Evaporation-driven paper-based microfluidic chip, detection system and application thereof

Abstract

The application discloses a paper-based microfluidic chip based on evaporation driving, which comprises a common channel (1), one end of the common channel (1) is respectively connected with a first channel (2) and a second channel (3), and the other end of the common channel (1) is respectively connected with a third channel (4) and a fourth channel (5); the upstream of the first channel (2) is connected with a first absorption area (6), and the upstream of the second channel (3) is connected with a second absorption area (8); the downstream of the third channel (4) is connected with a first evaporation area (9), and the downstream of the fourth channel (5) is connected with a second evaporation area (10). The application further discloses a detection system based on the paper-based microfluidic chip based on evaporation driving, and application of the paper-based microfluidic chip based on evaporation driving and the detection system in whole blood analysis. The application realizes pump-free, valve-free and controllable multi-path laminar flow driving, realizes online and efficient separation of red blood cells in whole blood, and realizes continuous sampling and real-time monitoring of the paper-based chip.

Description

Technical Field

[0001] This invention relates to the field of inspection and quarantine, and in particular to the application of an evaporation-driven paper-based microfluidic chip and its detection system in whole blood analysis. Background Technology

[0002] Point-of-care testing (POCT) is a promising technology for early disease diagnosis and personal health monitoring due to its speed, convenience, low cost, and lack of reliance on large instruments. Blood samples are rich in biological information and are the most widely studied human fluid. Blood-based analysis not only aids in disease diagnosis, treatment monitoring, and health status assessment but also predicts the risk of developing various diseases, enabling timely intervention. Therefore, blood analysis is considered the "gold standard" for clinical diagnosis. Developing POCT platforms suitable for blood analysis has become an important research direction in this field.

[0003] In recent years, paper-based microfluidic analytical devices (µPADs) have shown significant potential in POCT applications due to their portability, low cost, and the inherent porosity and capillary properties of paper materials (enabling self-driven fluid flow). Despite these advantages, µPADs still face challenges in achieving satisfactory analytical performance when used for blood testing. First, direct analysis of whole blood in µPADs remains difficult. The complexity and high abundance of blood components can severely interfere with analytical measurements, leading to reduced detection accuracy. Existing on-chip blood separation strategies, such as paper-based chemical modification or integration of commercial plasma separation membranes, suffer from limited separation efficiency and complex manufacturing processes. Second, fluid manipulation in most µPADs relies on passive capillary forces, making complex fluid control difficult. This severely hinders the realization of on-chip multifunctional integration, such as continuous sample introduction and on-chip target extraction. Furthermore, the detection modes are limited, lacking continuous monitoring capabilities. The vast majority of paper-based chips operate on a discrete detection mode of "single sample addition, single reading," unable to support continuous sample introduction and real-time dynamic monitoring, limiting their application in scenarios requiring frequent measurements. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a paper-based microfluidic chip and its detection system based on evaporation drive that is simple in structure, low in cost and easy to operate.

[0005] This invention also provides the application of evaporation-driven paper-based microfluidic chips and their detection systems in whole blood analysis.

[0006] A paper-based microfluidic chip based on evaporation drive includes a common channel, one end of which is connected to a first channel and a second channel, and the other end of which is connected to a third channel and a fourth channel. The upstream of the first channel is connected to a first absorption region, and a sample application point is provided on the first channel. The upstream of the second channel is connected to a second absorption region. The downstream of the third channel is connected to a first evaporation region, and the downstream of the fourth channel is connected to a second evaporation region, and a detection point is provided on the fourth channel.

[0007] The paper-based microfluidic chip based on evaporation drive of the present invention, wherein: the first absorption region and the first evaporation region are located on the same side of the common channel length direction axis, and the second absorption region and the second evaporation region are located on the other side of the common channel length direction axis;

[0008] The evaporation-driven paper-based microfluidic chip is made of hydrophilic filter paper.

[0009] A detection system comprising the aforementioned evaporation-driven paper-based microfluidic chip includes an electrochemical detection unit connected to a detection point in the evaporation-driven paper-based microfluidic chip; a first reservoir is disposed below a first absorption region, the first absorption region being immersed in a carrier solution in the first reservoir; a second reservoir is disposed below a second absorption region, the second absorption region being immersed in a carrier solution in the second reservoir; a first heating unit is disposed below the first evaporation region, and a second heating unit is disposed below the second evaporation region.

[0010] The detection system based on an evaporation-driven paper-based microfluidic chip of the present invention includes: both the first heating unit and the second heating unit include an adjustable temperature heating plate, wherein the adjustable temperature heating plate includes a temperature controller and a temperature sensor.

[0011] The detection system based on an evaporation-driven paper-based microfluidic chip of the present invention includes: an electrochemical detection unit comprising a detachable working electrode, a reference electrode, and an auxiliary electrode; the working electrode is a gold-plated plastic electrode; the working electrode is placed below the detection point, and the auxiliary electrode and the reference electrode of the electrosignal acquisition and transmission device are placed above the detection point, forming a three-electrode system; the electrodes are connected to an electrochemical workstation through a three-electrode interface to realize electrochemical signal transmission.

