Electrode test paper for detecting whole blood ketone body of dairy cow as well as preparation method and application of electrode test paper

Abstract

The invention discloses electrode test paper for cow whole blood ketone body detection and a preparation method and application thereof. The electrode test paper comprises a base material layer; the electrode layer is arranged on the base material layer and comprises a working electrode and a counter electrode; a sample collection port is formed in the label layer, the label layer is arranged on the electrode layer, the substrate layer, the working electrode and the counter electrode are partially exposed in the sample collection port, and a reaction reagent is loaded in an exposed area, so that a sample detection area is formed; the reaction reagent comprises enzyme, an electron mediator, a surfactant, a buffer solution and a high polymer. The preparation method comprises the following steps: S1, providing the substrate layer; s2, forming an electrode layer on the substrate layer by using a mask; s3, the label layer with the sample collection port is pasted on the base material layer, and the base material layer, the working electrode and the counter electrode are partially exposed in the sample collection port after pasting; and S4, loading a liquid reaction reagent to the exposure area, and drying to form a sample detection area. The application is that the electrode test paper is applied to cow whole blood ketone body detection.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to an electrode test paper for detecting whole blood ketone bodies in dairy cows, and a preparation method and application thereof. Background Art

[0002] Ketosis in dairy cows is an energy metabolism disorder characterized by elevated ketone bodies (β-hydroxybutyrate, acetoacetate, acetone) in dairy cows due to disordered carbohydrate and fat metabolism in dairy cows, and hypoglycemia. Ketosis is a common disease in dairy cows, with a high incidence in the peripartum period, especially in high-yielding dairy cows in the early lactation period with a daily milk production of more than 30 kg. Postpartum diseases of dairy cows in large-scale dairy farms have caused serious economic losses to the dairy industry, so it is of great significance to implement preventive monitoring measures for dairy cow diseases in the postpartum stage.

[0003] The method used for early diagnosis of ketosis in dairy cows is ketone body determination. At present, the main methods for ketone body detection include qualitative detection method of test strips, salicylaldehyde colorimetry, high performance liquid chromatography, and quantitative detection methods such as β-hydroxybutyric acid kits. However, all of the above methods have certain shortcomings, and their shortcomings are mainly manifested in poor accuracy, high false positives, high cost, cumbersome operation, and the need for expensive detection instruments. For example, Chinese patent CN218157942U discloses a screening diagnostic kit for dairy cow ketosis, which adopts standard colorimetry; Chinese patent CN103792230A discloses a dairy cow ketosis diagnostic kit and its detection method, and Chinese patent CN217484188U discloses a ketone body concentration detection test strip in dairy cow peripartum milk with multiple reaction zones, which has cumbersome operation, insufficient reading accuracy, semi-quantitative and other problems.

[0004] Existing technologies are difficult to meet the needs of portable precision detection products, and at the same time require feasible processes and mass production. Therefore, it is necessary to establish a simple and fast method to achieve portable precision detection and meet the needs of wide application in the fields of dairy cow ketone body detection. Summary of the invention

[0005] The purpose of the present invention is to design an electrode test paper for whole blood ketone body detection in dairy cows to solve the problems of poor accuracy, high cost, complicated operation and the like in existing ketone body detection methods, thereby solving the above problems.

[0006] To achieve the above object, the present invention is implemented by the following technical solutions:

[0007] The present invention designs an electrode test paper for detecting whole blood ketone bodies in dairy cows, and the electrode test paper comprises:

[0008] substrate layer;

[0009] An electrode layer is disposed on the substrate layer, and the electrode layer includes: a working electrode and a counter electrode;

[0010] and a label layer, on which a sample collection port is arranged, the label layer is laminated on the substrate layer, and the electrode layer is located between the substrate layer and the label layer, after lamination, the substrate layer, the working electrode and the counter electrode are partially exposed to the sample collection port, and reaction reagents are loaded in the exposed areas of the substrate layer, the working electrode and the counter electrode, thereby forming a sample detection area;

[0011] The reaction reagents include enzymes, electron mediators, surfactants, buffer solutions and polymers; the electrode layers are made of gold, platinum or palladium materials.

