Jain Priyamvada, Das Smita, Chakma Babina, Goswami Pranab
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India.
Anal Biochem. 2016 Dec 1;514:32-37. doi: 10.1016/j.ab.2016.09.013. Epub 2016 Sep 15.
A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I/I intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.
通过指数富集配体系统进化技术（SELEX）筛选出的一条针对恶性疟原虫乳酸脱氢酶（PfLDH）的90聚体单链DNA适配体（P38），以氧化石墨烯（GO）作为固定基质固定在玻碳电极（GCE）上，对修饰电极进行PfLDH检测研究。GO由粉末状铅笔石墨合成，并通过X射线衍射（XRD）表征，石墨层间距离从石墨的0.345nm增加到GO的0.829nm。通过拉曼光谱中的I/I强度比证实了P38在GO上的固定，其中石墨、GO和P38-GO的该比值分别为0.67、0.915和1.35。从电极的原子力显微镜（AFM）分析中表面高度的增加可以明显看出P38层在GO-GCE上的形成，从GO-GCE的约3.5nm增加到P38-GO-GCE的约27nm。当用目标物对所开发的适配体传感器进行检测时，证明其对PfLDH的检测下限低至0.5fM。通过对PfLDH催化产生的还原辅因子在0.65V下进行伏安测量，证实了适配体传感器的特异性。对一些常见蛋白质的干扰、储存稳定性、重复性以及实际样品分析的研究证明了该适配体传感器的实际应用潜力。