[0012] The detection system based on an evaporation-driven paper-based microfluidic chip of the present invention includes an electrochemical detection unit that is a carbon screen-printed electrode. The screen-printed electrode is placed below the detection point, and the electrode is connected to an electrochemical workstation through a three-electrode interface to realize electrochemical signal transmission.

[0013] The application of the evaporation-driven paper-based microfluidic chip or the detection system based on the evaporation-driven paper-based microfluidic chip described in this invention in whole blood analysis.

[0014] The method for detecting uric acid using the evaporation-driven paper-based microfluidic chip detection system described in this invention includes the following steps:

[0015] (1) Fabrication of paper-based chips and construction of electrochemical detection systems;

[0016] (2) Preparation of standard solutions: Uric acid standard solutions of different concentrations were prepared, with concentrations of 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L and 2 mmol / L, respectively;

[0017] (3) Electrochemical flow injection analysis:

[0018] The corresponding buffer solutions were introduced into the storage tanks. Without enhanced extraction, both the first and second storage tanks contained 0.01 M phosphate buffer solution with a pH of 7.4. With enhanced extraction, the first storage tank contained phosphate buffer solution, and the second storage tank contained phosphate buffer solution containing 5 mM NaOH. After the paper-based chip was fully saturated, the heaters were heated to the required temperatures: 100°C for the first heating unit and 95°C for the second heating unit. Simultaneously, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for uric acid detection was 0.45 V.

[0019] (4) Testing: After the baseline stabilizes, add 10 μL of uric acid standard solution of different concentrations at the sampling point. The addition method is as follows: start from the low concentration. Add 0.05 mmol / L standard solution for the first time. After the baseline stabilizes, add 0.1 mmol / L standard solution. After the baseline stabilizes, add 0.5 mmol / L standard solution. Continue this process until all the standard solutions are added. Obtain the electrochemical signal curve. The peak height is related to the concentration of uric acid solution.

[0020] The method for detecting acetaminophen using the evaporation-driven paper-based microfluidic chip detection system described in this invention includes the following steps:

[0021] (1) Fabrication of paper-based chips and construction of electrochemical detection systems;

[0022] (2) Preparation of standard samples: Acetaminophen standard solutions of different concentrations were prepared, with concentrations of 0.01 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L and 1 mmol / L, respectively;

[0023] (3) Electrochemical flow injection analysis:

[0024] The corresponding buffer solutions were introduced into the storage tanks. Without enhanced extraction, both the first and second storage tanks contained 0.01 M phosphate buffer solution with a pH of 7.4. With enhanced extraction, the first storage tank contained phosphate buffer solution, and the second storage tank contained phosphate buffer solution containing 5% ethanol. After the paper-based chip was completely saturated, the heaters were heated to the required temperatures: 100°C for the first heating unit and 95°C for the second heating unit. Simultaneously, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for acetaminophen detection was 0.3 V.

[0025] (4) Testing: After the baseline stabilizes, add 10 μL of acetaminophen standard solution of different concentrations at the sampling point. The addition method is as follows: start from the low concentration, add 0.01 mmol / L standard solution for the first time, add 0.05 mmol / L standard solution after the baseline stabilizes, add 0.1 mmol / L standard solution after the baseline stabilizes, and so on until all the standard solutions are added. The electrochemical signal curve is obtained, and its peak height is related to the concentration of acetaminophen solution.

[0026] The method for detecting uric acid in rat blood using the evaporation-driven paper-based microfluidic chip detection system described in this invention includes the following steps:

[0027] (1) Fabrication of paper-based chips and construction of electrochemical detection systems;

[0028] (2) Pretreatment of actual samples: The rat blood was diluted 10 times with phosphate buffer solution and the diluted rat blood was divided into 18 portions;

[0029] (3) Preparation of calibration curve by standard addition method: Take 4 portions of diluted rat blood and add uric acid standard solution to each portion so that the final concentrations of added uric acid are 0.05 mmol / L, 0.1 mmol / L, 0.3 mmol / L and 0.5 mmol / L, respectively;

[0030] (3) Electrochemical flow injection analysis:

[0031] The corresponding buffer solutions were introduced into the storage tanks. The first storage tank contained a phosphate buffer solution, and the second storage tank contained a phosphate buffer solution containing 5 mM NaOH. After the paper-based chip was completely saturated, the heater was heated to the required temperature. The temperature of the first heating unit was 100℃, and the temperature of the second heating unit was 95℃. At the same time, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for uric acid detection was 0.45 V.

