Homocysteine detection system for non-invasive body fluid sampling

Abstract

The application relates to the field of measurement technology for diagnostic purposes, and discloses a homocysteine detection system for non-invasive body fluid sampling, which comprises a non-invasive body fluid sampling module, a photoelectrochemical detection module and a signal processing and control unit integrated on a flexible substrate. The non-invasive body fluid sampling module performs reverse iontophoresis by applying a micro-current through an electroosmosis electrode, drives the directional migration of subcutaneous interstitial fluid and enriches the interstitial fluid in a hydrogel layer; the photoelectrochemical detection module is in fluid communication with the hydrogel layer, and a double-atom imprinting recognition layer is arranged on the surface of a photoelectrochemical electrode assembly of the photoelectrochemical detection module. The recognition layer is based on the double-site synergistic coordination of the sulfhydryl and Au sites and the amino and Zn sites of homocysteine molecules, and constructs a recognition cavity with a highly complementary spatial configuration and electronic structure. Without the need for invasive blood sampling, the application overcomes the interference of structure-approximate molecules in a complex body fluid matrix, and realizes the high selectivity, high sensitivity and continuous dynamic monitoring of homocysteine.

Description

Technical Field

[0001] This application relates to the field of measurement technology for diagnostic purposes, and particularly to sampling or measurement techniques for interstitial fluid. Background Technology

[0002] The descriptions in this section are intended only to provide background information for the implementation of this application and should not be construed as an admission or implication that they constitute prior art.

[0003] Homocysteine ​​(Hcy) is a key intermediate in the methionine cycle. Clinical studies have confirmed that high Hcy levels are an independent risk factor for cardiovascular and cerebrovascular diseases, and are closely related to the occurrence of atherosclerosis, myocardial infarction, and stroke. Therefore, regular and even continuous monitoring of Hcy is of great value for risk warning and chronic disease management in high-risk groups of cardiovascular disease.

[0004] Currently, Hcy testing primarily relies on venous blood collection in hospital settings, analyzed using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), or enzyme-linked immunosorbent assay (ELISA). While these methods offer high accuracy, they are complex to operate, require expensive equipment, and are significantly invasive, leading to poor patient compliance and hindering home-based and high-frequency dynamic monitoring. This discrete testing approach struggles to capture real-time fluctuations in human metabolic indicators, resulting in monitoring blind spots.

[0005] To address the aforementioned issues, non-invasive sensing technologies utilizing interstitial fluid (ISF) as a detection medium have attracted significant attention. ISF is considered a "blood-like fluid," and its small molecule concentrations show better correlations with indicators such as blood glucose and blood drug concentrations than those found in sweat or saliva. However, current technologies still face substantial challenges in ISF-based Hcy detection.

[0006] On the one hand, ISF is located below the stratum corneum of the skin, making it difficult to obtain. Although existing reverse iontophoresis technology can achieve non-invasive transdermal extraction using microcurrents, it is often limited by the high variability of skin impedance and the complex physiological environment, resulting in problems such as low sampling throughput and unstable extraction efficiency.

[0007] On the other hand, achieving highly specific recognition of homocysteine ​​(Hcy) in the complex matrix of bodily fluids is extremely difficult. Bioactive bodily fluids contain a wide range of structural analogs such as cysteine ​​(Cys), glutathione (GSH), and methionine, whose molecular structures and electrochemical activities are highly similar to those of Hcy. Existing photoelectrochemical or electrochemical sensors struggle to effectively distinguish these interfering molecules, easily leading to signal overlap and false alarms. Furthermore, when sensors are attached to the skin surface for extended periods, they are susceptible to non-specific adsorption of biomolecules (biofouling) and changes in environmental temperature and humidity, resulting in baseline drift and failing to meet the requirements for clinical-grade quantitative accuracy. Summary of the Invention

[0008] One objective of this application is to provide a non-invasive body fluid sampling homocysteine ​​detection system that can overcome the shortcomings of existing systems, such as low sampling efficiency, poor anti-interference ability, and insufficient stability, and achieve non-invasive, continuous, and accurate detection of Hcy.

[0009] This application discloses a non-invasive body fluid sampling homocysteine ​​detection system, comprising:

[0010] A non-invasive body fluid sampling module, suitable for attachment to the skin surface, includes a hydrogel layer and an electroosmotic electrode; the non-invasive body fluid sampling module is configured to apply a microcurrent through the electroosmotic electrode to perform reverse ion electroosmosis, thereby driving the migration of interstitial fluid under the skin and collecting it in the hydrogel layer;

[0011] A photoelectrochemical detection module is integrated with the non-invasive body fluid sampling module on the same flexible substrate and is fluidly connected to the non-invasive body fluid sampling module through the hydrogel layer to receive the interstitial fluid. The photoelectrochemical detection module includes a photoelectrochemical electrode assembly and a light window for allowing light to shine onto the electrode assembly. The photoelectrochemical electrode assembly includes a photosensitive semiconductor carrier and a single-atom imprinted recognition layer disposed on the photosensitive semiconductor carrier. The recognition layer contains Au single-atom sites and Zn single-atom sites, which together form imprinted recognition holes that are complementary to homocysteine ​​molecules in spatial configuration and electronic structure. These holes are used to specifically bind homocysteine ​​molecules through the dual-site coordination between the thiol group of homocysteine ​​and the Au single-atom site, and between the amino group and the Zn single-atom site.

[0012] A signal processing and control unit is electrically connected to the electroosmotic electrode and the photoelectrochemical electrode assembly; the signal processing and control unit is configured to control the reverse ion electroosmosis and measure the photocurrent signal generated by the photoelectrochemical detection module under illumination, and determine the concentration of homocysteine ​​in the interstitial fluid based on the photocurrent signal.

