Dual-mode paper-based sweat chloride ion sensor, preparation method and application

Abstract

The invention relates to a dual-mode paper-based sweat chloride ion sensor, a preparation method and application of the dual-mode paper-based sweat chloride ion sensor in rapid diagnosis of cystic fibrosis, and belongs to the crossing field of a biomedical detection technology and electrochemical / optical sensing materials and devices. The dual-mode paper-based sweat chloride ion sensor is composed of a colorimetric detection module and an electrochemical detection module. According to the invention, by optimizing the collaborative design of the colorimetric module and the electrochemical module and combining the integrated process of the silver nitrate / potassium chromate sensitive layer and the screen-printed electrode, the detection sensitivity limit is remarkably broken through, and the integrated process from preliminary screening to clinical definite diagnosis is realized. More importantly, a low-cost cellulose substrate and a silk-screen printing process are adopted, the single-time detection cost is lower than 0.1 dollar, the 180-second quick response characteristic is combined, the method completely meets the economical, practical, high-sensitivity, good-specificity, simple and quick to operate and repeatable diagnosis standards proposed by WHO, and the method is suitable for instant detection scenes of basic medical institutions.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of biomedical detection technology and electrochemical / optical sensing materials and devices, and relates to a dual-mode paper-based sweat chloride ion sensor, a preparation method and its application in the rapid diagnosis of cystic fibrosis. Background Art

[0002] Cystic fibrosis is a life-threatening, autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which disrupts chloride transport in epithelial cells, resulting in devastating multisystem consequences. The resulting electrolyte imbalance manifests as diagnostically elevated sweat chloride levels (>60 mmol / L, compared to a normal value of <40 mmol / L), making sweat testing the clinical gold standard for diagnosing cystic fibrosis. While genetic testing can provide mutation-specific information, its 3- to 5-day diagnostic turnaround time and high cost (US$200-500 per test) limit its application in time-sensitive clinical decision-making. In contrast, sweat chloride analysis provides accurate and reliable results within 30 minutes at an 80% lower cost, making it an essential tool for early intervention.

[0003] Current cystic fibrosis diagnostics primarily rely on laboratory-based sweat chloride testing, such as coulometric titration, ion chromatography, and inductively coupled plasma-mass spectrometry. These methods have significant limitations in sensitivity, cost, and operational complexity. Traditional methods have a coefficient of variation of 10% to 15% and are unable to meet the needs of testing low sample volumes (<10 μL). Ion chromatography and other methods require specialized operators and large equipment, making them incompatible with point-of-care testing.

[0004] Paper-based analytical devices, utilizing a cellulose matrix for fluid manipulation and reagent immobilization, have become a transformative solution for point-of-care diagnostics. Innovations in paper-based sweat chloride detection generally employ colorimetric or electrochemical transduction mechanisms. Colorimetric methods offer ease of operation, and the visual format is intuitive and easy to read. Electrochemical sensing, leveraging the inherent redox activity of chloride ions, offers high sensitivity. However, single-mode detection systems remain susceptible to matrix effects and instrument signal drift, particularly when analyzing complex biofluids such as sweat. Neither approach meets the clinical need for an integrated "screening to confirmation" workflow.

[0005] Therefore, the development of simpler, faster, more cost-effective, low-sample-volume, and self-calibrating dual-mode sensing devices is crucial to enable point-of-care cystic fibrosis diagnosis in resource-limited settings. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention aims to develop a dual-mode paper-based sweat chloride ion sensor with spatially isolated colorimetric and electrochemical detection modules. The sensor can simultaneously generate optical and electrical signals from a single sweat sample deposited in an integrated detection area, enabling synchronous dual-mode chloride analysis.

[0007] To achieve the objectives of the invention, the present invention adopts the following technical solution: a dual-mode paper-based sweat chloride ion sensor, which is composed of a colorimetric detection module and an electrochemical detection module; the colorimetric detection module is formed by impregnating silver nitrate and potassium chromate on filter paper; the electrochemical detection module is formed by printing a circular silver electrode as a working electrode on the filter paper impregnated with silver nitrate and potassium chromate, and then printing a three-quarter circular silver chloride reference electrode concentric with the working electrode and two parallel carbon wires, one carbon wire connected to the working electrode and the other carbon wire connected to one end of the reference electrode.

[0008] In some embodiments, the concentration of silver nitrate is 0.02-0.1 mol / L, and the concentration of potassium chromate is 0.015-0.055 mol / L.

