【摘要】：L-蘋果酸是普遍存在于動物、植物及微生物體內(nèi)的一種重要羧酸,在細(xì)胞內(nèi)參與多種生命活動,在食品、醫(yī)藥以及化工等領(lǐng)域都有重要的應(yīng)用。對于L-蘋果酸的定量檢測,目前主要包括色譜法、光譜法、酶法等,這些方法雖然能夠滿足測定精確度的要求,但一般步驟繁瑣、成本較高。蘋果酸脫氫酶(MDH)在存在其輔酶NAD(P)+時可在生理條件下催化蘋果酸的轉(zhuǎn)化及NAD(P)H的生成。利用對NAD(P)H敏感的電化學(xué)傳感器對其進(jìn)行檢測,可以實現(xiàn)對L-蘋果酸的定性定量分析。根據(jù)這一原理可以通過將蘋果酸脫氫酶固定于NADH敏感電極表面形成電流型酶電極的途徑解決L-蘋果酸的定量檢測問題。本文將蘋果酸脫氫酶同殼聚糖、戊二醛制成固定化酶制劑,并將其整合至由碳納米管修飾的電化學(xué)基底電極表面構(gòu)成酶電極,得到對L-蘋果酸敏感的酶生物傳感器:(1)利用魚精蛋白(Pr)與多壁碳納米管(CNT)形成穩(wěn)定的復(fù)合物,以此復(fù)合物對絲網(wǎng)印刷電極(SPE)的工作表面進(jìn)行修飾,獲得碳納米管功能化的絲網(wǎng)印刷電極Pr/CNT/SPE。通過表面微觀結(jié)構(gòu)表征發(fā)現(xiàn),魚精蛋白的應(yīng)用使碳納米管修飾電極表面形成了穩(wěn)定、致密的微觀結(jié)構(gòu),顯著提高了修飾電極對NADH的電催化性能。修飾電極對NADH在2-1500μmol/L的范圍內(nèi)存在線性響應(yīng),回歸方程i(μA)=-1.8764c(mmol/L)-0.0516,R2=0.984。響應(yīng)時間為6 s,靈敏度為1.8764μA·L/mmol/Lol,檢測限為0.05μmol/L(S/N=3)。實驗還確定了最佳的Pr/CNT質(zhì)量分?jǐn)?shù),確定Pr/CNT質(zhì)量分?jǐn)?shù)為0.5作為制備Pr/CNT/SPE的最佳方案。(2)以Pr/CNT/SPE作為基底電極構(gòu)建對L-蘋果酸靈敏的電化學(xué)酶電極。以戊二醛(GDI)對殼聚糖(CHIT)進(jìn)行改性處理,獲得表面保留有自由醛基的酶固定化載體。將MDH同此固定化載體以合適的比例混合可以得到固定化酶CHIT-GDI-MDH。以此固定化酶修飾Pr/CNT/SPE,并以核微孔膜防止固定化酶的泄漏,即得完整的蘋果酸脫氫酶電極CHIT-GDI-MDH/Pr/CNT/SPE。該酶電極對5-300μmol/L內(nèi)的L-蘋果酸存在線性響應(yīng),其可用以定量檢測的回歸方程為i(μA)=-0.69c(mmol/L)-0.0024(R2=0.995)。響應(yīng)時間為6-8 s,檢測靈敏度為0.69μA·L/mmol;檢測限為0.03μmol/L(S/N=3)。實驗還對酶電極的抗干擾性能進(jìn)行了評估,發(fā)現(xiàn)酶電極具備一定的抗干擾性能,適合用于實際樣本中L-蘋果酸的分析檢測。(3)酶電極的制備方法及檢測條件優(yōu)化。選取CHIT-GDI:MDH比例、pH、工作電位以及測試溫度4個因素作為實驗變量,首先通過單因素實驗分析了其各自對酶電極傳感性能的影響并確定出其各自的最佳水平。隨后,再以4個因素進(jìn)行正交試驗,進(jìn)一步尋找最優(yōu)方案。結(jié)果顯示,CHIT-GDI-MDH/Pr/CNT/SPE的最佳制備方法及工作條件為:以每30U MDH同100μL CHIT-GDI混合制備CHIT-GDI-MDH,選用pH為7.5的緩沖液,檢測溫度45℃,檢測電位0.36V。該酶酶電極性能穩(wěn)定,測試結(jié)果可靠,可用于實際樣本中L-蘋果酸的分析測試。其制作過程簡單,使用方便,適于工業(yè)化生產(chǎn)。
[Abstract]:L-malic acid is an important carboxylic acid in animals, plants and microbes. It is involved in many life activities in cells and has important applications in the fields of food, medicine and chemical industry. The quantitative determination of L-malic acid mainly includes chromatography, spectrometric method and enzymatic method. Although these methods can meet the requirements of accuracy, the general steps are tedious and the cost is high. Malate dehydrogenase (MDH) can catalyze the transformation of malic acid and the formation of NAD (P) H under physiological conditions in the presence of its coenzyme NAD (P). The qualitative and quantitative analysis of L- malic acid can be realized by using electrochemical sensor sensitive to NAD (P) H. According to this principle, the problem of quantitative detection of L-malic acid can be solved by immobilization of malate dehydrogenase on the surface of NADH sensitive electrode to form current-type enzyme electrode. In this paper, malate dehydrogenase and glutaraldehyde were prepared into immobilized enzyme preparation and integrated into the surface of electrochemical substrate electrode modified by carbon nanotubes to form enzyme electrode. An enzyme biosensor sensitive