Biosensor for detecting semen acid phosphatase activity and detection method

Abstract

The present invention provides a method for measuring semen acid phosphatase with high accuracy and high reliability and a semen acid phosphatase biosensor used in the method. An acid phosphatase detection part is formed in the semen acid phosphatase biosensor, and the acid phosphatase detection part has an electrode system and a reagent layer, thereby constituting a semen acid phosphatase biosensor. A method for detecting semen acid phosphatase by a semen acid phosphatase biosensor comprises the following steps: 1) taking an appropriate amount of semen into a reagent tube, mixing, and liquefying the semen; 2) inserting a semen acid phosphatase activity biosensor into a semen acid phosphatase activity detection instrument; 3) the siphon port of the semen acid phosphatase activity biosensor contacts the liquefied semen in the test tube, and the siphon port automatically sucks in semen to cover the reagent layer for reaction; 4) the instrument calculates and displays the result based on the obtained current.

Description

Technical Field

[0001] The present invention relates to a biosensor and a detection method for biochemical detection, in particular to a biosensor and a detection method for detecting the activity of semen acid phosphatase. Background Art

[0002] Acid phosphatase exists in all tissues and cells of the body, with the highest content in the prostate. In normal male serum, 2 / 3 of acid phosphatase comes from the prostate, and almost all acid phosphatase in semen comes from the prostate. Therefore, measuring acid phosphatase in semen helps to understand prostate function and diagnose prostate diseases.

[0003] Prostatitis is a clinical manifestation of pelvic pain or discomfort, urgency, frequency, pain during urination, and white urine at the end of the urine caused by infection of the prostate and / or stimulation of certain non-infectious factors. Prostatitis is a common disease in adult men, and the prevalence rate is higher in adult men under 50 years old. Patients with prostate enlargement generally show increased frequency of urination in the early stage, which is more obvious at night. Over time, it can lead to congestion and edema of the bladder neck. Urinating becomes even weaker. At this time, the prostate is filled with water and edema, making the urine stream thin and forked, and sometimes not in a line, dripping out. In the later stage, if the amount of residual urine is equal to the normal bladder capacity, urine will overflow automatically. The main factors causing the symptoms of prostate enlargement are the inability to urinate smoothly due to tension in the muscles of the bladder neck, or the inability to urinate due to the compression of the urethral outlet by the enlarged prostate. The activity of acid phosphatase is also different in prostate cancer and prostate hypertrophy. In addition, acid phosphatase can promote sperm activity. When the acid phosphatase in semen decreases, sperm activity will weaken, which may reduce the fertilization rate.

[0004] At present, some andrology departments in my country have carried out the detection of semen acid phosphatase activity, usually using photochemical colorimetry. The photochemical colorimetry is based on the fact that acid phosphatase decomposes disodium phenyl phosphate under acidic conditions to produce free phenol and phosphoric acid. Phenol reacts with 4-aminoantipyrine in an alkaline solution and is oxidized to a red quinone derivative by potassium ferrocyanide. The enzyme activity is measured according to the depth of red. Biochemical analyzers are large-scale detection equipment. If semen acid phosphatase needs to be detected, it can only be done in medical institutions, which is time-consuming and costly. Therefore, there is an urgent need for improved detection methods that can provide greater detection reliability under all conditions, are quick and convenient, and have high practical value. The electrochemical biosensor method has the advantages of being fast, simple, and highly sensitive. So far, there has been no report on the use of electrochemical biosensors for the detection of acid phosphatase activity in human semen. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the above-mentioned technology and to provide a biosensor and detection method for detecting acid phosphatase activity in semen which is easy to operate, has a detection time of less than 10 seconds, does not require semen dilution, and the instrument can directly give the detection results.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a biosensor for detecting the activity of acid phosphatase in semen, characterized in that it comprises an electrically insulating substrate, an electrode layer composed of a working electrode, a reference electrode and a short-circuit electrode arranged on the electrically insulating substrate, and a reagent layer composed of a reagent and a buffer. The reagent layer contains 3-carboxybenzenesulfonic acid, a reagent for improving reactivity and stability, and the content of the 3-carboxybenzenesulfonic acid is 0.5%-3%; the reagent layer contains disodium phenyl phosphate, which can be decomposed by acid phosphatase under acidic conditions, and the content of disodium phenyl phosphate is 1%-3%; and an electron mediator that can oxidize the free phenol produced by the decomposition of disodium phenyl phosphate by acid phosphatase.

