A method for real-time monitoring of intracellular NADH based on a nanowire-coupled photoelectrochemical sensor

Abstract

This invention provides a method for real-time monitoring of intracellular NADH based on a nanowire-coupled photoelectrochemical sensor, belonging to the field of biosensor technology. The method utilizes nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs that can spontaneously and non-destructively semi-embed in cells, reacting with intracellular NADH to generate electron transfer events. A photosensitive electrode Flex ITO-WO3 / BiVO4, coated on the cell surface, is used to gather the electrical signal from the nanowires, accelerating the intracellular oxidation reaction and improving the current response. This method enables real-time monitoring of intracellular NADH. It offers advantages such as low cost, simple operation, and high sensitivity, and is widely applicable to the detection of intracellular signaling molecules.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for real-time monitoring of intracellular NADH based on a nanowire-coupled photoelectrochemical sensor. Background Technology

[0002] Redox balance is fundamental to cellular homeostasis, and its disruption is associated with various pathophysiological conditions, such as inflammation, neurodegenerative diseases, and cancer. [1] In this process, nicotinamide adenine dinucleotide (NADH) plays an indispensable role as an important coenzyme in the cytoplasmic glycolysis pathway and the mitochondrial respiratory chain. [2] NADH is not only a common electron donor, providing energy for numerous intracellular biochemical reactions, but it also participates extensively in a series of important physiological processes such as DNA repair and epigenetic modification. [1] Furthermore, changes in NADH levels are closely associated with DNA damage caused by genotoxic substances, thus serving as a potential biomarker for genotoxicity to help assess cellular sensitivity and the extent of damage. These functions highlight NADH's central role in cellular metabolism and physiological regulation. Therefore, detecting endogenous NADH levels can not only provide a deeper understanding of cellular biochemical dynamics but also offer potential biomarkers for the early diagnosis and treatment of related diseases.

[0003] Currently, various methods for the detection of NADH have been established, such as colorimetric reactions based on WST-8, high-performance liquid chromatography, and fluorescence detection. [2,3] However, these methods require complex sample pretreatment steps and cannot monitor endogenous NADH in living cells in real time. Recent studies have found that NADH can undergo oxidation with PFBT Pdots, generating electron transfer events. [4,5] Based on this property, we propose using intracellular electrochemical sensors to monitor intracellular NADH in real time and non-destructively. However, current intracellular electrochemical sensing technology is still in its early stages of development, typically requiring the manual insertion of a single nanowire electrode into the cell using expensive micromanipulators. [6-9] The application scenarios are limited. Furthermore, because only a single nanowire is used as the medium, the electrochemical response is often weak, and the detection limit can only reach the μM level at its highest.

[10] Therefore, there is an urgent need to develop more efficient and sensitive electrochemical sensing technologies to improve the ability to monitor intracellular signaling molecules.

[0004] References

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[0006] [2] X. Zhao, R. Niu, S. Fan, X. Jing, R. Gao, H. Yang et al, A Dual-Mode NADH Biosensor Based on Gold Nanostars Decorated CoFe2 Metal–OrganicFrameworks to Reveal Dynamics of Cell Metabolism, ACS Sensor. 2022, 7: 2671-2679.

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[0008] [4] N. Wang, X. Cao, D. Sun, X. Li, G. Tian, J. Feng et al, A polymerdot-based NADH-sensitive electrochemiluminescence biosensor for analysis ofmetabolites in serum, Talanta. 2024, 267: 125149.

[0009] [5] H. Chen, J. Yu, X. Men, J. Zhang, Z. Ding, Y. Jiang et al,Reversible ratiometric NADH sensing using semiconducting polymer dots, AngewChem Int Edit. 2021, 60: 12007-12012.

[0010] [6] W. Wu, X. Chen, Y. Jiao, W. Fan, Y. Liu, W. Huang, VersatileConstruction of Biomimetic Nanosensors for Electrochemical Monitoring ofIntracellular Glutathione, Angew Chem Int Edit. 2022, 61: e202115820.

[0011] [7] A. N. Vaneev, P. V. Gorelkin, A. S. Garanina, H. V. Lopatukhina,S. S. Vodopyanov, A. V. Alova et al, In vitro and in vivo electrochemicalmeasurement of reactive oxygen species after treatment with anticancer drugs,Anal Chem. 2020, 92: 8010-8014.

[0012] [8] K. Hu, Y. Li, S. A. Rotenberg, C. Amatore, M. V. Mirkin,Electrochemical measurements of reactive oxygen and nitrogen species insidesingle phagolysosomes of living macrophages, J A Chem Soc. 2019, 141: 4564-4568.

