CN106483182A - A kind of dry type determines biology sensor of hemoglobin and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biochemical detection. In order to solve the problem of rapid determination of hemoglobin concentration, the invention proposes a dry-type biosensor for measuring hemoglobin. film, the sensor substrate is provided with a reaction electrode, an electrode output terminal, a conductive circuit connecting the reaction electrode and the electrode output terminal, an insulating layer is provided between the conductive circuit and the flow path layer, and the insulating layer is provided with a hollow groove corresponding to the reaction electrode. The flow path layer is provided with an inlet for the liquid to be tested, a hemolysis channel, and a reaction tank connected in sequence, wherein the hemolysis channel is decorated with a dry hemolysate that dissolves human blood cells, and a diversion film is provided on the hemolysis channel and the reaction tank. The invention can measure the hemoglobin concentration quickly, accurately, at low cost, portably and accurately, and has high application value.

Description

A dry biosensor for measuring hemoglobin and its preparation method

technical field

The invention relates to the technical field of biochemical detection, in particular to a dry-type biosensor for measuring hemoglobin and a preparation method thereof.

Background technique

Hemoglobin is a protein (Hemoglobin, abbreviated as Hb) responsible for carrying oxygen in higher organisms. It is the protein that makes blood red. Hemoglobin consists of four chains, two alpha chains and two beta chains, each chain has a ring of heme containing an iron atom. Oxygen binds to iron atoms and is transported by the blood. The characteristics of hemoglobin are: in places with high oxygen content, it is easy to combine with oxygen; in places with low oxygen content, it is easy to separate from oxygen. This characteristic of hemoglobin enables red blood cells to transport oxygen.

The clinical significance of hemoglobin determination is equivalent to red blood cell count. It can be used as a reference and standard for judging anemia and blood loss, and it can judge the type of anemia with the cooperation of other blood parameters. However, due to physiological reasons of strenuous exercise, or pathological reasons such as congenital heart disease, obstructive emphysema, pulmonary heart disease, pulmonary arteriovenous fistula, and abnormal hemoglobin disease with low oxygen-carrying capacity, high hemoglobin levels are all It can be used as an important basis for clinical diagnosis. Therefore, there is a need for a biosensor capable of rapid determination of hemoglobin concentration and a testing method thereof.

Most of the existing instruments for detecting hemoglobin use methemoglobin or hemoglobin complexes for absorbance detection. Although the toxic potassium cyanide binding method used by the gold standard has been phased out, the cyanide-free method currently used is not very stable and can be affected by the detection environment. The blood analyzers used in hospitals represented by this method are time-consuming, bulky and expensive. The commercially available portable hemoglobin analyzer uses the principle of light reflection for detection. Although it is small and portable, it is easily disturbed by the strong light environment, and the exposed probe head is easily polluted, resulting in errors in measurement results. However, the hemoglobin concentration obtained by the formula calculation method is susceptible to deviation due to the influence of the blood cell environment.

Contents of the invention

In order to solve the problem of rapid determination of hemoglobin concentration, the present invention proposes a dry-type biosensor for measuring hemoglobin and a preparation method thereof, which can quickly, accurately, low-cost, portable and accurately measure hemoglobin concentration, and has high application value.

The present invention is achieved through the following technical solutions: a dry-type biosensor for measuring hemoglobin, the biosensor sequentially includes a sensor substrate, an insulating layer, a flow path layer, and a diversion film, and the sensor substrate is provided with a reaction The electrode, the electrode output end, the conductive line connected between the reaction electrode and the electrode output end, an insulating layer is provided between the conductive line and the flow path layer, the insulating layer is provided with a hollow groove corresponding to the reaction electrode, and the flow path layer is provided with sequentially connected to-be-measured Liquid inlet, hemolysis channel, and reaction tank, wherein the hemolysis channel is modified with dry hemolysate that dissolves human blood cells, and a diversion film is provided on the hemolysis channel and the reaction tank.

The sensor base material is selected from one of polystyrene, polypropylene, polymethyl methacrylate, polyethylene and polyethylene terephthalate, with a thickness of 0.2-2 mm, preferably 0.2-0.5 mm.

The reaction electrodes are made of conductive carbon paste through screen printing, and the electrodes are parallel to each other. The reaction electrode is arranged at the bottom of the reaction groove in the flow path layer through the hollow groove of the insulating layer.

