CN102928468B - Whole blood urea bio-sensing test paper - Google Patents
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
本发明涉及一种全血尿素生物传感试纸，包括基片，所述基片上印有工作电极、参考电极和与所述工作电极及参考电极配套的连接导线、所述基片的一端印有用于与测试仪器相连的连接导线输出端子，所述工作电极和参考电极上有可与尿素进行反应的反应物层。采用本发明进行血尿素测试，相比于现有技术下需要半自动或全自动仪的酶动力比色法测试更为方便快捷，可以使得血尿素指标更为广泛地应用在体育监测或医疗分析中，也可以为普通人群提供一种简单快捷的测试方法。
The invention relates to a whole blood urea biosensing test paper, which comprises a substrate, on which a working electrode, a reference electrode and connecting wires matched with the working electrode and the reference electrode are printed, and one end of the substrate is printed with a The working electrode and the reference electrode have a reactant layer that can react with urea on the output terminal of the connecting wire connected with the test instrument. Using the present invention to test blood urea is more convenient and quicker than the enzyme kinetic colorimetry test that requires a semi-automatic or fully automatic instrument in the prior art, and can make the blood urea index more widely used in sports monitoring or medical analysis , can also provide a simple and quick test method for the general population.
Description
技术领域technical field
本发明涉及一种全血尿素生物传感试纸，属于生物传感技术领域。The invention relates to a whole blood urea biosensing test paper, which belongs to the technical field of biosensing.
背景技术Background technique
血尿素指标能反映运动时机体蛋白质的代谢情况，可作为评定运动量和监督过度训练的敏感指标，因此不论是在运动训练过程及之后的运动恢复效果评价中都是一项极为重要的监测指标。The blood urea index can reflect the body’s protein metabolism during exercise, and can be used as a sensitive index for evaluating exercise volume and supervising overtraining. Therefore, it is an extremely important monitoring index both in the process of exercise training and in the evaluation of exercise recovery after exercise.
目前，国内外关于测定血尿素的方法有缩合法和酶偶联速率法等。其中缩合法精度较差，酶偶联速率法相对快速准确。这两种方法都需要经过取血、离心血清、试剂加样、上机比色等过程，用时相对较长。如有学者报道，分光光度法测试一个样品的时间大约为40分钟，还需要有熟练的操作人员，不利于血尿素指标的广泛应用，特别不利于运动员的现场监测。At present, there are condensation method and enzyme coupling rate method for the determination of blood urea at home and abroad. Among them, the condensation method has poor accuracy, and the enzyme coupling rate method is relatively fast and accurate. These two methods need to go through the process of blood collection, centrifuged serum, reagent sample addition, and colorimetry on the machine, which takes a relatively long time. As some scholars reported, the spectrophotometric method takes about 40 minutes to test a sample and requires skilled operators, which is not conducive to the wide application of blood urea indicators, especially not conducive to the on-site monitoring of athletes.
酶电极法具有测试速度快、特异性高、灵敏度高的特点，是简化测试程序实现现场使用的最佳方法。酶电极法测试一个样品加上恢复时间大约为15分钟，而且血清样品不需要其它预处理，对于已经着色的分光光度法难以测定的样品，酶电极法仍然有效。传统的尿素酶电极测量法利用脲酶催化尿素，生成NH3，随着反应进行也会引起pH值的变化，尿素由脲酶催化生成的NH3由氨气敏电极响应，从而根据氨气的量推测出尿素的量。可使这种方法实际组成体系的复杂性也限制了其广泛的应用。The enzyme electrode method has the characteristics of fast test speed, high specificity and high sensitivity, and is the best method to simplify the test procedure and realize on-site use. The enzyme electrode method is about 15 minutes to test a sample plus the recovery time, and the serum sample does not need other pretreatments, and the enzyme electrode method is still effective for samples that are difficult to measure by spectrophotometry that has been stained. The traditional urease electrode measurement method uses urease to catalyze urea to generate NH 3 , which will also cause a change in pH value as the reaction progresses. The NH 3 generated by urease catalyzed by urea is responded to by an ammonia gas-sensing electrode, so it can be estimated based on the amount of ammonia gas The amount of urea produced. The complexity that allows this method to be practically composed also limits its broad applicability.