[0012] Specifically, the label layer is a thin sheet with a sample collection port formed by a single-sided adhesive with a certain thickness. The label layer is attached to the substrate layer to form a sample detection area that can accommodate the sample.

[0013] Furthermore, an electrode test paper for detecting whole blood ketone bodies in dairy cows: the substrate layer is made of glass or a polymer flexible material (such as polyethylene terephthalate, i.e., PET).

[0014] Furthermore, an electrode test paper for detecting ketone bodies in whole blood of dairy cows: the electrode layer is formed into corresponding working electrode and counter electrode patterns by printing, sputtering or etching.

[0015] Specifically, the electrode layer may be made of metal materials such as gold, platinum, palladium, or alloys, and a corresponding electrode pattern may be realized through processes such as printing, sputtering, and etching.

[0016] Furthermore, an electrode test paper for detecting whole blood ketone bodies in dairy cows: the loading amount of the reaction reagent is 0.01 to 10.0 mg.

[0017] Preferably, the loading amount of the reaction reagent is 1.0 mg.

[0018] Furthermore, an electrode test paper for detecting ketone bodies in whole blood of dairy cows: the reaction reagent comprises the following components in mass fractions:

[0019] 2.0-4.0wt% of beta-hydroxybutyrate dehydrogenase, 0.5-1.0wt% of hexaammineruthenium chloride, 0.01-0.02wt% of DCPIP, 10.0-20.0wt% of phosphate buffer solution, 1.0-3.0wt% of methylcellulose, and the balance of water.

[0020] Preferably, the reaction reagents include the following components in mass fractions: 3.0 wt % of β-hydroxybutyrate dehydrogenase, 0.9 wt % of hexaammineruthenium chloride, 0.015 wt % of DCPIP, 15.0 wt % of phosphate buffer solution, 1.5 wt % of methylcellulose, and the balance of water.

[0021] The present invention also provides a method for preparing an electrode test paper for detecting whole blood ketone bodies in dairy cows, the method comprising the following steps:

[0022] S1, providing a substrate and cleaning it to serve as a substrate layer;

[0023] S2, forming an electrode layer on the substrate layer using a mask;

[0024] S3, laminating the label layer with the sample collection port on the substrate layer, so that the electrode layer is located between the substrate layer and the label layer, and the substrate layer, the working electrode and the counter electrode are partially exposed in the sample collection port;

[0025] S4. Load the liquid reaction reagent to the above-mentioned exposure area by liquid dot method, and then dry it to form a sample detection area, thereby completing the preparation of the electrode test paper.

[0026] Furthermore, a method for preparing an electrode test paper for detecting whole blood ketone bodies in dairy cows is provided: the drying temperature is 40 to 80° C., and the drying time is 10 to 60 minutes.

[0027] A use of an electrode test paper for detecting whole-blood ketone bodies in dairy cows, and application of the electrode test paper in detecting whole-blood ketone bodies in dairy cows.

[0028] Further, a use of an electrode test paper for detecting whole blood ketone bodies in dairy cows: the whole blood ketone body detection process of dairy cows includes the following:

[0029] (a) preparing a plurality of cow whole blood samples in a simulated manner, and determining the concentration of whole blood ketones in each sample;

[0030] (b) dropping the whole blood samples into the sample detection area of ​​the electrode test paper, leaving the sample to stand, and then applying a voltage between the working electrode and the counter electrode of the electrode test paper, and collecting current data of the working electrode;

[0031] (c) performing linear fitting with the whole blood ketone concentration as the ordinate and the collected current data as the abscissa, thereby establishing a current-concentration calibration curve;

[0032] (d) Obtaining the corresponding current signal of the cow whole blood sample with unknown whole blood ketone concentration through the electrode test paper, and then obtaining the blood ketone concentration value of the cow whole blood sample by comparing with the current-concentration calibration curve established above.

