以矯正模組與奈米金於電流式生物感測器之研究

Researches of Amperometric Biosensors Using Correction Module and Gold Nanoparticles

林岳暉(Yueh-Hui Lin)

指導教授 ： 林志生 ； 張正

國立交通大學/生物科技學院/分子醫學與生物工程研究所/博士(2009年)

https://doi.org/10.6842/NCTU.2009.00692

摘要

以網印碳電極(screen-printed carbon electrode；SPCE)為架構的電流式生物感測器，是相當適合於以化學、生物或物理的工程技術，將酵素、氧化還原媒介物、抗體和奈米材料等，予以修飾其上，以強化其在作為醫療診斷、食品安全確保與環境監控時所需要的靈敏性。在本研究中，分別研究開發可運用在SPCE的一矯正模組與一奈米金(Au nanoparticles；AuNPs)修飾技術。
分別以有無葡萄糖氧化酶(glucose oxidase；GOD)之網印碳電極(GOD(+)-SPCE與GOD(-)-SPCE)作為複合電極，再以外加尿酸與調整血球容積比(hematocrit ratio；Hct%)方式進行葡萄糖測試溶液的電流法測定，進行矯正模組研究。在以YSI 2300 STAT Plus 作為參考法比較之下，全血中葡萄糖濃度受到尿酸干擾的關係性，以及Hct%，可藉本矯正模組使其更為準確。利用GOD(+)-SPCE與GOD(-)-SPCE之間的電流差值，ΔCurrent的計量，可降低大部分尿酸/生化物質的干擾。而所建立內插法方式可用於矯正Hct%在血糖檢測時的干擾；這內插法之Hct%矯正，在葡萄糖濃度高於110mg dL-1且同時Hct%低於35%時更顯重要。這樣的實用性可用於增進為糖尿病患族群服務時，全血量測的準確性。這方法也可提供給其他全血樣品，於進行電流式量測時降低干擾之用。
另一方面，該AuNPs修飾之SPCE，係以13-nm AuNPs搭配1,1-二茂鐵甲酸(ferrocenedicarboxylic acid；FeDC)以增強電流訊號；其可用於大腸桿菌O157：H7 (Escherichia coli O157:H7)電流式免疫電極，與偵測加保扶(carbofuran)/穀胱甘肽(glutathione)之酪胺酸酶(tyrosinase；Tyr)電極。在SPEC的工作極上，AuNPs、FeDC與抗體(antibody；Ab)的共修飾(SPCE/AuNPs/FeDC/Ab)，確實提昇了電流訊號；如此，可以偵測到在102~107 菌落形成單位(colony-forming units；CFU) mL-1範圍的E. coli O157:H7濃度。在磷酸緩衝液與牛奶樣品測試中的，可推算出每片AuNPs/FeDC-SPCE免疫電極的偵測極限，分別為6 CFU與50 CFU。此電流式免疫感測器的重點優勢為可於1小時完成牛奶樣品的檢測。相似地，AuNPs與FeDC在SPCE與Tyr的共修飾 (SPCE/AuNPs/FeDC/Tyr)，可以分別偵測到4.52-45.2 nM的加保扶與12.5~100 μM的穀胱甘肽。該SPCE/AuNPs/FeDC/Tyr試片的應用性與光譜法相較，毫不遜色。本研究所使用的酪胺酸酶為每試片2活性單位(unit；U)，相較於先前的電流式酪氨酸酶感測器研究，用量僅約1/30。矯正模組的基本架構與金奈米粒搭配二茂鐵的修飾，在網印碳電極之應用相當具潛力，將可整合成為一系統以應臨床檢測與環安監測的需求。

關鍵字

電流式生物感測器 ； 網印碳電極 ； 血糖 ； 奈米金 ； 大腸桿菌O157:H7 ； 酪胺酸酶

並列摘要

Amperometric biosensors constructed from screen-printed carbon electrode (SPCE) are amenable to chemical, biological or physiological engineering with modifying agents such as enzyme, redox mediators, antibodies and nano-material to impart the requisite sensitivity for medical diagnostics, food quality assurance and environmental monitoring in complex matrices of the biosensors. In this study, we developed a correction module and a modification technique with gold nanoparticles (AuNPs) for SPCE.
The SPCE test strips with and without glucose oxidase (i.e., GOD(+)-SPCEs and GOD(-)-SPCEs) were used for multiple test strips and the amperometric currents of glucose-testing solutions with various spiked uric acid concentrations and hematocrit ratios were measured for the study of correction module. By establishing the interference relationships between glucose concentrations and uric acid concentrations as well as Hct% values and with appropriate correction module, the whole blood glucose determinations could be made to be more accurate and comparable to those determined by the reference YSI method. Specifically, the use of ΔCurrent value, the current difference between GOD(+)-SPCE and GOD(-)-SPCE measurements, would reduce most of the uric acid/biochemical interferences. An interpolation method was also established to correct for the blood glucose determinations with Hct% interferences. The Hct% corrections using the interpolation method are especially important and necessary for those blood samples with glucose concentrations higher than 110 mg dL-1 and Hct% values lower than 35%. A practical solution can be developed to improve the accuracy of determinations for whole blood glucose and to serve the diabetic community better. This simplified approach also will be applied to reduce interferences for amperometric determinations of other analytes in the whole blood sample.
On the other hand, 13-nm diameter AuNPs coupled with ferrocenedicarboxylic acid (FeDC) are modified onto the SPCE for the enhancements of amperometric current for the Escherichia coli O157:H7 (E. coli O157:H7) immunosensors and the tyrosinase (Tyr)-based carbofuran/glutathione (GSH) biosensor. The modification of AuNPs, FeDC and antibodies (Ab) on the working electrode of SPCE (SPCE/AuNPs/FeDC/Ab) certainly enhances the mediated-amperometric signals. The concentrations of E. coli O157:H7 from 102 to 107 CFU mL-1 can be detected. The detection limit is 6 CFU per strip in phosphate buffer and 50 CFU per strip in milk, respectively. One of important advantages of our SPCE/AuNPs/FeDC/Abs immunosensing strips can be utilized for the determination of E. coli O157:H7 during 1 h. Similarly, the modification of AuNPs, FeDC and Tyr on the working electrode of SPCE (SPCE/AuNPs/FeDC/Tyr), can be enhance the efficiency for the determination of carbofuran and GSH; the linear concentration range of carbofuran and GSH is 4.52 to 45.2 nM and 12.5 to 100 M, respectively. The applications of SPCE/AuNPs/FeDC/Tyr strips were quite as good as the spectrophotometric methods of carbofuran and GSH. The using tyrosinase was only 2 Unit per strip that was 1/30 compared with the past studies. The basis of correction module and the modifications of AuNPs/FeDC provides a potential for further applications of amperometric SPCE, which will be incorporated into an integrated system for the requirements of diagnosis testing and environmental/safety monitoring.

並列關鍵字

amperometric biosensors ； screen-printed carbon electrode ； blood glucose ； gold nanoparticles ； Escherichia coli O157:H7 ； tyrosinase

參考文獻

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