Strategy method for detecting TP53 through octopus-shaped CdS QDs net and multivalent Au-S bond mediated photoelectrochemistry

Abstract

The invention discloses an octopus-shaped CdS QDs net and multivalent Au-S bond mediated photoelectrochemical detection strategy method for TP53. The method comprises the following steps: (1) preparing a TiO2-MXene (at) Au photoelectrode; (2) carrying out target identification and signal amplification reaction; and (3) adding a reaction solution obtained in the step (2) to the TiO2-MXene (at) Au photoelectrode obtained in the step (1) as a working electrode, and carrying out PEC measurement by taking a platinum wire as a counter electrode. An octopus-shaped CdS QDs net is formed based on a terminal deoxynucleotidyl transferase (TdT) chain extension reaction and a biotin-streptavidin coupling reaction, and a large number of CdS QDs can be enriched to the surface of the electrode through multivalent Au-S bond anchoring, so that the output of photoelectric signals is enhanced, and the sensitive detection of the cancer suppressor gene TP53 is realized.

Description

Technical Field

[0001] The present invention belongs to the field of detection, and specifically relates to an octopus-shaped CdS QDs network and a multivalent Au-S bond-mediated photoelectrochemical detection strategy method for TP53. Background Art

[0002] TP53 is a key tumor suppressor gene that protects genomic stability and prevents cancer development. Inherited TP53 mutations can affect cancer progression and treatment response by altering p53 protein function, potentially promoting tumor growth. Even subtle TP53 mutations can be clinically relevant. Therefore, establishing a simple, sensitive method for identifying and quantifying TP53 is crucial for early diagnosis and screening of the disease.

[0003] To date, there are only a few methods available for detecting hTR, including Northern blot analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. While these methods can achieve satisfactory sensitivity, they still suffer from drawbacks such as complex and time-consuming procedures. In recent years, with the advancement of nanotechnology, several new methods for detecting TP53 have emerged, including electrochemiluminescence (ECL), electrochemistry (EC), fluorescence (FL), surface-enhanced Raman scattering (SERS), and photoelectrochemistry (PEC). PEC stands out for its low background noise and high sensitivity, primarily due to its separation of photoexcitation and signal detection. However, current PEC biosensors still face numerous challenges in practical application. For example, confining both target recognition and signal generation to a single electrode surface complicates probe orientation control, limits hybridization efficiency, and increases the risk of interference. To overcome these limitations, innovative strategies are urgently needed to enhance the target capture and signal amplification capabilities of PEC biosensors to ensure their superior performance in complex biological environments.

[0004] In recent years, split photoelectrochemical biosensors have emerged as innovative detection platforms, separating target recognition from photoelectrode modification and signal transduction steps. This design minimizes interference and improves detection specificity. In such systems, efficient signal generation relies on the utilization of visible light, carrier mobility, and interfacial charge redistribution. Single semiconductors typically perform poorly due to the mismatch between photon absorption energy and redox potential, while inherent charge recombination and defect traps further limit efficiency. Notably, heterojunction photocatalysts (semiconductor-semiconductor / metal complexes) can address these limitations by enhancing charge separation, extending the light absorption range, and optimizing interfacial charge management. Compared to single-unit heterojunctions, multi-unit heterojunctions exhibit significant advantages in charge dynamics and solar energy utilization. Core-shell structures have shown great potential by maximizing the light absorption surface area and suppressing charge recombination through spatial confinement. The signal generation efficiency of PEC systems is highly dependent on the spatial configuration of the heterojunction components, interfacial charge transfer kinetics, and the density of photogenerated electrons. Against this backdrop, core-shell structures (comprising a semiconductor core and a photosensitive shell) have emerged as promising photoactive materials. These structures enhance light-harvesting capabilities through three mechanisms: (i) broadband light absorption, (ii) expanded active surface area, and (iii) suppressed charge recombination. Recent advances have employed DNA-guided assembly strategies, including hairpin probe-mediated interfacial assembly or solution-phase preassembly followed by DNA hybridization for immobilization on electrodes. Despite progress in charge separation and photocatalytic efficiency, constructing functional multi-heterojunctions remains challenging. Key limitations include sluggish DNA hybridization kinetics, insufficient coupling sites at the core-shell interface, and the lack of robust scaffolds for immobilizing high-density photoactive materials. These factors collectively hinder interfacial carrier redistribution and limit the strength of the PEC signal. Therefore, there is an urgent need to develop new photodetection platforms with a good detection range for the simultaneous detection of disease-associated TP53. Summary of the Invention

