基于纳米金修饰的核酸适体生物传感器用于环孢素浓度的体外测定

Detection of the Concentrations of Ciclosporin (CsA) with Aptamer Biosensor Modified by Nano Gold Solution in Vitro

利用环孢素(CsA)的两条高亲和力核酸适体生物传感器用于环孢素浓度的体外测定,在柠檬酸三钠为还原剂条件下,以氯金酸为原料,制备纳米金(Au)溶液,并对其进行表征,将aptamer-Ⅰ经酰胺化反应共价结合到磁性纳米颗粒表面,以纳米金(Au)修饰aptamer-Ⅱ,以固定在磁性纳米颗粒表面的aptamer-Ⅰ与环孢素孵育,再与Au-aptamer-Ⅱ作用,形成磁性纳米颗粒/环孢素/纳米金三明治夹心结构,磁分离技术分离三明治复合物,210 nm处检测分离前后紫外吸收强度的变化,对环孢素进行定量检测。结果紫外检测结果显示,制备的纳米金溶胶在520 nm处呈现典型的吸收峰,JEM-4 000EX电镜结果显示,纳米金溶胶颗粒粒径分布均匀、形貌、分散度较好,粒径大小在30 nm左右,此法对环孢素的响应范围是50~1 500 ng/m L,其线性回归方程为Y=4×10^(-3)X+5.769×10^(-1),R=0.997 3。此法可在1~2 h内完成检测,有望用于血液环孢素浓度的测定。

To make use of the two high affinitive DNA aptamers of CsA to build a kind of nucleic acid aptamer biosensor based on nanometer magnetic particles for CsA concentration determination. Under the condition of the sodium citrate as reducing agent, using gold chloride acid as raw material, the nanometer gold sol was prepared and its characterization was remarkable. The nucleic acid aptamer- I was covalently bonded to the surface of the magnetic nanoparticles. The aptamer- Ⅱ was modified with nanometer gold （Au） solution. The magnetic nanopart- icles fixed with aptamer- I and CsA was incubated together. The magnetic nanoparticles/CsA/nano gold sandwi- ches structure was formed to remove miscellaneous protein by magnetic separation technology. The change of UV absorption intensity in 210 nm before and after separation was detected, and quantitative analysis was carried out on ciclosporin （CsA）. Ultraviolet detection results showed that the absorption peak of the nanometer gold solution prepared was around 520 nm, and the results of JEM-4 000EX microscopy showed that the nano gold solution had good particle size distribution, morphology, neat dispersion and the particle size was around 50 nm, The response of this method was 50-1 500 ng/mL, the linear equation was Y=4×10-3X＋5.769×10-1, （R=0.997 3） and the detection limit was 30 ng/mL. This method can be completed within 1-2 h. It can be applied to ciclosporin （CsA） determination.