Multi-channel double-layer membrane anti-blood cell interference electrochemical test piece and application thereof

Abstract

The invention discloses a multi-channel double-layer membrane blood cell interference resisting electrochemical test piece and application thereof, and belongs to the technical field of electrochemical test pieces, the multi-channel double-layer membrane blood cell interference resisting electrochemical test piece comprises a base material, a sensing electrode, a top sealing layer and a double-layer membrane structure composed of an enzyme reaction membrane layer and a blood cell filtering membrane layer, and the double-layer membrane structure is located above the sensing electrode. According to the invention, a completely independent multi-channel microfluidic system is realized through physical structure partition, and a three-dimensional bulge structure is arranged at a blood inlet of each microfluidic channel, so that the blood suction starting speed and the sample entering efficiency are remarkably improved, and the problems of unstable blood suction efficiency and difficulty in avoiding cross interference of the existing multi-channel design are solved on the structural aspect. According to the invention, a double-layer membrane structure is adopted, a physical filter membrane layer with a stable aperture structure is constructed by blending PU / PVA, and the physical filter membrane layer is superposed on a glucose dehydrogenase reaction layer, so that red blood cells are prevented from entering a detection area from the source, and the influence of HCT on signals is reduced.

Description

Multichannel double-layer membrane electrochemical test strips for resisting blood cell interference and their applications Technical Field

[0001] This invention relates to the field of electrochemical test strip technology, specifically to a multi-channel double-layer membrane electrochemical test strip that resists blood cell interference and its application. Background Technology

[0002] Electrochemical test strips are point-of-care testing consumables that analyze body fluids based on electrochemical sensing principles, playing a significant role in the management of chronic diseases such as diabetes. These products typically utilize the current response between the working and counter electrodes, combined with glucose oxidase or glucose dehydrogenase immobilized in the reaction area of ​​the test strip, to achieve real-time measurement of blood glucose concentration. Compared to traditional colorimetric methods, electrochemical testing offers significant advantages such as faster detection speed, ease of operation, smaller sample size, and automated readings, making it the mainstream method for blood glucose monitoring in both home and hospital settings.

[0003] Currently, the typical structure of electrochemical test strips consists of multiple layers of bonded materials, forming a capillary-driven micro-reaction chamber. Blood samples enter the capillary channel through an inlet at one end of the strip, rapidly filling the reaction zone under capillary force and wetting the enzyme reaction layer covering the electrode surface. Electrode materials primarily use carbon paste or noble metals such as gold, platinum, and palladium to improve conductivity and signal stability. The reaction layer typically contains immobilized glucose enzymes and electron mediators (such as potassium ferricyanide) to catalyze the reaction and generate a measurable electrical signal. To ensure smooth blood sample filling of the reaction chamber without air bubbles, the strip design often includes micro-venting holes or a transparent observation window to release air and help users determine if blood absorption is sufficient.

[0004] With the continuous expansion of practical application scenarios, existing electrochemical test strips still face several technical bottlenecks: First, most mainstream products adopt a single-channel structure (e.g., CN200810081128.6), where blood samples enter through a single aspiration port and are transported to the electrode reaction zone along a single capillary path, ultimately generating a current signal output. However, the single-channel structure has significant shortcomings in terms of functional extensibility and detection stability: A) Firstly, it only supports a single sample introduction path, limiting its application capability in simultaneous detection of multiple items or repeated verification of results. Once the channel aspiration is incomplete, the fluid flow is interrupted, or the reaction is abnormal, the entire testing process cannot be compensated for, affecting the reliability of the detection results. This limitation is particularly prominent in clinical comparisons or monitoring complex conditions.

[0005] B) Secondly, regarding sample adaptability, traditional single-channel designs generally lack effective filtering structures to counter red blood cell interference, relying mostly on a single enzyme membrane for reaction signal generation. In samples with high hematocrit, red blood cells easily adhere to the reaction interface, causing electrical signal drift, reducing measurement accuracy, and even leading to sample aspiration failure due to red blood cell blockage. These structures have limited adaptability to changes in hematocrit and their stability is difficult to guarantee.

