TWI644101B - A test strip for electrochemical biosensor with high stability and manufacturing method thereof - Google Patents
A test strip for electrochemical biosensor with high stability and manufacturing method thereof Download PDFInfo
- Publication number
- TWI644101B TWI644101B TW106129252A TW106129252A TWI644101B TW I644101 B TWI644101 B TW I644101B TW 106129252 A TW106129252 A TW 106129252A TW 106129252 A TW106129252 A TW 106129252A TW I644101 B TWI644101 B TW I644101B
- Authority
- TW
- Taiwan
- Prior art keywords
- test piece
- ferricyanide
- electrochemical biosensor
- group
- electrode
- Prior art date
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
本發明提供一種具高穩定性電化學生物感測器試片,其具有鐵氰化物穩定劑以及光遮蔽劑成分,能有效克服試片易變質的缺點。因此,本發明之電化學生物感測器試片具有製造過程穩定、保存期限長、耐高溫以及耐光害之優勢。The invention provides a high stability electrochemical biosensor test piece which has a ferricyanide stabilizer and a light shielding agent component, and can effectively overcome the disadvantage that the test piece is easily deteriorated. Therefore, the electrochemical biosensor test piece of the present invention has the advantages of stable manufacturing process, long shelf life, high temperature resistance and light resistance.
Description
本發明提供一種具高穩定性的電化學生物感測器試片,特別係提供一種具耐高溫以及光的電化學生物感測器試片。The invention provides an electrochemical biosensor test piece with high stability, in particular to provide an electrochemical biosensor test piece with high temperature resistance and light.
電化學生物感測器中以酵素電極應用最早也最廣泛的。目前商業化電化學生物感測器以使用電子傳遞介質(mediators)協助電子傳遞的電化學生物感測器為主,其反應過程如圖1所示(以葡萄糖檢測做為例):第一步,血液樣品中的葡萄糖與葡萄糖氧化酶反應後形成葡萄糖酸和水,水與鐵氰化鉀反應變成亞鐵氰化鉀;第二步,外加電壓給此反應系統,使亞鐵氰化鉀氧化為鐵氰化鉀並釋放出電子,經由偵測所釋放出來的電流大小,及可計算出葡萄糖的濃度。The application of enzyme electrodes in electrochemical biosensors is the earliest and most widely used. At present, commercial electrochemical biosensors mainly use electrochemical biosensors that use electron transfer media to assist electron transfer. The reaction process is shown in Figure 1 (taking glucose detection as an example): the first step The glucose in the blood sample reacts with glucose oxidase to form gluconic acid and water, and the water reacts with potassium ferricyanide to become potassium ferrocyanide; in the second step, an external voltage is applied to the reaction system to oxidize potassium ferrocyanide. It is potassium ferricyanide and emits electrons. By detecting the amount of current released, and calculating the concentration of glucose.
影響使用電子傳遞介質協助電子傳遞的電化學生物感測器穩定性及壽命因素,以塗佈之酵素及電子傳遞介質的穩定性最為重要。由於酵素極易受溫度等因素影響其活性;以及電子傳遞介質易受溫度、光照等因素影響其化學性質。而鐵氰化鉀(Potassium ferricyanide)是各種使用氧化還原酵素的生物感測器中,非常常見的電子傳遞介質,其成本低適合用於製作成便宜的一次性的試片;且鐵氰化鉀在水中溶解度高,在輔助電極上能有足夠的相對反應以平衡工作電極上的反應。 相對反應提供足夠的電流以及化學位能,使得檢測時不需要參考電極,也不需要大於工作電極的輔助電極,表示使用鐵氰化鉀作為電子傳遞介質的電化學生物感測器可以簡化為具兩個電極的尺寸大小。The stability and lifetime factors of electrochemical biosensors that use electron transfer media to assist electron transport are most important for the stability of coated enzymes and electron transport media. Because enzymes are highly susceptible to temperature and other factors, and electron transfer media are susceptible to temperature, light and other factors affecting their chemical properties. Potassium ferricyanide is a very common electron transfer medium in various biosensors using redoxase, and its low cost is suitable for making disposable disposable test pieces; and potassium ferricyanide The solubility in water is high and there is sufficient relative reaction on the auxiliary electrode to balance the reaction on the working electrode. The relative reaction provides sufficient current and chemical potential energy so that the reference electrode is not required for the detection, and the auxiliary electrode larger than the working electrode is not required, indicating that the electrochemical biosensor using potassium ferricyanide as the electron transport medium can be simplified. The size of the two electrodes.
然而,使用鐵氰化鉀作為電子傳遞介質的電化學生物感測器具有以下之缺點,因鐵氰化鉀於其易受光、熱影響而轉變為亞鐵氰化鉀,而導致試片變質。尤其在當鐵氰化鉀與酵素混合成反應試劑時,即使是置於乾燥環境中保存,與酵素的反應或酵素自身氧化還原反應依然會進行。例如常見於檢測血糖的試片中,即便沒有葡萄糖參與,鐵氰化鉀與酵素混合反應試劑仍然會在儲存放置期間,兩者相互反應,而使得偵測的背景值提高,出現假性血糖高值。此種錯誤診斷結果會導致錯誤的醫療行為,譬如糖尿病人血糖檢測中,如誤診為高血糖而施予降血糖治療,會導致嚴重低血糖昏迷,甚至死亡。However, an electrochemical biosensor using potassium ferricyanide as an electron transporting medium has the following disadvantages, since potassium ferricyanide is converted into potassium ferrocyanide due to its vulnerability to light and heat, resulting in deterioration of the test piece. In particular, when potassium ferricyanide is mixed with an enzyme to form a reaction reagent, even if it is stored in a dry environment, the reaction with the enzyme or the auto-oxidation-reduction reaction of the enzyme proceeds. For example, in a test strip for detecting blood sugar, even if no glucose is involved, the potassium ferricyanide and enzyme mixed reaction reagent will still react with each other during storage and storage, so that the detected background value is increased and pseudo-hyperglycemia is high. value. Such misdiagnosis results in erroneous medical behaviors, such as blood glucose testing in diabetics, such as misdiagnosis of hyperglycemia and hypoglycemia treatment, which can lead to severe hypoglycemia coma and even death.
再者,商業化試片往往需要一年或較長的架上保存期限,在此期間也常遇到極冷、極熱或潮濕的環境;且用於電化學生物感測器的電子傳遞介質大多對光敏感,因而造成量測的不準確性。因此,一個可抵抗環境變化且具較常保存期限的電化學生物感測器試片,實有其存在的必要性。Furthermore, commercial test strips often require a shelf life of one year or longer, during which time they often encounter extremely cold, extremely hot or humid environments; and electronic transfer media for electrochemical biosensors. Most are sensitive to light, which results in inaccuracies in measurement. Therefore, an electrochemical biosensor test piece that is resistant to environmental changes and has a relatively long shelf life has its necessity.