[0009] Preferably, the optimal concentration is 0.04 mol / L silver nitrate reacting with 0.025 mol / L potassium chromate, which reduces heavy metal usage by 40% while maintaining a colorimetric contrast ratio of ≥95%. Higher concentrations result in darker coloration of the fiber matrix, but due to the more rapid kinetic reaction, the resulting white silver chloride precipitate ring becomes smaller.

[0010] In some embodiments, the working electrode has a diameter of 1.5 mm; the reference electrode has an outer diameter of 6 mm and an inner diameter of 5.5 mm.

[0011] The present invention also provides a method for preparing the dual-mode paper-based sweat chloride ion sensor, the method comprising the following steps:

[0012] S1 immersed Whatman 4 filter paper in a silver nitrate solution and dried it with air to obtain silver nitrate-modified filter paper. Whatman 4 filter paper was chosen as the substrate for the dual-mode sensor because of its low cost and light weight, suitability for disposable detection, high capillary-driven fluid transfer efficiency, structural compatibility, and support for the integration of colorimetric and electrochemical modules.

[0013] S2: immersing the silver nitrate modified filter paper described in step S1 in a potassium chromate solution and drying with forced air to obtain a sensing substrate covered with a silver chromate pre-deposition layer;

[0014] S3: screen printing the sensing substrate described in step S2 as a printing material, using silver paste to print a working electrode, heating to 100-120° C. and curing for 5-15 minutes to obtain a sensing element with a printed working electrode;

[0015] S4: screen printing the sensor element described in step S3 as a printing material, printing a reference electrode using silver / silver chloride paste, heating to 100-120° C. and curing for 5-15 minutes to obtain a sensor element with a printed working electrode and a reference electrode;

[0016] S5: screen printing the sensor element described in step S4 as a printing material, using carbon paste to print the conductor, and curing at 100-120° C. for 5-15 minutes to obtain a sensor element with a printed working electrode, a reference electrode, and a conductor;

[0017] S6: screen-printing the sensing element described in step S5 as a printing material, using UV-curable ink to print a hydrophobic circle with a diameter of 7 mm, and UV-curing to form a circular microfluidic channel to obtain a dual-mode paper-based sweat chloride ion sensor;

[0018] The present invention also provides an application of the dual-mode paper-based sweat chloride ion sensor in cystic fibrosis detection. The dual-mode paper-based sweat chloride ion sensor can perform colorimetric sensing for semi-quantitative detection and electrochemical sensing for quantitative detection in cystic fibrosis detection.

[0019] In some embodiments, the colorimetric sensing function performs semi-quantitative detection by:

[0020] S7: adding a chloride ion sample dropwise to the sensor detection area and allowing it to stand; the diameter of the sensor detection area is 5.5 to 6.5 mm; the detection range of the chloride ion sample is 20 to 100 mmol / L;

[0021] S8 measures the diameter of the AgCl precipitation ring with a ruler and compares it with the standard curve to obtain a semi-quantitative result;

[0022] S9 When the colorimetric result is ≥60mmol / L, it is preliminarily judged to be positive for cystic fibrosis.

[0023] In some embodiments, the electrochemical sensing function performs quantitative detection by:

[0024] S10 connects the sensor to the portable constant potential instrument and terminal equipment, opens the supporting software to set the voltage

[0025] Chemical test parameters;

[0026] S11: Add chloride ion sample to the sensor detection area and let it stand for 160-200 seconds;

[0027] S12 measures the diameter of the AgCl precipitation ring with a ruler and compares it with the standard curve to obtain a semi-quantitative result;

[0028] S13 simultaneously records the open circuit potential end potential value and calculates the chloride ion concentration according to the calibration equation;

[0029] S14 When both the colorimetric and electrochemical results are ≥60mmol / L, the diagnosis is positive for cystic fibrosis.

[0030] Preferably, in step S10, the terminal device is a smart phone or a computer, the electrochemical test parameters are that the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s.

[0031] Preferably, in step S11 , the amount of chloride ion sample added is 1.5 μL, and the standing time is 180 s.

[0032] Preferably, in step S12, the standard curve equation is y=0.031+1.968x.

[0033] Preferably, in step S13, when the dynamic range of chloride ion concentration is 10-40 mmol / L, the calibration equation is y=175.21-1.38x; when the dynamic range of chloride ion concentration is 40-150 mmol / L, y=130.94-0.23x.

[0034] Preferably, in step S14, the colorimetric result is obtained from step S12, and the electrochemical result is obtained from step S13, and the dual signals are cross-validated, so that the sensitivity and accuracy of the dual-mode sensor are effectively improved.