[0006] C) In addition, the blood aspiration port often adopts an opening structure flush with the plate body, without considering the auxiliary design for droplet contact angle and blood sample drainage. In micro-blood collection scenarios, droplets often adhere to the surface of the aspiration port, making it difficult to initiate capillary aspiration. If the blood viscosity is high or the sampling position is inaccurate, aspiration delay or failure is more likely to occur, causing difficulties for the user.

[0007] II. In existing electrochemical blood glucose test strips, to address the interference of hematocrit (HCT) in blood on detection accuracy, a blood cell filter membrane is often used to cover the inlet of the reaction chamber or the electrode surface to achieve preliminary separation of plasma and red blood cells. These filter membrane materials are mostly based on microporous polymer membranes or non-woven fiber materials, such as glass fiber membranes (GF), cellulose acetate membranes (CA), nylon membranes, and polyvinylidene fluoride (PVDF). Their mechanism of action mainly relies on physical pore size restriction, blocking large particles such as red blood cells from entering the enzyme reaction zone through size sieving, allowing only small glucose molecules to diffuse, thereby reducing signal interference. However, these anti-HCT membrane structures still have the following problems: A) Most of these anti-HCT membrane structures are single-layer designs, with simple structures and functions focused on the physical isolation of red blood cells, without systematic optimization for plasma diffusion efficiency, electrode interface protection, or complex interference mechanisms.

[0008] B) The membrane material has insufficient mechanical stability and flexibility, which can easily cause pore blockage or flow rate stagnation under conditions of high sample viscosity or high blood cell content (such as HCT>55%), affecting reaction efficiency and test time, and even causing sample injection failure.

[0009] C) In addition, single-layer membranes are prone to losing their filtration function due to deformation under pressure or adsorption saturation, and lack long-term repeatability.

[0010] III. With the increasing demand for multi-marker joint detection, some detection platforms are gradually trying to introduce multi-channel microfluidic structures into electrochemical test strips to support the simultaneous detection of multiple blood indicators or the repeated comparison of the same indicator. Currently, several large foreign manufacturers (such as Abbott and Roche) and research institutions have proposed corresponding solutions, mainly by setting multiple parallel channel structures, multiple blood absorption ports and independent detection electrodes on the test strip substrate to achieve multi-site reaction detection. However, these multi-channel test strips generally follow the single-channel design logic in terms of structure, and the channels are separated by bonding layers or boundary division methods. Multiple blood absorption ports are often distributed on one side of the test strip or arranged along the edge of the strip, and the blood sample enters each microfluidic channel through natural contact. The detection channel has independent capillary pathways and electrode areas, which theoretically have the potential to support the simultaneous reaction of multiple detection indicators. Although the above-mentioned multi-channel test strips provide a certain degree of parallel detection capability, they still have the following key defects: A) First, most of the multiple blood absorption ports adopt a "flat-mouth" structure flush with the strip body, lacking three-dimensional guidance or capillary initiation design. Under micro-volume blood collection conditions, droplets are difficult to form an effective contact surface. Especially when the blood sample is highly viscous or the finger touch point is misaligned, the blood often adheres to the periphery of the suction port and cannot be quickly drawn into the channel, which can easily lead to sampling failure or uneven flow rate.

[0011] B) Secondly, in most schemes, there is no physical barrier between multiple blood aspiration ports. Blood samples may cross-diffusion through edge gaps or interlayer micropores. This is especially likely to cause cross-flow problems between channels when the sample volume is large or the sheet is under pressure, which ultimately affects the independence and accuracy of the detection results of each microfluidic channel 2.

[0012] C) Furthermore, the channel body is often divided by simple die-cutting or screen printing, which limits the boundary sealing and structural stability. Blood may deviate in direction or have different flow rates before entering the downstream electrode area, resulting in problems such as asynchronous signal output and inconsistent response time between channels, which affects the consistency and comparison effectiveness of parallel detection.

