Double Photosystems-Based 'Z-Scheme' Photoelectrochemical

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Double Photosystems-Based 'Z-Scheme' Photoelectrochemical Sensing Mode for Ultrasensitive Detection of Disease Biomarker Accompanying 3D DNA Walker Shuzhen Lv, Kangyao Zhang, Yongyi Zeng, and Dianping Tang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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Analytical Chemistry

Double Photosystems-Based 'Z-Scheme' Photoelectrochemical Sensing Mode for Ultrasensitive Detection of Disease Biomarker Accompanying 3D DNA Walker Shuzhen Lv,† Kangyao Zhang,† Yongyi Zeng,‡ and Dianping Tang*,†

†

Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian Province), State Key Laboratory

of Photocatalysis on Energy and Environment, Department of Chemistry, Fuzhou University, Fuzhou 350116, People’s Republic of China ‡

Liver Disease Center, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: A new double photosystems-based 'Z-scheme' photoelectrochemical (PEC) sensing platform is designed for ultrasensitive detection of prostate-specific antigen (PSA) by coupling with three-dimensional (3D) DNA walker. Two photosystems consist of CdS quantum dots (photosystem I; PS I) and BiVO4 photoactive materials (photosystem II; PS II), whereas gold nanoparticles (AuNPs) photodeposited on high-active {010} facets of BiVO4 are used as the electron mediators to promote electron transfer from conduction band (CB) of PS II to valence band (VB) of PS I. 3D DNA walker-based amplification strategy is carried out between hairpin DNA1 conjugated onto the AuNP, hairpin DNA2 labeled with CdS quantum dot (QD-H2) and DNA walker complementary with the PSA aptamer modified to magnetic bead (Apt-MB). Upon addition of target, DNA walker strand is displaced from DNA walker/Apt-MB to open hairpin DNA1 on AuNP@BiVO4. In the presence of QD-H2, DNA walker induces the hybridization of DNA1 with DNA2 on the gold nanoparticles step by step, thereby resulting in the assembly of CdS QDs on the AuNP@BiVO4 to form 'Z-scheme' double photosystems with strong photocurrent. Under optimum conditions, the 'Z-scheme' PEC sensing system exhibits good photocurrent responses toward target PSA within the working range of 0.01 – 50 ng mL-1 at a low detection limit of 1.5 pg mL-1. Good reproducibility and accuracy are acquired for analysis of target PSA and human serum specimens relative to commercial PSA ELISA kit. Importantly, our strategy provides a new horizon for photoelectrochemical in vitro diagnostics.

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The photosynthesis of green plants occurs via a 'Z'-type electron transfer process to create enough energy for the water-splitting, which is known as the 'Z-scheme'.1-3 'Z-scheme' systems inspired by the light reaction of green plants with 'Z'-type photo-induced electron-transfer chain between two photosystems effectively depress electron-hole recombination and improve the photocurrent density. Specifically, photosystem I (PS I) and photosystem II (PS II) can collect the light energy by their reaction centers (P700 and P680 respectively) and pump electron to the excitation state.4,5 The exited electrons generated from P680 reaction center of photosystems II move to photosystems I by a series of electron transfer processes and the produced holes oxidize water into oxygen and protons.6,7 Moreover, the photo-induced electrons of P700 reaction center inside photosystems I reduce nicotinamide adenine dinucleotide phosphate.8 Significantly, the charge-separation quantum efficiency of these processes is close to 100%, which innovates an extremely attractive strategy for development of photoelectrochemical detection.9 The photoelectrochemical assay, a versatile sensing technique for various analysts, has emerged in recent years, with the advantages over the total separation of excitation source and the detection signal, featuring fast response time, the low background and good portability.10,11 However, the sensitivity is limited to the light-response electrode materials with high electron-hole recombination. Therefore, constructing an artificial 'Z-scheme' system for the photoelectrochemical detection is a good choice to dramatically depress the electron-hole recombination and evidently improve detectable sensitivity because of the high charge-separation quantum efficiency. In the artificial 'Z-scheme' systems, the excited electrons transfer from the conduction band (CB) of PS II to the valence band (VB) of PS I though the electron mediators at the interface between two photosystems.12,13 In this regard, the electron mediators are critical to effectively boost photogenerated electron transfer.14 The IO3-/I- and Fe3+/Fe2+ redox couples, the most commonly employed electron mediators, are not easily applicable for photoelectrochemical detection because their interference with the results could not ensure both accuracy and stability.15 Recently, in place of ionic mediators, solid electron mediators (e.g., reduced graphene oxide, nano gold and nano silver) have been found to effectively serve as the low-resistance electron pathways for two photosystems.16-18 Reduced graphene oxide was used as an efficient electron mediator for metal sulfides and CoOx-loaded BiVO4 in 'Z-scheme' systems for the water-splitting.19 Moreover, the all-solid-state CdS-Au-TiO2 visible-light 'Z-scheme' system exhibited high photocatalytic activity 3



