CN1710418A - A biosensor for rapidly detecting hemoglobin and its testing method - Google Patents

Abstract

The invention relates to a biosensor for rapidly detecting hemoglobin and a testing method thereof, and relates to the technical field of biochemical detection. It includes a hemoglobin biosensor and a portable biochemical detector for detecting hemoglobin; the hemoglobin biosensor includes a basic electrode, a sensitive film, an insulating layer, and an encapsulation layer, that is, a working electrode formed on an insulating substrate using micromachining technology and a dummy The reference electrode is covered with an insulating layer to make an electrode channel. After the electrode is treated with surface activation technology, a hydrophilic polymer solution is added dropwise to form a hydrophilic polymer layer, and then sensitive materials are fixed on the hydrophilic polymer layer to form Sensitive film layer, and then encapsulate the sensor with the encapsulation layer. When the test sample containing the analyte hemoglobin is added to the injection channel from the side, the test sample contacts with the sensitive membrane and reacts, and the current output signal generated by the reaction between the sensitive membrane and hemoglobin can be quickly detected by the portable biochemical detector.

Description

A biosensor for rapidly detecting hemoglobin and its testing method

technical field

The invention relates to the technical field of biochemical detection, and relates to a biosensor for rapidly detecting hemoglobin and a testing method thereof. The hemoglobin concentration in a test sample can be quickly obtained by using a portable biochemical detector and a biosensor to detect hemoglobin.

Background technique

Hemoglobin, commonly known as heme, is an iron-containing protein in red blood cells. It is the core substance of the oxygen transfer link. Its main function is to transport oxygen and carbon dioxide, buffer acidic substances, and participate in the regulation of acid-base balance in the body.

Generally, the normal range of hemoglobin concentration in the human body is 110-160g/L, of which the male content is 120-160g/L, and the female content is 110-150g/L. Determination of the hemoglobin concentration in a blood sample is very important for clinical diagnosis of anemia and evaluation of blood sampling quality. Therefore, there is a need for a sensor for rapidly determining the concentration of hemoglobin and a testing method thereof.

The test method of hemoglobin is usually the cyanide methemoglobin method, which converts hemoglobin into cyanide hemoglobin through the action of a solvent containing potassium ferricyanide and cyano group, and measures the absorbance near 540nm. Another common method is to convert hemoglobin into oxyhemoglobin, and also measure the absorbance of oxyhemoglobin method. Both methods require expensive analytical instruments and complex sample preparation. Therefore, a biosensor capable of simple, rapid and accurate determination of hemoglobin and its testing method has become an urgent problem to be solved.

Contents of the invention

An object of the present invention is to provide a simple and fast biosensor for measuring hemoglobin. The provided hemoglobin biosensor is disposable, avoids cross-infection, has good repeatability, does not need to process test samples, is simple to manufacture, can be produced in batches, and can reduce costs.

Another object of the present invention is to provide a testing method, which utilizes a portable biochemical detector and cooperates with the biosensor of the present invention to quickly detect the concentration of hemoglobin. This test method does not require professional and technical personnel to operate, and because the portable biochemical detector is small, portable, and cheap, it can be applied to clinical medicine, blood collection stations, plasma collection stations, family individuals, etc.

In order to achieve the above object, the technical solution of the present invention is to provide a biosensor for rapid detection of hemoglobin, including a basic electrode, an insulating layer and an encapsulation layer, and its basic electrode includes an insulating base material, a working electrode and a false reference electrode, and the base electrode There are two electrodes parallel to each other along the length direction on the upper surface of the material: a working electrode and a false reference electrode. On the two electrodes, an insulating layer is fixed. There is a groove in the insulating layer, which is a reaction channel. The upper surface of the insulating layer An encapsulation layer is solidly covered, and the encapsulation layer covers the reaction channel; there is a modification layer on the bottom surface of the reaction channel, and a sensitive film is fixed on the modification layer; the reaction channel and the inner surface of the encapsulation layer form a sample injection channel.