[0032] (4) Testing: After the baseline stabilizes, add 10 μL of spiked rat blood and the blood of the rat to be tested sequentially at the sampling point. The method of addition is as follows:

[0033] Spiked rat blood was added dropwise, starting with low concentrations. The first drop was 0.05 mmol / L solution, followed by 0.1 mmol / L solution after the baseline stabilized, then 0.3 mmol / L solution, and finally 0.5 mmol / L solution, for a total of 4 drops. After all the spiked rat blood had been added and the baseline stabilized, diluted rat blood was added dropwise, for a total of 14 drops. Each drop was added only after the previous baseline stabilized, until all 14 drops were added. Electrochemical signal curves were obtained.

[0034] The difference between this invention and existing technologies lies in the following:

[0035] The present invention, based on an evaporation-driven paper-based microfluidic chip and its detection system, has the following advantages:

[0036] (1) Achieved pump-free, valve-free, and controllable multi-channel laminar flow drive

[0037] By setting up two independently temperature-controlled evaporation zones downstream of the paper-based chip, local heating is used to enhance evaporation and generate a stable pressure gradient, replacing the traditional flow mode that relies on passive capillary action. This enables independent control of the flow rates of the two outlet channels, providing a novel multi-path laminar flow drive solution for complex fluid manipulation on paper-based chips (such as online sample pretreatment).

[0038] (2) Online and efficient separation of red blood cells from whole blood was achieved.

[0039] Based on the significant size difference between red blood cells and small molecule targets, and combined with the limitation of lateral diffusion time during the double-layer laminar flow process, red blood cells are basically retained in the original flow layer and flow towards the first evaporation zone, while small molecules diffuse rapidly to the fluid layer on one side of the detection point. Without the need for offline centrifugation or membrane filtration, online and continuous separation of whole blood is achieved, with a separation efficiency of over 92.5%.

[0040] (3) Significantly improved the extraction efficiency and detection sensitivity of small molecule targets.

[0041] Based on the solute-solvent interaction principle, by introducing a carrier solution with high solubility for the target small molecule into one inlet, the diffusion coefficient of the target molecule is significantly increased, thereby accelerating its lateral diffusion into the detection stream. Experiments have shown that this method reduces the detection limit for uric acid from 0.1 mmol / L to 0.01 mmol / L and the detection limit for acetaminophen from 0.03 mmol / L to 0.002 mmol / L, improving sensitivity by more than an order of magnitude.

[0042] (4) Continuous sample introduction and real-time monitoring of paper-based chips were realized.

[0043] This paper-based microfluidic device integrates multiple functions, including blood cell separation, target analyte extraction, and continuous sample introduction and detection. By continuously maintaining a localized unsaturated state in the evaporation zone, the same chip can perform more than 10 repeated sample additions (experiments have shown up to 18 times), breaking the limitations of traditional paper-based chips that are "single-use, single-read," making it suitable for scenarios requiring dynamic monitoring of concentration changes and significantly improving detection efficiency.

[0044] (5) Possesses good detection versatility and electrode compatibility

[0045] The paper-based chip can be adapted to different electrodes (such as gold-plated plastic electrodes and screen-printed carbon electrodes), making it suitable for the electrochemical detection of various target substances such as uric acid and acetaminophen, thus expanding the applicability of the device.

[0046] The following description, in conjunction with the accompanying drawings, further illustrates the evaporation-driven paper-based microfluidic chip, detection system, and applications of the present invention. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the structure of the X-shaped paper-based microfluidic chip of the present invention;

[0048] Figure 2 This is a schematic diagram of the evaporation-driven paper-based microfluidic chip and detection system of the present invention.

[0049] Figure 3 This is a diagram showing the effect of heating temperature on the dual-path laminar flow manifold on the X-type paper-based chip in this invention;

[0050] Figure 4 These are photographs of blood transport on a µPAD at different time points in this invention; wherein, the temperature of the first evaporation zone is 100 ℃, the temperature of the second evaporation zone is 95 ℃, and the channel length is 15 mm;

[0051] Figure 5 This is a diagram showing the relationship between the lateral diffusion distance of red blood cells and phenol red at the end of the common channel and the channel length in this invention; wherein, the first evaporation zone is heated to 100°C and the second evaporation zone is heated to 95°C.

[0052] Figure 6 This is an assembly diagram of different electrochemical detection systems and paper-based chips in this invention;

[0053] Figure 7The amperometric response curve (A) and corresponding calibration curve (B) of uric acid continuously added under enhanced extraction conditions are shown in this invention. The detection conditions are: potential 0.45 V; the amperometric response curve (C) and corresponding calibration curve (D) of acetaminophen continuously added under enhanced extraction conditions are shown in this invention. The detection conditions are: potential 0.3 V; (E) The amperometric response curve of uric acid in rat blood was continuously detected using the standard addition method. Detailed Implementation

[0054] 1. Device section

[0055] 1.1 Structure of X-shaped paper-based microfluidic chip

[0056] The paper-based microfluidic chip was prepared from DL42 polyester fiber filter paper. The paper-based microfluidic chip was designed using AutoCAD software and then printed using an HP LaserJet 1020 plus laser printer.