[0013] [9] Y. Jiao, H. Jiang, W. Wu, Y. Qi, M. Wen, X. Yang et al, Dual-channel nanoelectrochemical sensor for monitoring intracellular ROS and NADHkinetic variations of their concentrations, Biosens Bioelectron. 2023, 222:114928.

[0014]

[10] Y. Jiao, Y. Kang, M. Wen, H. Wu, X. Zhang, W. Huang, Fastantioxidation kinetics of glutathione intracellularly monitored by a dual-wire nanosensor, Angew Chem Int Edit. 2023, 135: e202313612. Summary of the Invention

[0015] This invention addresses the problems of existing technologies by providing a method for real-time monitoring of intracellular NADH based on a nanowire-coupled photoelectrochemical sensor. For the first time, this invention constructs an optoelectronically integrated nanowire (MWCNT@FITC-PFBTPdots-PDDA-Pt NWs), combined with a photosensitive electrode (Flex ITO-WO3 / BiVO4) covering the cell surface, to achieve real-time in-situ monitoring of intracellular NADH. Furthermore, addressing the shortcomings of existing intracellular electrochemical sensing technologies that heavily rely on micromanipulators and single nanowire electrodes, this invention proposes for the first time a nanowire that can spontaneously and non-destructively semi-embed in the cell as an artificial transmembrane electron tunnel, combining photoelectrochemical sensing and electrochemiluminescence technologies to provide a new strategy for sensing intracellular signal molecules.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0017] The first aspect of the present invention provides a nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs comprising multi-walled carbon nanotubes (MWCNT), fluorescein isothiocyanate (FITC), poly(9,9-dioctylfluorenyl-alt-benzothiadiazole) semiconductor polymer quantum dots (PFBT Pdots), polydiallyl dimethylammonium chloride (PDDA), and platinum (Pt).

[0018] A second aspect of the present invention provides a method for preparing the nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs, comprising the following steps:

[0019] (1) Fluorescein isothiocyanate (FITC) was linked to aminated multi-walled carbon nanotubes (MWCNTs) via a nucleophilic substitution reaction to obtain MWCNT@FITC;

[0020] (2) Poly(9,9-dioctylfluorenyl-alt-benzothiadiazole) semiconductor polymer quantum dots (PFBT Pdots) were bonded to MWCNT@FITC by amidation reaction to obtain MWCNT@FITC-PFBTPdots;

[0021] (3) Polydiallyldimethylammonium chloride (PDDA) was bound to MWCNT@FITC-PFBT Pdots by electrostatic interaction to obtain MWCNT@FITC-PFBT Pdots-PDDA;

[0022] (4) Coating MWCNT@FITC-PFBT Pdots-PDDA with a layer of platinum metal to obtain the nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs.

[0023] A third aspect of the present invention provides a photosensitive electrode Flex ITO-WO3 / BiVO4, wherein the photosensitive electrode is a type II n-heterojunction nanocomposite material formed by tungsten oxide (WO3) and bismuth vanadate (BiVO4) on a flexible indium tin oxide (FlexITO) thin film.

[0024] A fourth aspect of this invention provides a method for preparing the above-mentioned photosensitive electrode Flex ITO-WO3 / BiVO4, comprising the following steps:

[0025] (i) WO3 was prepared using Na2WO4·2H2O as a raw material;

[0026] (ii) BiVO4 was prepared using Bi(NO3)3·5H2O as a raw material;

[0027] (iii) WO3 and BiVO4 are sequentially drop-coated onto the surface of the Flex ITO film to obtain the photosensitive electrode FlexITO-WO3 / BiVO4.

[0028] The fifth aspect of this invention provides a method for real-time monitoring of intracellular NADH changes based on a nanowire-coupled photoelectrochemical sensor, comprising the following steps:

[0029] S1. Nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs were incubated with cells, and the nanowires were partially embedded into the cells through endocytosis.

[0030] S2. Cover the cell surface with the photosensitive electrode Flex ITO-WO3 / BiVO4, with the side carrying the WO3 / BiVO4 photosensitive material in contact with the cell. Place the working electrode, reference electrode, and counter electrode in contact with the other side of the photosensitive electrode. Under illumination, measure the intracellular NADH current response in real time.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] First, this invention provides a method for real-time monitoring of intracellular NADH levels based on a nanowire-coupled photoelectrochemical sensor. This breakthrough not only contributes to a deeper understanding of cellular energy metabolism and redox balance but also provides a new tool for assessing the degree of DNA damage. Second, this invention achieves electrochemical sensing of intracellular signaling molecules without relying on expensive micromanipulators. Utilizing nanowires as artificial transmembrane electron tunnels significantly improves detection sensitivity. Furthermore, the photoelectrochemical sensor is applied to the cell surface, accelerating intracellular oxidation reactions and promoting electron extraction, further enhancing the sensor's ability to detect trace intracellular signaling molecules, providing an efficient and sensitive analytical tool for biomedical research and clinical diagnosis. Attached Figure Description

[0033] Figure 1 This refers to the potential changes during the stepwise fabrication process of the nanowires in Example 1.