[0033] Furthermore, a use of an electrode test paper for detecting whole blood ketone bodies in dairy cows: the step (b) is carried out at room temperature for a standing time of not less than 30 minutes, and the applied voltage is 50 to 500 mV.

[0034] Preferably, the applied voltage is 300 mV.

[0035] Beneficial effects of the present invention:

[0036] (1) The electrode test paper for detecting whole blood ketone bodies in dairy cows provided by the present invention can conveniently detect whole blood ketones (β-hydroxybutyric acid) in dairy cows. It only needs to detect the current signal of the whole blood sample of the dairy cow through the electrode test paper, and then compare it with the calibration curve established by fitting to obtain the concentration value of whole blood ketones (β-hydroxybutyric acid) in the whole blood sample of the dairy cow, thereby assisting in the early diagnosis of dairy cow ketosis. At the same time, the electrode test paper of the present invention also has the advantages of simple manufacturing process and high detection accuracy.

[0037] (2) The electrode test paper for detecting whole blood ketone bodies in dairy cows provided by the present invention can further improve the accuracy of the test results and realize accurate detection within the shelf life; the manufacturing process of the electrode test paper of the present invention is simple, the raw materials are easily available, it is conducive to industrial production, and it is highly practical; in addition, the exhaust is unobstructed during the detection, the sample is quickly injected, and the test results can be quickly given, the detection efficiency is high, and cumbersome operation steps are avoided.

[0038] (3) The electrode test paper designed by the present invention for the detection of ketone bodies in the whole blood of dairy cows has a higher detection accuracy and better repeatability by optimizing the electrode layer material (selecting gold, platinum or palladium materials) and the reaction reagent. Under the specific collocation of the electrode layer material and the reaction reagent, the electrode test paper of the present invention has a higher detection accuracy and better repeatability, thereby significantly improving the detection performance, sensitivity and efficiency of the electrode test paper. Compared with conventional carbon electrodes, the fixed collocation of the electrode material and the reaction reagent selected by the present invention has the advantages of high conductivity, high stability, and high catalytic activity. The gold electrode test paper obtained by the preferred material of the present invention has the advantages of reasonable design structure, optimized design compatibility, high detection accuracy, and high detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0040] Figure 1 Schematic diagram of an explosion of an electrode test paper for detecting ketone bodies in whole blood of dairy cows designed for Example 1 of the present invention;

[0041] Figure 2 A schematic diagram of the structure of an electrode test paper for detecting ketone bodies in whole blood of dairy cows designed for Example 1 of the present invention;

[0042] Figure 3 The current-concentration calibration curve diagram established for Example 3 of the present invention;

[0043] Figures 4-5 It is the current curve diagram.

[0044] Markings in the figure: 1-substrate layer, 2-working electrode, 3-counter electrode, 4-label layer, 5-sample detection area, 41-sample collection port. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. 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.

[0046] In the description of the present invention, it should be understood that the terms "upper", "lower", "left", "right", "top", "bottom", etc., indicating the position or positional relationship, are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Moreover, the terms "first", "second", etc. are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein.

[0047] Example 1

[0048] like Figures 1-2 As shown, an electrode test paper for detecting ketone bodies in whole blood of dairy cows, the electrode test paper comprises:

[0049] The substrate layer 1 is made of polyethylene terephthalate (PET);

[0050] The electrode layer includes a working electrode 2 and a counter electrode 3. The electrode layer is formed on the substrate layer 1 by vacuum sputtering using a mask. The thickness of the electrode layer is set to 30 nm and the material is gold.