[0035] In some embodiments, the detection range of the chloride ion sample of the electrochemical detection module in S13 is 10-150 mmol / L.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The dual-mode paper-based sweat chloride ion sensor described in the present invention significantly breaks through the detection sensitivity limit by optimizing the coordinated design of colorimetric and electrochemical modules and combining the integrated process of silver nitrate / potassium chromate sensitive layer and screen-printed electrodes. The detection limit is as low as 2.13mmol / L, and the dynamic detection range covers 10-150mmol / L, realizing an integrated process from preliminary screening to clinical diagnosis.

[0038] (2) The dual-mode paper-based sweat chloride ion sensor described in the present invention, with its special design of microfluidic channel and capillary drive structure, reduces the sample volume requirement to 1.5 μL, which is only 1 / 30 of the traditional method, breaking through the bottleneck of scarce sweat sample detection and is particularly suitable for the scenario of collecting trace sweat from pediatric patients.

[0039] (3) The dual-mode paper-based sweat chloride ion sensor of the present invention uses a colorimetric-electrochemical dual signal cross-validation algorithm with a deviation threshold of ≤10%, reducing the false positive rate from 8% to 12% in a single mode to below 3%, significantly improving the clinical reliability of the test results and meeting the needs of precision medical diagnosis.

[0040] (4) The dual-mode paper-based sweat chloride ion sensor described in the present invention adopts a low-cost cellulose substrate and screen printing process, with a single detection cost of less than US$0.1. Combined with the rapid response characteristic of 180 seconds, it fully meets the WHO's economical, high-sensitivity, good specificity, simple operation, rapid and repeatable diagnostic standards, and is suitable for immediate detection scenarios in primary medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic structural diagram of the dual-mode paper-based sweat chloride ion sensor of the present invention;

[0042] Figure 2 The standard curve of the dual-mode paper-based sweat chloride ion sensor of the present invention is established: (a) colorimetric signal diameter-concentration curve; (b-c) electrochemical open circuit potential response characteristics;

[0043] Figure 3 This is the interfering substance test for the dual-mode paper-based sweat chloride ion sensor of the present invention;

[0044] Figure 4 This is a long-term stability test of the dual-mode paper-based sweat chloride ion sensor of the present invention;

[0045] Figure 5 The results of the spike recovery experiment of the dual-mode paper-based sweat chloride ion sensor of the present invention are compared with those of ion chromatography;

[0046] Figure 6 This is an application scenario of the dual-mode paper-based sweat chloride ion sensor of the present invention: a POC detection workflow integrating a portable potentiostat and a smartphone. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention, the technical process steps, and the specific implementation conditions. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Preparation Example 1

[0049] like Figure 1As shown, Whatman No. 4 filter paper was immersed in a 0.04 mol / L silver nitrate solution to completely wet it, and then dried in a 30°C forced air drying oven for 10 minutes to obtain a silver nitrate-modified filter paper; the silver nitrate-modified filter paper was immersed in a 0.025 mol / L potassium chromate solution for a second time to completely wet it, and dried at 30°C for 10 minutes to form a uniform silver chromate pre-deposition layer, thereby obtaining the sensing substrate of the dual-mode sensor. A working electrode with a diameter of 1.5 mm was screen-printed on the sensing substrate using silver paste and cured at 110°C for 10 min. A circular reference electrode with an outer diameter of 6.0 mm and an inner diameter of 5.5 mm was printed using Ag / AgCl mixed paste and cured at 110°C for 10 min. A counter electrode was then printed using carbon paste and cured at 110°C for 10 min. Finally, a hydrophobic ring with a diameter of 7 mm was screen-printed using UV-curable ink (model UV~320) and cured at 365 nm UV light for 10 min to form a hydrophobic boundary with a contact angle of 134.5°. This resulted in a dual-mode paper-based sweat chloride ion sensor, which was then stored in a dark and refrigerated place for future use.

[0050] Preparation Example 2

[0051] The sensor preparation method of this preparation example is the same as that of Preparation Example 1, except that the concentration of the silver nitrate solution is 0.02 mol / L, and the concentration of the potassium chromate solution is 0.015 mol / L.

[0052] Preparation Example 3

[0053] The sensor preparation method of this preparation example is the same as that of Preparation Example 1, except that the concentration of the silver nitrate solution is 0.06 mol / L, and the concentration of the potassium chromate solution is 0.035 mol / L.