[0005] The technical problem addressed by this invention is this: To overcome the limitations of single-unit heterojunction-mediated photoelectric detection platforms, we innovatively propose an octopus-inspired multi-heterojunction-mediated photoelectrochemical detection strategy for TP53. This multi-heterojunction exhibits precise spatial arrangement, robust material coupling, and efficient charge redistribution, significantly enhancing the PEC signal and boosting target detection sensitivity.

[0006] The technical solution of the present invention is: an octopus-shaped CdS QDs network and a multivalent Au-S bond-mediated photoelectrochemical detection strategy for TP53, comprising the following steps: (1) Preparation of TiO2-MXene@Au photoelectrode First, TiO2 nanofibers were modified on the ITO electrode by electrospinning, and MXene was integrated into the TiO2 surface to form an internal heterojunction TiO2-MXene. Then, Au NPs were reduced by ascorbic acid to prepare the TiO2-MXene@Au photoelectrode.

[0007] (2) Target recognition and signal amplification reaction a. Design a 3'-thiolated DNA probe SH-P1 targeting the tumor suppressor gene TP53. Mix SH-P1 with the sample to be tested, and the two hybridize to form a double-stranded structure. b. Using the double-stranded strand generated in step a as the initiator strand, terminal deoxynucleotidyl transferase (TdT)-mediated polymerase extension is triggered to generate thymine-rich DNA nanowires, which serve as "octopus tentacles"; c. 5'-Aminated thymine-rich (T-rich) single-stranded DNA (P2) was coupled with carboxyl-modified CdS QDs to generate CdS QDs-P2 conjugates; d. Bivalent biotinylated adenine-rich (A-rich) DNA (P3) is coupled with streptavidin to form an A-rich DNA network, which serves as the "octopus head." This network is then incubated with the products obtained in b and c to form an octopus-shaped CdS QD network based on AT base complementation. (3) The reaction solution of step (2) was added to the TiO2-MXene@Au photoelectrode of step (1) as the working electrode, and PEC measurement was performed using a platinum wire as the counter electrode.

[0008] Furthermore, the nucleotide sequence of the probe SH-P1 is shown in SEQ ID No. 1, and the 3' end is thiolated.

[0009] Furthermore, the nucleotide sequence of the probe P2 is shown in SEQ ID No. 2, and the 5' end is labeled with an amino group.

[0010] Furthermore, the nucleotide sequence of the probe P3 is shown in SEQ ID No. 3, and the 3' end and the 5' end are modified with biotin.

[0011] Furthermore, the nucleotide sequence of the target tumor suppressor gene TP53 is shown in SEQ ID No.4.

[0012] Furthermore, the nucleotide sequence of T1 is shown as SEQ ID No.5.

[0013] Furthermore, the nucleotide sequence of T2 is shown as SEQ ID No.6.

[0014] Furthermore, the nucleotide sequence of T3 is shown in SEQ ID No.7.

[0015] Furthermore, in the TdT-mediated chain extension reaction, the concentration of TdT polymerase was 10 U.

[0016] Furthermore, when performing target recognition, the concentration of the probe SH-P1 was 10 μM.

[0017] Furthermore, the chain extension reaction time was 2 h.

