201209411 六、發明說明： 【發明所屬之技術領域】 [0001] 本發明係關於-種酵素電極與其製k方法及應用 [先前技術3 [0002] 碳水化合物的分析方法包含旋光（optical rota_ tion)、比色（c〇l〇rimetry)、高效液相層析儀 (high-performance liquid chromatography, HPLC)，以及酵素電極（Qiu，J._D·，Zhou，W.-M.， Guo, J-, Wang, R., Liang, R.-P., 2009. Anal. ® Biochem. 385, 264-269; Sun, Y., Wang, H.,201209411 VI. Description of the Invention: [Technical Field] [0001] The present invention relates to an enzyme electrode and a method and application thereof [Prior Art 3 [0002] A method for analyzing carbohydrates includes optical rota tion, Colorimetric (c〇l〇rimetry), high-performance liquid chromatography (HPLC), and enzyme electrode (Qiu, J._D·, Zhou, W.-M., Guo, J-, Wang, R., Liang, R.-P., 2009. Anal. ® Biochem. 385, 264-269; Sun, Y., Wang, H.,
Sun, C., 2008. Biosens. Bioelectron. 24, 22-28;Zhang, S., Wang, N., Yu, H., Niu, Y., Sun, C., 2005. Bioelectrochemistry 67, 15-22)。多數科學家使用HPLC同時分析數種碳水化合物 ，可獲得準確度高與靈敏度良好的分析結果。然而， HPLC也具有許多缺點，它的設備體積龐大、昂貴，且分 Ο 析時間長；因此，對獠許多應用，例如臨床上糖尿病患 的葡萄糖分析而言，HPLC並非是最理想的方法。近年來 ，酵素電極因具有特定物質選擇性、操作容易、成本低 、方便攜帶、可即時快速分析等優點，成為分析特定碳 水化合物時比HPLC更好的選擇。 [0003] Clark與Lyons發展了第一代的酵素電極。5(^18卩- fer (Wang, J., 2008. Chem. Rev. 108, 814-825)之後做了改良，以人工媒介物（mediator)取 代氧，可避免受空氣中氧氣波動的缺點。從那時以來， 099137991 表單編號A0101 第3頁/共36頁 0992066199-0 201209411 二茂鐵（ferrocene)與其衍生物，因具各種所需特性， 例如低分子量、可逆性、低電位再生（regeneration at low potential)、產生穩定氧化還原反應（Ferr^ ndez, L., Carrero, Η., 2005. Electrochim. Acta 50，1233-1240)，被廣泛地作為葡萄糖酵素電極 的人工媒介物（Escorcia，A.，Dhirani, A.-A., 2007. J. Electroanal. Chem. 601, 260-268； Qian, W.L., Khan, Z., Watson, D.G., Fsarn-ley, J., 2008. J. Food Composit. Anal. 21, 78-83; Wang, S., Du, D. , 2004. Sens. Actuat-〇rs B 97，373-378)。通常’媒介物是溶於分析葡萄 糖的電解液中。然而，二茂鐵與其衍生物屬於致癌物質 ’測試後的電解液不能任意棄置，因此應用範圍受到限 制。為克服此缺點’ Q i u等（2 0 0 9)提出一種用於測量葡 萄糖的電極感應器（amperometric sensor)，其中媒介 物二茂鐵甲醛（ferrocene 被吸附在 奈米碳管的管壁上。另外，S—等(Yang，W.，Zh〇u， Η·. Sun, C., 2007. Macromol. Rapid Commun. 28，265-270)提出一種用於分析葡萄糖的酵素碳電極， 其中透過交聯作用（cross-linking)將二茂鐵媒介物固 疋於電極的幾丁質（chitosan)基材上。 [0004] 雖然習知技術提出許多方法，例如物理吸附、交聯 作用、封包（encapsulation)等，改善酵素與電極甚至 媒介物之間的固定性，至今仍需要提出一種具有高準確 性、高穩定性、寬量測濃度範圍，以及長期再使用性的 099137991 表單編號A0101 第4頁/共36頁 0992066199-0 201209411 酵素電極。 [0005] 另方面’丹麥科學家Ruzicka與Hansen (Zhi， Z. L.’ 1998. Trends Anal. Chem. 17， 411-417) 在提出流動注射分析系統（fl〇w injecti〇n ana-bsis，FIA)。由於具有可快速連續分析、設備簡單、 成本低等優點，該系統在環境偵測、製程分析、臨床診 斷等等許多領域，已被有效的實際利用。在操作上，流 動主射为析系統可以是線上（〇n_ 1 i ne )或非線上 〇 〇 (off-line)。非線上操作的程序繁雜，受測樣品也可能 在傳輸過程中流失，線上操作的方式可望解決此缺點。 近年來，科學家提出一些線上操作的FLA糸統（Kuniar， Μ·A., Thakur, M.S., Senthuran, A, T Senthur-an> V., Karanth, N.G., Hatti-Kaul, R., Mat-tiasson, B., 2001. World J. Microbio. Bio-technol. 17， 23-29; Nandakumar, M.P., Lali, A.M., Mattiasson, B., 1999. Bioseparation 8, 229-235 )，如利用酵素電極分淅微生物水解葡萄糖生物 ：.,： ： .·；：： ： ：：：. 轉換系統的葡萄糖（Gramsbergen，J.B.，Skj0th-Rasmussen, J., Rasmussen, C., Larabertsen, K.L., 2004. J. Neurosci. Methods 140, 93-101； Rhemrev-Boom, Μ.M., Jonker, M.A., Venema, K., Jobst, G., Tiessen, R., Korf, J., 2001. Analyst 126, 1073-1079; Yao, T., Yano, T., Nishino, H., 2004. Anal. Chimi. Acta 510，53-59)。然而，在這些已有的應用上有必 099137991 表單編號A0101 第5頁/共36頁 0992066199-0 201209411 要發展更方便使用的取«統，結合線上流動注射分析 系統，使分析程序更加簡易。 【發明内容】 [0006] [0007] [0008] 本發明的目的在於提供-種酵素電極的製造方法， 以製作-種具高準確性、高穩定性、寬量測濃度範圍， 以及可長期再使用的酵素電極。 根據上述目的’本發明實施例提供一種酵素電極的 製造方法，包含：提供具有—碳表面的_基底；在該破 表面上形成一金表面，藉此形成一電桎；共價鍵結一包 含醛基（aldehyde gr〇up)的媒介物於該電極表面；以 及共價鍵結一葡萄糖氧化酵素於該電極表面β 根據上述目的，本發明實施例提供一種酵素電極的 製造方法，包含：提供一基底，該基底具有一碳表面； 形成一金表面於該碳表面上，以形成一電極；以半胱胺 酸（L-cysteine)修飾該金表面，該金表面與半胱胺酸中 的硫醇基形成共價鍵結，並形成―具有一第一修飾表面 之第一電極；以一包含醛基（alde,de group)的媒介 物與半胱胺酸的胺基形成一席夫鹼（聚甲亞胺）（Schif f Base)共價鍵結’並形成一具有一第二修飾表面之第二電 極；以N, Ν’ -二環己基碳化二亞胺 (Ν, Ν’ -dicycl〇hexyicarb〇di imide)修飾該第二修飾 表面，N, Ν’ -二環己基碳化二亞胺與該金表面上的半胱 胺酸進行脫水反應形成共價鍵結，並形成一具有一第三 修飾表面之第三電極；以及該第三修飾表面與葡萄糖氧 化酵素接觸’使得葡萄糖氧化酵素與半耽胺酸基團的缓 099137991 表單編號Α0101 第6頁/共36頁 0992066199-0 201209411 [0009] Ο [0010] ο 基（carboxyl group)形成醯胺鍵（amide bond)共價鍵 結，以形成一具有一第四修飾表面之第四電極。 根據上述目的，本發明實施例提供一種酵素電極， 包含：一基材結構，具有一碳表面； 一金表面形成在該碳表面的至少一部份上；一胺基酸 (amino acid)包含一胺基（amino group)、一羧基 (carboxyl group)、一硫醇基（thiol group)，該胺 基酸透過該硫醇基與該金表面鍵結；一媒介物包含一醛 基（aldehyde group) ’該媒lih物與該胺基形成一席夫 鹼（聚甲亞胺）（Schiff Base);以及一葡萄糖氧化酵素 透過一具有二亞胺（d i i m i dft;)的肤鍵連接劑（peptide coupling agent)與該胺基蜂鍵結，其中該葡萄糖氧化 酵素與該肽鍵連接劑形成一醯胺鍵（amide bond)。 根據上述目的，本發明實施例提供__種酵素電極， 包含：一基材結構，該基材結構由内往外傘序包含一鉛 '·· ...J S 筆芯主體、一碳覆蓋層、一金覆蓋層；以及一修飾結構 ，該修飾結構係化學鍵結於該金覆蓋層上，該修飾結構 包含一半胱胺酸基團 '一包含醛基（aldehyde group) 的媒介物基團，與一葡萄糖氧化酵素基團，該金覆蓋層 與該半胱胺酸基團之間係為金—硫（Au_s)共價鍵結， 該半耽胺酸基團與該葡萄糖氧化酵素之間係為酿胺鍵 (amide bond)共價鍵結，該半胱胺酸基團與該媒介物基 團之間係為碳氡雙鍵（carbon-nitrogen double bond)共價鍵結。 099137991 【實施方式】 表單編號A0101 第7頁/共36頁 0992066199-0 201209411 [0011] 以下將詳述本案的各實施例，並配合圖式作為例示 。除了這些詳細描述之外，本發明還可以廣泛地實行在 其他的實施例中，任何所述實施例的輕易替代、修改、 等效變化都包含在本案的範圍内，並以之後的專利範圍 為準。在說明書的描述中，為了使讀者對本發明有較完 整的了解，提供了許多特定細節；然而，本發明可能在 省略部份或全部這些特定細節的前提下，仍可實施。此 外，眾所周知的程序步驟或元件並未描述於細節中，以 避免造成本發明不必要之限制。 [0012] 第1圖顯示根據本發明較佳實施例之酵素電極的製造 方法，其包含下列六個步驟。步驟（I )，在一鉛筆芯10的 表面上塗佈一層碳_嘗11，塗佈高度_大约_5 c m，製程條件 約為120°C、10 min。碳膏11的作用可避免酵素的活性 受鉛筆芯10雜質的干擾。使用碳純度愈高的碳膏11可預 期有愈高的酵素活性。步驟（II)，將上述鉛筆芯10置於 四氣金酸（tetrachloroaurate)水溶液，施加電壓0. 2V ，使四氣金酸還原成金粒子沈積在碳膏11表面上，高度 約0.8 cm，形成具有一金表面12的電極13。此步驟的製 程條件約為28 °C、2 hour。接著，在同溫度下，以水清 洗電極13，清除電極13表面上未還原的金化合物。步驟 (III)，將電極13浸沒於25°C、濃度20 mM的半胱胺酸 (L-cysteine)溶液中1小時，使金粒子與半胱胺酸中的 硫醇基（-SH, sulphydryl or thiol group)形成共 價鍵結之第一修飾表面12A，並且形成具有第一修飾表面 12A的第一電極13A。接著，在25°C下，以去離子水清洗 099137991 表單編號A0101 第8頁/共36頁 0992066199-0 201209411 第一電極13A，清除金表面12上以物理吸附的半胱胺酸。 步驟（IV)，將具有第一修飾表面12Α的第一電極13Α沉浸 在75 C、濃度0. 1 mM的二茂鐵甲搭（ferrocene car-boxaldehyde，FcAld，溶於體積比99. 5/0. 5的乙醇/氣 化氫）溶液中1小時，使媒介物（FcAld)與半耽胺酸的胺 基（amino group)形成一席夫鹼（聚曱亞胺）（Schif f base)共價鍵結的第二修飾表面12B，並且形成具有第二 修飾表面12B的第二電極1 3B。接著，在75°C下，以去離 子水清洗第二電極13B。步驟（V)，將第二電極13B沉浸 在溶於40 C甲醇、濃度5· 〇 mM的N，Ν’ -二環己基碳化二 亞胺（Ν，Ν’ -dicyclohexylcarbodi iiaide)溶液中 1 小 時，使N，Ν’ -二環己基碳化二亞胺與半胱胺酸的羧基 (carboxyl group)進行脫水反應形成共價鍵結，形成 第三修飾表面12C ’並形成具有第三修飾表面的第三電極 13C。接著可依序以甲醇與去離子水，在4(rc下清洗第三 電極13C約5 min，以移除以物理吸附的二亞胺 (diimide)。步驟(VI)，將第三電極13C沉浸在以0. 1 Μ 、pH 7. 0的碟酸納缓衝溶液（s〇dium ph〇Sphate buffer solution， NaPB) 所製備的濃度 50 “Μ 的葡萄糖氧 化酵素（glucose oxidase) 25°C溶液中 24 hour，使 葡萄糖氧化酵素與第三修飾表面12c的半胱胺酸基團之間 形成醢胺鍵（amide bond or peptide bond)共價鍵結 而固定在第三修飾表面12(：上，形成第四修飾表面121)與 具有第四修飾表面12D的第四電極13D。至此，第四電極 13D即為所需的酵素電極，可保存於〇 1 m、pH 7.0、4 °C的磷酸鈉緩衝溶液中待用。 099137991 表單編號A0101 第9頁/共36頁 0992066199-0 201209411 [0013] 上述較佳實施例可做適當變更。例如，一基底其部 份或全部表面具有一碳表面，可取代錯筆怎塗佈碳赏。 步驟（IV)可在步驟（V)或（VI)之後執行。基底的形狀可 為棒狀、片狀、或其他任意形狀；材質可包含任何金屬 或非金屬或其組合。形成金表面12的方法可包含沈積 (deposition)、喷墨（ink-inject)、電鑛 (e 1 ectrodepos i t i on) ' 網印（screen-pr i nt i ng)， 或其他方法。其他具有胺基（amino group)、叛基 (carboxyl group)、硫醇基（thiol group)的胺基酸 f'， (amino acid)可取代半胱胺酸。其他具有二亞胺結構的 肽鍵連接劑（peptide coupling agent)可取代N，Ν’ -二環己基碳化二亞胺。其他具有一醛基（aldehyde group)的媒介物可取代二茂鐵甲搭（ferrocene car-boxaldehyde)。此外，製程溫度與各溶液濃度亦可做適 度變更。 [0014] 以下將驗證上述較佳實施例所製備酵素電極的特性 ，並且將所製備酵素電極應用於一線上流動注射分析系 統（FIA)，以長期、連續地分析一生物反應器 (bioreactor)的葡萄糖濃度。 [0015] 第2A圖顯示根據本發明一實施例的流動注射分析系 統的架構圖。