The reaction electrode is coated with a sensitive material to form a sensitive film. The sensitive material is made of an electronic medium, a surfactant, and a plasticizer. The preparation method is: coating the reaction electrode with an electronic medium, a surfactant, and a plasticizer. Sensitive materials composed of solvents are placed in a constant temperature drying oven to dry to form a sensitive film. The electron mediator is selected from one of methylene blue, potassium ferricyanide, ferrocene and its derivatives, and phenol violet, and the molar volume ratio of the sensitive material is 0.04-0.2mol: 1L, preferably 0.08 - 0.14mol: 1L. The surfactant is selected from one of Triton X-100, Tween 20, quaternary ammonium salts, and sodium laurate, and the dosage is 0.001-3g:1L of the mass-volume ratio of sensitive materials. The plasticizer is selected from one of sodium humate, carboxymethyl cellulose, polyvinyl alcohol, and phthalate, and the molar volume ratio of the usage amount to the sensitive material is 1-30mmol: 1L, preferably 1-10mmol: 1L . The invention detects the hemoglobin concentration through the electrochemical conduction of the sensitive membrane.

The electrode output terminal and the conductive circuit are printed with conductive silver paste through screen printing technology. The transmission of electrical signals uses silver electrodes to avoid the inaccuracy of current signals caused by high impedance during long-distance transmission. The electrode output end is arranged outside the ends of the insulating layer and the conduction layer to interface with the detection instrument. The electrode output terminal includes a working electrode and a counter electrode. The electrode output terminal can be two electrodes: it is composed of a working electrode and a counter electrode, and the reference electrode and the counter electrode are shared. Composed of electrode and reference electrode, the electrodes are parallel to each other.

The insulating layer is directly printed on the surface of the conductive circuit on the base of the sensor, and the reaction electrode is left through the hollow groove, and is contacted and reacted in the reaction layer of the flow path layer.

The hemolysis channel of the flow path layer is decorated with dry hemolysis substances that dissolve human blood cells. The dry hemolysis substances are selected from solidified non-ionic active agent solutions, and the concentration is 1-40g/L, preferably 1-10g/L . The dry hemolytic substance is selected from dodecylpyridinium ammonium chloride, dodecylpyridinium ammonium bromide, sodium dodecylsulfonate, saponin, polyethylene glycol octylphenyl ether, cetyl tris One of methyl ammonium bromide, preferably saponin. The modified dry hemolyzed substance in the hemolyzed channel of the flow path layer is placed in a constant temperature drying oven to dry to form a dry hemolyzed modified layer. The measurement uses fresh human blood obtained from the hospital to obtain the current response of different concentrations of hemoglobin.

The diversion film is hydrophilic and covers the hemolysis channel and the reaction tank of the flow path layer to form a semi-closed chamber and bring driving force to the liquid to be tested.

The sensor first coats the reaction electrode with a mixed solution composed of an electronic mediator, a surfactant, and a plasticizer, and puts it in a constant temperature drying oven to dry to form a sensitive film, and then coats the prepared flow path layer The hemolyzed substance is modified in the hemolyzed channel, and dried in a constant temperature drying oven to form a hemolyzed modified layer.

The dry-type biosensor for measuring hemoglobin is a kind of electrochemical biosensor that can directly detect human whole blood and quickly give results without pretreatment. Through a portable blood multi-parameter detector and a biosensor Detecting hemoglobin can quickly obtain the concentration of hemoglobin in the test sample. The method for detecting hemoglobin by the dry-type rapid detection hemoglobin biosensor is as follows: directly inject the whole blood of the person to be tested into the inlet of the solution to be tested, and the electrochemical instrument applies the working voltage to the output terminal of the electrode, and after 30 seconds to 2 minutes, The electrochemical detection instrument detects the concentration of hemoglobin together. Preferably, the amount of human blood to be tested is 6-10 uL.

When the blood to be tested enters the inlet of the liquid to be tested, the blood to be tested first reacts with the hemolyzed substance in the hemolyzed channel, releases hemoglobin, and then reacts with the sensitive membrane on the reaction electrode. Electrochemical instruments can be used to quickly detect the reaction. The current signal is used to obtain the concentration of hemoglobin.

The hemoglobin biosensor of the present invention is disposable, avoids cross-infection between different samples, has good repeatability, avoids cumbersome sample pretreatment process, and can realize large-scale production at low cost.

