of mesocrystals leads to improved light-scattering ability and provides abundant surface areas for effective interfacial charge collection and fast electrolyte diffusion.20 Furthermore, the mesopores in TiO2 mesocrystals making selective incorporation of various guest materials inside their frameworks and pore walls.21-22 To facilitate the subsequent immobility of the antigen or antibody in PEC immune biosensing, polyamidoamine dendrimers (PAAD)@CAM nanocomposite prepared by the high porosity of CAM was used as good PEC sensing matrix. The amino groups in PAAD could be used as hole acceptors to reduce the surface defects of semiconductors (the photogenerated electrons would be captured by surface detects) reported by previous researches, which dramatically enhanced the PEC performance of CAM.23-24 Besides, the amine groups in PAAD provided the convenience for the subsequent immobilization of antigen in this sensing. Herein, two important biomarker, prostate specific antigen (PSA) and human interleukin-6 (IL-6), were immobilized onto different disks of PAAD@CAM modified electrode through a classic glutaraldehyde (GLD) coupling reaction. And then gC3N4 and CS-AgI labeled different antibodies were selfassembled onto corresponding disks of the dual disk electrode for different antigens by competitive immune recognition reaction, which was elaborated in Scheme 1. Exhilaratingly, the critical voltage of PAAD@CAM/g-C3N4 and PAAD@CAM/CS-AgI was -0.135 V and 0.12 V, respectively, and the immune recognition process didn’t change the critical voltages. When the applied bias was set to 0.12 V, the photocurrent was totally produced by the PAAD@CAM/gC3N4 in the immune recognition reaction of prostate-speciﬁc antigen (PSA). Similarly, the human Interleukin-6 (IL-6) concentration can be detected under -0.135 V applied bias and it was completely not affected by the PSA immune recognition process. Accordingly, this biosensor fabricated on one individual electrode can be used to detect PSA and IL-6 by controlling the external applied bias.


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Scheme 1 The Schematic illustration of the fabricate process of potentiometric addressable photoelectrochemical immunosensor and detect process of double biomarkers.

EXPERIMENTAL SECTION Materials and Reagents. Sodium dodecyl sulphate (SDS), HCl, Melamine, silver nitrate (AgNO3) and KI were purchased from Sinopham Chemical Reagent Co. (Shanghai, China). Titanium (IV) isopropoxide (TIP) and chitosan (CS) were received from Sigma (St. Louis, MO, USA). Glutaraldehyde (GLD, 25% aqueous solution) was obtained from Shanghai Jinshan Tingxin Chemical Plant (China). Bovine serum albumin (BSA, 96–99%) were purchased from Biss Inc. (Beijing, China). IL-6, IL-6 antibody (1 mg mL-1), PSA, PSA antibody (1.9 mg mL-1), α-fetoprotein antigen (AFP), and carcinoembryonic antigen (CEA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). The phosphate buffer solution (PBS, 0.1 M) as the supporting electrolyte was prepared by mixing stock solution of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 and adjusting the pH. Other reagents were of analytical regent grade. The water used for the preparation of the solution was purified using a Water purifier (China) purification system. Apparatus. The morphology and structural characterization were observed by Scanning electron microscopy (SEM, S8010instrument) and Transmission electron microscopy (TEM, FEI F20 S-TWIN instrument). X-ray diffraction (XRD) pattern was carried on a Rigaku X-ray diffractometer using Cu Kα (λ = 1.5418 Å) radiation. The PEC measurements were performed with a homemade PEC system. And all the experiments were measured on a CHI 760 electrochemical workstation (Shanghai Chenhua Instrument Co., China) with a three-electrode cell. A modified dual-disk glassy carbon electrode (3 mm in diameter), a platinum wire and An Ag/AgCl electrode (sat. KCl) were used as working electrode, counter electrode and reference electrode respectively. The excitation source of homogeneous light (420 nm) was filtered from xenon lamp (86 nmW cm-2, Beijing, China) by monochromator before using (Scheme S1).

