g-C3N4 Nanoheterostructures-Based Signal-Generation

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Carbon Dots/g-C3N4 Nanoheterostructures-Based SignalGeneration Tags for Photoelectrochemical Immunoassay of Cancer Biomarkers Coupling with Copper Nanoclusters Shuzhen Lv, Yi Li, Kangyao Zhang, Zhenzhen Lin, and Dianping Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13272 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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ACS Applied Materials & Interfaces

Carbon Dots/g-C3N4 Nanoheterostructures-Based Signal-Generation Tags for Photoelectrochemical Immunoassay of Cancer Biomarkers Coupling with Copper Nanoclusters

Shuzhen Lv, Yi Li,* Kangyao Zhang, Zhenzhen Lin, and Dianping Tang*

Key Laboratory of 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 35011168, People's Republic of China

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

KEYWORDS: carbon

quantum dots, graphitic carbon nitride, nanoheterostructures, copper

nanoclusters, photoelectrochemical immunoassay

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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 class of 0-dimensional/2-dimensional (0D/2D) nanoheterostructures based on carbon quantum dots (CQDs) and graphitic carbon nitride (g-C3N4) was designed as the signal-generation tags for the sensitive photoelectrochemical (PEC) immunoassay of prostate-specific antigen (PSA) coupling with the copper nanoclusters (CuNCs). Combination of CQDs with g-C3N4 promoted the photoexcited electron/hole separation, and largely increased the photocurrents of the nanoheterostructures. Initially, a sandwich-type immunoreaction was carried out on monoclonal anti-PSA antibody-coated microplate by using PSA aptamer linked with CuNCs as the tracer. Accompanying the immunocomplex, the carried CuNCs were dissolved under acidic conditions. The as-released copper ions from the CuNCs could be captured onto the CQDs/g-C3N4 nanoheterostructures via the amino-group on the CQD surface as well as the -NHx (x = 1,2,3) of g-C3N4 nanosheets. The strong coordination of the Lewis basic sites on the CQDs/g-C3N4 with Cu2+ decreased the photocurrent of the nanoheterostructures. Under optimal conditions, CQDs/g-C3N4 nanoheterostructures displayed good photocurrent responses for the detection of PSA within the dynamic linear range of 0.02 – 100 ng mL-1 and a limit of detection (LOD) of 5.0 pg mL-1. This method was also evaluated for quantitative screening of human PSA serum specimens by using the referenced electrochemiluminescent enzyme-linked immunoassay (ECL-ELIA), and gave good matched results between two methods. Additionally, this system is beneficial to explore the charge-separation and photo-induced electron transfer mechanism in the photoelectrochemical sensing protocols. ■ INTRODUCTION Two-dimensional (2D) nanomaterials (e.g., MoS2, WS2, NbSe2, MnO2, graphene and g-C3N4) have been extensively applied in the sensing field owing to atomic layer thickness, extraordinary physicochemical and biocompatible unique properties.1 Recently, 2D nanostructures-based photoelectrochemical (PEC) detection strategies have gained vigorous attention because the excitation light source and the electrical readout signal in the PEC method are part of different energy forms, endowing it with a low background signal and high sensitivity. Graphitic-phase carbon nitride (g-C3N4) is a cost-effective and environmentfriendly metal-free photoactive semiconductor with a narrow band gap of ~2.7 eV for the absorption of visible light.2 Nevertheless, 2D g-C3N4 nanocrystals alone usually display a low sensitivity in the PEC sensing systems because of the relatively high recombination of photoexcited electro-hole pairs in the nanosheets. An alternative approach to construct the heterojunctions with different nanostructures would 2