[0057] The structure of a paper-based microfluidic chip is as follows Figure 1 As shown, it includes a common channel 1, one end of which is connected to the first channel 2 and the second channel 3 respectively, and the other end is connected to the third channel 4 and the fourth channel 5 respectively; the upstream of the first channel 2 is connected to the first absorption area 6, and a sample application point 7 is set on the first channel 2; the upstream of the second channel 3 is connected to the second absorption area 8; the downstream of the third channel 4 is connected to the first evaporation area 9; the downstream of the fourth channel 5 is connected to the second evaporation area 10, and a detection point 11 is set on the fourth channel 5.

[0058] The first absorption region 6 and the first evaporation region 9 are located on the same side of the length axis of the common channel 1, while the second absorption region 8 and the second evaporation region 10 are located on the other side of the length axis of the common channel 1. The evaporation-driven paper-based microfluidic chip is formed by cutting hydrophilic filter paper. The diameter of the first evaporation region 9 and the second evaporation region 10 is 20 mm. The width of the common channel 1, the first channel 2, the second channel 3, the third channel 4, the fourth channel 5, the first absorption region 6, and the second absorption region 8 is 5 mm. The length of the common channel 1 is 15 mm, and the lengths of the first channel 2, the second channel 3, the third channel 4, and the fourth channel 5 are all 10 mm. The lengths of the first absorption region 6 and the second absorption region 8 are 15 mm.

[0059] 1.2 Evaporation-driven paper-based microfluidic device

[0060] like Figure 2As shown, a first liquid reservoir 13 is disposed below the first absorption region 6 of the X-shaped paper-based microfluidic chip, and the first absorption region 6 is immersed in the carrier solution in the first liquid reservoir 13; a second liquid reservoir 14 is disposed below the second absorption region 8, and the second absorption region 8 is immersed in the carrier solution in the second liquid reservoir 14. The two absorption regions are used to introduce different carrier solutions; a first heating unit 15 is disposed below the first evaporation region 9, and a second heating unit 16 is disposed below the second evaporation region 10. Both the first heating unit 15 and the second heating unit 16 include independent adjustable temperature heating plates, which include a temperature controller and a temperature sensor, enabling precise temperature control within ±0.5℃. An electrochemical detection unit 12 is connected to the detection point 11 in the X-shaped paper-based microfluidic chip.

[0061] 1.2.1 The electrochemical detection unit 12 in the uric acid detection is self-made, and specifically includes the following structure:

[0062] Three-electrode systems for uric acid detection, such as Figure 6 As shown in Figure A, it includes a flat working electrode and its electrical signal acquisition and transmission device.

[0063] 1) Flat working electrode: Gold was sputtered onto a polycarbonate (PC) film under a constant current of 30 mA for 80 seconds. The gold-plated PC film was then cut into sheets 2 cm long and 1 cm wide. To define the working area, an insulating tape with a 5 mm diameter hole was tightly wrapped around the electrode.

[0064] 2) Electrical signal acquisition and transmission device: a platinum wire (0.5 mm in diameter) as an auxiliary electrode, an Ag / AgCl wire (0.5 mm in diameter) as a reference electrode, a copper connection probe as the working electrode interface, and a printed circuit board (PCB) as the conductive connection layer. See the patent application "Electrical signal acquisition and transmission device for a flat working electrode" (Invention Patent, Patent No.: ZL201610479208.5), which discloses a method for manufacturing an electrical signal acquisition and transmission device for a flat working electrode.

[0065] 3) Three-electrode system: The prepared flat working electrode is placed below the detection point 11 in the X-shaped paper-based microfluidic chip, and the auxiliary electrode and reference electrode of the electrical signal acquisition and transmission device are placed above the detection point 11 in the X-shaped paper-based microfluidic chip to form a three-electrode system.

[0066] 1.2.2 In the detection of acetaminophen, the electrochemical detection unit 12 is a commercially available product. Product information is as follows:

[0067] A commercially available screen-printed electrode for acetaminophen detection was purchased from Nanjing Tengsen Analytical Instruments Co., Ltd. The working electrode and counter electrode are both carbon, and the reference electrode is silver / silver chloride. During detection, the screen-printed electrode is placed below detection point 11 in the X-shaped paper-based microfluidic chip.

[0068] 2. Working principle

[0069] The working principle of this invention is as follows:

[0070] Localized enhanced evaporation driving fluid:

[0071] The first absorption zone 6 and the second absorption zone 8 are respectively immersed in the first storage tank 13 and the second storage tank 14 containing the carrier solution. The heating plates at the bottom of the first evaporation zone 9 and the second evaporation zone 10 are activated, and the heating temperature is set. In the evaporation zone, the liquid in the paper substrate evaporates due to heating, forming locally unsaturated regions, which leads to the generation of a gas-liquid interface within the pores of the paper substrate, generating a capillary pressure difference. This pressure difference acts as a driving force, causing the liquid to flow from the inlet storage tank to the evaporation zone. Since the heating temperature of the two evaporation zones can be independently controlled, the flow rates of the two outlet channels can be adjusted separately.