[0013] In summary, although the number of multi-channel test chips has been expanded, there are still significant shortcomings in ensuring the sampling stability, signal accuracy, and anti-interference capability of each microfluidic channel 2.

[0014] Based on this, the present invention designs a multi-channel double-layer membrane electrochemical test strip that resists blood cell interference and its application to solve the above problems. Summary of the Invention

[0015] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a multi-channel double-layer membrane electrochemical test strip that resists blood cell interference and its application.

[0016] To achieve the above objectives, the present invention provides the following technical solution: a multi-channel, double-layer membrane electrochemical test strip for resisting blood cell interference, comprising a substrate, a sensing electrode, a top sealing layer, and a double-layer membrane structure composed of an enzyme reaction membrane layer and a blood cell filtration membrane layer, wherein the double-layer membrane structure is located above the sensing electrode, and the upper end of the substrate is encapsulated by the top sealing layer; the substrate is provided with multiple independent microfluidic channels, each microfluidic channel having an independent blood inlet, a liquid channel, and a detection area, and structural partitions are provided between the microfluidic channels to isolate them from each other; a three-dimensional protrusion structure is provided at the blood inlet at one end of each microfluidic channel; the three-dimensional protrusion structure is a step-shaped protrusion formed in the horizontal direction of the blood inlet; two sensing electrode mounting holes are provided in the detection area at the other end of each microfluidic channel; the sensing electrode is inserted and installed in the sensing electrode mounting hole.

[0017] Furthermore, when two microfluidic channels are provided on the substrate, the three-dimensional protrusion structure is a three-dimensional protrusion with a pointed opening; when three microfluidic channels are provided on the substrate, the three-dimensional protrusion structures on both sides are three-dimensional protrusions with a pointed opening; and the three-dimensional protrusion structure in the middle is a trapezoidal three-dimensional protrusion with a flat opening in the middle.

[0018] Furthermore, the depth of the microfluidic channel is 0.08 mm; the thickness of the three-dimensional protrusion structure is 0.37 mm, and the upper surface of the three-dimensional protrusion structure is flush with the bottom surface of the microfluidic channel.

[0019] Furthermore, by die-cutting, mutually isolated microfluidic channels, pre-reserved sensor electrode mounting holes, a three-dimensional protrusion structure at the blood inlet of the microfluidic channel, and mounting through holes are formed on the surface of the PET substrate.

[0020] Furthermore, the surface of the PET substrate is subjected to plasma treatment.

[0021] Furthermore, the sensing electrode is a copper-based gold-plated electrode, formed by electroplating a uniform and dense Au layer on the surface of a copper substrate.

[0022] Furthermore, the copper-based gold-plated electrode is assembled by inserting it into the pre-cut sensor electrode mounting holes on the substrate. After assembly, the upper surface of the copper-based gold-plated electrode is higher than the microfluidic channel.

[0023] Furthermore, the enzyme reaction membrane uses GDH as the core reaction component, and the preparation process is as follows: GDH is dissolved in phosphate buffer, and electron mediators, stabilizers and thickeners are added and stirred to form a homogeneous enzyme solution; then the enzyme solution is dropped onto the surface of a copper-based gold-plated electrode using a micro-dispensing instrument, with a controlled amount of 10-30 μL / dip, and after drying, a firmly attached enzyme reaction membrane is formed.

[0024] Furthermore, the preparation process of the blood cell filtration membrane is as follows: PU prepolymer and PVA are weighed and thoroughly dissolved in N,N-dimethylformamide by stirring to form a homogeneous and transparent solution; the solution is then irradiated with an electron beam at a dose of 15-25 kGy; subsequently, a crosslinking agent, fumaric acid or citric acid, and a surfactant are added; the solution is then irradiated with gamma rays at a dose of 25-35 kGy; the resulting membrane solution is coated onto the surface of a solidified enzyme reaction membrane, controlling the membrane thickness to be within the range of 30-50 μm; drying and curing are then performed to ensure the crosslinking reaction is fully completed, forming a dense and uniform blood cell filtration membrane with a microporous structure.

