Photoelectrochemical Biosensor Based on 1D In₂O₃ Tube Decorated with 2D ZnIn₂S₄ Nanosheets for Sensitive PSA Detection

Abstract

In photoelectrochemical (PEC) biosensing, efficient electron-hole separation is crucial to obtain preferred photocurrent response and analytical performance; thus, constructing developed heterointerfaces with high carrier transfer efficiency is an effective method for sensitive evaluation of analytes. Herein, a 1D ZnIn₂S₄ nanosheet-decorated 2D In₂O₃ tube was developed to integrate with a prostate antigen (PSA)-sensitive aptamer for sensitive PSA antigen detection. 1D In₂O₃ tubes were first prepared by two-step hydrothermal and annealing methods, followed by the in-situ growth of ZnIn₂S₄ nanosheets. Morphology, optical properties, structure, and PEC performance of prepared In₂O₃-ZnIn₂S₄ were characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet–visible spectrophotometry, X-ray diffraction, X-ray photoelectron spectroscopy, and an electrochemical workstation. Benefiting from the photoelectric effect and specific 1D/2D hierarchical structure, In₂O₃-ZnIn₂S₄ displayed enhanced optical absorption and photocarrier separation, thus a superior photoelectrochemical response. Proposed bioassay protocol possessed a linear range from 0.001 to 50 ng/mL and a detection limit at 0.00037 ng/mL. In addition, this biosensor exhibited satisfactory anti-interface ability and stability, which also could be extended to other quantitative platforms for detecting other proteins.

1. Introduction

Photoelectrochemical (PEC) bioanalysis, featuring high sensitivity and simple equipment, has been used to quantify various analytes through photocurrent variation [1,2]. Essentially, sensing photocurrent signals strongly depends on the light–electricity conversion process and carrier separation efficiency of photoactive materials. Thus, besides recognition units, photoactive materials with superior PEC performance are crucial factors when designing sensitive PEC biosensors. Indium oxide (In₂O₃), as a typical moderate-band-gap n-type semiconductor, has been widely used in photoelectrochemistry due to its high chemical stability, low resistivity, low toxicity, and easy preparation [3,4]. However, pure In₂O₃ has always suffered from poor PEC performance, which mainly contributed to high carrier recombination rate, limited photon utilization, and deficient surface active sites. Multiple reports have demonstrated heterojunction construction by coupling other semiconductors with different band gaps, which could prominently promote the photogenerated electrons and hole migration at the heterojunction interface, thus enhancing the PEC performance of In₂O₃-based materials [5,6,7,8].

Up to now, multiple In₂O₃-based heterostructures, such as In₂O₃/Co₃O₄ [6], In₂O₃/g-C₃N₄ [9], In₂O₃/CdS [10], and In₂O₃/In₂S₃ [11], have been prepared with improved photoelectric transformation efficiency. However, mismatched lattice between these semiconductors and In₂O₃ usually induced heterointerface impedance and restricted carrier separation. Compared with the above mentioned semiconductors, 2D layered ZnIn₂S₄ has drawn increasing attention due to high specific surface areas, short carrier migration pathways, rich active sites, and similar lattice parameters [12,13]. The high lattice matching degree of In₂O₃ and ZnIn₂S₄ is conducive to the formation of a compact interface and thus greatly decreases the charge immigration impedance of the heterointerface, and finally, facilitates spatial charge separation [14]. A significant challenge is that ZnIn₂S₄ sheets tend to agglomerate into nanoclusters, causing a low specific surface area and reduced active sites [15,16]. Supporting carriers, including CdS nanocube, NiMoOx nanorod, FeWO₄ flower, and Co₉S₈ tube, favor lamellar growth and inhibit agglomerates [17,18,19,20]. Thus, it is of great favor to employ a 1D In₂O₃ tube as the supporter to offer ana abundant area for nanosheet growth and conduction paths for photogenerated carrier transport. With such a design, branched ZnIn₂S₄ nanosheets on In₂O₃ tubes could facilitate solar-light harvesting, and an intimate heterointerface could modulate the migration pathway for extending the photogenerated charge lifetime.