Synthesis of CAM, g-C3N4 nanosheets, CS-AgI nanoparticles. A typical experiment procedure for the preparation of CAM was follows25: 3 g SDS was introduced into 100 mL of 2 M HCl solution and stirred for a few minutes. Afterward, 3 mL of TIP was added into the above suspension and kept at 80 °C with stirring for 48 h. Finally, the resulting products were dried at 60 °C for 12 h after washed thoroughly with distilled water, and then calcined for 30 min at 400 °C for later use. g-C3N4 nanosheets were synthesized by a similarly approach in previous report.26 Briefly, 10.0 g of white melamine powder was heated at 600 °C for 4 h in a tube furnace under open air condition and the primrose yellow product can be obtained after it was cooled to room temperature. And then the 10 mg mL-1 stock solution was prepared by dispersing 30 mg of g-C3N4 nanosheets into 3 mL redistilled water with the aid of ultrasonication for 8 h. CS-AgI nanoparticles were obtained by a facial precipitation method.27 Firstly, 30 mL of 0.5 % (m/v) CS solution was mixed with 30 mL of 0.1 M AgNO3 aqueous solution with stirring for 0.5 h. Successively, 30 mL of 0.15 M KI aqueous solution were introduced with stirring for 3 h and then the dispersion containing CS-AgI nanoparticles were collected for the future use. Preparation of PAAD@CAM nanocomposite, CS-g-C3N4, CS-g-C3N4 labeled PSA antibody (Ab1@g-C3N4) and CSAgI labeled IL-6 antibody (Ab2@CS-AgI). Briefly, 100 µL of 0.5% PAAD solution was added into 100 µL of 6 mg mL-1 CAM solution and sonicated for 3 h. Subsequently, the resulting solution was centrifuged at 3000 rpm for 10 min to obtain the precipitate (PAAD@CAM nanocomposite) and then added distilled water to make final volume achieve 200 µL for subsequent application. CS-g-C3N4 were prepared by mixing 100 µL of 0.5% CS solution with 1 mL of 3 mg mL-1 g-C3N4 solution with the aid of ultrasonication for 48 h. Afterwards, the resulting solution was centrifuged at 6000 rpm for 20 min to obtain the precipitate. Finally, distilled water was added into the precipitate to make final volume achieve 1 mL (which defined as 3 mg mL-1) for subsequent application. The preparation process of Ab1@g-C3N4 was as follows. Firstly, 120 µL of 0.3 mg mL-1 CS-g-C3N4 was mixed with 20 µL of anti-PSA solution (pH 7.4 PBS). Then 30 µL of 0.5% GLD solution as coupling agent was added into the above solution. Subsequently, the free antibodies were removed after the solution being stirred for 3 h at 37 °C by centrifugation at 6000 rpm, and then the bioconjugates were washed with PBS (pH 7.4) for several times to obtain anti-PSA modified CS-gC3N4. Following that, 1.0 wt% BSA solution was added to block possible remaining active sites, and then the resulting Ab1@g-C3N4 were washed with PBS and redispersed in PBS. The preparation of Ab2@CS-AgI was similar to the above process except that CS-g-C3N4 and anti-PSA were replaced with CS-AgI and anti-IL-6, respectively. Fabrication of the PAPEC biosensor. A dual-disk glassy carbon electrode (DDCE) was polished carefully with 0.3 and 0.05 µm alumina slurry on chamois leather to produce a mirror-like surface, then washed ultrasonically with anhydrous alcohol and distilled water and dried in air before use. With a micropipette, 4 µL of PAAD@CAM solution was firstly dropped onto diskⅠof the fresh prepared DDCE surface and