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be advantageous.3-6 Nanoheterostructures (as a kind of multiphase solid nanomaterials) have significantly broadened to encompass a large variety of systems (e.g., quantum dots and nanosheets; 0D/2D) made of distinctly dissimilar components.7-9 Ye et al. found that BiVO4 QDs/g-C3N4-based nanoheterostructures could exhibit excellent organic dye degradation and catalytic efficiency under visible light driven.10 Liu et al. used CdS QDs/g-C3N4-based heterojunctions as the highly efficient photoactive species to enhance the photocurrent of PEC aptasensor.11 In contrast with heavy metal-containing semiconductors, carbon quantum dots (CQDs) possess distinctive advantages (e.g., eco-friendly, non-toxicity, stability and easy modification with other materials).12 Combination of CQDs with g-C3N4 nanosheets for the preparation of 0D/2D nanoheterostructures is conducive for photoexcited charge separation and transfer in the PEC measurement. For the successful development of PEC sensing system, introduction of electron receptors with high efficiency should be indispensable. Copper ion (Cu2+) can be used as a fluorescent quencher to achieve the energy/electronic transmission (e.g., binding noble metal nanoclusters, QDs and fluorescent organic moieties).13-15 Sukwattanasinittb and Rashatasakhon developed Cu2+-bond fluorescent organic molecules to trigger resonance energy transfer under the NIR excitation.16 Tian et al. utilized ultrathin g-C3N3 nanosheets for Cu2+ detection via photoinduced electron transfer.17 In this regard, Cu2+ can employed as an good electron receptor of photoactive materials for the construction of PEC sensing scheme due to its simple instrumentation and low cost especially for resource-limited areas.18-20 As is well-known, copper ion can be acquired via the acid-treated copper nanostructures. Undoubtedly, different nanocrystals such as nanospheres, nanotubes, nanobelts and nanoclusters usually contain the various-amount copper ions. Unfavorably, pure copper nanostructures without any-group modifications are difficultly labeled onto the biomolecules (e.g., antibody or aptamer). Inspiringly, the emergence on DNA-templated synthesis of metal nanoclusters (e.g., gold nanoclusters, silver nanoclusters and copper nanoclusters) opens a new horizon for the use of nanomaterial labels for signal amplification.21-24 The Yang's group constructed a fluorescent enzyme immunoassay via enzyme-triggered synthesis of poly(thymine)-templated copper nanoclusters.25 To this end, our motivation in this work is to utilize copper nanoclusters (attached onto single-stranded DNA molecules) for design of CQDs/g-C3N4 nanoheterostructures-based PEC sensing platform. Prostate-specific antigen (PSA; a glycoprotein enzyme encoded in humans by the kallikrein-3 gene) is present at minor amounts leaked out into circulation from the normal prostates, but is often increased 3



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in prostatic diseases. To demonstrate the capability of our design, we herein employed PSA as a model analyte for the development of PEC immunosensing system by using CQDs/g-C3N4 heterojunctions as the signal-generation tags and the released Cu2+ ions from copper nanoclusters (CuNCs) as the electron receptors (Scheme 1). The photocurrents are enhanced through the heterojunction of the high-efficiency CQDs and g-C3N4 nanosheets owing to their unique PEC properties. The sandwich-type immunoreaction is carried out on monoclonal anti-human PSA antibody-coated microplate using the PSA aptamer conjugated with DNA-templated CuNCs as the tracer. Accompanying the aptamers, the carried out CuNCs are dissolved under acidic conditions. In this case, the as-generated Cu2+ ions from the nanoclusters can doubly quench/weaken the photocurrents of CQDs/g-C3N4 nanoheterostructures, thereby resulting in the signal amplification of PEC immunoassay.

Scheme 1. Schematic illustration of carbon quantum dots-functionalized g-C3N4 (CQDs/g-C3N4) nanoheterostructuresbased photoelectrochemical (PEC) immunoassay toward target PSA: (A) Mechanism of Cu2+-quenched photocurrent of CQDs/g-C3N4 nanoheterostructures, and (B) immunoreaction on monoclonal anti-PSA antibody-coated microplate using the PSA aptamer conjugated with DNA-templated copper nanoclusters (Apt-CuNCs) as the tracer. 4