流動注射分析系統2 0透過電化學分析次系 統22與停流（stop-flow)取樣次系統23，連接生物反應 器21，並分析其產物。在本實施例，生物反應器21以固 定化纖維水解酵素將廢棄竹筷進行水解，其產物包含葡 萄糖。另外，亦以線上高效液相層析儀配合折射率偵測 099137991 表單編號A0101 第10頁/共36頁 0992066199-0 201209411 器（HPL〇RI)對產物進行分析，以做比較。Sun, C., 2008. Biosens. Bioelectron. 24, 22-28; Zhang, S., Wang, N., Yu, H., Niu, Y., Sun, C., 2005. Bioelectrochemistry 67, 15-22 ). Most scientists use HPLC to simultaneously analyze several carbohydrates for high accuracy and sensitivity. However, HPLC also has a number of disadvantages, its apparatus is bulky, expensive, and has a long time to decompose; therefore, HPLC is not the most ideal method for many applications, such as glucose analysis in clinically diabetic patients. In recent years, enzyme electrodes have been selected as a better choice than HPLC for the analysis of specific carbohydrates due to their specific material selectivity, ease of operation, low cost, ease of carrying, and instant and rapid analysis. [0003] Clark and Lyons developed the first generation of enzyme electrodes. 5 (^18卩-fer (Wang, J., 2008. Chem. Rev. 108, 814-825) has been modified to replace oxygen with a mediator to avoid the disadvantage of oxygen fluctuations in the air. Since then, 099137991 Form No. A0101 Page 3 / Total 36 Page 0992066199-0 201209411 Ferrocene and its derivatives, due to various desirable properties, such as low molecular weight, reversibility, low potential regeneration (regeneration at Low potential), producing a stable redox reaction (Ferr^ ndez, L., Carrero, Η., 2005. Electrochim. Acta 50, 1233-1240), widely used as an artificial vehicle for glucose enzyme electrodes (Escorcia, A. , Dhirani, A.-A., 2007. J. Electroanal. Chem. 601, 260-268; Qian, WL, Khan, Z., Watson, DG, Fsarn-ley, J., 2008. J. Food Composit. Anal. 21, 78-83; Wang, S., Du, D., 2004. Sens. Actuat-〇rs B 97, 373-378). Usually the 'vehicle is dissolved in the electrolyte for the analysis of glucose. However, The electrolyte after the test that ferrocene and its derivatives belong to carcinogens cannot be disposed of arbitrarily, so the application range is limited. To overcome This shortcoming 'Q iu et al. (2000) proposed an amperometric sensor for measuring glucose, in which the ferrocene formaldehyde (ferrocene is adsorbed on the wall of the carbon nanotube). S- et al (Yang, W., Zh〇u, Η. Sun, C., 2007. Macromol. Rapid Commun. 28, 265-270) propose an enzyme carbon electrode for the analysis of glucose, through cross-linking Cross-linking immobilizes a ferrocene vehicle on a chitosan substrate of an electrode. [0004] Although the prior art proposes many methods, such as physical adsorption, cross-linking, encapsulation, and the like. To improve the immobilization between enzymes and electrodes and even vehicles, it is still necessary to propose a high accuracy, high stability, wide measurement concentration range, and long-term reusability of 099137991 Form No. A0101 Page 4 of 36 Page 0992066199-0 201209411 Enzyme electrode. [0005] On the other hand, the Danish scientist Ruzicka and Hansen (Zhi, Z. L.' 1998. Trends Anal. Chem. 17, 411-417) proposed a flow injection analysis system (fl〇w injecti〇n ana-bsis, FIA). Due to its advantages of rapid and continuous analysis, simple equipment, and low cost, the system has been effectively utilized in many fields such as environmental detection, process analysis, and clinical diagnosis. In operation, the flow main shot system can be either online (〇n_ 1 i ne ) or non-line off off-line. The procedures for non-line operations are cumbersome, and the sample to be tested may also be lost during transmission. The way of online operation is expected to solve this shortcoming. In recent years, scientists have proposed some online FLA systems (Kuniar, Μ·A., Thakur, MS, Senthuran, A, T Senthur-an) V., Karanth, NG, Hatti-Kaul, R., Mat-tiasson , B., 2001. World J. Microbio. Bio-technol. 17, 23-29; Nandakumar, MP, Lali, AM, Mattiasson, B., 1999. Bioseparation 8, 229-235), such as the use of enzyme electrodes Microbial Hydrolysis of Glucose Creature: .,: : .·;:: : :::. Converting System Glucose (Gramsbergen, JB, Skj0th-Rasmussen, J., Rasmussen, C., Larabertsen, KL, 2004. J. Neurosci. Methods 140, 93-101; Rhemrev-Boom, Μ.M., Jonker, MA, Venema, K., Jobst, G., Tiessen, R., Korf, J., 2001. Analyst 126, 1073-1079; Yao , T., Yano, T., Nishino, H., 2004. Anal. Chimi. Acta 510, 53-59). However, there are certain applications in these applications. 099137991 Form No. A0101 Page 5 of 36 0992066199-0 201209411 To develop a more convenient use of the system, combined with the online flow injection analysis system, the analysis program is easier. SUMMARY OF THE INVENTION [0007] [0008] [0008] [0008] [0008] The object of the present invention is to provide a method for producing an enzyme electrode, to produce - high accuracy, high stability, wide measurement concentration range, and long-term re- The enzyme electrode used. According to the above object, an embodiment of the present invention provides a method for manufacturing an enzyme electrode, comprising: providing a substrate having a carbon surface; forming a gold surface on the broken surface, thereby forming an electric raft; and covalent bonding An aldehyde-based vehicle is disposed on the surface of the electrode; and a covalently bonded glucose-oxidase is applied to the surface of the electrode. According to the above object, an embodiment of the present invention provides a method for producing an enzyme electrode, comprising: providing a method a substrate having a carbon surface; forming a gold surface on the carbon surface to form an electrode; modifying the gold surface with a cysteine (L-cysteine), the gold surface and sulfur in the cysteine The alcohol group forms a covalent bond and forms a first electrode having a first modified surface; forming a Schiff base with a carrier comprising an alde, de group and an amine group of cysteine (Imiamine) (Schif f Base) covalently bonded 'and forms a second electrode having a second modified surface; with N, Ν'-dicyclohexylcarbodiimide (Ν, Ν'-dicycl〇hexyicarb 〇di imide) a second modified surface, N, Ν'-dicyclohexylcarbodiimide is dehydrated by the dehydration reaction of the cysteine on the gold surface to form a third electrode having a third modified surface; The third modified surface is in contact with glucose oxidase to make glucose oxidase and hemi-amino acid group slow 099137991 Form No. 1010101 Page 6 of 36 Page 0992066199-0 201209411 [0009] Ο [0010] ο Group) forming an amide bond covalently bonded to form a fourth electrode having a fourth modified surface. According to the above object, an embodiment of the present invention provides an enzyme electrode comprising: a substrate structure having a carbon surface; a gold surface formed on at least a portion of