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then the same volume of PAAD@CAM solution was also dropped onto disk Ⅱ of the DDCE, and dried at room temperature, which named as PAAD@CAM. To immobilize the PSA and IL-6 on modified DDCE, PAAD@CAM was firstly immersed into 100 µL of 5 % GLD solution for 50 min. Subsequently, 20 µL of 1 ng mL-1 PSA solution and 20 µL of 1 ng mL-1 IL-6 solution were placed onto the disk Ⅱ and disk Ⅱ of modified DDCE for 45 min, respectively, which the electrode named as PAAD@CAM/Ag. Afterwards, the PAAD@CAM/Ag was soaked into 100 µL of 1% BSA solution for 50 min to block the remaining active groups. Subsequently, the above obtain electrode was dipped into 50 µL of mixed solution including 25 µL of PSA and 25 µL of IL-6. And then 25 µL of Ab1@g-C3N4 and 25 µL of Ab2@CSAgI solution were immediately dropped into the above mixed solution incubating for 45 min, which the PAPEC biosensor (PAAD@CAM/Ag/Ab) was constructed. Between each step in this self-assemble process, the electrode was washed thoroughly with distilled water to remove the non-specificity absorption. RESULTS AND DISCUSSION Characterizations of CAM. To explore the phase structure and crystallite size of synthesized products, the XRD pattern was firstly investigated in Figure S1. The narrow sharp diffraction peaks at 2θ =25.3°, 37.8°, and 48.1° can be indexed to the (101), (004), and (200) planes of tetragonal anatase TiO2 (JCPDS 73-1764), implying that high crystalline and only pure anatase TiO2 formed in the samples. By using the Scherer equation, the average crystallite size was calculated to be about 11 nm based on (101) diffraction peak. Furthermore, the morphology and structure of the TiO2 sample were scrutinized by typical SEM, TEM, HRTEM and FFT patterns in Figure 1. As exhibited in Figure 1A, largescale monodisperse nanoparticles with the size of 40-60 nm were obtained. Besides, the distinct rough surface and porous nature of the TiO2 nanoparticles could be clearly observed in Figure 1B. Further investigation was carried out by TEM to reveal the porous structure of such anatase TiO2 nanoparticles in Figure 1C, which can be verified by the result of BarrettJoyner-Halenda pore distribution (2.7 nm), demonstrating the very uniform mesopores can be obtained in this product. The typical TEM pattern of an individual nanoparticle in Figure 1D depicted that the TiO2 nanoparticle with cube-like morphology was constructed from tiny nanoparticle subunits. The characteristic property of the prepared TiO2 forecasted its large surface area, which agreed well with the Brunauer– Emmett–Teller surface area of TiO2 (199 m2 g-1). In addition, the HRTEM image inserted in Figure 1D demonstrated a clear lattice fringe with a spacing of 0.35 nm well correspond to the spacing of the (101) planes of the anatase structure. As depicted in the light inset of Figure 1D, the FFT image behaved as single-crystal-like diffraction, implying the building of nanoparticle subunits were highly crystalline. From aforementioned phenomena, it is obvious that the

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prepared TiO2 sample not only owns high crystallinity but also possesses high porosity, which predicated the good PEC performance.

Figure 1 SEM (A, B) and TEM (C, D) images of CAM. The insets in D are related the HRTEM and FFT patterns from the whole nanoparticle.

Excellent PEC performance of PAAD@CAM. Although CAM possessed good photoelectrochemical property (Figure S2 and S3), the using of CAM as photoactive material in PEC sensing has not attracted attention. To open the door to PEC biosensing using CAM as a good matrix, PAAD with 64 primary amines was absorbed into CAM by virtue of large surface area and high porosity of mesocrystals. Exhilaratingly, the obtained PAAD@CAM nanocomposite (Figure S4) depicted enhanced PEC performance compared with pure CAM (Figure 2). To evaluate the better PEC response of PAAD@CAM nanocomposite, a series of PEC experiments were carried out in 0.1 M PBS (pH 7.0). As exhibited in Figure 2A, a distinct photocurrent density enhancement was observed on the PAAD@CAM nanocomposite film under the chopped on/off illumination. Additionally, the open-circuit voltage (Voc) changes of CAM and PAAD@CAM were examined in Figure 2B. When the light was on, a sharp dropped photovoltage was observed because of the accumulation of photogenerated electrons in the semiconductor films under the light illumination.28 After cut off the illumination, the accumulated electrons were slowly scavenged by the redox species in the electrolyte which lead to slowly increase of Voc.29 Compared to CAM, the significant decrease in the Voc for PAAD@CAM predicted that more electrons were accumulation in nanocomposite film, which indicated more charge separation or less charge recombination for the nanocomposite thin film.