[0034] Figure 2 Fluorescent images of the nanowires prepared in Example 1.

[0035] Figure 3 Cyclic voltammetry curves of glassy carbon electrodes (GCE) modified with different nanowires in Example 1 in 1 mM NADH solution and 0.1 M pH 7.4 PBS solution.

[0036] Figure 4 The electrochemiluminescence curves of GCE modified with different nanowires in Example 1 in 1 mM NADH solution and 0.1 M pH 7.4 PBS solution are shown.

[0037] Figure 5 The X-ray diffraction (XRD) analysis results are for the photosensitive material prepared in Example 2.

[0038] Figure 6 The X-ray photoelectron spectroscopy (XPS) analysis results are for the photosensitive material prepared in Example 2.

[0039] Figure 7 Scanning electron microscope (SEM) image of the photosensitive electrode prepared in Example 2.

[0040] Figure 8High-resolution transmission electron microscope (HRTEM) image of the photosensitive electrode prepared for Example 2.

[0041] Figure 9 The light and dark current density curves of the photosensitive electrode prepared in Example 2.

[0042] Figure 10 Electrochemical impedance spectroscopy of the photosensitive electrode prepared in Example 2.

[0043] Figure 11 This is a linear relationship graph obtained when the nanowire-coupled photoelectrochemical sensor of Example 3 detects the concentration of NADH solution in vitro.

[0044] Figure 12 This is a laser confocal scanning image of nanowires non-destructively semi-embedded in cells, as shown in Example 4. In the image, red represents the cytoskeleton, blue represents the cell nucleus, and green represents the nanowires.

[0045] Figure 13 Example 5 shows the results of monitoring changes in intracellular NADH levels using a nanowire-coupled photoelectrochemical sensor after cells were exposed to different concentrations of genotoxin (lansoprazole chloride, chemical name 2-chloromethyl-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride, abbreviated as Lanchlor).

[0046] Note: Figure 3 and Figure 4 The electrochemical data were obtained using the MPI-E analysis system (Xi'an Ruimai Analytical Instruments Co., Ltd., Xi'an, China), while all other electrochemical data were measured using the CHI 660E electrochemical workstation (Chenhua Instruments Co., Ltd., Shanghai, China). Detailed Implementation

[0047] The present invention will now be described in detail.

[0048] Terminology

[0049] NADH (Reduced Nicotinamide Adenine Dinucleotide): NADH is an important intracellular coenzyme, primarily acting as an electron donor in cellular energy metabolism. It plays a role in processes such as glycolysis and the tricarboxylic acid cycle, and is also widely involved in physiological activities such as DNA repair and antioxidation. Changes in NADH levels are closely related to cellular redox balance and metabolic state, and it serves as a biomarker for many pathophysiological states.

[0050] Nanowires: Nanowires are one-dimensional structures confined to less than 100 nm in the lateral direction (with no longitudinal confinement), including metallic nanowires, semiconductor nanowires, and insulator nanowires. In this invention, specially designed nanowires can spontaneously semi-embed in cells through endocytosis, undergo oxidation reactions with intracellular NADH, and act as artificial transmembrane electron tunnels, exporting electrons related to intracellular NADH levels.

[0051] Photoelectrochemical sensor: A photoelectrochemical sensor is a sensor that combines the photoelectric effect and electrochemical reaction. It utilizes the electrochemical reaction under light stimulation to generate an electrical signal. In this invention, the photosensitive electrode generates photogenerated holes under light stimulation, stimulating the oxidation reaction of NADH in cells, and aggregating the electrical signal of nanowires to achieve real-time monitoring of intracellular NADH.

[0052] PFBT Pdots (Poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] Polymer dots): PFBT Pdots are semiconductor polymer quantum dots with excellent photoelectric properties. They can undergo oxidation reactions with NADH, triggering electron transfer events. They are the recognition elements in the nanowire structure of this invention, enhancing the sensor's anti-interference capability.

[0053] PDDA (polydiallyl dimethylammonium chloride): PDDA is a positively charged polymer that enhances the interaction between nanowires and cell membranes through electrostatic interactions, thereby promoting the uptake of nanowires by cells.