the carbon surface; and an amino acid comprising a An amino group, a carboxyl group, a thiol group, the amino acid is bonded to the gold surface through the thiol group; and a vehicle comprises an aldehyde group 'The medium lih forms a Schiff Base with the amine group; and a glucose oxidase is passed through a peptide coupling agent having a diimidate; Bonded to the amine group, wherein the glucose oxidase forms an amide bond with the peptide bond. According to the above object, an embodiment of the present invention provides an __ an enzyme electrode, comprising: a substrate structure comprising a lead '···JS refill body, a carbon cover layer, from the inner to the outer umbrella sequence, a gold coating; and a modifying structure chemically bonded to the gold coating layer, the modified structure comprising a cysteine group, a carrier group comprising an aldehyde group, and a a glucose oxidase group, wherein the gold coating layer and the cysteine group are covalently bonded to gold-sulfur (Au_s), and the semi-proline group and the glucose oxidase are brewed An amide bond is covalently bonded, and the cysteine group and the vehicle group are covalently bonded to a carbon-nitrogen double bond. 099137991 [Embodiment] Form No. A0101 Page 7 of 36 0992066199-0 201209411 [0011] Hereinafter, various embodiments of the present invention will be described in detail with reference to the drawings. In addition to the detailed description, the present invention may be widely practiced in other embodiments, and any alternatives, modifications, and equivalent variations of the described embodiments are included in the scope of the present invention, and the scope of the following patents is quasi. In the description of the specification, many specific details are set forth in the description of the invention, and the invention may be practiced otherwise. In addition, well-known program steps or elements are not described in detail to avoid unnecessarily limiting the invention. 1 shows a method of manufacturing an enzyme electrode according to a preferred embodiment of the present invention, which comprises the following six steps. In the step (I), a layer of carbon-taste 11 is applied to the surface of a pencil lead 10, and the coating height is about _5 c m, and the process conditions are about 120 ° C for 10 min. The action of the carbon paste 11 prevents the activity of the enzyme from being disturbed by the impurities of the pencil lead 10. The higher the purity of the carbon, the higher the purity of the enzyme, the higher the enzyme activity is expected. In step (II), the above-mentioned pencil lead 10 is placed in a tetrachloroaurate aqueous solution, and a voltage of 0.2 V is applied to reduce the four gas gold acid to gold particles deposited on the surface of the carbon paste 11 at a height of about 0.8 cm. An electrode 13 of a gold surface 12. The process conditions for this step are approximately 28 °C, 2 hours. Next, the electrode 13 is washed with water at the same temperature to remove the unreduced gold compound on the surface of the electrode 13. In step (III), the electrode 13 is immersed in a solution of 20-mM cysteine (L-cysteine) at 25 ° C for 1 hour to make the gold particles and the thiol group in the cysteine (-SH, sulphydryl). Or thiol group) forms a covalently bonded first modified surface 12A and forms a first electrode 13A having a first modified surface 12A. Next, it was washed with deionized water at 25 ° C. 099137991 Form No. A0101 Page 8 of 36 0992066199-0 201209411 The first electrode 13A was removed to physically adsorb the cysteine on the gold surface 12. The ferrocene car-boxaldehyde (FcAld) is dissolved in a volume ratio of 99.5/0. The ferrocene car-boxaldehyde (FcAld) is dissolved in a volume ratio of 99.5/0. 1 hour in a solution of 5 ethanol/hydrogenated hydrogen), the vehicle (FcAld) forms a Schiff base (covalent bond) with the amino group of the semi-proline. The second modification surface 12B and forms the second electrode 13B having the second modification surface 12B. Next, the second electrode 13B was washed with deionized water at 75 °C. In step (V), the second electrode 13B is immersed in a solution of N, Ν'-dicyclohexylcarbodiimide (溶于, Ν'-dicyclohexylcarbodi iiaide) dissolved in 40 C methanol at a concentration of 5 mM mM for 1 hour. The N,Ν'-dicyclohexylcarbodiimide is dehydrated with a carboxyl group of cysteine to form a covalent bond to form a third modified surface 12C' and form a third surface having a third modified surface. Electrode 13C. Then, the third electrode 13C may be washed with methanol and deionized water at 4 (rc) for about 5 minutes to remove the physically adsorbed diimide. Step (VI), immersing the third electrode 13C In a concentration of 50 Μ glucose oxidase 25 ° C solution prepared with 0.1 Μ, pH 7.0 s sdium ph〇Sphate buffer solution (NaPB) 24 hours, the glucose oxidase and the cysteine group of the third modified surface 12c form a amide bond or peptide bond covalently bonded to the third modified surface 12 (:, formed The fourth modified surface 121) and the fourth electrode 13D having the fourth modified surface 12D. Up to this point, the fourth electrode 13D is a desired enzyme electrode, which can be stored in a sodium phosphate buffer of 〇1 m, pH 7.0, 4 °C. The solution is ready for use. 099137991 Form No. A0101 Page 9/36 Page 0992066199-0 201209411 [0013] The above preferred embodiment can be modified as appropriate. For example, a part or all of the surface of a substrate has a carbon surface, which can be replaced. How to apply a carbon reward to the wrong pen. Step (IV) can be in step (V) or VI) is performed thereafter. The shape of the substrate may be a rod shape, a sheet shape, or any other shape; the material may include any metal or non-metal or a combination thereof. The method of forming the gold surface 12 may include deposition, inkjet (ink -inject), e 1 ectrodepos iti on 'screen-pr i nt i ng', or other methods. Others have an amino group, a carboxyl group, a thiol group ( The amino acid of thiol group) can replace cysteine. Other peptide coupling agents with diimine structure can replace N,Ν'-dicyclohexylcarbodiimide. Other hydrocarbons having an aldehyde group can be substituted for ferrocene car-boxaldehyde. In addition, the process temperature and the concentration of each solution can be changed moderately. [0014] The characteristics of the enzyme electrode prepared in the examples, and the prepared enzyme electrode was applied to a one-line flow injection analysis system (FIA) for long-term, continuous analysis of the glucose concentration of a bioreactor. [0015] Figure 2A display An architectural diagram of a flow injection analysis system in accordance with an embodiment of the present invention. The flow injection analysis system 20 is coupled to the bioreactor 21 through an electrochemical analysis subsystem 22 and a stop-flow sampling subsystem 23, and analyzes the product. In the present embodiment, the bioreactor 21 hydrolyzes the discarded bamboo chopsticks with a fixed fiber hydrolyzing enzyme, and the product thereof contains glucose. In addition, the product was analyzed by on-line high performance liquid chromatography with refractive index detection 099137991 Form No. A0101 Page 10 of 36 0992066199-0 201209411 (HPL〇RI) for comparison.