when gold nanoparticles (as the electron mediator) transferred the photo-induced electron from TiO2 to CdS.20 These works provide good operation of the solid-state electron mediators in artificial 'Z-scheme' systems for the development of photoelectrochemical biosensing protocols. DNA-based molecular machines (e.g., DNA tweezers, DNA robots and DNA walkers) inspired by the intracellular protein motors have attracted extensive attention because of their potential applications in material assembly, drug delivery and various biosensor.21 In particular, DNA walkers can be controlled to convert chemical energy to precise mechanical motion along a designated path on the nanoscale though the well-defined Watson-Crick base pairing rule.22,23 Compared with one-dimensional (1D) DNA footpaths and 2D DNA origami, 3D DNA tracks guiding the movement of DNA walkers possess powerful DNA enrichment capacity for the merit of signal amplification.24 The movements in 3D DNA tracks are acquired by the fuel strand-assisted partial base-pairing, dissociation and branch migration reactions with various conformational changes.25 Li and his co-workers described a stepwise walking scheme by using a nicking endonucleases to propel 3D walker operation on the surface of gold nanoparticle (AuNP).26 The Li's group has addressed the challenge that the motion of DNA walker propelled by a specific endonuclease and designed an enzyme-free DNA walker driven by a single catalytic or double catalytic DNA moving on the surface of functionalized magnetic beads for the detection of DNA and the enzymatic activity of T4 polynucleotide kinase.27 Due to highly local DNA concentration for the signal readout, 3D DNA walker has been demonstrated as a good choice for efficient signal amplification of interest. In this work, we aim to construct an all-solid-state artificial 'Z-scheme' system for the design of photoelectrochemical (PEC) biosensing platform inspired by the light reaction of green plants with 3D DNA walkers for the signal amplification (Scheme 1). In the all-solid-state artificial 'Z-scheme' system, bismuth vanadate (BiVO4) with the narrow band gap of 2.43 eV for visible light absorption replaces the role of P680 as the PS II, serving as an electron provider for PS I. Gold nanoparticle (AuNP; an efficient electron mediator) is selectively photodeposited on the {010} facets of BiVO4 to promote the photogenerated electron flow, which is functionalized with thiolated hairpin DNA1 (H1) for the 3D track for DNA walker. Initially, the aptamers of prostate-specific antigen (PSA; a classical cancer biomarker) are covalently conjugated to magnetic bead (MB), and the aptamers are then partially complementary with catalytic strands of DNA walkers. Upon addition of target PSA, the catalytic strand of DNA walker is displaced from magnetic bead to open the conjugated hairpin 4


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DNA1 on AuNP. In this case, the motion of the DNA walkers is triggered through the addition of CdS quantum dot-labeled hairpin (fuel) DNA2 (QD-H2). Accompanying the repeatedly progression of DNA walkers on AuNPs (H1 opening ↔ displacement with H2 ↔ walker releasing), numerous CdS QDs are conjugated onto the surface of gold nanoparticles for the formation of the 'Z-scheme' double photosystems. The carried CdS QDs are used as photosystem I in place of P700 of the green plant photosynthesis for the signal amplification. During PEC measurement, the excited electrons transfer from the conduction band of BiVO4 to the valence band of CdS QD through the electron mediator (AuNP) at the interface between two photosystems, thereby resulting in the increasing photocurrent because of the separation efficiency of the photogenerated carriers evidently improved.

Scheme 1. Schematic illustration of photoelectrochemical (PEC) sensing platform for prostate-specific antigen (PSA) by coupling with 'Z-scheme' double photosystems and three-dimensional (3D) DNA walker amplification strategy: (A) Target PSA-induced release of DNA walker strand from the PSA aptamer-conjugated magnetic bead (Apt-MB) (note: DNA walker strand was partially complementary with the aptamer); (B) DNA walker-triggered walking reaction on hairpin DNA1 (H1)-functionalized AuNPs@BiVO4 with the aid of CdS quantum dot-labeled hairpin DNA2 (QD-H2); and (C) The photogenerated electron transfer between BiVO4 photosystem II (PS II) and CdS QD photosystem I (PS I) in the artificial 'Z-scheme' system.