[0054] Preparation Example 4

[0055] The sensor preparation method of this preparation example is the same as that of Preparation Example 1, except that the concentration of the silver nitrate solution is 0.08 mol / L, and the concentration of the potassium chromate solution is 0.045 mol / L.

[0056] Preparation Example 5

[0057] The sensor preparation method of this preparation example is the same as that of Preparation Example 1, except that the concentration of the silver nitrate solution is 0.1 mol / L, and the concentration of the potassium chromate solution is 0.055 mol / L.

[0058] Example 1

[0059] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. It uses colorimetric sensing for semi-quantitative detection of chloride ions. 1.5 μL of a standard sample with a chloride ion content of 60 mmol / L is added to the sensor detection area and allowed to stand for 180 seconds to produce an AgCl precipitate ring.

[0060] Example 2

[0061] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 2. It uses colorimetric sensing for semi-quantitative detection of chloride ions. 1.5 μL of a standard sample with a chloride ion content of 60 mmol / L is added dropwise to the sensor detection area and allowed to stand for 180 seconds to produce an AgCl precipitate ring.

[0062] Example 3

[0063] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 3. It uses colorimetric sensing for semi-quantitative detection of chloride ions. 1.5 μL of a standard sample with a chloride ion content of 60 mmol / L is added dropwise to the sensor detection area and allowed to stand for 180 seconds to produce an AgCl precipitate ring.

[0064] Example 4

[0065] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 4. It uses colorimetric sensing for semi-quantitative detection of chloride ions. 1.5 μL of a standard sample with a chloride ion content of 60 mmol / L is added to the sensor detection area and allowed to stand for 180 seconds to produce an AgCl precipitate ring.

[0066] Example 5

[0067] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 5. It uses colorimetric sensing for semi-quantitative detection of chloride ions. 1.5 μL of a standard sample with a chloride ion content of 60 mmol / L is added to the sensor detection area and allowed to stand for 180 seconds to produce an AgCl precipitate ring.

[0068] Example 6

[0069] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. Colorimetric sensing is used for semi-quantitative detection of chloride ions, while electrochemical sensing is used for quantitative detection. The sensor is connected to a portable potentiostat and a smartphone. The accompanying software is opened and the electrochemical test parameters are set as follows: the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. 1.5μL of a standard sample with a chloride ion content of 10mmol / L is added to the sensor detection area. The sample is allowed to stand for 180s. The diameter of the AgCl precipitation ring is measured using a ruler, and the end potential value of the open circuit potential is simultaneously recorded.

[0070] Example 7

[0071] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 20 mmol / L.

[0072] Example 8

[0073] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 30 mmol / L.

[0074] Example 9

[0075] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 40 mmol / L.

[0076] Example 10

[0077] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 50 mmol / L.

[0078] Example 11

[0079] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 60 mmol / L.

[0080] Example 12

[0081] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 70 mmol / L.

[0082] Example 13

[0083] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 80 mmol / L.

[0084] Example 14

[0085] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 90 mmol / L.

[0086] Example 15

[0087] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 100 mmol / L.

[0088] Example 16

[0089] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 110 mmol / L.

[0090] Example 17

[0091] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 120 mmol / L.

[0092] Example 18

[0093] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 130 mmol / L.

[0094] Example 19

[0095] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 140 mmol / L.

[0096] Example 20

[0097] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 150 mmol / L.

[0098] Example 21

[0099] The sensor preparation method and detection method of this embodiment are the same as those of Example 6, except that the chloride ion content of the standard sample is 160 mmol / L.

[0100] Example 22

[0101] This embodiment is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1 and the regression curve calibration equation obtained in Examples 6 to 21. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. The sensor is connected to a portable potentiostat and a smart phone, and the supporting software is opened to set the electrochemical test parameters as follows; the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. 1.5 μL of a sample containing 60 mmol / L chloride ions and 3 mmol / L lactic acid is added dropwise to the sensor detection area, allowed to stand for 180 seconds, and the diameter of the AgCl precipitation ring is measured by a ruler, and the open circuit potential end potential value is recorded simultaneously. Substitute the regression curve calibration equations obtained in Examples 6 to 21 respectively to obtain the chloride ion concentration.

[0102] Example 23

[0103] The sensor preparation method and detection method described in this embodiment are the same as those in Example 22, except that the sample contains 60 mmol / L chloride ions and 500 μmol / L uric acid.

[0104] Example 24

[0105] The sensor preparation method and detection method described in this embodiment are the same as those in Example 22, except that the sample contains 60 mmol / L chloride ions and 0.5 mmol / L glucose.

