Photoelectrochemical sensing array and wearable sensor based on self-supplying electron donors and their use in monitoring neuropeptide y

Abstract

The application discloses a photoelectrochemical sensing array for detecting neuropeptide Y based on self-provided electron donors, which takes a polyimide film as a base material and constructs a graphene-based three-electrode array through a laser induction technology; wherein, after modifying ytterbium-doped bismuth sulfide nanomaterials coated with phosphomolybdic acid in the working electrode area, coupling neuropeptide Y aptamers as molecular recognition elements, and then blocking non-specific recognition sites, the photoelectrochemical sensing array is prepared; wherein, the ytterbium-doped bismuth sulfide nanomaterials coated with phosphomolybdic acid generate a photocurrent under near-infrared light excitation, and the neuropeptide Y aptamers can specifically bind with neuropeptide Y; and according to the linear relationship between the photocurrent drop degree of the working electrode area and the concentration of neuropeptide Y, the monitoring of neuropeptide Y is realized. The excitation and detection signals of the photoelectrochemical sensing array and the sensor thereof are separated, near-infrared light is used as an excitation source, the application of voltage can avoid the expression of unnecessary substances in sweat, the anti-interference ability is strong, excellent advantages such as high sensitivity and low background signal are exhibited, a wide linear range (1fM-100pM) and an ultra-low detection limit (0.39fM) are possessed, and the in-situ monitoring of trace neuropeptide Y in sweat can be realized without stimulation and with ultra-sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical sensing technology and smart wearable devices, specifically relating to a near-infrared excited self-supplied electron donor photoelectrochemical sensing array and wearable sensor and its application in monitoring sweat neuropeptide Y. Background Technology

[0002] Currently, wearable sensors based on "smart healthcare" are booming, bringing broad application prospects to fields such as personal health management and telemedicine diagnosis. Among these sensors, wearable electrochemical (W-EC) sensors aim to replace centralized detection systems that are overwhelmed by medical demands, shifting towards more easily accessible personalized self-monitoring methods, thereby reducing medical costs and improving diagnostic efficiency. To date, scientists have successfully designed a series of advanced W-EC sensors to monitor highly abundant targets (such as metabolites, electrolytes, and hormones) in human sweat in an in situ, non-invasive, and real-time manner (Chem. Rev. 2023, 123, 5049). Meanwhile, the development of other low-abundance but clinically valuable sweat biomarkers (e.g., peptides) is of great significance for W-EC sensors. Neuropeptide Y (NPY) is a trace biomarker of sweat associated with stress and depression (approximately pM level), mainly distributed in the central and peripheral nervous systems of mammals. Furthermore, clinical studies have shown that NPY levels in sweat play a crucial role in the diagnosis of conditions such as depression, anxiety, and post-traumatic stress disorder (Science 2023, 382, ​​eabq8173). However, current sweat-electro-electro-detection sensors typically employ an electrostimulation-electro-detection mode, which suffers from high background signals, making it difficult to meet the high-sensitivity detection requirements for trace NPY in sweat. Additionally, current W-EC sensors require a bias voltage as the excitation source, inevitably leading to abnormal NPY expression during in-situ monitoring, thus affecting the accuracy of the actual monitoring results. Therefore, existing W-EC sensors cannot yet achieve non-stimulatory, ultrasensitive, and in-situ monitoring of the dynamic levels of trace NPY in human sweat.

[0003] Photoelectrochemical (PEC) sensors have attracted much attention due to their ability to combine the advantages of optical and electrochemical sensors, representing a rapidly developing new technology in the field of electrochemical sensing. Currently, most PEC sensors use ultraviolet-visible light (UV-Vis) as the excitation source, achieving good photoelectric conversion efficiency between a rigid photoelectrode and an external electron donor (e.g., ascorbic acid). However, these PEC sensors are not suitable for wearable sensing applications, primarily because: (i) high-energy-density UV-Vis light can damage biosensing interfaces or human skin; (ii) human sweat cannot provide the necessary electron donor for the photoelectric conversion system; and (iii) rigid photoelectrodes cannot be directly attached to human skin. Furthermore, most currently reported wearable electronic devices are based on silicone support substrates, which rely on expensive microfabrication equipment and complex processing techniques. Therefore, developing an innovative wearable PEC sensor for non-irritating, ultrasensitive, in-situ monitoring of trace NPY kinetics in sweat is a highly challenging task. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by proposing a photoelectrochemical sensing array with a near-infrared excited self-supplied electron donor. The excitation and detection signals are separated, and near-infrared light is used as the excitation source instead of a bias voltage. This avoids the possibility that applying voltage may cause the expression of unnecessary substances in sweat. It has strong anti-interference ability and exhibits excellent advantages such as high sensitivity and low background signal. It has a wide linear range (1 fM to 100 pM) and an ultra-low detection limit (0.39 fM), which can realize non-irritating and ultra-sensitive in-situ monitoring of trace NPY in sweat.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0006] A photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor is disclosed. It uses a polyimide film as a substrate and constructs a graphene-based three-electrode array using laser-induced technology, comprising a counter electrode, a reference electrode, and a working electrode. Specifically, the working electrode region is modified with phosphomolybdic acid (PMA)-coated ytterbium-doped bismuth sulfide nanomaterials (Yb-Bi2S3 / PMA), which are then coupled with a neuropeptide Y (NPY) aptamer as a molecular recognition element. The non-specific recognition sites on the working electrode surface are then blocked with bovine serum albumin, resulting in a near-infrared light-excited photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor. The phosphomolybdic acid-coated ytterbium-doped bismuth sulfide (Yb-Bi2S3 / PMA) nanomaterials possess near-infrared light excitation and self-supplied electron donor characteristics.