為了解決這樣的問題,本發明一種添加鐵氰化物穩定劑以及光遮蔽劑的電化學生物感測器試片,以提升利用氧化還原酵素並使用鐵氰化物作為電子傳遞介質的電化學生物感測器對光以及熱之穩定性,以提供製程穩定性、架上保存期限延長、耐高溫環境保存的試片。In order to solve such a problem, the present invention relates to an electrochemical biosensor test piece incorporating a ferricyanide stabilizer and a light shielding agent to enhance electrochemical biosensing using oxidoreductase and using ferricyanide as an electron transport medium. The stability of the light and heat to provide process stability, extended shelf life, and high temperature environment.
本發明之一目的在於提供一種檢測樣本中待測物的電化學生物感測器試片,包含:一基板;一電路層,設置於該基板之上,其中該電路層至少包含一第一電極以及一第二電極;一試劑層,設置於該電路層之上,其中該第一電極與該第二電極交互而成、與該試劑層重疊形成一電極反應區;一黏著層,設置於該試劑層之上,並部分露出底下之該電極反應區,以利進行一待測物之檢測;以及一覆蓋層,設置於該黏著層之上,並部分露出底下之該試劑層及該電極反應區;其中該試劑層包含一試劑層組合物,且該試劑層組合物包含:一鐵氰化物、一氧化還原酵素、一鐵氰化物穩定劑以及一光遮蔽劑;且該氧化還原酵素對於該待測物是具專一性的。An object of the present invention is to provide an electrochemical biosensor test piece for detecting a sample to be tested in a sample, comprising: a substrate; a circuit layer disposed on the substrate, wherein the circuit layer comprises at least a first electrode And a second electrode; a reagent layer disposed on the circuit layer, wherein the first electrode and the second electrode are formed to overlap with the reagent layer to form an electrode reaction zone; an adhesive layer is disposed on the electrode Above the reagent layer, partially exposing the electrode reaction zone underneath to facilitate detection of a test object; and a cover layer disposed on the adhesive layer and partially exposing the underlying reagent layer and the electrode reaction Wherein the reagent layer comprises a reagent layer composition, and the reagent layer composition comprises: a ferricyanide, a oxidoreductase, a ferricyanide stabilizer, and a light shielding agent; and the oxidoreductase The object to be tested is specific.
在本發明之一實施例中,該鐵氰化物穩定劑係選自於由聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚丙烯醯胺(polyacrylamides)、明膠(gelatin)、糊精(dextrin)、纖維素鹼金族鹽(alkali metal salts of cellulose)、水解聚乙烯醇(hydrolyzed polyvinyl alcohol)及其任意組合所組成之群組。In one embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, cellulose. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.
在本發明之另一實施例中,該鐵氰化物係為鐵氰化鉀或鐵氰化鈉。In another embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.
在本發明之一實施例中,該光遮蔽劑係選自於由硫酸鋇(BaSO 4)、二氧化鈦(TiO 2)、碳酸鈣(CaCO 3)、三氧化二鋁 (Al 2O 3)、二氧化矽(SiO 2) 、高嶺土、煅燒高嶺土、黏土以及瓷土所組成之群組。 In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO 4 ), titanium dioxide (TiO 2 ), calcium carbonate (CaCO 3 ), aluminum oxide (Al 2 O 3 ), a group consisting of cerium oxide (SiO 2 ), kaolin, calcined kaolin, clay, and china clay.
在本發明之一實施例中,其中該待測物係葡萄糖時,該氧化還原酵素係為葡萄糖氧化酶或葡萄糖脫氫酶;該待測物係尿酸時,該氧化還原酵素係為尿酸酶;該待測物係膽固醇時,該氧化還原酵素係為膽固醇酯酶或膽固醇氧化酶;該待測物係三酸甘油酯時,該氧化還原酵素係酯酶、甘油激酶或磷酸甘油氧化酶;該待測物係肌酸酐時,該氧化還原酵素係該肌酸酐酶、肌酸酶或肌氨酸氧化酶。In one embodiment of the present invention, wherein the analyte is glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and when the analyte is uric acid, the oxidoreductase is uricase; When the test substance is cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test substance is triglyceride, the oxidoreductase esterase, glycerol kinase or phosphoglycerol oxidase; When the test substance is creatinine, the oxidoreductase is the creatininase, creatase or sarcosine oxidase.
在本發明之一實施例中,其中該試劑層組合物進一步包含一非離子界面活性劑、一消泡劑、一酵素穩定劑、一高分子酵素結構穩定劑、一纖維素増稠劑以及一緩衝劑。In an embodiment of the invention, the reagent layer composition further comprises a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and a Buffer.
在本發明之一實施例中,該緩衝劑係為0.1 M磷酸緩衝劑;該非離子界面活性劑係為采酮(Triron X-100)。In one embodiment of the invention, the buffer is a 0.1 M phosphate buffer; the nonionic surfactant is a ketol (Triron X-100).
在本發明之一實施例中,其中該酵素穩定劑係選自於由海藻糖(trehalse)、萄糖酸鈉(sodium gluconate)、甘露糖(mannose)、環糊精(cyclodextrin)、蔗糖(sucrose)、甘氨酸 (glycine)、丙氨酸(alanine)、絲氨酸(serine)、***糖(arabinose)、山梨聚糖(sorbitan)、2-去氧葡萄糖(2-deoxy-D-glucose)、木糖(xylose)、果糖(fructose)以及谷氨酸(glutamic acid)所組成之群組。In one embodiment of the invention, the enzyme stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose ), glycine, alanine, serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose A group consisting of xylose), fructose, and glutamic acid.
在本發明之一實施例中,其中該高分子酵素結構穩定劑係選自於由聚乙烯醇(polyvinyl alcohol,PVA)、聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚氧乙烯(polyethylene oxide)、聚乙烯醇吡咯烷酮與聚醋酸乙烯共聚物(copolymers of polyvinyl pyrrolidone and polyvinyl acetate)以及聚乙烯醇與聚醋酸乙烯共聚物(copolymers of polyvinyl alcohol and polyvinyl acetate)所組成之群組。In an embodiment of the invention, the polymer enzyme structure stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly A group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate.
在本發明之一實施例中,其中該纖維素増稠劑係為羧甲基纖維素(carboxymethyl cellulose,CMC)或羥乙基纖維(hydroxymethyl cellulose,HEC)。In an embodiment of the invention, the cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC).
在本發明之一實施例中,其中該試劑層藉由塗佈方式設置於該電路層之上,該塗佈方式包含空氣噴槍(air brush)、刮刀 (doctor blade)或網版印刷(screen printing)。In an embodiment of the invention, the reagent layer is disposed on the circuit layer by coating, and the coating method comprises an air brush, a doctor blade or a screen printing. ).