[0054] Technical solution

[0055] The first aspect of the present invention provides a nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs comprising multi-walled carbon nanotubes (MWCNTs), fluorescein isothiocyanate (FITC), poly(9,9-dioctylfluorenyl-alt-benzothiadiazole) semiconductor polymer quantum dots (PFBT Pdots), polydiallyl dimethylammonium chloride (PDDA), and platinum (Pt).

[0056] The various components of the nanowires play a crucial role in the real-time monitoring of intracellular NADH. Specifically, the positive charge of PDDA allows it to interact with the negative charge on the cell membrane surface, promoting partial uptake of the nanowires; PFBT Pdots undergo a specific oxidation reaction with NADH, triggering electron transport events; MWCNTs, with their excellent conductivity, can effectively extract free electrons from the cell; and the introduction of Pt further enhances electron transport efficiency and strengthens the sensor's current response.

[0057] A second aspect of the present invention provides a method for preparing the nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs, comprising the following steps:

[0058] (1) Fluorescein isothiocyanate (FITC) was linked to aminated multi-walled carbon nanotubes (MWCNTs) via a nucleophilic substitution reaction to obtain MWCNT@FITC;

[0059] (2) Poly(9,9-dioctylfluorenyl-alt-benzothiadiazole) semiconductor polymer quantum dots (PFBT Pdots) were bonded to MWCNT@FITC by amidation reaction to obtain MWCNT@FITC-PFBTPdots;

[0060] (3) Polydiallyldimethylammonium chloride (PDDA) was bound to MWCNT@FITC-PFBT Pdots by electrostatic interaction to obtain MWCNT@FITC-PFBT Pdots-PDDA;

[0061] (4) Coating MWCNT@FITC-PFBT Pdots-PDDA with a layer of platinum metal to obtain the nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs.

[0062] In some embodiments of the present invention, the aminated MWCNTs can be prepared using conventional methods of the prior art or can be purchased directly. The aminated MWCNTs used in the present invention were purchased from Xianfeng Nanomaterials Technology Co., Ltd.

[0063] In some embodiments of the present invention, step (1) specifically involves: adding FITC solution dropwise to an aminated MWCNT suspension, stirring at room temperature in the dark for 20-30 h, and filtering and washing the reactants after stirring to obtain MWCNT@FITC.

[0064] In some embodiments of the present invention, the mass ratio of FITC to aminated MWCNT is (1-3):1.

[0065] In some embodiments of the present invention, the solvent in the FITC solution is selected from one of dimethyl sulfoxide (DMSO), tetrahydrofuran, acetonitrile, dimethylformamide, ethylene glycol, and acetone, and the dispersant in the aminated MWCNT suspension is one of PBS buffer solution (pH 7.4), deionized water, HEPES buffer, and Tris buffer.

[0066] In some embodiments of the present invention, the PFBT Pdots are prepared as follows: PFBT and styrene-co-maleic anhydride copolymer (PSMA) are added to tetrahydrofuran (THF), and the precursor solution is obtained by ultrasonic treatment for 10-40 min. The precursor solution is injected into ultrapure water, and ultrasonic treatment is performed again for 2-5 min. THF is removed by rotary evaporator, and the reactants are filtered to obtain PFBT Pdots.

[0067] In some embodiments of the present invention, the mass ratio of PFBT to PSMA is (3-7):1.

[0068] In some embodiments of the present invention, step (2) specifically involves: mixing PFBT Pdots with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) to obtain a PFBT Pdots solution; mixing MWCNT@FITC with N-hydroxysuccinimide (NHS) to obtain a MWCNT@FITC dispersion; adding the PFBT Pdots solution to the MWCNT@FITC dispersion; stirring for 10-20 h; and after stirring, filtering and washing the reactants to obtain MWCNT@FITC-PFBTPdots.

[0069] In some embodiments of the present invention, the mass ratio of PFBT Pdots, MWCNT@FITC, EDC, and NHS is 1:(4-6):(4-6):(4-6).

[0070] In some embodiments of the present invention, step (3) specifically involves dispersing MWCNT@FITC-PFBT Pdots in an aqueous solution containing PDDA and NaCl, stirring at room temperature in the dark for 20-30 h, and filtering and washing the reactants after stirring to obtain MWCNT@FITC-PFBT Pdots-PDDA.

[0071] In some embodiments of the present invention, the mass ratio of MWCNT@FITC-PFBT Pdots, PDDA, and NaCl is (25-35) mg: 1 g: (1-3) g.

[0072] In some embodiments of the present invention, step (4) specifically involves dispersing MWCNT@FITC-PFBT Pdots-PDDA in water, adding platinum nanoparticle colloid, stirring for 4-6 h, filtering and washing the reactants after stirring to obtain MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs.