[0072] Due to the low Reynolds number of paper-based microchannels (10... - (On the order of 3), only molecular diffusion occurs between the two fluid streams, with no convective mixing. Within a common flow channel, the fluids from the two inlets flow side-by-side in laminar flow, forming a clear liquid-liquid interface. For example... Figure 3 As shown.

[0073] Red blood cell separation:

[0074] A whole blood sample (e.g., 10 μL) is added to the sample application point. The blood is propelled downstream by the carrier solution on one side of the first absorption zone 6. During laminar flow, red blood cells (approximately 6–8 μm in diameter) have a very low diffusion coefficient and hardly leave the original fluid layer during the brief lateral diffusion time. Therefore, they mainly flow towards the first evaporation zone 9 and are collected there. Small molecule target substances (such as uric acid and acetaminophen, with a diameter <1 nm) have a high diffusion coefficient and can rapidly diffuse into the adjacent carrier liquid layer, flowing towards the detection point with the fluid in the second absorption zone 8. The movement of red blood cells in the X-shaped paper-based channel is as follows: Figure 4 As shown.

[0075] Enhanced extraction:

[0076] By introducing a solvent with high solubility for the target small molecule into the second absorption region 8, based on the principle of enhanced solute-solvent interaction (i.e., increasing the effective association coefficient), the diffusion coefficient of the target small molecule is significantly improved, thereby enabling it to enter adjacent fluids and flow through the detection point faster and more within a limited channel length, achieving "enhanced extraction".

[0077] Using phenol red as a model compound, its solubility in water is 0.77 g / L and its solubility in 10 mm NaOH is 1.3 g / L. Figure 5 The diffusion distance of erythrocytes and phenol red at the end of common channel 1, perpendicular to the flow direction, is represented. Without enhanced extraction (both inlets are immersed in water), the diffusion distance of phenol red at the liquid-liquid interface is slightly greater than that of erythrocytes. When the carrier solution in absorption zone 2 is replaced with 10 mM NaOH, which has higher solubility for phenol red, the diffusion distance of phenol red into the highly soluble mobile phase increases significantly.

[0078] Continuous electrochemical detection:

[0079] When the solution containing the target small molecule flows through the detection point, it comes into contact with the three-electrode system. A constant potential is applied (e.g., 0.45 V for uric acid detection, 0.3 V for acetaminophen detection), and the current response is recorded. The change in the current signal over time reflects the dynamic change in the concentration of the target molecule.

[0080] 3. Application of the Evaporation-Driven Paper-Based Microfluidic Chip and Detection System of this Invention

[0081] 3.1 A method for detecting uric acid in blood using a detection system based on an evaporation-driven paper-based microfluidic chip, comprising the following steps:

[0082] (1) Fabrication of paper-based chips and construction of electrochemical detection system: Refer to the device section of this invention;

[0083] (2) Preparation of standard solutions: Uric acid standard solutions of different concentrations were prepared, with concentrations of 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L and 2 mmol / L, respectively.

[0084] (3) Electrochemical flow injection analysis

[0085] The corresponding buffer solutions were introduced into the storage tanks (without enhanced extraction, both the first storage tank 13 and the second storage tank 14 contained 0.01 M phosphate buffer solution with a pH of 7.4; with enhanced extraction, the first storage tank 13 contained phosphate buffer solution, and the second storage tank 14 contained phosphate buffer solution containing 5 mM NaOH). After the paper-based chip was completely saturated, the heaters were heated to the required temperatures (the temperature of the first heating unit 15 was 100℃, and the temperature of the second heating unit 16 was 95℃). Simultaneously, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for uric acid detection was 0.45 V.

[0086] (4) Testing: After the baseline stabilizes, add 10 μL of uric acid standard solution of different concentrations sequentially at sampling point 7. The addition method is as follows: start with the lowest concentration, add 0.05 mmol / L standard solution for the first time, and after the baseline stabilizes, add 0.1 mmol / L standard solution, and after the baseline stabilizes, add 0.5 mmol / L standard solution, and so on, until the highest concentration standard solution is added. The result is as follows. Figure 7 The peak height of the electrochemical signal curve shown in Figure A is related to the concentration of the uric acid solution.

[0087] result: Figure 7 A shows a comparison of transient current signals for different concentrations of uric acid with and without enhanced extraction at a constant potential of 0.45 V. Without enhanced extraction, both fluid streams were 0.01 M PBS (pH 7.4); with enhanced extraction, the carrier solution in the second reservoir 14 was replaced with PBS containing 5 mM NaOH, which has higher solubility for uric acid. The linear relationship between uric acid concentration and peak current is shown below. Figure 7 As shown in B, within the concentration range of 0.05–2 mmol / L, the linear regression equation for extraction without enhancement is i = 0.20434C. UA – 0.01424, R² = 0.982, the calculated limit of detection (LOD) is 0.1 mmol / L. In comparison, the linear regression equation with enhanced extraction is i = 2.06234C. UA +0.47719, R² = 0.990, LOD is 0.01 mmol / L.