The described biosensor for rapid detection of hemoglobin, the insulating substrate is made of polyvinyl chloride, polystyrene, polyester, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, Polyethylene terephthalate and acrylonitrile-butadiene-styrene polymers of any one.

In the biosensor for rapid detection of hemoglobin, the working electrode and the pseudo-reference electrode are composed of a two-layer structure, and the lower layer is a transition layer made of any one of chromium, titanium, nickel and nickel-chromium metal, The upper layer is made of any one of gold, silver, platinum and palladium precious metals.

In the biosensor for rapid detection of hemoglobin, the sensitive membrane is made of sensitive materials, and the sensitive materials are composed of hydrophilic polymers, electronic mediators, surfactants, stabilizers, and buffers; wherein,

Hydrophilic polymers are made from carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyethyl methyl cellulose, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methyl Acrylic acid and its salts can be made from one of the polymers, the amount is 0.2-2wt%, preferably 0.5wt%;

The electron mediator is composed of methylene blue, toluene saddle blue, cresyl solid violet, ferricyanate, ferrocene and its derivatives, p-benzoquinone, phenazine methyl sulfate, indophenol and its derivatives and β-naphthoquinone - any one of potassium 4-sulfonate, preferably methylene blue, the amount is 0.1-10mM, preferably 0.5-2mM; or ferricyanate, the amount is 20-500mM, preferably 50-200mM;

Surfactant is selected from triton X-100, diethanolamide, alkyl alcohol amidophosphoester salt, alcohol ether carboxylate, monoalkyl phosphate, sodium laurylsulfonate and alkylphenol polyoxyethylene Optionally made from ether, the amount is 0.02~3wt%, preferably 0.05~0.5wt%;

The stabilizer is selected from sodium succinate, trisodium citrate, bovine serum albumin, trehalose, chitin, and silk fibroin, and its amount is 1-50mM, preferably 1-10mM;

The buffer for dissolving the above reagents is made from any one of phosphate buffer, citric acid buffer, acetic acid buffer and Tris hydrochloric acid buffer, and its preferred pH value is 7.4.

In the biosensor for rapid detection of hemoglobin, the base material has a thickness of 0.1-1mm, preferably 0.2-0.6mm; the thickness of the insulating layer and the packaging layer are different, and the thickness of both is between 0.1-0.5mm. Preferably it is 0.2 to 0.4 mm.

The biosensor for rapid detection of hemoglobin, the modification layer and the sensitive film, are manufactured by the following steps: first, modify a layer of hydrophilic polymer solution with hydrophilic effect in the reaction channel on the electrode surface, and place it in Dry in an electric blast drying oven to form a surface modification layer, take it out; add a reaction solution containing an electronic mediator dropwise on the modification layer, then place it in an electric blast drying oven to dry to form a sensitive film, take it out, and set aside.

A test method for rapid detection of hemoglobin, the equipment used includes a biosensor for rapid detection of hemoglobin and a portable biochemical detector, wherein the panel of the biochemical detector is provided with a power key, a menu key, a test strip slot and a liquid crystal display; First connect the detector to the working electrode of the sensor and the false reference electrode electrically, apply a voltage to the two electrodes, and then measure the current generated by the biochemical reaction between the reagent and the test sample to determine the concentration of hemoglobin in the sample value.

In the test method described above, it first turns on the power button to start the machine, presses the menu button to browse the measurement information, and when the menu button is set to measure hemoglobin, operate according to the prompts on the liquid crystal display; One end of the lead wire is inserted into the test strip slot of the tester. After insertion, it is electrically connected to the test strip. The LCD display enters the sample dropping state. After dropping the sample, the LCD display immediately enters the countdown test state. The concentration value of hemoglobin in the measured sample is displayed on the display.