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■ EXPERIMENTAL SECTION Material and Reagent. Prostate-specific antigen (PSA) standards, monoclonal mouse anti-human PSA antibody (mAb; Clone no.: PSA28/A4; Preservative: 15 mM sodium azide + 1.0% BSA in PBS; 1.0 µg mL-1) and other disease biomarkers (e.g., cancer antigen 15-3: CA 15-3; alpha-fetoprotein: AFP; carcinoembryonic antigen: CEA; and human IgG: HIgG) were purchased from Abcam (Hong Kong, China). The sequence of oligonucleotide used in this work was 5'-poly (30T) TTA TTA TTA AAT TAA AGC TCG CCA TCA AAT AGC TTT-3' (note: The underlined letters stands for the aptamer of PSA). Copper(II) sulfate pentahydrate (CuSO4·5H2O), melamine, catechol, ethanediamine, sodium ascorbate, and 3-morpholinopropanesulfoinc acid (MOPS) buffer were acquired from Sinopharm Chem. Inc. (Shanghai, China). Phosphate-buffered saline (PBS) solution came from the products of Sigma-Aldrich. All other chemicals used in this work were of analytical grade and used without further purification. Ultrapure water obtained from a water purification system (18.2 MΩ·cm-1, Milli-Q, Millipore) was used throughout this work. Synthesis of CQDs/g-C3N4 Nanoheterostructures. At the first step, bulk g-C3N4 nanostructures were synthesized by heating melamine to 550 °C for 130 min and then kept at this temperature for another 3 h in air.26 The obtained yellow agglomerates were milled into powder carefully with a pestle and mortar. After that, the bulk g-C3N4 nanostructures were dispersed into ultrapure water and sonicated for 12 h. The resulting suspension was centrifuged for 10 min (4500g) to get the supernatant of g-C3N4 nanosheets. Further, the supernatant was centrifuged for 10 min at 10,000g to get the precipitation of g-C3N4 nanosheets. The obtained precipitation was finally dried at 50 °C in a vacuum oven for use. At the second step, the aminated carbon quantum dots (CQDs) were prepared via typical one-pot synthesis method.27 Briefly, catechol (0.28 g) and ethanediamine (500 µL, original concentration) were initially dissolved into ultrapure water (25 mL) to form a transparent solution. Then the mixture was put into a 50-mL Teflon equipped stainless steel autoclave and heated at 180 °C for 12 h. After that, the nigger-brown product solution was centrifuged in order to remove the impurities and further dialyzed in a dialysis bag to remove small molecules. Finally, the aminated CQDs were collected from the dialysis bag and concentrated for further use. At the third step, CQDs-functionalized g-C3N4 nanoheterostructures were prepared consulting to the literature with minor modification.28 32 mL of ethanol and 32 mL of ultrapure water were initially 5



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mixed together. Then, 0.12 g of g-C3N4 nanosheets and 2.4 mg of the aminated CQDs were thrown into the resulting ethanol aqueous solution under vigorous stirring. The suspension was transferred into a 100-mL Teflon-lined autoclave after sonicating for 30 min and then heated at 180 °C for 4 h. Finally, the CQDs/g-C3N4 nanoheterostructures were washed with ultrapure water twice by centrifugation and dried at 50 °C in a vacuum oven. Functionalization of PSA Aptamer with Fluorescent Copper Nanoclusters (Apt-CuNCs). 5 µL of 25 µM aptamer-T30 single-stranded DNA was first dissolved into 10 µL of 20 mM ascorbate acid and then the MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.8) was added to the mixture to give a volume of 40 µL. Following that, 10 µL of 1.0-mM copper sulfate was introduced at room temperature and the T30 as templates for Cu2 + reduction to produce fluorescent copper nanoclusters (note: The mechanism of Cu2+ reduction to produce fluorescent copper nanoclusters templated by poly T was due to binding interactions between thymine and Cu2+ ions, and the thymine-complexed Cu2+ ions were reduced to Cu0 by ascorbic acid along the contour of the poly-T template).29 Fabrication of CQDs/g-C3N4-Based Sensing Platform and Immunoreaction Protocol. Initially, the FTO electrode was ultrasonically cleaned by water/alcohol for 15 min in turn. Then, the waterproof tape with a round hole was affixed onto the electrode and the area of the round hole was 0.196 cm2. Subsequently, 30 µL of 0.5 mg mL-1 CQDs/g-C3N4 was dropped onto the electrode and dried at room temperature. The immunoreaction was carried out in a 96-well microtiter plate coated with monoclonal anti-PSA antibody, as described in our previously published work.30 The PSA samples or standards (50 µL) were added into the as-prepared microplate and incubated for 100 min at 37 °C. Following that, the microplate was washed three times with the washing buffer (10 mM PBS, 0.05% Tween 20, pH 7.4) to remove the unbound PSA. Afterwards, 50 µL of the above-prepared Apt-CuNCs suspension was added. After incubation for 100 min at 37 °C, the microplate was washed three times with the washing buffer. Subsequently, 200 µL of 0.1 M HNO3 was injected into the microplate to release copper ions from the Apt-CuNCs. Finally, the released copper ions were transferred to a homemade detection cell for PEC measurement in 0.1 M Na2SO4 (Please see the detailed measurement procedure in our previous work30). ■ RESULTS AND DISCUSSION Characterization of CQDs/g-C3N4 Nanoheterostructures. To achieve a high-efficiency photoanode