[0013] In a preferred embodiment, the single-atom imprinted recognition layer is prepared by the following method:

[0014] Homocysteine ​​template molecules, gold ion precursors and zinc ion precursors are mixed in solution to form a coordination precursor solution.

[0015] The photosensitive semiconductor carrier is immersed in the coordination precursor solution for incubation.

[0016] The reaction system containing the photosensitive semiconductor carrier is frozen to a solid state;

[0017] In the frozen state, a reduction potential is applied to the photosensitive semiconductor carrier to perform electrochemical deposition, so as to form dispersed Au single atoms and Zn single atoms on the surface of the photosensitive semiconductor carrier;

[0018] Thaw the reaction system and remove the homocysteine ​​template molecule.

[0019] In a preferred embodiment, the freezing to solid temperature range is from -80°C to -20°C to suppress the aggregation of metal atoms; and the electrochemical deposition is performed by a constant potential.

[0020] In a preferred embodiment, the reduction potential is in the range of -9 to -11V.

[0021] In a preferred embodiment, the gold ion precursor is chloroauric acid, the zinc ion precursor is zinc sulfate, and the molar ratio of the homocysteine ​​template molecule, gold ions, and zinc ions is 1:1:1.

[0022] In a preferred embodiment, the photosensitive semiconductor carrier is titanium dioxide material;

[0023] The signal processing and control unit is configured to control the microcurrent applied by the electroosmotic electrode within the range of 0.2 mA to 0.4 mA.

[0024] In a preferred embodiment, the system employs a planar multilayer stacked structure; the non-invasive body fluid sampling module and the photoelectrochemical detection module are arranged side by side on the flexible substrate; the hydrogel layer covers the sensing areas of the non-invasive body fluid sampling module and the photoelectrochemical detection module, forming a continuous liquid channel so that interstitial fluid diffuses laterally from the non-invasive body fluid sampling module to the photoelectrochemical detection module.

[0025] In a preferred embodiment, a conductive circuit layer is disposed on the flexible substrate for connecting the electroosmotic electrode and the photoelectrochemical electrode assembly, respectively; the conductive circuit layer is a silver paste circuit formed by screen printing.

[0026] In a preferred embodiment, the system further includes an environmental sensor for detecting at least one parameter including ambient temperature, humidity, and skin contact resistance; the signal processing and control unit is configured to establish a correction model using the at least one parameter to correct the photocurrent signal.

[0027] In a preferred embodiment, the flexible substrate is made of one or more materials selected from polydimethylsiloxane and thermoplastic polyurethane; and the signal processing and control unit includes a wireless communication module for transmitting a determined homocysteine ​​concentration to an external terminal device.

[0028] In the embodiments of this application, a non-invasive detection system integrating a reverse iontophoresis non-invasive body fluid sampling module and a photoelectrochemical detection module based on a gold (Au) and zinc (Zn) dual single-atom imprinted recognition layer is constructed, achieving non-invasive, continuous, and highly selective quantitative monitoring of homocysteine ​​in subcutaneous interstitial fluid. This technical solution utilizes reverse iontophoresis to effectively extract interstitial fluid under non-invasive conditions, solving the pain points of traditional venous blood collection being highly invasive and unable to be monitored in real time. More importantly, by constructing Au and Zn dual single-atom imprinted sites on a photosensitive semiconductor, and utilizing their specific dual-site coordination with the thiol and amino groups in the homocysteine ​​molecule, respectively, a recognition hole highly complementary to the spatial configuration and electronic environment of the target molecule is constructed at the atomic scale. This "dual anchoring" mechanism significantly enhances the molecular recognition specificity in complex body fluid matrices (such as those containing structurally similar interfering substances like cysteine ​​and glutathione), greatly improving the signal-to-noise ratio and accuracy of photoelectric detection signals. This provides a dynamic early warning tool for high-risk cardiovascular populations that combines non-invasive convenience with medical-grade detection accuracy.

[0029] Furthermore, by employing a preparation process that includes template molecule coordination, freeze curing, and low-temperature electrochemical deposition, the free diffusion and aggregation of metal precursors can be restricted by ice crystal lattices, enabling the in-situ anchoring of highly dispersed metal single atoms on the surface of a photosensitive semiconductor carrier, thus ensuring atomic-level precision and high catalytic activity of the imprint recognition sites.

[0030] Furthermore, by precisely controlling the freezing temperature within the range of -80 ℃ to -20 ℃ and combining it with constant potential deposition, the nucleation growth and aggregation behavior of metal atoms can be suppressed both thermodynamically and kinetically, thereby maximizing the stable existence of bimetallic sites in single-atom form and improving the structural uniformity of the recognition layer.

[0031] Furthermore, by applying a high reduction potential of around -10 V, sufficient overpotential driving force can be provided in the high-impedance frozen solid medium to ensure that metal ions can overcome the solid-phase mass transfer resistance and be effectively reduced, thereby achieving controllable adjustment of the single-atom loading.

[0032] Furthermore, by selecting chloroauric acid and zinc sulfate as precursors and strictly controlling their molar ratio with the template molecule to be 1:1:1, the number of coordination complexes formed can be maximized from a stoichiometric perspective. This ensures that sufficient and structurally complete effective recognition sites are distributed on the electrode surface after template removal, thereby improving detection sensitivity.

[0033] Furthermore, by selecting titanium dioxide as the photosensitive semiconductor carrier and limiting the electroosmotic current to a safe threshold of 0.2 mA to 0.4 mA, it is possible to ensure photoelectric conversion efficiency and chemical stability while taking into account the tolerance of human skin, avoiding skin stinging or redness caused by excessive current, and ensuring the safety of long-term wear.