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Figure 2 (A) Applied potential bias-dependent photocurrent densities, (B)open-circuit voltage response, (C) M-S plots and (D) IPCE spectra of CAM (a) and PAAD@CAM (b) in 0.1 M PBS (pH 7.0).

Therefore, the bias voltage applied in external circuit may be changed the value or even the polarity of photocurrent. To confirm the above inferences, the photocurrent responses of PAAD@CAM/g-C3N4 and PAAD@CAM/CS-AgI under different bias voltages were investigated in Figure 3. An anodic photocurrent was yielded on PAAD@CAM/g-C3N4 modified electrode and a cathodic photocurrent was generated on PAAD@CAM/CS-AgI modified electrode without the assistance of bias voltage (the data were not demonstrated). By adjusting the bias voltage, the photocurrent of PAAD@CAM/CS-AgI almost completely disappeared under 0.12 V applied bias, but PAAD@CAM/g-C3N4 exhibited a great anodic photocurrent (Figure 3A). Similarly, although PAAD@CAM/CS-AgI displayed a great photocurrent response under -0.135 V applied bias, PAAD@CAM/g-C3N4 didn’t showed obvious photocurrent (Figure 3B). Excitingly, such the phenomenon certified our presumption that the photocurrent of photoactive materials can be controlled by mediating the applied bias, which afforded a key precondition for the development of the potentiometric addressable PEC sensor.

In order to investigate the carrier concentration in semiconductor, Mott-Schottky curve were displayed in Figure 2C. Obviously, in the linear parts of the curves, the slopes of the tangent lines were all positive, which indicated that the CAM and PAAD@CAM were n-type semiconductors. The Mott-Schottky plots were obtained using the following equation (1): 30

C -2 =

2 ( E -E

FB

-

kT e

)

εε 0eN D (1) Where ε, ε0 , e, ND refers to the relative dielectric constant of the semiconductor (60 in the case of TiO2), the vacuum permittivity (8.85×10-14 F cm-1), the unique charge (1.602×1019 C) and the apparent electron donor density (cm3), respectively. The ND of CAM and PAAD@CAM were calculated to be 1.56×1016 F cm-3 and 1.66×1016 F cm-3, respectively. Therefore, the incorporation of PAAD into CAM can contribute to the increase of electron density. Furthermore, IPCE as a better parameter to inspect the photocurrent density under monochromatic light excitation was recorded in Figure 2C. IPCE at every wavelength was calculated according to the equation (2):28 1240 × j

IPCE % =

λ× P

× 100%

(2) where j refers to the measured photocurrent density at the specific measurement wavelength in mA cm-2, λ refers to the wavelength of the incident light in nm, and P refers to the power density of measured irradiance in mW cm-2. As expected, the PAAD@CAM nanocomposite displayed higher IPCE values at the wavelength range from 280nm to 460 nm than that of CAM film. Accordingly, a conclusion can be drawn according to the aforementioned findings that PAAD can enhance the PEC performance of CAM, which may be resulted from that the amine groups in PAAD worked as hole scavengers to reduce the surface defects of TiO2 mesocrystals. Feasibility evaluation of potentiometric addressable strategy. In the photoelectrochemical process, electron-hole pairs can be produced in photoactive materials under light illumination and then the electrons or holes transferred to the electrode, which led to the generation of photocurrent.

Figure 3 Photocurrent densities of PAAD@CAM/g-C3N4 (a) and PAAD@CAM/CS-AgI (b) under 0.12 V (A) and -0.135 V (B) applied bias.