[0018] Furthermore, the preparation method of the probe P2 connected to CdS QDs via amino groups is as follows: 30 μL of CdS QDs with a concentration of 0.5 mg / mL is added to 3.75 μL of a mixed solution of EDC / NHS, activated at 37°C for 1 h, and then 10 μL of the P2 probe shown in SEQ ID No. 2 with a concentration of 20 μM is added, and the mixture is placed in a shaker at 37°C for 12 h to obtain a CdS QDs-P2 conjugate. Beneficial effects:

[0019] We innovatively propose an octopus-shaped CdS QD network and multivalent Au-S bonds-mediated photoelectrochemical detection of TP53. In this system, titanium dioxide nanofibers (TiO2) were synthesized by electrospinning to serve as a PEC signal generator. MXene was incorporated into the TiO2 to form an internal heterojunction (TiO2-MXene), and AuNPs were subsequently grafted onto the TiO2-MXene surface to create a Schottky junction. The octopus-shaped CdS QD structure and multivalent Au-S bonds together ensure precise spatial alignment, robust material coupling, and efficient charge redistribution, enhancing the PEC signal. Electron paramagnetic resonance studies reveal that photogenerated electrons, holes, and free radicals contribute to the photocatalytic process. The biosensor can detect TP53 as low as 0.019 pM, exhibits excellent stability, and operates reliably in real samples. This platform provides a powerful tool for diagnosing and monitoring TP53-related diseases. Given the demand for early disease analysis and diagnosis, this strategy shows great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of the strategy for photoelectrochemical detection of TP53 based on octopus-shaped CdS QDs network and multivalent Au-S bonds.

[0021] Figure 2:(A) Polyacrylamide gel electrophoresis characterization of DNA nanostructure assembly;(B) Agarose gel electrophoresis characterization of DNA nanostructure assembly;(C) Photocurrent response of TiO2-MXene@Au photoelectrode in the presence and absence of TP53;(D) Photocurrent response during electrode assembly;(E) Impedance diagrams of different electrodes in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 M KCl.

[0022] Figure 3 : Scanning electron microscopy (SEM) images of (A) TiO2, (B) TiO2-Mxene, (C) TiO2-Mxene@Au, and (D) TiO2-Mxene@Au@CdS QDs.

[0023] Figure 4 : High-resolution transmission electron microscopy (HRTEM) images of TiO2-MXene@Au@CdS QDs (A and B), and (C) their corresponding TiO2-MXene@Au@CdS QDs mapping.

[0024] Figure 5 : (A) XRD spectra of TiO2, TiO2-MXene, TiO2-MXene@Au and TiO2-MXene@Au@CdS QDs; (B) XPS spectra of TiO2-MXene@Au@CdS QDs; (C) Ti 2p; (D) O 1s; (E) C 1s; (F) Au 4f; (G) Cd3d; (H) S 2p orbitals.

[0025] Figure 6 : MXene concentration (A); effect of AA concentration during Au NPs deposition (B); TdT concentration (C); incubation time (D); P2 concentration (E) and CdS QDs concentration (F) on the photoelectrochemical response of the biosensor.

[0026] Figure 7 : (A) PEC spectra at different TP53 concentrations; (B) PEC response as a function of TP53 concentration; the inset shows the linear relationship between PEC intensity and the logarithm of TP53 concentration; (C) Stability of the proposed strategy in TP53 determination; (D) Selectivity.

[0027] Figure 8(A) Possible transfer mechanism of photogenerated electrons at the (TiO2-MXene@Au@CdS QDs) electrode. EPR spectra of CdS QDs, TiO2, TiO2-MXene, TiO2-MXene@Au, and TiO2-MXene@Au@CdS QDs under irradiation: (B) TEMPO-h+; (C) TEMPO-e-; (D) DMPO-·OH; (E) DMPO-·O2-. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0029] 1. Materials and Methods 1. Reagents All DNA oligonucleotides were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.; their sequences are shown in Table 1. Streptavidin, chloroauric acid (HAuCl₄), tetrabutyl titanate, and polyvinylpyrrolidone (PVP) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Acetylacetone and ascorbic acid (AA, analytical grade) were obtained from Aladdin Biochemical Technology Co., Ltd. Cobalt chloride (CoCl₂), terminal deoxyribonucleotidyl transferase (TdT), and TdT buffer were purchased from New England Biotechnology (Beijing) Co., Ltd. Enzyme-free sterile water and deoxythymidine nucleotides (dTTPs) were provided by Sangon Biotech (Shanghai) Co., Ltd. Monolayer colloidal aqueous MXene materials were purchased from Beike Nanotechnology Co., Ltd. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Thermo Fisher Scientific (China). Water-soluble carboxyl-modified cadmium sulfide quantum dots (CdS QDs, λem = 570 nm) were obtained from Ruixi Biotechnology Co., Ltd. (Xi'an, China). Ethanol and acetic acid were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Blood was obtained from Liaocheng People's Hospital. All other reagents were of analytical grade. All aqueous solutions were prepared with ultrapure water (≥18 MΩ cm-1). Ultrapure water (≥18 MΩ cm-1) was prepared using a UP water purification system.