[0034] Furthermore, by designing a planar multilayer stacked structure and a continuous hydrogel channel covering the sampling and detection areas, the lateral automatic diffusion and transport of the collected samples can be achieved by utilizing concentration difference and capillary action without the need for external pumps or valves, thus realizing seamless fluid coupling between the sampling unit and the detection unit on a miniaturized flexible device.

[0035] Furthermore, by using screen printing silver paste to prepare the conductive circuit layer, the manufacturing cost of the device can be significantly reduced while ensuring the reliability of circuit connection and bending stability on the flexible substrate. This facilitates the large-scale production and one-time use promotion of the wearable patch.

[0036] Furthermore, by integrating environmental sensors to monitor temperature, humidity, and contact impedance in real time and introducing a multi-parameter correction model, baseline drift and signal fluctuations caused by sweat secretion, changes in environmental temperature, or changes in skin contact conditions can be effectively compensated, the influence of environmental noise on photocurrent readings can be eliminated, and the accuracy and consistency of detection data under different scenarios can be ensured.

[0037] Furthermore, by using polydimethylsiloxane (PDMS) or thermoplastic polyurethane (TPU), which have excellent breathability and biocompatibility, as the encapsulation substrate and combining it with a wireless communication module, the comfort of long-term wear can be greatly improved, skin allergic reactions can be reduced, and remote real-time transmission of detection data can be achieved, meeting the practical application needs of home medical care and remote chronic disease management. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of an integrated reverse ion electroosmosis-photoelectric detection patch structure according to an embodiment of this application;

[0039] Figure 2 This is a schematic diagram of the fabrication process of a single-atom imprint recognition layer according to an embodiment of this application;

[0040] Figure 3This is a schematic diagram of the single-atom imprinting principle (molecular-level structural principle diagram) according to an embodiment of this application.

[0041] Figure 4 This is a timing flowchart of electroosmotic sampling photoelectric detection according to an embodiment of this application;

[0042] Figure 5A This is a real-time photoelectrochemical response curve of in vivo Hcy according to an embodiment of this application;

[0043] Figure 5B This is a dynamic distribution diagram of cardiac troponin I (cTnI) over time according to an embodiment of this application;

[0044] Figure 5C This is a pairwise comparison diagram of individuals at the time points when Hcy and cTnI reach significant changes, according to an embodiment of this application;

[0045] Figure 5D This is a distribution characteristic diagram of the time difference (Δt) between cTnI and Hcy according to an embodiment of this application. Detailed Implementation

[0046] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0047] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0048] The first embodiment of this application relates to a non-invasive body fluid sampling homocysteine ​​detection system, comprising:

[0049] A non-invasive body fluid sampling module, suitable for attachment to the skin surface, comprises a hydrogel layer and an electroosmotic electrode. The module is configured to apply a microcurrent through the electroosmotic electrode to perform reverse iontophoresis, thereby driving the migration and collection of subcutaneous interstitial fluid within the hydrogel layer.

[0050] The photoelectrochemical detection module is integrated with the non-invasive body fluid sampling module on the same flexible substrate and is fluidly connected to the non-invasive body fluid sampling module through a hydrogel layer to receive interstitial fluid. The photoelectrochemical detection module includes a photoelectrochemical electrode assembly and a light window for allowing light to illuminate the electrode assembly. The photoelectrochemical electrode assembly includes a photosensitive semiconductor carrier and a single-atom imprinted recognition layer disposed on the photosensitive semiconductor carrier. The recognition layer contains Au single-atom sites and Zn single-atom sites, which together form imprinted recognition holes that are complementary to homocysteine ​​molecules in spatial configuration and electronic structure. These holes are used to specifically bind homocysteine ​​molecules through two-site coordination interactions between the thiol group of homocysteine ​​and the Au single-atom site, and between the amino group and the Zn single-atom site. Optionally, the photosensitive semiconductor carrier is made of titanium dioxide.

[0051] A signal processing and control unit is electrically connected to the electroosmotic electrode and photoelectrochemical electrode assembly. The signal processing and control unit is configured to control the reverse ion electroosmosis process and measure the photocurrent signal generated by the photoelectrochemical detection module under illumination, determining the concentration of homocysteine ​​in the interstitial fluid based on the photocurrent signal. Optionally, the signal processing and control unit is configured to control the microcurrent applied by the electroosmotic electrode within the range of 0.2 mA to 0.4 mA. Optionally, the signal processing and control unit may also include a wireless communication module for transmitting the determined homocysteine ​​concentration to an external terminal device.

[0052] Optionally, in one embodiment, the single-atom imprinted recognition layer is prepared by:

[0053] Homocysteine ​​template molecules, gold ion precursors, and zinc ion precursors are mixed in solution to form a coordination precursor solution. The gold ion precursor can be chloroauric acid, and the zinc ion precursor can be zinc sulfate. The molar ratio of the homocysteine ​​template molecule, gold ions, and zinc ions can be 1:1:1.

[0054] The photosensitive semiconductor carrier is immersed in a coordination precursor solution for incubation.

[0055] The reaction system containing the photosensitive semiconductor carrier is frozen to a solid state. The freezing temperature range can be from -80 °C to -20 °C (preferably -78 °C) to suppress the aggregation of metal atoms. Electrochemical deposition is performed using a constant potential.

[0056] In a frozen state, an electrochemical deposition is performed by applying a reduction potential to the photosensitive semiconductor carrier to form dispersed Au and Zn single atoms on the surface of the photosensitive semiconductor carrier. The reduction potential can be -9 to -11 V, preferably -10 V.

[0057] Thaw the reaction system and remove homocysteine ​​template molecules.