[0007] In the aforementioned photoelectrochemical sensing array with a self-supplied electron donor, under near-infrared light (780–850 nm, e.g., 808 nm) excitation, valence band electrons in the Yb-Bi2S3 / PMA nanomaterial transition to the conduction band, and the coated PMA layer further provides electrons, amplifying the generated photocurrent. The NPY aptamer can specifically recognize the target NPY. When the NPY aptamer specifically captures and recognizes NPY in sweat, due to the insulating nature of NPY, the conductivity of the working electrode area decreases, the electron transfer efficiency deteriorates, and the photocurrent decreases. The degree of decrease corresponds to the concentration of the target NPY, that is, the photocurrent signal is converted into NPY concentration information, thereby realizing the detection of trace concentrations of NPY in sweat.

[0008] According to the above scheme, in the phosphomolybdic acid-coated ytterbium-doped bismuth sulfide (Yb-Bi2S3 / PMA) nanomaterial, the mass ratio of phosphomolybdic acid to ytterbium-doped bismuth sulfide is 0.5:1 to 3:1; the molar percentage of Yb doping relative to Bi2S3 in the ytterbium-doped bismuth sulfide is 10% to 40%.

[0009] According to the above scheme, the preparation method of Yb-Bi2S3 / PMA nanomaterials specifically includes the following steps:

[0010] ① Preparation of ytterbium-doped bismuth sulfide (Yb-Bi2S3) nanorods: First, add 0.7-0.9 g Bi(NO3)3·5H2O and 0.1-0.2 g YbCl3·6H2O to 10-15 mL of ethylene glycol and sonicate until completely dissolved, denoted as solution A; add 0.5-0.8 g Na2S·9H2O to 14-16 mL of ethylene glycol, denoted as solution B; add 0.9-1 g CO(NH2)2 to 8-12 mL of ethylene glycol and sonicate until completely dissolved, denoted as solution C; add solution B dropwise to solution A while stirring to form a black suspension, then add solution C, and stir vigorously at room temperature for 15-25 minutes to obtain a mixture; transfer the resulting mixture to a high-pressure reactor and heat at 170-190℃ for 20-25 h, centrifuge to separate the black solid product, which is Yb-Bi2S3 nanorods, wash and dry for later use;

[0011] ②Preparation of Yb-Bi2S3 / PMA nanomaterials: Using Yb-Bi2S3 nanorods as a precursor, 0.05–0.2 g of Yb-Bi2S3 nanorods were first dispersed in 3–8 mL of anhydrous ethanol and stirred. After dispersion, 0.05–0.2 g of phosphomolybdic acid (PMA) was added, and the mixture was stirred in a light-protected environment for 8–12 h. Then, it was heated in a N2 atmosphere until the solvent was completely evaporated. Finally, the Yb-Bi2S3 / PMA nanomaterials were collected at the bottom of the container, ground with an agate mortar, and stored in a dry, light-protected environment for later use.

[0012] According to the above scheme, the aptamer sequence of neuropeptide Y (NPY) is: 5′-NH2-C6-AGC AGC ACA GAG GTC AGATGC AAA CCA CAG CCT GAG TGG TTA GCG TAT GTC ATT TAC GGA CCT ATG CGT GCT ACCGTG AA-3′.

[0013] The main steps of the above-mentioned method for preparing the photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor are as follows:

[0014] (1) A graphene (LIG) three-electrode array is prepared by laser engraving using polyimide film as substrate, including three regions: counter electrode, reference electrode and working electrode; the LIG three-electrode array is both conductive and flexible, and can also be called a conductive flexible LIG three-electrode array.

[0015] (2) The reference electrode region was modified with Ag / AgCl paste and dried at room temperature to serve as the reference electrode; the counter electrode region did not require any treatment and was directly used as the counter electrode.

[0016] (3) A Yb-Bi2S3 / PMA nanomaterial solution is drop-coated onto the working electrode area. After drying, the aptamer (Apt) of neuropeptide Y is further modified to specifically recognize the target analyte NPY. Then, bovine serum albumin (BSA) is used to block the non-specific recognition site to avoid non-specific binding of the target analyte, thereby obtaining the LIG-Yb-Bi2S3 / PMA@Apt-BSA electrode array, which is the photoelectrochemical sensing array for detecting neuropeptide Y based on self-supplied electron donors described in this invention.

[0017] According to the above scheme, the process of step (1) is as follows: Polyimide (PI) tape is pasted onto a transparent and flexible polyethylene terephthalate (PET) film. Using a pre-set three-electrode array pattern, a matching laser engraving machine is used to engrave the graphene electrode film of the three-electrode array on the surface of the PI tape, thus obtaining a conductive flexible LIG three-electrode array. The specific steps are as follows: ① Design a batch of three-electrode array patterns using drawing software, where the diameter of the working electrode is controlled between 2 and 5 mm, and then output a high-resolution three-electrode array image for engraving; ② Paste the polyimide (PI) tape onto a transparent and flexible polyethylene terephthalate (PET) film, drag the three-electrode array image into the laser engraving software Scarve 4, set the laser power to 3-5 W, and the engraving depth to 50-70%, and use a matching laser engraving machine to engrave the graphene electrode film of the three-electrode array on the surface of the PI tape; ③ After engraving, rinse the LIG three-electrode array with ultrapure water and air dry.

[0018] According to the above scheme, step (2) specifically involves brushing an appropriate amount of Ag / AgCl slurry onto the reference electrode region of the LIG three-electrode array, with a dosage of approximately 0.6–0.9 μL / mm. -2 The reference electrode of the photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor, as described in this invention, can be obtained by drying at room temperature.