[0073] In some embodiments of the present invention, the mass concentration of MWCNT@FITC-PFBT Pdots-PDDA in platinum nanoparticle colloid is (0.2-0.6) g / L.

[0074] In some embodiments of the present invention, the preparation method of the platinum nanoparticle colloid includes: mixing H2PtCl6·6H2O with sodium citrate, diluting with water, adding 0.1 M sodium borohydride, stirring for 0.5-2 h, and letting stand at room temperature for 20-30 h to form platinum nanoparticle colloid.

[0075] In some embodiments of the present invention, the mass ratio of H2PtCl6·6H2O to sodium citrate is 1:(1-3).

[0076] A third aspect of the present invention provides a photosensitive electrode Flex ITO-WO3 / BiVO4, wherein the photosensitive electrode is a type II n-heterojunction nanocomposite material formed by tungsten oxide (WO3) and bismuth vanadate (BiVO4) on a flexible indium tin oxide (FlexITO) thin film.

[0077] A fourth aspect of this invention provides a method for preparing the above-mentioned photosensitive electrode Flex ITO-WO3 / BiVO4, comprising the following steps:

[0078] (i) WO3 was prepared using Na2WO4·2H2O as a raw material;

[0079] (ii) BiVO4 was prepared using Bi(NO3)3·5H2O as a raw material;

[0080] (iii) WO3 and BiVO4 are sequentially drop-coated onto the surface of the Flex ITO film to obtain the photosensitive electrode FlexITO-WO3 / BiVO4.

[0081] In some embodiments of the present invention, step (i) specifically involves: dissolving Na2WO4·2H2O in ultrapure water, adding hydrochloric acid while stirring to maintain the pH at 1-2, then adding NaCl and H2C2O4, stirring for 10-40 min, adding ultrapure water, heating the mixture to 120-170°C, reacting for 1-3 h, cooling to room temperature after the reaction is complete, collecting the precipitate, washing, and drying to obtain a yellow powder, which is WO3.

[0082] In some embodiments of the present invention, the mass ratio of Na2WO4·2H2O, NaCl, and H2C2O4 is (3-4):(1-1.5):1.

[0083] In some embodiments of the present invention, step (ii) specifically involves: dissolving Bi(NO3)3·5H2O in HNO3, adding polyvinylpyrrolidone (PVP) and stirring for 20-40 min to obtain solution A; dissolving NH4VO3 in NaOH solution and stirring for 20-40 min to obtain solution B; adding solution B dropwise to solution A; and then continuing to stir for 0.5-2 h. After stirring is complete, heating the reactants to 180-220°C and continuing to stir the reaction for 20-30 h. After the reaction is complete, cooling to room temperature, collecting the precipitate, washing, and drying, the resulting yellow powder is BiVO4.

[0084] In some embodiments of the present invention, the mass ratio of Bi(NO3)3·5H2O, PVP, and NH4VO3 is (20-30):(3-7):(3-7).

[0085] In some embodiments of the present invention, step (iii) specifically involves: drop-coating WO3 onto a Flex ITO electrode and maintaining it at 200-300°C for 2-4 hours. After the electrode cools to room temperature, it is washed with a buffer solution to obtain a Flex ITO-WO3 electrode. Then, BiVO4 is drop-coated onto the Flex ITO-WO3 electrode and maintained at 50-100°C for 1-2 hours to form a WO3 / BiVO4 heterojunction, thereby obtaining the photosensitive electrode Flex ITO-WO3 / BiVO4.

[0086] The fifth aspect of this invention provides a method for real-time monitoring of intracellular NADH changes based on a nanowire-coupled photoelectrochemical sensor, comprising the following steps:

[0087] S1. Nanowires MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs were incubated with cells, and the nanowires were partially embedded into the cells through endocytosis.

[0088] S2. Cover the cell surface with the photosensitive electrode Flex ITO-WO3 / BiVO4, with the side carrying the WO3 / BiVO4 photosensitive material in contact with the cell. Place the working electrode, reference electrode, and counter electrode in contact with the other side of the photosensitive electrode. Under illumination, measure the intracellular NADH current response in real time.

[0089] In some embodiments of the present invention, the cells are HepaRG cells.

[0090] In some embodiments of the present invention, the working electrode is a glassy carbon electrode, the reference electrode is a silver / silver chloride electrode, and the counter electrode is a platinum electrode.

[0091] In some embodiments of the present invention, the light source is a xenon lamp.

[0092] In some embodiments of the present invention, S2 uses a CHI 660E electrochemical workstation to record the intracellular NADH current response.

