West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.
School of Chemical Engineering, Sichuan University, Chengdu 610065, China.
Analyst. 2018 Feb 7;143(3):700-708. doi: 10.1039/c7an01566f. Epub 2018 Jan 4.
Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 μL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 μmol L, in which the determination limit and detection limit were 2.5 and 0.75 μmol L, respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 μmol L (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.
本文开发了一种荧光毛细管生物传感器，用于定量检测微体积细胞内丙酮酸（PA）。该传感器通过自组装技术将金纳米粒子（AuNPs）和乳酸脱氢酶（LDH）修饰到氨化毛细管（20 μL）的内表面。通过将样品和 NADH 的混合溶液吸入生物传感器后，NADH 荧光值的变化来定量 PA 浓度。本研究考察了影响传感器性能的因素，包括毛细管表面氨基的质子化程度、AuNP 自组装的浓度和时间、LDH 自组装的活性浓度和时间、LDH 固定化的流速和酸度、pH 值、温度以及 NADH/PA/LDH 反应体系的反应时间。在优化条件下，该生物传感器对 PA 的线性响应范围为 2.5-120 μmol L，检测限和定量限分别为 2.5 和 0.75 μmol L。当相对标准偏差（RSD）控制在 1.5%以下时，该生物传感器可重复使用超过 41 次。在室温（约 25°C）下，使用该生物传感器测量了健康人红细胞内的细胞内 PA，PA 含量为 241.76 ± 68.05 μmol L（n = 8）。标准加入回收率为 95-106%。在 PA 生物传感器中使用 AuNPs 不仅提高了固定化 LDH 对 PA 的亲和力及其稳定性，而且显著延长了 PA 生物传感器的使用寿命。