发明内容Contents of the invention
为克服现有技术的上述缺陷，本发明提供了一种全血尿素生物传感试纸，可快速简便地进行全血血尿素测试，有利于广泛应用。In order to overcome the above-mentioned defects of the prior art, the present invention provides a whole blood urea biosensing test paper, which can quickly and easily perform the whole blood urea test, and is beneficial to wide application.
本发明所采用的技术方案为：一种全血尿素生物传感试纸，包括基片，所述基片上印有工作电极、参考电极和与所述工作电极及参考电极配套的连接导线，所述基片的一端印有用于与测试仪器相连的连接导线输出端子，所述工作电极和参考电极上有可与尿素进行反应的反应物层。The technical solution adopted in the present invention is: a whole blood urea biosensing test paper, including a substrate, on which a working electrode, a reference electrode and connecting wires matched with the working electrode and the reference electrode are printed, and the One end of the substrate is printed with an output terminal of a connecting wire used to connect with a test instrument, and the working electrode and the reference electrode have a reactant layer that can react with urea.
所述反应物层的反应物可以包括α-酮戊二酸和NADH。The reactants of the reactant layer may include α-ketoglutarate and NADH.
优选地，所述反应物层含α-酮戊二酸7～26.28ug，含NADH36.64～119.4ug。Preferably, the reactant layer contains 7-26.28 ug of α-ketoglutarate and 36.64-119.4 ug of NADH.
所述连接导线和工作电极及参考电极优选由印在所述基片上的导电层和印在所述导电层上的碳涂层构成，所述参考电极的碳涂层与所述反应物层之间优选印有Ag/AgCl层。The connecting wire, the working electrode and the reference electrode are preferably composed of a conductive layer printed on the substrate and a carbon coating printed on the conductive layer, and the carbon coating of the reference electrode is connected to the reactant layer. Preferably an Ag/AgCl layer is printed between them.
所述工作电极和参考电极上可以通过双面胶带纸粘贴覆盖有塑料薄膜，所述双面胶带纸优选为两条，每条均横向粘贴在所述工作电极和参考电极上，两条所述双面胶带纸之间优选留有间隙，构成横向的反应池通道。The working electrode and the reference electrode can be pasted and covered with a plastic film by a double-sided adhesive tape. The double-sided adhesive tape is preferably two, each of which is horizontally pasted on the working electrode and the reference electrode. A gap is preferably left between the double-sided adhesive tapes to form a transverse reaction pool channel.
所述反应物层还可以含有用于对尿素与所述反应物的反应起催化作用的酶。The reactant layer may also contain enzymes for catalyzing the reaction of urea with the reactant.
所述起催化作用的酶可以包括脲酶、谷氨酸脱氢酶和谷氨酸氧化酶。The catalytic enzymes may include urease, glutamate dehydrogenase and glutamate oxidase.
优选地，所述酶中含脲酶0.12～0.36U，含谷氨酸脱氢酶0.24～0.72U，含谷氨酸氧化酶0.24～0.72U。Preferably, the enzyme contains 0.12-0.36 U of urease, 0.24-0.72 U of glutamate dehydrogenase, and 0.24-0.72 U of glutamate oxidase.
所述反应物层还可以含有在反应时用于电子传递的电子媒介物质、用于固定酶的物质和缓冲液。The reactant layer may also contain an electron mediator substance for electron transfer during the reaction, a substance for immobilizing enzymes, and a buffer.
所述电子媒介物质可以为铁氰化钾，所述反应物层优选含铁氰化钾18.19～59.26ug；所述用于固定酶的物质可以为明胶、丝素蛋白和牛血清白蛋白与戊二醛交联中的任意一种，所述反应物层优选含用于固定酶的物质20～40ug；所述缓冲液可以为咪唑缓冲液或磷酸缓冲液，所述反应物层含缓冲液优选为40～100ug，所述缓冲液的pH值为6.5～7.5。The electron mediator substance may be potassium ferricyanide, and the reactant layer preferably contains 18.19-59.26ug of potassium ferricyanide; the substance used for immobilizing enzymes may be gelatin, silk fibroin, bovine serum albumin and pentadiene Any one of aldehyde cross-linking, the reactant layer preferably contains 20-40ug of substances for immobilizing enzymes; the buffer can be imidazole buffer or phosphate buffer, and the reactant layer contains buffer preferably 40-100ug, and the pH value of the buffer solution is 6.5-7.5.
