Nano-polyaniline / Prussian blue electrode, preparation method, device and method for detecting biomarkers in exhaled breath

Abstract

The present invention relates to a nano-polyaniline / Prussian blue electrode for detecting biomarkers in exhaled breath, a preparation method, an apparatus and a method thereof. The preparation method comprises the following steps: providing an AAO template and an electrode body, fixing the AAO template on the electrode body to obtain an assembly; immersing the assembly in an acid solution of aniline, and performing a first electrodeposition thereof using cyclic voltammetry to obtain an intermediate assembly; wherein the concentration of aniline in the acid solution is 10-50 mg / mL; placing the intermediate assembly in an alkaline solution, and obtaining a nano-polyaniline electrode after the AAO template is completely dissolved; immersing the nano-polyaniline electrode in an aqueous solution containing Fe 3+ and Fe(CN)6 3‑ The nano-polyaniline / Prussian blue electrode is prepared by performing a second electrodeposition on the nano-polyaniline / Prussian blue electrode using cyclic voltammetry, taking out the nano-polyaniline / Prussian blue electrode, and washing and drying the nano-polyaniline / Prussian blue electrode.

Description

Technical Field

[0001] The present invention relates to a nano-polyaniline / Prussian blue electrode, a preparation method, a device and a method for detecting biomarkers in exhaled breath, and in particular to a nano-polyaniline / Prussian blue electrode, a preparation method, a device and a method for detecting hydrogen peroxide in exhaled breath, belonging to the field of electrochemical detection. Background Art

[0002] The number of people suffering from lung and respiratory diseases such as asthma, lung cancer and other chronic lung diseases is increasing. Since most of these lung diseases are chronic diseases, they need to be tested frequently. The content of biomarkers in exhaled breath is closely related to these chronic lung diseases, so it is extremely necessary to develop sensors that can sensitively and quickly monitor the concentration of biomarkers in exhaled breath. Exhaled breath contains a large amount of water vapor and hundreds of trace organic compounds, some of which are biomarkers directly related to diseases. Since the low concentration measurement of biomarkers in exhaled breath is inaccurate, it may pose a challenge to establish the relationship between the marker concentration and health status, so higher requirements are placed on the collection efficiency and sensing sensitivity of exhaled breath condensate.

[0003] Currently, the detection of biomarkers in exhaled breath is generally performed by first collecting a certain amount of exhaled breath condensate, and then analyzing and detecting the substances therein using electrochemical or optical methods. A portable detection device is then constructed. Chinese invention patent application specification CN202010157459.8 discloses a mask that can detect the novel coronavirus pathogen. This mask needs to be worn for 30 minutes to obtain the results, and its collection efficiency is low. This also means that the concentration of biomarkers in the condensate measured based on this method may be lower than the actual concentration, resulting in a false negative result.

[0004] Chinese invention patent application specification CN201880019387.1 discloses an analyzer for collecting and analyzing exhaled breath condensate. The device combines a condensation zone with a detection zone, and constructs a horseradish peroxidase electrode in the detection zone to detect hydrogen peroxide. The performance of the enzyme-modified electrochemical sensor is unstable and the detection sensitivity is low. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, one of the objectives of the present invention is to provide a nano-polyaniline / Prussian blue electrode with high detection sensitivity for detecting biomarkers in exhaled breath and its preparation method. By using an AAO template to construct a three-dimensional columnar structure, the conductive material polyaniline is added to improve the conductivity of the sensor and enhance its detection sensitivity. The second objective of the present invention is to provide a device and method for detecting biomarkers in exhaled breath.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A method for preparing a nano-polyaniline / Prussian blue electrode for detecting biomarkers in exhaled breath comprises the following steps:

[0008] S1. Providing an AAO template and an electrode body, and fixing the AAO template (porous alumina template) on the electrode body to obtain an assembly;

[0009] S2, immersing the assembly in an acid solution of aniline, and performing a first electrodeposition on the assembly using cyclic voltammetry to obtain an intermediate assembly;

[0010] The concentration of aniline in the acid solution is 80-90 mg / mL; the scanning voltage of the first electrodeposition is -0.8-2 V, the scanning rate is 40-60 mV / s, and the number of cycles is 10-20;

[0011] S3, placing the intermediate assembly in an alkaline solution, and obtaining a nano-polyaniline electrode after the AAO template is completely dissolved;

[0012] S4, immersing the nano-polyaniline electrode in a solution containing Fe 3+ and Fe(CN)6 3- a solution of , and performing a second electrodeposition thereof using cyclic voltammetry, and then taking out, washing, and drying to obtain a nano-polyaniline / Prussian blue electrode;

[0013] Among them, in the solution, Fe 3+ The concentration of Fe(CN)6 is 1.5-4mM (mmol / L), 3- The concentration is 1.5-4 mM; the scanning voltage of the second electrodeposition is -0.5-1 V, the scanning rate is 40-60 mV / s, and the number of cycles is 15-25 circles.

[0014] Furthermore, in S1, the electrode body includes one of a glassy carbon electrode and a screen-printed electrode.

[0015] Furthermore, in S1, the pore size of the AAO template is 20-40 nm.

[0016] Furthermore, in S2, the acid in the acid solution is one or more of hydrochloric acid and sulfuric acid; preferably, H + The concentration is 0.5-2mol / L.