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material for development of PEC sensing platform, the successful preparation of photoactive materials is very crucial. Figure 1A-C gives high-resolve transmission electron microscope (HRTEM; H-7650, Hitachi, Japan) images of different nanostructures. As shown in Figure 1A, the as-synthesized g-C3N4 nanosheets displayed planar sheet-like morphology with an irregular shape. Also, we observed the Tyndall phenomenon of g-C3N4 aqueous solution under the red laser light (Figure 1A, inset). Moreover, the synthesized CQDs exhibited regular well-dispersed spherical nanoparticles with a size-distribution range from 3 nm to 5 nm (Figure 1B). Such the lattice in the pristine CQDs (Figure 1B) could be obviously achieved after the formation of CQDs/g-C3N4 nanoheterostructures (Figure 1C, inset). Logically, one question arises as to whether the phase structures of g-C3N4 nanosheets were changed after conjugation with CQDs. To demonstrate this issue, X-ray diffraction (XRD; PANalytical X'Pert Spectrometer) was utilized during the synthesis (Figure 1D). As seen from curve 'a', two characteristic peaks at 13.1° and 27.6° were observed, which were corresponded to (100) and (002) diffraction planes of g-C3N4 nanosheets, respectively.31 The sharp peak at 27.6° was attributed to the conjugated aromatic units with the typical graphite-like periodic repeated stacking.32 Significantly, the phase structures of g-C3N4 nanosheets after formation of CQDs/g-C3N4 nanoheterostructures were not disturbed, and a weak diffraction signal for CQDs at 25.6° was discerned in the CQDs/g-C3N4 (curve 'b'), which was ascribed to the low-mass CQDs in the nanoheterostructures.33 Furthermore, we also used the diffuse reflection spectra (DRS) to monitor the corresponding bandgap change of g-C3N4 nanosheets before and after conjugation with the CQDs. As indicated from Figure 1E, the bandgap of CQDs/g-C3N4 nanoheterostructures (carve 'b') decreased in comparison with that of bare g-C3N4 nanosheets (carve 'a'), demonstrating that there might be the photoexcited charge separation states.34 The prepared Apt-CuNCs were characterized by fluorescence spectrum. The maximum emission wavelength of Apt-CuNCs was at 626 nm under 348 nm excitation wavelength (Figure 1F). Elsewhere, a bright red fluorescence could be observed (Figure 1F, insert) under ultraviolet light at 365 nm. These results elucidated that the CQDs/g-C3N4 nanoheterostructures and fluorescent copper nanoclusters were successfully synthesized.

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Figure 1. HRTEM images of (A) g-C3N4 (inset: Tyndall phenomenon for g-C3N4 suspension under the red laser light ), (B) CQDs (inset: HRTEM image within the small-sized scale) and (C) CQDs/g-C3N4 (inset: magnification image); (D) XRD patterns of (a) g-C3N4 and (b) CQDs/g-C3N4; (E) DRS plots of (ɑhv)2 vs. photon energy (hv) of (a) g-C3N4 and (b) CQDs/g-C3N4 (inset: UV-vis DRS spectra); and (F) fluorescence spectra of Apt-CuNCs (inset: fluorescence photograph under ultraviolet light at 365 nm).

The photoexcited charge-carrier interfacial transition of g-C3N4 nanosheets after integration with the CQDs under the light excitation was investigated by using the photoluminescence (PL) spectrometry. As shown in Finger 2A, pure g-C3N4 nanosheets (curve 'a') at the excitation wavelength of 353 nm had high PL emission intensity, implying high recombination possibility of electron-hole pairs. In contrast, the weak PL emission intensity of CQDs/g-C3N4 nanoheterostructures (curve 'b') at the same excitation wavelength indicated that the nanoheterostructures could greatly suppress the charge recombination and promote the photoexcited charge-carrier interfacial transition.35 Typically, the conduction band (CB) of g-C3N4 nanosheets (ca. -1.16 eV in the potential) is more negative than that of CQDs (ca. -0.5 eV). In this case, the photoinduced electrons of g-C3N4 nanosheets were moved to CQDs. Meanwhile, the high valence band (VB) of CQDs (ca. 2.25 eV) could trigger its holes to transform the valence band of the 8