本發明另之一目的在於提供一種電化學生物感測器試片的製造方法,包含:提供一基板;配置一電路層於該基板上,其中該電路層至少包含一第一電極以及一第二電極;塗佈一試劑層在該電路層上,其中該第一電極和該第二電極交互而成、與該試劑層重疊形成一電極反應區,且該試劑層包含一鐵氰化物、一氧化還原酵素、一鐵氰化物穩定劑以及一光遮蔽劑;塗佈一黏著層於該試劑層上,並部分露出底下之電極反應區,以利進行一待測物檢測;以及塗佈一覆蓋層於該黏著層上,並部分露出底下之試劑層及該電極反應區。Another object of the present invention is to provide a method for manufacturing an electrochemical biosensor test strip, comprising: providing a substrate; and disposing a circuit layer on the substrate, wherein the circuit layer comprises at least a first electrode and a second An electrode is coated on the circuit layer, wherein the first electrode and the second electrode are alternately formed to overlap with the reagent layer to form an electrode reaction zone, and the reagent layer comprises a ferricyanide and an oxidation a reducing enzyme, a ferricyanide stabilizer and a light shielding agent; coating an adhesive layer on the reagent layer, partially exposing the underlying electrode reaction zone to facilitate detection of a test object; and coating a coating layer On the adhesive layer, the underlying reagent layer and the electrode reaction zone are partially exposed.
在本發明之一實施例中,其中該塗佈包含空氣噴槍(air brush)、刮刀 (doctor blade)或網版印刷(screen printing)方式。In an embodiment of the invention, wherein the coating comprises an air brush, a doctor blade or a screen printing.
在本發明之一實施例中,其中該鐵氰化物穩定劑係選自於由聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚丙烯醯胺(polyacrylamides)、明膠(gelatin)、糊精(dextrin)、纖維素鹼金族鹽(alkali metal salts of cellulose)、水解聚乙烯醇(hydrolyzed polyvinyl alcohol)及其組合所組成之群組。In an embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, and fiber. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and combinations thereof.
在本發明之另一實施例中,其中該光遮蔽劑係選自於由硫酸鋇(BaSO 4)、二氧化鈦(TiO 2)、碳酸鈣(CaCO 3)、三氧化二鋁 (Al 2O 3)、二氧化矽(SiO 2) 、高嶺土、煅燒高嶺土、黏土以及瓷土所組成之群組。 In another embodiment of the present invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO 4 ), titanium dioxide (TiO 2 ), calcium carbonate (CaCO 3 ), and aluminum oxide (Al 2 O 3 ). a group consisting of cerium oxide (SiO 2 ), kaolin, calcined kaolin, clay, and china clay.
本發明再之一目的在於提供一種檢測樣本中待測物的試劑層,包含:一鐵氰化物;一氧化還原酵素;一鐵氰化物穩定劑;以及一光遮蔽劑。在本發明之一實施例中,其中該鐵氰化物穩定劑係選自於由聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚丙烯醯胺(polyacrylamides)、明膠(gelatin)、糊精(dextrin)、纖維素鹼金族鹽(alkali metal salts of cellulose)、水解聚乙烯醇(hydrolyzed polyvinyl alcohol)及其任意組合所組成之群組。Still another object of the present invention is to provide a reagent layer for detecting a sample to be tested in a sample, comprising: a ferricyanide; a oxidoreductase; a ferricyanide stabilizer; and a light shielding agent. In an embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, and fiber. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.
在本發明之一實施例中,其中該鐵氰化物係為鐵氰化鉀或鐵氰化鈉。In an embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.
在本發明之一實施例中,其中該光遮蔽劑係選自於由硫酸鋇(BaSO 4)、二氧化鈦(TiO 2)、碳酸鈣(CaCO 3)、三氧化二鋁 (Al 2O 3)、二氧化矽(SiO 2) 、高嶺土、煅燒高嶺土、黏土以及瓷土所組成之群組。 In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO 4 ), titanium dioxide (TiO 2 ), calcium carbonate (CaCO 3 ), aluminum oxide (Al 2 O 3 ), A group consisting of cerium oxide (SiO 2 ), kaolin, calcined kaolin, clay, and china clay.
在本發明之一實施例中,其中該待測物係葡萄糖時,該氧化還原酵素係為葡萄糖氧化酶或葡萄糖脫氫酶;該待測物係尿酸時,該氧化還原酵素係為尿酸酶;該待測物係膽固醇時,該氧化還原酵素係為膽固醇酯酶或膽固醇氧化酶;該待測物係三酸甘油酯時,該氧化還原酵素係酯酶、甘油激酶或磷酸甘油氧化酶;該待測物係肌酸酐時,該氧化還原酵素係該肌酸酐酶、肌酸酶或肌氨酸氧化酶。In one embodiment of the present invention, wherein the analyte is glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and when the analyte is uric acid, the oxidoreductase is uricase; When the test substance is cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test substance is triglyceride, the oxidoreductase esterase, glycerol kinase or phosphoglycerol oxidase; When the test substance is creatinine, the oxidoreductase is the creatininase, creatase or sarcosine oxidase.
在本發明之一實施例中,其中該試劑層組合物進一步包含:一非離子界面活性劑、一消泡劑、一酵素穩定劑、一高分子酵素結構穩定劑、一纖維素増稠劑以及一緩衝劑。In an embodiment of the invention, the reagent layer composition further comprises: a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and A buffer.
在本發明之一實施例中,其中該酵素穩定劑係選自於由海藻糖(trehalse)、萄糖酸鈉(sodium gluconate)、甘露糖(mannose)、環糊精(cyclodextrin)、蔗糖(sucrose)、甘氨酸 (glycine)、丙氨酸(alanine)、絲氨酸(serine)、***糖(arabinose)、山梨聚糖(sorbitan)、2-去氧葡萄糖(2-deoxy-D-glucose)、木糖(xylose)、果糖(fructose)以及谷氨酸(glutamic acid)所組成之群組。In one embodiment of the invention, the enzyme stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose ), glycine, alanine, serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose A group consisting of xylose), fructose, and glutamic acid.
在本發明之一實施例中,其中該高分子酵素結構穩定劑係選自於由聚乙烯醇(polyvinyl alcohol,PVA)、聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚氧乙烯(polyethylene oxide)、聚乙烯醇吡咯烷酮與聚醋酸乙烯共聚物(copolymers of polyvinyl pyrrolidone and polyvinyl acetate)以及聚乙烯醇與聚醋酸乙烯共聚物(copolymers of polyvinyl alcohol and polyvinyl acetate)所組成之群組。In an embodiment of the invention, the polymer enzyme structure stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly A group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate.
在本發明之一實施例中,其中該纖維素増稠劑係為羧甲基纖維素(carboxymethyl cellulose,CMC)或羥乙基纖維(hydroxymethyl cellulose,HEC)。In an embodiment of the invention, the cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC).