[0017] Furthermore, in S3, the alkali solution is an aqueous solution of one or more of sodium hydroxide, potassium hydroxide, and barium hydroxide.

[0018] Based on the same inventive concept, the present invention also provides a nano-polyaniline / Prussian blue electrode for detecting biomarkers in exhaled breath, which is prepared by the preparation method described above.

[0019] Based on the same inventive concept, the present invention also provides a device for detecting biomarkers in exhaled breath, comprising:

[0020] A gas collection module, used to collect exhaled air and guide the exhaled air to the condensation module;

[0021] The condensation module has a condensation channel, the two ends of the condensation channel are an air inlet end and an exhaust end respectively, and the air inlet end is connected to the gas collection module;

[0022] a detection module comprising a single-chip microcomputer and a three-electrode detection circuit electrically connected to the single-chip microcomputer, wherein the working electrode of the three-electrode detection circuit is the nano-polyaniline / Prussian blue electrode as described above, and the working electrode, counter electrode, and reference electrode of the three-electrode detection circuit all extend into the exhaust port; and

[0023] The display module is electrically connected to the single chip computer and is used to display the detection results.

[0024] Furthermore, the gas collection module includes a gas collection hood and a tube, and the gas collection hood, the tube and the air inlet end are connected in sequence.

[0025] Furthermore, the condensation module includes a metal sheet, the condensation channel is arranged in the metal sheet, at least one surface of the metal sheet is provided with a semiconductor refrigeration sheet, and a heat dissipation mechanism is provided on a side of the semiconductor refrigeration sheet away from the metal sheet.

[0026] Optionally, the heat dissipation mechanism is a fan.

[0027] Optionally, the metal sheet is an aluminum sheet.

[0028] Optionally, the thickness of the metal sheet is 2-8 mm, further 3-5 mm (preferably 3.5 mm, further optionally 2.5-4.5 mm).

[0029] Furthermore, the condensation channel includes at least two parallel curved sub-channels, and a plurality of secondary flow structures are provided on the sub-channels. Preferably, the secondary flow structures are semi-cylindrical. In this way, the condensation efficiency of the exhaled air is improved, and at the same time, the biomarkers in the exhaled air are enriched to a certain extent (see Figure 15 It can be seen that the channel with a two-flow structure does have a certain enrichment effect on the same concentration of hydrogen peroxide, indicating that it has a certain enrichment effect on biomarkers in exhaled breath).

[0030] Optionally, the cross section of the sub-channel is rectangular, preferably square. Optionally, the length and width of the cross section of the sub-channel are 1-2 mm and 1-2 mm respectively.

[0031] Optionally, the radius of the inner circle of the sub-channel (ie, the side surface close to the center of curvature) is 0.6-1.2 mm, and the radius of the outer circle (ie, the side surface away from the center of curvature) is 2-2.66 mm.

[0032] Furthermore, the diameter of the secondary flow structure is 1-1.4 mm.

[0033] Furthermore, the detection module also includes a three-electrode detection circuit' electrically connected to the single-chip computer, the working electrode' of the three-electrode detection circuit' is the electrode body (i.e., the electrode not modified with nano-polyaniline / Prussian blue), and the working electrode', counter electrode' and reference electrode' of the three-electrode detection circuit all extend into the exhaust end.

[0034] Optionally, the display module is an LED display screen or a computer.

[0035] Optionally, the biomarker is hydrogen peroxide.

[0036] The method for detecting biomarkers in exhaled breath, using the device as described above, comprises the following steps:

[0037] The gas collection module collects the exhaled gas and guides the exhaled gas to the condensation module;

[0038] The exhaled air condenses in the condensation channel of the condensation module to form condensate;

[0039] The condensate contacts the working electrode, counter electrode and reference electrode of the three-electrode detection circuit. The three-electrode detection circuit detects the current signal and transmits the current signal to the microcontroller. The microcontroller converts the current signal into a voltage signal, and converts the voltage signal into the concentration value of the biomarker, and controls the display module to display the concentration value of the biomarker.

[0040] The method further includes the following steps: the condensate contacts the working electrode, counter electrode, and reference electrode of the three-electrode detection circuit; the three-electrode detection circuit detects a current signal and transmits the current signal to a single-chip microcomputer; the single-chip microcomputer converts the current signal into a voltage signal, performs a differential calculation on the voltage signal and the voltage signal to obtain a differential voltage result; then, the differential voltage result is converted into a biomarker concentration value, and a display module is controlled to display the biomarker concentration value. This helps improve the accuracy of the detection results.

[0041] Furthermore, the condensed liquid after detection can flow out from the exhaust port.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The nano-polyaniline / Prussian blue electrode of the present invention has high detection sensitivity. By constructing a low-cost three-dimensional columnar nano-conductive electrode substrate on the surface of an ordinary electrode body, the present invention has better prospects for promotion and use compared to electrode bodies using expensive two-dimensional materials (such as carbon nanotubes, gold nanoparticles, etc.), and its sensitivity is significantly improved compared to ordinary electrodes.

[0044] (2) The device of the present invention can improve the condensation efficiency of exhaled breath, achieve the enrichment and concentration effect of biomarkers in exhaled breath, and avoid false negative detection;

[0045] (3) Compared with the existing technology, the present invention can still meet the actual detection needs within a low-cost range.