本发明的有益效果：采用本发明进行测试时，将试纸插入测试仪器的插口，形成测试回路，再将血液滴加在反应物层上，反应物使工作电极和参考电极连通，即可通过测试仪器获得工作电极和参考电极之间的电阻特性或其他电特性，由于这些电特性由反应物同血滴接触后发生的反应特性决定，体现血液中血尿素的含量，由此测试仪器根据该特性进行运算后可获得相应的血尿素测试结果。采用本发明进行血尿素的测试，相比于现有技术下需要半自动或全自动仪的酶动力比色法测试更为方便快捷，可以使得血尿素指标更为广泛地应用在体育监测或医疗分析中，可以为普通人群或者没有大型测试仪器条件的人群提供一种简单快捷的测试方法；本发明反应池通道的设计还方便左右手不同手指的取血。Beneficial effects of the present invention: when using the present invention for testing, the test paper is inserted into the socket of the test instrument to form a test circuit, and then the blood is dripped on the reactant layer, and the reactant connects the working electrode and the reference electrode, and the test can be passed The instrument obtains the resistance characteristics or other electrical characteristics between the working electrode and the reference electrode. Since these electrical characteristics are determined by the reaction characteristics of the reactant after contact with the blood drop, it reflects the content of blood urea in the blood. Therefore, the testing instrument is based on this characteristic. After calculation, the corresponding blood urea test result can be obtained. Using the present invention to test blood urea is more convenient and quicker than the enzyme kinetic colorimetry test that requires a semi-automatic or fully automatic instrument in the prior art, and can make the blood urea index more widely used in sports monitoring or medical analysis Among them, it can provide a simple and fast test method for the general population or those without large-scale testing equipment; the design of the reaction pool channel of the present invention is also convenient for blood collection from different fingers of the left and right hands.
附图说明Description of drawings
图1为本发明的结构示意图；Fig. 1 is a structural representation of the present invention;
图2为本发明的结构分解图。Figure 2 is an exploded view of the structure of the present invention.
具体实施方式detailed description
参见图1和图2，本发明提供了一种全血尿素生物传感试纸，包括基片1，所述基片可以采用PVC板材，所述基片上可以采用丝网印刷法印有工作电极、参考电极和与所述工作电极及参考电极配套的连接导线，所述基片的一端印有用于与测试仪器相连的连接导线输出端子2，所述工作电极和参考电极上有可与尿素进行反应的反应物层3。Referring to Fig. 1 and Fig. 2, the present invention provides a kind of whole blood urea biosensing test paper, comprise substrate 1, described substrate can adopt PVC sheet material, can adopt screen printing method to be printed with working electrode, reference on the described substrate. electrode and the supporting connecting wire with the working electrode and the reference electrode, one end of the substrate is printed with a connecting wire output terminal 2 for connecting with the test instrument, and the working electrode and the reference electrode have a Reactant Layer 3.
所述反应物层的反应物可以包括α-酮戊二酸和NADH，所述反应物层优选含α-酮戊二酸7～26.28ug，如：7ug、10ug、15ug、20ug或26.28ug；所述反应物层优选含NADH36.64～119.4ug，如36.64ug、50ug、80ug、100ug或119.4ug。The reactants of the reactant layer may include α-ketoglutarate and NADH, and the reactant layer preferably contains 7-26.28ug of α-ketoglutarate, such as: 7ug, 10ug, 15ug, 20ug or 26.28ug; The reactant layer preferably contains 36.64-119.4ug of NADH, such as 36.64ug, 50ug, 80ug, 100ug or 119.4ug.