在本發明之一實施例中,該緩衝劑係為0.1 M磷酸緩衝劑;該非離子界面活性劑係為采酮(Triron X-100)。In one embodiment of the invention, the buffer is a 0.1 M phosphate buffer; the nonionic surfactant is a ketol (Triron X-100).
在本發明之一實施例中,其中該試劑層藉由塗佈方式設置於該電路層之上,該塗佈方式包含空氣噴槍(air brush)、刮刀 (doctor blade)或網版印刷(screen printing)。因此,本發明一種添加鐵氰化物穩定劑以及光遮蔽劑的電化學生物感測器試片,以提高例如是鐵氰化物的電子傳遞介質對光、熱穩定性。故本發明之電化學生物感測器試片適合商業上需要一年或較長的架上保存期限、能抵抗極熱或潮濕的環境以及能抵抗光害之需求。In an embodiment of the invention, the reagent layer is disposed on the circuit layer by coating, and the coating method comprises an air brush, a doctor blade or a screen printing. ). Accordingly, the present invention provides an electrochemical biosensor test piece incorporating a ferricyanide stabilizer and a light shielding agent to improve the optical and thermal stability of an electron transport medium such as ferricyanide. Therefore, the electrochemical biosensor test piece of the present invention is suitable for a commercial one year or longer shelf life, resistant to extreme heat or humidity, and resistant to light damage.
以下將配合圖式進一步說明本發明的實施方式,下述所列舉的實施例係用以闡明本發明,並非用以限定本發明之範圍,任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.
本發明提供一種具高穩定性電化學生物感測器試片,其具有鐵氰化物穩定劑以及光遮蔽劑成分,能有效克服使用鐵氰化物作為電子傳遞介質易受熱影響而轉變為亞鐵氰化物,而導致試片變質的缺點,並能長時間維持酵素活性。本發明利用以下實施例證實該電化學生物感測器試片具有良好之耐高溫穩定性、光穩定性以及測量穩定性。 實施例 1 本發明之具高穩定性的電化學生物感測器試片的製造方法 The invention provides a high stability electrochemical biosensor test piece with a ferricyanide stabilizer and a light shielding agent component, which can effectively overcome the conversion of ferricyanide to ferrocyanide by using iron cyanide as an electron transport medium. The compound, which causes the test piece to deteriorate, and can maintain the activity of the enzyme for a long time. The present invention demonstrates that the electrochemical biosensor test piece has good high temperature stability, light stability, and measurement stability using the following examples. Embodiment 1 Method for manufacturing electrochemical biosensor test piece with high stability of the present invention
如圖2所示,本發明提供一種具有對光以及熱穩定性的用於電化學生物感測器的試片的製造方法,其步驟包含:提供一基板110;配置一電路層120於該基板110上,其中該電路層120至少包含一第一電極121以及一第二電極122;塗佈一試劑層130在該電路層120上,其中該第一電極121和該第二電極122交互而成、與該試劑層130重疊形成一電極反應區131;塗佈一黏著層140於該試劑層130上,並部分露出底下之電極反應區131,以利進行一待測物檢測;以及塗佈一覆蓋層150於該黏著層140上,並部分露出底下之試劑層130及該電極反應區131,其中該塗佈包含空氣噴槍(air brush)、刮刀 (doctor blade)或網版印刷(screen printing)方式。之後,以40 ℃乾燥30分鐘,再裝入避光之乾燥罐中,以完成試片。As shown in FIG. 2, the present invention provides a method for manufacturing a test piece for an electrochemical biosensor having light and thermal stability, the method comprising: providing a substrate 110; and disposing a circuit layer 120 on the substrate 110, wherein the circuit layer 120 includes at least a first electrode 121 and a second electrode 122; a reagent layer 130 is coated on the circuit layer 120, wherein the first electrode 121 and the second electrode 122 are formed. An electrode reaction zone 131 is formed by overlapping with the reagent layer 130; an adhesive layer 140 is coated on the reagent layer 130, and the bottom electrode reaction zone 131 is partially exposed to facilitate detection of a sample to be tested; The cover layer 150 is on the adhesive layer 140, and partially exposes the underlying reagent layer 130 and the electrode reaction zone 131, wherein the coating comprises an air brush, a doctor blade or a screen printing the way. Thereafter, it was dried at 40 ° C for 30 minutes, and then placed in a dry tank in the dark to complete the test piece.
在本發明之試劑層130中,包含:一鐵氰化物、一氧化還原酵素、一鐵氰化物穩定劑以及一光遮蔽劑。The reagent layer 130 of the present invention comprises: a ferricyanide, a oxidoreductase, a ferricyanide stabilizer, and a light shielding agent.
在本發明之一實施例中,該鐵氰化物穩定劑係選自於由聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚丙烯醯胺(polyacrylamides)、明膠(gelatin)、糊精(dextrin)、纖維素鹼金族鹽(alkali metal salts of cellulose)、水解聚乙烯醇(hydrolyzed polyvinyl alcohol)及其任意組合所組成之群組。In one embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, cellulose. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.
在本發明之一實施例中,該光遮蔽劑係選自於由硫酸鋇(BaSO 4)、二氧化鈦(TiO 2)、碳酸鈣(CaCO 3)、三氧化二鋁 (Al 2O 3) 、二氧化矽(SiO 2) 、高嶺土、煅燒高嶺土、黏土以及瓷土所組成之群組,其中硫酸鋇的折射率為1.64,其具有良好的折射力,可扮演最佳的光擴散劑,且具有特殊的正立,文體外形,更可加強反射光線,達到透光不透明的作用,為優先選擇。 In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO 4 ), titanium dioxide (TiO 2 ), calcium carbonate (CaCO 3 ), aluminum oxide (Al 2 O 3 ), a group consisting of cerium oxide (SiO 2 ), kaolin, calcined kaolin, clay and china clay, wherein barium sulfate has a refractive index of 1.64, which has good refractive power, can play an optimal light diffusing agent, and has special The erect, stylistic shape, can enhance the reflection of light, to achieve the role of light transmission opacity, is preferred.
在本發明之一實施例中 ,該鐵氰化物係為鐵氰化鉀或鐵氰化鈉。In one embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.
在本發明之一實施例中 ,其中該試片係欲測定葡萄糖時,該氧化還原酵素係為葡萄糖氧化酶或葡萄糖脫氫酶;該試片係欲測定尿酸時,該氧化還原酵素係為尿酸酶;該試片係欲測定膽固醇時,該氧化還原酵素係為膽固醇酯酶或膽固醇氧化酶;該試片係欲測定三酸甘油酯時,該氧化還原酵素係酯酶、甘油激酶或磷酸甘油氧化酶;以及該試片係欲測定肌酸酐時,該氧化還原酵素係該肌酸酐酶、肌酸酶或肌氨酸氧化酶。In an embodiment of the invention, wherein the test piece is to measure glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and the test piece is uric acid, the oxidoreductase is uric acid Enzyme; when the test piece is to measure cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test piece is to measure triglyceride, the oxidoreductase esterase, glycerol kinase or glycerol phosphate An oxidase; and when the test piece is to measure creatinine, the oxidoreductase is the creatinine, creatase or sarcosine oxidase.