[0025] To better achieve the objectives of this invention, this invention also provides an application of the aforementioned multi-channel double-layer membrane anti-blood cell interference electrochemical test strip in the parallel detection of multiple biomarkers or the repeated detection of the same biomarker.

[0026] Compared to existing technologies, the advantages of this invention are as follows: By introducing a multi-channel structure and a prominent blood inlet design into the electrochemical test strip, and setting a double-layer membrane structure in the electrode area, this invention achieves a comprehensive improvement in sampling stability, signal accuracy, and anti-interference capability at the overall design level. Specifically, this is reflected in the following aspects: 1. Multi-channel microfluidic structure design: In this invention, each microfluidic channel is completely physically isolated. Each microfluidic channel is equipped with an independent blood inlet, liquid transport path, and electrode detection area, enabling parallel detection of multiple biomarkers (such as glucose, lactate, etc.) on the same strip, or repeated measurement of the same biomarker. This avoids the problems of single-channel structures, limited functionality, and poor fault tolerance, enhancing the reliability and adaptability of the detection system under complex sample conditions. Simultaneously, it eliminates the risk of sample crosstalk and electrical signal interference, ensuring independent signal output from each microfluidic channel, thus improving the accuracy and stability of measurement data. The channel width and depth of the microfluidic channels can be optimized according to sample volume requirements, ensuring smooth flow of blood samples driven by capillary force.

[0027] 2. Design of the Blood Inlet Protrusion Structure: This invention features a blood inlet at the beginning of each microfluidic channel. A stepped, three-dimensional protrusion structure is designed in the horizontal direction of the inlet area. This structure significantly disrupts the surface tension of the blood droplet, improving the wettability and drainage of the blood-channel interface. This significantly improves the initiation efficiency of blood contact and entry into the microchannel, enabling rapid blood drainage in peripheral sampling. It is particularly suitable for micro-volume blood collection in the extremities, effectively reducing the test error rate caused by delayed or failed blood absorption. The improvement in blood absorption efficiency is especially significant under extreme sample conditions such as high HCT or high blood viscosity, ensuring the test strip's universal applicability to a wide range of blood samples. This geometrically guided structure requires no additional power or complex surface treatment, has a simple manufacturing process, strong adaptability, and good industrial feasibility.

[0028] 3. The bilayer membrane design provides a physical barrier and functional division of labor to combat blood cell interference. The bottom layer is an enzyme reaction layer immobilized with glucose dehydrogenase, responsible for specifically recognizing glucose and generating an electrical signal. The upper layer is a PU / PVA blended microporous filter membrane, which, through its physical porous structure, blocks red blood cells from entering the enzyme reaction zone. It stably blocks red blood cells with a diameter of approximately 6-8 μm, preventing them from entering the enzyme reaction zone and ensuring that the bottom electrode only responds to target small molecules. This solves the interference problems such as red blood cell deposition and electrode shielding when HCT increases. Compared to traditional single-layer membranes, this membrane system has stronger flexibility and permeability stability, improving filtration efficiency while maintaining a rapid diffusion rate and reducing the probability of membrane clogging, thereby enhancing detection consistency and accuracy.

[0029] Furthermore, compared to existing electrochemical correction methods that rely on algorithms to compensate for HCT interference, the bilayer membrane structure proposed in this invention achieves interference suppression through physical means, simplifying the detection system structure and avoiding multiple measurements and complex signal fitting processes. This method does not rely on dynamic calculations and corrections at the instrument end, reducing system development complexity and instrument costs, while improving the consistency and clinical interpretability of measurement data under extreme HCT sample conditions. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0031] Figure 1 is a schematic diagram of the structure of the two-channel double-layer membrane electrochemical test strip for resisting blood cell interference of the present invention after removing the top sealing layer.

[0032] Figure 2 is a magnified view of part A in Figure 1.

[0033] Figure 3 is a schematic diagram of the structure of the three-channel double-layer membrane anti-blood cell interference electrochemical test strip of the present invention after removing the top sealing layer.

[0034] Figure 4 is a magnified view of part B in Figure 3.