[0106] Example 25

[0107] The sensor preparation method and detection method described in this embodiment are the same as those in Example 22, except that the sample contains 60 mmol / L chloride ions and 90 mmol / L sodium ions.

[0108] Example 26

[0109] The sensor preparation method and detection method described in this embodiment are the same as those in Example 22, except that the sample contains 60 mmol / L chloride ions and 20 mmol / L potassium ions.

[0110] Example 27

[0111] This embodiment is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. Before the test, the sensor was stored in the dark for 2 days at room temperature (25°C) and refrigerated (4°C). The sensor was connected to a portable potentiostat and a smart phone, and the supporting software was opened to set the electrochemical test parameters as follows; the test method was open circuit potential, the test voltage range was -1V to 1V, and the test time was 180s. 1.5 μL of a standard sample containing 60 mmol / L chloride ions was added to the sensor detection area, allowed to stand for 180 seconds, and the diameter of the AgCl precipitation ring was measured by measuring with a ruler, and the end potential value of the open circuit potential was recorded simultaneously. Substitute the regression curve calibration equations obtained in Examples 6 to 21 respectively to obtain the chloride ion concentration.

[0112] Example 28

[0113] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor is stored in the dark for 4 days at room temperature (25°C) and refrigerated (4°C).

[0114] Example 29:

[0115] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 6 days at room temperature (25°C) and refrigerated (4°C).

[0116] Example 30

[0117] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 8 days at room temperature (25°C) and refrigerated (4°C).

[0118] Example 31

[0119] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 10 days at room temperature (25°C) and refrigerated (4°C).

[0120] Example 32

[0121] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 12 days at room temperature (25°C) and refrigerated (4°C).

[0122] Example 33

[0123] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 14 days at room temperature (25°C) and refrigerated (4°C).

[0124] Example 34

[0125] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 16 days at room temperature (25°C) and refrigerated (4°C).

[0126] Example 35

[0127] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 18 days at room temperature (25°C) and refrigerated (4°C).

[0128] Example 36

[0129] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 20 days at room temperature (25°C) and refrigerated (4°C).

[0130] Example 37

[0131] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 22 days at room temperature (25°C) and refrigerated (4°C).

[0132] Example 38

[0133] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 24 days at room temperature (25°C) and refrigerated (4°C).

[0134] Example 39

[0135] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 26 days at room temperature (25°C) and refrigerated (4°C).

[0136] Example 40

[0137] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 28 days at room temperature (25°C) and refrigerated (4°C).

[0138] Example 41

[0139] The sensor preparation method and detection method described in this example are the same as those in Example 27, except that the sensor can be stored in the dark for 30 days at room temperature (25°C) and refrigerated (4°C).

[0140] Example 42

[0141] This embodiment is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. The sensor is connected to a portable potentiostat and a smart phone, and the supporting software is opened to set the electrochemical test parameters as follows: the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. 1.5μL of artificial sweat sample No. 1 is added to the sensor detection area, allowed to stand for 180s, and the diameter of the AgCl precipitation ring is measured by a ruler, and the open circuit potential end potential value is recorded simultaneously. Substitute the regression curve calibration equations obtained in Examples 6 to 21 respectively to obtain the chloride ion concentration. The chloride ion content in artificial sweat sample No. 1 is tested using an ion chromatograph.

[0142] Example 43

[0143] The sensor preparation method and detection method described in this embodiment are the same as those in Example 42, except that the sample is artificial sweat No. 2.

[0144] Example 44

[0145] The sensor preparation method and detection method described in this embodiment are the same as those in Example 42, except that the sample is artificial sweat No. 3.

[0146] Example 45

[0147] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. The sensor is connected to a portable potentiostat and a smartphone, and the accompanying software is opened to set the electrochemical test parameters as follows: the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. A standard amount of 20mmol / L of Cl is added to artificial sweat No. 1. - Prepare a spiked solution. Add 1.5 μL of the sample to the sensor detection area and let it sit for 180 seconds. Measure the diameter of the AgCl precipitation ring using a ruler and simultaneously record the open circuit potential end potential. Substitute the values ​​into the regression curve calibration equations obtained in Examples 6 to 21 to determine the chloride ion concentration. Analyze the chloride ion content in the spiked sample using an ion chromatograph.

[0148] Example 46

[0149] The sensor preparation method and detection method of this embodiment are the same as those of embodiment 45, except that Cl is added to the sample. - The standard amount is 40mmol / L.