[0007] The buffer is preferably selected from the group consisting of acetic acid, acetate, citric acid, and citrate.

[0008] In the present invention, the reagent for improving reactivity and stability in the reagent layer is preferably selected from sulfonic acid and its derivatives, such as benzenesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, p-toluenesulfonic acid, p-aminobenzenesulfonic acid, 4-hydroxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid. The present invention preferably uses 3-carboxybenzenesulfonic acid. 3-Carboxybenzenesulfonic acid and its sodium salt are non-volatile, have strong acidity, have excellent solubility in water and have small moisture absorption characteristics. 3-Carboxybenzenesulfonic acid and its sodium salt can provide an acidic environment for the reagent system, accelerate the decomposition speed of disodium phenyl phosphate by acid phosphatase, thereby making the semen acid phosphatase biosensor of the present invention have good reactivity, and can stabilize potassium ferrocyanide in the reaction system, so that the semen acid phosphatase biosensor of the present invention has a good storage period, and plays a role as a stabilizer. Preferably, the content of sulfonic acid and its derivatives is 0.5%-3%.

[0009] In the present invention, the reagent layer contains phosphate esters that can be decomposed by acid phosphatase, which can be selected from disodium phenyl phosphate, disodium phenylphosphate, sodium phenolphthalein phosphate, sodium glycerophosphate, α-naphthol sodium phosphate, etc. Preferably, the phosphate ester content is 0.5%-5%, and the optimal phosphate ester content is 1%-3%.

[0010] The reagent layer preferably also contains an electron mediator. Dissolved oxygen in the test solution can also be used as an electron mediator, but it is best to put an electron mediator in the reagent layer. As electron mediators, for example, metal complexes such as potassium ferrocyanide, osmium-tris(bipyridinium), ferrocene derivatives, benzoquinone derivatives such as p-benzoquinone, phenazinium derivatives such as N-methylphenazinium sulfate monomethyl ester salt, phenothiazinium derivatives such as methylene blue, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, hexaammineruthenium chloride, etc. These electron mediators can be used alone or in combination of two or more. Preferably, the content of the electron mediator is 3%-15%.

[0011] The reagent layer preferably also contains a hydrophilic polymer. The following substances are preferably used as the hydrophilic polymer: carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polylysine and other polyamino acids, polystyrene sulfonic acid, gelatin and its derivatives, polymers of acrylic acid or its salts, polymers of methacrylic acid or its salts, starch and its derivatives, polymers of maleic anhydride or its salts, agarose gel and its derivatives. If the reagent layer contains a hydrophilic polymer, the measurement result of the seminal acid phosphatase biosensor will not be affected by external vibrations. Furthermore, the hydrophilic polymer has the advantage of being able to act as a binder that slowly fixes the enzyme on the electrode. Preferably, the hydrophilic polymer content is 0.2%-5%, and the optimal hydrophilic polymer content is 0.5%-3%.

[0012] The electron mediator, reagent and buffer for improving reactivity and stability for detecting the acid phosphatase of semen and the buffer can be loaded on the biosensor in a mixed state. In this way, the manufacturing process of the biosensor is simple, and the reagent layer can be easily made. In addition, because disodium phenyl phosphate and the electron mediator are mixed together, the free phenol produced by the acid phosphatase decomposing disodium phenyl phosphate and the electron mediator are in a very close position, and the free phenol produced by the acid phosphatase decomposing disodium phenyl phosphate can be oxidized by the electron mediator very quickly. Thus, the reactivity of the biosensor for detecting the activity of acid phosphatase of semen is improved, and even when the measuring time is shorter, enough responsiveness can be obtained.

[0013] The buffer loading in the biosensor for detecting the activity of acid phosphatase in semen is preferably below 0.05mmol / L, so that the solubility of the electron mediator will not be reduced. In addition, since the response value corresponding to 0U / ml acid phosphatase, i.e., the blank response value, will not rise under the above loading, the obtained biosensor for detecting the activity of acid phosphatase in semen can have high reliability. The reagent layer contains a buffer so that the reagent layer formed by solution development and drying can be smoothed. In the case of forming the reagent layer by drying the reagent aqueous solution, the reagent layer will have bumps and depressions due to the coarsening of the reagent crystals. This is more significant when the electron mediator potassium ferrocyanide is contained. After the reagent layer contains a buffer, the reagent layer will not have bumps and depressions and will become smooth. The reason is believed to be that the buffer makes the crystals generated by the drying of the solution finer.