[0093] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, it is worth noting that the raw materials involved in the present invention are all commercially available products unless otherwise specified. Example 1

[0094] This embodiment provides a method for preparing MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs nanowires, including the following steps:

[0095] (1) Dissolve 50 mg FITC in 15 mL DMSO to obtain FITC solution. Disperse 50 mg aminated MWCNT in 50 mL PBS buffer solution (pH = 7.4) to obtain aminated MWCNT solution. Add FITC solution dropwise to aminated MWCNT solution and stir at room temperature in the dark for 24 h. Then, filter with a 0.45 μm microporous membrane and wash thoroughly to obtain MWCNT@FITC.

[0096] (2) 5 mg PFBT and 1 mg styrene-maleic anhydride copolymer (Poly (styrene-co-maleicanhydride) (PSMA)) were added to tetrahydrofuran (THF), and the precursor solution was obtained by sonication for 30 min. Then, the precursor solution was rapidly injected into ultrapure water and sonicated again for 3 min. After that, THF was removed by rotary evaporator. Finally, it was filtered through a 0.22 μm microporous membrane to obtain PFBT Pdots.

[0097] 25 mg of EDC was added to PFBT Pdots to obtain a PFBT Pdots solution. 25 mg of NHS was added to MWCNT@FITC and stirred for 1 h to obtain a MWCNT@FITC dispersion. The PFBT Pdots solution was added to the MWCNT@FITC dispersion and stirred for 12 h. After filtration through a 0.45 μm microporous membrane and thorough washing, MWCNT@FITC-PFBT Pdots were obtained.

[0098] (3) 20 mg MWCNT@FITC-PFBT Pdots were dispersed in an aqueous solution containing 0.75 g PDDA and 1.5 g NaCl, stirred at room temperature in the dark for 24 h, and then filtered through a 0.45 μm microporous membrane. After thorough washing, MWCNT@FITC-PFBT Pdots-PDDA was obtained.

[0099] (4) Mix 36 mg H2PtCl6·6H2O with 64 mg sodium citrate, dilute with water, add 0.1 M sodium borohydride dropwise, stir gently for 1 h, and let stand at room temperature for 24 h to form platinum nanoparticle colloids;

[0100] MWCNT@FITC-PFBT Pdots-PDDA was dispersed in 50 mL of water, platinum nanoparticle colloid was added, and the mixture was stirred continuously for 5 h. After stirring, the reaction mixture was filtered through a 0.45 μm microporous membrane and thoroughly washed to obtain MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs. Furthermore, to promote partial uptake of the nanowires by cells, the size of the nanowires needed to be controlled using a cell disruptor.

[0101] The characterization results of the prepared nanowires are as follows: Figures 1 to 4 As shown in the figure. The results show that FITC, PFBT Pdots, PDDA, and Pt were successfully modified onto MWCNTs. MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs exhibited good current and electrochemiluminescence signals for NADH. Example 2

[0102] This embodiment provides a method for preparing a photosensitive electrode Flex ITO-WO3 / BiVO4, including the following steps:

[0103] (i) Dissolve 1.8 g Na2WO4·2H2O in ultrapure water and stir continuously. Then, add 6 M hydrochloric acid dropwise to the above solution to maintain the pH at 1.5. Subsequently, add 0.6 g NaCl and 0.5 g H2C2O4 to the mixture and stir for 30 min. Then add ultrapure water and pour the resulting mixture into a high-pressure reactor lined with polytetrafluoroethylene. Keep it at 150 °C for 2 h. After the reactor cools to room temperature, collect the precipitate by centrifugation and wash it alternately with deionized water and ethanol. After drying, the resulting yellow powder is labeled as WO3.

[0104] (ii) Dissolve 1.0 g Bi(NO3)3·5H2O in HNO3, add 0.25 g PVP and stir for 30 min to form solution A, dissolve 0.3 g NH4VO3 in NaOH solution and stir for 30 min to form solution B, add solution B dropwise to solution A, and then continue stirring for 1 h. Transfer the mixture to a high-pressure reactor lined with polytetrafluoroethylene and heat at 200 °C for 24 h. After the reactor cools to room temperature, wash the yellow precipitate alternately with deionized water and anhydrous ethanol, and dry it under vacuum. The resulting yellow powder is labeled as BiVO4.

[0105] (iii) The Flex ITO electrode was washed sequentially with acetone, ethanol and ultrapure water and dried in air. WO3 was then dropped onto the ITO electrode and kept at 300°C for 180 min to ensure that the particles adhered tightly to the electrode. After the electrode cooled to room temperature, it was washed three times with washing buffer (10 mM Tris-HCl and 50 mM NaCl, pH 7.4). Then, BiVO4 was dropped onto the FlexITO-WO3 electrode and kept at 80°C for 120 min to form a WO3 / BiVO4 heterojunction, thus obtaining the photosensitive electrode Flex ITO-WO3 / BiVO4.