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention integrates the electrochemical detection of hemoglobin into a dry one-step operation, eliminating the process of blood pretreatment;

(2) Use stable and low-cost screen printing as a support to provide fast and reliable reference for medical staff's diagnosis and rescue, especially suitable for non-hospital testing places.

Description of drawings

Fig. 1 is the structural schematic diagram of the biosensor of dry type measuring hemoglobin of the present invention;

Fig. 2 is a structural disassembly diagram of a biosensor for dry-type determination of hemoglobin of the present invention;

Fig. 3 is the response curve of the dry hemoglobin sensor detecting human hemoglobin;

Among them, 1: sensor substrate, 2: electrical insulation layer, 3: flow path layer, 4: diversion film, 5: reaction electrode, 6: conductive line, 7: electrode output terminal, 8: liquid inlet to be tested, 9: Hemolysis channel, 10: Reaction tank.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Example 1

The structural schematic diagram of the biosensor for dry-type determination of hemoglobin of the present invention is shown in Figure 1, and the structural disassembly diagram of the dry-type measurement of hemoglobin biosensor is shown in Figure 2, which sequentially includes a sensor substrate 1, an insulating layer 2, and a flow path layer 3 , diversion film 4, the sensor substrate 1 is provided with a reaction electrode 5, an electrode output terminal 7, a conductive circuit 6 connected between the reaction electrode and the electrode output terminal, and an insulating layer 2 is provided between the conductive circuit 6 and the flow path layer 3 The insulating layer 2 is provided with a hollow groove corresponding to the reaction electrode 5, and the flow path layer 3 is provided with a test liquid inlet 8, a hemolysis channel 9, and a reaction tank 10 connected in sequence, and the hemolysis channel 9 and the reaction tank 10 are provided with guides. flow film4.

Firstly, a sensor substrate 1 with a thickness of 0.4mm is made of polypropylene, and then two parallel reaction electrodes 5 are printed on the surface of the sensor substrate 1 by screen printing technology from carbon paste, and at the end of the sensor substrate 1 The surface is printed with two parallel electrode output terminals 7 by silver paste through screen printing technology, one is the working electrode and the other is the counter electrode, and the silver paste is printed on the surface of the sensor substrate between the reaction electrode 5 and the electrode output terminal 4. conductive line 6,

Secondly, potassium ferricyanide, triton x-100, and sodium humate are mixed to make a sensitive material, wherein the concentration of potassium ferricyanide in the sensitive material is 0.14mol/L, and the mass volume concentration of triton x-100 is 0.1g/L, the concentration of sodium humate is 8mmol/L. Take the hemoglobin biosensor that has not been loaded with the flow path layer, first use alcohol to hydrophilize the reaction electrode 5, then place it in a drying oven to dry until it is completely dry, and then drop 5ul of sensitive material onto the surface of the working electrode and the counter electrode , and then place it in a drying oven at 40 degrees Celsius for 30 minutes. An insulating layer 2 is printed on the conductive circuit 6, and the insulating layer 2 is bonded to the flow path layer 3 through an adhesive.

Then, 10 ul of dry hemolysate solution containing 5 g/L saponin was drip-coated in the hemolysis channel 9 of the flow channel layer 3, and then dried in a 50-degree Celsius drying oven for 20 minutes. Then, the flow guide film 4 is used to seal the hemolysis channel 9 and the reaction tank 10 of the flow path layer 3 to make a dry biosensor 1 for measuring hemoglobin.

Embodiment 2-4

Using the structure and preparation method of Example 1, the composition of the sensitive material and the composition of the hemolyzed substance are shown in Table 1, and biosensors 2 to 4 for dry measurement of hemoglobin were made respectively.