[0014] The biosensor for detecting acid phosphatase activity of semen provided by the present invention is provided with an insulating substrate, and a covering member is formed between the insulating substrate and the covering member to form a passage for supplying a test solution to an electrode layer. With such a structure, there is an advantage that the amount of the test solution can be determined at a certain amount.

[0015] The sample of the biosensor described in the present invention is semen. Under normal circumstances, the semen of men is jelly-like just after being ejected from the body. After about 10-30 minutes, the semen will gradually liquefy into a watery liquid. This process is called the liquefaction of semen, but some men's semen is not liquefied within 60 minutes. If it is not liquefied, the semen becomes a paste, and the siphon port of the biosensor cannot automatically siphon the semen into the reaction zone. The test can only be carried out smoothly after the semen liquefies itself, but some men's semen liquefies only after 30-60 minutes, and even still does not liquefy after 60 minutes, requiring the user to wait for such a long time, which is very inconvenient. In addition, if the semen is not liquefied thoroughly, the semen will not be uniform enough, which may lead to inaccurate test results. In order to detect in time when the semen is fresh and ensure the activity of acid phosphatase, the present invention also provides a reagent tube for promoting semen liquefaction, and the reagent tube provides a reagent for accelerating semen liquefaction, which can be trypsin, chymotrypsin, preferably chymotrypsin, because chymotrypsin has a stronger ability to decompose proteins, making the semen liquefaction faster.

[0016] The present invention provides a detection method of a biosensor for detecting the activity of semen acid phosphatase, the method comprising the following steps:

[0017] 1) Use a dropper or pipette to take an appropriate amount of semen into the reagent tube and mix it to liquefy the semen;

[0018] 2) inserting the semen acid phosphatase activity biosensor into a semen acid phosphatase activity detection instrument;

[0019] 3) The siphon port of the semen acid phosphatase activity biosensor contacts the liquefied semen in the test tube, and the siphon port automatically sucks in the semen to cover the reagent layer for reaction;

[0020] 4) The instrument calculates and displays the result based on the current obtained.

[0021] The protease contained in the reagent tube to promote semen liquefaction may preferably be trypsin or chymotrypsin.

[0022] The present invention provides a biosensor and a detection method for detecting acid phosphatase activity in semen. Compared with the prior art, the biosensor and the detection method using the biosensor can be operated more simply and the detection time is less than 10 seconds. There is no need to dilute the semen, and the instrument can directly give the result. The present invention is very suitable for personal detection at home and bedside detection in medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the structure of a semen acid phosphatase biosensor according to an embodiment of the present invention;

[0024] Figure 2 This is a structural exploded view of a semen acid phosphatase biosensor according to an embodiment of the present invention;

[0025] Figure 3 This is a graph showing the concentration-current curve of semen acid phosphatase tested by the semen acid phosphatase biosensor in an embodiment of the present invention.

[0026] Among them: insulating substrate 1, conductive electrode 2, working electrode 2-1, reference electrode 2-2, short-circuit electrode 2-3, insulating isolation layer 3, reaction window 4, reagent layer 5, siphon sample guide groove 6, siphon port 7, adhesive spacer 8, air guide hole 9.

[0027] For example:

[0028] The present invention is described below with reference to embodiments, but the present invention is not limited to these embodiments.

[0029] Example 1

[0030] This example provides a preparation example of a biosensor for detecting acid phosphatase activity in semen.

[0031] The present embodiment provides a biosensor for detecting the activity of acid phosphatase in semen, which comprises an electrically insulating substrate, an electrode layer composed of a working electrode, a reference electrode and a short-circuit electrode arranged on the electrically insulating substrate, and a reagent layer composed of a reagent and a buffer; the reagent layer contains 3-carboxybenzenesulfonic acid, a reagent for improving reactivity and stability, with a 3-carboxybenzenesulfonic acid content of 0.5%-3%; the reagent layer contains disodium phenyl phosphate, which can be decomposed by acid phosphatase under acidic conditions, with a disodium phenyl phosphate content of 1%-3%; and an electron mediator capable of oxidizing free phenol produced by the decomposition of disodium phenyl phosphate by acid phosphatase. The buffer is preferably selected from: acetic acid, acetate, citric acid, and citrate.