[0058] Optionally, in one embodiment, the system employs a planar multilayer stacked structure. The non-invasive body fluid sampling module and the photoelectrochemical detection module are arranged side-by-side on a flexible substrate. A hydrogel layer covers the sensing areas of both the non-invasive body fluid sampling module and the photoelectrochemical detection module, forming a continuous liquid channel to allow interstitial fluid to diffuse laterally from the non-invasive body fluid sampling module to the photoelectrochemical detection module. A conductive circuit layer is disposed on the flexible substrate for connecting the electroosmotic electrode and the photoelectrochemical electrode assembly, respectively. The flexible substrate can be made of one or more materials selected from polydimethylsiloxane and thermoplastic polyurethane. The conductive circuit layer is a silver paste circuit formed by screen printing.

[0059] Optionally, in one embodiment, the system further includes an environmental sensor for detecting at least one parameter including ambient temperature, humidity, and skin contact resistance. The signal processing and control unit is configured to use the detected parameters to build a correction model to correct the photocurrent signal.

[0060] Compared with existing homocysteine ​​detection technologies and wearable body fluid sensing technologies, the embodiments of this application have significant advantages in terms of detection method, identification mechanism, signal stability and system integration.

[0061] First, the embodiments of this application achieve truly non-invasive detection. Traditional Hcy detection requires venous blood collection and relies on laboratory analysis, a highly invasive process with a long testing cycle, making continuous monitoring impossible. Existing wearable devices are mostly limited to acquiring physical signals (heart rate, blood oxygen, etc.) and lack the ability to quantitatively analyze chemical indicators. The embodiments of this application, by constructing a non-invasive body fluid sampling layer on the skin surface and using interstitial fluid instead of blood as the detection medium, can complete the acquisition of biochemical indicators without puncture or blood collection, providing a new technical approach for molecular-level health monitoring.

[0062] Secondly, the embodiments of this application possess the dual advantages of structural precision and chemical selectivity in molecular recognition mechanisms. Existing Hcy detection methods often rely on enzymatic reactions, immunorecognition, or non-specific electrochemical signals, which are significantly affected by environmental interference and lack selectivity. The embodiments of this application employ a single-atom imprinting strategy, using the Hcy molecule itself as a template, and constructing Au–Zn dual single-atom sites in situ on a TiO2 photosensitive semiconductor support through a freeze-electrodeposition chemical reduction process. This design can simultaneously utilize the coordination characteristics of Hcy's thiol and amino groups to form "dual-anchored recognition" sites, which are highly matched to the target molecule in both spatial configuration and electronic environment, thereby achieving highly selective recognition of Hcy in complex body fluids. Compared to traditional molecularly imprinted polymers, the recognition sites in the embodiments of this application are uniformly distributed, have short electron transport paths, and high recognition efficiency, avoiding template residue and non-specific adsorption problems.

[0063] Third, the embodiments of this application exhibit excellent anti-interference and environmental stability. In biological fluid environments, the concentrations of small molecules such as cysteine ​​(Cys), glutathione (GSH), and methionine (Met) are typically much higher than those of Hcy, easily causing signal interference. Existing detection systems generally cannot distinguish these structurally similar sulfur-containing compounds. The embodiments of this application, through a coordination-templated bimetallic single-atom recognition structure, effectively eliminate the adsorption of non-target molecules, resulting in interference signals of less than 10%. Simultaneously, a stable bond is formed between the imprinted layer and the photosensitive semiconductor carrier through electrochemical in-situ reduction, preventing desorption or inactivation due to prolonged detection or multiple cycles. Signal attenuation after long-term use is less than 5%, significantly superior to conventional modified electrode systems.

[0064] Fourth, the detection interface and flexible packaging design of this application combine to form a wearable, reusable integrated system. The imprinted photoelectrode and the flexible hydrogel sampling layer are directly coupled in space and connected to the micro signal acquisition unit through conductive channels. The entire system is encapsulated in a polydimethylsiloxane (PDMS) or thermoplastic polyurethane (TPU) substrate, ensuring both mechanical flexibility and photoelectric response stability. This structure is suitable for adhesion to the skin surface and can operate stably in human environments such as 37°C and high humidity, achieving integrated "sampling-detection-transmission" operation. Compared with existing electrochemical patches or microneedle sensors, this application does not require skin penetration, offers stable signals, is safe to use, and has higher user compliance and clinical translation potential.

[0065] Fifth, the design scheme of this application has good scalability and versatility. The single-atom imprinting strategy does not depend on a specific molecule type; only the template molecule needs to be changed to obtain a specific recognition interface for different small molecules, which is suitable for the detection of amino acids, drug metabolites, and other cardiovascular-related biomarkers. This versatility makes this application not only applicable to the early myocardial infarction risk prediction of Hcy, but also provides a technical foundation for building a wearable health management platform for multi-indicator joint monitoring.

[0066] In summary, the embodiments of this application significantly outperform existing technologies in terms of non-invasive sampling methods, single-atom imprint recognition mechanisms, anti-interference performance, and flexible wearable integrated design. Its innovation lies in the first-ever fusion of single-atom-level molecular recognition with a wearable optoelectronic detection system, achieving a complete closed loop of "near-blood fluid—highly selective recognition—low-noise signal—continuous monitoring." This solves the long-standing problem of traditional detection technologies failing to balance non-invasiveness and accuracy, providing a new technical solution for molecular-level health monitoring and early warning of cardiovascular diseases.

[0067] To better understand the technical solution of this application, a specific embodiment will be used for illustration below. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of this application.