Table 1

test case

Take the dry-type biosensor 1 for measuring hemoglobin as an example. When in use, use a pipette gun to suck 10uL of blood to be tested and drop it on the inlet 8 of the liquid to be tested. 8 enters the hemolysis channel 9, and carries out the hemolysis reaction within 10-20 seconds of flowing in the hemolysis channel 9. Afterwards, the lysed blood reaches the reaction tank 10, and the hemoglobin flowing out from the blood cells contacts the sensitive membrane, and undergoes electron exchange reaction, generating a current positively related to the hemoglobin concentration. The current is detected by the electrochemical workstation, the working electrode of the electrochemical workstation is connected to the electrode output terminal 7 of the sensor, the counter electrode and the reference electrode of the workstation are connected to the counter electrode of the electrode output terminal 7, and a reaction voltage (0.2 v-0.6v, preferably 0.5v), use the hemoglobin concentration of the people's whole blood calibrated by the standard instrument as a standard, establish the calibration curve of the hemoglobin concentration and the current output signal, as shown in Figure 3, the correlation curve between hemoglobin and the current response is y=0.0362x+1.3755 (x is the hemoglobin concentration g/L; y is the response current μA of the electrochemical workstation), and the correlation reaches 0.9945.

Claims (10)

1. A dry biosensor for measuring hemoglobin, characterized in that, the biosensor comprises a sensor substrate, an insulating layer, a flow path layer, and a diversion film, and the sensor substrate is provided with a reaction electrode and an electrode output terminal , The conductive line connecting the reaction electrode and the electrode output end, an insulating layer is provided between the conductive line and the flow path layer, the insulating layer is provided with a hollow groove corresponding to the reaction electrode, and the flow path layer is provided with the inlet of the liquid to be tested and the hemolysis channel connected in sequence 1. A reaction tank, wherein the hemolysis channel is modified with a dry hemolysis substance, and a diversion film is arranged on the hemolysis channel and the reaction tank. 2. A kind of biosensor for dry measuring hemoglobin according to claim 1, characterized in that, said sensor base material is selected from polystyrene, polypropylene, polymethyl methacrylate, polyethylene, polyethylene One of the terephthalate esters, the thickness is 0.2-2mm. 3. A kind of biosensor for dry measuring hemoglobin according to claim 1, characterized in that the reaction electrode is formed by screen printing from conductive carbon paste, and the reaction electrode is arranged in the flow path layer through the hollow groove of the insulating layer bottom of the reaction tank. 4. a kind of biosensor of dry measuring hemoglobin according to claim 3, is characterized in that, is coated with sensitive material to form sensitive film on the reaction electrode, and sensitive material is made of electron mediator, surfactant, plasticizer. become. 5. a kind of dry-type biosensor for measuring hemoglobin according to claim 4, is characterized in that, described electron mediator is selected from methylene blue, potassium ferricyanide, ferrocene and derivatives thereof, phenol violet One of them, the dosage is that the molar volume ratio of the sensitive material is 0.04-0.2mol: 1L, and the surfactant is selected from one of Triton x-100, Tween 20, quaternary ammonium salts, and sodium laurate , the dosage is that the mass volume ratio of the sensitive material is 0.001-3g: 1L, and the plasticizer is selected from one of sodium humate, carboxymethyl cellulose, polyvinyl alcohol, and phthalate, and the usage amount is the same as that of the sensitive material The molar volume ratio is 1-30mmol: 1L. 6. A kind of biosensor for dry measuring hemoglobin according to claim 1, characterized in that, the electrode output end and the conductive circuit are formed by screen printing with conductive silver paste, and the electrode output end is arranged on the insulating layer and the conduction line. In addition to the end of the layer, the electrode output includes the working electrode and the counter electrode. 7. A dry biosensor for measuring hemoglobin according to claim 1, characterized in that, the dry hemolyzed substance is modified in the hemolyzed channel of the flow path layer, and the dry hemolyzed substance is selected from non-ionic active agent solutions, and the concentration content 1-40g/L. 8. A kind of dry-type biosensor for measuring hemoglobin according to claim 7, characterized in that the dry-type hemolysate is selected from dodecylpyridinium ammonium chloride, dodecylpyridinium ammonium bromide, dodecyl One of sodium sulfonate, saponin, polyethylene glycol octylphenyl ether, and cetyltrimethylammonium bromide. 9. A method for detecting hemoglobin with a dry biosensor for measuring hemoglobin as claimed in claim 1, wherein the detection method is: directly inject the whole blood of the people to be tested into the inlet of the solution to be tested, and the electrochemical instrument will work The voltage is applied to the electrode output terminal, and after 30 seconds to 2 minutes, the electrochemical detection instrument detects the concentration of hemoglobin. 10 . The method for detecting hemoglobin with a dry biosensor for measuring hemoglobin according to claim 9 , wherein the amount of human blood to be tested is 6-10 uL. 11 .