[0088] The results showed that the enhanced extraction strategy significantly improved the sensitivity of uric acid detection. Within the same concentration range, the slope of the calibration curve with enhanced extraction (2.06234) was approximately one order of magnitude higher than that without enhanced extraction (0.20434), indicating that the strategy effectively enhanced the electrochemical response signal. Simultaneously, the detection limit decreased from 0.1 mmol / L to 0.01 mmol / L, a 10-fold improvement. This improvement is attributed to the enhanced diffusion coefficient of uric acid by the highly soluble solvent (PBS containing 5 mM NaOH) in the second reservoir, promoting accelerated mass transfer of uric acid to the detection zone. These results validate the feasibility and effectiveness of the solubility-controlled accelerated extraction strategy in paper-based microfluidic electrochemical detection.

[0089] 3.2 A method for detecting acetaminophen in blood using a detection system based on an evaporation-driven paper-based microfluidic chip, comprising the following steps:

[0090] (1) Fabrication of paper-based chips and construction of electrochemical detection system: Refer to the device section of this invention;

[0091] (2) Preparation of standard samples: Acetaminophen standard solutions of different concentrations were prepared, with concentrations of 0.01 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L and 1 mmol / L, respectively.

[0092] (3) Electrochemical flow injection analysis

[0093] The corresponding buffer solutions were introduced into the storage tanks (without enhanced extraction, both the first storage tank 13 and the second storage tank 14 contained 0.01 M phosphate buffer solution with a pH of 7.4; with enhanced extraction, the first storage tank 13 contained phosphate buffer solution, and the second storage tank 14 contained phosphate buffer solution containing 5% ethanol). After the paper-based chip was completely saturated, the heaters were heated to the required temperatures (the temperature of the first heating unit 15 was 100℃, and the temperature of the second heating unit 16 was 95℃). Simultaneously, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for acetaminophen detection was 0.3 V.

[0094] (4) Testing: After the baseline stabilizes, add 10 μL of acetaminophen standard solution of different concentrations sequentially at sampling point 7. The addition method is as follows: start with the lowest concentration, adding 0.01 mmol / L standard solution for the first time, and after the baseline stabilizes, add 0.05 mmol / L standard solution, and after the baseline stabilizes, add 0.1 mmol / L standard solution, and so on, until the highest concentration standard solution is added. The result is as follows. Figure 7 The peak height of the electrochemical signal curve shown in C is related to the concentration of the acetaminophen solution.

[0095] Results: This function of the paper-based microfluidic device was further verified using acetaminophen. Figure 7 C, 7D). Within the concentration range of 0.01–1 mmol / L, the linear regression equation for extraction without enhancement is i = 2.89869C. ACP –0.01047, R² = 0.996, LOD = 0.03 mmol / L. When the carrier solution in the second reservoir 14 was replaced with PBS containing 5% ethanol (this solvent has higher solubility for acetaminophen), the linear regression equation for enhanced extraction was i = 6.2473C. ACP +0.09487, R² = 0.999, LOD determined to be 0.002 mmol / L.

[0096] The results showed that the enhanced extraction strategy also significantly improved the detection sensitivity of acetaminophen. Within the concentration range of 0.01–1 mmol / L, the slope of the calibration curve with enhanced extraction (6.2473) was approximately 2.2 times higher than that without enhanced extraction (2.89869), and the limit of detection decreased from 0.03 mmol / L to 0.002 mmol / L, an improvement of 15 times. This result is consistent with the uric acid detection results, further validating the universality and effectiveness of the strategy of enhancing the diffusion coefficient of the target analyte by introducing a highly soluble solvent in different small molecule systems. Furthermore, this strategy does not require complex chip structure modifications; a significant improvement in detection performance can be achieved simply by changing the carrier solution in the second reservoir, providing a simple and universal method for high-sensitivity detection on paper-based microfluidic platforms.

[0097] 3.3 Actual Sample Testing

[0098] A method for detecting uric acid in rat blood using a detection system based on an evaporation-driven paper-based microfluidic chip includes the following steps:

[0099] (1) Fabrication of paper-based chips and construction of electrochemical detection system: Refer to the device section of this invention;

[0100] (2) Pretreatment of actual samples: The rat blood was diluted 10 times with phosphate buffer solution and the diluted rat blood was divided into 18 portions;

[0101] (3) Preparation of calibration curve by standard addition method: Take 4 portions of diluted rat blood and add uric acid standard solution to each portion so that the final concentrations of added uric acid are 0.05 mmol / L, 0.1 mmol / L, 0.3 mmol / L and 0.5 mmol / L, respectively.