[0019] According to the above scheme, step (3) specifically involves: ① dispersing Yb-Bi2S3 / PMA nanomaterials in ultrapure water and ultrasonically preparing a Yb-Bi2S3 / PMA aqueous dispersion with a concentration of 10–18 mg / mL. -1 ② Apply approximately 0.4–0.6 μL of Yb-Bi2S3 / PMA aqueous dispersion to the working electrode area. -2 The electrode was dried in a drying oven to obtain the Yb-Bi2S3 / PMA modified working electrode region; ③ Use 40-60 mg / mL -1 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 40–60 mg / mL -1 A mixture of N-hydroxysuccinimide (NHS) was used to activate the Yb-Bi2S3 / PMA modified working electrode region (approximately 0.6–0.9 μL / mm²). -2 The activation time is 20–40 min; ④ After activation, the working electrode region is functionalized by adding Apt solution through an amidation reaction, and incubated in a humid environment at 30–40 °C for 3–6 h. The unimmobilized aptamer is then washed away to form the aptamer-modified working electrode region (abbreviated as LIG-Yb-Bi2S3 / PMA@Apt); The concentration of Apt solution is 1–3 μM, and the amount added is approximately 0.1–0.3 μL. -2 ⑤ Drop a BSA solution (mass fraction 0.04-0.06%) onto the surface of the above-mentioned LIG-Yb-Bi2S3 / PMA@Apt working region, then incubate at room temperature for 20-40 min, and rinse away unbound BSA molecules with ultrapure water to obtain the working electrode of the photoelectrochemical sensing array for detecting neuropeptide Y based on self-supplied electron donor as described in this invention (abbreviated as LIG-Yb-Bi2S3 / PMA@Apt-BSA), and store it at 2-8℃ for later use.

[0020] This invention also provides a method for detecting neuropeptide Y using the above-mentioned photoelectrochemical sensing array, the main steps of which are:

[0021] (1) Dissolve the neuropeptide Y standard sample in a buffer solution to prepare a gradient concentration NPY standard solution, preferably in the range of 1 fM to 100 pM;

[0022] (2) NPY standard solutions of different concentrations were respectively drop-coated in parallel onto the working electrode region of the photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor as described in this invention, with a drop volume of approximately 0.2–0.4 μL / mm. -2 Then place it in a sealed environment at 30-40℃ and 60%-90% humidity for 20-40 minutes;

[0023] (3) After incubation in step (2), the three electrodes (counter electrode, working electrode and reference electrode) of the photoelectrochemical sensing array based on self-supplied electron donor detection of neuropeptide Y described in this invention are connected to the electrochemical workstation through wires. Photocurrent data are collected by the electrochemical workstation under near-infrared light excitation. Then, a standard curve is established with the logarithm of NPY concentration in NPY standard solution as the abscissa and the photocurrent value as the ordinate.

[0024] (4) Under conditions parallel to steps (2) and (3), the photocurrent of the test liquid is detected, and then the standard curve method is used to detect the target neuropeptide Y in the test liquid; wherein, the test liquid of the present invention is human sweat containing trace amounts of NPY.

[0025] Building upon the above, this invention also provides an intelligent, flexible, wearable photoelectrochemical sensor capable of in-situ, non-irritating, and sensitive monitoring of the dynamic levels of trace NPY in sweat. This intelligent, flexible, wearable sensor comprises a near-infrared light-excited photoelectrochemical sensing array, a signal acquisition and processing circuit module, a power supply module, and a display module, as described in this invention. The photoelectrochemical sensing array is in contact with an absorption layer that collects sweat in real time (the absorption layer is used to collect sweat from the subject's forearm). The photoelectrochemical sensing array, the signal acquisition and processing circuit module, and the display module are all electrically connected to the power supply module. The photoelectrochemical sensing array, electrically connected to the signal acquisition and processing circuit module, generates an electrical signal and outputs it in the form of photocurrent, converting it into the concentration of neuropeptide Y in sweat. The display module is used to display the concentration of neuropeptide Y in sweat.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) The present invention employs laser engraving to prepare a flexible LIG three-electrode array. By sensitizing the working electrode region with Yb-Bi2S3 / PMA nanomaterials, an NPY aptamer is coupled as a molecular recognition element. Then, bovine serum albumin is used to block the non-specific recognition sites on the working electrode surface, thereby obtaining a near-infrared excitation-enabled photoelectrochemical sensing array. The modified aptamer can specifically capture and recognize the target analyte NPY in the sample. After recognition, the electron transport rate on the electrode surface is blocked, and the photocurrent decreases, thus establishing a quantitative relationship between the photocurrent signal and the concentration of the target analyte NPY. The photoelectrochemical sensing array and sensor of the present invention utilize photoelectrochemical technology, separating the excitation and detection signals. Near-infrared light is used as the excitation source instead of a bias voltage, avoiding the possibility that applying voltage might cause the expression of unnecessary substances in sweat. It exhibits strong anti-interference ability and high sensitivity, demonstrating excellent advantages such as high sensitivity and low background signal, and possesses a wide linear range (1 fM to 100 pM) and an ultra-low detection limit (0.39 fM).

[0028] (2) The photoelectrochemical sensing array described in this invention can be excited under near-infrared (780-850nm) conditions, which can effectively avoid the stimulation and damage to the human body caused by high energy density excitation light. At the same time, it can achieve self-sufficiency of electrons through the excellent multi-electron redox properties of the coating layer PMA of nanomaterial Yb-Bi2S3, and has good photoelectric conversion efficiency, which makes up for the lack of human sweat in providing the necessary electron donors for the photoelectric conversion system during wearable process.

[0029] (3) The photoelectrochemical sensing array described in this invention is further integrated with the signal acquisition and processing circuit module to realize the in-situ signal acquisition and processing, and output to the display module (for example, it can be wirelessly transmitted to a customized client, mobile APP via Bluetooth) for display. The difference in electrical signal corresponds to the dynamic change of target concentration, thereby realizing in-situ, sensitive and non-irritating dynamic monitoring of trace biomarkers in human sweat. Moreover, the photoelectrochemical sensing array has excellent mechanical stability (300 bending cycles), which has broad application prospects in the field of personalized smart healthcare. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the construction process of the PEC sensor in the embodiment. Integration and packaging of the three-electrode array (A); photoelectric material sensitization (B); schematic diagram of the manufacturing principle of the flexible sensor with specific NPY aptamer biofunctionalization (C).

[0031] Figure 2 This is a schematic diagram of the three electrodes used to prepare LIG using PI as an example.