Description of drawings

Figure 1 is an exploded perspective view of a hemoglobin biosensor;

Fig. 2 is a vertical sectional view of the hemoglobin biosensor;

Figure 3 is the cyclic voltammetry curves of different concentrations of hemoglobin, wherein methylene blue is the electron mediator;

Fig. 4 is the current response curve of different concentrations of hemoglobin, wherein potassium ferricyanate is an electron mediator;

Fig. 5 is a linear correlation curve for detecting the hemoglobin biosensor, and potassium ferricyanate is an electronic mediator;

Fig. 6 is a schematic diagram of hemoglobin biosensor testing;

Fig. 7 is an appearance diagram of the portable biochemical detector.

Detailed ways

According to one embodiment of the present invention, a method for manufacturing a hemoglobin biosensor is provided.

Please refer to FIG. 1 and FIG. 2 , the hemoglobin biosensor a includes a basic electrode, a sensitive film 9 , an insulating layer 4 and an encapsulation layer 5 . The basic electrode includes an insulating substrate 1 , a working electrode 2 and a false reference electrode 3 . The electrodes 2 and 3 are made of precious metals and formed by sputtering. This method is simple and can greatly improve the performance of the sensor a, and can be mass-produced. The details of the production of the electrodes 2 and 3 can refer to the applied for patents. The number is 200410030214.X, and will not be repeated here.

The insulating substrate 1 is made of polyvinyl chloride, polystyrene, polyester, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, polyethylene terephthalate and acrylonitrile - Made of a polymer selected from butadiene-styrene, etc., its thickness is between 0.1~1mm, preferably 0.2~0.6mm, the insulating layer 4 and the encapsulation layer 5 are the same material as the insulating substrate 1 , the thickness is different, the thickness is between 0.1 ~ 0.5mm, preferably 0.2 ~ 0.4mm.

The working electrode 2 and the false reference electrode 3 are formed of noble metals of the same material, and the electrodes 2 and 3 are composed of a two-layer structure, including a transition layer made of a metal selected from chromium, titanium, nickel and nickel chromium. and an upper layer made of one of the precious metals selected from gold, silver, platinum and palladium. The shape of electrodes 2 and 3 refers to the patent application, the application number is 02160271.9, it can adopt electrodes of various shapes such as circle, square, bar. The electrode system can be two-electrode, that is, working electrode, pseudo-reference electrode (shared counter electrode and reference electrode), or three-electrode, that is, working electrode, counter electrode and reference electrode.

In the hemoglobin biosensor a, the sensitive membrane 9 is made of sensitive materials. The sensitive material is composed of a hydrophilic polymer, an electronic mediator, a surfactant, a stabilizer, and a buffer, and two kinds of sensitive materials are provided in the embodiment. First, modify a layer of hydrophilic macromolecule with hydrophilic effect in the reaction channel 7 on the surface of the electrodes 2 and 3, place it in an electric blast drying oven to dry to form a surface modification layer 8, take it out; Add the reaction solution containing the electronic mediator etc., and then place it in an electric blast drying oven to dry to form a sensitive film 9, take it out, and save it for later use. Standard hemoglobin solution was used in the test, and different current response curves and related results were obtained.

Hydrophilic polymers are derived from carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyethyl methyl cellulose, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid A composition selected from extremely salty polymers, etc., the amount of the hydrophilic polymer is 0~2wt%, preferably 0.5wt% in the embodiment.

The electron mediator is composed of methylene blue, toluene saddle blue, cresyl solid violet, ferricyanate, ferrocene and its derivatives, p-benzoquinone, phenazine methyl sulfate, indophenol and its derivatives and β-naphthoquinone - One selected from 4-potassium sulfonate, etc. In the embodiment, methylene blue and ferricyanate are used as electronic mediators to make two sensitive materials, wherein the amount of methylene blue is 0.1~10mM, preferably 0.5~2mM , preferably 1 mM in the embodiment; the amount of ferricyanate is 20 ~ 500 mM, preferably 50 ~ 200 mM, preferably 100 mM in the embodiment.