[0051] and a label layer 4, the label layer 4 is a sheet having a sample collection port 41 formed by a single-sided adhesive having a certain thickness, the label layer 4 is pasted on the substrate layer 1 to form a sample detection area 5 that can accommodate a sample, the electrode layer is located between the label layer 4 and the substrate layer 1, and after pasting, the substrate layer 1, the working electrode 2 and the counter electrode 3 are partially exposed in the sample collection port 41 (such as Figure 2 As shown), and 1.0 mg of reaction reagent is loaded in the exposed area to form a sample detection area 5;

[0052] The reaction reagent comprises the following components in mass fractions: 3.0wt% of beta-hydroxybutyrate dehydrogenase, 0.9wt% of hexaammineruthenium chloride, 0.015wt% of DCPIP, 15.0wt% of phosphate buffer solution (0.5mM, pH 7.1), 1.5wt% of methylcellulose, and the balance of water.

[0053] Example 2

[0054] The method for preparing the electrode test paper for detecting ketone bodies in whole blood of dairy cows designed in the above-mentioned embodiment 1 comprises the following specific steps:

[0055] S1, providing a polyethylene terephthalate sheet and cleaning it to serve as a substrate layer 1;

[0056] S2, using a mask plate, vacuum sputtering a 30 nm thick working electrode 2 and a counter electrode 3 on the substrate layer 1 as an electrode layer, wherein the material of the electrode layer (including the working electrode 2 and the counter electrode 3) is gold;

[0057] S3, laminating a label layer 4 having a thickness of about 0.125 mm and a sample collection port 41 on the substrate layer 1, wherein the electrode layer is located between the substrate layer 1 and the label layer 4 after the label layer 4 is laminating, and the substrate layer 1, the working electrode 2 and the counter electrode 3 are partially exposed to the sample collection port 41;

[0058] S4, then use a dispensing machine to load the liquid reaction reagent into the above-mentioned exposure area in a dispensing manner, and then place it in an oven at 50° C. for drying for 30 minutes to form a sample detection area 5, thereby completing the preparation of the gold electrode test paper.

[0059] Example 3

[0060] The electrode test paper designed in the above embodiment 1 is used to detect ketone bodies in the whole blood of dairy cows, and the detection process is as follows:

[0061] (a) First, 8 cow whole blood samples (respectively recorded as samples 1 to 8) were simulated and prepared in the concentration range of 0 to 5 mmol / l, and then the whole blood ketone (β-hydroxybutyrate) concentration value in each sample was measured by Abbott's Assistant Sunbetter to New Blood Ketone Meter. The specific test results are shown in Table 1;

[0062] (b) Then, the whole blood samples (samples 1 to 8) were added dropwise to the sample detection area 5 of the electrode test paper, and then allowed to stand at room temperature for 30 minutes. Then, an excitation voltage of 300 mV was applied between the working electrode 2 and the counter electrode 3 of the electrode test paper, and the current data of the working electrode at 10 seconds was collected; each whole blood sample was tested with three identical electrode test papers (referred to as electrode test papers 1 to 3), and then the average value of the current data measured by the three electrode test papers was taken. For specific test results, see Table 1;

[0063] (c) A linear fit was performed using the whole blood ketone (β-hydroxybutyrate) concentration value measured in each whole blood sample as the ordinate and the average current data of each sample collected as the abscissa to establish a current-concentration calibration curve, R 2 >0.99, such as Figure 3 As shown;

[0064] (d) The corresponding current data of the whole blood sample of the cow with unknown whole blood ketone (β-hydroxybutyrate) concentration is obtained by electrode test paper, and then the blood ketone concentration value of the whole blood sample of the cow is obtained by comparing with the current-concentration calibration curve established above, which can meet the detection requirements of whole blood ketone bodies of the cow.

[0065] Table 1 shows the whole blood ketone concentration test results and current data acquisition results of samples 1 to 8 in Example 3

[0066]

[0067]

[0068] In Table 1, the unit of whole blood ketone concentration is mmol / l, and the unit of current is μA.