在本發明之試劑層中,進一步包含:一非離子界面活性劑、一消泡劑、一酵素穩定劑、一高分子酵素結構穩定劑、一纖維素増稠劑以及一緩衝劑,其中該酵素穩定劑係選自於由海藻糖(trehalse)、萄糖酸鈉(Sodium gluconate)、甘露糖(mannose)、環糊精(cyclodextrin)、蔗糖(sucrose)、甘氨酸 (glycine)、丙氨酸(alanine)、絲氨酸(serine)、***糖(arabinose)、山梨聚糖(sorbitan)、2-去氧葡萄糖(2-deoxy-D-glucose)、木糖(xylose)、果糖(fructose)以及谷氨酸(glutamic acid)所組成之群組;且該高分子酵素結構穩定劑係選自於由聚乙烯醇(polyvinyl alcohol,PVA)、聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚氧乙烯(polyethylene oxide)、聚乙烯醇吡咯烷酮與聚醋酸乙烯共聚物(copolymers of polyvinyl pyrrolidone and polyvinyl acetate)以及聚乙烯醇與聚醋酸乙烯共聚物(copolymers of polyvinyl alcohol and polyvinyl acetate)所組成之群組;且該纖維素増稠劑係為羧甲基纖維素(carboxymethyl cellulose,CMC)或羥乙基纖維(hydroxymethyl cellulose,HEC);且該緩衝劑係為0.1 M磷酸緩衝劑;該非離子界面活性劑係為采酮(Triron X-100)。The reagent layer of the present invention further comprises: a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and a buffer, wherein the enzyme The stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose, glycine, alanine. ), serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose, fructose, and glutamic acid ( a group consisting of glutamic acid); and the polymerase structural stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly a group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate; The cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC); and the buffer is 0.1 M phosphate buffer; the nonionic surfactant is Ketone (Triron X-100).
在本發明之一實施例,該基板為一電絕緣基板,其材質可包含但不限於聚氯乙烯、聚苯乙烯、聚酯、聚碳酸酯、聚醚、聚乙烯、聚丙烯、聚乙烯對苯二甲酸酯、或聚對苯二甲酸乙二醇酯。In an embodiment of the invention, the substrate is an electrically insulating substrate, and the material thereof may include, but not limited to, polyvinyl chloride, polystyrene, polyester, polycarbonate, polyether, polyethylene, polypropylene, polyethylene. Phthalate or polyethylene terephthalate.
在本發明之一實施例,該電路層的第一電極與第二電極分別為工作電極及輔助電極,其相對位置沒有限制。在本發明之又一實施例中,第一電極及第二電極結構可經由絲網印刷方式印刷出所需圖案,而工作電極及輔助電極可包括但不限於碳、單一金屬、合金及/或其他導電材料。此外,本發明對工作電極與輔助電極的相對位置、形狀及尺寸沒有限制。In an embodiment of the invention, the first electrode and the second electrode of the circuit layer are respectively a working electrode and an auxiliary electrode, and the relative positions thereof are not limited. In still another embodiment of the present invention, the first electrode and the second electrode structure may print a desired pattern via screen printing, and the working electrode and the auxiliary electrode may include, but are not limited to, carbon, a single metal, an alloy, and/or Other conductive materials. Further, the present invention is not limited to the relative position, shape and size of the working electrode and the auxiliary electrode.
在本發明之一實施例,該黏著層可為絕緣具有中隔層之雙面膠。In an embodiment of the invention, the adhesive layer may be a double-sided tape having an insulating interlayer.
在本發明之一實施例,該覆蓋層為具有親水性質的透明電絕緣膜,其可包括但不限於聚氯乙烯、聚苯乙烯、聚酯、聚碳酸酯、聚醚、聚乙烯、聚丙烯、聚乙烯對苯二甲酸酯、或聚對苯二甲酸乙二醇酯。 實施例 2 本發明之用於電化學生物感測器的試片的耐熱穩定性 In an embodiment of the invention, the cover layer is a transparent electrical insulating film having hydrophilic properties, which may include, but is not limited to, polyvinyl chloride, polystyrene, polyester, polycarbonate, polyether, polyethylene, polypropylene. , polyethylene terephthalate, or polyethylene terephthalate. Example 2 Heat resistance stability of a test piece for an electrochemical biosensor of the present invention
在本發明之一實施例中,用於電化學生物感測器的試片係使用鐵氰化鉀作為電子傳遞介質,但使用鐵氰化鉀具有易受熱影響而轉變為亞鐵氰化鉀,而導致試片變質的缺點。尤其在當鐵氰化鉀與酵素混合成反應試劑時,就算是置於乾燥環境中保存,此種轉變依然會進行。因此,本發明針對該試片的耐熱性進行測試。In one embodiment of the present invention, a test piece for an electrochemical biosensor uses potassium ferricyanide as an electron transport medium, but potassium ferricyanide is converted to potassium ferrocyanide by being susceptible to heat. The disadvantage of causing the test piece to deteriorate. Especially when potassium ferricyanide is mixed with an enzyme to form a reagent, even if it is stored in a dry environment, this transformation will still proceed. Therefore, the present invention tests for the heat resistance of the test piece.
本實施例以葡萄糖氧化酶為例。首先,將葡萄糖氧化酶溶於0.1 M磷酸鹽緩衝液中,添加上述實施例1中所有鐵氰化物穩定劑之混合物,其中包含:聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、聚丙烯醯胺(polyacrylamides)、明膠(gelatin)、糊精(dextrin)、纖維素鹼金族鹽(alkali metal salts of cellulose)以及水解聚乙烯醇(hydrolyzed polyvinyl alcohol),於40 ℃真空乾燥。另配製一無添加鐵氰化物穩定劑者做為對照組。兩者置放於40 ℃箱,每週取出測量活性並與置放第一日者做比對,計算其殘留活性百分率。This example is exemplified by glucose oxidase. First, glucose oxidase was dissolved in 0.1 M phosphate buffer, and a mixture of all the ferricyanide stabilizers in the above Example 1 was added, which contained: polyvinyl pyrrolidone, polyacrylamides. Gelatin, dextrin, alkali metal salts of cellulose, and hydrolyzed polyvinyl alcohol were dried under vacuum at 40 °C. Another person who did not add ferricyanide stabilizer was used as a control group. The two were placed in a 40 ° C box, and the activity was measured weekly and compared with those placed on the first day, and the percentage of residual activity was calculated.
