| 摘要: | 人類血清白蛋白是人類體內運送物質重要的蛋白質，並維持體內的滲透壓和緩衝血液中pH值。在健康的腎臟中，人類血清白蛋白不會排出體外到尿液中。但在某些腎臟病中，一旦腎臟出了狀況會導致尿液中出現白蛋白的成分。所以在人體尿液中的人類血清白蛋白濃度是一個診斷腎功能的一個重要指標。一般的人類血清白蛋白感測電極是由鉑或金製作而成，這些金屬非常的昂貴，而且難以製造。本研究利用網版印刷技術搭配表面粗糙化技術，建構出一種多孔指叉碳電極，用於人類血清白蛋白感測。透過在網印碳漿中均勻混合碳酸鈣(CaCO3)粉末和硬酯酸(Stearic acid)，並且以鹽酸溶解碳酸鈣粉末，在電極表面製作出許多細微孔洞，增加電極的檢測表面積，並增強其量測電流響應，以及創造羧基於電極表面上，使抗體得以固定在電極表面上。本研究利用計時伏安法(chronoamperometry)，量測電極的電化學響應。根據實驗結果顯示，電極在純系統溶液的人類血清白蛋白量測中，有著廣大的線性量測區間，為10 ~ 300 mg/L，R2=0.99812。涵蓋了人體尿液正常到微量蛋白尿的量測範圍。電極也有1.68 μA mg-1 mL的優良靈敏度。透過表面粗糙化的方法，也使電極在純系統量測下無論在靈敏度和線性區間上，相較於傳統網印碳電極，皆有所提升。電極同時在含有人類尿液中的各種干擾物質的樣本接受測試，實驗結果也顯示以計時伏安法量測的電極，受到干擾物影響造成的量測偏差皆小於5%。最後在實際的人體樣本檢驗中，和臨床的檢驗方法也有著良好的相關性，檢測濃度100 mg/L以下的HSA誤差小於16%，檢測濃度大於100 mg/L以上的HAS誤差皆小於9%。透過這種電極，提高了網印碳電極在人類血清白蛋白的感測效能，也為人類血清白蛋白診斷提供了一個更加便宜方便的選擇。;Human serum albumin is the most abundant protein in human blood plasma. Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains osmotic pressure. When the kidneys are working properly, albumin is not present in the urine. But when the kidneys are damaged, trace amount of albumin leaks into the urine, called albuminuria. If early kidney damage is not treated, large amount of albumin may leak into the urine and this can lead to chronic kidney disease (CKD). Therefore, the concentration of human serum albumin in human urine is an important indicator of a diagnosis of renal function. The clinical detection method of particle-enhanced turbidimetric inhibition immunoassay (PETINIA) instrument is very expensive. We want to create a low cost, easy-to-use biosensor for HSA detection to improve the modern measurement techniques. In this study, the porous carbon electrode with carboxylic surface was fabricated by screen-printing through uniform mixing of calcium carbonate (CaCO3) powder and stearic acid in the screen printing carbon paste. Then hydrochloric acid was used to dissolve the calcium carbonate powder to increase the surface area of the detection electrodes by porous structures. The obtained screen-printed porous carbon electrode (SPPCE) with carboxylic group surface was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). CV measurement showed the enhanced current response of SPPCE. Using EDC and sulfo-NHS crosslinking, anti-HSA antibodies were immobilized on SPPCE. In this study, we used anti-HSA/EDC+sulfo-NHS/ SPPCE immunosensor to measure HSA by chronoamperometry (CA) electrochemical technique. The electrochemical measurements of the urine samples with interferences demonstrated a high specificity and selectivity of this biosensor in detecting has. The Scanning Electron Microscope/Energy Dispersive Spectrometer (SEM/EDS) mapping was used to check every immobilization step. In optimal conditions, the immunosensor could detect HSA in a high linear range from 10 to 300 mg/L with a 1.68 μA mg-1 mL sensitivity. The calibration curve equation isI=2.10514-0.00175[HSA(mg/L)] with a high coefficient of determination (R2 = 0.99703). Finally, SPPCE based immunosensor was used to measure HSA in clinical urine samples from hospital. Measurement results showed a good correlation with clinical turbidimetric testing and error values were less than 12%. These results suggest that the HSA immunosensor is user-friendly, reliable, and highly specific for the detection of urinary albumin at the range of microalbuminuria. |