to L-malic acid was obtained. (1) A stable complex of protamine (Pr) and multiwalled carbon nanotube (CNT) was obtained to modify the working surface of screen printing electrode (SPE). Preparation of carbon Nanotube functionalized screen Printing electrode Pr/CNT/SPE. It was found that the application of protamine made the surface of carbon nanotube modified electrode form stable and compact microstructure, and the electrocatalytic performance of modified electrode for NADH was improved significantly. The modified electrode has a linear response to NADH in the range of 2-1500 渭 mol/L. The regression equation I (渭 A) = -1.8764c (mmol/L) -0.0516C R2O 0.984. The response time is 6 s, the sensitivity is 1.8764 渭 A / mol / L ol / L and the detection limit is 0. 05 渭 mol/L (S/N=3). The experiment also determined the best Pr/CNT mass fraction, The best method for the preparation of Pr/CNT/SPE was determined as the mass fraction of Pr/CNT was 0.5. (2) the electrochemical enzyme electrode sensitive to L-malic acid was constructed by using Pr/CNT/SPE as the substrate electrode. Chitosan (CHIT) was modified with glutaraldehyde (GDI) to obtain the immobilized carrier with free aldehyde group on the surface. The immobilized enzyme CHIT-GDI-MDH. can be obtained by mixing MDH with the immobilized carrier in an appropriate proportion. The Pr/CNT/SPE, was modified by immobilized enzyme and the leakage of immobilized enzyme was prevented by nuclear microporous membrane. The complete malate dehydrogenase electrode CHIT-GDI-MDH/Pr/CNT/SPE. was obtained. The enzyme electrode has a linear response to L-malic acid in 5-300 渭 mol/L. The regression equation for quantitative detection is I (渭 A) = -0.69c (mmol/L) -0.0024 (R2O0.995). The response time was 6-8 s, the detection sensitivity was 0.69 渭 A / L / mmol and the detection limit was 0.03 渭 mol/L (S/N=3). The anti-interference performance of the enzyme electrode was also evaluated. It was found that the enzyme electrode had certain anti-interference performance and was suitable for the analysis and detection of L- malic acid in practical samples. (3) the preparation method and detection conditions of enzyme electrode were optimized. Four factors, CHIT-GDI:MDH ratio, pH, working potential and test temperature, were selected as experimental variables. Firstly, the effects of CHIT-GDI:MDH ratio, pH, working potential and test temperature on the sensing performance of enzyme electrode were analyzed by single factor experiments and their optimal levels were determined. Then, the orthogonal experiment was carried out with four factors to find the best scheme. The results showed that the optimal preparation method and working conditions of CHIT-GDI-MDH/Pr/CNT/SPE were as follows: CHIT-GDI-MDH, was prepared by mixing every 30 U MDH with 100 渭 L CHIT-GDI in buffer solution of pH 7.5, detection temperature was 45 鈩，

本文編號：2423399