[0035] Figure 5 is a cross-sectional view of the electrochemical test piece of the present invention.

[0036] Figure 6 is a schematic diagram of the liquid to be tested entering the electrochemical test piece of the present invention.

[0037] Figure 7 shows the fitted curve of the lactic acid signal.

[0038] Figure 8 shows the blood glucose signal fitting curve. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Example 1: Please refer to Figures 1-6 in the accompanying drawings of the instruction manual. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference includes a substrate 1, a sensing electrode, a top sealing layer, and a double-layer membrane structure composed of an enzyme reaction membrane layer and a blood cell filtration membrane layer. The double-layer membrane structure is located above the sensing electrode. The upper end of the substrate 1 is encapsulated by the top sealing layer. Two or three independent microfluidic channels 2 are provided on the substrate 1. Each microfluidic channel 2 is equipped with an independent blood inlet, liquid channel, and detection area. Structural partitions are provided between the microfluidic channels 2 to isolate them from each other, ensuring that each microfluidic channel 2 can effectively absorb, transport, and... The liquids in the reaction process are completely isolated, thus avoiding cross-contamination and signal interference; the top sealing layer has an exhaust port at the end of each microfluidic channel 2; a three-dimensional protrusion structure 3 is provided at the blood inlet at one end of each microfluidic channel 2; the three-dimensional protrusion structure 3 is a step-shaped protrusion formed in the horizontal direction of the blood inlet, which makes it easier for blood to be quickly drawn into the channel by capillary force when it comes into contact with the suction port due to the change in contact angle. This structure improves the problems of blood suction delay, insufficient sample or failure in traditional flat-mouth blood suction in fingertip blood collection, and is especially suitable for use scenarios with micro-sampling or high blood viscosity.

[0041] When two microfluidic channels 2 are provided on the substrate 1, the three-dimensional protrusion structure 3 is a three-dimensional protrusion with a pointed opening; when three microfluidic channels 2 are provided on the substrate 1, the three-dimensional protrusion structures 3 on both sides are three-dimensional protrusions with a pointed opening; the three-dimensional protrusion structure 3 in the middle is a trapezoidal three-dimensional protrusion with a flat opening in the middle; the groove depth of the microfluidic channel 2 is 0.08 mm; the thickness of the three-dimensional protrusion structure 3 is 0.37 mm, and the upper surface of the three-dimensional protrusion structure 3 is flush with the bottom surface of the groove of the microfluidic channel 2; two sensor electrode mounting holes 4 are provided in the detection area at the other end of each microfluidic channel 2; the sensor electrodes (working electrode and reference / counter electrode) are inserted and installed in the sensor electrode mounting holes 4 as the basic conductor for signal acquisition; the sensor electrode mounting holes 4 can be pre-cut to ensure that the subsequent sensor electrodes are accurately aligned with the microfluidic path, ensuring stable signal acquisition and accurate response in the detection area.

[0042] Preferably, the substrate 1 is also provided with a number of mounting through holes 5 for processing and positioning, which facilitates processing and positioning.

[0043] The substrate 1 is made of PET (polyethylene terephthalate) sheet. PET substrate, as the load-bearing structure, possesses good dimensional stability, electrical insulation, and optical transmittance, making it suitable for large-scale continuous processing. The following can be formed on the surface of the PET substrate by die-cutting: mutually isolated microfluidic channels 2, pre-reserved sensor electrode mounting holes 4, a three-dimensional protrusion structure 3 at the blood inlet of the microfluidic channel 2, and mounting through holes 5. Simultaneously, plasma treatment is performed on the surface of the PET substrate to improve its surface tension, enhance the adhesion of the bilayer membrane structure and sample wetting performance, laying the foundation for subsequent bilayer membrane structure fixation and microfluidic flow.

[0044] The sensing electrode is a copper-based gold-plated electrode, formed by electroplating a uniform and dense layer of metallic gold (Au) on the surface of a copper substrate. The copper-based gold-plated electrode is assembled by inserting it into the sensing electrode mounting holes 4 pre-cut in the substrate 1. After assembly, the upper surface of the sensing electrode is slightly higher than the microfluidic channel 2.