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g-C3N4 nanosheets (ca. 1.62 eV). The electrochemical impedance spectroscopy (EIS) was utilized to investigate the fabrication process of CQDs/g-C3N4-based photoanode in pH 7.4 PBS containing 5.0 mM Fe(CN)64−/3− and 0.1 KCl at an alternate voltage of 5 mV within the range from 10−2 to 105 Hz (Figure 2B). The parameter (Rct) represents the charge transfer resistance of redox probe, which equals the arc radii of Nyquist plots, and it can reflect the charge transfer property of photoanode surface. As shown in Finger 2B, the arc radii of CQDs/g-C3N4- modified FTO electrode (plots 'd') was smaller than that of g-C3N4-modified electrode (plots 'c'), suggesting that CQDs/g-C3N4 afforded smaller resistance for the charge transfer. This result indicated that CQDs/g-C3N4 nanoheterostructures had superior to g-C3N4 nanosheets alone in the photoexcited charge separation, and could accelerate the electron communication between the solution and the photoanode. Figure 2C shows the photoelectrochemical responses of g-C3N4 nanosheets in the absence and presence of CQDs in 0.1 M Na2SO4. It could be observed that the CQDs/g-C3N4-modified electrode (carve 'd') had a bigger photocurrent density than those of CQDs/FTO (carve 'b') and g-C3N4/FTO (carve 'c') alone. The reason might be attributed to the fact that the CQDs/g-C3N4 nanoheterostructures greatly suppressed the electro-hole recombination.

Figure 2. (A) Fluorescence spectra of (a) g-C3N4 and (b) CQDs/g-C3N4 (insets: the corresponding photograph on the modified FTO electrodes under ultraviolet light at 365 nm); (B) Nyquist diagrams and (C) photocurrent densities at (a) FTO, (b) CQDs/FTO, (c) g-C3N4/FTO and (d) CQDs/g-C3N4/FTO.

Feasibility of CQDs/g-C3N4-Based PEC Photoanode. As mentioned above, the photocurrent of the as-synthesized CQDs/g-C3N4 nanoheterostructures decreases in the presence of copper ions (which can be released from the subsequent Apt-CuNCs under acidic conditions). As control tests, we first studied the fluorescence properties of the as-prepared CQDs or g-C3N4 nanosheets alone in the absence and 9



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presence of copper ion (Figure 3A-B). As shown from curve 'a', a strong fluorescence emission intensity was acquired at g-C3N4 nanosheets (Figure 3A) or CQDs (Figure 3B) alone. However, the fluorescence intensity decreased upon addition of copper ions into these two system, respectively (Figure 3A-B, curve 'b'). The reason was ascribed to the fact that the captured Cu2+ ions by the N on the g-C3N4 nanosheets and the -NHx (x = 1,2,3) on the aminated CQDs could cause the photo-induced electron transfer between g-C3N4 (CQDs) and Cu2+.17 Furthermore, the Fourier transform infrared (FTIR) spectroscopy was employed to investigate the chelated reaction of the -NHx (x = 1,2,3) groups, C-N and C=N heterocycles with Cu2+ ions (Figure 3C). The peaks of g-C3N4 nanosheets between 3400 and 3000 cm -1 were assigned to the stretching vibration of terminal -NHx groups bonds of the defect sites and the stretching vibration of O-H from the physically adsorbed water (Figure 3C, curve 'b'). In contrast, the characteristic bands at 1240, 1320, 1400, 1568 and 1610 cm-1 were attributed to the typical stretching vibration modes of C-N and C=N heterocycles, while that at 810 cm-1 was assigned to the triazine units breathing mode.36 As seen from curve 'b' in Figure 3B, the peaks at 3240, and 3040 cm-1 suggested the existence of -NH2 and -NH3+ at the aminated CQDs, while the peaks at 1662, 1514 and 1384 cm-1 were corresponded to the stretching vibrations of amide I, C=O, amide II, N-H, and amide III, C-N groups, respectively.37 The FTIR spectrum of CQDs/g-C3N4 (Figure 3C, curve 'c') was similar to that of bare g-C3N4 because of the generally identical peak positions for CQDs and g-C3N4. Further, we also investigated the photocurrent density of CQDs/g-C3N4-modified FTO electrode in 0.1 M Na2SO4 before and after addition of Cu2+ ion (Figure 3D). Obviously, a decrease in the photocurrent density was observed in the presence of copper ion (curve 'b' vs. curve 'a'). The reason was attributed to the fact that the photoinduced electrons were transferred from the conduction band (CB) of the g-C3N4 nanosheets and CQDs to copper ion.38 Hence, CQDs/g-C3N4-modified photoanode electrode could be utilized for the detection of copper ion. By using copper nanoclusters-conjugated PSA aptamer, this system could be employed for the determination of PSA, accompanying the sandwich-type immunoassay format in the subsequent work.