[0150] Example 47

[0151] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. The sensor is connected to a portable potentiostat and a smartphone, and the accompanying software is opened to set the electrochemical test parameters as follows: the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. A standard amount of 20mmol / L of Cl is added to artificial sweat No. 2. - Prepare a spiked solution. Add 1.5 μL of the sample to the sensor detection area and let it sit for 180 seconds. Measure the diameter of the AgCl precipitation ring using a ruler and simultaneously record the open circuit potential end potential. Substitute the values ​​into the regression curve calibration equations obtained in Examples 6 to 21 to determine the chloride ion concentration. Analyze the chloride ion content in the spiked sample using an ion chromatograph.

[0152] Example 48

[0153] The sensor preparation method and detection method of this embodiment are the same as those of embodiment 47, except that Cl is added to the sample. - The standard amount is 40mmol / L.

[0154] Example 49

[0155] This example is based on the dual-mode paper-based sweat chloride ion sensor described in Preparation Example 1. The colorimetric sensing function is used for semi-quantitative detection of chloride ions, and the electrochemical sensing function is used for quantitative detection. The sensor is connected to a portable potentiostat and a smartphone, and the accompanying software is opened to set the electrochemical test parameters as follows: the test method is open circuit potential, the test voltage range is -1V to 1V, and the test time is 180s. 20mmol / L standard amount of Cl is added to artificial sweat No. 3. - Prepare a spiked solution. Add 1.5 μL of the sample to the sensor detection area and let it sit for 180 seconds. Measure the diameter of the AgCl precipitation ring using a ruler and simultaneously record the open circuit potential end potential. Substitute the values ​​into the regression curve calibration equations obtained in Examples 6 to 21 to determine the chloride ion concentration. Analyze the chloride ion content in the spiked sample using an ion chromatograph.

[0156] Example 50

[0157] The sensor preparation method and detection method described in this embodiment are the same as those in Example 49, except that the standard amount of Cl~ added to the sample is 40 mmol / L.

[0158] The images and colorimetric signals of the sensors described in Examples 1 to 5 are shown in Table 1, and the colorimetric signals and electrochemical signals of the sensors described in Examples 6 to 21 are shown in Table 2.

[0159] Table 1 Images and colorimetric signals of the sensors described in Examples 1 to 5.

[0160]

[0161] Table 2 Colorimetric and electrochemical signals of the sensors described in Examples 6 to 21.

[0162]

[0163]

[0164] According to the experimental data obtained in Examples 6 to 21, the chloride ion concentration and the colorimetric signal (AgCl precipitation ring diameter) were fitted respectively (as Figure 2 a) and the chloride ion concentration and electrochemical signal (open circuit potential voltage value) (such as Figure 2 b~c), the following conclusions were drawn: in colorimetric sensing, the diameter of the AgCl precipitation ring increases with the increase of chloride ion concentration, and a good linear relationship is shown in the dynamic detection range of 20~100mmol / L. The regression equation is y=0.031+1.968x, and the correlation coefficient R 2The electrochemical sensing results show that the terminal potential decreases with the increase of chloride ion concentration. The dynamic detection ranges are 10-40 mmol / L and 40-150 mmol / L, respectively. The regression equations are y=175.21-1.38x and y=130.94-0.23x, respectively. The correlation coefficient R 2 are 0.982 and 0.995 respectively. The detection limit (LOD) is calculated as LOD=3S / N, where S is the standard deviation of the low concentration solution signal and N is the slope of the calibration curve. After measurement, the detection limits of the colorimetric method and the electrochemical detection method are 13.2mmol / L and 2.13mmol / L, respectively. Compared with the colorimetric method, the electrochemical method has a wider detection range and a lower detection limit, which can meet different detection requirements under different backgrounds. The colorimetric signals and electrochemical signals of the sensors described in Examples 22 to 26 are shown in Table 3.

[0165] Table 3 Colorimetric and electrochemical signals of the sensors described in Examples 22 to 26.

[0166]

[0167] From the test data in Table 3, it can be seen that the dual-mode sensor of Examples 22 to 26 of the present invention can be used in the presence of various interfering substances (lactate 3mmol / L, uric acid 500μmol / L, glucose 0.5mmol / L, Na + 90mmol / L, K + 20mmol / L) simulated sweat matrix, the deviation of the colorimetric signal AgCl precipitation ring diameter is ≤5.1% (such as Figure 3 a), electrochemical open circuit potential potential offset ≤ ± 2.3mV (such as Figure 3 b) The dual-mode comprehensive relative error is ≤10%. Experimental results show that the present invention, through the synergistic design of electrochemical screen-printed electrodes and the Ag2CrO4 / AgCl selective replacement reaction, significantly inhibits the cross-interference of anions and cations in complex matrices, significantly reducing the "false positive" effect (inhibition rate >85%), and significantly outperforming existing technologies in terms of test result repeatability (RSD <3.5%) and reliability (clinical compliance rate ≥97%).