[0045] The double-layer membrane structure includes an enzyme reaction membrane at the bottom and a blood cell filtration membrane at the top, with clear division of functions and synergistic effect between the two.

[0046] The enzyme reaction membrane uses glucose dehydrogenase (GDH) as the core reaction component. The preparation process is as follows: GDH is dissolved in phosphate buffer (0.1 mol / L, pH 7.4) according to the required activity units. A certain proportion of electron mediator (such as potassium ferricyanide), stabilizer (such as BSA or PEG), and thickener (sodium carboxymethyl cellulose) are added and stirred to form a homogeneous enzyme solution. Then, the enzyme solution is dropped onto the surface of a copper-based gold-plated electrode using a micro-dispensing instrument at a controlled volume of 10-30 μL / dip. The electrode is then dried in a low-temperature drying oven at 25°C and 20-30% relative humidity for 30-60 minutes to form a firmly attached enzyme reaction membrane.

[0047] The blood cell filtration membrane uses a blend of polyurethane (PU) and polyvinyl alcohol (PVA) as the matrix, and constructs a microporous structure through cross-linking. The preparation process is as follows: Weigh PU prepolymer (20 wt%) and PVA (10 wt%) into N,N-dimethylformamide (DMF) and stir thoroughly to dissolve, forming a homogeneous and transparent solution; then irradiate the solution with an electron beam dose of 15-25 kGy; subsequently add crosslinking agent fumaric acid (FA) or citric acid (CA) at a dose of 1-3 wt%, and add a small amount of surfactant (such as Tween-20, at a dose of 0.1 wt%) to improve the uniformity of the coating; then irradiate the solution with gamma rays at a dose of 25-35 kGy; coat the obtained membrane solution onto the surface of the cured enzyme reaction membrane layer using a doctor blade coating method, controlling the membrane layer thickness in the range of 30-50 μm; after coating, cure in an oven at 60℃ for 1 hour to ensure that the crosslinking reaction is fully completed, forming a dense and uniform blood cell filtration membrane layer with a microporous structure.

[0048] This invention achieves a comprehensive improvement in sampling stability, signal accuracy, and anti-interference capability at the overall design level by introducing a multi-channel structure and a prominent blood inlet design in the electrochemical test strip, and setting a double-layer membrane structure in the electrode area. Specifically, this is reflected in the following aspects: 1. Multi-channel microfluidic structure design: In this invention, each microfluidic channel 2 is completely physically isolated. Each microfluidic channel 2 is equipped with an independent blood inlet, liquid transport path, and electrode detection area, enabling parallel detection of multiple biomarkers (such as glucose, lactate, etc.) on the same strip, or repeated measurement of the same biomarker. This avoids the problems of single-channel structures, limited functionality, and poor fault tolerance, enhancing the reliability and adaptability of the detection system under complex sample conditions. Simultaneously, it eliminates the risk of sample crosstalk and electrical signal interference, ensuring independent signal output from each microfluidic channel 2, improving the accuracy and stability of measurement data. The channel width and depth of the microfluidic channel 2 can be optimized according to sample volume requirements, ensuring smooth flow of blood samples driven by capillary force.

[0049] 2. Design of the Blood Inlet Protrusion Structure: This invention features a blood inlet at the beginning of each microfluidic channel 2. A stepped, three-dimensional protrusion structure 3 is designed in the horizontal direction of the inlet area. This structure significantly disrupts the surface tension of the blood droplet, improving the wettability and drainage of the blood-channel interface. This significantly improves the initiation efficiency of blood contact and entry into the microchannel, enabling rapid blood drainage in peripheral sampling. It is particularly suitable for micro-volume blood collection in the extremities, effectively reducing the test error rate caused by delayed or failed blood absorption. The improvement in blood absorption efficiency is especially significant under extreme sample conditions such as high HCT or high blood viscosity, ensuring the test strip's universal applicability to a wide range of blood samples. This geometrically guided structure requires no additional power or complex surface treatment, has a simple manufacturing process, strong adaptability, and good industrial feasibility.