[0068] This application relates to a wearable Hcy detection system and method based on non-invasive body fluid sampling and photoelectrochemical recognition. The system generally consists of a non-invasive body fluid sampling module, a photoelectrochemical detection module, a signal processing and control unit, and a flexible encapsulation and attachment structure, as shown in the figure. Figure 1 As shown, this is embodied in a reverse ion electroosmosis-photoelectric detection integrated patch. Figure 1 The top left image shows the overall layout of the wearable patch, which also shows the relative positions of the non-invasive body fluid sampling module and the photoelectrochemical detection module on the flexible substrate. Figure 1 The sub-diagrams below and on the right correspond to the unfolded diagrams of different structural units in the main diagram: among which, Figure 1 The lower left corner shows a hydrogel cavity diagram, illustrating the liquid channel structure covering the electroosmotic and photoelectric regions in the main image; Figure 1 The lower right corner shows the silver paste circuit diagram, which corresponds to the conductive wiring inside the main diagram; Figure 1 The upper right corner shows the light window diagram, where the small squares represent light windows, corresponding to the upper encapsulation openings in the photoelectrochemical detection module area on the right side of the main image; while Figure 1 The outlines of the non-invasive body fluid sampling module and the photoelectrochemical detection module in the upper left main image are composed of the stacked structures of these sub-images. Figure 1 The green rectangle in the middle represents the electrode. Therefore, the four sub-diagrams are used to illustrate the specific graphics and structure of each functional layer in the main diagram, together forming a layered view of the main patch structure.

[0069] Figure 1 The synergistic action of its components enables real-time detection and quantitative analysis of hemocytosis (Hcy) in the interstitial fluid of human skin, featuring non-invasiveness, high selectivity, wearability, and continuous monitoring. Previous in vivo studies in mice have demonstrated the temporal association between Hcy and acute myocardial infarction, indicating the feasibility of a non-invasive sampling approach. See also... Figures 5A-5D .

[0070] Figure 5A This is the in vivo photoelectrochemical real-time response curve of Hcy, which shows the results of in vivo real-time monitoring of Hcy by photoelectrochemical (PEC) in an acute myocardial injury model. Figure 5A The upper half of the figure shows a continuous record of the original photocurrent changing over time, and its stable photoelectric response can be observed. Figure 5AThe lower half of the figure shows the evolution trend of the extracted photocurrent over time. The results show that in the early stages of the model, the Hcy-related photoelectric signal began to rise continuously and then exhibited a monotonically increasing trend. The red shaded area marks the time window when the Hcy signal began to deviate significantly from the baseline, a point that is significantly earlier than the significant increase stage of the traditional myocardial injury marker cTnI. These results indicate that abnormal changes in Hcy not only occur statistically earlier than cTnI, but can also be captured in vivo in real time by photoelectrochemical methods, providing a direct signal basis for early warning.

[0071] Figure 5B This is a dynamic distribution graph of cTnI over time, showing the dynamic changes in circulating cTnI over 0–24 hours in an acute myocardial injury model using an enzyme-linked immunosorbent assay (ELISA). The results show that cTnI remains at a low level in the early time points (0–2 hours), gradually increases in the following hours, reaches a peak in the middle and late stages, and then slowly declines. This result is consistent with the delayed release characteristics of cTnI as a classic marker of myocardial necrosis after acute myocardial infarction (AMI), suggesting that it is more suitable for post-mortem diagnosis than early warning.

[0072] Figure 5C This is a paired comparison plot of individuals at the time points when Hcy and cTnI reached significant changes. Figure 5C The time points at which Hcy and cTnI reached significant change thresholds were compared within the same animal individual. It was observed that in all individuals, the time point of Hcy change significantly preceded the time point of cTnI increase. Paired analysis showed a significant time difference between the two (p<0.001), indicating that abnormal changes in Hcy systematically preceded cTnI changes over time, rather than being random fluctuations.

[0073] Figure 5D This is a distribution characteristic diagram of the time difference (Δt) between the changes in cTnI and Hcy. Figure 5D The time difference (Δt=t) between the changes in cTnI and Hcy was further statistically analyzed. cTnI -t Hcy ), where t cTnI This is the time point at which the cTnI signal first significantly deviates from the individual baseline, t HcyThis is the time point at which the Hcy signal first significantly deviates from the individual baseline. The results show that Δt exhibits a stable distribution across the population, with a median on an hourly scale and significantly greater than zero overall (p<0.0001). These results indicate that Hcy provides a stable and repeatable early response window during acute myocardial injury, offering a potential temporal advantage for early risk identification.

[0074] comprehensive Figures 5A-5D The results show that the dynamic changes in Hcy occurred significantly earlier than the increase in cTnI during acute myocardial injury, revealing a clear temporal association between Hcy and acute myocardial infarction, and defining a time window that can be used for early warning.

[0075] Structurally, the lower layer of the system is a non-invasive body fluid sampling module. Its function is to obtain interstitial fluid (ISF), a component similar to blood, from the skin surface, providing a detectable sample for the detection unit. The non-invasive body fluid sampling module uses a hydrogel with good biocompatibility and high water content as its main material. The hydrogel absorbs a small amount of ISF through skin micropermeability and maintains a moist environment, thus completing body fluid extraction without damaging the skin integrity. This module can passively achieve sampling without external current, or it can improve the sampling rate by applying a microcurrent to promote the migration of charged small molecules across the stratum corneum. The current during this process is typically controlled within the range of 0.2 to 0.4 mA to ensure safety and comfort. The hydrogel sampling layer is both conductive and permeable, enabling direct transfer of ISF to the detection unit above after sampling, achieving seamless contact between the body fluid and the sensing interface.