[0069] The present invention can simplify the detection process of whole-blood ketone bodies in dairy cows by designing an electrode test paper for detecting whole-blood ketone bodies in dairy cows. It is only necessary to use the electrode test paper to detect the current signal of the whole-blood sample of dairy cows, and then accurately obtain the blood ketone concentration value of the whole-blood sample of dairy cows through linear curve comparison, thereby simply, quickly and accurately meeting the detection needs of whole-blood ketone bodies in dairy cows, and assisting in the early diagnosis of dairy cow ketosis according to the detection situation.

[0070] Example 4

[0071] The difference between Example 4 and Example 1 is that the material used for the electrode layer in Example 4 is platinum, and the reaction reagent in Example 4 includes the following components in mass fractions: 2.0wt% of β-hydroxybutyrate dehydrogenase, 0.5wt% of hexaammineruthenium chloride, 0.01wt% of DCPIP, 20.0wt% of phosphate buffer solution, 2.0wt% of methyl cellulose, and the remainder of water, and the rest is the same as Example 1.

[0072] Example 5

[0073] The difference between Example 5 and Example 1 is that the material used for the electrode layer in Example 5 is palladium, and the reaction reagent in Example 5 includes the following components in mass fractions: 4.0wt% of β-hydroxybutyrate dehydrogenase, 0.7wt% of hexaammineruthenium chloride, 0.02wt% of DCPIP, 11.0wt% of phosphate buffer solution, 2.5wt% of methyl cellulose, and the remainder of water, and the rest is the same as Example 1.

[0074] Comparative Example 1

[0075] The difference between Comparative Example 1 and Example 1 is that the material of the electrode layer in the carbon electrode test paper provided in Comparative Example 1 is carbon, that is, the working electrode 2 and the counter electrode 3 in the electrode test paper of Comparative Example 1 are both carbon electrodes, and the rest is the same as Example 1.

[0076] Test: 7 cow whole blood samples (respectively recorded as samples 9 to 15) were simulated and prepared. Under the same environment, the electrode test papers of Example 1 and Comparative Example 1 were used to detect the current of cow whole blood samples 9 to 15 and blank control samples (i.e., samples with a whole blood ketone concentration of 0). Each sample was tested using 3 identical electrode test papers (respectively recorded as electrode test papers 1 to 3). An excitation voltage of 300 mV was used, and the current value at 10 seconds was read, thereby obtaining the test results of the electrode test paper of Example 1 (as shown in Tables 2 and 3). Figure 4 As shown in Table 3 and Table 4, the test results of the electrode test paper of Comparative Example 1 are shown in Table 3 and Table 4. Figure 5 shown).

[0077] Table 2 is the current data (μA) test results of the electrode test paper of Example 1 for blank control samples and cow whole blood samples 9 to 15

[0078]

[0079] Table 2 is the current data (μA) test results of the electrode test paper of comparative example 1 for blank control samples and cow whole blood samples 9 to 15

[0080]

[0081] From the current data in Table 2 and Table 3, it can be seen that the current data of the electrode test paper designed by the present invention when detecting the blank control sample is 0.3μA, while the current data of the carbon electrode test paper in comparative example 1 when detecting the blank control sample is 1.2μA. This shows that the detection limit of the electrode test paper of the present invention is lower. Compared with the carbon electrode test paper in comparative example 1, it can detect the whole blood ketone concentration of cow whole blood samples within the current data range of 0.3-1.2μA, while the carbon electrode test paper in comparative example 1 cannot detect it. Therefore, it can be said that the detection accuracy of the electrode test paper of the present invention is higher. Figure 4 and Figure 5 It can be seen from the current fitting curve that Figure 4 The linear fit is better, indicating that its accuracy is better, reflecting that the accuracy of the electrode test paper of the present invention is better than that of the carbon electrode test paper.

[0082] Therefore, the present invention optimizes the electrode layer material and the reaction reagent compatibility, and under the specific combination of the electrode layer material and the reaction reagent, the detection accuracy of the electrode test paper of the present invention is higher, and the detection performance, sensitivity and efficiency of the electrode test paper are significantly improved.