[0050] 3. The bilayer membrane design provides a physical barrier and functional division of labor to combat blood cell interference. The bottom layer is an enzyme reaction layer immobilized with glucose dehydrogenase, responsible for specifically recognizing glucose and generating an electrical signal. The upper layer is a PU / PVA blended microporous filter membrane, which, through its physical porous structure, blocks red blood cells from entering the enzyme reaction zone. It stably blocks red blood cells with a diameter of approximately 6-8 μm, preventing them from entering the enzyme reaction zone and ensuring that the bottom electrode only responds to target small molecules. This solves the interference problems such as red blood cell deposition and electrode shielding when HCT increases. Compared to traditional single-layer membranes, this membrane system has stronger flexibility and permeability stability, improving filtration efficiency while maintaining a rapid diffusion rate and reducing the probability of membrane clogging, thereby enhancing detection consistency and accuracy.

[0051] Furthermore, compared to existing electrochemical correction methods that rely on algorithms to compensate for HCT interference, the bilayer membrane structure proposed in this invention achieves interference suppression through physical means, simplifying the detection system structure and avoiding multiple measurements and complex signal fitting processes. This method does not rely on dynamic calculations and corrections at the instrument end, reducing system development complexity and instrument costs, while improving the consistency and clinical interpretability of measurement data under extreme HCT sample conditions.

[0052] Experiment 1: Different concentrations of glucose and lactic acid were added to a serum sample, along with red blood cells, to obtain serum with a 40% hematocrit. After mixing, the serum sample to be tested was obtained. The serum sample was dropped onto the inlet of the electrochemical test strip, and the serum sample was absorbed into microfluidic channel 2 by capillary force. A current signal was obtained from the reaction. Ten parallel experiments were performed to verify the repeatability of the test strip. The experimental results are shown in Table 1-2.

[0053] Table 1. Test results of lactic acid current signal (unit: μA) Table 2. Results of blood glucose current signal test (40% HCT) (unit: μA) The average current signal of lactate / glucose at the same concentration was calculated, and the fitted curves of lactate signal (Figure 7) and glucose signal (Figure 8) were obtained. It can be seen that the fitted curve R... 2 A value above 0.99 indicates a good fit, suggesting that the electrochemical test strip can simultaneously detect blood glucose and lactate.

[0054] Experimental Example 2: Different concentrations of glucose were added to serum samples, and different concentrations of red blood cells were added to serum solutions of the same glucose concentration to obtain serums with different hematocrits. After mixing, the serum samples to be tested were obtained. The obtained serum samples were dropped onto electrochemical test strips containing a double-membrane structure (enzyme reaction membrane and blood cell filtration membrane) and electrochemical test strips without a double-membrane structure, respectively. The sensing results of the two types of electrochemical test strips are shown in Table 3-4, verifying that the electrochemical test strip containing a double-membrane structure has anti-HCT function.

[0055] Table 3 Current signals of electrochemical test pieces without bilayer membrane structure (unit: μA) Table 4 Current signals of electrochemical test pieces with double-layer film structure (unit: μA) Using the current value of the 40% hematocrit test as a benchmark, the percentage deviation of the current signal at other HCT values ​​at the same blood glucose concentration relative to the current signal at 40% HCT was calculated. The results are shown in Table 5-6.