EXPERIMENTAL SECTION Fabrication of 'Z-Scheme' Double Photosystem Sensing Platform. Prior to fabrication of the photoelectrochemical sensing platform, PSA aptamer-conjugated magnetic bead (Apt-MB) and CdS 5



quantum dot-labeled hairpin DNA2 (QD-H2) were synthesized via a typical carbodiimide coupling, whereas AuNPs-deposited BiVO4 nanohybrids were prepared by coupling hydrothermal synthesis28 with the photo-reduction method29 (Please see the detailed process in the Supporting Information). To construct target-triggered release of DNA walker from magnetic beads, DNA walker/Apt-MB was synthesized in pH 7.5 TNaKT buffer by adding DNA walker (15 µL, 50 µM) into the prepared Apt-MB (300 µL, 25 mg mL-1). After incubation for 2 h at 37 °C with slight shaking on a rotator, the resulting mixture was magnetically separated by an external magnet to remove the unconjugated DNA walkers. During this process, DNA walker partially hybridized with the labeled PSA aptamer on magnetic beads. The collected DNA walker/Apt-MB was used for the subsequent detection of target PSA in pH 7.5 TNaKT buffer. Next, AuNPs@BiVO4 nanohybrid-coated fluorine-doped tin oxide (FTO) electrode was prepared as follows. Prior to modification, FTO electrode was cleaned by sonication in acetone and ultrapure water. Thereafter, the cleaned FTO electrode was stuck by the waterproof transparent tape with a hole (r = 2.5 mm) to obtain an active area of 19.625 mm2. Following that, the above-prepared AuNPs@BiVO4 aqueous suspension (30 µL, 10 mg mL-1) was thrown on the active surface of FTO electrode, and dried at room temperature. After being washed with ultrapure water, the resulting FTO electrode was immersed into pH 7.5 TNaKT buffer containing hairpin DNA1, and incubated for 2 h at room temperature. During this process, hairpin DNA1 (H1) was assembled onto the AuNPs@BiVO4 by the Au-S bond. The unbound active sites on gold nanoparticles were blocked by incubation with MCH for 60 min at room temperature. Finally, hairpin DNA1-conjugated AuNPs@BiVO4/FTO (H1/AuNPs@BiVO4/FTO) photosensitive electrode was used for progression of 3D DNA walker. Photoelectrochemical Measurement Based on 3D DNA Walker-Based Double Photosystems. Scheme 1 represents schematic illustration of the PEC sensing system toward target PSA based on 'Z-scheme' double photosystems accompanying 3D DNA walker amplification strategy. The assay was carried out as follows. Initially, PSA standard or human serum sample (30 µL) was added in pH 7.5 TNaKT buffer containing 25 mg mL-1 DNA walker/Apt-MB (30 µL), and incubated for 80 min at 37 °C in order to release the DNA walker strand. Following that, the resulting suspension was magnetically separated by an external magnet, and the supernatant was transferred into a 1.5-mL centrifugal tube loading with QD-H2 (30 µL, 10 mg mL-1). After being gently mixed, the 6


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H1/AuNPs@BiVO4/FTO electrode was dipped into the solution, and reacted for 150 min at 37 °C. During this process, DNA walker strand readily executed the walker-displacement reaction step by step on the surface of AuNPs@BiVO4 by the interaction of hairpin DNA1 with DNA walker and QD-H2. Subsequently, the photocurrent of the resulting electrode was measured in the detection cell (Please see the process for photoelectrochemical measurement in the Supporting Information). RESULTS AND DISCUSSION Construction of 'Z-Scheme' PEC Sensing Platform with 3D DNA Walker. In this system, the photocurrent is enhanced through the formation of 'Z-Scheme' system on the AuNPs@BiVO4 with the introduction of CdS QDs. DNA walker/PSA aptamer-conjugation magnetic beads can be used for the rapid separation and purification upon addition of target PSA. The assay mainly consists of magneto-controlled reaction system with DNA walker, hairpin DNA1-modified AuNPs@BiVO4 and QD-labeled hairpin DNA2. DNA walker strand is immobilized on magnetic bead by partial hybridization

with

PSA

aptamer.

Before

formation

of

'Z-Scheme'

system,

H1/AuNPs@BiVO4-modified FTO electrode displays a relatively low background photocurrent. In the presence of target PSA, the analyte reacts with the labeled aptamer on magnetic bead, and releases DNA walker strand. After that, the walker strand opens hairpin DNA1 immobilized on the AuNPs@BiVO4 nanohybrids.

Upon QD-H2

introduction, hairpin

DNA2 executes the

strand-displacement reaction between hairpin DNA1/DNA walker, thereby resulting in the release of DNA walker again to trigger the next-step reaction one by one. Similar with human-walking, numerous QD-H2 conjugates are assembled onto the surface of AuNPs@BiVO4 nanohybrids to form the 'Z-Scheme' double photosystems. CdS QDs are used as photosystem I (PS I), whereas BiVO4 photoactive materials are utilized as photosystem II (PS II). Gold nanoparticles (AuNPs) photodeposited on the high-active {010} facets of BiVO4 are utilized as the electron mediators to promote electron transfer from the conduction band (CB) of PS II to the valence band (VB) of PS I. 3D DNA walker-based amplification strategy is carried out between hairpin DNA1, hairpin DNA2 and walker strand, accompanying formation of 'Z-Scheme' CdS/AuNPs@BiVO4 double photosystems (PS I@AuNP@PS II). In this case, the photocurrent of 'Z-Scheme' double photosystems heavily enhances relative to that of AuNPs@BiVO4 nanohybrids. 7