[0046] (4) The device of the present invention can be integrated into a mask (such as an N95 mask) to achieve portable detection of biomarkers in respiratory gas, or it can be integrated into a fixed detection platform to achieve on-the-go detection and meet the detection needs of multiple people. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It curves of the nano-polyaniline / Prussian blue electrode in Example 1 and other electrodes.

[0048] Figure 2 is the fitting curve of the it curve of the nano-polyaniline / Prussian blue electrode and other electrodes in Example 1;

[0049] Among them, Prussian blue electrode: Prussian blue is directly electrodeposited on the electrode.

[0050] Polyaniline + Prussian blue electrode: Polyaniline and Prussian blue are co-electrodeposited on the electrode.

[0051] Polyaniline / Prussian blue electrode: Polyaniline is first electrodeposited on the electrode, and then Prussian blue is electrodeposited.

[0052] Figure 3 This is the stability test curve of the nano-polyaniline / Prussian blue electrode in Example 1.

[0053] Figure 4 This is the selectivity test curve of the nano-polyaniline / Prussian blue electrode in Example 1.

[0054] Figure 5 It curves of the nano-polyaniline / Prussian blue glass screen-printed electrode and the carbon electrode of Example 1 and Example 20.

[0055] Figure 6 for Figure 5 The fitting curve of .

[0056] Figure 7Schematic diagram of the preparation process of Example 1.

[0057] Figure 8 This is a simplified structural diagram of the device of Example 21.

[0058] Figure 9 Schematic diagrams of two integrated modes of the device for detecting biomarkers in exhaled breath of the present invention: portable (left) and fixed (right).

[0059] Figure 10 This is a schematic structural diagram of the sub-channel of Example 21.

[0060] Figure 11 This is a schematic structural diagram of the condensation channel of Example 21.

[0061] Figure 12 Velocity cloud diagrams of five condensation channel structures at high and low speeds.

[0062] Figure 13 Images of the condensate volume generated at different times for five condensation channel structures.

[0063] Figure 14 It is the time required for the five condensation channel structures to be generated and fill the entire condensation structure at a low exhaled air velocity.

[0064] Figure 15 These are the amperometric response curves of hydrogen peroxide in the condensate collected after simulation experiments using the same concentration of hydrogen peroxide for five condensation channel structures.

[0065] Figure 16 This is the relationship curve between the blank electrode, the working electrode and different concentrations of hydrogen peroxide in Example 21.

[0066] Figure 17 This is a relationship curve between the actual output voltage value obtained in Example 21 and different concentrations of hydrogen peroxide.

[0067] Figure 18 These are SEM images of the products of each step in the preparation process of the nano-polyaniline / Prussian blue electrode in Example 1. DETAILED DESCRIPTION

[0068] The present invention will be described in detail below with reference to the following embodiments. It should be noted that the embodiments and features of the embodiments may be combined unless they conflict. For ease of description, the words "upper," "lower," "left," and "right" appearing below merely indicate the directions of upper, lower, left, and right relative to the accompanying drawings and do not limit the structure.

[0069] Mask: The mask in the embodiment of the present application is an N95 mask, which can be used to cover the user's mouth or nose when the user breathes.

[0070] Hydrogen peroxide: A biomarker found in exhaled breath that is closely associated with chronic lung diseases such as lung cancer and asthma.

[0071] Polyaniline: An inexpensive conductive material.

[0072] Prussian blue: can be used for the specific detection of hydrogen peroxide.

[0073] AAO template: an aluminum oxide thin plate with a single pore size and uniform arrangement. In the specific implementation, a 30 nm AAO template is used.

[0074] Example 1

[0075] See also Figure 7 A method for preparing a nano-polyaniline / Prussian blue electrode for detecting a biomarker (hydrogen peroxide) in exhaled breath comprises the following steps:

[0076] S1, providing an AAO template and an electrode body, and fixing the AAO template on the electrode body with a chitosan solution to obtain an assembly;

[0077] S2, immersing the assembly in an acid solution of aniline, and performing a first electrodeposition on the assembly using cyclic voltammetry to obtain an intermediate assembly;

[0078] The concentration of aniline in the acid solution is 85 mg / mL; the scanning voltage of the first electrodeposition is -0.4-1 V, the scanning rate is 50 mV / s, and the number of cycles is 16; depositing the conductive material polyaniline first not only facilitates the subsequent deposition of Prussian blue and facilitates the effectiveness of Prussian blue, but also effectively improves the detection sensitivity of the resulting nano-polyaniline / Prussian blue electrode;

[0079] S3, placing the intermediate assembly in a sodium hydroxide solution, and obtaining a nano-polyaniline electrode after the AAO template is completely dissolved;

[0080] S4, immersing the nano-polyaniline electrode in a solution containing FeCl3 and K3Fe(CN)6, performing a second electrodeposition on the electrode using cyclic voltammetry, taking it out, washing it, and drying it in a drying oven at 60°C for 10 minutes, then dropping 5 μL of Nafion solution on the electrode for fixing the electrode to obtain a nano-polyaniline / Prussian blue electrode. The electron microscope images of the products obtained in each step are shown in FIG. Figure 18 ;

[0081] Wherein, in the solution, Fe 3+ The concentration of Fe(CN)6 is 2.5mmol / L,3- The concentration was 2.5 mmol / L; the scanning voltage of the second electrodeposition was -0.1-0.5 V, the scanning rate was 50 mV / s, and the number of cycles was 20.