Prostate antigen (PSA) level, as an early portent of prostate dysfunction, is associated with prostate cancer, underscoring the importance of sensitive PSA detection (<4 ng/mL—traditionally considered normal). Multiple sensing strategies have been developed for PSA quantification. For example, a sandwich-type electrochemical immunosensor with delaminated MXene@AuNPs as signal amplification was reported for PSA ultra-sensitive analysis [21]. Another peptide–antibody sandwich electrochemical biosensor based on the MnO₂-functionalized COF was successfully constructed [22]. A meta-nano-channel silicon field-effect biosensor fabricated on silicon-on-insulator wafers was proposed for label-free PSA sensing [23]. These studies achieved ultra-sensitive PSA detection, which is usually accompanied by complex sensor modification processes and cumbersome sensing procedures.

In this work, a 1D/2D heterostructure was designed by decorating ZnIn₂S₄ nanosheets onto In₂O₃ tubes to integrate with the PSA-sensitive aptamer for PSA antigen quantification. The overall heterostructure preparation process involved a template method and in-situ growth strategy where MIL-68 (Materials of Institute Lavoisier MOF materials) was transformed into tubular In₂O₃ and further modified with ZnIn₂S₄ nanosheets (Figure 1A). Under visible light, nanosized heterogeneous interface favored the effective separation and migration of electron-hole pairs, endowing the hybrid with desirable PEC performance (Figure 1B). During this process, the photogenerated electrons accumulated in the conduction band of In₂O₃ while photogenerated holes accumulated in the valence band of ZnIn₂S₄. Efficient separation of electrons and holes was achieved with ascorbic acid as the electron donor to scavenge holes. Furthermore, a modified aptamer on the PEC electrodes could specifically bind with the target; thus, a relevance between PSA concentration and photocurrent response was established. Finally, the PEC sensing platform displayed a linear response range from 0.001 to 50 ng/mL and a detection limit at 0.00037 ng/mL. This platform also possessed satisfying selectivity and stability. Such a simple construction process and efficient sensing protocol have great potential in other biomolecule quantifications.

2. Materials and Methods

2.1. Reagents and Apparatus

All reagents are of analytical reagent grade and are directly used for all experiments. Ultrapure water (resistivity ≥ 18.25 MΩ cm) was obtained from a Lichun water purification system. Glass with a 150 nm FTO layer (~15 Ω/square resistance) was purchased from Xiamen FTO Photoelectricity Industry (Xiamen, China). Nitrate hydrate, N. N-dimethylformamide, chitosan, bovine serum albumin (BSA), and glutaraldehyde were offered by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); 1,4-benzenedicarboxylic acid, zinc chloride, indium chloride, and thioacetamide were bought from Macklin Reagent Co., Ltd. (Shanghai, China); 0.01 M PBS (pH = 7.4) buffer was employed as incubation buffer.

PSA aptamer: (5′-NH2-(CH2)6-ATTAAAGCTCGCCATCAAATAGC-3′)

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were obtained using the QUANTA FEG 250 thermal field emission scanning electron microscopy (FEI Co., Hillsboro, OR, USA) and Hitachi H600 with 200 kV acceleration voltage. Elemental mapping images were recorded using an EDX spectroscope attached to a TEM. X-ray diffraction (XRD) patterns and X-ray photoelectron spectroscopy (XPS) spectra were collected by a D8 advance diffractometer system equipped with Cu Ka radiation (Bruker Co., Bremen, Germany) and an ESCALAB 250Xi photoelectron spectrometer, respectively. UV-vis absorption measurements were achieved by a UH-4150 ultraviolet–visible spectrophotometer (Hitachi, Tokyo, Japan). And the photocurrent and electrochemical impedance spectroscopy (EIS) were measured on a CHI 660D electrochemical workstation (Shanghai Chenhua Instruments Corporation, Shanghai, China) with a three-electrode system. Photocurrents were recorded under a 500 W xenon lamp (100 mW·cm⁻²) illumination.

