新型血清CYFRA21-1检测电化学生物传感器的研制

doi:10.3969/j.issn.1673-8640.2020.06.017

摘要/Abstract

摘要：

目的构建一种用于检测细胞角蛋白19片段（CYFRA21-1）的高灵敏电化学生物传感器。方法合成三维石墨烯@金纳米粒子（3D-G@Au）复合材料,用其修饰玻碳电极,通过壳聚糖、戊二醛与抗CYFRA21-1抗体交联,构建用于检测CYFRA21-1的3D-G@Au的电化学生物传感器。对构建成功的3D-G@Au的电化学生物传感器的电化学性能及检测性能进行初步评价。结果循环伏安图显示裸玻碳电极、3D-G修饰玻碳电极和3D-G@Au修饰玻碳电极的氧化还原峰逐渐升高,提示3D-G@Au电化学生物传感器构建成功。传感器抗原-抗体相互作用的最佳温育时间为60 min,最佳孵育温度为35 ℃。采用3D-G@Au电化学生物传感器检测CYFRA21-1,线性范围为0.1~300.0 ng/mL,检测限为0.1 ng/mL（信噪比=3）,10 mmol/L 维生素C、10 mmol/L 多巴胺和10 mmol/L 尿酸对检测无干扰,重现性较好[相对标准偏差（RSD）分别为1.08%、3.11%],与化学发光法比较,RSD为1.96%~4.78%。制备好的3D-G@Au电化学生物传感器可在4 ℃条件下稳定30 d。结论构建的3D-G@Au电化学生物传感器可用于血清CYFRA21-1的检测。

关键词: 细胞角蛋白19片段, 三维石墨烯, 金纳米粒子, 电化学生物传感器

Abstract:

Objective To construct a highly sensitive electrochemical biosensor for the detection of cytokeratin 19-fragment（CYFRA21-1）. Methods Three-dimensional graphene @gold nanoparticles（3D-G@Au）nanocomposite was modified on glassy carbon electrode surface to enhance the conductivity of electrochemical biosensor. Anti-CYFRA21-1 was captured and fixed on the modified 3D-G@Au glassy carbon electrode through the cross-linking of chitosan and glutaraldehyde. The 3D-G@Au electrochemical biosensor had been completed as to determine CYFRA21-1. The electrochemical and determination performance of successfully constructed 3D-G@Au electrochemical biosensor was evaluated preliminarily. Results The cyclic voltammetry showed that the redox peaks of bare glassy carbon electrode,3D-G modified glassy carbon electrode and 3D-G@Au modified glassy carbon electrode increased gradually,indicating that the 3D-G@Au electrochemical biosensor was successfully constructed. The optimal incubation time of antigen-antibody interaction was 60 min,and the optimal incubation temperature was 35 ℃. CYFRA21-1 was detected by 3D-G@Au electrochemical biosensor,the linear range was 0.1-300.0 ng/mL,and the determination limit was 0.1 ng/mL（signal-to-noise ratio=3）. 10 mmol/L Vit C,10 mmol/L DA and 10 mmol/L UA had no interference to the determination,and the reproducibility was good [relative standard deviations（RSD） were 1.08% and 3.11%], compared with that of chemiluminescence method (RSD was 19.6%-4.78%). The 3D-G@Au electrochemical biosensor can be stable for 30 d at 4 ℃. Conclusions It is potential for the determination of serum CYFRA21-1 with 3D-G@Au electrochemical biosensor.

Key words: Cytokeratin 19-fragment, Three-dimensional graphene, Gold nanoparticle, Electrochemical biosensor

中图分类号:

R446.1

王建富, 李小平, 陈文虎, 樊凯, 钱苗苗. 新型血清CYFRA21-1检测电化学生物传感器的研制[J]. 检验医学, 2020, 35(6): 595-600.

WANG Jianfu, LI Xiaoping, CHEN Wenhu, FAN Kai, QIAN Miaomiao. A novel electrochemical biosensor for the detection of serum CYFRA21-1[J]. Laboratory Medicine, 2020, 35(6): 595-600.

图/表 5

图1 三维石墨烯修饰玻碳电极和3D-G@Au修饰玻碳电极的CV图注：（a）0.5 mol/L NaCl溶液中三维石墨烯修饰玻碳电极的CV图;（b）0.5 mol/L NaCl溶液中3D-G@Au修饰玻碳电极的CV图,箭头表示在0.42处出现的还原峰

图2 5 mmol/L [FeCN6]3-/4-+0.1 mol/L KCl溶液中不同修饰玻碳电极的CV图注：1为裸玻碳电极;2为三维石墨烯修饰玻碳电极;3为3D-G@Au修饰玻碳电极

图3 在35 ℃条件下孵育时间与DPV峰电流关系曲线

图4 3D-G@Au电化学生物传感器的重复性注：（a）同一支3D-G@Au电化学生物传感器对1 ng/mL CYFRA21-1溶液连续测量5次;（b）3支不同的3D-G@Au电化学生物传感器对1 ng/mL CYFRA21-1溶液连续测量5次

表1 回收率和RSD结果

加入浓度/（ng/mL）	检测结果均值/（ng/mL）	回收率/%	RSD/%
0.5	0.51	102.00	2.8
1	1.03	103.00	2.1
10	9.86	98.60	1.8
20	20.58	102.90	1.0
50	49.86	99.72	1.9
100	103.40	103.40	2.5
200	204.40	102.20	3.1
300	298.65	99.53	2.6

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