Surfactant, which is derived from Triton X-100, diethanolamide, alkyl alcohol amide phosphoester salt, alcohol ether carboxylate, monoalkyl phosphate, sodium lauryl sulfonate and alkylphenol poly One selected from oxyethylene ether, etc., the amount of surfactant is 0~3wt%, preferably 0.05~0.5wt%, preferably 0.1wt% in the embodiment.

A stabilizer for maintaining enzyme activity, which is selected from sodium succinate, trisodium citrate, bovine serum albumin, trehalose, chitin, silk fibroin, etc., and its amount is 1 ~ 50mM, preferably 1 to 10 mM, preferably 3.4 mM in the examples.

Solvent for dissolving enzymes and electronic mediators, it can use various buffers such as water, phosphate buffer, citrate buffer, acetate buffer, Tris hydrochloric acid buffer, etc., the purpose is to be used to maintain a constant pH value, embodiment The preferred pH value is 7.4.

According to another embodiment of the present invention, there is provided a test method for rapidly detecting hemoglobin using a portable biochemical detector, in the hemoglobin biosensor system, it includes a hemoglobin biosensor and a detector for displaying the hemoglobin concentration in the test sample , the detector is connected to the working electrode and the false reference electrode of the sensor, and the working voltage is applied to the two electrodes, and the current generated due to the reaction between the reagent and the test sample is measured to determine the concentration of hemoglobin.

Example 1

The basic composition of the hemoglobin biosensor a is shown in Figure 1, which is an exploded perspective view of the sensor a, which includes an insulating substrate 1, and the upper surface of the substrate 1 has two electrodes parallel to each other along the length direction: a working electrode 2 and a dummy electrode. The reference electrode 3 is fixed with an insulating layer 4 of the same material as the insulating substrate 1 on the two electrodes. There is a groove in the insulating layer 4, which is a reaction channel 7. The upper surface of the insulating layer 4 is covered with an encapsulation layer 5. , the encapsulation layer 5 covers the reaction channel 7 . As shown in FIG. 2 again, it is a vertical cross-sectional view of the hemoglobin sensor a. There is a layer of modification layer 8 on the bottom surface of the reaction channel 7, and a sensitive film 9 is affixed on the modification layer 8; the inner surface of the reaction channel 7 and the packaging layer 5 constitutes a sampling channel 10.

The sensitive film 9 is made of sensitive material. The production steps of the sensitive material are as follows: take the semi-finished product of the hemoglobin biosensor a that has not been packaged with the encapsulation layer 5, and first activate the surfaces of the electrodes 2 and 3 exposed on the bottom of the reaction channel 7. The surface activation technology refers to the patent that has been applied for, and the application number is 02154524.3 , without going into details here, then drop 7 μl of aqueous solution of 0.5wt% carboxymethylcellulose (hereinafter referred to as CMC) to the bottom surface of the reaction channel 7, and then place it in a blast drying oven at 37° C. to dry for 20 minutes , taken out to form a CMC modification layer 8 . The electrodes 2 and 3 modified by CMC have a good hydrophilic effect, and when the test sample is added by capillary force, the test sample can quickly contact with the sensitive membrane 9 for reaction. Take the surface-modified semi-finished sensor a, drop 4 μl of sensitive material containing electronic mediators on the modified layer 8, and also place it in a blast drying oven at 37°C for 20 minutes to form a sensitive film 9, take it out and save it for later use . The sensitive material includes, besides the electronic medium, a surfactant with a content of 0.1wt%, a stabilizer with a content of 3.4mM, and a buffer for adjusting the pH value with a pH value of 7.4. Different electronic mediators have different contents, and two electronic mediators are provided here as preferred embodiments.