[0056] Table 5 Percentage Deviation of Current Values ​​Without Double-Layer Membrane Structure Test Piece Table 6 Percentage Deviation of Current Values ​​for Test Pieces with Double-Layer Membrane Structure Comparative analysis of the results in Tables 5 and 6 shows that the current value deviation of the test piece without the double-layer membrane structure is generally above 15%, with the highest deviation reaching 100%, indicating that HCT has a significant impact on current measurement. In contrast, the current value deviation of the test piece with the double-layer membrane structure is mostly very small, except in a few cases where the deviation exceeds 15%, indicating that the double-layer membrane structure has good anti-HCT effect.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-channel, double-layer membrane electrochemical test strip resistant to blood cell interference, characterized in that, The substrate (1) includes a sensing electrode, a top sealing layer, and a double-layer membrane structure consisting of an enzyme reaction membrane layer and a blood cell filtration membrane layer. The double-layer membrane structure is located above the sensing electrode. The upper end of the substrate (1) is encapsulated by the top sealing layer. Multiple independent microfluidic channels (2) are provided on the substrate (1). Each microfluidic channel (2) is equipped with an independent blood inlet, a liquid channel, and a detection area. Structural partitions are provided between the microfluidic channels (2) to isolate them from each other. A three-dimensional protrusion structure (3) is provided at the blood inlet of one end of each microfluidic channel (2). The three-dimensional protrusion structure (3) is a step-shaped protrusion formed in the horizontal direction of the blood inlet. Two sensing electrode mounting holes (4) are provided in the detection area at the other end of each microfluidic channel (2). The sensing electrode is inserted and installed in the sensing electrode mounting hole (4).

2. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 1, characterized in that, When two microfluidic channels (2) are provided on the substrate (1), the three-dimensional protrusion structure (3) is a three-dimensional protrusion with a pointed opening; when three microfluidic channels (2) are provided on the substrate (1), the three-dimensional protrusion structures (3) on both sides are three-dimensional protrusions with a pointed opening; the three-dimensional protrusion structure (3) in the middle is a trapezoidal three-dimensional protrusion with a flat opening in the middle.

3. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 1, characterized in that, The groove depth of the microfluidic channel (2) is 0.08 mm; the thickness of the three-dimensional protrusion structure (3) is 0.37 mm, and the upper surface of the three-dimensional protrusion structure (3) is flush with the bottom surface of the groove of the microfluidic channel (2).

4. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 3, characterized in that, The microfluidic channels (2), pre-reserved sensor electrode mounting holes (4), three-dimensional protrusion structure (3) at the blood inlet of the microfluidic channel (2) and mounting through holes (5) are formed on the surface of the PET substrate by die cutting.

5. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 4, characterized in that, The PET substrate surface is subjected to plasma treatment.

6. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 1, characterized in that, The sensing electrode is a copper-based gold-plated electrode, which is formed by electroplating a uniform and dense Au layer on the surface of a copper substrate.

7. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 6, characterized in that, The copper-based gold-plated electrode is assembled by inserting it into the sensing electrode mounting hole (4) pre-cut on the substrate (1). After assembly, the upper surface of the copper-based gold-plated electrode is higher than the microfluidic channel (2).

8. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 1, characterized in that, The enzyme reaction membrane uses GDH as the core reaction component, and the preparation process is as follows: GDH is dissolved in phosphate buffer, and electron mediators, stabilizers and thickeners are added and stirred to form a homogeneous enzyme solution; then the enzyme solution is dropped onto the surface of a copper-based gold-plated electrode using a micro-dispensing instrument, with a controlled amount of 10-30 μL / dip, and after drying, a firmly attached enzyme reaction membrane is formed.

9. The multi-channel double-layer membrane electrochemical test strip for resisting blood cell interference according to claim 1, characterized in that, The preparation process of the blood cell filtration membrane is as follows: PU prepolymer and PVA are weighed and thoroughly dissolved in N,N-dimethylformamide by stirring to form a homogeneous and transparent solution; the solution is then irradiated with an electron beam at a dose of 15-25 kGy; subsequently, a crosslinking agent, fumaric acid or citric acid, and a surfactant are added; the solution is then irradiated with gamma rays at a dose of 25-35 kGy; the resulting membrane solution is coated onto the surface of a solidified enzyme reaction membrane, controlling the membrane thickness to be within the range of 30-50 μm; the membrane is then dried and solidified to ensure the crosslinking reaction is fully completed, forming a dense and uniform blood cell filtration membrane with a microporous structure.

10. The application of an electrochemical test strip with multi-channel double-layer membrane anti-blood cell interference as described in any one of claims 1 to 9 in the parallel detection of multiple biomarkers or the repeated detection of the same biomarker.

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