[0082] In S1, the electrode body is a glassy carbon electrode.

[0083] In S2, the acid in the acid solution is hydrochloric acid; H + The concentration is 1 mol / L.

[0084] A nano polyaniline / Prussian blue electrode for detecting a biomarker (hydrogen peroxide) in exhaled breath is prepared by the preparation method described above.

[0085] The electrode was tested using an electrochemical workstation with an IT curve at a test voltage of 0.2 V. Figure 1 The electrode obtained in this example has a high sensitivity to hydrogen peroxide detection. Compared with the existing electrochemical sensor electrode using horseradish peroxidase, it greatly improves the stability of hydrogen peroxide detection. Compared with the enzyme probe hydrogen peroxide sensor, due to the use of enzymes, it requires a harsh detection environment and the enzyme is easily inactivated, resulting in great instability in the detection. The data fitting of the IT curve is obtained. Figure 2 , the detection limit of the sensor electrode of this embodiment is as low as 2.52μM, and the detection range is 0-1mM. To verify the long-term stability of the electrode, it is tested once a day. Figure 3 The amperometric response of the electrode remained basically unchanged within five days, proving that the electrode has long-term stability. At the same time, the specificity of the electrode was tested by adding three common interfering substances: vitamin C, dopamine and urea. The results are as follows Figure 4 It was shown that it has good specificity for hydrogen peroxide.

[0086] Figure 1 The preparation methods of the other three electrodes are as follows:

[0087] The preparation method of Prussian blue electrode is as follows:

[0088] S1. Provide an electrode body.

[0089] S2, immersing the electrode in a solution containing FeCl3 and K3Fe(CN)6, performing a second electrodeposition on the electrode using cyclic voltammetry, taking it out, washing it, and drying it in a drying oven at 60°C for 10 minutes, then dropping 5 μL of Nafion solution on the electrode for fixing the electrode to obtain a Prussian blue electrode;

[0090] Wherein, in the solution, Fe 3+ The concentration of Fe(CN)6 is 2.5mmol / L,3- The concentration was 2.5 mmol / L; the scanning voltage of the first electrodeposition was -0.1-0.5 V, the scanning rate was 50 mV / s, and the number of cycles was 20.

[0091] In S1, the electrode body is a glassy carbon electrode.

[0092] The preparation method of polyaniline / Prussian blue electrode is as follows:

[0093] S1. Provide an electrode body.

[0094] S2, immersing the electrode body in an acid solution of aniline, and performing a first electrodeposition thereof using cyclic voltammetry to obtain an intermediate assembly;

[0095] The concentration of aniline in the acid solution is 85 mg / mL; the scanning voltage of the first electrodeposition is -0.4-1 V, the scanning rate is 50 mV / s, and the number of cycles is 16;

[0096] S3, immersing the electrode in a solution containing FeCl3 and K3Fe(CN)6, performing a second electrodeposition on the electrode using cyclic voltammetry, taking the electrode out, washing it, and drying it in a drying oven at 60°C for 10 minutes, then dropping 5 μL of Nafion solution on the electrode for fixing the electrode to obtain a polyaniline / Prussian blue electrode;

[0097] Wherein, in the solution, Fe 3+ The concentration of Fe(CN)6 is 2.5mmol / L, 3- The concentration was 2.5 mmol / L; the scanning voltage of the second electrodeposition was -0.1-0.5 V, the scanning rate was 50 mV / s, and the number of cycles was 20.

[0098] In S1, the electrode body is a glassy carbon electrode.

[0099] In S2, the acid in the acid solution is hydrochloric acid; H + The concentration is 1 mol / L.

[0100] The preparation method of polyaniline + Prussian blue electrode is as follows:

[0101] S1. Provide an electrode body.

[0102] S2. Immerse the electrode body in an acid solution containing aniline, FeCl3 and K3Fe(CN)6, and perform electrodeposition on it using cyclic voltammetry to obtain a polyaniline + Prussian blue electrode.

[0103] The concentration of aniline in the acid solution is 85 mg / mL; Fe 3+ The concentration of Fe(CN)6 is 2.5mmol / L,3- The concentration of 2.5mmol / L; the scanning voltage of electrodeposition was -0.1-0.5V, the scanning rate was 50mV / s, and the number of cycles was 20;

[0104] In S1, the electrode body is a glassy carbon electrode.

[0105] In S2, the acid in the acid solution is hydrochloric acid; H + The concentration is 1 mol / L.

[0106] Comparative Example 1

[0107] Example 1 was repeated, except that in S2, the concentration of aniline was 75 mg / mL.

[0108] After testing, the detection limit of the electrode for hydrogen peroxide was 3.01 μM.

[0109] Example 2

[0110] Example 1 was repeated, except that in S2, the concentration of aniline was 80 mg / mL.

[0111] After testing, the detection limit of the electrode for hydrogen peroxide was 2.61 μM.