Successful construction of the PAPEC biosensor. As a proof-of-concept application, a PAPEC biosensor for PSA and IL-6 detection was firstly developed. The stepwise construct process of this PAPEC biosensor was characterized by electrochemical impedance spectroscopy (EIS), an effective strategy for monitoring the changes in the assembly process, in Figure 4A. Compared to DDCE (curve a), the semicircle of PAAD@CAM modified electrode (curve c) was apparently decreased, revealing the good electrical conductivity of PAAD@CAM composite. Subsequently, the semicircle continuously enlarged after further modification with Ag (PSA and IL-6, curve d), BSA (curve e), and photoactive materials (g-C3N4 and AgI) labeled Ab1 (anti-PSA and anti-IL-6, curve f), indicating the successful fabrication of this biosensor. To evaluate the feasibility of this PAPEC biosensor for the PSA and IL-6 detection, the photocurrent densities of different modified electrodes were recorded. As demonstrated in Figure 4B, there was a negligible photocurrent response on DDCE (curve a), but an obvious photocurrent can be observed on CAM modified electrode (curve b). Compared with curve b, PAAD@CAM modified electrode (curve c) exhibited great enhanced photocurrent because of that PAAD can eliminated the surface defect of CAM, implying the PAAD@CAM nanocomposite can be employed as a good photoactive material for PEC sensing application. Then the photocurrent density subsequently decreased after further immobilized Ag (curve d) and BSA (curve e), resulting from the elevated hindrance effect of these proteins. After photoactive materials labeled antibodies were assembled onto the modified electrode, the anodic photocurrent changed to cathodic



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photocurrent (curve f), illustrating the successful fabrication of this PAPEC biosensor. Besides, the photocurrent responses of this PAPEC biosensor at 0.12 V (curve g) and -0.135V (curve h) displayed in Figure 4B further prove the successful recognition between Ag and photoactive material labeled Ab.

Figure 4 EIS spectra (A) and PEC responses (B) of different modified electrodes. (a) DDCE, (b) CAM, (c) PAAD@CAM, (d) PAAD@CAM electrode after PAAD@CAM/Ag, (e) PAAD@CAM/Ag/BSA, (f) PAAD@CAM/Ag/Ab at 0 V, PAAD@CAM/Ag/Ab at 0.12 V (g) and -0.135 V (h). The concentration of PSA and IL-6 was 10-4 ng mL-1 and 1 pg mL-1. The inset in Figure A is the equivalent circuit.

Feasibility evaluation of this PAPEC biosensor for double biomarkers detection on an individual dual-disk electrode. To validate the feasibility of this PAPEC biosensor for PSA and IL-6 simultaneous detection, the effects of different IL-6 concentration to PSA detection and different PSA concentration to IL-6 detection were investigated. As shown in Figure 5A, different concentrations of IL-6 displayed negligible photocurrent change to the PSA detection at 0.12 V, which the photocurrent value was well accordance with that of blank experiment (curve e, disk Ⅱwas used to fabricate the PAPEC biosensor for PSA detection and disk Ⅱ was not modified). Similarly, the presence of variable concentrations of PSA did not change the photocurrent response of IL-6 at 0.135V (Figure 5B), which the photocurrent value was well accordance with that of blank experiment (curve e). The results not only demonstrated that the cross-reactivity between PSA antibody, IL-6 antibody and their noncognate antigens was negligible but also certified that the amount of photoactive component at its critical voltage would not affected the photocurrent response of other photoactive component. Accordingly, this biosensor can be used to simultaneous detect PSA and IL-6 by potentiometric addressable strategy in this PEC measurement.