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Figure 3. (A) Fluorescence spectra of (a) g-C3N4 and (b) g-C3N4 + Cu2+; (B) Fluorescence spectra of (a) CQDs and (b) CQDs + Cu2+; (C) FTIR spectra of (a) g-C3N4, (b) CQDs and (c) CQDs/g-C3N4; (D) Photoelectrochemical responses of (a) CQDs/g-C3N4/FTO and (b) CQDs/g-C3N4/FTO + Cu2+ in 0.1 M Na2SO4.

Optimization of Experimental Conditions. In this work, the photocurrent density of CQDs/g-C3N4-based PEC immunosensing platform decreased with the increasing target PSA concentration. To achieve a wide linear range and a high sensitivity in this system, a strong initial (background) photocurrent on the CQDs/g-C3N4-modified FTO electrode would be preferable. Usually, the nanoheterostructures exhibit different photocurrent density with various doping ratios. As so, the photocurrents at CQDs/g-C3N4-modified FTO electrodes with different CQD mass percentages were separately investigated in the absence of immunoreaction. As seen from Figure 4A, the photocurrent density first increased with the increasing CQD mass percentage, and then decreased. An optimal CQD mass percentage was 2.0 wt%. The reason was ascribed to the fact that a low-mass CQD percentage

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could not effectively inhibit the electro-hole pairs high recombination of g-C3N4 nanosheets, whilst a high-amount CQD hold the excited area of g-C3N4 nanosheets by light. Thus, g-C3N4 nanosheets (0.12 g) and the aminated CQDs (2.4 mg) were used for preparation of CQDs/g-C3N4 nanoheterostructures. At this condition, we investigated the effect of different immunoreaction times on the photocurrents of CQDs/g-C3N4-modified FTO electrode by using 0.1 ng mL-1 PSA as an example in 0.1 M Na2SO4 (note: To avoid confusion, the immunoreaction times of mAb-coated microplate with target PSA were paralleled with those with Apt-CuNCs). As shown in Figure 4B, the photocurrent densities decreased with the incubation time aged, and tended to level off after 100 min. To save the assay time, 100 min was selected for the mAb-PSA-Apt-CuNCs reaction.

Figure 4. Dependence of photocurrent CQDs/g-C3N4-based immunosensing platform on (A) CQD mass percentage in the CQDs/g-C3N4 nanoheterostructures (note: This case did not contain the immunoreaction with Apt-CuNCs) and (B) the incubation time for the mAb-PSA-Apt-CuNCs reaction in 0.1 M Na2SO4 (note: 0.1 ng mL−1 PSA used as an example). Each data represents the average value obtained from three independent measurements.

Analytical Performance of CQDs/g-C3N4-Based PEC Immunosensing Platform. By using the as-synthesized CQDs/g-C3N4 nanoheterostructures as the photoactive materials, a novel immunosensing protocol was designed for the photoelectrochemical detection of PSA using the in-situ prepared CuNCs on the PSA aptamer (Apt-CuNCs) as the tracer with a sandwich-type assay format under optimal conditions. Figure 5A gives the photocurrent responses of this system in 0.1 M Na2SO4 toward PSA standards with different concentrations. Owing to the overpowering efficiency of the as-released copper ions from the Apt-CuNCs toward the CQDs/g-C3N4 photoanode, the photocurrent density on the

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modified electrode decreased with the increasing PSA concentration in the detection solution. As shown in Figure 5B, a good linear relationship between the photocurrent density and the logarithm of PSA level was achieved within the dynamic working range from 0.02 ng mL-1 to 100 ng mL-1. The linear regression equation for the derived calibration curve is ∆J (nA cm-2) = - 389.45 × LogCPSA (ng mL-1) + 1051.2 (note: ∆J stands for the change in the photocurrent density, J = I/0.196, while 0.196 is the active area of the modified electrode). The limit of detection (LOD) was 5.0 pg mL−1, which was calculated by the expression of 3S/K (note: S stands for the standard deviation of 11 assays for blank solution, while K is the slope of the calibration plot).