Figure 1. (A) HRTEM image of CdS QDs (inset: The corresponding HRTEM image); (B) SEM image of BiVO4; (C) FESEM image of AuNPs-functionalized BiVO4; (D) XRD patterns of (a) BiVO4 and (b) AuNPs@BiVO4; (E) XPS spectra of AuNPs@BiVO4 [note: High-resolution XPS spectra of (inset top) Bi element, and (inset bottom) V and O elements]; and (F) High-resolution XPS spectra of Au element.

To realize our design, the all-solid-state artificial 'Z-scheme' system was firstly constructed by the photodepositing gold nanoparticles on semiconductor BiVO4 to form a linker between PS II (BiVO4) and PS I (CdS QD). CdS QDs hydrothermally grown for 120 min at 100 °C were characterized by the high-solution transmission electron microscope (HRTEM; H-7650, Hitachi, Japan). As shown in Figure 1A, the as-prepared CdS QDs capped with 3-mercaptopropionic acid exhibited an average diameter of 3.5 nm, and the lattice planes of quantum dots could be obviously observed (Figure 1A, inset). The corresponding fluorescence spectra with a fluorescence emission at 620 nm (λex = 385 nm) is shown in Figure S1 of the Support Information. The as-synthesized BiVO4 obtained by the hydrothermal procedure was imaged by the scanning electron microscopy (SEM; Hitachi 4800, Japan). As shown in Figure 1B, BiVO4 with the well-developed crystal facets possessed a decahedral shape and the size varied from 1.5 µm to 2.0 µm. The top and bottom square facets of BiVO4 were corresponded to be {010} facets, which was favorable for accumulation of the photogenerated electrons. Moreover, the isosceles trapezoidal sides were the hole-accumulated facet {110}. In this regard, AuNPs@BiVO4 nanohybrids could be prepared via a photo-reduction method 8


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since gold nanoparticles would be reduced by the photo-generated electrons on the {010} facets of BiVO4. Figure 1C displays high-resolution field emission scanning electron microscope (FESEM) of the as-synthesized AuNPs@BiVO4 nanohybrids. As seen from Figure 1C, many nanoparticles were coated on the {010} facets of BiVO4, indicating that gold nanoparticles could be selectively photo-reduced on the electron-accumulated {010} facets. Moreover, the crystallographic structure of AuNPs@BiVO4 nanohybrids at different stages were characterized by powder X-ray diffraction (XRD) in Finger 1D. The XRD pattern of the as-prepared pure BiVO4 (Finger 1D, carve 'a') was well-matched with JCPDS No. 14-0688, indicating that the sample was crystalline with single monoclinic scheelite structure.30 Additionally, the diffraction peaks at 44.3° and 38.2° (Finger 1D, carve 'b') assigned to the (200) and (111) plane of Au according to the JCPDS No. 01-089-3697 could confirmed the existence of Au phase in the AuNPs@BiVO4 nanohybrids.31 Furthermore, the chemical component and chemical status of the samples were analyzed by X-ray photoelectron spectroscopy (XPS) spectrum. Finger 1E shows the XPS survey spectrum of the AuNPs@BiVO4 nanohybrids with the elements of Bi, V, O, and Au. The photoelectron peaks appeared at binding energies of 516.8, 524.4, and 530.0 eV were attributed to V 2p3/2, V 2p1/2 and O 1s bands (Figure 1E, insets). Obviously, the characteristic peaks located at 159.1 and 164.3 eV corresponded to Bi 4f7/2 and Bi 4f5/2, indicating the Bi3+ in AuNPs@BiVO4 nanohybrids (Figure 1E, inset). Meanwhile, the high-resolution spectrum of Au element (Figure 1F) with peaks at 84.0 eV (Au 4f7/2) and 89 eV (Au 4f5/2) confirmed the existence of metallic gold on the surface of the BiVO4.32 Feasibility Evaluation of DNA Walker-Based 'Z-Scheme' Double-Photosystem PEC Sensing Platform. Based on the advantages that the photo-generated electrons flow from PS II to PS I bridged by AuNP electron mediator to depress the electron-hole recombination, the all-solid-state artificial 'Z-scheme' system was firstly developed in PEC sensing platform. As a proof-of-principle, CdS QDs (playing the role of the PS I as well as the signal reporter) were introduced by the process of DNA walker into this system. To trigger the process of DNA walker, the formation of the binding peptide linkages between CdS QDs and H2 were of great importance, which were characterized by fourier-transform infrared (FTIR) spectroscopy. Figure 2A shows FTIR spectra of CdS QDs before (carve 'a') and after (carve 'b') modification with aminated hairpin DNA2. Two strong characteristic absorption peaks at 1394 and 1550 cm-1 were assigned to the symmetric and antisymmetric stretches of carboxyl groups, suggesting the existence of carboxyl groups on the surface of CdS QDs.33 Three 9