[0168] The colorimetric and electrochemical signals of the sensors described in Examples 27 to 41 are as follows: Figure 4As shown. Analysis of test data shows that during the 30-day storage period, the dual-mode sensors described in Examples 27 to 41 of the present invention exhibited a maximum colorimetric signal offset of 5.8% (n=5) at room temperature (25°C), and an RSD of the AgCl precipitation ring diameter of ≤3.2%. This was attributed to photooxidative degradation of the paper-based silver chromate indicator (decay rate 0.19% / day). Under refrigerated conditions (4°C), the colorimetric signal offset was <1.5% (n=5), and the color uniformity retention rate was ≥98.7%, confirming that low-temperature environments can effectively inhibit the decomposition of the colorimetric indicator. The electrochemical signal open-circuit potential drift was ≤±0.8 mV (equivalent Cl- concentration error <1.2%). Experimental results demonstrate that the dual-mode sweat chloride ion sensor of the present invention maintains performance stability during long-term storage (30-day decay rate <6%), which is more than 60% higher than that of traditional paper-based sensors (decay rate >15%), meeting the shelf-life standards of clinical diagnostic reagents. The colorimetric signals and electrochemical signals of the sensors described in Examples 42 to 44 are shown in Table 4.

[0169] Table 4 Colorimetric and electrochemical signals of the sensors described in Examples 42 to 44.

[0170]

[0171] The colorimetric and electrochemical signals of the sensors described in Examples 45 to 50 are as follows: Figure 5 shown.

[0172] As shown in the test data in Table 4, the error of the colorimetric method is less than 3.3%, and the error of the electrochemical method is less than 1.27%. Statistical analysis shows no significant difference between the sensor's measured values ​​and the gold standard method, indicating that the sensor of the present invention has potential application value in cystic fibrosis screening. Figure 5 The results of the spike recovery experiments are presented, with recoveries ranging from 99.4% to 102.3%, further verifying the accuracy and reliability of the sensor.

[0173] As the instruction manual Figure 6 As shown, the dual-mode sensor of the present invention adopts an integrated colorimetric-electrochemical detection module, which is characterized by: a colorimetric module: a visual interpretation mechanism based on the change of the diameter of the AgCl precipitation ring (dynamic range 20-100mM, resolution 5mM), suitable for rapid screening scenarios without instrument support (detection time 180s); an electrochemical module: an open circuit potential measurement system (detection limit 2.13mM, linear range 10-150mM, R 2 =0.998), equipped with a portable potentiostat (power consumption <5mW), and an interference suppression ratio >90%. The integrated process of the silver nitrate / potassium chromate sensitive layer and screen-printed electrodes enables simultaneous dual-signal acquisition and cross-validation (deviation threshold ≤10%).

[0174] The sensor is integrated with a commercial sweat collection device and terminal equipment, and its workflow exhibits the following advantages: (1) Ultra-low sample requirement: the sample consumption for a single test is ≤1.5 μL (in compliance with the CLSI EP17-A2 micro-sample standard), which is suitable for pediatric sweat secretion rates (0.5-10 μL / min / gland); (2) Fast response: the stabilization time of the color reaction and electrochemical signal is controlled within 180 s; (3) Multi-signal multiplexing: spatial colorimetric signals (resolution 0.1 mm) and electrochemical signals (accuracy ±0.5 mV) are collected simultaneously, and the data fusion algorithm improves diagnostic accuracy.

[0175] The present invention achieves the following through a modular layered detection architecture (claim 10):

[0176] Primary care scenarios: The colorimetric mode supports instrument-free screening (operation training time <30 minutes), meeting the WHO's diagnostic criteria for affordability, high sensitivity, good specificity, simplicity, speed, and repeatability.

[0177] Precision medicine scenarios: Cloud-based analysis of electrochemical data (encrypted transmission compliant with HIPAA) supports long-term health monitoring (data storage period ≥ 5 years). Compared with traditional single-function sensors, this solution reduces the misdiagnosis rate from 12% to below 3% (P < 0.01, t-test), increasing detection efficiency by 2.3 times.

