Photoelectrochemical Biosensor Based On 1D ZnIn₂S₄ Nanosheet Decorated 2D In₂O₃ Tube For Sensitive PSA Detection

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 signals strongly depend on 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), 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 suffered from poor PEC performance, which was mainly contributed to high carrier rate, limited photon utilization, and deficient surface active sites. Multiple reports have been demonstrated heterojunction construction by coupling else semiconductor with different band gaps could prominently enhance PEC performance of In₂O₃-based materials [5,6,7,8].

Up to now, multiple In2O3-based heterostructures, such as In₂O₃/Co₃O₄ [9], In₂O₃/g-C₃N₄ [10], In₂O₃/CdS [11], and In₂O₃/In₂S₃ [12], have been prepared with improved photoelectric transformation efficiency. However, mismatched lattice between these semiconductors and In₂O₃ usually increase heterointerface impedance and restrict carriers separation. Compared with above mentioned semiconductors, 2D layered ZnIn₂S₄ has drawn increasing attention due to short carrier migration pathways, rich active sites, and similar lattice parameters [13,14]. High lattice matching degree of In₂O₃ and ZnIn₂S₄ is conducive to the formation of compact interface and thus greatly decreases charge immigration impedance of the heterointerface, and finally facilitates spatial charge separation [15]. A significant challenge is that ZnIn₂S₄ sheets tend to agglomerate into nanoclusters, causing low specific surface area and reduced active sites [16,17]. Supporting carriers including CdS nanocube, NiMoOx nanorod, FeWO₄ flower, and Co₉S₈ tube, favor lamellar growth and inhibit agglomerate [18,19,20,21]. Thus, it is of great favor to employ 1D In₂O₃ as the supporter to offer abundant area for nanosheet growth and conduction paths for photogenerated carrier transport. With such design, branched ZnIn₂S₄ nanosheets on In₂O₃ tubes could facilitate solar-light harvesting and intimate heterointerface could modulate the migration pathway for extending the photogenerated charge lifetime.

In this work, a 1D/2D heterostructure was designed by decorating ZnIn2S4 nanosheets onto In₂O₃ tubes to integrate with the prostate antigen (PSA)-sensitive aptamer for PSA quantification. The overall heterostructure preparation process involved template method and in-situ growth strategy where MIL-68 was transformed into tubular In₂O₃ and further modification of ZnIn₂S₄ nanosheets (Figure 1A). Under visible light, nanosized heterogeneous interface favored the effective separation and migration of electron-hole pairs, endowing hybrid with desirable PEC performance (Figure 1B). Furthermore, modified aptamer on the PEC electrodes could specifically binding with target, thus a relevance between PSA concentration and photocurrent response was established. Such simple and efficient sensing protocol has great potential in other biomolecule quantification.

2. Materials and Methods

2.1. 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. Resultant solution was heated at 120 °C for 30 min, followed by filtrated 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.

2.2. 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.

2.3. Fabrication of Sensing Platform and Analysis Protocol

FTO glass was pre cleaned sequentially with acetone, ethanol, and deionized water under violent ultrasonication. In order to obtain an attractive PEC signal, 1 mL prepared In₂O₃-ZnIn₂S₄ solution was spin-coated onto the FTO glass, followed by drying under an infrared lamp for 30 min. 50 μL chitosan aqueous solution (0.08 wt%) in 1% acetic acid was dropped onto 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 dealt with 20 µL 1 µM aptamer for 70 min at 4℃, followed by the addition of 10 μL of 2wt% bovine serum albumin (BSA) to block non-specific binding sites. Obtained working electrode was stored at 4℃ and denoted as FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA. Before PEC measurements, FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA was incubated with 20 μL PSA at room temperature for 30 min. Notably, FTO electrodes were thoroughly cleaned by 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.