所述连接导线和工作电极及参考电极优选由印在所述基片上的导电层5和印在所述导电层上的碳涂层6构成。所述导电层的材料可以为导电银浆油墨，所述碳涂层的材料可以为碳油墨，所述参考电极的碳涂层与所述反应物层之间可以印有Ag/AgCl层4。The connecting wires and working and reference electrodes are preferably formed by a conductive layer 5 printed on the substrate and a carbon coating 6 printed on the conductive layer. The material of the conductive layer may be conductive silver paste ink, the material of the carbon coating may be carbon ink, and an Ag/AgCl layer 4 may be printed between the carbon coating of the reference electrode and the reactant layer.
所述反应物层还可以含有用于对尿素与所述反应物的反应起催化作用的酶，所述起催化作用的酶可以包括脲酶、谷氨酸脱氢酶和谷氨酸氧化酶。所述酶中优选含脲酶0.12～0.36U，如：0.12U、0.15U、0.2U、0.25U、0.3U或0.36U；所述酶中优选含谷氨酸脱氢酶0.24～0.72U，如：0.24U、0.3U、0.5U、0.6U或0.72U；所述酶中优选含谷氨酸氧化酶0.24～0.72U，如：0.24U、0.3U、0.5U、0.6U或0.72U。The reactant layer may also contain enzymes for catalyzing the reaction between urea and the reactants, and the catalytic enzymes may include urease, glutamate dehydrogenase and glutamate oxidase. The enzyme preferably contains 0.12-0.36U of urease, such as: 0.12U, 0.15U, 0.2U, 0.25U, 0.3U or 0.36U; the enzyme preferably contains 0.24-0.72U of glutamate dehydrogenase, such as 0.24U, 0.3U, 0.5U, 0.6U or 0.72U; the enzyme preferably contains 0.24-0.72U of glutamic acid oxidase, such as: 0.24U, 0.3U, 0.5U, 0.6U or 0.72U.
所述反应物层还可以含有在反应时用于电子传递的电子媒介物质、用于固定酶的物质和缓冲液。所述电子媒介物质可以为铁氰化钾，所述反应物层优选含铁氰化钾18.19～59.26ug，如：18.19ug、20ug、30ug、50ug或59.26ug；所述用于固定酶的物质可以为明胶、丝素蛋白和牛血清白蛋白与戊二醛交联中的任意一种，所述反应物层优选含用于固定酶的物质20～40ug，如：20ug、25ug、30ug、35ug或40ug；所述缓冲液可以为咪唑缓冲液或磷酸缓冲液，所述反应物层优选含缓冲液40～100ug，如：40ug、60ug、80ug、90ug或100ug，所述缓冲液的pH值为6.5～7.5。The reactant layer may also contain an electron mediator substance for electron transfer during the reaction, a substance for immobilizing enzymes, and a buffer. The electron mediator substance can be potassium ferricyanide, and the reactant layer preferably contains 18.19-59.26ug of potassium ferricyanide, such as: 18.19ug, 20ug, 30ug, 50ug or 59.26ug; the substance used to immobilize the enzyme It can be any one of gelatin, silk fibroin and bovine serum albumin cross-linked with glutaraldehyde, and the reactant layer preferably contains 20-40ug of substances for immobilizing enzymes, such as: 20ug, 25ug, 30ug, 35ug or 40ug; the buffer can be imidazole buffer or phosphate buffer, the reactant layer preferably contains buffer 40 ~ 100ug, such as: 40ug, 60ug, 80ug, 90ug or 100ug, the pH of the buffer is 6.5 ~7.5.