本實施例是以標準溶液添加法分析葡萄糖濃度，以 補償酵素電極的衰退’並去除溶液之基質效應(matrix effect)。在竹筷水解期間，起初i6小時的反應，每隔4 J時以螺動栗（peristahic p⑽p)233對生物反應器u 的水解產物進行取樣’之後取樣週期為8小時。固定化水 解酵素的水解產物樣品’以注射器過濾器（syringe filter)235進行過濾。水解產物樣品的取樣、裝載、注 射等程序描述如下：（1)以手動方式，利用停流取樣次系 統23的取樣注射器231 r自生物反應器21中取樣2 mL ; (2)如第2B圖所示，將停流取樣次系統23的六孔注射閥 232定位在「裝載」的位置（如第2B圖所示）’使1 mL的 樣品注入六孔注射閥232的樣品承載管，其容積為50 eL ;(3)將六孔注射閥232定位在「注射丄的位置（如第2B 圖所示），使位於閥内的5〇 yL樣品，經由蠕動泵223注 射進入電化學分析次系統22 ; (4)將六孔注射閥232定 位在「裝載」的位置；（5)以標準品注射器234取1 mL的 葡萄糖標準溶液；（6)自標舉品注射器234透過三孔閥 238人與三孔閥2386，注入0.11111的葡萄糖標準溶液於樣 品注射器231 ; (7)自樣品注射器231，透過三孔閥238B ，注入大約1.1 mL已添加0.1 mL標準溶液的樣品 (spiked sampie)於六孔注射閥232的50 取樣環内 ;(8)將六孔注射閥232定位在「注射」的位置’使位於 閥内的50 已添加標準溶液的樣品，經由蠕動泵223注 射進入電化學分析次系統22進行分析；（9)當分析完成， 099137991 表單編號A0101 第11 1/共36頁 0992066199-0 201209411 將六孔注射賴找位在「裝載」的位置；（1G)以去離子 K237取代葡萄糖標準溶液236，以清洗停流取樣次系統 23兩注㈣231 /234與相關管路，以便進行下-次分析 /主意上述葡萄糖標準溶液的濃度，視水解產物的葡萄 糖濃度而^ ’通常添加的標準品濃度是使添加標準品之 樣品的測試電流強度大於原始樣品的丨.5倍^此水解 產物的葡萄糖含量可以由標準添加方程式獲得(H町is， 2007. Quantitative chemical analysis, *t h ed. , W. H. Freeman and Company, New York, p. 87-90) 〇 ；·；；：' .. .- . ...This example analyzes the glucose concentration by standard solution addition to compensate for the decay of the enzyme electrode and removes the matrix effect of the solution. During the hydrolysis of the bamboo chopsticks, the reaction was initially i6 hours, and the hydrolyzate of the bioreactor u was sampled every 4 J with a peristalic p (10) p) 233. The sampling period was 8 hours. The hydrolyzed product sample of the immobilized hydrolysate was filtered by a syringe filter 235. The procedures for sampling, loading, and injection of the hydrolyzed product sample are described as follows: (1) Manually sampling 2 mL from the bioreactor 21 using the sampling syringe 231r of the stop flow sampling subsystem 23; (2) Figure 2B As shown, the six-hole injection valve 232 of the stop flow sampling subsystem 23 is positioned at the "loading" position (as shown in Figure 2B) 'sample the 1 mL sample into the sample carrier tube of the six-hole injection valve 232. 50 eL; (3) Position the six-hole injection valve 232 at the position of the injection port (as shown in Figure 2B), so that the 5〇yL sample located in the valve is injected into the electrochemical analysis subsystem via the peristaltic pump 223. 22; (4) Positioning the six-hole injection valve 232 at the "loading" position; (5) taking 1 mL of the glucose standard solution with the standard syringe 234; (6) passing the three-hole valve 238 from the standard injector 234 With the three-hole valve 2386, a 0.11111 glucose standard solution is injected into the sample injector 231; (7) from the sample injector 231, through the three-hole valve 238B, and about 1.1 mL of a sample (spiked sampie) to which 0.1 mL of the standard solution has been added is injected into the six-hole Injection valve 232 within 50 sampling loops; (8) injection of six holes 232 is positioned at the "injection" position to cause a sample of 50 added standard solution located in the valve to be injected into the electrochemical analysis subsystem 22 via the peristaltic pump 223 for analysis; (9) when the analysis is completed, 099137991 Form No. A0101 No. 11 1/36 pages 0992066199-0 201209411 Place the six-hole injection in the "loading" position; (1G) replace the glucose standard solution 236 with deionized K237 to clean the stop-flow sampling system 23 two notes (four) 231 / 234 and Related piping for the next-stage analysis/intention of the concentration of the above-mentioned glucose standard solution, depending on the glucose concentration of the hydrolysate, the standard concentration of the standard is usually such that the test current intensity of the sample to which the standard is added is greater than that of the original sample. .5 times the glucose content of this hydrolyzate can be obtained by the standard addition equation (Hori is, 2007. Quantitative chemical analysis, *th ed., WH Freeman and Company, New York, p. 87-90) 〇; ;:' .. .- . ...
[0017] [0018] 作為舉例而非限制，第2A圖所示的系統，使用美國 德州CH儀器公司製造的CHI611B電化學工作站222配合特 殊設計的三電極流動室（fl〇w cell)221，並利用蠕動泵 223輸送樣品，以進行線上葡萄糖含量分析。三電極流動 至221具有一#導線作為逆:電極（e〇unter electrode, CE)、一銀/氣化銀（Ag/AgC1y作為參考電極（reference electrode，RE) ’以及冬發明實砗例所製備的酵素電極 作為工作電極（working electrode)。 另外’停流取樣次系統23利用美國海克力斯Bi〇-Rad公司型號MV-6的樣品注射閥作為上述系統的六孔注射 閥232。水解產物的取樣工作還包含運用蠕動泵233與兩 個三孔閥239A/B。生物反應器21使用台灣台中Bi〇top process & Equipment公司型號BTF-A3L的3L生物反應 器，其包含pH電極211與溫度探針212透過控制器213分 別控制生物反應器21内反應液的pH值與溫度。另外，取 099137991 表單編號A0101 第12頁/共36頁 0992066199-0 201209411 [0019] [0020] Ο [0021] Ο [0022] [0023] 樣官214用於導出生物反應器21的產物。 注意在本發明其他實施例，上述線上流動注射分析 系統20可用於分析其他非葡萄糖的分析物，只要將三電 極流動室22中的酵素電極，置換成可分析該分析物的酵 素電極即可°另外’三電極流動室22也可以設計成兩電 極式的流動室。 以HPLC作葡萄糖的定量分析 在廢竹‘滨水解成葡萄糖期間，亦以HPLC-RI進行線 上分析水解產物。取樣週期與之前酵素電極流動注射分 析所述相同。 II* BP" 循環伏安分析 由德環伏安分析（cyclic volta.m:inetrie)可獲得 本發明實施例所製備酵素電極的氧化電位。分析時，電 位掃描範圍從〇. 〇至〇. 7 V，掃描速度為50 mVs_1。分 析樣品為三種葡萄糖標準溶液，濃度分別mM、4 mM 、10 mM，利用〇, 1 μ、plf 7· 0的磷酸鈉緩衝溶液 (NaPB)配製。 第3A圖顯示上述三種標準溶液的循環伏安分析圖， 其t曲線（a)(b)(c)分別為〇 mM、4 mM、10 mM的循環 伏安曲線，而箭號表示掃描的方向。 由圖可觀察到曲線（b)(c)在0. 45V時具有明顯的陽 極電流，而曲線（a)沒有陽極電流。此表示媒介物以八“ 的氧化電位是0.45 V,且媒介物與葡萄糖氧化酵素成功 地固定在碳電極的修飾表面上。 099137991 表單編號A0101 第13頁/共36頁 0992066199-0 201209411 [0024] [0025] [0026] 標準品添加校正檢量線法 發明是以標準添加校正檢量線法（…时㈣漆 1〇11 Calibrati〇n method, Pijanowska d G Sprenkels A τ ni+, . ’ . ·， ，A.J·，Olthvus，w·，Bergveld I: :Γ. Μ· B 91，98,2)定量分析葡 …示準品添加校正檢量線可獲得酵素電極用於偵 測葡萄糖的靈敏度、偵測極限值（limit 〇f detec_ h〇n’ _ ’以及線性濃度债測範圍。在35乂下製作找 準品添加校正檢量線。每次測量前，利料氮氣_鐘不 使磷酸鈉緩衝溶液（NaPB)脫氧。資先，以Q. i Μ、邱 7.0的NaPB製備濃度50祕與250 mM的葡萄糖標準溶液 ’並放置整夜使兩種不同旋光活性的a—與卜形式的葡萄 糖自然轉換（mutarotation)。為製備標準品添加校正檢 量線，將8 mL的0. 1 Μ、PH 7. 0的NaPB溶液置入電化與 電池當作背景溶液。3分鐘後，每隔1 · 5分鐘添加體積約2 至285 /^L的葡萄糖標準溶液v直至溶液的濃度為％ 6 mM。取每次標準品添加後最後3秒的時間讀取電流值。 第3B圖顯示根據本發明實施例製備的酵素電極以 上述方法製備標準添品加校正檢量線時的電流、時門曲 線。每次添加葡萄糖時的電流變化量，表示所添加葡萄 糖所對應之葡萄糖氧化的量。在時間為6〇〇至18〇〇秒時， 圖中電流變化的幅度較小，這是因為此階段所添加的葡 萄糖量很小，將圖示放大之後仍可以明顯看到電流變化 第3 C圖顯示本發明實施例製備之酵素電極的標準品 099137991 表單編號A0101 第14頁/共36頁 0992066199- 201209411 添加校正檢量線，其根據上財法製作独線性最小平 方避知法獲得-直線型檢量線。結果顯*其線性濃度範 圍為〇至55.5Π1Μ，線性相關係數（Γ2)為〇 9951。在文獻 中葡肖糖標準品添加校正檢量線最佳的線性濃度範圍 為〇· 05 mM至26 mM，線性相關係數（Γ2)為 °-9948(Tanj X.-c., Tian, Y.-X. , Cai, P.-X., Z〇u, X.-Y., 2005. Anal. Bioanal. Chem. 381, 500-507)。 [0027] 〇 而標準品添加校正檢量線的斜率即為偵測靈敏度， 亦即75.4 nA mM 1，此靈敏度對葡萄糖分析而言，已經 相當優異。