基于核酸杂交-酶联桥接的悬浮芯片技术同时定量反义寡核苷酸药物原型及其代谢物方法的初步建立

目的 利用悬浮芯片技术，建立基于核酸杂交-酶联桥接的悬浮芯片技术对反义寡核苷酸药物原型及其代谢产物同时定量的分析方法。方法 本研究在确证探针与微球偶联的基础上，考察生物基质效应、偶联微球浓度、检测探针浓度、S1核酸酶用量以及荧光显色液浓度等因素对方法定量的影响。最终在同一生物样品中对反义寡核苷酸原型及其代谢产物进行同时定量分析。结果 通过考察生物基质效应，确定稀释10倍的PBS作为条件优化及样品定量分析的生物学基质；微球浓度为150个/μl，检测探针浓度为400 nmol/L，S1核酸酶用量为1 U/孔，荧光显色液浓度为1 μg/ml。在猕猴血浆基质中该方法针对原型与代谢产物序列的定量范围均为7.81～2000 nmol/L。在同时含有低浓度（20 nmol/L）和高浓度（1500 nmol/L）AI-ON1原型（Ai）与AI-ON1缩短代谢产物（Am）的混合血浆样品中，该方法可同时特异性地定量分析Ai与Am。结论 本研究成功建立基于核酸杂交-酶联桥接的悬浮芯片定量分析方法，可在微量生物样品中实现对反义寡核苷酸药物原型及其代谢产物的同时定量分析。

Simultaneous quantification of antisense oligonucleotides and its metabolite in biological samples by a nucleic acid hybridization combined enzyme-linked bridging method based on suspension array technology

Objective To establish a nucleic-acid hybridization-enzyme ligation method based on suspension array for detection of antisense oligonucleotide drug prototype and its metabolites. Methods On the basis of confirming the couple of probe and microspheres, we investigated matrix effect and optimized the experiment conditions as follows： concentration of microspheres, detecting probe, S1 nuclease and fluorescent report substrate. The method was established for detection of antisense oligonucleotide drug prototype and its metabolites at the same time with a trace of sample. Results Biological matrix effect investigation determined the dilution 10 times by PBS as a proper condition for experiment optimization and quantification analysis. Concentration of microspheres was 50 beads/μl. Dectecting probe concentration was 400 nmol/L, S1 nuclease amount was 1 U/well and fluorescent report liquid concentration was 1 μg/ml. The range of the quantitative method for the prototype or metabolites in the macaque plasma matrix was 7.81-2000 nmol/L. In the same sample, the concentration of 20 nmol/L or 1500 nmol/L antisense oligonucleotides prototype and its metabolites can be quantified specifically through this method. Conclusion We have successfully established a nucleic-acid hybridization-enzyme ligation based on suspension array to simultaneously quantify the antisense oligonucleotide prototype and its metabolites with a trace of sample.