[0032] Figure 3A and E represent the morphological characterization of the flexible LIG three-electrode array (labeled LIG) in the embodiment; B and F represent the morphological characterization of LIG-Bi2S3; C and G represent the morphological characterization of LIG-Yb-Bi2S3; D, H, I, J, K, L, M, N, O, and P represent the morphological characterization of LIG-Yb-Bi2S3 / PMA; AD represents SEM images, EH represents TEM images, and IP represents EDS characterization.

[0033] Figure 4 The following is a SEM characterization of LIG(A), LIG-Bi2S3(B), LIG-Yb-Bi2S3(C), and LIG-Yb-Bi2S3 / PMA(D) in the examples, with a scale bar of 2 μM.

[0034] Figure 5 The XRD patterns (A), Raman spectra (B), Fourier transform infrared spectra (C), and XPS full spectra (D) of (Ⅰ) LIG, (Ⅱ) LIG-Bi2S3, (Ⅲ) LIG-Yb-Bi2S3, and (Ⅳ) LIG-Yb-Bi2S3 / PMA in the examples are shown below. The high-resolution XPS spectrum (E) of Bi 4f in LIG-Yb-Bi2S3 / PMA is also shown. The DRS spectra (F) and corresponding Tacu curves (G) of Bi2S3 and Yb-Bi2S3 are shown below. The band structure (H) and density of states (I) of Yb-Bi2S3 are calculated using DFT.

[0035] Figure 6 The high-resolution XPS spectra of C1s(A) in LIG; C1s(B) and Bi 4f(C) in LIG-Bi2S3; and C1s(D), Bi 4f(E) and Yb 4d(F) in LIG-Yb-Bi2S3 are shown in the examples.

[0036] Figure 7 The high-resolution XPS spectra of C1s(A), Yb 4d(B), P 2p(C) and Mo 3d(D) in LIG-Yb-Bi2S3 / PMA in the examples are shown.

[0037] Figure 8 The diagram shows the optimized conditions in the example, including the Yb doping amount (A), the mass ratio of PMA to Yb-Bi2S3, and the Yb-Bi2S3 / PMA concentration (C).

[0038] Figure 9 This is an optimized graph showing the aptamer concentration in the examples.

[0039] Figure 10The sensing performance and biocompatibility of W-PECA were tested in the examples. Cyclic voltammetry (A) and AC impedance (B) of (Ⅰ) LIG, (Ⅱ) LIG-Bi2S3, (Ⅲ) LIG-Yb-Bi2S3, (Ⅳ) LIG-Yb-Bi2S3 / PMA, (Ⅴ) LIG-Yb-Bi2S3 / PMA@Apt, (Ⅵ) LIG-Yb-Bi2S3 / PMA@Apt-BSA, and (Ⅶ) LIG-Yb-Bi2S3 / PMA@Apt-BSA-NPY in 5 mM K3 / K4[Fe(CN)6] + 0.1 M KCl solution; photocurrent response (C) of electrodes Ⅰ~Ⅶ in 0 V, 0.1 M PBS (pH=7.4); 1 pM... Reproducibility of the NPY response (D); photocurrent response (E) and corresponding linearity (F) at different NPY concentrations (1 fM–100 pM); selectivity (G) and 30-day storage stability (H); confocal microscopy images of HUVEC cells stained with Calcein-AM (live cells) and PI (dead cells), control group, and LIG-Yb-Bi2S3 / PMA@Apt-BSA electrodes (I). Scale bar: 50 μm.

[0040] Figure 11 This example demonstrates the flexibility and mechanical stability testing of the flexible three-electrode sensing array. The changes in current (A) and resistance (B) of a 0.5×1cm strip-shaped LIG during 7000 cycles of stretching and bending are shown.

[0041] Figure 12 This is a customized mobile app for data display and analysis. The app's opening page (A), the app page during Bluetooth connection (B), and real-time sweat NPY concentration data analysis (C) are shown.

[0042] Figure 13 To investigate the dynamic testing of sweat NPY under different conditions using the intelligent W-PECA system. The mechanism of NPY 1R's regulation of eating behavior (A); photocurrent ratio (I / I0) curves of sweat NPY changes before and after lunch for three subjects (Subject 1-3) (BD), corresponding NPY concentration changes (E); I / I0 values ​​(F) of the three subjects undergoing physiological stress CPT testing at different times (0-30 min) and corresponding NPY levels (G); schematic diagram of NPY circadian rhythm (H); NPY concentration records of the same subject at different times on the same day (I); circadian rhythm of sweat NPY established by two subjects (sweat samples were collected and monitored each morning and afternoon) over a 7-day period (J). Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0044] Example

[0045] 1. Mass fabrication of flexible LIG three-electrode arrays

[0046] (a) Use drawing software to design batch three-electrode array patterns, where the working electrode is a circular electrode working area with a diameter of 3mm, and output it as a high-resolution image for engraving.

[0047] (b) Adhere a 5cm wide PI tape to a 21cm × 29.7cm (A4 size) PET film. In the Scarve 4 laser engraving software, import a three-electrode array image, adjust the laser power to 40% of 10W, and the engraving depth to 60%. Use a laser engraving machine to engrave on the PI tape surface. (c) After engraving, rinse the three-electrode array with ultrapure water and dry it to obtain batches of flexible LIG three-electrode arrays (e.g., ...). Figure 2 As shown, it includes a working electrode region, a reference electrode region, and a counter electrode region, wherein the counter electrode region does not require any processing and is directly used as the counter electrode.