[0030] 2. Instruments Agarose gel electrophoresis was performed on an electrophoresis instrument, and the corresponding gel images were captured using a GelDoc™ XR+ imaging system (Bio-RAD Laboratories Inc., USA). Photoelectrochemical measurements were performed on a Zahner Cimp-2 workstation (Zahner, Germany) using a white light source at a wavelength of 570 nm. Electrochemical impedance spectroscopy (EIS) was measured using a CHI660E electrochemical workstation (Shanghai Chenhua) using a standard three-electrode system. The morphology of the prepared materials was characterized using transmission electron microscopy (TEM, JEM-F200JEOL, Japan) and scanning electron microscopy (SEM, Thermo Fisher Scientific FIB-SEM GX4). The elemental distribution and composition of the photoactive substances were analyzed using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). X-ray powder diffraction measurements were performed using a Kigaka D / max 2500 advanced X-ray diffractometer with Cu-Ka radiation and a scan step size of 0.02° (2θ range of 5 to 90°). X-ray photoelectron spectroscopy measurements were performed using a K-Alpha spectrometer (Thermo Science Ltd., USA).

[0031] 3. Preparation of TiO2-MXene and Au NPs modified electrodes The TiO2 electrode was prepared by spinning one-dimensional self-supporting titanium dioxide nanofibers (TiO2) onto an ITO substrate using electrospinning technology. The specific steps are as follows: Figure 1 As shown. First, 0.8 g of polyvinylpyrrolidone (PVP) was dissolved in a mixed solvent (1.4 mL of acetic acid + 8.4 mL of ethanol) at room temperature and magnetically stirred for 4 h. Subsequently, 1.8 mL of tetrabutyl titanate and 0.4 mL of acetylacetone were added, and stirring was continued for 2 h to obtain a uniform precursor solution. Spinning was performed using a standard electrospinning apparatus (voltage 19 kV, flow rate 5 mL / h, distance from nozzle to collector 15 cm) for 30 min. The spun fibers were collected on a receiving plate covered with aluminum foil and calcined in air at a heating rate of 5 °C / min, followed by sintering at 490 °C for 90 min. The MXene dispersion was coated on TiO2 by drop coating and dried at 60 °C for 1 h. For the deposition of Au NPs, the electrode was immersed in a 10 mM HAuCl4 solution with the addition of 1 mM AA, immersed for 2 h and then dried at 60 °C for 10 min to obtain the TiO2-MXene@Au photoelectrode.

[0032] 4. Target recognition and signal amplification reaction The target recognition reaction involves identifying the tumor suppressor gene TP53, while the signal amplification reaction involves constructing an octopus-shaped CdS QD network. 30 μL of CdS QDs were added to 3.75 μL of an EDC / NHS mixture and activated at 37°C for 1 hour. Then, 10 μL of 20 μM P2 was added. The mixture was incubated at 37°C in a shaker (600 rpm) for 12 hours to produce the CdS QDs-P2 conjugate. TP53 and SH-P1 were mixed in TdT buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, pH 7.90). The mixture was heated to 90°C for 5 minutes and then cooled to room temperature to produce a partially complementary double-stranded probe. Subsequently, 1 μL of CoCl₂, 25 mM deoxythymidine triphosphate (dTTP), and TdT were added to the reaction system, followed by incubation at 37°C for 1 hour to initiate polymerization and extension reactions, generating thymine-rich DNA nanowires that serve as "octopus tentacles." Simultaneously, the P3 probe self-assembled with streptavidin at 37°C for 30 minutes to form the "octopus head." Subsequently, the CdS QDs-P2 conjugate was added and incubated at 37°C for 2 hours to form an octopus-shaped CdS QDs network.