2.4. Detection Limit Calculation

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

3. Results and Discussion

3.1. Morphology and Structure Characterization

Hybrid In₂O₃-ZnIn₂S₄ was synthesized by 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 amount of nanosheets (Figure 2B and Figure S1). After loading nanosheets onto In₂O₃, 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 and D). Significantly, compared self-assembled ZnIn₂S₄ nanosheet clusters, interconnected nanosheets is 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) [22]. These images demonstrated the successful fabrication of 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, respectively. As shown in Figure 3A, a typical absorption edge at ~425 nm and poor light-harvesting capacity in visible light region were demonstrated for pure In₂O₃. For hybrid In₂O₃-ZnIn₂S₄, a robust photo-absorption in UV and visible light region to ~542 nm was obtained. Using Tauc plots method [23], 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 above materials were 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°, 60.7° in black curve were well-matched with characteristic standard In₂O₃ data (JCPDS No. 06-0416) [24]. Except diffraction peaks of In₂O₃, additional peak at 47.2° gathered from In₂O₃-ZnIn₂S₄ was assigned to (1 1 0) crystal plane of pure ZnIn₂S₄. To probe the elemental valence details, X-ray photoelectron spectroscopy (XPS) was performed with C 1s peak as the standard reference. In the survey spectrum, the presence of In, O, Zn, and S elements was confirmed, consistent with above mentioned mapping diagram. In specific, two binding energies at 1022.3 and 1045.5 eV were attributed to Zn 2p3/2 and Zn 2p1/2 of Zn²⁺ chemical state. 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⁺. S 2p spectrum could be deconvoluted into two characteristic S²⁻ peaks at 161.8 and 163.0 eV. In a word, above mentioned results matched each other, illustrating the successful fabrication of tubular In₂O₃-ZnIn₂S₄.

3.2. PEC and EIS Behaviors

Stepwise modification process of PSA sensing platform was estimated by electrochemical impedance spectroscopy (EIS) where the larger radius always means lower charge transfer rate (Figure 4A). It could be observed that the electron-transfer resistance (Ret) elevated dramatically after immobilization of In₂O₃-ZnIn₂S₄ complex onto the FTO surface due to their low conductivity. With the identify element and blocking agent modification progress, Ret witnessed an upward trend. This is because large steric hindrance of aptamer and BSA diminished charge transfer capacity. Moreover, the transient photocurrent responses were also measured using 0.1 M ascorbic acid solution as the sacrificial agent (Figure 4B). As expected, FTO/In₂O₃-ZnIn₂S₄ (red curve) showed a significant photocurrent enhancement compared with FTO/In₂O₃ (black curve) thanks to the heterointerfaces promoting photogenerated electron-hole separation. Under visible light radiation, photoinduced electrons and holes were generated on the In₂O₃ and ZnIn₂S₄ surface. Driven by the internal electric field, photoinduced electrons and holes were accumulated on the conduction band of ZnIn₂S₄ and valance band of In₂O₃, respectively. An enhanced anodic photocurrent was obtained and holes were captured by electron donor (ascorbic acid). After photoelectrode incubated with non-conductive aptamer and BSA, the continuously decreasing photocurrent value was obtained (blue and green curves). EIS and PEC response demonstrated continuous fixation of biomolecules on the FTO electrode.

3.3. Analytical Performance

PSA level, as an early portent of prostate dysfunction, is associated with prostate cancer, underscoring the importance of sensitive PSA detection. Thus, based on this well-designed PEC biosensor, we further explored its capability for the quantification of PSA antigen. According to analysis protocol mentioned in experimental section, PSA at different concentrations was applied onto photoelectrodes and acquired PEC signals were analyzed. After incubation with 0.001 ng/mL PSA, the photocurrent intensity abated (red curve in Figure 5A). This may because aptamer-PSA binding events enlarged steric hindrance and inhibited the diffusion of ascorbic acid to electrode surface. As PSA concentration increased from 0.001 to 50 ng/mL, the photocurrent intensity gradually reduced. In other words, there was an excellent negative correlation between photocurrent and logarithmic value of PSA concentrations and corresponding regression curve was -ΔI=1.441+0.349lgC_PSA (ng/mL) (Figure 5B). 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 rapid photoinduced charges separation and specific biometric events.

To further assess proposed PEC biosensors, both selectivity and stability were monitored. As illustrated in Figure 5C, significant photocurrent change was only appeared in the presence of 1 ng/mL PSA not other substances including carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), and immunoglobulin G (IgG). This result indicated those interferences had almost no impact on sensing performance. Moreover, the photocurrent response of FTO/In₂O₃-ZnIn₂S₄/aptamer/BSA electrodes on 4th week maintained 85% of original value. Results indicated acceptable storage stability.

4. Conclusions

In summary, we successfully constructed an effective PEC biosensor based on FTO/In₂O₃-ZnIn₂S₄ sensitization structure and PSA-sensitive aptamer for sensitive analysis of PSA. In₂O₃ tubes were firstly prepared by two-step hydrothermal and annealing methods, followed by the in-situ growth of ZnIn₂S₄ nanosheets. In this process, generated tubular composite established efficient energy level matching between In₂O₃ and ZnIn₂S₄. This intimate interface contacts inhibited the photocarrier recombination and made effective migration of photoinduced electrons and holes, thus obtaining desirable initial PEC signals. Besides, biological binding sites on hybrid contributed to aptamer-protein event and further achieved PSA sensing. The constructed PEC biosensor. 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. Developed PEC sensing platform has high sensitivity, satisfying selectivity and stability, and guide the optimal PEC electrode construction for PSA detection.