features at 1650, 1560 and 1300 cm-1 located in the zone consistent with the amide bond (-CONH-), respectively, corresponding to the stretching vibrations of C=O (amide I), N-H (amide II), and C-N (amide III).34 By the same token, the carboxylated magnetic beads before and after conjugation with PSA aptamer were characterized by using FTIR spectroscopy (Figure S2). The peaks at 1656 and 3190 cm

-1

(carve 'a') were -C=O and -OH stretching vibrations of carboxylate group, respectively,

with the bending vibration of -OH located at 938 cm -1. By the characteristic absorption peaks of amide I at 1660 cm-1, amide II at 1540 cm−1 and amide III vibration at 1278 cm-1, conjugation of the carboxylated magnetic beads with PSA aptamer could be confirmed. Also, gel electrophoresis was used to investigate the DNA walker progression (Figure 2B). All the sequences of oligonucleotides were designed referring to the literature.35,27 As shown in Figure 2B, hairpin DNA1 could be opened through the hybridization reaction with DNA walker when reaction DNA walker strand with the equal-amount hairpin DNA1. The formation of DNA walker/hairpin DNA1 duplex (lane 'd') in this process could be proved by the fact that the migration of the band (lane 'd') was slower than that of DNA walker (lane 'a') or hairpin DNA (lane 'b'). Afterwards, the opened hairpin DNA1 exposed a new long single-stranded DNA region, which could interact with hairpin DNA2, triggering branch migration and forming hairpins DNA1/DNA2 duplex (lane 'e'). Compared with DNA walker/hairpin DNA1 complex (lane 'd'), the formed hairpins DNA1/DNA2 duplex migrated slowly (lane 'e') since the base number of hairpins DNA1/DNA2 was more than that of DNA walker/hairpin DNA1. As control test, the incubation of hairpin DNA1 with hairpin DNA2 in the absence of DNA walker was investigated by gel electrophoresis. As seen from lane 'f', two bands for hairpin DNA1 and hairpin DNA2 could be obviously observed, and no complex was achieved. These results indicated that the progression of DNA walker could be readily implemented in the presence of hairpin DNA1 and hairpin DNA2 with the assistance of DNA walker strand. On the basis of these results, the as-prepared DNA walker/Apt-MB and H1/AuNPs@BiVO4/FTO were preliminarily used for the detection of target PSA (1.0 ng mL-1 PSA used as an example in this case). Prior to PEC measurement, one question arises to whether CdS QD-labeled hairpin DNA2 could be readily conjugated onto H1/AuNPs@BiVO4/FTO by DNA walker strand. Electrochemical impedance spectroscopy (EIS; an extremely useful analytical tool to monitor the interfacial property in the electrochemical sensing systems) enables to monitor physicochemical characteristics during the electrode-modified processes by sampling the charge transfer at high frequencies and following 10


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mass transfer at low frequencies. Typically, the semicircle diameter of the Nyquist diagram in EIS measurement stands for the resistance of the electron transfer (Ret; Ω) since it usually relies on the insulating features between the electrode and the electrolyte interface. Figure 2C gives the Nyquist diagrams at FTO electrode after modification with different materials or components in PBS (0.1 M, pH 7.4) containing 10 mM Fe(CN)64-/3- within the range of 10-2 – 105 Hz at an alternate voltage of 5 mV. Nyquist 'a' represents electrochemical impedance spectroscopy of the unmodified FTO, and the resistance was 113.7 Ω. After modification of AuNPs@BiVO4 on the FTO electrode, the resistance increased to 327.9 Ω (Nyquist 'b'). To further clarify the advantage of gold nanoparticles, pure BiVO3-modified FTO electrode was also monitored by electrochemical impedance spectroscopy. As shown in Nyquist 'c', the absence of gold nanoparticles caused the increasing resistance relative to Nyquist 'b'. The reason was attributed to the fact that gold nanoparticles could act as a so-called promoter and facilitated the electron transfer between the electrode and the solution. Moreover, the resistance increased again when hairpin DNA1 was covalently conjugated to AuNPs@BiVO3/FTO (Nyquist 'd') because of the electrostatic repulsion between Fe(CN6)3-/4- probe and the negatively charged oligonucleotides. Upon reaction of DNA walker/Apt-MB with 1.0 ng mL-1 PSA and the incubation of supernatant with H1/AuNPs@BiVO4/FTO in the presence of QD-H1, the resistance of the modified electrode further increased (Nyquist 'e'), which derived from the formation of the negatively charged DNA duplexes. All the results verified that the double-photosystem sensing platform at different steps were successfully constructed as expected. Next, the as-prepared H1/AuNPs@BiVO4/FTO electrode was used for PEC detection of target PSA coupling with DNA walker-based amplification strategy in 0.1 M Na2SO4 (Figure 2D). Curves 'a-c' show the photocurrents of BiVO4/FTO, AuNPs@BiVO4/FTO and H1/AuNPs@BiVO4/FTO photosensitive electrodes, respectively. Relative to BiVO4/FTO (curve 'a'), introduction of nanogold particles on the {010} facets of BiVO4 could cause the increasing photocurrent (curve 'b'), which was ascribed to the depression of the photo-generated electron-hole pairs recombination on BiVO4 surface by AuNPs. In contrast with curve 'b', the assembly of hairpin DNA1 on the AuNPs@BiVO4 slightly decreased the photocurrent because of DNA skeleton. When the photosensitive electrode was incubated with DNA walker/Apt-MB, 1.0 ng mL-1 PSA and QD-H2, significantly, the photocurrent largely enhanced (curve 'd'). Upon CdS QDs introduction on the AuNPs@BiVO4, the photo-generated electron transferred from the CB of BiVO4 to the VB of CdS through the AuNPs 11