2.2. Preparation of Hollow Tubular In₂O₃

Tubular In₂O₃ was synthesized by sequential hydrothermal and thermal methods. Initially, indium nitrate hydrate (0.06 g) and 1,4-benzenedicarboxylic acid (0.06 g) were dissolved in 40 mL N,N-dimethylformamide, and further stirred for 5 min at room temperature. The resultant solution was heated at 120 °C for 30 min, followed by filtration and washed with ethanol to obtain white MIL-68. Finally, an annealing procedure at 500 °C was performed for 2 h in a muffle furnace, yielding light yellow In₂O₃ tubes.

2.3. Synthesis of Branched-Sheet Embedded Tubular In₂O₃-ZnIn₂S₄

Briefly, as-prepared In₂O₃ tubes (0.1 g) and ZnIn₂S₄ precursor (0.05 g of zinc chloride, 0.23 g indium chloride, and 0.24 g thioacetamide) were fully dissolved in 30 mL deionized water and then stirred continuously for 30 min at 80 °C in an oil bath. The obtained precipitate In₂O₃-ZnIn₂S₄ was collected, centrifugated, washed with deionized water, and dried under vacuum. Nanosheet-based ZnIn₂S₄ clusters were synthesized using the same method without the addition of In₂O₃.

2.4. Fabrication of Sensing Platform and Analysis Protocol

FTO glass was pre-cleaned sequentially with acetone, ethanol, and deionized water under violent ultrasonication before use. In order to obtain an attractive PEC signal, 1 mL of prepared In₂O₃-ZnIn₂S₄ solution was spin-coated onto the FTO glass, followed by drying under an infrared lamp for 30 min. A volume of 50 μL of chitosan aqueous solution (0.08 wt.%) in 1% acetic acid was dropped onto the FTO electrode, and then, 5 wt.% glutaraldehyde solution was applied onto the electrode to trigger amino groups for subsequent biomolecule modification. After that, the electrode was incubated with 20 µL of 1 µM aptamer for 70 min at 4 °C by adding the adapter solution onto the electrode surface, followed by the addition of 10 μL of 2 wt.% BSA to block non-specific binding sites. During the 70-min adapter incubation process, the adapter was connected to the electrode via glutaraldehyde-mediated Schiff base. The obtained working electrode was stored at 4 °C and denoted as FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA. Before PEC measurements, the FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA was incubated with 20 μL PSA at room temperature for 30 min. Notably, FTO electrodes were thoroughly cleaned with PBS buffer (pH = 7.4, 0.01 M) after each step. All PEC signals were generated by a typical three-electrode system (FTO working electrode, counter electrode, and reference electrode) in 0.1 M ascorbic acid (AA) solution.

2.5. Detection Limit Calculation

The detection limit was obtained by the formula I_LOD = I_blank + 3S_blank, where I_blank and S_blank are the average photocurrent of 10 independent samples (without PSA) and the corresponding standard deviation, respectively. Then, I_LOD was brought into the regression curve to obtain the detection limit.

3. Results and Discussion

3.1. Morphology and Structure Characterization

Hybrid In₂O₃-ZnIn₂S₄ was synthesized by the template method and in-situ growth technique and further investigated by scanning electron microscope (SEM), transmission electron microscope (TEM), and high-resolution TEM (HRTEM). Apparently, a highly dispersed tube microstructure with well-defined tube walls and cavities was found for In₂O₃ (Figure 2A), providing adequate space for ZnIn₂S₄ growth. While pure ZnIn₂S₄ displayed irregular clusters assembled by a large number of nanosheets (Figure 2B and Figure S1). After loading the nanosheets onto In₂O₃, the as-prepared composite exhibited uniform and dense coverage of ultrathin nanosheets, and O, In, S, and Zn elements were evenly distributed on the single tube (Figure 2C,D,(D1–D4)). Significantly, compared to self-assembled ZnIn₂S₄ nanosheet clusters, interconnected nanosheets are beneficial for increasing specific surface area and providing sufficient active sites. Furthermore, a tight interfacial junction between In₂O₃ and ZnIn₂S₄ was successfully constructed where lattice spacing of 0.29 and 0.32 nm were ascribed to the In₂O₃ (2 2 2) and ZnIn₂S₄ (1 0 2) crystal planes, respectively (Figure 2E) [24]. These images demonstrated the successful fabrication of a branched sheet embedded tubular hybrid and heterostructure.