[0112] Example 3

[0113] Example 1 was repeated, except that in S2, the concentration of aniline was 90 mg / mL.

[0114] After testing, the detection limit of the electrode for hydrogen peroxide was 2.63 μM.

[0115] Comparative Example 2

[0116] Example 1 was repeated, except that in S2, the concentration of aniline was 95 mg / mL.

[0117] After testing, the detection limit of the electrode for hydrogen peroxide was 4.03 μM.

[0118] It can be concluded from Examples 1-3 and Comparative Examples 1 and 2 that when the concentration of aniline in the acid solution is controlled at 80-90 mg / mL, the detection sensitivity of the obtained electrode is higher.

[0119] Example 4

[0120] Example 1 was repeated except that the scanning voltage of the first electrodeposition was -0.8-1 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.58 μM.

[0121] Example 5

[0122] Example 1 was repeated except that the scanning voltage of the first electrodeposition was -0.4-2 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.56 μM.

[0123] Comparative Example 3

[0124] Example 1 was repeated except that the scanning voltage of the first electrodeposition was -1-1 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.03 μM.

[0125] Comparative Example 4

[0126] Example 1 was repeated except that the scanning voltage of the first electrodeposition was -0.4-2.2 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.05 μM.

[0127] It can be seen that controlling the scanning voltage range of the first electrodeposition within -0.8-2 V is helpful to improve the detection sensitivity of the electrode.

[0128] Example 6

[0129] Example 1 was repeated except that the scan rate of the first electrodeposition was 40 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.64 μM.

[0130] Example 7

[0131] Example 1 was repeated except that the scan rate of the first electrodeposition was 60 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.66 μM.

[0132] Comparative Example 5

[0133] Example 1 was repeated except that the scan rate of the first electrodeposition was 35 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.05 μM.

[0134] Comparative Example 6

[0135] Example 1 was repeated except that the scan rate of the first electrodeposition was 65 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.07 μM.

[0136] The scanning rate range of the first electrodeposition can be controlled within 40-60 mV / s, which helps to improve the detection sensitivity of the electrode.

[0137] Example 8

[0138] Example 1 was repeated except that the number of cycles of the first electrodeposition was 10 and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of the electrode for hydrogen peroxide was found to be 2.65 μM.

[0139] Example 9

[0140] Example 1 was repeated except that the number of cycles of the first electrodeposition was 20 and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of the electrode for hydrogen peroxide was found to be 2.67 μM.

[0141] Comparative Example 7

[0142] Example 1 was repeated except that the number of cycles of the first electrodeposition was 8 and the other parameters were the same. The electrode was tested using an electrochemical workstation and the detection limit of hydrogen peroxide was 4.11 μM.

[0143] Comparative Example 8

[0144] Example 1 was repeated except that the number of cycles of the first electrodeposition was 22 and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of the electrode for hydrogen peroxide was found to be 4.13 μM.

[0145] The number of cycles of the first electrodeposition can be controlled within a range of 10-20 cycles, which helps to improve the detection sensitivity of the electrode.

[0146] Example 10

[0147] Repeat Example 1, except that: Fe 3+ and Fe(CN)6 3- The concentration of the electrode was 1.5 mM. The detection limit of the electrode for hydrogen peroxide was 2.64 μM using an electrochemical workstation.

[0148] Example 11

[0149] Repeat Example 1, except that: Fe 3+ and Fe(CN)6 3- The concentration of the electrode was 4 mM. The detection limit of the electrode for hydrogen peroxide was 2.62 μM using an electrochemical workstation.

[0150] Comparative Example 9

[0151] Repeat Example 1, except that: Fe 3+ and Fe(CN)6 3- The concentration of each was 1 mM. Using an electrochemical workstation, the electrode was tested and its detection limit for hydrogen peroxide was found to be 4.15 μM.

[0152] Comparative Example 10

[0153] Repeat Example 1, except that: Fe 3+ and Fe(CN)6 3- The concentration of the electrode was 4.5 mM. The detection limit of the electrode for hydrogen peroxide was 4.17 μM using an electrochemical workstation.

[0154] The second electrodeposited Fe 3+ and Fe(CN)6 3- The concentration range of α-aminobutyric acid is controlled in 1.5-4 mM, which helps to improve the detection sensitivity of the electrode.

[0155] Example 12

[0156] Example 1 was repeated except that the scanning voltage of the second electrodeposition was -0.5-0.5 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.68 μM.

[0157] Example 13

[0158] Example 1 was repeated except that the scanning voltage of the second electrodeposition was -0.1-1 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of hydrogen peroxide was found to be 2.66 μM.

[0159] Comparative Example 11

[0160] Example 1 was repeated except that the scanning voltage of the second electrodeposition was -0.7-0.5 V. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.13 μM.

[0161] Comparative Example 12

[0162] Example 1 was repeated except that the scanning voltage for the second electrodeposition was -0.5-0.7 V. All other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was 4.15 μM.

[0163] The scanning voltage range of the second electrodeposition can be controlled within -0.5-1V, which helps to improve the detection sensitivity of the electrode.

[0164] Example 14

[0165] Example 1 was repeated, except that the scan rate of the second electrodeposition was 40 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit was found to be 2.69 μM.