另外，偵測極限值（L〇d)為15. 〇 ，比其 他文獻提出的葡萄糖偵測電極為佳（Asav，E:，AkyU_ maz, E., 2010. Biosens. Bioelectron. 25, 1014-1018; Barsan, Μ.M., Klin ar, J. , Bati .M., Brett, C.M.A., 2007. Talanta 71, 1893-1900; Ghica, M.E. , Brett, C. M. A. , 2006. 〇 Electroanalysis 18, 748-756; Tsai, M.-C., Tsai, Y.-C., 2009. Sens. Actuators, B 141, 592-598)。 [0028] 酵素電極的穩定度 第4圖顯示本發明實施例所製備酵素電極經長期使用 後的穩疋性’包含葡萄糖標準品添加校正檢量線的線性 相關係數、靈敏度、偵測極限值。經過3 8天測試後，酵 素電極的靈敏度降低到約32.4 nA mM-1，約為初始靈敏 度的43% ;然而’酵素電極仍具有良好的線性濃度範圍（〇 099137991 表單編號A0101 第15頁/共36頁 0992066199-0 201209411 至44. 4 mM)與線性相關係數〇. 9922。另外，偵測極限值 (L0D)由15以Μ增加至90 #M。與文獻（Asav and Akyilmaz, 2010; Ghica and Brett, 2006; Sun et al.，2008 ; Yang et al.，2006)所提出的葡萄 糖電極比較，其在經過10天或1個月的使用後，靈敏度分 別降低至0.15"八11^-1(降低50%)、0.20/^111»1_1( mM 1(降低90%)。此表示酵素電極靈敏度的衰減，是無可 避免的現象；然而，只要酵素電極仍具有寬廣的線性濃 度範圍與良好線性相關儀數，則分析仍具有準確性。因 此，在實際應用上，葡萄糖偵測電極的線性濃度範圍與 偵測極限值是兩個最重要的規格參數。 [0029]流動注射分析系統的最佳流量值 在流動注射分析系統’前述實施例所製備的酵素電 極作為三電極流動室的工作電極。另外，使肢i [pH 7.0的NaPB料做為流躲射分料統的載體溶液與三電 極流動㈣電解液。由於酵素t歸生的電流訊號會受 載體溶液《科，g此將制溶液的流量分別設定為 I 5 2‘ 〇、2. 5、3. 0 ' 4. 5 niL mirT1 ’ 以濃度 〇·2 _的葡萄糖標準溶液作測試，試圖找出最佳流量值 [0030] 々IL /射分析系統在不同流量時 ，酵素電極產生的電流訊號。第咖.本發明流動注 射分析系統在不同流量時，酵素電極連續測量5次產生的 電流訊號值，其中曲線⑷（b)(c)分別為流量2 ml 099137991 表單編號A0101 第16頁/共36頁 0992066199-0 201209411 min 、L 5 mL min i、1. 0 mL· mirT1 的5次測量值曲 線’並且於施加電壓.為〇. 45V ;載體溶液〇. i μ、PH 7.0的NaPB溶液；注射量為50;葡萄糖濃度為〇2 mM之環境下進行測量。 [0031] Ο [0032] 由第5A圖可看出隨著流量增加，訊號減弱。當流量 超過2. 5 mL min —1，訊號過低，不利於分析程序。雖然 第5八圖顯示流量1〇11^111丨11_1時具有最大訊號值136.3 nC’但第5B圖顯示當流量為2.〇 mL min—1時（曲線a)， 產生最穩定的訊號；因此.，選用.2· 〇 mL mi η-1作為流動 注射分析系統的最徐流量傳。 流動注射分析系統的干擾 、 測量葡萄糖含量時，通常會:受到其他非葡萄糖物質 ’特別是與葡萄糖具有類似結構的碳水化合物的干擾。 〇 例如’由HPLC分析獲知竹筷水解產物包含至少三種碳水 化合物：葡萄糖、木糖（Xy/l〇se)、纖維雙糖 (cellobiose)。然而，發明人先前的研究（Cheng, C.， Chen, C.-S. , Hsieh, P.-Η., 2010. J. Chroma-togr. A 1217, 2104-2110; Cheng, C., Tsai, H.-R., Chang, K.-C., 2006. J. Chroraatogr. A 1 1 1 9, 188-196)指出植物纖維的水解產物還包含一些微 量的糖類，例如阿拉伯糖（arabinose)、甘露糖 (mannose)、半乳糖（galactose)。 為了分析干擾物質的影響，分別製備1.66 mM的葡 萄糖標準溶液與具有濃度為3.33 mM(Barsan et al.， 2007)的碳水化合物干擾物質的1.66 mM葡萄糖溶液，並 099137991 表單編號A0101 第17頁/共36頁 0992066199-0 [0033] 201209411 以上述流動注射分析系統分別測試，以計算出酵素電極 對干擾物質的選擇係數（selectivity coefficient) [0034] 表一列出本發明所製備酵素電極應用於流動注射分 析系統量測葡萄糖時，各種干擾物質的選擇係數（k)與干 擾程度。結果顯示對於纖維雙糖、木糖、阿拉伯糖，其 選擇係數k均為0、干擾程度均為0 %。另外，甘露糖與半 乳糖分別有7%與5%的干擾程度。 表一 碳水化合物 信號比0 干擾程度(％) 選擇係數β 纖維雙糖 1.00±0.01 0.0 0.00 木糖 1.00±0.01 0.0 0.00 阿拉伯糖 1.00±0.01 0.0 0.00 甘露糖 1.07±0·01 7.0 0.07 半乳糖 1.05±0.01 5.0 0.05 3量測次數(》) = 3 b訊號比=(總訊號)*» +刊Hd/(總訊號)88修 CA=(訊號)千*·/(訊號)g*« [0035] 酵素電極與HPLC定量分析結果比較 本發明實例以應用酵素電極的流動注射分析系統， 與高效液相層析儀連接折射率彳貞測器（HPLC-RI)，分別 分析竹筷水解產物的葡萄糖濃度。表二列出兩種分析方 法的比較結果。結果顯示，除了水解反應第4小時之外， 其餘各反應時間的葡萄糖分析結果，以流動注射分析系 統測得的葡萄糖濃度，皆大於以高效液相層析儀測得的 099137991 表單編號A0101 第18頁/共36頁 0992066199-0 201209411 濃度。由此結果看來兩系統之間似乎存在有系統誤差； 然而，若考慮甘露糖（mannose)、半乳糖（galactose) ，與其他水解產物含有的未知干擾物質對於酵素電極的 影響，以及基質效應（matrix effect)對於使用外標準 品校正曲線法（external standard calibration quantification method)之高效液相層析儀折射率偵 測器的影響，則表二所列是合理的測試結果。 [0036][0018] By way of example and not limitation, the system shown in FIG. 2A uses a CHI611B electrochemical workstation 222 manufactured by CH Instruments, Texas, USA, in conjunction with a specially designed three-electrode flow cell (221), and Samples were delivered using a peristaltic pump 223 for on-line glucose content analysis. The three-electrode flow to 221 has a # wire as a counter: an electrode (e〇unter electrode, CE), a silver/vaporized silver (Ag/AgC1y as a reference electrode (RE)', and a winter invention example The enzyme electrode serves as a working electrode. In addition, the 'stop flow sampling system 23' uses a sample injection valve of the Hercules Bi〇-Rad model MV-6 of the United States as the six-hole injection valve 232 of the above system. The sampling work also includes the use of a peristaltic pump 233 and two three-hole valves 239A/B. The bioreactor 21 uses a 3L bioreactor of Taichung Process & Equipment Model BTF-A3L from Taichung, Taiwan, which includes a pH electrode 211 and The temperature probe 212 controls the pH and temperature of the reaction liquid in the bioreactor 21 through the controller 213. In addition, it takes 099137991 Form No. A0101 Page 12 / Total 36 Page 0992066199-0 201209411 [0019] [0020] Ο [0021 [0023] The sample 214 is used to derive the product of the bioreactor 21. Note that in other embodiments of the invention, the above-described inline flow injection analysis system 20 can be used to analyze other non-glucose analytes. As long as the enzyme electrode in the three-electrode flow chamber 22 is replaced with an enzyme electrode capable of analyzing the analyte, the 'three-electrode flow chamber 22 can also be designed as a two-electrode flow chamber. Quantitative analysis of glucose by HPLC. During the hydrolysis of waste bamboo into the glucose, the hydrolyzed product was also analyzed by HPLC-RI. The sampling period was the same as that of the previous enzyme electrode flow injection analysis. II* BP" Cyclic voltammetry analysis by German voltammetric analysis ( Cyclic volta.m:inetrie) The oxidation potential of the enzyme electrode prepared in the examples of the present invention can be obtained. When analyzing, the potential scanning range is from 〇. 〇 to 〇. 7 V, and the scanning speed is 50 mVs_1. The analysis sample is three glucose standard solutions. The concentration was mM, 4 mM, 10 mM, respectively, and was prepared by using sodium phosphate buffer solution (NaPB) of μ, 1 μ, pfl 7·0. Figure 3A shows the cyclic voltammetric analysis of the above three standard solutions, the t curve ( a) (b)(c) are cyclic voltammetry curves of 〇 mM, 4 mM, 10 mM, respectively, and arrows indicate the direction of the scan. It can be observed from the graph that the curve (b) (c) has a Obvious anode current Whereas the curve (a) no anode current. This represents a vehicle with eight "oxidation potential is 0.45 V, and the vehicle successfully with glucose oxidase immobilized on the modified surface of the carbon electrode. 099137991 Form No. A0101 Page 13 / Total 36 Page 0992066199-0 201209411 [0024] [0026] The standard addition correction calibration line method is based on the standard addition calibration calibration line method (... when (4) paint 1〇11 Calibrati〇n method, Pijanowska d G Sprenkels A τ ni+, . ' . ·, AJ·, Olthvus, w·, Bergveld I: :Γ. Μ· B 91,98,2) Quantitative analysis of Portuguese... The calibration curve can be used to detect the sensitivity of the enzyme electrode, detect the limit value (limit 〇f detec_ h〇n' _ ' and the linear concentration debt measurement range. Create a calibration calibration line at 35 制作Before each measurement, the nitrogen gas _ clock does not deoxidize the sodium phosphate buffer solution (NaPB). First, prepare a concentration of 50 secret and 250 mM glucose standard solution with Q.i Μ, Qiu 7.0 NaPB' and place the whole At night, two different optically active a- and b-forms of glucose are naturally converted (mutarotation). A calibration calibration curve is added for the preparation of the standard, and 8 mL of 0.11 Μ, pH 7. 