[0178] Technical personnel should note: Although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention. Any modification using the inventive concept will be included in the scope of protection of this patent.

Claims

1. A dual-mode paper-based sweat chloride ion sensor, characterized in that: The dual-mode paper-based sweat chloride ion sensor consists of a colorimetric detection module and an electrochemical detection module; the colorimetric detection module is formed by impregnating silver nitrate and potassium chromate on filter paper; the electrochemical detection module is formed by printing a circular silver electrode as a working electrode on the filter paper impregnated with silver nitrate and potassium chromate, and then printing a three-quarter circular silver chloride reference electrode concentric with the working electrode and two parallel carbon wires, one carbon wire connected to the working electrode and the other connected to one end of the reference electrode.

2. The dual-mode paper-based sweat chloride ion sensor according to claim 2, characterized in that: The concentration of the silver nitrate is 0.02-0.1 mol / L, and the concentration of the potassium chromate is 0.015-0.055 mol / L.

3. The dual-mode paper-based sweat chloride ion sensor according to claim 4, characterized in that: The diameter of the working electrode is 1.5 mm; the outer diameter of the reference electrode is 6 mm and the inner diameter is 5.5 mm.

4. The method for preparing the dual-mode paper-based sweat chloride ion sensor according to any one of claims 1 to 3, wherein: The preparation method comprises the following steps: S1: Soak Whatman 4 filter paper in silver nitrate solution and air dry it to obtain silver nitrate-modified filter paper; S2: immersing the silver nitrate modified filter paper described in step S1 in a potassium chromate solution and drying with forced air to obtain a sensing substrate covered with a silver chromate pre-deposition layer; S3: screen printing the sensing substrate described in step S2 as a printing material, using silver paste to print a working electrode, heating to 100-120° C. and curing for 5-15 minutes to obtain a sensing element with a printed working electrode; S4: screen printing the sensor element described in step S3 as a printing material, printing a reference electrode using silver / silver chloride paste, heating to 100-120° C. and curing for 5-15 minutes to obtain a sensor element with a printed working electrode and a reference electrode; S5: screen printing the sensor element described in step S4 as a printing material, using carbon paste to print the conductor, and curing at 100-120° C. for 5-15 minutes to obtain a sensor element with a printed working electrode, a reference electrode, and a conductor; S6: The sensing element described in step S5 is used as a printing material for screen printing, and a hydrophobic circle with a diameter of 7 mm is printed using UV-curable ink. The circular microfluidic channel is formed by UV-curing to obtain a dual-mode paper-based sweat chloride ion sensor.

5. Application of the dual-mode paper-based sweat chloride ion sensor according to any one of claims 1 to 4 in cystic fibrosis detection, characterized in that: The dual-mode paper-based sweat chloride ion sensor can perform colorimetric sensing for semi-quantitative detection and electrochemical sensing for quantitative detection in cystic fibrosis detection.

6. Application of the dual-mode paper-based sweat chloride ion sensor in cystic fibrosis detection as claimed in claim 5, characterized in that: The steps of performing semi-quantitative detection using the colorimetric sensing function are as follows: S7: adding a chloride ion sample dropwise to the sensor detection area and allowing it to stand; the diameter of the sensor detection area is 5.5-6.5 mm; the detection range of the chloride ion sample is 20-100 mmol / L; S8 measures the diameter of the AgCl precipitation ring with a ruler and compares it with the standard curve to obtain a semi-quantitative result; S9 When the colorimetric result is ≥60 mmol / L, it is preliminarily judged to be positive for cystic fibrosis.

7. Application of the dual-mode paper-based sweat chloride ion sensor in cystic fibrosis detection as claimed in claim 5, characterized in that: The steps of performing quantitative detection using the electrochemical sensing function are as follows: S10 connects the sensor to the portable potentiostat and terminal device, and opens the supporting software to set the electrochemical test parameters; S11 adds chloride ion sample to the sensor detection area and allows it to stand; S12 measures the diameter of the AgCl precipitation ring with a ruler and compares it with the standard curve to obtain a semi-quantitative result; S13 simultaneously records the open circuit potential end potential value and calculates the chloride ion concentration according to the calibration equation; S14 When both the colorimetric and electrochemical results are ≥60 mmol / L, the diagnosis is positive for cystic fibrosis.

8. Application of the electrochemical sensing function of the dual-mode paper-based sweat chloride ion sensor according to claim 7 in the quantitative detection of cystic fibrosis, characterized in that: The detection range of the chloride ion sample of the electrochemical detection module in S13 is 10~150 mmol / L.

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