[0166] Example 15

[0167] Example 1 was repeated except that the scan rate of the second electrodeposition was 60 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.67 μM.

[0168] Comparative Example 13

[0169] Example 1 was repeated except that the scan rate of the second electrodeposition was 35 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.22 μM.

[0170] Comparative Example 14

[0171] Example 1 was repeated except that the scan rate of the second electrodeposition was 65 mV / s. Other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 4.20 μM.

[0172] It can be seen that the scanning rate range of the first electrodeposition is controlled in 40-60 mV / s, which helps to improve the detection sensitivity of the electrode.

[0173] Example 16

[0174] Example 1 was repeated, except that the number of cycles of the second electrodeposition was 15, and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of the electrode for hydrogen peroxide was found to be 2.58 μM.

[0175] Example 17

[0176] Example 1 was repeated, except that the number of cycles of the first electrodeposition was 25 and the other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit was found to be 2.56 μM.

[0177] Comparative Example 15

[0178] Example 1 was repeated, except that the number of cycles of the second electrodeposition was 10, and other parameters were the same. The electrode was tested using an electrochemical workstation, and its detection limit was found to be 4.19 μM.

[0179] Comparative Example 16

[0180] Example 1 was repeated except that the number of cycles of the second electrodeposition was 30, and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit of hydrogen peroxide was 4.14 μM.

[0181] It can be seen that controlling the number of cycles of the first electrodeposition in the range of 15-25 cycles is helpful to improve the detection sensitivity of the electrode.

[0182] Example 18

[0183] Repeat Example 1, except that: the first electrodeposition acid solution H + The concentration was 0.5 moL / L, and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.55 μM.

[0184] Example 19

[0185] Repeat Example 1, except that: the first electrodeposition acid solution H + The concentration was 2 moL / L, and other parameters were the same. The electrode was tested using an electrochemical workstation, and the detection limit for hydrogen peroxide was found to be 2.59 μM.

[0186] Comparative Example 17

[0187] Repeat Example 1, except that: the first electrodeposition acid solution H + The concentration was 0.3 moL / L, and other parameters were the same. The electrode was tested using an electrochemical workstation, and its detection limit for hydrogen peroxide was found to be 4.60 μM.

[0188] Comparative Example 18

[0189] Repeat Example 1, except that: the first electrodeposition acid solution H + The concentration was 2.2 moL / L, and other parameters were the same. The electrode was tested using an electrochemical workstation, and its detection limit for hydrogen peroxide was found to be 4.58 μM.

[0190] The acid solution H of the first electrodeposition can be + The concentration range is controlled within 0.5-2moL / L, which helps to improve the detection sensitivity of the electrode.

[0191] Example 20

[0192] Example 1 is repeated, with the only difference being that the electrode body is a screen-printed electrode.

[0193] The nano-polyaniline / Prussian blue modified on the screen-printed electrode can be easily integrated and can conveniently realize the portable detection of hydrogen peroxide. To this end, the performance of the nano-polyaniline / Prussian blue electrode based on the screen-printed electrode in Example 2 was further verified, and the obtained it curve Figure 5 , and fitting it to get Figure 6 As can be seen from the figure, the performance of the nano-polyaniline / Prussian blue electrode with the screen-printed electrode as the main body in Example 2 is consistent with that of the nano-polyaniline / Prussian blue electrode in Example 1, and the linear fit is very good, which can be used to detect hydrogen peroxide.

[0194] Example 21

[0195] See also Figure 8 , a device for detecting a biomarker (hydrogen peroxide) in human exhaled breath, comprising:

[0196] Gas collection module 1, used to collect exhaled air and guide the exhaled air to the condensation module;

[0197] The condensation module has a condensation channel 4, the two ends of the condensation channel are respectively an air inlet end 13 and an exhaust end 14, and the air inlet end is connected to the gas collection module;

[0198] A detection module comprising a single-chip microcomputer 8 and a three-electrode detection circuit 6 electrically connected to the single-chip microcomputer 8, wherein the working electrode of the three-electrode detection circuit 6 is the nano-polyaniline / Prussian blue electrode as described in Example 2, and the working electrode, counter electrode (carbon electrode, the same below) and reference electrode (silver / silver chloride, the same below) of the three-electrode detection circuit 6 are all extended into the exhaust end; and

[0199] The display module 9 (LED display screen) is electrically connected to the single chip computer 8 and is used to display the detection results.

[0200] The gas collection module includes a gas collection cover 101 and a tube 102 , and the gas collection cover 101 , the tube 102 and the gas inlet end are connected in sequence.

[0201] The condensation module includes a metal sheet 2, within which the condensation channel 4 is disposed. A semiconductor cooling sheet 3 is provided on at least one surface of the metal sheet 2, and a heat dissipation mechanism 7 is provided on the side of the semiconductor cooling sheet 3 facing away from the metal sheet 2. During condensation, exhaled air is input into the condensation channel on the aluminum sheet through a tube 1. The semiconductor cooling sheet 3 cools the aluminum sheet, causing the exhaled air to condense into exhaled air condensate. To optimize the performance of the cooling sheet, a heat dissipation mechanism 7 is provided on its backside to dissipate heat from the semiconductor cooling sheet.