[0033] 5. Photoelectrochemical detection TP53 biosensor platform The TiO2-MXene@Au photoelectrode was incubated with 30 μL of the prepared octopus-shaped CdS QD mesh at 37°C for 2 h, forming a multi-component heterojunction via multivalent Au-S bonding. PEC experiments were performed in 0.2 M Na2SO4 solution (pH 7.40) at a potential of 0 V under white LED illumination. Measurements were performed on an electrochemical workstation (Zahner Zennium, Germany) using the TiO2-MXene@Au photoelectrode as the working electrode and a platinum wire as the counter electrode. Control experiments were performed under the same conditions, excluding TP53, to verify the specificity and efficiency of the PEC response.

[0034] 2. Results and Discussion 1. Feasibility verification of PEC sensing system To verify the TP53-mediated formation of thymine-rich (T-rich) DNA nanowires and octopus-shaped CdS QDs networks, the polymerization products were analyzed by polyacrylamide gel electrophoresis and 3% agarose gel electrophoresis. Figure 2As shown, distinct migration pattern differences were observed for TP53 (lane 2), SH-P1 (lane 3), and the SH-P1 / TP53 duplex (lane 4). In the presence of SH-P1 / TP53 and TdT (lane 5), only a single, slower-migrating band was observed, confirming the successful synthesis of high-molecular-weight thymine-rich DNA nanowires that can serve as "octopus tentacles." Figure 2 As shown, bivalent biotinylated adenine-rich (A-rich) P3 was coupled to streptavidin to form an A-rich DNA network, which served as the "octopus head." Its migration rate was slower than that of free P3 (lane 2), confirming the successful coupling of biotin and streptavidin. Further anchoring CdS QDs-P2 (lane 6) produced an octopus-shaped CdS QDs network.

[0035] In order to verify the feasibility of the constructed PEC biosensor for detecting TP53, a series of photoelectrochemical experiments were conducted ( Figure 2 ). In the experiment, TiO2 was assembled onto the electrode surface by electrospinning technology, MXene was integrated into TiO2 and AuNPs (Au) were grafted to form a core and Schottky junction. TP53 can mediate the generation of an octopus-shaped CdS QDs network and further assemble it onto the electrode surface with the help of multivalent Au-S bonds, significantly enhancing the photocurrent signal. In the absence of TP53, the thiol group occupies the 3' end of the SH-Primer, preventing the terminal deoxynucleotidyl transferase (TdT)-mediated polymerization extension and the formation of thymine-rich DNA chains. Therefore, the system cannot achieve CdS QDs anchoring. After constructing the TiO2-MXene@Au photoelectrode, only SH-P1 is anchored on the electrode surface, and free CdS QDs cannot be enriched on the above-mentioned photoelectrode surface. Due to the lack of high-density charge carriers in the detection area, the photoelectrochemical signal is very low ( Figure 2 , light green curve). These results confirmed the reliability of the developed PEC biosensor in TP53 detection.

[0036] In addition, the photoelectrochemical signal response and electrochemical impedance spectroscopy (EIS) were monitored simultaneously ( Figure 2 ). Compared with the TiO2 electrode alone ( Figure 2 , curve a), compared with the TiO2-MXene electrode ( Figure 2 , curve b) shows a lower charge transfer resistance (Rct), indicating enhanced conductivity and faster kinetics. In situ deposition of Au NPs by ascorbic acid (AA) reduction further reduced Rct ( Figure 2 , curve c), indicating that it has higher conductivity.

[0037] 2. Characterization of TiO2-MXene@Au@CdS QDs Scanning electron microscopy (SEM) was used to characterize the microstructure and morphology of TiO2-MXene@Au@CdS QDs. Figure 3 As shown in Figure 2, TiO2 exhibits an interwoven structure with a relatively rough surface and fiber diameters ranging from 100 to 300 nm. Figure 3 ), the fiber diameter increased slightly and the surface became smoother, which is mainly due to the higher conductivity of TiO2-MXene than pure TiO2, which is consistent with the impedance test results. There is no obvious aggregation of Au NPs distributed uniformly along the TiO2 surface ( Figure 3 ), which is conducive to the extensive capture of the octopus-shaped CdS QDs network through Au-S bonds. After the CdS QDs network is fixed ( Figure 3 ), the fibers are slightly thickened and the morphology is well maintained.