結果如圖3所示,相較於無添加鐵氰化物穩定劑的對照組在40 ℃環境下持續10週葡萄糖氧化酶的活性已降低接近無殘留活性;本發明之添加有鐵氰化物穩定劑的電化學生物感測器試片在40 ℃環境下持續10週葡萄糖氧化酶的活性皆維持在90%。證實本發明之電化學生物感測器試片因添加鐵氰化物穩定劑而顯示具有良好之耐熱穩定性,儲存10週之後仍保留約90%之酵素活性。The results are shown in Fig. 3. Compared with the control group without the addition of ferricyanide stabilizer, the activity of glucose oxidase has been reduced to near no residual activity for 10 weeks at 40 °C; the addition of ferricyanide stabilizer to the present invention The electrochemical biosensor test strips were maintained at 90% for 10 weeks at 40 °C. It was confirmed that the electrochemical biosensor test piece of the present invention showed good heat resistance stability by the addition of the ferricyanide stabilizer, and retained about 90% of the enzyme activity after 10 weeks of storage.
進一步地,本實施例以葡萄糖氧化酶為例,並依據實施例1的步驟製造電化學生物感測器試片,完成後於40 ℃真空乾燥後,放入乾燥罐中,並置於50 ℃烘箱中,每隔七日取出,以恆電位儀(Logic SP-150)測量不同濃度之葡萄糖溶液(50、100、250 mg/dL)之電流值。Further, in this embodiment, glucose oxidase is taken as an example, and an electrochemical biosensor test piece is manufactured according to the procedure of Example 1. After completion, it is vacuum dried at 40 ° C, placed in a drying tank, and placed in an oven at 50 ° C. In the middle, every seven days, the current value of different concentrations of glucose solution (50, 100, 250 mg / dL) was measured with a potentiostat (Logic SP-150).
結果如圖4所示,經50 ℃高溫環境儲存,添加鐵氰化物穩定劑之葡萄糖檢測試片,測試50、100、250 mg/dL不同濃度之葡萄糖溶液,28天後依然顯示穩定之測量值。證實本發明之電化學生物感測器試片因添加鐵氰化物穩定劑而顯示具有良好之耐高溫穩定性。 實施例 3 本發明之用於電化學生物感測器的試片的光穩定性 The results are shown in Figure 4. After storage in a high temperature environment of 50 °C, a glucose test strip containing a ferricyanide stabilizer was added to test 50, 100, 250 mg/dL glucose solutions of different concentrations, and stable measurements were still displayed after 28 days. . It was confirmed that the electrochemical biosensor test piece of the present invention showed good high temperature stability due to the addition of the ferricyanide stabilizer. Example 3 Photostability of a test piece for an electrochemical biosensor of the present invention
因本發明之用於電化學生物感測器的試片係使用鐵氰化鉀作為電子傳遞介質,但使用鐵氰化鉀作為電子傳遞物之電化學檢測試片在曝露於光照環境中,鐵氰化鉀會轉換成亞鐵氰化鉀,此種轉換常發生於試片製程中從試劑塗佈到乾燥裝罐的過程或試片長期保存中,會造成檢測試片時的背景值昇高,導致檢測值偏高。因此,本發明針對該試片的耐光性進行測試。The test piece for the electrochemical biosensor of the present invention uses potassium ferricyanide as an electron transport medium, but an electrochemical test piece using potassium ferricyanide as an electron transporter is exposed to a light environment, iron Potassium cyanide is converted to potassium ferrocyanide. This conversion often occurs during the test strip process from reagent coating to dry canning or long-term storage of the test piece, which will cause an increase in the background value of the test piece. , resulting in a high detection value. Therefore, the present invention tests the light resistance of the test piece.
本實施例以葡萄糖氧化酶為例,依據實施例1的步驟製造添加光遮蔽劑的電化學生物感測器試片作為實驗組,其中試劑層包含:0.75 mg/mL 葡萄糖氧化酶 (250 U/mg)、100 mg/mL 鐵氰化鉀、1.5 g/mL 光遮蔽劑硫酸鋇 (BaSO 4)、0.5 mg/mL 羧甲基纖維素(carboxymethyl cellulose)、0.1 mg/mL 采酮(Triron X-100) 以及上述所有鐵氰化物穩定劑之混合物;另製造無添加光遮蔽劑的電化學生物感測器試片作為對照組,其中試劑層包含:0.75 mg/mL 葡萄糖氧化酶 (250 U/mg)、100 mg/mL 鐵氰化鉀、0.5 mg/mL 羧甲基纖維素(carboxymethyl cellulose)、0.1 mg/mL 采酮(Triron X-100) 以及上述所有鐵氰化物穩定劑之混合物。兩重試片皆以空氣噴槍(air brush)塗佈在反應區上,置於40 ℃乾燥箱中,乾燥30分鐘,分別以螢光紫外燈曝曬0小時、0.5 小時、1 小時、6 小時、 12小時、24 小時,再裝入避光之乾燥罐中,於室溫保存,隔日取出以恆電位儀測量背景電流值。 In this example, an example of the glucose oxidase was used to prepare an electrochemical biosensor test piece with a light shielding agent as an experimental group according to the procedure of Example 1, wherein the reagent layer contained: 0.75 mg/mL glucose oxidase (250 U/ Mg), 100 mg/mL potassium ferricyanide, 1.5 g/mL light screening agent barium sulfate (BaSO 4 ), 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X- 100) and a mixture of all the above-mentioned ferricyanide stabilizers; another electrochemical biosensor test piece without added light shielding agent was used as a control group, wherein the reagent layer contained: 0.75 mg/mL glucose oxidase (250 U/mg) ), 100 mg/mL potassium ferricyanide, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X-100), and a mixture of all the above-mentioned ferricyanide stabilizers. The two test pieces were coated on the reaction zone with an air brush, placed in a 40 ° C dry box, dried for 30 minutes, and exposed to a fluorescent UV lamp for 0 hours, 0.5 hours, 1 hour, 6 hours, respectively. 12 hours, 24 hours, and then placed in a dry can without light, stored at room temperature, and taken out every other day to measure the background current value with a potentiostat.