[0106] The characterization results of the prepared WO3, BiVO4 and Flex ITO-WO3 / BiVO4 are as follows: Figures 5 to 10 As shown in the figure. The results show that the WO3 / BiVO4 heterojunction has been tightly attached to the Flex-ITO surface, forming a photosensitive electrode that is more sensitive to light and has better conductivity. Example 3

[0107] This embodiment provides a method for using a nanowire-coupled photoelectrochemical sensor to detect NADH solution in vitro, including the following steps:

[0108] S1. Prepare NADH solutions of different concentrations and add them to the grooves of the confocal dish;

[0109] S2. Add 50 μL of 40 μg / mL MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs nanowires;

[0110] S3. Cover the solution surface with the photosensitive electrode Flex ITO-WO3 / BiVO4, with the side carrying the WO3 / BiVO4 photosensitive material in contact with the solution;

[0111] S4. Using a glassy carbon electrode as the working electrode, a silver / silver chloride electrode as the reference electrode, and a platinum electrode as the counter electrode, contact the other side of the photosensitive electrode to receive an electrical signal related to the NADH concentration.

[0112] Current response of different concentrations of NADH as follows Figure 11 As shown, a good linear relationship is demonstrated between the current response and the logarithm of NADH concentrations from 0.100 ng / mL to 10.0 μg / mL. Example 4

[0113] This embodiment provides a research method for the partial uptake of nanowires by cells, including the following steps:

[0114] S1. A certain amount of MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs was co-incubated with HepaRG cells for 16 h;

[0115] S2.HepaRG cells were washed twice with preheated PBS (phosphate-buffered saline) and then fixed in 4% paraformaldehyde fixative for 30 min.

[0116] S3.HepaRG cells were washed three times with PBS and then incubated with immunostaining blocking solution (purchased from Beyotime Biotechnology Co., Ltd.) for 20 min.

[0117] S4.HepaRG cells were washed three times with PBS and then incubated with rhodamine-labeled phalloidin-TRITC solution for 30 min.

[0118] S5.HepaRG cells were washed three times with PBS and then incubated with DAPI (4',6-diamidinyl-2-phenylindole) solution for 3 min. After rinsing with PBS, the relative positions of the nanowires and the cytoskeleton were observed under a laser confocal scanning microscope.

[0119] The result of nanowires being partially taken up by cells is as follows Figure 12 As shown. Example 5

[0120] This embodiment provides a method for detecting intracellular NADH using a nanowire-coupled photoelectrochemical sensor, comprising the following steps:

[0121] S1. 50 μL of 40 μg / mL nanowires were treated with different concentrations (0 μM, 10 μM, 50 μM, 80 μM, 100 μM) of lansoprazole chloride (a known genotoxic agent reported in the literature) for 16 h.

[0122] S2. Place the photosensitive electrode, working electrode, reference electrode, and counter electrode according to the method described in Example 3;

[0123] The S3.CHI 660E electrochemical workstation records the intracellular NADH current response.

[0124] like Figure 13 The figure shows the current-time curve data obtained after the current stabilized. The results indicate that in HepaRG cells, NADH levels initially increased and then decreased with increasing lansoprazole chloride concentration. This is likely because the genotoxin first triggers the intracellular DNA damage response (DDR) mechanism, requiring the synthesis of large amounts of NADH to address DNA damage in the early stages. However, once the genotoxin concentration reaches a threshold, the rate of NADH consumption exceeds the rate of synthesis, leading to a gradual decrease in NADH levels.

[0125] The experimental results above demonstrate that the nanowire-coupled photoelectrochemical sensor provided by this invention can non-destructively and in real-time measure the intracellular NADH electrical signal. Compared with traditional intracellular electrochemical sensing methods, it eliminates the need for expensive micromanipulators and single nanowire electrodes, simplifying the operation and improving detection sensitivity. Furthermore, the introduction of the photosensitive electrode accelerates intracellular oxidation reactions and electron transfer, significantly amplifying the current response. Additionally, the examples used are limited to HepaRG cells; however, this invention can be applied to any cell line containing NADH.