[0038] Further analysis by high-resolution transmission electron microscopy (HRTEM) confirmed the formation of TiO2-MXene@Au@CdSQDs multi-heterojunction ( Figure 3 ).exist Figure 4 In the figure, the lattice fringe spacings are 0.35 nm and 0.28 nm, corresponding to the (101) crystal plane of TiO2 and the (002) crystal plane of MXene, respectively, confirming the formation of the inner heterojunction. In addition, the lattice spacing of the Au (111) crystal plane is 0.23 nm, and the lattice spacing of the CdS QDs (101) crystal plane is 0.32 nm, indicating that the outer heterojunction interface is clear and there is a clear interface between TiO2-MXene and Au@CdS QDs. The above results preliminarily confirm the successful construction of the multi-heterojunction. It is worth noting that a large number of CdS QDs are uniformly anchored on the TiO2-MXene@Au surface, which proves the feasibility of the proposed strategy based on Au-S bonding CdS QD network. Element distribution map ( Figure 4 ) confirmed the presence and spatial distribution of Ti, O, C, Au, Cd, and S, further demonstrating the formation of a multi-layer heterojunction. The outer layer of quantum dots enhances visible light utilization and increases the local photogenerated electron density. Furthermore, the multi-layer heterojunction promotes rapid charge transfer and suppresses electron-hole pair recombination, thereby improving the performance of the photoelectrochemical sensor.

[0039] This study also characterized the proposed electrode structure by X-ray diffraction (XRD). Figure 5As shown in Figure 2, the diffraction peaks at 25.3°, 37.8°, 48.0°, 54.3°, 62.7° and 75.1° correspond to the (101), (004), (200), (105), (204) and (215) crystal planes of anatase TiO2 (JCPDS No. 21-1272), respectively. In addition, the diffraction peaks at 9.3°, 18.9° and 60.4° correspond to the (002), (006) and (110) crystal planes of MXene, respectively. For TiO2-MXene composite materials ( Figure 5 , curve c), the XRD pattern retains the diffraction peak of TiO2, and the MXene diffraction peak appears at the same time, confirming the successful composite of TiO2 and MXene. After the introduction of Au NPs, new diffraction peaks at 38.2°, 44.4°, 64.5° and 77.6° appear, corresponding to the (111), (200), (220) and (331) crystal planes of metallic Au (JCPDS No. 04-0784), respectively ( Figure 5 , curve d). In addition, the diffraction peaks at 27.5° and 47.8° correspond to the (101) and (102) planes of hexagonal CdS (JCPDS No. 72-0677), respectively. Figure 5 , curve e), indicating that the TiO2-MXene@Au@CdS QDs heterojunction has high crystallinity.

[0040] To further confirm the elemental composition and chemical valence state of the material, this study also used X-ray photoelectron spectroscopy (XPS) to analyze MXene-TiO2@Au@CdS. Figure 5The full XPS spectrum of MXene-TiO2@Au@CdS QDs, as well as high-resolution spectra of Ti, O, C, Au, Cd, and S, are presented. In the Ti 2p spectrum, the peaks at 464.1 eV and 458.5 eV correspond to the Ti-O bond of Ti4+ in TiO2, while the peaks at 460.7 eV and 454.5 eV are attributed to the Ti-C bond in the MXene. The O 1s spectrum shows peaks at 531.7 eV and 529.9 eV, attributed to the Ti-OH bond and the Ti-O-Ti bond, respectively. The C 1s spectrum reveals three components: C-C (286.3 eV), CO (284.7 eV), and C-Ti (288.7 eV). For Au, the peaks at 83.4 eV and 87.1 eV correspond to the Au 4f7 / 2 and Au 4f5 / 2 of metallic Au, respectively. In the Cd 3d spectrum, peaks at 405.4 eV and 412.2 eV confirm the presence of Cd2+, while the S 2p spectrum shows peaks at 164.5 eV and 169.1 eV, consistent with S2- in CdS QDs. These results are consistent with literature reports on MXene, Au NPs, Cd, and S. Overall, these results confirm the successful preparation of TiO2 / MXene@Au@CdS.