1) When methylene blue is used as the electron mediator, the concentration is 1mM, and the cyclic voltammetry curves of different concentrations of hemoglobin measured by using it as the base phase solution are shown in Figure 3. I, II, III, and IV represent 0μM, 50μM, and 100μM, respectively. , cyclic voltammetry curve of 200 μM hemoglobin. The reaction principle is:

2) When potassium ferricyanate is used as the electronic medium, its concentration is 100mM, and the cyclic voltammetry curves of different concentrations of hemoglobin measured with it as the base phase solution are shown in Figure 4, wherein 1, 2, 3, 4, 5 represents the current response curves of 0 μM, 500 μM, 1000 μM, 2000 μM and 3000 μM hemoglobin, respectively. This sensor has good linear correlation (as shown in Figure 5) and repeatability, which can reach 0.995 and 2.3% respectively, and the response can reach a steady state within 60 seconds. It is suitable for clinical medicine, blood collection stations, plasma collection stations, and family individuals and other rapid detection.

The reaction principle is:

Finally, encapsulation is performed on the insulating layer 4 with the encapsulation layer 5 to complete the fabrication of the hemoglobin biosensor a. The test sample is supplied to the sampling channel 10 from the side of the sensor a by capillary force, and the sample quickly covers the entire reaction area. The hemoglobin in the test sample contacts the sensitive membrane 9 and reacts to generate a current corresponding to the hemoglobin concentration. In order to obtain Experimental response curve, adopt electrochemical workstation to detect (use the test method described in embodiment 2 in actual clinical application), the working electrode of electrochemical workstation is connected with the working electrode 2 of sensor a, the reference electrode of electrochemical workstation and counter electrode Short circuit, and then connect with the false reference electrode 3 of the sensor a, add a working voltage to the test system, and detect the hemoglobin concentration according to the generated current output signal.

Example 2

As shown in Figure 6, it is a schematic diagram of a method for testing with a portable biochemical detector b connected to a hemoglobin biosensor a, and the sensor a fixed with a sensitive film 9 is connected to the portable biochemical detector b, wherein, see Figure 7, the biochemical detector The panel of b is provided with power key 14, menu key 13, test strip slot 11 and liquid crystal display 12; The menu key 13 can flip through the measurement information, and when the menu key 13 is set to measure hemoglobin, operate according to the prompts of the liquid crystal display 12 . When prompted to insert the test strip, insert one end of the electrode lead wire 6 of the hemoglobin biosensor a into the test strip slot 11 of the detector b. , the liquid crystal display screen 12 immediately displays the countdown test state, and after the test is over, the liquid crystal display screen 12 displays the concentration value of hemoglobin in the sample to be tested.

The above invention discloses several methods and materials of the present invention. A preferred embodiment of the present invention, however, can be better understood with reference to the accompanying drawings. The preferred embodiments are for the purpose of illustration only, not limitation of the invention. Many modifications and variations of the present invention are possible on the basis of the above description. Therefore, within the scope of the appended claims, the invention may be implemented in other ways than those described above. For example, using a three-electrode system, using noble metal platinum as an electrode material, and directly fixing sensitive materials.

Claims (8)

1. The utility model provides a detect biosensor of hemoglobin fast, includes basic electrode, insulating layer and packaging layer, its characterized in that: the basic electrode comprises an insulating base material, a working electrode and a pseudo-reference electrode, wherein two electrodes which are parallel to each other along the length direction are arranged on the upper surface of the base material: the device comprises a working electrode and a pseudo-reference electrode, wherein an insulating layer is fixedly connected above the working electrode and the pseudo-reference electrode, a groove is formed in the insulating layer and is a reaction channel, and an encapsulating layer is fixedly covered on the upper surface of the insulating layer and covers the reaction channel; a modification layer is arranged on the bottom surface of the reaction channel, and a sensitive film is fixedly connected on the modification layer; the reaction channel and the inner surface of the packaging layer form a sample feeding channel.

2. The biosensor for rapid detection of hemoglobin according to claim 1, wherein: the insulating base material is made of any one polymer of polyvinyl chloride, polystyrene, polyester, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate and acrylonitrile-butadiene-styrene.