Optical properties, crystalline phases, and chemical states were also measured by UV-vis absorption spectra, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS), respectively. As shown in Figure 3A, a typical absorption edge at ~425 nm and poor light-harvesting capacity in the visible light region were demonstrated for pure In₂O₃. For hybrid In₂O₃-ZnIn₂S₄, a robust photo-absorption in the UV and visible light region to ~542 nm was obtained. Using the Tauc plot method [25], the bandgap energies (Eg) of In₂O₃, ZnIn₂S₄, In₂O₃-ZnIn₂S₄ were calculated to be 2.9, 2.58, and 2.29 eV, respectively. Furthermore, XRD patterns of the above-mentioned materials are shown in Figure 3B. Nine distinct diffraction peaks at 21.5°, 30.6°, 35.5°, 37.7°, 41.8°, 43.7°, 51.1°, 55.9°, and 60.7° in black curve were well-matched with characteristic standard In₂O₃ data (JCPDS No. 06-0416) [14]. Except for diffraction peaks of In₂O₃, an additional peak at 47.2° gathered from In₂O₃-ZnIn₂S₄ was assigned to the (1 1 0) crystal plane of pure ZnIn₂S₄. To probe the elemental valence details, XPS was performed with the C 1s peak as the standard reference. In the survey spectrum, the presence of In, O, Zn, and S elements was confirmed, consistent with the above-mentioned mapping diagram. Specifically, two binding energies at 1022.3 and 1045.5 eV were attributed to the Zn 2p3/2 and Zn 2p1/2 of Zn²⁺ chemical states. Two prominent peaks presented at 445.2 eV (In 3d5/2) and 452.8 eV (In 3d3/2) were recorded, which were similar to that of standard In³⁺. The S 2p spectrum could be deconvoluted into two characteristic S²⁻ peaks at 161.8 and 163.0 eV. In a word, the above-mentioned results matched each other, illustrating the successful fabrication of tubular In₂O₃-ZnIn₂S₄.

3.2. PEC and EIS Behaviors

To obtain a desirable initial signal for subsequent PEC biosensing, a 1D ZnIn₂S₄ nanosheet-decorated 2D In₂O₃ tube served as the photosensitive material. The interconnected nanosheets grown in situ on the tube exhibit a high specific surface area and provide more carrier migration pathways, thus endowing the photoactive material with a high photocurrent. The entire sensing platform was constructed after modifying the PSA-sensitive aptamer for PSA-sensitive detection. During the platform construction and sensing process, two key factors, including the In₂O₃-ZnIn₂S₄ concentration and PSA antigen incubation time, were investigated, and the results are described in Figure 4. With the increase in In₂O₃-ZnIn₂S₄ concentration, the photocurrent value displayed an inverted V-shaped pattern, and the maximal signal was obtained at 1.5 mg mL⁻¹ In₂O₃-ZnIn₂S₄. Additionally, Figure 4B exhibited the effect of PSA incubation time on PEC response. The photocurrent response decreased continuously from 10 to 50 min, and 30 min was chosen as the incubation time.