[0048] 2. Fabrication of near-infrared light-excited photoelectrochemical sensing arrays

[0049] S1. Apply an appropriate amount of Ag / AgCl slurry (approximately 0.6–0.9 μL) to the reference electrode region of the aforementioned flexible LIG three-electrode array. -2 It is dried at room temperature and used as a reference electrode;

[0050] S2-1, ①Preparation of Bi2S3 nanorods:

[0051] First, 0.9095 g of Bi(NO3)3·5H2O was added to 12.5 mL of ethylene glycol, denoted as solution A; 0.6761 g of Na2S·9H2O was added to 15.0 mL of ethylene glycol, denoted as solution B; and 0.9610 g of CO(NH2)2 was added to 10.0 mL of ethylene glycol, denoted as solution C. Solutions A, B, and C were sonicated until completely dissolved. Then, solution B was added dropwise to solution A while stirring, followed by solution C. The mixture was then vigorously stirred at room temperature for 20 minutes. Finally, the mixture was transferred to a high-pressure reactor and heated at 180 °C for 24 h. The resulting black solid product (Bi2S3) was centrifuged and washed at least six times with ultrapure water and ethanol, then dried in an oven at 60 °C for 6 h to obtain Bi2S3 nanorods with lengths concentrated in the range of 62–142 nm and diameters concentrated in the range of 20–65 nm.

[0052] ②Preparation of Yb-Bi2S3 nanorods:

[0053] First, 0.8186 g of Bi(NO3)3·5H2O and 0.1454 g of YbCl3·6H2O were added to 12.5 mL of ethylene glycol, denoted as solution A; 0.6761 g of Na2S·9H2O were added to 15.0 mL of ethylene glycol, denoted as solution B; and 0.9610 g of CO(NH2)2 were added to 10.0 mL of ethylene glycol, denoted as solution C. Solutions A, B, and C were sonicated until completely dissolved. Then, solution B was added dropwise to solution A while stirring, forming a black suspension. Solution C was then poured into the mixture, and the mixture was vigorously stirred at room temperature for 20 minutes. Finally, the mixture was transferred to a high-pressure reactor and heated at 180°C for 24 hours. The obtained black solid product (i.e. Yb-Bi2S3) was centrifuged and washed with ultrapure water and ethanol at least 6 times, and dried in an oven at 60°C for 6 hours to obtain Yb-Bi2S3 nanorods with a length in the range of 90-360 nm and a diameter in the range of 17-50 nm. The Yb doping amount was 20% relative to Bi2S3.

[0054] ③ Preparation of Yb-Bi2S3 / PMA nanomaterials: Using Yb-Bi2S3 nanorods as precursors, 0.1g of Yb-Bi2S3 was first dispersed in 5mL of anhydrous ethanol and stirred. After dispersion, 0.1g of phosphomolybdic acid (PMA) was added and stirred in a light-protected environment for 10h. Then, the mixture was heated in a N2 atmosphere until the solvent was completely evaporated. Finally, the Yb-Bi2S3 / PMA nanomaterials were collected at the bottom of the container, ground with an agate mortar, and stored in a dry, light-protected environment for later use.

[0055] ④ Preparation of Yb-Bi2S3 / PMA aqueous dispersion: 0.016 g of Yb-Bi2S3 / PMA nanomaterials were weighed and dispersed in 1 mL of ultrapure water, and sonicated for 20 min to obtain a concentration of 16 mg / mL. -1 Yb-Bi2S3 / PMA aqueous dispersion.

[0056] ⑤ Drop 3.5 μL of Yb-Bi2S3 / PMA aqueous dispersion onto the surface of the working electrode of the above LIG electrode array, and dry it in a drying oven to obtain the LIG-Yb-Bi2S3 / PMA electrode array.

[0057] By replacing Yb-Bi2S3 / PMA nanomaterials with Bi2S3 and Yb-Bi2S3 respectively as controls, the resulting sensing sequences were labeled as LIG-Bi2S3 electrode array and LIG-Yb-Bi2S3 electrode array respectively.

[0058] Yb-Bi2S3 / PMA nanomaterials with a Yb-to-Bi2S3 doping ratio of 20% and a PMA-to-Yb-Bi2S3 mass ratio of 1:1 were dispersed in ultrapure water and ultrasonicated for 10 min to obtain a concentration of 16 mg / mL. -1 A uniform aqueous dispersion was obtained. 3.5 μL of the aqueous dispersion was uniformly drop-coated onto the working electrode area of ​​the flexible LIG three-electrode array and dried in an electric oven at 60 °C for 30 min. After drying, the LIG-Yb-Bi2S3 / PMA electrode array was obtained.

[0059] S2-2. Based on the LIG-Yb-Bi2S3 / PMA electrode array obtained in S2-1, the NPY aptamer was further modified to achieve NPY aptamer functionalization. The aptamer sequence is: 5′-NH2-C6-AGC AGC ACA GAG GTC AGA TGC AAACCA CAG CCT GAG TGG TTA GCG TAT GTC ATT TAC GGA CCT ATG CGT GCT ACC GTG AA-3′. The modification process is as follows: 5 μL EDC (50 mg / mL) was used. -1 ) and NHS (50mg mL) -1 The LIG-Yb-Bi2S3 / PMA surface was activated for 30 min with a mixture of NPY solution (2 μM) for 30 min; then 2 μL of NPY solution (2 μM) was added for modification. After incubation at 37 °C in a humid environment for 4 h, the unimmobilized aptamer was washed away, and an aptamer-modified working electrode region (abbreviated as LIG-Yb-Bi2S3 / PMA@Apt) was formed through an amidation-like reaction.

[0060] S2-3. Add 0.5 μL of BSA solution (concentration 0.05%) to the surface of the above-mentioned LIG-Yb-Bi2S3 / PMA@Apt working region, incubate at room temperature for 30 min, and then rinse away the unbound BSA molecules with ultrapure water to obtain the working electrode of the near-infrared light-excited photoelectrochemical sensing array described in this invention (abbreviated as LIG-Yb-Bi2S3 / PMA@Apt-BSA), and store it at 4°C for later use.

[0061] In the above steps, the LIG-Yb-Bi2S3 / PMA electrode array in S2-1 is replaced with a LIG-Bi2S3 electrode array and a LIG-Yb-Bi2S3 electrode array, respectively, and subsequent steps are performed to compare it with the near-infrared light-excited photoelectrochemical sensing array described in this invention.