[0202] The detection module also includes a three-electrode detection circuit electrically connected to the single-chip microcomputer. The working electrode of the three-electrode detection circuit is the electrode itself (i.e., the electrode not modified with nano-polyaniline / Prussian blue). The working electrode, counter electrode, and reference electrode of the three-electrode detection circuit all extend into the exhaust port. This eliminates the influence of background current and improves the accuracy of detection results.

[0203] Metal sheet 2 is an aluminum sheet (length 55mm, width 25mm, thickness 4mm)

[0204] See also Figure 10 and Figure 11 The condensation channel comprises four wavy, curved subchannels 401 connected in parallel between the inlet and exhaust ports. Several semi-cylindrical secondary flow structures 402 are provided on these subchannels. As shown in the figure, the subchannel has a square cross-section with a side length of 1.5 mm. The radius of the inner circle 11 of the subchannel is 0.83 mm, and the radius of the outer circle 12 is 2.33 mm. The secondary flow structures are arranged on the same side of the subchannel, with two opposing semi-circular secondary flow structures located on the inner side of the subchannel. The diameter 9 is 1.3 mm.

[0205] The method for detecting biomarkers in exhaled breath, using the device as described above, comprises the following steps:

[0206] The gas collection module 1 collects the exhaled gas and guides the exhaled gas to the condensation module;

[0207] The exhaled air condenses in the condensation channel of the condensation module to form condensate;

[0208] The condensate contacts the working electrode, the counter electrode and the reference electrode of the three-electrode detection circuit 6, and the three-electrode detection circuit 6 detects the current signal and transmits the current signal to the single chip microcomputer;

[0209] At the same time, the condensate comes into contact with the working electrode, counter electrode, and reference electrode of the three-electrode detection circuit. The three-electrode detection circuit detects a current signal and transmits it to the microcontroller. The microcontroller converts the current signal into a voltage signal and calculates the difference between the voltage signal and the voltage signal to obtain a differential voltage result. This differential voltage result is then converted into a biomarker concentration value, which is then displayed on the display module. In this way, the weak current signal generated in the detection area is amplified and converted from digital to analog by the three-electrode detection circuit before being transmitted to the microcontroller. After data processing, a concentration value is output and displayed on the LED display.

[0210] See also Figure 12-15To prove that adding a secondary flow structure can improve the condensation efficiency of exhaled air, the inventors designed five different condensation channel structures (i.e., the condensation channel of the present embodiment, a linear condensation channel, an elliptical condensation channel, a curved condensation channel without a secondary flow structure, and a curved condensation channel with a secondary flow structure on the opposite side). Simulation analysis was performed using Ansys, using the built-in exhaled air condensation model, at high speeds of 0.5 m / s and low speeds of 0.1 m / s. The results are shown below. Figure 12 As shown, Figure 12 What is shown is the velocity cloud diagram of different structures at high and low speeds. It can be seen from the figure that after the secondary flow structure is added, the velocity direction of the exhaled air changes after flowing through the secondary flow structure, which has an impact on the exhaled air in the main channel of the main channel curve, reducing the speed of the exhaled air flow, and indirectly increasing the contact time between the exhaled air and the metal sheet, thereby improving the condensation efficiency of the exhaled air. However, under high speed conditions, the exhaled air in the secondary flow structure will form vortices, which will cause the exhaled air condensate to exist in large quantities in the secondary flow structure, while it is perfectly applicable under low speed conditions. In order to further improve the condensation efficiency of the exhaled air, 4 identical sub-channels with secondary flow structures are constructed on the aluminum sheet. The structure is as follows Figure 11 As shown, they are all connected in parallel between the air inlet 13 and the air outlet 14 to achieve the diversion and merging of the exhaled air. The provision of multiple sub-channels for gas diversion not only increases the contact area between the exhaled air and the aluminum sheet, but also reduces the gas flow rate, thereby increasing the condensation efficiency of the exhaled air.

[0211] In order to verify that the condensation channel of this embodiment has the best condensation effect compared with the other types of condensation channels mentioned above, an air pump and a gas washing bottle were used to simulate the exhalation process of the lungs. The exhaled air was simulated by the air pump, and the solution was placed in a constant temperature water bath with an aqueous solution containing a certain concentration of hydrogen peroxide to simulate the exhaled air. The results are as follows: Figure 13 and Figure 14 As shown, the condensation channel of this embodiment has the highest efficiency in generating condensate, which can fill the entire channel in about 10 minutes. Compared with the existing technology, the condensation efficiency is greatly improved. At the same time, this structure has a certain enrichment effect on hydrogen peroxide. The exhaled breath simulation experiment was carried out using the same concentration of hydrogen peroxide. The results are shown in Figure 2. Figure 15 As shown, the condensate collected by the condensation channel with the secondary flow structure has the highest hydrogen peroxide concentration, which reduces the sensitivity requirement of subsequent sensor detection.

[0212] Using the device and method described in Example 21, experiments were conducted using hydrogen peroxide solutions of varying concentrations to simulate condensate. The linearity of the curve obtained was 0.99, which is very good, demonstrating the effectiveness of the detection circuit and method. It can be used to detect hydrogen peroxide or other biomarkers in exhaled breath. The relationship between the blank and nano-polyaniline / Prussian blue screen-printed electrodes and the voltage at each concentration was obtained as shown in the following figure. Figure 16 As shown, the relationship curve between the actual voltage value and the hydrogen peroxide concentration is obtained by differential calculation. Figure 17 , its R 2 =0.98782, with good linearity, indicating that the device and method of the present invention can be used for real-time and accurate detection of hydrogen peroxide in exhaled breath.