所述工作电极和参考电极上可以通过双面胶带纸7粘贴覆盖有塑料薄膜8，所述双面胶带纸优选为两条，每条均横向粘贴在所述工作电极和参考电极上，两条所述双面胶带纸之间留有间隙，构成横向的反应池通道9，便于通过虹吸现象引导待测血液与所述反应物层充分接触，此种设计，需要进行测试时，只需将人体取血位置接触反应池通道任意一端的开口即可，依靠反应池通道的虹吸作用即可将血液吸入所述反应池通道，所述反应池通道两端的开口适用于左右手不同的手指取血。所述塑料薄膜还可防止所述工作电极和参考电极暴漏在空气中，对测试结果造成影响。The working electrode and the reference electrode can be pasted and covered with a plastic film 8 by a double-sided adhesive tape 7, and the double-sided adhesive tape is preferably two, each of which is horizontally pasted on the working electrode and the reference electrode. There is a gap between the double-sided adhesive tapes to form a horizontal reaction pool channel 9, which is convenient to guide the blood to be tested to fully contact the reactant layer through the siphon phenomenon. With this design, when testing is required, only the human body The blood collection position only needs to be in contact with the opening at any end of the reaction pool channel, and the blood can be sucked into the reaction pool channel by the siphon effect of the reaction pool channel. The plastic film can also prevent the working electrode and the reference electrode from being exposed to the air, which will affect the test results.
所述基片设置所述工作电极和参考电极的一端可以设有向外延伸的手持端10，所述手持端的长度优选为10～18mm，所述手持端的设计，有利于试纸的拿取，避免了拿取试纸时对反应区域造成污染而影响测试效果。One end of the substrate on which the working electrode and the reference electrode are set can be provided with a hand-held end 10 extending outwards. The length of the hand-held end is preferably 10-18mm. The design of the hand-held end is conducive to the taking of test paper and avoids In order to pollute the reaction area when taking the test paper and affect the test effect.
实施例1：Example 1:
用丝网印刷方法在PVC基片上用导电银浆油墨印刷连接导线导电层、工作电极导电层和参考电极导电层，在连接导线导电层、工作电极导电层和参考电极导电层上用碳油墨印刷连接导线碳涂层、工作电极碳涂层和参考电极碳涂层，再用银浆油墨与AgCl的混合物印刷在参考电极碳涂层上构成Ag/AgCl层，银浆油墨与AgCl的混合比例为10/1（W/W）。在工作电极和参考电极上横向粘贴两条双面胶带纸，两条双面胶带纸之间留有间隙，构成反应池通道。将含有α-酮戊二酸26.28ug、NADH119.4ug、脲酶0.36U、谷氨酸脱氢酶0.72U、谷氨酸氧化酶0.72U、铁氰化钾59.26ug、明胶20ug、pH值为7的咪唑缓冲液40ug的混合液5ul滴加在反应池通道的工作电极上，在干燥的室温下形成反应物层。两条双面胶带纸上覆盖一整片塑料薄膜，与反应池通道配合构成双侧开口的反应池。PVC基片设有工作电极和参考电极的一端设有向外延伸12mm的空白端作为手持端。Use the screen printing method to print the conductive layer of the connecting wire, the conductive layer of the working electrode and the conductive layer of the reference electrode on the PVC substrate with conductive silver paste ink, and print with carbon ink on the conductive layer of the connecting wire, the conductive layer of the working electrode and the conductive layer of the reference electrode Connect the wire carbon coating, the working electrode carbon coating and the reference electrode carbon coating, and then use the mixture of silver paste ink and AgCl to print on the reference electrode carbon coating to form an Ag/AgCl layer. The mixing ratio of silver paste ink and AgCl is 10/1 (W/W). Paste two double-sided tapes horizontally on the working electrode and the reference electrode, leaving a gap between the two double-sided tapes to form the channel of the reaction pool. Containing α-ketoglutarate 26.28ug, NADH119.4ug, urease 0.36U, glutamate dehydrogenase 0.72U, glutamate oxidase 0.72U, potassium ferricyanide 59.26ug, gelatin 20ug, pH value 7 5 ul of the mixed solution of 40 ug of imidazole buffer solution was added dropwise on the working electrode of the channel of the reaction cell, and a reactant layer was formed at a dry room temperature. The two double-sided adhesive tapes are covered with a whole piece of plastic film, which cooperates with the channel of the reaction pool to form a reaction pool with openings on both sides. One end of the PVC substrate provided with the working electrode and the reference electrode is provided with a blank end extending outward for 12 mm as a hand-held end.