[0032] In this embodiment, the reagent for improving reactivity and stability in the reagent layer is preferably selected from sulfonic acid and its derivatives, such as benzenesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, p-toluenesulfonic acid, p-aminobenzenesulfonic acid, 4-hydroxybenzenesulfonic acid, and 3-carboxylbenzenesulfonic acid. 3-Carboxylbenzenesulfonic acid is preferred in this embodiment. 3-Carboxylbenzenesulfonic acid and its sodium salt are non-volatile, have strong acidity, have excellent solubility in water and have small moisture absorption characteristics. 3-Carboxylbenzenesulfonic acid and its sodium salt can provide an acidic environment for the reagent system, accelerate the decomposition speed of disodium phenyl phosphate by acid phosphatase, thereby making the semen acid phosphatase biosensor of the present invention have good reactivity, and can stabilize potassium ferrocyanide in the reaction system, so that the semen acid phosphatase biosensor of the present invention has a good storage period, and plays a role as a stabilizer. Preferably, the content of sulfonic acid and its derivatives is 0.5%-3%.

[0033] In this embodiment, the reagent layer contains phosphate esters that can be decomposed by acid phosphatase, which can be selected from disodium phenyl phosphate, disodium phenylphosphate, sodium phenolphthalein phosphate, sodium glycerophosphate, α-naphthol sodium phosphate, etc. In this embodiment, the phosphate ester content is preferably 0.5%-5%, and the optimal phosphate ester content is 1%-3%.

[0034] The reagent layer preferably also contains an electron mediator. Dissolved oxygen in the test solution can also be used as an electron mediator, but it is best to put an electron mediator in the reagent layer. As the electron mediator, for example, it can be a metal complex such as potassium ferrocyanide, osmium-tris(bipyridinium), ferrocene derivatives, benzoquinone derivatives such as p-benzoquinone, phenazinium derivatives such as N-methylphenazinium sulfate monomethyl ester salt, phenothiazinium derivatives such as methylene blue, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, hexaammine ruthenium chloride, etc. These electron mediators can be used alone or in combination of two or more. In this embodiment, it is preferred that the electron mediator content is 3%-15%.

[0035] The reagent layer preferably also contains hydrophilic polymer. The following materials are preferably used as hydrophilic polymer: polyamino acids such as carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polylysine, polystyrene sulfonic acid, gelatin and its derivatives, polymers of acrylic acid or its salts, polymers of methacrylic acid or its salts, starch and its derivatives, polymers of maleic anhydride or its salts, agarose gel and its derivatives. If the reagent layer contains hydrophilic polymer, the measurement result of the seminal acid phosphatase biosensor will not be affected by vibration from the outside. Moreover, hydrophilic polymer has the advantage of being able to play the role of the binder that enzyme is slowly fixed on the electrode. The present embodiment is preferred, and the hydrophilic polymer content is 0.2%-5%, and the optimal hydrophilic polymer content is 0.5%-3%.

[0036] The electron mediator, reagent and buffer for improving reactivity and stability for detecting the acid phosphatase of semen can be loaded on the biosensor in a mixed state. In this way, the manufacturing process of the biosensor is simple, and the reagent layer can be easily made. In addition, because disodium phenyl phosphate and the electron mediator are mixed together, the free phenol produced by the acid phosphatase decomposing the disodium phenyl phosphate is in a very close position to the electron mediator, and the free phenol produced by the acid phosphatase decomposing the disodium phenyl phosphate can be oxidized by the electron mediator very quickly. Thus, the reactivity of the acid phosphatase biosensor of semen is improved, and even when the measuring time is shorter, enough responsiveness can be obtained.

[0037] The buffer loading amount in the sensor is preferably below 0.05mmol / L, so that the solubility of the electron mediator will not be reduced. In addition, since the response value corresponding to 0U / ml acid phosphatase, that is, the blank response value, will not increase under the above loading amount, the biosensor can have high reliability. The reagent layer contains a buffer to make the reagent layer formed by solution development and drying smooth. In the case of forming a reagent layer by drying an aqueous reagent solution, the reagent layer will have bumps and depressions due to the coarsening of the reagent crystals. This is particularly significant when it contains the electron mediator potassium ferrocyanide. After the reagent layer contains a buffer, the reagent layer will not have bumps and depressions and will become smooth. The reason is believed to be that the buffer makes the crystals generated by the drying of the solution finer.