[0213] In summary, the present invention, by constructing a curved condensation channel with a secondary flow structure, can not only accelerate the condensation efficiency of exhaled breath, but also have a certain enrichment effect on the biomarkers therein. At the same time, adding cheap conductive materials to construct a three-dimensional nanoelectrode greatly improves the detection performance of the electrochemical sensor. Finally, the condensation module, detection module and display module can be integrated into an N95 mask, which can be used for portable real-time detection of biomarkers in exhaled breath, or it can be fixed on a detection platform (see Figure 9 ), realizing fast real-time detection of multiple people and meeting the detection application needs of different scenarios.

[0214] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention. The contents of the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, modifications to various equivalent forms of the present invention by those skilled in the art all fall within the scope defined by the claims appended to this application.

Claims

1. A method for preparing a nano-polyaniline / Prussian blue electrode for detecting biomarkers in exhaled breath, characterized in that: The steps include: S1. Providing an AAO template and an electrode body, and fixing the AAO template on the electrode body to obtain an assembly; S2, immersing the assembly in an acid solution of aniline, and performing a first electrodeposition on the assembly using cyclic voltammetry to obtain an intermediate assembly; The concentration of aniline in the acid solution is 80-90 mg / mL; the scanning voltage of the first electrodeposition is -0.8-2 V, the scanning rate is 40-60 mV / s, and the number of cycles is 10-20; S3, placing the intermediate assembly in an alkaline solution, and obtaining a nano-polyaniline electrode after the AAO template is completely dissolved; S4, immersing the nano-polyaniline electrode in a solution containing Fe 3+ and Fe(CN)6 3- a solution of , and performing a second electrodeposition thereof using cyclic voltammetry, and then taking out, washing, and drying to obtain a nano-polyaniline / Prussian blue electrode; Wherein, in the solution, Fe 3+ The concentration of Fe(CN)6 is 1.5-4mM, 3- The concentration is 1.5-4 mM; the scanning voltage of the second electrodeposition is -0.5-1 V, the scanning rate is 40-60 mV / s, and the number of cycles is 15-25 circles.

2. The preparation method according to claim 1, characterized in that In S1, the electrode body includes one of a glassy carbon electrode and a screen-printed electrode.

3. The preparation method according to claim 1, characterized in that In S1, the pore size of the AAO template is 20–40 nm.

4. The preparation method according to claim 1, characterized in that In S2, the acid in the acid solution is one or more of hydrochloric acid and sulfuric acid; H + The concentration is 0.5-2mol / L.

5. The preparation method according to claim 1, characterized in that In S3, the alkali solution is an aqueous solution of one or more of sodium hydroxide, potassium hydroxide, and barium hydroxide.

6. A nano-polyaniline / Prussian blue electrode for detecting biomarkers in exhaled breath, characterized in that: Prepared by the preparation method according to any one of claims 1 to 5.

7. A device for detecting biomarkers in exhaled breath, characterized in that include: A gas collection module (1) for collecting exhaled air and directing the exhaled air to a condensation module; The condensation module has a condensation channel (4), the two ends of the condensation channel (4) are an air inlet end (13) and an air outlet end (14), and the air inlet end is connected to the air collection module; A detection module, comprising a single-chip microcomputer (8) and a three-electrode detection circuit (6) electrically connected to the single-chip microcomputer (8), wherein the working electrode of the three-electrode detection circuit (6) is the nano-polyaniline / Prussian blue electrode as claimed in claim 6, and the working electrode, the counter electrode and the reference electrode of the three-electrode detection circuit (6) are all extended into the exhaust end; and The display module (9) is electrically connected to the single chip computer (8) and is used to display the detection results.

8. The device according to claim 7, characterized in that The condensation module comprises a metal sheet (2), the condensation channel (4) is arranged in the metal sheet (2), at least one surface of the metal sheet (2) is provided with a semiconductor refrigeration sheet (3), and a heat dissipation mechanism (7) is provided on a side of the semiconductor refrigeration sheet (3) away from the metal sheet (2).

9. The device according to claim 7, characterized in that The condensation channel (4) comprises at least two parallel curved sub-channels (401), and a plurality of secondary flow structures (402) are provided on the sub-channels (401).

10. The device according to claim 9, characterized in that The secondary flow structure is semi-cylindrical.

11. A method for detecting biomarkers in exhaled breath, characterized in that The method is carried out using the device according to any one of claims 7 to 10, comprising the steps of: The gas collection module (1) collects the exhaled gas and guides the exhaled gas to the condensation module; The exhaled air condenses in the condensation channel of the condensation module to form condensate; The condensate contacts the working electrode, the counter electrode and the reference electrode of the three-electrode detection circuit (6). The three-electrode detection circuit (6) detects the current signal and transmits the current signal to the single-chip microcomputer. The single-chip microcomputer converts the current signal into a voltage signal, and converts the voltage signal into a concentration value of the biomarker, and controls the display module to display the concentration value of the biomarker.

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