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electron mediator, thus greatly depressing the photo-generated electron-hole pair recombination in the 'Z-scheme' system. Maybe, one question to be produced was whether the strong photocurrent originated from the nonspecific absorption toward DNA walker and QD-H2. To investigate this issue, the H1/AuNPs@BiVO4/FTO electrode was incubated with DNA walker/Apt-MB and QD-H2 in the absence of target PSA. As shown in curve 'e', the photocurrent of the resulting electrode was almost the same as the background signal of curve 'c', indicating that the increasing photocurrent derived from target-triggered DNA walker progression and the specific reaction of the aptamer with PSA analyte.

Figure 2. (A) FTIR spectra of (a) carboxylated CdS QDs and (b) CdS QDs-labeled hairpin DNA2. (B) Agarose gel electrophoresis images for DNA walker products: (a) DNA walker, (b) hairpin DNA1, (c) hairpin DNA2, (d) DNA walker + hairpin DNA1, (e) DNA walker + hairpin DNA1 + hairpin DNA2, and (f) hairpin DNA1 + hairpin DNA2.

(C)

Nyquist

diagrams

for

(a)

FTO,

(b)

AuNPs@BiVO4/FTO,

(c)

BiVO4/FTO,

(d)

H1/AuNPs@BiVO4/FTO, and (e) H1/AuNPs@BiVO4/FTO after incubation with DNA walker/Apt-MB + 1.0 ng mL-1 PSA + QD-H2 in pH 7.4 PBS containing 10 mM Fe(CN)64-/3- with the range from 10-2 Hz to 105 Hz at an alternate voltage of 5 mV. (D) Photocurrent responses of (a) BiVO4/FTO, (b) AuNPs@BiVO4/FTO, (c) H1/AuNPs@BiVO4/FTO, and (d,e) H1/AuNPs@BiVO4/FTO after incubation with DNA walker/Apt-MB + QD-H2 in the (d) presence and (e) absence of 1.0 ng mL-1 PSA in 0.1 M Na2SO4. 12


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Optimization of Experimental Conditions. As described above, the photocurrent of 3D DNA walker-based PEC sensing platform was amplified through the formation of the 'Z-scheme' double photosystems on the AuNPs@BiVO4 with the aid of CdS QDs. In this regard, the size of CdS QDs would directly affect the photocurrent density of the 'Z-scheme' CdS QDs@AuNPs@BiVO3 system. A small-sized CdS QD was unfavorable for the photocurrent enhancement, whereas a big-sized QD has a relatively low photoconversion efficiency.36 Three kinds of CdS QDs with different sizes including 2.7 nm (carve 'a'), 3.1 nm (carve 'b') and 3.5 nm (carve 'c') were monitored in this system for the detection of 1.0 ng mL-1 PSA (used as an example) (Figure 3A). As seen in Figure 3A, use of 3.5-nm CdS QDs with the orange fluorescence could exhibit a stronger photocurrent that those of 2.7 nm and 3.1 nm. Thus, CdS QDs with 3.5 nm in diameter were used for the construction of the 'Z-scheme' double photosystems in this work. Typically, target-induced release of DNA walker strand from Apt-MB also affects the formation of 'Z-scheme' double photosystems. A too-short reaction time between DNA walker/Apt-MB and target PSA does not facilitate the release of DNA walker, thereby resulting in the low photocurrent. As shown in Figure 3B, the photocurrent density increased with the increasing reaction time, and reached a maximum value after 80 min. By the same token, the walking time of DNA walker strand on the H1/AuNPs@BiVO4 was monitored because it would directly affect the formation of 'Z-scheme' double photosystems, and an optimum photocurrent was acquired after 150 min (Figure 3C). To save the assay time, 80 min and 150 min were chosen for the target-based reaction time and the walking time, respectively.