[0076] The patch adopts a planar multi-layer stacked structure, with non-invasive body fluid sampling modules and photoelectrochemical detection modules arranged side by side on the same flexible substrate, and electrical connection and functional partitioning achieved through silver paste conductive circuit. Figure 1 The area shown in the lower right corner is the silver paste circuit layer, which is formed into linear conductive patterns on a flexible substrate using screen printing. The left side shows the electrode leads for the non-invasive body fluid sampling module, and the right side shows the electrode leads for the photoelectrochemical detection module. Both circuits have pre-installed solder pads or connectors at their ends for connection to external control circuits and light source driver boards. To avoid mutual interference, the leads of the electroosmotic electrode and the photoelectrochemical electrode are routed parallel to each other on the layout, maintaining an insulating gap in between, ensuring that the electric and optical fields of the two modules do not couple when they are working.

[0077] Functional graphic layers are stacked sequentially on top of the silver paste circuit layer. Figure 1The top left corner shows the layout of the "Non-invasive Body Fluid Sampling Module - Photoelectrochemical Detection Module", which shows the planar outline of the functional electrode area: the left side is the working electrode and counter electrode area of ​​the non-invasive body fluid sampling module, which is shaped like a large sheet electrode with a narrow neck, used to form a relatively uniform electric field on the skin; the right side is the electrode area of ​​the photoelectrochemical detection module, where the electrode pattern is surrounded by multiple conductive finger-like structures along the longitudinal direction, leaving a photosensitive electrode and reaction area in the middle, which facilitates the integration of photosensitive semiconductor and recognition layer in this area. Figure 1 The graphic in the lower left corner is the outline of the hydrogel cavity, which covers the sensing areas of the non-invasive body fluid sampling module and the photoelectrochemical detection module, forming a continuous liquid channel: the interstitial fluid extracted from the skin under electroosmotic drive first accumulates in the left electroosmotic area, and then diffuses laterally through the hydrogel to the right photoelectrochemical detection area, thus completing the closed-loop process of "sampling-transportation-detection" in the same patch.

[0078] Figure 1 The upper right corner features a light window structure, an opening within the overlay encapsulation layer. This layer covers the silver paste circuitry and hydrogel, providing mechanical protection and electrical insulation. The light window is only located in the corresponding area of ​​the photoelectrochemical detection module, allowing light from an external light source or the built-in LED to directly illuminate the photosensitive electrode surface, preventing obstruction by the opaque encapsulation material. The light window is precisely aligned with the central reaction area of ​​the photoelectrochemical detection module to ensure uniform illumination and high photoelectric conversion efficiency. The non-invasive body fluid sampling module is covered by a closed encapsulation layer, with the hydrogel exposed only on the side in contact with the skin. This ensures a stable electroosmotic pathway for current to pass through the hydrogel-skin interface.

[0079] pass Figure 1 As shown in the structure, this application achieves a collaborative design of the electroosmotic electrode pattern, hydrogel cavity, silver paste conductive circuit, and optical window position on the same flexible patch. On the one hand, it clearly divides the electroosmotic sampling area and the photoelectric detection area in space and makes them continuously connected in the liquid path. On the other hand, through the layout optimization of the silver paste circuit and the encapsulation layer, it ensures that each functional module achieves a compact layout and reliable electrical connection within a small patch, providing a specific structural basis for stable and repeatable interstitial fluid sampling and Hcy photoelectric detection in a wearable state.

[0080] The photoelectrochemical detection module, located above the sampling layer, has a core single-atom imprinted recognition layer, the preparation method of which is as follows: Figure 2 As shown, it includes the following steps:

[0081] First, a suitable metal ion precursor is selected based on the coordination characteristics of the functional groups in the Hcy molecule. The Hcy molecule contains both thiol (–SH) and amino (–NH2) groups, which can form stable coordination groups with metal ions such as gold and zinc. Therefore, chloroauric acid (HAuCl4) and zinc sulfate (ZnSO4) are preferred as bimetallic precursors to achieve synergistic imprinting of two single-atom sites. Second, Hcy is mixed with the aforementioned metal ion precursor in a specific ratio and dissolved in an acidic aqueous solution (preferably 0.5 mol / L dilute sulfuric acid) to form a precursor solution. The ratio is determined based on the number of coordinating groups in Hcy, generally Hcy:Au:Zn = 1:1:1.

[0082] Subsequently, the pretreated photosensitive semiconductor carrier (e.g., titanium dioxide electrode sheet) is placed into the precursor solution and incubated for several hours under stirring conditions to promote full coordination between Hcy molecules and metal ion precursors.

[0083] Subsequently, the reaction system containing the photosensitive semiconductor carrier is frozen to a solid state, preferably in the temperature range of -80°C to -20°C.

[0084] Subsequently, electrochemical reduction is performed under this frozen state. The electrochemical reduction process can be carried out using a constant potential, with the preferred reduction potential being -10 V. The process automatically stops when the cumulative charge of the system reaches a preset value, thereby controlling the loading of metal single atoms. Low-temperature freezing effectively suppresses the aggregation of metal atoms, thus achieving dual single-atom dispersion of Au and Zn on the surface of the photosensitive semiconductor carrier.

[0085] After electrochemical reduction, the reaction system is thawed naturally and repeatedly washed with deionized water to remove residual Hcy template molecules and unreduced metal ions. If necessary, it can be dried under an inert atmosphere to obtain a photoelectrode loaded with a dual single-atom imprinted structure of Au and Zn. The surface of the obtained material contains imprinted recognition sites complementary to the Hcy molecular structure, and their spatial configuration and electronic environment highly match the thiol-amino coordination characteristics of Hcy. When Hcy re-enters the detection interface, it can undergo specific adsorption and electronic coupling on this recognition layer, significantly altering the interfacial charge transfer efficiency of the photoelectrode, thereby generating a measurable signal response in photoelectrochemical detection.