[0041] 3. TP53 sensing performance of PEC biosensor platform TiO2 is a promising anode material for photoelectrochemical (PEC) sensors. However, its low electronic conductivity limits its practical application. Ti3C2-MXene was introduced to enhance the performance of TiO2 due to its excellent structural stability and high conductivity. In order to optimize the effect of MXene on the performance of TiO2, the effect of MXene concentration was systematically studied ( Figure 6 ). Furthermore, key experimental parameters influencing the analytical performance of the PEC sensor were optimized, including AA concentration, TdT concentration, P2 concentration, CdS QD dosage, and incubation time. The optimal experimental conditions ultimately determined were: MXene concentration of 3 mg / mL, AA concentration of 6 × 10⁻³ M, TdT of 10 U, CdS QDs concentration of 0.6 mg / mL⁻¹, P2 concentration of 10 μM, and an incubation time of 1 h.

[0042] Under optimized experimental conditions, the constructed PEC biosensor platform achieved sensitive detection of TP53 with a wide linear range. Figure 7As shown, the photocurrent increases linearly with increasing TP53 concentration. This enhancement arises from the target-triggered binding of the octopus-shaped CdS QD network to the electrode via multivalent Au-S bonds, forming a multi-heterojunction that enhances the photocurrent. The photocurrent difference (ΔI, the difference in photocurrent between the presence and absence of TP53) is linearly correlated with TP53 concentration within the concentration range of 0.1 pM to 1.0 μM (R² = 0.990). The linear regression equation is ΔI = 249.43 lg CTP53 + 167.0, where ΔI represents the photocurrent change (nA) and C represents the TP53 concentration (mol / L, M). The detection limit (LOD) of this method is 0.019 pM, which is superior to most previous methods. This performance highlights the advantages of the octopus-shaped CdS QD network and the multivalent Au-S bonding strategy.

[0043] To evaluate the stability of the biosensor, intra- and inter-batch testing was performed at TP53 concentrations of 1 pM, 0.1 μM, and 1 μM. The intra-batch relative standard deviations (RSDs) were 2.13%, 1.98%, and 1.46%, respectively, while the inter-batch RSDs were all below 5.23%, confirming the high reproducibility of the method. To assess the selectivity of the PEC sensor for a specific target, several non-target analytes were introduced for testing. Figure 7 The PEC responses of the constructed biosensor were demonstrated in the presence of TP53, single-base mismatch (T1), double-base mismatch (T2), and non-complementary (T3) DNA. The signals for non-target sequences were similar to those for blank samples, demonstrating extremely high specificity. This selectivity stems from the higher thermodynamic stability of the SH-P1 probe when base-pairing with the target is complementary compared to mismatched sequences.

[0044] 4. Inspection of actual samples To evaluate the practical application potential of this biosensor, recovery testing was performed in human serum using the standard addition method. TP53 at varying concentrations was spiked into healthy human serum (diluted 30-fold in PBS), and the biosensor was then used to detect TP53. The average recoveries of TP53 in real samples ranged from 95.2% to 105.4%, with relative standard deviations (RSDs) ranging from 1.79% to 3.32% (n=3). These results confirm the reliability of this biosensor in real-world sample analysis.

[0045] 5. Charge transfer mechanism of photoelectrochemical PEC sensor In order to further explore the mechanism of photogenerated carriers (electrons, holes and free radicals) in the photocatalytic process, this study used electron paramagnetic resonance (EPR) technology. Figure 8The working mechanism of the constructed PEC biosensor is demonstrated. CdS QDs have attracted much attention due to their narrow band gap, enabling efficient absorption of visible light and generation of electron-hole pairs. In this system, the synergistic effect of the octopus-like CdS QD network and multivalent Au-S bonds enables high-density carrier redistribution within the TiO2-MXene electrode.