Figure 5. (A) Photocurrents and (B) the calibration plots of CQDs/g-C3N4-based photoelectrochemical immunosensing platform toward PSA standards in 0.1 M Na2SO4, (C) the stability of the CQDs/g-C3N4-modified FTO electrode, and (D) the specificity of CQDs/g-C3N4-based PEC immunoassay (note: PSA: 0.1 ng mL−1, AFP: 10 ng mL−1, CEA: 10 ng mL−1, HIgG: 10 ng mL−1, CA 15-3: 10 ng mL−1). Each data represents the average value obtained from three independent measurements. 13



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The method reproducibility is also another important analytical performance during the measurement. Generally, the light irradiation affects the photocurrent of the photoactive materials during the multiple determinations. To investigate this issue, the photocurrents of CQDs/g-C3N4-modified FTO electrode were measured under the 500-W Xe lamp with a repeated 'on-off' light irradiation. As seen from Figure 5C, the background photocurrent density at the 'off' switch and the response photocurrent density at the 'on' state were almost stable. The relative standard deviation (RSD) values were 5.6% (on) and 4.7% (off) within the continuous 15 'on ↔ off' switches, respectively. The results indicated that CQDs/g-C3N4-modified FTO electrode could be repeatedly used for the photocurrent measurement. Another important concern for this system is whether other biomolecules/cancer biomarkers interfere with the photocurrent of the CQDs/g-C3N4-based PEC immunosensing platform. To verify this point, different non-target standards (i.e., other cancer biomarkers) including AFP, CEA, CA 15-3 and HIgG were detected under the different conditions by using our strategy. As indicated from Figure 5D, almost the same photocurrents were obtained at these non-target analytes alone in comparison with background signal. Moreover, the co-existence of the high-concentration non-target analyte with the low-level PSA did not cause the significant change in the photocurrent relative to pure target PSA. These results further revealed that our system had a good selectivity toward target PSA. To meet the requirements of accurate detection and the method reliability for real practical samples based on the above-mentioned results, the CQDs/g-C3N4-based photoelectrochemical immunosensing platform was employed for the quantitative monitoring of 6 human serum samples containing target PSA, which were gifted from the local Fujian Provincial Hospital (Fuzhou, China). These samples were collected following the Rules and Ethics of the local Committee. For comparison, these samples were first detected by using electrochemiluminescent enzyme-linked immunoassay (ECL-ELIA) (note: All the ECL-ELIA-based detection processes were finished by Clinical Laboratory and Medical Diagnostic Laboratory (Dr. Jianxin Huang, Fujian Provincial Hospital, China, Acknowledgement!). Following that, these samples were determined using CQDs/g-C3N4-based PEC immunoassay, respectively. The results obtained from two methods are summarized in Table 1. As shown in Table 1, all texp values based on an independent two-sample t-test39 were below 2.77 (note: tcrit[0.05,4] = 2.77), indicating a good method accuracy between two methods. Furthermore, we also evaluated the regression equation on the basis of the average values between two methods as follows: y = 0.9752 x + 0.1732 (R2 = 0.9981, n = 8; x-axis: by PEC immunoassay; y-axis: by referenced ECL-ELIA). As analyzed from the equation, the slope and 14


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intercept were close to the ideal values '0' and '1', respectively. Hence, the method accuracy of the CQDs/g-C3N4-based PEC immunosensing was satisfactory, and could be preliminarily utilized for the analysis of human serum samples with good results. Table 1. Comparison of the Results Obtained by CQDs/g-C3N4-Based PEC Immunosensing Platform and the Referred ECL-ELIA Method for Human PSA Serum Specimens. Method Accuracy [conc.: mean ± SD (RSD), ng mL-1, n = 3] Sample no.

PEC immunoassay

ECL-ELIA

texp

1

5.09 ± 0.33 (6.48%)

4.92 ± 0.28 (5.69%)

0.68

2

2.84 ± 0.25 (8.80%)

2.90 ± 0.31 (10.69%)

0.26

3

0.83 ± 0.05 (6.02%)

0.91 ± 0.03 (3.30%)

2.38

4

9.61 ± 0.97 (10.09%)

9.13 ± 0.55 (6.02%)

0.74

5

20.14 ± 1.18 (5.86%)

19.78 ± 1.40 (7.08%)

0.34

6

1.62 ± 0.09 (5.56%)

1.55± 0.12 (7.74%)

0.81

7

0.36 ± 0.04 (11.11%)

0.40 ± 0.03 (7.50%)

1.39

8

0.14 ± 0.01 (7.14%)

0.17± 0.02 (11.76%)