[0086] The structural principle of single-atom imprinting at the molecular level is shown in the diagram below. Figure 3As shown. The single-atom imprinted recognition layer prepared by the above method possesses both structural template memory and electronic coordination specificity, retaining the high activity and high utilization of single-atom materials while significantly improving the selectivity and anti-interference performance for the target molecule Hcy. Unlike traditional molecularly imprinted polymers or single-metal active sites, the Au–Zn dual single-atom imprinted system of this application can simultaneously recognize the thiol and amino coordination centers in the Hcy molecule, forming a "coordination dual anchoring effect," thereby generating a larger signal response and lower background noise during detection.

[0087] In terms of signal acquisition and data processing, the output of the photoelectrochemical detection module is connected to the signal acquisition and processing circuit. The circuit includes a microcontroller unit, signal amplifier, filter, and analog-to-digital converter, which amplifies and digitizes the photocurrent signal. While acquiring the Hcy signal, the system simultaneously detects parameters such as ambient temperature, humidity, and skin contact impedance to establish a multi-dimensional correction model and eliminate measurement errors caused by environmental changes. The processed signal is then converted to concentration using an embedded algorithm to obtain the real-time Hcy concentration value. The detection data can be wirelessly transmitted to external terminal devices, such as mobile phones or computing platforms, via Bluetooth Low Energy or Near Field Communication for subsequent health management and risk assessment.

[0088] The entire system is encapsulated on a flexible wearable carrier, which can be made of polydimethylsiloxane (PDMS), thermoplastic polyurethane (TPU), or other polymers with good breathability and skin compatibility. The overall thickness of the patch does not exceed one millimeter, allowing it to adhere tightly to the skin on areas such as the forearm, upper arm, or neck. The patch surface is designed with a breathable and sweat-resistant membrane structure to reduce interference from sweat on the signal and prevent poor electrical contact caused by moisture evaporation. To ensure wearing comfort, the patch can be fixed with medical adhesive and has a certain degree of reusability, with each patch capable of stable operation more than ten times.

[0089] The workflow of the system is as follows Figure 4 As shown, specifically: after the user wears the patch on the skin, the hydrogel sampling layer begins to absorb a small amount of interstitial fluid, and the sampling phase lasts for approximately ten to fifteen minutes. Subsequently, the photoelectrochemical detection module initiates a photoelectric reaction under light illumination, generating a photocurrent signal related to Hcy concentration. After amplification and filtering, the signal enters the microcontroller unit for processing and correction, and Hcy concentration data is output after linear regression and fitting. The entire process can be set to cycle periodically, achieving continuous dynamic monitoring. The detection cycle is generally 20 minutes to 1 hour and can be automatically repeated as needed. Monitoring results can be displayed in real time or uploaded to external devices for early warning of cardiovascular risks.

[0090] In summary, this embodiment integrates a non-invasive body fluid sampling layer and a photoelectrochemical detection module on a wearable flexible platform to construct a novel Hcy monitoring system with molecular selectivity, signal stability, and real-time performance. Compared with existing technologies, this system requires no blood sampling, is easy to operate, and can directly achieve quantitative detection of Hcy on the skin surface, making it suitable for home health monitoring and early disease intervention for high-risk cardiovascular populations.

[0091] This application establishes a non-invasive detection system based on interstitial fluid of the skin for continuous monitoring of homocysteine. The system uses interstitial fluid from the skin surface as the detection medium, extracting a small amount of body fluid without breaking the skin through a flexible hydrogel sampling layer, thus achieving truly non-invasive sampling. The hydrogel layer possesses both good conductivity and permeability, maintaining the moisture of the skin microenvironment and the patency of ion channels, ensuring the stability and repeatability of body fluid sampling. Compared with traditional venous blood sampling, this sampling system eliminates the need for needle puncture, making the detection process safe, painless, and repeatable, suitable for continuous health monitoring and clinical follow-up. Furthermore, the sampling layer and the upper detection electrode are directly coupled to form an integrated structure, allowing sampling and detection to be completed within the same patch, avoiding errors and contamination caused by sample transfer.

[0092] This application employs a single-atom imprinting method to construct a bimetallic single-atom recognition layer specific to Hcy. This method uses the Hcy molecule itself as a template and constructs gold (Au) and zinc (Zn) dual single-atom sites in situ on the surface of a photosensitive semiconductor carrier through three steps: template molecule coordination, cryo-electrochemical deposition, and template removal. During the imprinting process, the thiol groups of Hcy form coordination bonds with Au ions, and the amino groups form coordination bonds with Zn ions. After template removal, dual-coordinate recognition holes with complementary spatial and electronic structures to Hcy are left on the surface of the photosensitive semiconductor carrier. This structure enables dual-site synergistic recognition of Hcy molecules in complex bodily fluid environments, exhibiting extremely high selectivity and stability. Compared with traditional molecularly imprinted polymers or single-metal catalytic sites, the single-atom imprinted layer of this application achieves precise distribution and electronic control of recognition sites at the atomic scale, significantly improving signal response sensitivity and anti-interference capability.

[0093] This application presents a wearable homocysteine ​​(Hcy) detection system integrating reverse iontophoresis sampling and photoelectrochemical detection. This system enables non-invasive extraction and real-time detection of interstitial fluid on the skin surface, forming an integrated "sampling-detection-signal output" system. The system employs a flexible patch structure, consisting of a reverse iontophoresis non-invasive body fluid sampling module, a photoelectrochemical detection module, and a control circuit. After the patch is attached to the skin surface, a weak constant current (approximately 0.2–0.4 mA) drives charged small molecules to migrate from the subcutaneous interstitial fluid to the hydrogel layer inside the patch, achieving non-invasive body fluid sampling. This hydrogel simultaneously acts as an ion conductor and sample enrichment medium, collecting sufficient body fluid for detection within 10–20 minutes. The photoelectrochemical detection module is directly integrated with the sampling layer, utilizing a semiconductor electrode to generate a photocurrent response under illumination to detect the Hcy concentration in the collected fluid. The acquisition, amplification, and conversion of the detection signal are completed by a micro-control circuit and transmitted to an external terminal for real-time display via Bluetooth or a wired interface. The entire system features small size, low power consumption, and stable signal, enabling dynamic monitoring while being worn.