0 NaPB solution is placed in the electrochemical Use the battery as a background solution. After 3 minutes, add every 1 · 5 minutes A glucose standard solution v of about 2 to 285 /^L is accumulated until the concentration of the solution is % 6 mM. The current value is read for the last 3 seconds after each standard addition. Figure 3B shows a preparation according to an embodiment of the present invention. The current and time gate curves of the standard electrode and the calibration curve are prepared by the above method. The amount of current change when glucose is added each time indicates the amount of glucose oxidation corresponding to the added glucose. The time is 6〇〇. At 18 sec, the magnitude of the current change in the graph is small, because the amount of glucose added at this stage is small, and the current change can still be clearly seen after the enlargement of the figure. FIG. 3C shows an embodiment of the present invention. Prepared enzyme electrode standard 099137991 Form No. A0101 Page 14 / Total 36 page 0992066199- 201209411 Add the calibration check line, which is obtained according to the method of making the linear least squares avoidance method to obtain the - linear type calibration curve. * The linear concentration range is 〇 to 55.5Π1Μ, and the linear correlation coefficient (Γ2) is 〇9951. In the literature, the optimal linear concentration range for the addition of the calibration curve of the glucomannan standard is 〇 From 05 mM to 26 mM, the linear correlation coefficient (Γ2) is °-9948 (Tanj X.-c., Tian, Y.-X., Cai, P.-X., Z〇u, X.-Y., 2005. Anal. Bioanal. Chem. 381, 500-507). [0027] 斜率 The slope of the calibration curve added to the standard is the detection sensitivity, which is 75.4 nA mM 1, which is quite good for glucose analysis. In addition, the detection limit value (L〇d) is 15. 〇, which is better than the glucose detection electrode proposed in other literatures (Asav, E:, AkyU_ maz, E., 2010. Biosens. Bioelectron. 25, 1014-1018 Barsan, Μ.M., Klin ar, J., Bati.M., Brett, CMA, 2007. Talanta 71, 1893-1900; Ghica, ME, Brett, CMA, 2006. 〇Electroanalysis 18, 748-756; Tsai, M.-C., Tsai, Y.-C., 2009. Sens. Actuators, B 141, 592-598). [0028] Stability of the enzyme electrode Fig. 4 is a graph showing the linear correlation coefficient, sensitivity, and detection limit value of the calibration curve of the glucose standard added by the enzyme electrode prepared in the examples of the present invention after long-term use. After 38 days of testing, the sensitivity of the enzyme electrode was reduced to approximately 32.4 nA mM-1, approximately 43% of the initial sensitivity; however, the 'enzyme electrode still has a good linear concentration range (〇099137991 Form No. A0101 Page 15 of 36 pages 0992066199-0 201209411 to 44. 4 mM) and linear correlation coefficient 〇. 9922. In addition, the detection limit value (L0D) is increased from 15 to 90 #M. Compared with the glucose electrode proposed in the literature (Asav and Akyilmaz, 2010; Ghica and Brett, 2006; Sun et al., 2008; Yang et al., 2006), the sensitivity is after 10 days or 1 month of use. Decrease to 0.15 "eight 11^-1 (50% reduction), 0.20/^111»1_1 (mM 1% reduction). This indicates that the attenuation of the sensitivity of the enzyme electrode is inevitable; however, as long as The enzyme electrode still has a wide linear concentration range and a good linear correlation meter, so the analysis is still accurate. Therefore, in practical applications, the linear concentration range and detection limit of the glucose detection electrode are the two most important specifications. [0029] The optimum flow rate of the flow injection analysis system in the flow injection analysis system 'the enzyme electrode prepared in the previous example is used as the working electrode of the three-electrode flow chamber. In addition, the limb i [pH 7.0 NaPB material is used as The flow of the carrier solution and the three electrodes flow (4) electrolyte. The current signal due to the enzyme t will be subjected to the carrier solution, and the flow rate of the solution is set to I 5 2' 〇, 2. 5,3. 0 ' 4. 5 niL mirT1 'tested with a glucose standard solution with a concentration of 〇·2 _ to try to find the optimum flow value [0030] 々IL / ray analysis system at different flow rates, the current signal generated by the enzyme electrode. Analyze the system at different flow rates, the enzyme electrode continuously measures the current signal value generated 5 times, wherein the curve (4)(b)(c) is the flow rate 2 ml 099137991 Form No. A0101 Page 16 / Total 36 Page 0992066199-0 201209411 min , L 5 mL min i, 1.0 mL · mirT1 5 times measurement curve 'and applied voltage. 〇 45V; carrier solution 〇. i μ, pH 7.0 NaPB solution; injection volume is 50; glucose concentration is Measured in an environment of 〇 2 mM [0031] 003 [0032] It can be seen from Figure 5A that the signal is weakened as the flow rate increases. When the flow rate exceeds 2. 5 mL min —1, the signal is too low, which is not conducive to the analysis procedure. Although Figure 5 shows that the flow rate is 1〇11^111丨11_1 with the maximum signal value of 136.3 nC', but Figure 5B shows that when the flow rate is 2.〇mL min-1 (curve a), the most stable signal is generated; Therefore, use .2· 〇mL mi η-1 as the flow injection Xu most traffic transmission system interference flow injection analysis system, measuring the glucose content, generally: by other non-glucose substances' particular interference with glucose having a similar structure of the carbohydrate. 〇 For example, it is known from HPLC analysis that the bamboo chop hydrolysate contains at least three kinds of carbohydrates: glucose, xylose (Xy/l〇se), and cellobiose. However, the inventor's previous research (Cheng, C., Chen, C.-S., Hsieh, P.-Η., 2010. J. Chroma-togr. A 1217, 2104-2110; Cheng, C., Tsai , H.-R., Chang, K.