[0062] Performance Characterization

[0063] (1) Characterization of LIG, LIG-Bi2S3, LIG-Yb-Bi2S3, and LIG-Yb-Bi2S3 / PMA

[0064] Figure 3 In the diagram, A and E show that the flexible LIG three-electrode array prepared by laser engraving has a three-dimensional layered porous structure; B and F show that the Bi2S3 nanorods are uniformly distributed in size on the LIG surface; C and G show that the Yb-Bi2S3 nanorods are uniformly distributed in size on the LIG surface; D and H show that the Yb-Bi2S3 / PMA nanomaterials are uniformly distributed on the LIG substrate surface; and IP shows the successful preparation of the working electrode, the LIG-Yb-Bi2S3 / PMA electrode array.

[0065] Figure 4 This indicates that Bi2S3, Yb-Bi2S3, and Yb-Bi2S3 / PMA nanomaterials are uniformly distributed on the flexible LIG three-electrode array.

[0066] Figure 5In the study, XRD patterns (A), Raman spectra (B), and Fourier transform infrared spectra (C) further confirmed the successful preparation of LIG, LIG-Bi2S3, LIG-Yb-Bi2S3, and LIG-Yb-Bi2S3 / PMA; XPS spectra (D and E) showed that Yb-Bi2S3 / PMA nanorods were successfully modified on the LIG surface; F and G showed that the near-infrared absorption of Yb-Bi2S3 material was superior to that of Bi2S3, and its band gap was 1.23 eV; H and I calculated the band gap of Yb-Bi2S3 by density functional theory (DFT) to be 1.2 eV, which is close to the band gap calculated by the Tacu curve, and its density of states diagram showed that the valence band was mainly contributed by the 3p orbitals of S and the 4f orbitals of Yb, and the conduction band was mainly contributed by the 6p orbitals of Bi. After Yb-Bi2S3 nanomaterials are coated with PMA, the PMA layer can provide electron donors for optoelectronic materials under photoexcitation conditions, improve photoelectric separation efficiency, and amplify photocurrent effect. At the same time, the acidic functional groups in PMA can combine with the amino groups in the NPY aptamer sequence, playing a role in aptamer immobilization.

[0067] Figure 6 , Figure 7 XPS spectroscopy confirmed the successful preparation of LIG, LIG-Bi2S3, LIG-Yb-Bi2S3, and LIG-Yb-Bi2S3 / PMA.

[0068] Figure 8 By adjusting the doping amount of Yb relative to Bi2S3 in the nanomaterial of S2-1 to 10%, 20%, 30%, and 40%, respectively, the photocurrent values ​​of Yb-Bi2S3 nanomaterials with different doping amounts were recorded using an electrochemical workstation. By comparison, the photocurrent effect of Yb-Bi2S3 nanomaterials reached its best when the molar percentage of Yb relative to Bi2S3 was 20%. Figure 8 By adjusting the mass ratio of Yb-Bi2S3 nanomaterials to PMA in S2-1 to 0, 1:0.5, 1:1, 1:2, and 1:3, B found through photocurrent testing that the photocurrent of the composite material reached its maximum value when the mass ratio was 1:1. However, when there were no phosphomolybdic acid-coated ytterbium-doped bismuth sulfide nanorods, the electron-hole pairs transitioning in the photoelectric material easily recombine under near-infrared light excitation, resulting in unsatisfactory photoelectric conversion efficiency. Figure 8 C represents the concentrations of Yb-Bi2S3 / PMA nanomaterials dropped onto S2-1 at 10, 12, 14, 16, and 18 mg / mL, respectively. -1 A comparison of photocurrent values ​​at different times shows that the concentrations of Yb-Bi2S3 / PMA nanomaterials are 16 mg / mL. -1 The current is at its maximum during time.

[0069] 3. The main steps of the method for detecting neuropeptide Y in sweat based on the above photoelectrochemical sensing array are as follows:

[0070] ① Prepare gradient concentration NPY standard solutions: Dissolve the human neuropeptide Y standard sample in buffer solution according to the usage requirements to prepare 1 mg / mL solutions. -1 The samples were serially diluted as required to prepare NPY standard solutions with concentrations of 1 fM, 5 fM, 20 fM, 100 fM, 1 pM, 5 pM, 20 pM, and 100 pM.

[0071] ② Incubate NPY in the working electrode area in a humid environment at 30-40℃: Take 2μL of NPY standard solution of different concentrations and drop it onto the working electrode area of ​​LIG-Yb-Bi2S3 / PMA@Apt-BSA. Place the electrode array in a humid and sealed environment in a constant temperature chamber and incubate for 20-40 min.

[0072] ③ Connect to the electrochemical workstation to collect photocurrent data of different concentrations of NPY and fit linearity: Connect the three electrodes of the photoelectrochemical sensor array to the electrochemical workstation through external wires. Under the condition of NIR 808nm excitation, the current data is collected by the electrochemical workstation. The logarithm of the NPY concentration C in the standard solution is used as the abscissa and the photocurrent value I is used as the ordinate to establish a linear regression equation. Figure 10 E represents the photocurrent response of the prepared photoelectrochemical sensing array to different concentrations of NPY, and it exhibits a linear correlation. Figure 10 F is the fitted linear equation, I = -1.172logC - 4.276(R) 2 =0.9986).

[0073] Figure 9 This refers to the comparison of the recognition ability of the sensor arrays prepared with incubation aptamer concentrations of 0.5, 1, 2, 4, and 6 μM in S2-2 for 1 pM NPY. The recognition of 1 pM NPY is optimal when the NPY aptamer concentration is 2 μM, and the difference in photocurrent before and after recognition is the largest.

[0074] Figure 10 A, B, and C indicate that the electrochemical performance of LIG-Yb-Bi2S3 / PMA is superior to that of LIG-Yb-Bi2S3, LIG-Bi2S3, and LIG. The electrochemical performance of LIG-Yb-Bi2S3 / PMA gradually decreases with the gradual modification of Apt, BSA, and NPY. Figure 10 D indicates that the electrochemical performance of different batches of the prepared electrode arrays varies little, with an RSD value of 3.5%.