[0083] The above are preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. An electrode test paper for detecting ketone bodies in whole blood of dairy cows, characterized in that: The electrode test strips include: A substrate layer (1); An electrode layer, which is arranged on the substrate layer (1), and the electrode layer comprises: a working electrode (2) and a counter electrode (3); and a label layer (4) on which a sample collection port (41) is arranged, the label layer (4) is laminated on the substrate layer (1) and the electrode layer is located between the two, after lamination, the substrate layer (1), the working electrode (2) and the counter electrode (3) are partially exposed to the sample collection port (41), and a reaction reagent is loaded in the exposed area, thereby forming a sample detection area (5); The reaction reagents include enzymes, electron mediators, surfactants, buffer solutions and polymers; the electrode layers are made of gold, platinum or palladium materials.

2. The electrode test paper for detecting whole blood ketone bodies in dairy cows according to claim 1, characterized in that: The substrate layer (1) is made of glass or a flexible polymer material.

3. The electrode test paper for detecting ketone bodies in whole blood of dairy cows according to claim 1, characterized in that: The electrode layer is formed into corresponding working electrode (2) and counter electrode (3) patterns by printing, sputtering or etching.

4. The electrode test paper for detecting ketone bodies in whole blood of dairy cows according to claim 1, characterized in that: The loading amount of the reaction reagent is 0.01 to 10.0 mg.

5. The electrode test paper for detecting ketone bodies in whole blood of dairy cows according to claim 1, characterized in that: The reaction reagent includes the following components by mass fraction: 2.0-4.0wt% of beta-hydroxybutyrate dehydrogenase, 0.5-1.0wt% of hexaammineruthenium chloride, 0.01-0.02wt% of DCPIP, 10.0-20.0wt% of phosphate buffer solution, 1.0-3.0wt% of methylcellulose, and the balance of water.

6. A method for preparing an electrode test paper for detecting whole blood ketone bodies in dairy cows according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: S1, providing a substrate and cleaning it to serve as a substrate layer (1); S2, forming an electrode layer on the substrate layer (1) using a mask; S3, laminating the label layer (4) having the sample collection port (41) onto the substrate layer (1), so that the electrode layer is located between the substrate layer (1) and the label layer (4), and the substrate layer (1), the working electrode (2) and the counter electrode (3) are partially exposed to the sample collection port (41); S4, loading the liquid reaction reagent onto the above-mentioned exposed area by liquid dot method, and then drying it to form a sample detection area (5), thereby completing the preparation of the electrode test paper.

7. The method for preparing an electrode test paper for detecting whole blood ketone bodies in dairy cows according to claim 6, characterized in that: The drying temperature is 40-80°C and the drying time is 10-60 minutes.

8. A use of an electrode test paper for detecting whole blood ketone bodies in dairy cows, characterized in that: Use of the electrode test paper according to any one of claims 1 to 5 in the detection of ketone bodies in the whole blood of dairy cows.

9. The use of an electrode test paper for detecting whole blood ketone bodies in dairy cows according to claim 8, characterized in that: The whole blood ketone body detection process of dairy cows includes the following: (a) preparing a plurality of cow whole blood samples in a simulated manner, and determining the concentration of whole blood ketones in each sample; (b) dropping the whole blood samples into the sample detection area (5) of the electrode test paper respectively, leaving the sample to stand, and then applying a voltage between the working electrode (2) and the counter electrode (3) of the electrode test paper, and collecting the current data of the working electrode (2); (c) performing linear fitting with the whole blood ketone concentration as the ordinate and the collected current data as the abscissa to establish a current-concentration calibration curve; (d) Obtaining the corresponding current signal of the cow whole blood sample with unknown whole blood ketone concentration through the electrode test paper, and then obtaining the blood ketone concentration value of the cow whole blood sample by comparing with the current-concentration calibration curve established above.

10. The use of an electrode test paper for detecting whole blood ketone bodies in dairy cows according to claim 9, characterized in that: The standing in step (b) is carried out at room temperature for no less than 30 minutes, and the applied voltage is 50 to 500 mV.

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