[0038] A biosensor for detecting the activity of semen acid phosphatase in a preferred embodiment of the present invention is as follows: Figure 1 , Figure 2As shown, a biosensor for detecting the activity of acid phosphatase in semen provided in this embodiment includes an insulating substrate 1, on which three strip-shaped conductive electrodes 2 are arranged, namely a working electrode 2-1, a reference electrode 2-2 and a short-circuit electrode 2-3, wherein the short-circuit electrode 2-3 is connected to the working electrode 2-1 or the reference electrode 2-2, and an insulating isolation layer 3 is arranged on the upper part of the conductive electrode 2, one end of the insulating isolation layer 3 is flush with the insulating substrate 1, and the other end is between the conductive part of the strip-shaped conductive electrode 2 and the flush end of the insulating isolation layer 3, so that the electrode conductive part at the front end of the conductive electrode 2 is exposed to the outside; a reaction window 4 is opened on the insulating isolation layer 3, and the span of the reaction window 4 is to cover the working electrode 2-1 and the reference electrode 2-2 at the same time. The short-circuit electrode 2-3 is used to sense the presence of the sample to be measured and start the measuring instrument; all electrodes can be prepared by screen printing technology or sputtering technology.

[0039] A reagent layer 5 is pasted in the reaction window 4; the reagent layer 5 contains biological reagents that react with the substance to be tested, including phosphates that react with the substance to be tested and an electron transfer mediator, and also contains a buffer, an adhesive and a surfactant; the reagent layer 5 reacts with acid phosphatase to obtain electrons and transfers them to the electron transfer media, and the electron transfer media is oxidized on the electrode surface to generate current. The instrument detects the generated current and converts it into the concentration of acid phosphatase.

[0040] An adhesive spacer 8 is provided on the insulating isolation layer 3, and a strip-shaped siphon sample guide groove 6 is provided on the adhesive spacer 8 and at the corresponding position on the insulating isolation layer 3 and the reaction window 4. An air guide hole 9 is provided at one end of the siphon sample guide groove 6 close to the electrode conduction part, so as to discharge gas when the sample enters the reaction window 4. The air guide hole 9 is a strip-shaped groove that runs through the narrow end of the adhesive spacer 8; a siphon port 7 is provided at one end of the siphon sample guide groove 6 away from the electrode conduction part, and the siphon port 7, the siphon sample guide groove 6 and the air guide hole 9 are interconnected.

[0041] The sample of the biosensor for detecting the acid phosphatase activity of semen provided in this embodiment is semen. Under normal circumstances, the semen of men is jelly-like just after being ejaculated from the body. After about 10-30 minutes, the semen will gradually liquefy into a watery liquid. This process is called the liquefaction of semen, but some men's semen is not liquefied within 60 minutes. If it is not liquefied, the semen becomes a paste, and the siphon port of the biosensor cannot automatically siphon the semen into the reaction area. The test can only be carried out smoothly after the semen liquefies itself, but some men's semen liquefies only after 30-60 minutes, and even still does not liquefy after 60 minutes, requiring the user to wait for such a long time, which is very inconvenient. In addition, if the semen is not liquefied thoroughly, the semen will not be uniform enough, which may lead to inaccurate test results. In order to detect the semen in time when it is fresh and ensure the activity of acid phosphatase, the present invention also provides a reagent tube for promoting the liquefaction of semen. The reagent tube provides a reagent for accelerating the liquefaction of semen, which can be trypsin or chymotrypsin, preferably chymotrypsin, because chymotrypsin has a stronger ability to decompose proteins, making the semen liquefaction faster.

[0042] In the specific implementation, the working electrode, reference electrode and short-circuit electrode were made on a 0.25 mm thick PET plastic substrate by screen printing technology. The working electrode, reference electrode and short-circuit electrode were made of conductive carbon ink purchased from Acheson. After a thin layer of conductive ink was evenly spread on the PET plastic substrate by screen printing, it was placed in an 80°C oven for drying for 60 minutes.

[0043] Secondly, screen printing technology is used to print insulating varnish as an electrical insulating isolation layer on the substrate printed with the electrode system. The insulating varnish is purchased from Acheson. After the insulating varnish is spread on the PET plastic substrate by screen printing to form a thin layer of ink dispersed evenly, it is placed in a UV dryer for one minute to dry.

[0044] Next, a reagent layer was fixed on the electrode surface for the detection of semen acid phosphatase. The components and weight percentages of the reagent layer reaction solution are shown in Table 1:

[0045] Table 1

[0046] Material Weight (w / v) Potassium ferrocyanide 5％ Disodium phenyl phosphate 4％ 3-Carboxybenzenesulfonic acid 2％ Hydroxyethyl Cellulose 1％ Citric acid buffer (PH = 6.0) 88％

[0047] Take 4 μL of the reagent layer reaction solution and drop it onto the electrode surface, dry it at 50°C for 20 min, and set aside.