实施例2：Example 2:
用丝网印刷方法在PVC基片上用导电银浆油墨印刷连接导线导电层、工作电极导电层和参考电极导电层，在连接导线导电层、工作电极导电层和参考电极导电层上用碳油墨印刷连接导线碳涂层、工作电极碳涂层和参考电极碳涂层，再用银浆油墨与AgCl的混合物印刷在参考电极碳涂层上构成Ag/AgCl层，银浆油墨与AgCl的混合比例为10/1（W/W）。在工作电极和参考电极上横向粘贴两条双面胶带纸，两条双面胶带纸之间留有间隙，构成反应池通道。将含有α-酮戊二酸26.28ug、NADH119.4ug、脲酶0.36U、谷氨酸脱氢酶0.72U、谷氨酸氧化酶0.72U、铁氰化钾59.26ug、丝素蛋白40ug、pH值为7的磷酸缓冲液100ug的混合液5ul滴加在反应池通道的工作电极上，在干燥的室温下形成反应物层。两条双面胶带纸上覆盖一整片塑料薄膜，与反应池通道配合构成双侧开口的反应池。PVC基片设有工作电极和参考电极的一端设有向外延伸12mm的空白端作为手持端。Use the screen printing method to print the conductive layer of the connecting wire, the conductive layer of the working electrode and the conductive layer of the reference electrode on the PVC substrate with conductive silver paste ink, and print with carbon ink on the conductive layer of the connecting wire, the conductive layer of the working electrode and the conductive layer of the reference electrode Connect the wire carbon coating, the working electrode carbon coating and the reference electrode carbon coating, and then use the mixture of silver paste ink and AgCl to print on the reference electrode carbon coating to form an Ag/AgCl layer. The mixing ratio of silver paste ink and AgCl is 10/1 (W/W). Paste two double-sided tapes horizontally on the working electrode and the reference electrode, leaving a gap between the two double-sided tapes to form the channel of the reaction pool. Will contain α-ketoglutarate 26.28ug, NADH119.4ug, urease 0.36U, glutamate dehydrogenase 0.72U, glutamate oxidase 0.72U, potassium ferricyanide 59.26ug, silk fibroin 40ug, pH 5 ul of a mixture of 100 ug of phosphate buffer solution of 7 was added dropwise on the working electrode of the channel of the reaction cell, and a reactant layer was formed at a dry room temperature. The two double-sided adhesive tapes are covered with a whole piece of plastic film, which cooperates with the channel of the reaction pool to form a reaction pool with openings on both sides. One end of the PVC substrate provided with the working electrode and the reference electrode is provided with a blank end extending outward for 12 mm as a hand-held end.
本发明的生物化学反应过程如下：Biochemical reaction process of the present invention is as follows:
需要进行测试时，将试纸设有所述连接导线输出端子的一端插入测试仪器的插口，所述全血尿素生物传感试纸的连接导线输出端子与相应的测试仪器输入端子连接，形成测试回路，再将血液滴加在反应物层上，反应物使工作电极和参考电极连通，即可通过测试仪器获得工作电极和参考电极之间的电阻特性或其他电特性，由于这些电特性由反应物同血滴接触后发生的反应特性决定，体现血液中血尿素的含量，由此测试仪器根据该特性进行运算后可获得相应的血尿素测试结果，并显示在显示屏上，因此可以从测试仪器上直接读取测试数据。When testing is required, the end of the test paper provided with the output terminal of the connecting wire is inserted into the socket of the test instrument, and the output terminal of the connecting wire of the whole blood urea biosensing test paper is connected to the corresponding input terminal of the test instrument to form a test loop. Then the blood is dripped on the reactant layer, and the reactant connects the working electrode and the reference electrode, and the resistance characteristics or other electrical characteristics between the working electrode and the reference electrode can be obtained through the test instrument, because these electrical characteristics are determined by the reactant and the reference electrode. The characteristics of the reaction that occurs after the contact of the blood drop is determined, reflecting the content of blood urea in the blood, so the test instrument can obtain the corresponding blood urea test result after calculating according to this characteristic, and display it on the display screen, so it can be read from the test instrument. Read test data directly.
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