[0126] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs, comprising multi-walled carbon nanotubes, fluorescein isothiocyanate, poly (9, 9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots, polydiallyldimethylammonium chloride and platinum; the nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs is prepared by the following method: (1) connecting fluorescein isothiocyanate to the amino-functionalized multi-walled carbon nanotubes by nucleophilic substitution reaction to obtain MWCNT@FITC; (2) combining poly (9, 9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots to MWCNT@FITC by amidation reaction to obtain MWCNT@FITC-PFBT Pdots; (3) combining polydiallyldimethylammonium chloride to MWCNT@FITC-PFBT Pdots by electrostatic interaction to obtain MWCNT@FITC-PFBT Pdots-PDDA; (4) wrapping a layer of platinum on MWCNT@FITC-PFBT Pdots-PDDA to obtain the nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs.

2. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 1, wherein, Specifically, step (1) is: preparing fluorescein isothiocyanate solution and amino-functionalized multi-walled carbon nanotube dispersion respectively, adding the fluorescein isothiocyanate solution into the amino-functionalized multi-walled carbon nanotube dispersion, stirring at room temperature for 20-30 hours in the dark, and then filtering and washing the reaction product to obtain MWCNT@FITC; And / or, specifically, step (2) is: mixing poly (9, 9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots with 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidazole to obtain PFBT Pdots solution, mixing MWCNT@FITC with N-hydroxysuccinimide to obtain MWCNT@FITC dispersion, adding the PFBT Pdots solution into the MWCNT@FITC dispersion, stirring for 10-20 hours, and then filtering and washing the reaction product to obtain MWCNT@FITC-PFBT Pdots.

3. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 2, wherein, The mass ratio of fluorescein isothiocyanate to amino-functionalized multi-walled carbon nanotubes is (1-3) :

1.

4. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 2, wherein, The preparation method of the poly (9, 9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots is: adding poly (9, 9-dioctylfluorene-alt-benzothiadiazole) and styrene-maleic anhydride copolymer into tetrahydrofuran, obtaining a precursor solution after ultrasonic treatment for 10-40 min, injecting the precursor solution into ultrapure water, ultrasonic treating again for 2-5 min to remove tetrahydrofuran, and filtering the reaction product to obtain poly (9, 9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots.

5. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 4, wherein, The mass ratio of the poly(9,9-dioctylfluorene-alt-benzothiadiazole) and styrene-maleic anhydride copolymer is (3-7):

1.

6. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 2, wherein, The mass ratio of the poly(9,9-dioctylfluorene-alt-benzothiadiazole) semiconductor polymer quantum dots, MWCNT@FITC, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide is 1:(4-6):(4-6):(4-6).

7. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 1, wherein, Step (3) is specifically: dispersing the MWCNT@FITC-PFBT Pdots in an aqueous solution containing polydiallyldimethylammonium chloride and NaCl, stirring in the dark at room temperature for 20-30 h, and then filtering and washing the reaction product to obtain MWCNT@FITC-PFBT Pdots-PDDA. And / or, step (4) is specifically: dispersing the MWCNT@FITC-PFBT Pdots-PDDA in water, adding platinum nanoparticle colloid, stirring for 4-6 h, and then filtering and washing the reaction product to obtain MWCNT@FITC-PFBT Pdots-PDDA-PtNWs.

8. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 7, wherein, The mass ratio of the MWCNT@FITC-PFBT Pdots, polydiallyldimethylammonium chloride, and NaCl is (25-35) mg:1 g:(1-3) g.

9. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 7, wherein, The mass concentration of the MWCNT@FITC-PFBT Pdots-PDDA in the platinum nanoparticle colloid is (0.2-0.6) g / L.

10. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 9, wherein, The preparation method of the platinum nanoparticle colloid comprises the following steps: mixing H2PtCl6·6H2O with sodium citrate, diluting with water, adding 0.1 M sodium borohydride, stirring for 0.5-2 h, and then standing at room temperature for 20-30 h to form the platinum nanoparticle colloid.

11. The nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of claim 10, wherein, The mass ratio of H2PtCl6·6H2O to sodium citrate is 1:(1-3). 12.A method for real-time monitoring of intracellular NADH changes based on nanowire-coupled photoelectrochemical sensors, comprising the following steps: S1.incubating the nanowire MWCNT@FITC-PFBT Pdots-PDDA-Pt NWs of any one of claims 1-11 with cells, and the nanowires are partially embedded in the cells through endocytosis; S2.covering a photosensitive electrode Flex ITO-WO3 / BiVO4 on the surface of the cells, and the side carrying the WO3 / BiVO4 photosensitive material is in contact with the cells, and the working electrode, reference electrode and counter electrode are in contact with the other side of the photosensitive electrode, and the current response of intracellular NADH is determined in real time under light conditions. 13.The use of the method of claim 12 in electrochemical real-time monitoring of intracellular signaling molecules, which comprises monitoring cell metabolism, oxidative stress, drug screening, toxicology evaluation, and environmental toxicity monitoring.

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