[0048] After fixing the reagent layer, the PET substrate is bonded with an adhesive spacer, and a groove is provided on the adhesive spacer, corresponding to the electrode reaction area, and exposing the end of the electrode with the enzyme reaction reagent, as well as the reference electrode and the short-circuit electrode. The PET substrate and the adhesive spacer form a siphon sample guide groove and a siphon port.

[0049] Example 2

[0050] This example illustrates the linear relationship between the concentration of semen acid phosphatase and the current, and a method for detecting semen acid phosphatase using the semen acid phosphatase biosensor prepared in Example 1. The method comprises the following steps:

[0051] 1) Use a dropper or pipette to take an appropriate amount of semen into the reagent tube and mix it to liquefy the semen;

[0052] 2) inserting the semen acid phosphatase activity biosensor into a semen acid phosphatase activity detection instrument;

[0053] 3) The siphon port 7 of the semen acid phosphatase activity biosensor contacts the liquefied semen in the test tube, and the siphon port 7 automatically sucks in the semen to cover the reagent layer 5 for reaction;

[0054] 4) The instrument calculates and displays the result based on the current obtained.

[0055] The protease contained in the reagent tube to promote semen liquefaction may preferably be trypsin or chymotrypsin.

[0056] The specific operation is as follows: collect fresh semen, add different amounts of high-concentration prostatic acid phosphatase standard to the fresh semen, the addition of high-concentration prostatic acid phosphatase should not change the matrix, and use a biochemical analyzer to test the acid phosphatase concentration after preparation, which are 21U / ml, 53U / ml, 97U / ml, 214U / ml, 469U / ml, and 982U / ml semen samples respectively. Use a dropper to take the prepared semen sample and add it to the reagent tube containing chymotrypsin and mix for one minute. The semen acid phosphatase biosensor prepared in Example 1 is placed in a matching tester and a 300mV working voltage is applied to both ends of the working electrode and the reference electrode; take the semen in the reagent tube and add it to the siphon port. The semen automatically absorbs about 2μL of the sample through the siphon sample guide groove 6, enters the reaction zone and reacts with the reagent in the reagent layer to form an electric current. The current corresponding to the semen acid phosphatase concentration can be quickly detected on the matching tester. The linear relationship between the semen acid phosphatase concentration and the current is shown in Figure 1 After calibration using the above method, the obtained linear curve is input into the storage device of the matching tester, and the concentration of semen acid phosphatase of unknown samples can be directly read from the matching tester. Figure 1 It can be seen that, since the sample is not diluted, the semen acid phosphatase biosensor of the present invention has high reactivity and good linearity.

[0057] The above examples are for illustrative purposes only and are not intended to be limiting. Any equivalent modifications and changes made to the present invention without departing from the spirit and scope of the present invention, including but not limited to the original components described in the embodiments of the present invention being replaced by software, hardware, firmware, or any combination thereof, shall be included within the scope of the present invention.

Claims

1. A biosensor for detecting acid phosphatase activity in semen, characterized in that: It includes an electrical insulating substrate, an electrode layer composed of a working electrode, a reference electrode and a short-circuit electrode arranged on the electrical insulating substrate, and a reagent layer composed of a reagent and a buffer; The reagent layer contains 3-carboxybenzenesulfonic acid for improving reactivity and stability, wherein the content of the 3-carboxybenzenesulfonic acid is 0.5%-3%; disodium phenyl phosphate which can be decomposed by acid phosphatase under acidic conditions, wherein the content of disodium phenyl phosphate is 1%-3%; and an electron mediator which can oxidize the acid phosphatase to decompose disodium phenyl phosphate to produce free phenol; The buffer is acetic acid, acetate, citric acid or citrate.

2. A detection method using the biosensor for detecting semen acid phosphatase activity according to claim 1, characterized in that At least the following steps are included: 1) Take an appropriate amount of semen into a reagent tube and mix it to liquefy the semen; 2) inserting the semen acid phosphatase activity biosensor into a semen acid phosphatase activity detection instrument; 3) The siphon port of the semen acid phosphatase activity biosensor contacts the liquefied semen in the reagent tube, and the siphon port automatically sucks in the semen to cover the reagent layer for reaction; 4) The instrument calculates and displays the result based on the current obtained.

3. The detection method of a biosensor for detecting semen acid phosphatase activity according to claim 2, characterized in that: The reagent tube contains a protease for promoting semen liquefaction, and the protease is trypsin or chymotrypsin.

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