[0075] Anti-interference test: Highly representative ions, small molecules, and proteins from human sweat were used as interfering agents to test the anti-interference ability of the prepared photoelectric sensor array. Standards of the interfering agents were dissolved in buffer solutions to prepare solutions of 10mM sodium chloride (NaCl), 2mM potassium chloride (KCl), 100μM glucose (Glu), 50μM uric acid (UA), 50μM urea (Ur), 1μM ascorbic acid (AA), and 1μM cortisol (Cort). These solutions were then incubated on the electrode array, and the photocurrent of a 1pM NPY solution was compared. Stability test: The prepared photoelectric sensor array was subjected to a 1pM solution every 5 days. NPY solution was subjected to photocurrent testing to assess its storage lifespan; Biocompatibility experiment: Considering that the prepared photoelectric sensing array comes into contact with human skin, cytotoxicity studies were conducted on the LIG-Yb-Bi2S3 / PMA electrode array using human umbilical vein endothelial cells (HUVECs). After co-culturing the electrode array with HUVECs for 48 h, live cells were stained with Calcein-AM and dead cells were stained with propidium iodide (PI). Confocal microscopy was used to observe the control group (without electrode array) and the experimental group (with electrode array) to assess its biocompatibility in practical wearable biosensing applications. Figure 10 The anti-interference test of G shows that the prepared photoelectric sensor array is not affected by the high concentration of ions, proteins and other substances in sweat, and has good accuracy in the specific recognition of NPY. Figure 10 H indicates that the prepared photoelectric sensor array has good storage stability. After 30 days of storage, the performance of the photoelectric sensor array can still reach 80% of the initial performance. Figure 10 I indicates that the prepared photoelectric sensing array has good biocompatibility and will not produce toxic or other adverse effects on the skin surface.

[0076] Figure 11 A and B indicate that in dynamic tensile testing, the electrochemical performance of the long strip-shaped LIG is not affected by the tensile bending process, and the electrochemical performance tends to be stable.

[0077] 4. Smart wearable sensors

[0078] The smart wearable sensor includes a flexible three-electrode sensing array (i.e., the photoelectrochemical sensing array prepared above), a signal acquisition and processing circuit system, a power supply module, and a display module; wherein the electrode sensing array, signal acquisition and processing circuit system, and display module are all electrically connected to the power supply module. The three-electrode sensing array comes into contact with an absorbent sponge pad that collects sweat. As the concentration of the target compound NPY in the sweat changes, a corresponding current response signal is generated. Subsequently, the target compound concentration signal is intelligently processed and wirelessly transmitted to a customized mobile app, where the NPY concentration is presented in the form of the mobile app. External connecting wires that enable electrical connections need to be insulated to prevent potential electrical contact between their circuitry and skin or sweat.

[0079] Figure 13 A is a schematic diagram illustrating the regulation of basal feeding behavior by NPY. Figure 13 BE indicates that the NPY concentration decreased in volunteers after lunch, which is consistent with the changes in NPY concentration during eating behavior; Figure 13 F and G indicate the dynamic changes in NPY concentration in volunteers at different times during the physiological stress CPT test, which is consistent with the pattern that NPY concentration increases due to stress in the CPT test. Figure 13 H is a schematic diagram of the NPY diurnal rhythm. In an individual, the NPY concentration reaches its peak in the morning of the same day, then decreases, and reaches its lowest point in the evening. Figure 13 I indicates that the tested individual conforms to a circadian rhythm; Figure 13 J indicates the sensor's measurement results of the dynamic changes in NPY of the tested object over a week.

[0080] Taking eating behavior as an example (eating behavior of 3 healthy subjects before and after lunch), the accuracy and practicality of the smart wearable sensor constructed in this embodiment are verified. As shown in Table 1, NPY standard was added to these original sweat samples, and then analyzed according to the above procedure, with a recovery rate of 93.57%–105.90%. This demonstrates that the smart wearable sensor is accurate and feasible for in-situ detection of NPY in sweat.

[0081] Table 1 shows the spiked detection of NPY in the sweat of three healthy volunteers before and after meals using a smart wearable sensor.

[0082]

[0083] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor, characterized in that, The photoelectrochemical sensing array uses polyimide film as substrate and constructs a graphene-based three-electrode array using laser-induced technology, including a counter electrode region, a reference electrode region, and a working electrode region. Among them, after modifying the working electrode region with phosphomolybdic acid-coated ytterbium-doped bismuth sulfide (Yb-Bi2S3 / PMA) nanomaterials, a neuropeptide Y aptamer is coupled as a molecular recognition element. Then, bovine serum albumin is used to block non-specific recognition sites, thereby obtaining a photoelectrochemical sensing array based on the detection of neuropeptide Y using a self-supplied electron donor. Among them, Yb-Bi2S3 / PMA nanomaterials have the characteristics of near-infrared light excitation and self-electron donor. Under near-infrared light excitation, Yb-Bi2S3 / PMA nanomaterials generate photocurrent, and neuropeptide Y aptamer can specifically bind to the target neuropeptide Y. The degree of decrease in photocurrent of the working electrode is linearly related to the concentration of the target neuropeptide Y, thereby realizing the detection of neuropeptide Y.

2. The photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor according to claim 1, characterized in that, In Yb-Bi2S3 / PMA nanomaterials, the mass ratio of PMA to Yb-Bi2S3 is 0.5:1 to 3:1; the molar percentage of Yb doping in Yb-Bi2S3 relative to Bi2S3 is 10% to 40%; Yb-Bi2S3 is in the form of nanorods with a length of 90 to 360 nm and a diameter of 17 to 50 nm.