結果如表一所示,無添加光遮蔽劑之電化學生物感測器試片,隨曝曬時間增加,顯示逐漸昇高之背景電流值,添加光遮蔽劑之電化學生物感測器的試片則在曝曬12小時間以上,才出現顯著昇高之背景電流值。證實本發明之電化學生物感測器試片因添加光遮蔽劑而顯示具有良好的光穩定性。 表一、添加光遮蔽劑對電化學生物感測器的試片背景電流之影響 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> 背景電流 (uA) </td></tr><tr><td> 曝曬時間 (小時) </td><td> 無添加BaSO<sub>4</sub> (-) </td><td> 添加BaSO<sub>4</sub> (+) </td></tr><tr><td> </td><td> </td><td> </td></tr><tr><td> 0 </td><td> 2.55 </td><td> 2.55 </td></tr><tr><td> 0.5 </td><td> 4.27 </td><td> 2.57 </td></tr><tr><td> 1 </td><td> 7.76 </td><td> 2.71 </td></tr><tr><td> 6 </td><td> 11.42 </td><td> 2.82 </td></tr><tr><td> 12 </td><td> 12.92 </td><td> 4.50 </td></tr><tr><td> 24 </td><td> 13.30 </td><td> 7.73 </td></tr></TBODY></TABLE>實施例 4 本發明之用於電化學生物感測器的試片的酵素穩定性 The results are shown in Table 1. The electrochemical biosensor test piece without the added light shielding agent showed a gradually increasing background current value as the exposure time increased, and the test piece of the electrochemical biosensor with the light shielding agent added. A significant increase in background current value occurs only after exposure for more than 12 hours. It was confirmed that the electrochemical biosensor test piece of the present invention showed good photostability due to the addition of a light shielding agent. Table 1. Effect of adding light shielding agent on background current of electrochemical biosensor <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td></td><td> background current (uA) </td></tr><tr><td> exposure time (hours) </td><td> no added BaSO<sub>4</sub> ( -) </td><td> Add BaSO<sub>4</+> (+) </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 0 </td><td> 2.55 </td><td> 2.55 </td></tr><tr><td> 0.5 </td><td> 4.27 </td><td> 2.57 </td></tr><tr><td> 1 </td><td> 7.76 </td><td> 2.71 </td></tr ><tr><td> 6 </td><td> 11.42 </td><td> 2.82 </td></tr><tr><td> 12 </td><td> 12.92 </td ><td> 4.50 </td></tr><tr><td> 24 </td><td> 13.30 </td><td> 7.73 </td></tr></TBODY></ TABLE> Example 4 Enzyme stability of the test piece for electrochemical biosensor of the present invention
過去雖已知可利用高分子聚合物保護包括鐵氰化物等血紅素檢測物質,避免光照之不穩定性。此保護作用推測為高分子聚合物包覆作用,可避免直接與光接觸。而本發明利用此種包覆作用穩定電流,以避免電化學試片中電子傳遞物鐵氰化物與酵素長期直接接觸而反應,造成檢測電流背景值昇高之不穩定性。In the past, it has been known that macromolecular polymers can be used to protect heme detecting substances, such as ferricyanide, from being unstable. This protective effect is presumed to be a high molecular polymer coating to avoid direct contact with light. The present invention utilizes such a coating to stabilize the current, so as to avoid long-term direct contact between the electron-transporting material, ferricyanide and the enzyme, in the electrochemical test piece, and the instability of the background value of the detected current is increased.
本實施例以葡萄糖氧化酶為例,依據實施例1的步驟的電化學生物感測器試片作為實驗組,其中試劑層另包含:0.75 mg/mL 葡萄糖氧化酶 (250 U/mg)、100 mg/mL 鐵氰化鉀、5 mg/mL 高分子酵素結構穩定劑聚乙烯醇吡咯烷酮(polyvinyl pyrrolidone)、0.5 mg/mL 羧甲基纖維素(carboxymethyl cellulose)、0.1 mg/mL 采酮(Triron X-100)以及添加上述所有鐵氰化物穩定劑之混合物;另製造無添加高分子酵素結構穩定劑的電化學生物感測器試片作為對照組,其中試劑層包含:0.75 mg/mL 葡萄糖氧化酶 (250 U/mg)、100 mg/mL 鐵氰化鉀、0.5 mg/mL 羧甲基纖維素(carboxymethyl cellulose)、0.1 mg/mL 采酮(Triron X-100) 以及添加上述所有鐵氰化物穩定劑之混合物。兩重試片皆以空氣噴槍(air brush)塗佈在反應區上,置於40 ℃乾燥箱中,乾燥30分鐘,再裝入避光之乾燥罐中。兩組試片試片置於40 ℃烘箱中,每隔三日取出,以恆電位儀(Logic SP-150)測量不同濃度之葡萄糖溶液之電流值,檢測其穩定性。In this embodiment, an electrochemical biosensor test piece according to the procedure of Example 1 is taken as an experimental group, wherein the reagent layer further comprises: 0.75 mg/mL glucose oxidase (250 U/mg), 100. Mg/mL potassium ferricyanide, 5 mg/mL polymer enzyme structural stabilizer polyvinyl pyrrolidone, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X -100) and adding a mixture of all the above-mentioned ferricyanide stabilizers; and preparing an electrochemical biosensor test piece without adding a polymerase structural stabilizer as a control group, wherein the reagent layer comprises: 0.75 mg/mL glucose oxidase (250 U/mg), 100 mg/mL potassium ferricyanide, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X-100), and all the ferricyanide added above a mixture of agents. Both test pieces were coated on the reaction zone with an air brush, placed in a 40 ° C dry box, dried for 30 minutes, and placed in a dry tank protected from light. The test pieces of the two sets of test pieces were placed in an oven at 40 °C and taken out every three days. The current values of different concentrations of glucose solution were measured by a potentiostat (Logic SP-150) to test the stability.
結果如圖5及6所示,無添加鐵氰化物穩定劑之電化學生物感測器試片,顯示隨時間逐漸昇高之測量背景值(圖5),添加鐵氰化物穩定劑之電化學生物感測器試片則無此現象(圖6)。證實本發明之電化學生物感測器試片因添加鐵氰化物穩定劑而顯示具有測量穩定性。The results are shown in Figures 5 and 6. Electrochemical biosensor test strips without added ferricyanide stabilizer showed a measured background value that gradually increased with time (Fig. 5). Electrochemistry with addition of a ferricyanide stabilizer This is not the case with biosensor test strips (Figure 6). It was confirmed that the electrochemical biosensor test piece of the present invention showed measurement stability by the addition of a ferricyanide stabilizer.
綜上所述,本發明提供一種具高穩定性電化學生物感測器試片,其具有鐵氰化物穩定劑以及光遮蔽劑成分,其中鐵氰化物穩定劑較佳為高分子聚合物,光遮蔽劑較佳為硫酸鋇(BaSO 4)。該電化學生物感測器試片可克服使用當鐵氰化物作為電子傳遞介質時,在保存期間與酵素的反應或自身氧化還原反應,而導致診斷結果錯誤的缺點;以及克服電子傳遞介質對光敏感,因而造成量測的不準確性的缺點。因此,本發明之電化學生物感測器試片具有製造過程穩定、保存期限長、耐高溫以及耐光害之優勢,適合商業上使用,達到患者能自我健康管理的目的,有助於預防及減緩相關疾病併發症的發生,以及醫療資源的龐大負擔。 In summary, the present invention provides a high stability electrochemical biosensor test piece having a ferricyanide stabilizer and a light shielding agent component, wherein the ferricyanide stabilizer is preferably a high molecular polymer, light. The masking agent is preferably barium sulfate (BaSO 4 ). The electrochemical biosensor test piece overcomes the shortcomings of using the reaction of the ferricyanide as an electron transport medium with the enzyme during the storage or the auto-oxidation-reduction reaction, resulting in a wrong diagnosis result; and overcoming the electron transfer medium to the light. Sensitive, thus causing the disadvantage of inaccuracies in measurement. Therefore, the electrochemical biosensor test piece of the invention has the advantages of stable manufacturing process, long shelf life, high temperature resistance and light resistance, is suitable for commercial use, achieves the purpose of self-health management of the patient, and helps prevent and slow down. The occurrence of related disease complications and the enormous burden of medical resources.