3. The biosensor for rapid detection of hemoglobin according to claim 1, wherein: the working electrode and the pseudo-reference electrode are composed of two layers, the lower layer is a transition layer made of any one of chromium, titanium, nickel and nickel-chromium metals, and the upper layer is made of any one of gold, silver, platinum and palladium noble metals.

4. The biosensor for rapid detection of hemoglobin according to claim 1, wherein: the sensitive membrane is made of a sensitive material, and the sensitive material consists of a hydrophilic polymer, an electronic mediator, a surfactant, a stabilizer and a buffer solution; wherein,

the hydrophilic polymer is prepared from one selected from carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyethyl methyl cellulose, polyvinyl alcohol, gelatin and derivatives thereof, acrylic acid and salts thereof, and methacrylic acid and polymers of salts thereof, and the amount of the hydrophilic polymer is 0.2-2 wt%;

the electron mediator is prepared from any one of methylene blue, toluene saddle blue, cresol purple, ferricyanate, ferrocene and derivatives thereof, para-benzoquinone, phenazine methyl sulfate, indophenol and derivatives thereof and β -naphthoquinone-4-potassium sulfonate, and is preferably the methylene blue, the amount of which is 0.1-10 mM, and is preferably 0.5-2 mM, or the ferricyanate, the amount of which is 20-500 mM;

the surfactant is prepared from one of triton X-100, diethanolamide, alkylolamide phosphate, alcohol ether carboxylate, monoalkyl phosphate, sodium dodecyl sulfate and alkylphenol polyoxyethylene ether in an amount of 0.02-3 wt%;

the stabilizer is prepared from one of sodium succinate, trisodium citrate, bovine serum albumin, trehalose, chitin and silk fibroin, and the amount of the stabilizer is 1-50 mM;

the buffer solution for dissolving the reagent is prepared from one of phosphate buffer solution, citric acid buffer solution, acetic acid buffer solution and Tris hydrochloric acid buffer solution, and the pH value is preferably 7.4.

5. The biosensor for rapid detection of hemoglobin according to claim 1, wherein: the thickness of the base material is 0.1-1 mm; the thickness of the insulating layer is different from that of the packaging layer, and the thickness of the insulating layer is 0.1-0.5 mm.

6. The biosensor for rapid detection of hemoglobin according to claim 1, wherein: the manufacturing method of the modifying layer and the sensitive film comprises the following steps: firstly, modifying a layer of hydrophilic polymer solution with a hydrophilic effect in a reaction channel on the surface of an electrode, placing the hydrophilic polymer solution in an electrothermal blowing drying box for drying to form a surface modification layer, and taking out the surface modification layer; and dropwise adding reaction liquid containing an electronic mediator on the modification layer, placing the modification layer in an electrothermal blowing drying oven for drying to form a sensitive film, and taking out for later use.

7. A test method for rapidly detecting hemoglobin is characterized in that: the device comprises a biosensor for rapidly detecting hemoglobin and a portable biochemical detector, wherein a panel of the biochemical detector is provided with a power key, a menu key, a test strip slot and a liquid crystal display screen; the detector is electrically connected with the working electrode and the pseudo-reference electrode of the sensor, voltage is applied to the two electrodes, and then current generated by biochemical reaction between the reagent and the test sample is measured to determine the concentration value of hemoglobin inthe sample.

8. The test method of claim 7, wherein: firstly, turning on a power key to start the machine, pressing a menu key to browse measurement information, and when the menu key is arranged for measuring hemoglobin, operating according to the prompt of a liquid crystal display screen; when the examination strip is inserted in the suggestion, insert the examination strip slot of detector with hemoglobin biosensor's electrode lead wire one end, insert back and examination strip electricity intercommunication, liquid crystal display shows to get into the suggestion and drips the appearance state, drips the appearance after, liquid crystal display shows immediately and gets into count-down test state, after the test, shows the concentration value of hemoglobin in the sample of surveying on liquid crystal display.

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