-C., 2006. J. Chroraatogr. A 1 1 1 9, 188-196) indicates that the hydrolysate of plant fibers also contains some traces of sugars, such as arabinose. , mannose, galactose. To analyze the effects of interfering substances, a 1.66 mM glucose standard solution and a 1.66 mM glucose solution with a carbohydrate interfering substance concentration of 3.33 mM (Barsan et al., 2007) were prepared, respectively, and 099137991 Form No. A0101 Page 17 of 36 pages 0992066199-0 [0033] 201209411 The flow injection analysis system described above was separately tested to calculate the selectivity coefficient of the enzyme electrode to the interfering substance. [0034] Table 1 lists the enzyme electrode prepared by the present invention for flow injection. The selection coefficient (k) and the degree of interference of various interfering substances when the analysis system measures glucose. The results showed that for fiber disaccharide, xylose, and arabinose, the selection coefficient k was 0 and the degree of interference was 0%. In addition, mannose and galactose have a degree of interference of 7% and 5%, respectively. Table 1 Carbide signal ratio 0 interference degree (%) Selection coefficient β fiber disaccharide 1.00±0.01 0.0 0.00 xylose 1.00±0.01 0.0 0.00 arabinose 1.00±0.01 0.0 0.00 mannose 1.07±0·01 7.0 0.07 galactose 1.05± 0.01 5.0 0.05 3 measurement times (》) = 3 b signal ratio = (total signal) *» + publication Hd / (total signal) 88 repair CA = (signal) thousand * · / (signal) g * « [0035] Comparison of Enzyme Electrode and HPLC Quantitative Analysis Results The present invention uses a flow injection analysis system using an enzyme electrode and a high performance liquid chromatography coupled with a refractive index detector (HPLC-RI) to analyze the glucose concentration of the bamboo chop hydrolysate. . Table 2 lists the comparison results of the two analytical methods. The results showed that, except for the fourth hour of the hydrolysis reaction, the glucose analysis results of the other reaction times were greater than those measured by the flow injection analysis system, which was greater than that of the high-performance liquid chromatograph. 099137991 Form No. A0101 No. 18 Page / Total 36 pages 0992066199-0 201209411 Concentration. From this result, it seems that there is a systematic error between the two systems; however, if mannose, galactose, the influence of unknown interfering substances contained in other hydrolysates on the enzyme electrode, and the matrix effect are considered ( Matrix effect) For the effect of the high performance liquid chromatography refractive index detector using the external standard calibration quantification method, the results listed in Table 2 are reasonable test results. [0036]
反應時 分析方法 間 線上HPLCa 線上FIAa ⑻ 纖維雙糖 葡萄糖 木糖 葡萄糖 (mM) (mM) (mM) (mM) 0 0.02 0.02 0.11 0.04 4 0.05 0.27 0.69 0.25 8 0.04 0.44 0.76 0.45 12 0.03 0.54 0.85 0.61 16 0.03 0.57 0.85 0.62 24 0.02 0.64 0.90 0.67 32 0.03 0.69 0.96 0.71 40 0.03 0.65 0.94 0.70 48 0,02 0.33 0.84 0.37 56 0.02 0.16 0.76 0.21 64 0.00 0.00 0.51 0.00 3量測次數(《) = 2 為驗證是否兩種分析方法存在系統誤差，以統計學 的線性迴歸線方法分析。第6圖顯示在相同取樣時間，兩 099137991 表單編號A0101 第19頁/共36頁 0992066199-0 201209411 種分析方法測量水解產物之葡萄糖濃度的線性迴歸分析 結果，可得到一直線，其斜率為1. 0451、Y座標截距為 0. 0110、線性廻歸係數（r2)為0. 9920。在95%的信心水 準下，線性迴歸直線的斜率範圍是從0. 9744至1. 1158 ; 這個範圍涵蓋了理想斜率值1. 0。同樣，在95%的信心水 準下，Y座標截距的範圍是從-0. 0019至0. 0239，也涵蓋 了理想截距值0，亦即原點。因此，兩種分析方法的結果 是相當的，且不存在系統誤差。 [0037] 本發明上述實施例製備一種新穎的酵素電極，利用 化學鍵結，使氧化還原媒介物與葡萄糖氧化酵素連接至 電極的修飾表面，據此完成的酵素電極用於偵測葡萄糖 濃度時，具有最寬廣的偵測線性濃度範圍、極低的偵測 極限值，與長期的穩定性。所製備的酵素電極可應用於 一流動注射分析系統，作為該系統的三電極流動室的工 作電極，該分析系統還包含一停流取樣次系統，使分析 程序更加簡易。流動注射分析系統可連接一生物反應器 ，可進行線上分析生物反應器的生物轉換產物。當用於 測量葡萄糖濃度時，流動注射分析系統的分析結果可與 高效液相層析儀的結果相比擬，其分析精確性的相對標 準偏差（RSD)小於3. 7 %。 [0038] 另外，本發明實施例所製備的酵素電極，可用於構 成一種血糖試片，其連接一血糖機，以讀取血糖試片產 生的信號，據此判斷試片上血液樣本的葡萄糖濃度。第7 圖顯示根據本發明實施例的一血糖試片30，用於一血糖 機。血糖試片30包含毛細管段31、偵測段32、電極接觸 099137991 表單編號A0101 第20頁/共36頁 0992066199-0 201209411 段33。偵測段32至少包含工作電極（we)、逆電極（CE)、 參考電極（RE)，其中本發明的酵素電極作為工作電極 (WE )。毛細管段31至少包含樣品孔34，利用毛細引力將 血液樣品吸入並傳％至偵測段32，使血液樣品與偵測段 32的電極接觸。工作電極（WE)具有一部份表面鍵結有氧 化還原媒介物與葡萄糖氧化酵素。例如，整個工作電極 的表面可沈積有金表面’但只有在金表面的中間或末端 部分，鍵結有媒介物與葡萄糖氧化酵素。 [0039] 〇 偵測段32透過分佈奉毛細管段31下方的導線（未圖 示）電性連接電極接觸·33。在測試時，電極接觸段33被 插入血糖機，而偵測段32產％的信號經由電極接觸段33 由灰糖機讀取，藉此決定血液樣本中的血糖濃度。 [0040] 在本發明另一實施例’省略上述電極接觸段33。因 此，偵測段32被插入血糖機，其直接讀取偵測段32各電 極的信號。另外，除了被配置於*争間部位，樣品孔34可 配置於毛細管段31的端部或侧邊。 〇 W [0041] 傳統的血糖試片皆為使用過即丟棄。由於工作電極 ，亦即酵素電極的長期穩定性極佳，本發明的血糖試片 可設計成一種可重複使用的血糖試片。為達此目的， 毛細管段31可設計成可置換式；在每次血糖測試完成後 將偵測& 3 2浸沒於—或多個溶液或溶劑中，使去除殘 留在電極上的血液樣品與反應產物，接著以—新的毛細 s &31取代現有的毛細管段3丨，如此即可進行下一次測 量0 099137991 表單編號A0101 第21頁/共36頁 0992066199-0 201209411 [0042] 以上所述僅為本發明之較佳實施例而已，並非用以 限定本發明之申請專利範圍；凡其他未脫離發明所揭示 之精神下所完成之等效改變或修飾，均應包含在下述之 申請專利範圍内。 【圖式簡單說明】 [0043] 第1圖顯示根據本發明較佳實施例之酵素電極的製造方法 第2A圖顯示根據本發明一實施例的流動注射分析系統的 架構圖， 第2 B圖顯示第2 A圖之流動注射分析系統的六孔注射閥； 第2C圖顯示2A圖之流動注射分析系統的三電極流動室的 上視圖與側視圖； 第3A圖顯示以本發明實施例製備的酵素電極測量三種標 準溶液的循環伏安分析圖； 第3B圖顯示根據本發明實施例製備的酵素電極，以上述 方法製備標準品添加校正檢量線時的電流-時間曲線； 第3 C圖顯示本發明實施例製備之酵素電極的標準品添加 校正檢量線； 第4圖顯示本發明實施例所製備酵素電極經長期使用後的 穩定性； 第5A圖顯示本發明流動注射分析系統在不同流量時，酵 素電極產生的電流訊號； 第5B圖顯示本發明流動注射分析系統在不同流量時，酵 素電極連續測量5次產生的電流訊號值； 第6圖顯示在相同取樣時間，以HPLC與本發明流動注射分 析系統兩種分析方法測量水解產物之葡萄糖濃度的線性 099137991 表單編號A0101 第22頁/共36頁 0992066199-0 201209411 迴·歸分析曲線； 第7圖顯示根據本發明實施例的一多次使用血糖試片，用 於一血糖機。 【主要元件符號說明】 [0044] Ο ο 099137991 10鉛筆芯 11碳膏 12金表面 12Α第一修飾表面 12Β第二修飾表面 12C第三修飾表面 12D第四修飾表面 13電極 13Α第一電極 13Β第二電極 13C第三電極 13D第四電極 20流動注射分析系統 21生物反應器 22電化學分析次系統 23停流取樣次系統 30血糖試片 31毛細管段 32偵測段 33電極接觸段 34樣品孔 211 pH電極 表單編號A0101 第23頁/共36頁 0992066199-0 201209411 212溫度探針 213控制器 214取樣管 2 21三電極流動室 222電化學工作站 223蠕動泵 231取樣注射器 232六孔注射閥 233蠕動泵 234標準品注射器 235注射器過濾器 236葡萄糖標準溶液 237去離子水 238A三孔閥 238B三孔閥 239A三孔閥 2 3 9 B三孔閥 099137991 表單編號A0101 第24頁/共36頁 0992066199-0Reaction time analysis method on the line HPLCa line FIAa (8) Cellobiose glucose xylose glucose (mM) (mM) (mM) (mM) 0 0.02 0.02 0.11 0.04 4 0.05 0.27 0.69 0.25 8 0.04 0.44 0.76 0.45 12 0.03 0.54 0.85 0.61 16 0.03 0.57 0.85 0.62 24 0.02 0.64 0.90 0.67 32 0.03 0.69 0.96 0.71 40 0.03 0.65 0.94 0.70 48 0,02 0.33 0.84 0.37 56 0.02 0.16 0.76 0.21 64 0.00 0.00 0.51 0.00 3 Measurement times (") = 2 To verify whether two The analytical method has systematic errors and is analyzed by statistical linear regression method. Figure 6 shows the results of a linear regression analysis of the glucose concentration of the hydrolysate at the same sampling time, two 099137991 Form No. A0101, page 19/36 pages 0992066199-0 201209411, and a straight line with a slope of 1. 0451 0。 The Y coordinate intercept is 0. 0110, the linear coefficient of return (r2) is 0. 9920. In the 95% confidence level, the slope of the linear regression line ranges from 0. 9744 to 1. 1158; this range covers the ideal slope value of 1.0. Similarly, at 95% confidence level, the Y coordinate intercept range is from -0. 0019 to 0. 