[0094] In animal experiments, the device has achieved stable operation on the skin of mice. The patch can work continuously for several hours, demonstrating good skin compatibility and signal repeatability. A significant advantage of this integrated design is that sampling and detection are completed on the same platform, eliminating the need for sample transfer or complex operations, thus greatly improving detection speed and accuracy. In a further embodiment, this application can introduce a single-atom imprinted recognition layer on the surface of the photoelectrochemical detection module to enhance the selective recognition of Hcy molecules, realizing a complete non-invasive detection system of "reverse ion electroosmosis sampling + single-atom imprinted photoelectrochemical detection," providing a new wearable detection solution for early warning of cardiovascular risks.

[0095] It should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0096] This specification includes combinations of various embodiments described herein. Individual references to embodiments (e.g., “one embodiment”, “some embodiments”, or “preferred embodiments”) do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless indicated to be mutually exclusive or are readily apparent to those skilled in the art. It should be noted that the word “or” is used in a non-exclusive sense throughout this specification unless the context explicitly indicates or requires it.

[0097] All references to this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the contents of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A non-invasive body fluid sampling homocysteine ​​detection system, characterized in that, include: A non-invasive body fluid sampling module, suitable for attachment to the skin surface, includes a hydrogel layer and an electroosmotic electrode; The non-invasive body fluid sampling module is configured to apply a microcurrent through the electroosmotic electrode to perform reverse ion electroosmosis, thereby driving the interstitial fluid under the skin to migrate and be collected in the hydrogel layer; The photoelectrochemical detection module is integrated with the non-invasive body fluid sampling module on the same flexible substrate, and is fluidly connected to the non-invasive body fluid sampling module through the hydrogel layer to receive the interstitial fluid; The photoelectrochemical detection module includes a photoelectrochemical electrode assembly and a light window for allowing light to shine onto the electrode assembly; wherein, the photoelectrochemical electrode assembly includes a photosensitive semiconductor carrier and a single-atom imprinted recognition layer disposed on the photosensitive semiconductor carrier; the recognition layer contains Au single-atom sites and Zn single-atom sites, which together form imprinted recognition holes that are complementary to homocysteine ​​molecules in spatial configuration and electronic structure, for specifically binding homocysteine ​​molecules through the two-site coordination between the thiol group of homocysteine ​​and the Au single-atom site, and the amino group and the Zn single-atom site; A signal processing and control unit is electrically connected to the electroosmotic electrode and the photoelectrochemical electrode assembly; the signal processing and control unit is configured to control the reverse ion electroosmosis and measure the photocurrent signal generated by the photoelectrochemical detection module under illumination, and determine the concentration of homocysteine ​​in the interstitial fluid based on the photocurrent signal.

2. The system according to claim 1, characterized in that, The single-atom imprinted recognition layer is prepared by the following method: Homocysteine ​​template molecules, gold ion precursors and zinc ion precursors are mixed in solution to form a coordination precursor solution. The photosensitive semiconductor carrier is immersed in the coordination precursor solution for incubation. The reaction system containing the photosensitive semiconductor carrier is frozen to a solid state; In the frozen state, a reduction potential is applied to the photosensitive semiconductor carrier to perform electrochemical deposition, so as to form dispersed Au single atoms and Zn single atoms on the surface of the photosensitive semiconductor carrier; Thaw the reaction system and remove the homocysteine ​​template molecule.

3. The system according to claim 2, characterized in that, The freezing temperature range is from -80°C to -20°C to suppress the aggregation of metal atoms; and the electrochemical deposition is performed by a constant potential.

4. The system according to claim 3, characterized in that, The reduction potential is -9 to -11 V.

5. The system according to claim 2, characterized in that, The gold ion precursor is chloroauric acid, and the zinc ion precursor is zinc sulfate; and the molar ratio of the homocysteine ​​template molecule, gold ions, and zinc ions is 1:1:

1.

6. The system according to claim 1, characterized in that, The photosensitive semiconductor carrier is titanium dioxide material; The signal processing and control unit is configured to control the microcurrent applied by the electroosmotic electrode within the range of 0.2 mA to 0.4 mA.

7. The system according to claim 1, characterized in that, The system adopts a planar multilayer stacked structure; the non-invasive body fluid sampling module and the photoelectrochemical detection module are arranged side by side on the flexible substrate; the hydrogel layer covers the sensing area of ​​the non-invasive body fluid sampling module and the sensing area of ​​the photoelectrochemical detection module, forming a continuous liquid channel so that the interstitial fluid diffuses laterally from the non-invasive body fluid sampling module to the photoelectrochemical detection module.

8. The system according to claim 7, characterized in that, A conductive circuit layer is disposed on the flexible substrate for connecting the electroosmotic electrode and the photoelectrochemical electrode assembly respectively; the conductive circuit layer is a silver paste circuit formed by screen printing.

9. The system according to claim 1, characterized in that, The system also includes an environmental sensor for detecting at least one parameter, including ambient temperature, humidity, and skin contact resistance; the signal processing and control unit is configured to establish a correction model using the at least one parameter to correct the photocurrent signal.

10. The system according to any one of claims 1-9, characterized in that, The flexible substrate is made of one or more materials selected from polydimethylsiloxane and thermoplastic polyurethane; and the signal processing and control unit includes a wireless communication module for transmitting the determined homocysteine ​​concentration to an external terminal device.

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