2.32

■ CONCLUSIONS In summary, the work successfully constructed a new photoelectrochemical sensing platform, based on CQDs/g-C3N4 nanoheterostructures, for the sensitive detection of disease-related biomarkers (PSA used as an example) by coupling with copper nanoclusters-assembled PSA aptamer. Compared with CQDs and g-C3N3 nanosheets alone, the CQDs/g-C3N4 heterojunction could display strong photocurrent and good photoexcited electron/hole separation. Introduction of copper nanoclusters-functionalized PSA aptamer in the sandwich-type immunoassay format could efficiently avoid the participation of natural enzymes (e.g., horseradish peroxidase and alkaline phosphatase) and reduce the assay cost. Meanwhile, the in-situ as-synthesized CuNCs on the aptamers could escape the complex process of enzyme labels. Relative to single copper nanoparticle, the aptamer-concatemerized copper nanoclusters could release numerous copper ions under acidic conditions to decrease doubly the photocurrents of CQDs/g-C3N4 nanoheterostructures, thus resulting in the signal amplification of the immunoassay. Nevertheless, one disadvantage of our strategy is that the detectable photocurrent decreases with the increasing PSA level in the sample. Favorably, this 'signal-off' assay protocol can be changed by adopting a competitive-type 15



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immunoassay mode, thereby causing the 'signal-on' detection scheme. Therefore, future works should focus on the change in the assay formats for the development of the competitive-type immunoassays, especial for the detection of small-molecular biotoxins or mycotoxins. ■ AUTHOR INFORMATION Corresponding Authors *Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mails: [email protected] (Y.L.) & [email protected] (D.T.).

■ ACKNOWLEDGEMENTS Authors thank the National Natural Science Foundation of China (21475025, 21675029 & 21402028), the National Science Foundation of Fujian Province (2014J07001) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) for financial assistance. ■ REFERENCES (1) Yu, S.; Wu, X.; Wang, Y.; Guo, X.; Tong, L. 2D Materials for Optical Modulation: Challenges and Opportunities. Adv. Mater. 2017, 29, 1606128 (pp. 1-26). (2) Zhuang, J.; Lai, W.; Xu, M.; Zhou, Q.; Tang, D. Plasmonic AuNP/g-C3N4 Nanohybrid-Based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying DNAzyme-Catalyzed Precipitation Amplification. ACS Appl. Mater. Interfaces 2015, 7, 8330-8338. (3) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA Nanostructures for Theranostic Application. Acc. Chem. Res. 2014, 47, 550-559. (4) Abi, A.; Lin, M.; Pei, H.; Fan, C.; Ferapontava, E.; Zuo, X. Electrochemical Switching with 3D DNA Tetrahedral Nanostructures Self-Assembled at Gold electrode. ACS Appl. Mater. Interfaces 2014, 6, 8928-8931. (5) Chen, L.; Chao, J.; Qu, X.; Zhang, H.; Zhu, D.; Su, S.; Aldalbahi, A.; Wang, L.; Pei, H. Probing Cellular Molecules with PolyA-Based Engineered Aptamer Nanobeacon. ACS Appl. Mater. Interfaces 2017, 9, 8014-8020. (6) Qu, X.; Wang, S.; Ge, Z.; Wang, J.; Yao, G.; Li, J.; Zuo, X.; Shi, J.; Song, S.; Wang, L.; Li, L.; Pei, H.; Fan, C. Programming Cell Adhesion for On-Chip Sequential Boolean Logic Funtions. J. Am. Chem. Soc. 2017, 139, 10176-10179. (7) Guo, C.; Yang, H.; Sheng Z.; Lu, Z.; Song, Q.; Li, C. Layered Graphene/Quantum Dots for Photovoltaic Devices. Angew. Chem., Int. Ed. 2010, 49, 3014-3017. (8) Qi, L.; Xiao, M.; Wang, X.; Wang, C.; Wang, L.; Song, S.; Qu, X.; Shi, J.; Pei, H. DNA-Encoded Raman-Active Anisotropic Nanoparticles for microRNA Detection. Anal. Chem. 2017, 89, 9850-9858. (9) Qu, X.; Zhu, D.; Yao, G.; Su, S.; Chao, J.; Liu, H.; Zuo, X.; Wang, L.; Shi, J.; Wang, L.; Huang, W.; Pei, H.; Fan, C. An Exonuclease III-Powered, On-Particle Stochasctic DNA Walker. Angew. Chem., Int. Ed. 2017, 56, 1855-1858. 16


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2005; Chapter 5, 115-118.

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■ For TOC only

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g-C3N4 Nanoheterostructures-Based Signal-Generation

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