Figure 3. Influences of (A) different-size CdS QDs (a: 2.7 nm; b: 3.1 nm; c: 3.5 nm) (inset: the corresponding fluorescence images), (B) reaction time of the immobilized aptamer on magnetic bead with target PSA, and (C) the walking time of 3D DNA walker with the assembly of CdS QDs on the photocurrent of 3D DNA walker-based 'Z-scheme' PEC sensing platform (1.0 ng mL-1 PSA used in this case). 13



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Photocurrent Responses of 'Z-Scheme' Double Photosystems toward PSA. By coupling with the as-prepared DNA walker/Apt-MB and H1/AuNPs@BiVO4/FTO, PSA standards with different concentrations were determined by the developed strategy under the optimum conditions. Figure 4A represents the photocurrent responses of PEC sensing system after reaction with PSA standards, and the photocurrent density increased with the increasing PSA concentration, indicating the gradual formation of 'Z-scheme' double photosystems. Meanwhile, a good linear relationship between the photocurrent density and the logarithm of PSA levels could be obtained within the working range of 10 pg mL-1 – 50 ng mL-1 (Figure 4B). The linear regression equation could be fitted to y (µA cm-2) = 1.2219 × logC[PSA] + 0.9989 (pg mL-1) (R2 = 0.994, n = 7), and the limit of detection (LOD) was 1.5 pg mL-1 estimated from the expression of 3S/K (where K and S stand for the slope of the calibration plot and the standard deviation of blank solution, respectively). Obviously, the LOD of our system was lower than those of existed commercial human PSA ELISA kits (e.g., 10 pg mL-1 for Sigma, Cat# no.: RAB0331; 9.4 pg mL-1 for Abcam, Cat# no.: ab188388; 4.9 pg mL-1 for Abcam, Cat# no.: ab188389; 0.5 ng mL-1 for CusaBio, Cat# no.: CSB-E04768h; 40 pg mL-1 for MultiSciences, Cat# no.: 70-EK1212-96) and other PEC detection schemes, e.g., in-situ enzymatic ascorbic acid production as electron donor for CdS QDs enquipped TiO2 nanotubes (0.5 ng mL-1),37 enzymatic

oxydate-triggered

self-illuminated

PEC

sensing

system

(3.0

pg

mL-1),38

bio-bar-code-based PEC immunoassay (1.8 pg mL-1),39 CdS@g-C3N4 heterojunction-based PEC biosensor (4.0 pg mL-1),40 and carbon dots/g-C3N4 nanoheterostructures-based PEC immunoassay (5.0 pg mL-1).41 Reproducibility and Specificity of 'Z-Scheme' Double Photosystems. As a newly developed detection method, the reproducibility and specificity of this system are very important for the future applications. Firstly, the batch-to-batch reproducibility of the as-prepared DNA walker/Apt-MB and H1/AuNPs@BiVO4/FTO was monitored by assaying 1.0 ng mL-1 PSA standard, respectively. The relative standard deviation (RSD) by using the same-batch H1/AuNPs@BiVO4/FTO and DNA walker/Apt-MB was 8.7% (n = 3), whilst that of using different batches was 10.3% (n = 3). Further, the reproducibility of H1/AuNPs@BiVO4/FTO photosensitive electrode was studied by evaluating the photocurrents at continuous multiple assays. During this process, the switch was automatically controlled by the 'on-off' light irradiation with a 500-W Xe lamp. As shown in Figure 4C, all the baselines and response currents (n = 10) were relatively stable at the 'on' and 'off' state, respectively. 14


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The results also revealed that AuNPs@BiVO4 photoactive materials were reproducible and could be used for the batch-preparation.

Figure 4. (A) Photocurrent responses of 3D DNA walker-based PEC sensing platform with double photosystems toward the different-concentration PSA standards under a 500-W Xe lamp excitation. (B) The corresponding calibration curve. (C) The reproducibility of H1/AuNPs@BiVO4/FTO under continuous 'on-off' light irradiation with a 500-W Xe lamp. (D) The specificity of 'Z-scheme' double photosystems against 100 ng mL-1 CEA, 100 ng mL-1 HIgG, 100 ng mL-1 CA 15-3, 1.0 ng mL-1 PSA and their mixture at three times (1,2,3) (note: The mixture contained the above-mentioned all analytes with the same concentrations).