3. The photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor according to claim 2, characterized in that, The preparation method of Yb-Bi2S3 / PMA nanomaterials specifically includes the following steps: ①Preparation of Yb-Bi2S3 nanorods: Bismuth nitrate and ytterbium chloride were dissolved in ethylene glycol to obtain solution A; sodium sulfide was dissolved in ethylene glycol to obtain solution B; CO(NH2)2 was dissolved in ethylene glycol to obtain solution C; solution B was added dropwise to solution A while stirring to form a black suspension, and then solution C was added and stirred until homogeneous to obtain a mixture; the resulting mixture was transferred to a reaction vessel and heated at 170~190 ℃ for 20~25 h, and the black solid product was separated, which is Yb-Bi2S3 nanorods, washed and dried for later use; ②Preparation of Yb-Bi2S3 / PMA nanomaterials: Yb-Bi2S3 nanorods were used as precursors and dispersed in anhydrous ethanol. PMA was then added, and the mixture was stirred in a light-protected environment for 8-12 h. The mixture was then heated in a N2 atmosphere until the solvent was completely evaporated. Finally, the solid product was collected at the bottom of the container, which is the Yb-Bi2S3 / PMA nanomaterial. After grinding, it was stored in a dry, light-protected environment for later use.

4. The photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor according to claim 1, characterized in that, The sequence of the neuropeptide Y aptamer is: 5′-NH2-C6-AGC AGC ACA GAG GTC AGA TGC AAA CCA CAGCCT GAG TGG TTA GCG TAT GTC ATT TAC GGA CCT ATG CGT GCT ACC GTG AA-3′.

5. The method for preparing the photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor as described in claim 1, characterized in that, The main steps are as follows: (1) A graphene three-electrode array, comprising a counter electrode, a reference electrode, and a working electrode, was prepared by laser engraving using polyimide film as the substrate. (2) The reference electrode region was modified with Ag / AgCl paste and dried to serve as the reference electrode; the counter electrode region was directly used as the counter electrode. (3) A water dispersion of Yb-Bi2S3 / PMA nanomaterials was drop-coated onto the working electrode area, dried, and then modified with neuropeptide Y aptamers. Then, bovine serum albumin was used to block non-specific recognition sites to obtain a photoelectrochemical sensing array for detecting neuropeptide Y based on a self-supplied electron donor.

6. The method for fabricating the photoelectrochemical sensing array according to claim 5, characterized in that, Step (3) specifically includes the following steps: ① Disperse Yb-Bi2S3 / PMA nanomaterials in ultrapure water and prepare Yb-Bi2S3 / PMA aqueous dispersion by sonication; ② Take Yb-Bi2S3 / PMA aqueous dispersion and drop it onto the working electrode area. After drying, obtain the Yb-Bi2S3 / PMA modified working electrode area; ③ Activate the Yb-Bi2S3 / PMA modified working electrode area with a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 20-40 min; ④ Add neuropeptide Y aptamer solution to the activated working electrode area and incubate for 3-6 h in a humid environment at 30-40 ℃ to form aptamer modified working electrode area; ⑤ Continue to add bovine serum albumin solution to the surface of the working electrode area and incubate for 20-40 min to block non-specific recognition sites, and obtain the working electrode of the photoelectrochemical sensing array based on self-supplied electron donor detection of neuropeptide Y.

7. The method for fabricating the photoelectrochemical sensing array according to claim 6, characterized in that, In step ①, the concentration of the Yb-Bi2S3 / PMA aqueous dispersion is 10~18 mg / mL. -1 In step ②, the amount of Yb-Bi2S3 / PMA aqueous dispersion dropped into the working electrode area is 0.4~0.6 μL / mm. -2 In step ③, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the mixture solution is 40~60 mg / mL. -1 The concentration of N-hydroxysuccinimide is 40-60 mg / mL. -1 The droplet loading of the mixture solution in the Yb-Bi2S3 / PMA modified working electrode region was 0.6–0.9 μL / mm. -2 In step ④, the concentration of the neuropeptide Y aptamer solution is 1–3 μM, and the drop volume is 0.1–0.3 μL. -2 In step ⑤, the mass fraction of the BSA solution is 0.04~0.06%.

8. The method for detecting neuropeptide Y using the photoelectrochemical sensing array as described in claim 1, characterized in that, The main steps are as follows: (1) Dissolve the neuropeptide Y standard sample in a buffer solution to prepare a gradient concentration neuropeptide Y standard solution; (2) Different concentrations of neuropeptide Y standard solutions were respectively drop-coated in parallel onto the working electrode area of ​​the photoelectrochemical sensing array for detecting neuropeptide Y based on self-supplied electron donor as described in claim 1, and then placed in a closed environment with a temperature of 30~40 ℃ and a humidity of 60%~90% for 20~40 min. (3) After incubation in step (2), the three electrodes of the photoelectrochemical sensing array for detecting neuropeptide Y based on self-supplied electron donor as described in claim 1 are connected to the electrochemical workstation through wires, and photocurrent data are collected under near-infrared light excitation; then, a standard curve is established with the logarithm of the concentration of neuropeptide Y in the standard solution of neuropeptide Y as the abscissa and the photocurrent value as the ordinate. (4) Under conditions parallel to steps (2) and (3), the photocurrent of the test liquid is detected, and then the concentration of the target neuropeptide Y in the test liquid is obtained by combining the standard curve of step (3).

9. The method for detecting neuropeptide Y using a photoelectrochemical sensing array according to claim 8, characterized in that, The linear range of the standard curve is 1 fM ~ 100 pM; the drop volume of the neuropeptide Y standard solution in step (2) is 0.2~0.4 μL mm. -2 The test fluid is human sweat.

10. A smart, flexible, wearable photoelectrochemical sensor, characterized in that... The device includes the photoelectrochemical sensing array, signal acquisition and processing circuit module, power supply module, and display module as described in claim 1; the photoelectrochemical sensing array is in contact with the absorption layer that collects sweat in real time, and the photoelectrochemical sensing array, signal acquisition and processing circuit module, and display module are all electrically connected to the power supply module; the photoelectrochemical sensing array is electrically connected to the signal acquisition and processing circuit module, outputting the generated sensing signal in the form of photocurrent and converting it into the concentration of neuropeptide Y in sweat; the display module is used to display the concentration of neuropeptide Y in sweat.

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