Figure 5 (A) Photocurrent densities of this PAPEC biosensor for PSA (1 pg mL-1) detection in the presence of different IL-6 concentrations (from curve a to curve e were 1, 0.1, 0.01, 0.001, 0 pg mL-1). (B) Photocurrent densities of this PAPEC biosensor for IL-6 (0.1 pg mL-1) detection in the presence of different PSA concentrations (from curve a to curve e were 1, 0.1, 0.01, 0.001, 0 pg mL-1). Analytical performance. Under the optimum conditions (Figure S8-S10), the photocurrent density of this PAPEC

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biosensor for both IL-6 and PSA all increased with decreasing concentrations of targets (Figure 6). Besides, the photocurrent densities were proportional to logarithmic value of targets concentrations with the liner range from 10-5 to 90 pg mL-1 for IL-6 and 10-6 to 90 ng mL-1 for PSA, respectively. Compared with previous single-analyte immunosensor, the proposed PAPEC biosensor demonstrated lower detection limit for both analytes (Table S1). To evaluate the specificity of the biosensor, the measurements were undergone by incubating the target IL-6 and PSA against the other interferences in Figure S11. Obviously, although the existence of a large excess of the interferences, the photocurrent signal changes were negligible compared with the blank test, indicating the PEC biosensor possessed good discrimination ability.

Figure 6 The photocurrent responses of the PAPEC biosensor towards different concentrations of IL-6 (A) and PSA (C). The corresponding calibration curves of IL-6 (B) and PSA (D).

As depicted in Figure S12, the photocurrent densities of both IL-6 and PSA were high stable without obvious descended under continuous light on and off, implying the good chemical and structural stability of this biosensor. After storing at 4 °C in darkness for 15 days, there was no evident photocurrent response change, validating the developed biosensor owned good storage stability. Furthermore, six electrodes were prepared for the detection of IL-6 (0.1 pg mL1 ) and PSA (1 pg mL-1) to explore the reproducibility of this biosensor. The relative standard deviation (RSD) was 1.8 % for IL-6 and 2.3 % for PSA, suggesting its precision and reproducibility. To evaluate the applicability of this fabricated biosensor for IL-6 and PSA in real samples, six 10-fold diluted serum samples from prostate cancer patients (samples 1-3) and prostate cancer-free patients (samples 4-6) were investigated and compared with the results from single-target ELISA. As exhibited in Figure S13 and S14, there was no significant difference in PSA and IL-6 values between the two methods, which demonstrated that the fabricated PAPEC biosensor owned great potential as a reliable strategy for the detection of PSA and IL-6 in serum sample. CONCLUSIONS Taking the advantage of the potentiometric addressable strategy, a robust PEC immunosensor was firstly constructed to selectively and quantitatively detect PSA and IL-6 on a dual


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disk electrode by control the applied voltage. The proposed biosensor demonstrated satisfactory performances including wide linear range, low detection limit and good selectively. Besides, this ultrasensitive biosensor was successful applied to IL-6 and PSA determination in human serum. Furthermore, the utilization of PAAD not only provided a new strategy for the anodic photocurrent enhancement of semiconductor but also enabled the biosensor suitable for the detection of other biomarkers. Moreover, the employment of g-C3N4 and AgI expand the photoresponse in visible light region for avoiding the destructive effect of UV light to biomolecules. Unquestionably, the successful application of this potentiometric addressable technology in the dual-analyte PEC detection will pave a new channel for fabricating high throughput PEC analytical devices.

ASSOCIATED CONTENT Supporting Information. XRD patterns of CAM; Excellent PEC performance of CAM; FT-IR spectra of CAM, PAAD@CAM, CS, g-C3N4, CS-g-C3N4, AgI, AgI-CS and CS ; Parameter optimization; Stability tests of the PEC biosensor; Figures of merits of comparable methods for determination of PSA and IL-6. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone/fax: +86-591-83713866. E-mail: [email protected] (H. Dai) ACKNOWLEDGMENT This project was financially supported by the NSFC (21205016, 21575024), National Science Foundation of Fujian Province (2016J06003, 2016J05026), Education Department of Fujian Province (JA14071, JB14036, JA13068) and Foundation of Fuzhou Science and Technology Bureau (2015-S-160, 2015-G72) and New Century Talent Project of Fujian Province.

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