The specificity of 'Z-scheme' double photosystems was studied since the photocurrent stemmed from the interaction of the aptamer with target PSA. As is well-known, many proteins or cancer biomarkers are existed in normal human serum, which might interfere the developed PEC sensing system. To clarify this concern, several common biomarkers including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA 15-3) and human IgG (HIgG) were measured by our strategy. In this case, the photocurrent density of 100-fold higher non-target was comparative with low-concentration target PSA. As shown in Figure 4D, these non-target analytes did not cause the significant increase in the photocurrent relative to background signal. Favorably, 15



coexistence of non-targets with target PSA did not also increase the photocurrent of this system. So, the specificity and selectivity of 'Z-scheme' double photosystem was satisfactory for PSA detection. Detection of Human Serum Specimens and Interlaboratory Validation. To investigate the potential application of 'Z-scheme' double photosystems to complex samples, eight human serum specimens were collected from patients at our First Affiliated Hospital of Fujian Medical University (Fuzhou, China), and determined by using 'Z-scheme' double photosystems. PSA concentrations in the samples were evaluated using the signals generated by the samples in the 'Z-scheme' double photosystems in combination with the regression equation shown in Figure 4B. As the reference, these samples were also analyzed by using commercial human PSA ELISA kit (Abcam, Cat#: ab188388; sensitivity: 9.4 pg mL-1; linear range: 31.25 – 2000 pg mL-1). The results obtained by these two methods are listed in Table 1. The method accuracy and interlaboratory validation were executed on the basis of a Student's t-test method (Please see the Supporting Information).42 Therefore, no significant differences were encountered in the analysis of 8 clinical serum samples between two methods since all texp values were below 2.77 (tcrit[0.05,4] = 2.77). CONCLUSIONS In summary, a new photoelectrochemical sensing platform by coupling with 'Z-scheme' double photosystems has been successfully developed for sensitive detection of low-abundance biomarker. The photocurrent was amplified through an unconventional DNA walker reaction. Compared with traditional photoelectrochemical sensing systems, highlights of this works lie in the following issues: (i) the increasing photocurrent derived from the formation of 'Z-scheme' double photosystems in the presence of target analyte; (ii) introduction of analyte readily triggered the release of DNA walker and the progressive assembly of CdS QDs on the photo-deposited AuNPs on the high-active {010} facets of BiVO4; and (iii) the all-solid-state artificial 'Z-scheme' system inspired by green plant from PS II (BiVO4), PS I (CdS) and electron mediators (AuNP) can effectively depress the electron-hole recombination to enhance the photocurrent density. Importantly, this work opens a new horizon for the construction of signal-on photoelectrochemical sensing platform on the basis of 'Z-scheme' double photosystems. Actually, this system is not only suitable for the detection of macromolecules, but also is favorable for the screening of small molecules (e.g., mycotoxins and marine toxins) by changing the sequence of the corresponding aptamer. 16


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.0000. Materials and reagents, preparation of carboxylated CdS quantum dots, synthesis of CdS QD-labeled hairpin DNA2 (QD-H2), conjugation of carboxylated magnetic beads with PSA aptamer (Apt-MB), preparation of AuNPs-deposited BiVO4 nanohybrids, photoelectrochemical (PEC) measurement, Analysis of real sample, calculation method for t-test statistics, fluorescence spectra of the carboxylated CdS QDs with an average diameter of 3.5 nm (Figure S1), FTIR spectra of PSA aptamer-conjugated magnetic beads carboxylated magnetic beads (Figure S2) (PDF).

AUTHOR INFORMATION Corresponding Author * Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected] (D. Tang). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT The authors thank the National Natural Science Foundation of China (21675029, 21475025), the National Science Foundation of Fujian Province (2014J07001), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) for financial assistance.

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Table 1. Screening of Human Serum Specimens and Interlaboratory Validation between DNA Walker-Based Double-Photosystem PEC Sensing System and the Referred PSA ELISA Kit Method Accuracy [conc.: mean ± SD (RSD), ng mL-1, n = 3] Sample no.

PEC Sensing Platform

PSA ELISA Kit

texp

1

3.16 ± 0.13 (4.11%)

3.52 ± 0.26 (7.39%)

2.14

2

0.81 ± 0.07 (8.64%)

0.87± 0.05 (5.75%)

1.21

3

26.71 ± 1.99 (7.45%)

26.33 ± 1.28 (4.86%)

0.28

4

18.32 ± 1.91 (10.43%)

17.81 ± 1.66 (9.32%)

0.35

5

7.71 ± 0.42 (5.45%)

7.13 ± 0.33 (4.63%)

1.88

6

11.32 ± 0.51 (4.51%)

11.87± 0.47 (3.96%)

1.37

7

0.15 ± 0.01 (6.67%)

0.16 ± 0.02 (12.50%)

0.77

19



FOR ONLY TOC

20


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Double Photosystems-Based 'Z-Scheme' Photoelectrochemical

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