[0102] (3) Electrochemical flow injection analysis

[0103] The corresponding buffer solutions were introduced into the storage tanks (the first storage tank 13 contained a phosphate buffer solution, and the second storage tank 14 contained a phosphate buffer solution containing 5 mM NaOH). After the paper-based chip was completely saturated, the heaters were heated to the required temperatures (the first heating unit 15 was at 100°C, and the second heating unit 16 was at 95°C). Simultaneously, a constant potential was applied for amperometric detection, and the time-current response curve was continuously recorded. The constant potential for uric acid detection was 0.45 V.

[0104] (4) Testing: After the baseline stabilizes, add 10 μL of spiked rat blood and the blood of the rat to be tested sequentially at sampling point 7. The method of addition is as follows:

[0105] First, spiked rat blood was added dropwise, starting with low concentrations. The first drop was 0.05 mmol / L solution, followed by 0.1 mmol / L solution after baseline stabilization, then 0.3 mmol / L solution, and finally 0.5 mmol / L solution, for a total of four drops. After all the spiked rat blood drops were added and the baseline stabilized, diluted rat blood was added dropwise, for a total of 14 drops. Each drop was added only after the previous baseline had stabilized, until all 14 drops were added. The results are as follows: Figure 7 The electrochemical signal curve shown in E is an example.

[0106] result: Figure 7 E is the amperometric curve for continuous measurement of blood samples using the standard addition method. It can be seen that, using the same paper-based microfluidic chip, blood can be continuously added up to 18 times. The relative standard deviation (RSD) for 14 consecutive measurements is 19.3%. Further repeated additions may cause red blood cells to accumulate in the evaporation zone, thereby clogging the channel and hindering fluid flow.

[0107] The results show that the paper-based microfluidic chip has good continuous injection capability and acceptable repeatability. Up to 18 consecutive injections can be completed using the same chip, indicating that the device can maintain stable fluid drive performance during long-term operation. The RSD of 14 consecutive measurements is 19.3%, which is within the acceptable range for paper-based continuous analysis systems.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A paper-based microfluidic chip driven by evaporation, characterized in that: It includes a common channel (1), one end of which is connected to the first channel (2) and the second channel (3), and the other end is connected to the third channel (4) and the fourth channel (5); the upstream of the first channel (2) is connected to the first absorption area (6), and a sample addition point (7) is provided on the first channel (2); the upstream of the second channel (3) is connected to the second absorption area (8); the downstream of the third channel (4) is connected to the first evaporation area (9), the downstream of the fourth channel (5) is connected to the second evaporation area (10), and a detection point (11) is provided on the fourth channel (5).

2. The evaporation-driven paper-based microfluidic chip according to claim 1, characterized in that: The first absorption zone (6) and the first evaporation zone (9) are located on the same side of the length axis of the common channel (1), and the second absorption zone (8) and the second evaporation zone (10) are located on the other side of the length axis of the common channel (1). The evaporation-driven paper-based microfluidic chip is made of hydrophilic filter paper.

3. A detection system comprising the evaporation-driven paper-based microfluidic chip as described in claim 1 or 2, characterized in that: It includes an electrochemical detection unit (12) connected to the detection point (11) in the evaporation-driven paper-based microfluidic chip; a first storage tank (13) is provided at the lower part of the first absorption region (6), and the first absorption region (6) is immersed in the carrier solution in the first storage tank (13); a second storage tank (14) is provided at the lower part of the second absorption region (8), and the second absorption region (8) is immersed in the carrier solution in the second storage tank (14); a first heating unit (15) is provided at the lower part of the first evaporation region (9), and a second heating unit (16) is provided at the lower part of the second evaporation region (10).

4. The detection system based on an evaporation-driven paper-based microfluidic chip according to claim 3, characterized in that: Both the first heating unit (15) and the second heating unit (16) include an adjustable temperature heating plate, which includes a temperature controller and a temperature sensor.

5. The detection system based on an evaporation-driven paper-based microfluidic chip according to claim 4, characterized in that: The electrochemical detection unit (12) includes a detachable working electrode, a reference electrode and an auxiliary electrode. The working electrode is a gold-plated plastic electrode. The working electrode is placed below the detection point (11), and the auxiliary electrode and reference electrode of the electrical signal acquisition and transmission device are placed above the detection point (11) to form a three-electrode system. The electrode is connected to the electrochemical workstation via a three-electrode interface to achieve electrochemical signal transmission.

6. The detection system based on an evaporation-driven paper-based microfluidic chip according to claim 4, characterized in that: The electrochemical detection unit (12) is a carbon screen printed electrode. The screen printed electrode is placed below the detection point (11). The electrode is connected to the electrochemical workstation through a three-electrode interface to realize electrochemical signal transmission.

7. The application of the detection system based on the evaporation-driven paper-based microfluidic chip as described in claim 1 or 2, or any one of claims 3 to 6, in whole blood analysis.