110‧‧‧基板110‧‧‧Substrate
120‧‧‧電路層120‧‧‧ circuit layer
121‧‧‧第一電極121‧‧‧First electrode
122‧‧‧第二電極122‧‧‧second electrode
130‧‧‧試劑層130‧‧‧Reagent layer
131‧‧‧電極反應區131‧‧‧Electrode reaction zone
140‧‧‧黏著層140‧‧‧Adhesive layer
150‧‧‧覆蓋層150‧‧‧ Coverage
圖1係為以使用電子傳遞介質(mediators)協助電子傳遞的電化學生物感測器之反應過程示意圖。 圖2係為本發明之具高穩定性的電化學生物感測器試片的結構圖。 圖3係為添加鐵氰化物穩定劑與葡萄糖氧化之組合物的耐熱穩定性曲線圖。 圖4係為本發明之添加鐵氰化物穩定劑的電化學生物感測器試片的耐熱穩定性曲線圖。 圖5係為無添加高分子酵素結構穩定劑的電化學生物感測器試片的反應電流曲線圖。 圖6為本發明之添加高分子酵素結構穩定劑的電化學生物感測器試片的反應電流曲線圖。Figure 1 is a schematic illustration of the reaction process of an electrochemical biosensor using electron transport media to assist electron transport. 2 is a structural view of a test piece of an electrochemical biosensor having high stability according to the present invention. Fig. 3 is a graph showing the heat resistance stability of a composition in which a ferricyanide stabilizer and glucose oxidation are added. Fig. 4 is a graph showing the heat resistance stability of an electrochemical biosensor test piece to which a ferricyanide stabilizer is added according to the present invention. Figure 5 is a graph showing the reaction current of an electrochemical biosensor test piece without the addition of a polymerase structural stabilizer. Fig. 6 is a graph showing the reaction current of an electrochemical biosensor test piece incorporating a polymerase structural stabilizer according to the present invention.
Claims (22)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW106129252A TWI644101B (en) | 2017-08-29 | 2017-08-29 | A test strip for electrochemical biosensor with high stability and manufacturing method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW106129252A TWI644101B (en) | 2017-08-29 | 2017-08-29 | A test strip for electrochemical biosensor with high stability and manufacturing method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
TWI644101B true TWI644101B (en) | 2018-12-11 |
TW201913086A TW201913086A (en) | 2019-04-01 |
Family
ID=65431644
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW106129252A TWI644101B (en) | 2017-08-29 | 2017-08-29 | A test strip for electrochemical biosensor with high stability and manufacturing method thereof |
Country Status (1)
Country | Link |
---|---|
TW (1) | TWI644101B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112870382A (en) * | 2021-01-28 | 2021-06-01 | 复旦大学附属中山医院 | Arabinose and silicone oil type antifoaming agent combination for bowel preparation |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1874720A (en) * | 2003-09-03 | 2006-12-06 | 生命修复国际股份有限公司 | Personal diagnostic devices and related methods |
CN101883982A (en) * | 2008-05-09 | 2010-11-10 | 松下电器产业株式会社 | Method, device and apparatus for measuring the concentration of creatinine, and method, device and apparatus for measuring the amount of salt in urine using the same |
CN106124595A (en) * | 2016-06-17 | 2016-11-16 | 浙江亿联健医疗器械有限公司 | A kind of biosensor and preparation method thereof and reaction reagent |
-
2017
- 2017-08-29 TW TW106129252A patent/TWI644101B/en not_active IP Right Cessation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1874720A (en) * | 2003-09-03 | 2006-12-06 | 生命修复国际股份有限公司 | Personal diagnostic devices and related methods |
CN101883982A (en) * | 2008-05-09 | 2010-11-10 | 松下电器产业株式会社 | Method, device and apparatus for measuring the concentration of creatinine, and method, device and apparatus for measuring the amount of salt in urine using the same |
CN106124595A (en) * | 2016-06-17 | 2016-11-16 | 浙江亿联健医疗器械有限公司 | A kind of biosensor and preparation method thereof and reaction reagent |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112870382A (en) * | 2021-01-28 | 2021-06-01 | 复旦大学附属中山医院 | Arabinose and silicone oil type antifoaming agent combination for bowel preparation |
CN112870382B (en) * | 2021-01-28 | 2023-05-26 | 复旦大学附属中山医院 | Combination of arabinose and silicone oil defoamer for intestinal tract preparation |
Also Published As
Publication number | Publication date |
---|---|
TW201913086A (en) | 2019-04-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
ES2383905T3 (en) | Self-limiting compositions in terms of dimension and test devices for measuring analytes in biological fluids | |
US8673127B2 (en) | Biosensor | |
KR100293873B1 (en) | Biosensor | |
ES2343184T3 (en) | METER FOR USE IN A METHOD OF REDUCING INTERFERENCES IN AN ELECTROCHEMICAL SENSOR USING TWO DIFFERENT POTENTIALS APPLIED. | |
JP5781127B2 (en) | Biosensor | |
ES2224614T3 (en) | SUBSTRATE AND BIOSENSOR ANALYSIS PROCEDURE. | |
TW201423100A (en) | Electrochemical-based analytical test strip with bare interferent electrodes | |
JPH06506351A (en) | Improved methods and reagents for analyte measurement | |
CN115266865B (en) | Method for improving stability of electrochemical sensor | |
ES2402578T3 (en) | Test sensor reagent that has cellulose polymers | |
TWI644101B (en) | A test strip for electrochemical biosensor with high stability and manufacturing method thereof | |
JP4627912B2 (en) | Biosensor | |
WO2023246298A1 (en) | Electrochemical biosensor and preparation method therefor | |
US8658012B2 (en) | Biosensor for electrochemical measurement of 1,5-anhydroglucitol, and measuring method and measuring kit using the same | |
CA1134911A (en) | Halide ion-selective devices | |
JP2000081408A (en) | Biosensor | |
WO2015002184A1 (en) | Biosensor | |
JP4856697B2 (en) | Biosensor manufacturing method and biosensor | |
KR20150052868A (en) | Improved matrix stability compositions and methods | |
JP2002221508A (en) | Biosensor | |
KR20140033668A (en) | Electrode element for detecting glucose with impurity blocking structure and sensor for detecting glucose | |
JP2001343350A (en) | Biosensor | |
JP5798044B2 (en) | Biosensor | |
JPS58216947A (en) | Enzyme electrode for measuring sucrose concentration | |
Liao et al. | Effects of tin oxide sputtered on a carbon electrode for fabricating glucose biosensor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MM4A | Annulment or lapse of patent due to non-payment of fees |