[0046] The introduced Au NPs have a Fermi level lower than the conduction band (CB) of CdS QDs. Under visible light irradiation, the octopus-shaped CdS QDs network can efficiently generate high-density carriers (electrons and holes). Among them, the generated holes (h + ) can effectively oxidize OH - , while electrons (e-) preferentially transfer from the CB of CdS QDs to the CB of Au NPs and the inner layer of TiO2-MXene. The Schottky barrier formed by the Au-CdS QDs interface and TiO2-MXene effectively inhibits the backflow of electrons, acts as an electron "trap", significantly suppresses charge recombination, and thus significantly amplifies the photocurrent response ( Figure 8 ). EPR results show that the generation of free radicals is significantly enhanced in the TiO2-MXene@Au@CdS QDs system. The generation of superoxide radicals (·O2-) is particularly significant ( Figure 8 ), which is attributed to the conduction band potential ratio of the material O2 / ·O2 - The redox potential of the ions is more negative, which promotes electron-mediated oxygen reduction. At the same time, the generation of hydroxyl radicals (·OH) is also very significant ( Figure 8 ), showing excellent redox activity and photoelectric efficiency. This multi-component heterojunction provides a powerful strategy for developing advanced PEC sensing platforms with higher sensitivity and broad applicability.

Claims

1. A strategy for photoelectrochemical detection of TP53 based on an octopus-shaped CdS QDs network and multivalent Au-S bonds, characterized in that: The steps include: (1) Preparation of TiO2-MXene@Au photoelectrode First, TiO2 nanofibers (TiO2) were modified on the surface of an ITO electrode by electrospinning, and MXene was integrated into the TiO2 surface to form an internal heterojunction TiO2-MXene. Then, Au NPs (Au) were reduced by ascorbic acid to prepare a TiO2-MXene@Au photoelectrode. (2) Target recognition and signal amplification reaction a. Design a 3'-thiolated DNA probe SH-P1 targeting the tumor suppressor gene TP53. Mix SH-P1 with the sample to be tested, and the two hybridize to form a double-stranded structure. b. Using the double-stranded strand generated in step a as the initiator strand, terminal deoxynucleotidyl transferase (TdT)-mediated polymerase extension is triggered to generate thymine-rich DNA nanowires, which serve as "octopus tentacles"; c. 5'-Aminated thymine-rich (T-rich) P2 was coupled with carboxyl-modified CdS QDs (CdS QDs) to generate CdS QDs-P2 conjugates; d. Bivalent biotinylated adenine-rich (A-rich) DNA (P3) is coupled with streptavidin to form an A-rich DNA network, which serves as the "octopus head." This network is then incubated with the products obtained in b and c to form an octopus-shaped CdS QDs network based on AT base complementation. (3) The reaction solution from step (2) was added to the TiO2 / MXene@Au photoelectrode from step (1) as the working electrode, and PEC measurement was performed using a platinum wire as the counter electrode.

2. The method according to claim 1, characterized in that The nucleotide sequence of the SH-P1 is shown in SEQ ID No. 1, and the 3' end is thiolated.

3. The method according to claim 1, characterized in that The nucleotide sequence of the probe P2 is shown in SEQ ID No. 2, and the 5' end is labeled with an amino group; the nucleotide sequence of the probe P3 is shown in SEQ ID No. 3, and the 3' end and the 5' end are modified with biotin.

4. The method according to claim 1, wherein The nucleotide sequence of the target tumor suppressor gene TP53 is shown in SEQ ID No.

4.

5. The method according to claim 1, wherein For TdT-mediated chain extension reactions, the concentration of TdT polymerase was 10 U.

6. The method according to claim 1, wherein The concentration of SH-P1 was 10 μM for target recognition.

7. The method according to claim 1, characterized in that The chain extension reaction time was 2 h.

8. The method according to claim 1, characterized in that The preparation method of the probe P2 connected to CdS QDs via amino groups is as follows: 30 μL of CdS QDs with a concentration of 0.5 mg / mL is added to 3.75 μL of a mixed solution of EDC / NHS, and the mixture is activated at 37°C for 1 h. Then, 10 μL of the P2 probe shown in SEQ ID No. 2 with a concentration of 20 μM is added, and the mixture is placed in a shaker at 37°C for 12 h to obtain a CdS QD-P2 conjugate.

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