8. A method for detecting uric acid using the evaporation-driven paper-based microfluidic chip detection system as described in claim 5, characterized in that: Includes the following steps: (1) Fabrication of paper-based chips and construction of electrochemical detection systems; (2) Preparation of standard solutions: Uric acid standard solutions of different concentrations were prepared, with concentrations of 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L and 2 mmol / L, respectively; (3) Electrochemical flow injection analysis: The corresponding buffer solution is introduced into the storage tank. Without enhanced extraction, the first storage tank (13) and the second storage tank (14) are both 0.01 M phosphate buffer solution with a pH of 7.

4. With enhanced extraction, the first storage tank (13) is a phosphate buffer solution and the second storage tank (14) is a phosphate buffer solution containing 5 mM NaOH. After the paper-based chip is completely saturated, the heater is heated to the required temperature. The temperature of the first heating unit (15) is 100℃ and the temperature of the second heating unit (16) is 95℃. At the same time, a constant potential is applied for amperometric detection and the time-current response curve is continuously recorded. The constant potential for uric acid detection is 0.45 V. (4) Testing: After the baseline stabilizes, add 10 μL of uric acid standard solution of different concentrations at the sampling point (7). The method of addition is as follows: start from the low concentration. Add 0.05 mmol / L standard solution for the first time. After the baseline stabilizes, add 0.1 mmol / L standard solution. After the baseline stabilizes, add 0.5 mmol / L standard solution. Add in this way until all the standard solutions are added. Obtain the electrochemical signal curve. The peak height is related to the concentration of uric acid solution.

9. A method for detecting acetaminophen using the detection system based on an evaporation-driven paper-based microfluidic chip as described in claim 6, characterized in that: Includes the following steps: (1) Fabrication of paper-based chips and construction of electrochemical detection systems; (2) Preparation of standard samples: Acetaminophen standard solutions of different concentrations were prepared, with concentrations of 0.01 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.5 mmol / L and 1 mmol / L, respectively; (3) Electrochemical flow injection analysis: The corresponding buffer solution is introduced into the storage tank. Without enhanced extraction, the first storage tank (13) and the second storage tank (14) are both 0.01 M phosphate buffer solution with a pH of 7.

4. With enhanced extraction, the first storage tank (13) is a phosphate buffer solution and the second storage tank (14) is a phosphate buffer solution containing 5% ethanol. After the paper-based chip is completely saturated, the heater is heated to the required temperature. The temperature of the first heating unit (15) is 100°C and the temperature of the second heating unit (16) is 95°C. At the same time, a constant potential is applied for amperometric detection and the time-current response curve is continuously recorded. The constant potential for acetaminophen detection is 0.3 V. (4) Testing: After the baseline stabilizes, add 10 μL of acetaminophen standard solution of different concentrations at the sample addition point (7). The addition method is as follows: start from the low concentration, add 0.01 mmol / L standard solution for the first time, add 0.05 mmol / L standard solution after the baseline stabilizes, add 0.1 mmol / L standard solution after the baseline stabilizes, and so on until all the standard solutions are added. The electrochemical signal curve is obtained, and its peak height is related to the concentration of acetaminophen solution.

10. A method for detecting uric acid in rat blood using the detection system based on an evaporation-driven paper-based microfluidic chip as described in claim 5, characterized in that: Includes the following steps: (1) Fabrication of paper-based chips and construction of electrochemical detection systems; (2) Pretreatment of actual samples: The rat blood was diluted 10 times with phosphate buffer solution and the diluted rat blood was divided into 18 portions; (3) Preparation of calibration curve by standard addition method: Take 4 portions of diluted rat blood and add uric acid standard solution to each portion so that the final concentrations of added uric acid are 0.05 mmol / L, 0.1 mmol / L, 0.3 mmol / L and 0.5 mmol / L, respectively; (3) Electrochemical flow injection analysis: The corresponding buffer solutions are introduced into the storage tanks. The first storage tank (13) contains a phosphate buffer solution, and the second storage tank (14) contains a phosphate buffer solution containing 5 mM NaOH. After the paper-based chip is fully saturated, the heater is heated to the required temperature. The temperature of the first heating unit (15) is 100°C, and the temperature of the second heating unit (16) is 95°C. At the same time, a constant potential is applied for amperometric detection, and the time-current response curve is continuously recorded. The constant potential for uric acid detection is 0.45 V. (4) Testing: After the baseline stabilizes, add 10 μL of spiked rat blood and the blood of the rat to be tested sequentially at the sampling point (7). The method of addition is as follows: Spiked rat blood was added dropwise, starting with low concentrations. The first drop was 0.05 mmol / L solution, followed by 0.1 mmol / L solution after the baseline stabilized, then 0.3 mmol / L solution, and finally 0.5 mmol / L solution, for a total of 4 drops. After all the spiked rat blood had been added and the baseline stabilized, diluted rat blood was added dropwise, for a total of 14 drops. Each drop was added only after the previous baseline stabilized, until all 14 drops were added. Electrochemical signal curves were obtained.

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