0239, which also covers the ideal intercept value of 0, which is the origin. Therefore, the results of the two analytical methods are comparable and there is no systematic error. [0037] The above embodiment of the present invention prepares a novel enzyme electrode, which uses chemical bonding to connect a redox medium and glucose oxidase to the modified surface of the electrode, and the enzyme electrode thus completed is used for detecting the glucose concentration. The widest range of detection linear concentrations, extremely low detection limits, and long-term stability. The prepared enzyme electrode can be applied to a flow injection analysis system as a working electrode of the three-electrode flow chamber of the system, and the analysis system also includes a stop-flow sampling subsystem to make the analysis procedure easier. The flow injection analysis system can be connected to a bioreactor for on-line analysis of bioreactor bioconverter products. When the glucose concentration is measured, the analysis results of the flow injection analysis system can be compared with the results of the high performance liquid chromatography, and the relative standard deviation (RSD) of the analytical accuracy is less than 3.7 %. Further, the enzyme electrode prepared in the embodiment of the present invention can be used to construct a blood glucose test piece which is connected to a blood glucose meter to read a signal generated by the blood glucose test piece, and thereby determines the glucose concentration of the blood sample on the test piece. Figure 7 shows a blood glucose test strip 30 for use in a blood glucose meter in accordance with an embodiment of the present invention. The blood glucose test strip 30 includes a capillary section 31, a detection section 32, and an electrode contact. 099137991 Form No. A0101 Page 20 of 36 0992066199-0 201209411 Section 33. The detecting section 32 includes at least a working electrode (we), a counter electrode (CE), and a reference electrode (RE), wherein the enzyme electrode of the present invention functions as a working electrode (WE). The capillary section 31 contains at least a sample well 34 which is inhaled by capillary force and passed to the detection section 32 to bring the blood sample into contact with the electrode of the detection section 32. The working electrode (WE) has a portion of the surface bonded with a redox mediator and glucose oxidase. For example, the surface of the entire working electrode may be deposited with a gold surface 'but only in the middle or end portion of the gold surface, with a vehicle and glucose oxidase bound. [0039] The detecting section 32 is electrically connected to the electrode contact 33 through a wire (not shown) distributed under the capillary section 31. During the test, the electrode contact section 33 is inserted into the blood glucose meter, and the signal of the detection section 32% is read by the glucose machine via the electrode contact section 33, thereby determining the blood glucose concentration in the blood sample. [0040] The above-described electrode contact portion 33 is omitted in another embodiment of the present invention. Therefore, the detection section 32 is inserted into the blood glucose meter, which directly reads the signals of the electrodes of the detection section 32. Further, the sample hole 34 may be disposed at the end or side of the capillary section 31 in addition to being disposed at the intervening portion. 〇 W [0041] Traditional blood glucose test strips are discarded after use. Since the long-term stability of the working electrode, i.e., the enzyme electrode, is excellent, the blood glucose test piece of the present invention can be designed as a reusable blood glucose test piece. To this end, the capillary section 31 can be designed to be replaceable; after each blood glucose test is completed, the detection & 3 2 is immersed in - or a plurality of solutions or solvents to remove the blood sample remaining on the electrode and The reaction product, followed by the replacement of the existing capillary section 3丨 with the new capillary s & 31, so that the next measurement can be made. 0 099137991 Form No. A0101 Page 21 / Total 36 Page 0992066199-0 201209411 [0042] The present invention is not intended to limit the scope of the present invention; any equivalent changes or modifications made without departing from the spirit of the invention should be included in the following claims. Inside. BRIEF DESCRIPTION OF THE DRAWINGS [0043] FIG. 1 is a view showing a method of manufacturing an enzyme electrode according to a preferred embodiment of the present invention. FIG. 2A is a block diagram showing a flow injection analysis system according to an embodiment of the present invention, and FIG. Figure 6A shows a six-hole injection valve of a flow injection analysis system; Figure 2C shows a top view and a side view of a three-electrode flow chamber of the flow injection analysis system of Figure 2A; Figure 3A shows an enzyme prepared in accordance with an embodiment of the present invention The electrode measures the cyclic voltammetry analysis chart of the three standard solutions; FIG. 3B shows the current-time curve when the enzyme electrode prepared according to the embodiment of the present invention is prepared by the above method, and the calibration curve is added; The standard of the enzyme electrode prepared in the embodiment of the invention is added with a calibration calibration line; FIG. 4 shows the stability of the enzyme electrode prepared by the embodiment of the present invention after long-term use; FIG. 5A shows the flow injection analysis system of the present invention at different flow rates. The current signal generated by the enzyme electrode; Figure 5B shows that the flow injection analysis system of the present invention continuously measures the enzyme electrode 5 times at different flow rates. The current signal value; Figure 6 shows the linear measurement of the glucose concentration of the hydrolysate by HPLC and the flow injection analysis system of the present invention at the same sampling time. Form 09107991 Form No. A0101 Page 22 / Total 36 Page 0992066199-0 201209411 Back-to-back analysis curve; Figure 7 shows a multi-use blood glucose test piece for a blood glucose meter according to an embodiment of the present invention. [Main component symbol description] [0044] ο ο 099137991 10 pencil lead 11 carbon paste 12 gold surface 12 Α first modified surface 12 Β second modified surface 12C third modified surface 12D fourth modified surface 13 electrode 13 Α first electrode 13 Β second Electrode 13C Third Electrode 13D Fourth Electrode 20 Flow Injection Analysis System 21 Bioreactor 22 Electrochemical Analysis Sub System 23 Stop Flow Sampling Subsystem 30 Blood Glucose Test Strip 31 Capillary Section 32 Detection Section 33 Electrode Contact Section 34 Sample Well 211 pH Electrode Form No. A0101 Page 23 / Total 36 Page 0992066199-0 201209411 212 Temperature Probe 213 Controller 214 Sample Tube 2 21 Three Electrode Flow Chamber 222 Electrochemical Workstation 223 Peristaltic Pump 231 Sampling Syringe 232 Six Hole Injection Valve 233 Peristaltic Pump 234 Standard syringe 235 syringe filter 236 glucose standard solution 237 deionized water 238A three-hole valve 238B three-hole valve 239A three-hole valve 2 3 9 B three-hole valve 099137991 form number A0101 page 24 / total 36 page 0992066199-0