The stepwise construction process of the PSA-sensing platform was estimated by electrochemical impedance spectroscopy (EIS,) where the larger radius always means a lower charge transfer rate (Figure 5A). The corresponding equivalent circuit, including solution resistance (R_s), double-layer capacitance (C_d), the electrode transfer resistance (R_et), and the Warburg impedance (Z_W) (Table S1), is shown as an inset. Warburg impedance reflects charge diffusion from solution to electrode interface. During successive modification processes, the Warburg impedance gradually increased, indicating a continuous decrease in diffusion rate. R_et is a critical signal revealing the interfacial properties of the modified procedure. It could be observed that the electron-transfer resistance (Ret) elevated dramatically after immobilization of the In₂O₃-ZnIn₂S₄ complex onto the FTO surface due to their low conductivity. With the identify element and blocking agent modification progress, Ret showed an upward trend. This is because the large steric hindrance of the aptamer and BSA diminished the charge transfer capacity. Moreover, the transient photocurrent responses were also measured using 0.1 M ascorbic acid solution as the sacrificial agent (Figure 5B). As expected, FTO/In₂O₃-ZnIn₂S₄ (red curve, ~4.13 µA) showed significant photocurrent enhancement compared with FTO/In₂O₃ (black curve, ~1.33 µA) thanks to the heterointerfaces promoting photogenerated electron-hole separation. Under visible light radiation, photoinduced electrons and holes were generated on the In₂O₃ and ZnIn₂S₄ surfaces. Driven by the internal electric field, photoinduced electrons and holes accumulated on the conduction band of ZnIn₂S₄ and valence band of In₂O₃, respectively. An enhanced anodic photocurrent was obtained, and holes were captured by an electron donor (ascorbic acid). After the photoelectrode was incubated with a non-conductive aptamer and BSA, the continuously decreasing photocurrent value was obtained (blue curve, ~3.41 µA; green curve, ~2.76 µA). The EIS and PEC response demonstrated continuous fixation of biomolecules on the FTO electrode.

3.3. Analytical Performance

Based on this well-designed PEC biosensor, we further explored its capacity for the quantification of PSA antigen. According to the analysis protocol mentioned in the experimental section, PSA at different concentrations was applied to photoelectrodes and acquired PEC signals were analyzed. After incubation with 0.001 ng/mL PSA, the photocurrent intensity abated (red curve in Figure 6A). This may be because aptamer–PSA binding events enlarged steric hindrance and inhibited the diffusion of ascorbic acid to the electrode surface. As the PSA concentration increased from 0.001 to 50 ng/mL, the photocurrent intensity gradually decreased. In other words, there was an excellent negative correlation between photocurrent and logarithmic value of PSA concentrations, and the corresponding regression curve was −ΔI = 1.441 + 0.349lgC_PSA (ng/mL) (Figure 6B). This proposed PEC sensing platform possessed a linearity (R²) at 0.991 with a detection limit at 0.00037 ng/mL (S/N = 3). Such performance is primarily because of rapid photoinduced charge separation and specific biometric events between the aptamer and the PSA antigen.

To further assess proposed PEC biosensors, both selectivity and stability were monitored. As illustrated in Figure 6C, significant photocurrent change only appeared in the presence of 1 ng/mL PSA, not other 1 ng/mL substances, including carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), and immunoglobulin G (IgG). This result indicates that those interferences had almost no impact on the sensing performance. Moreover, the photocurrent response of the FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA electrodes at the 4th week maintained 85% of its original value (Figure 6D). Results indicate acceptable storage stability.

4. Conclusions

In summary, we successfully constructed an effective PEC biosensor based on FTO/In₂O₃-ZnIn₂S₄ sensitization structure and a PSA-sensitive aptamer for sensitive analysis of PSA. In₂O₃ tubes were first prepared by a two-step hydrothermal and annealing method, followed by the in-situ growth of ZnIn₂S₄ nanosheets. In this process, the generated tubular composite established efficient energy-level matching between In₂O₃ and ZnIn₂S₄. These intimate interface contacts inhibited the photocarrier recombination and made effective migration of photoinduced electrons and holes, thus obtaining desirable initial PEC signals. Additionally, biological binding sites on the hybrid In₂O₃-ZnIn₂S₄ contributed to the aptamer–protein event and further achieved PSA sensing. The constructed PEC biosensor presented a wide detection range from 0.001 to 50 ng/mL, with a detection limit down to 0.00037 ng/mL. The developed PEC sensing platform has high sensitivity, satisfying selectivity and stability, and guides the optimal PEC electrode construction for PSA detection.