肽核酸生物传感器检测系统构建时靶序列浓度的影响及线性范围的确定

Effects of target concentration and determination of its linear range on construction of PNA biosensor detection system

目的 探索肽核酸生物传感器检测系统构建时靶序列浓度值对杂交效应的影响 ,以及靶序列浓度线性范围的确定。方法 石英谐振式生物传感器阵列表面先固定上 1.5 μM的bis PNA探针 ,观察终浓度为 1pg/L至 10 0 μg/L的HBVDNA与探针进行杂交所引起的频率变化及反应所需时间。并对频率下降值与靶序列浓度做线性回归 ,确定其线性范围。结果 靶序列浓度为 1pg/L时 ,传感器未能检测出任何频率的改变。随着浓度从 10 pg/L增加到 10 0 μg/L ,杂交反应引起的频率下降值呈先增加后趋于缓和的趋势 ,以 10 μg/L为分界线 ,而杂交平衡时间并没有显现出一定的变化趋势。在 10pg/L到 10 μg/L的浓度范围内 ,浓度与频率下降值的线性回归方程为lgC =- 2 .74 5 5 +0 .0 6 91×△F ,相关系数r=0 .992 3。结论 随着靶序列浓度的升高 ,杂交反应引起的频率下降值呈典型饱和曲线趋势 ,靶序列浓度的线性检测范围为 :10 pg/L～ 10 μg/L。

Objective To investigate the effects of target concentration on hybridization efficiency and to determine its linear range during the construction of peptide nucleic acid (PNA) biosensor detection system. Methods 1.5 μM bis-PNA probe was firstly immobilized on the surface of biosensor array. Then target HBV genomic DNA varied from 1 pg/L to 100 μg/L was hybridized with the probe, and the decrease of frequency and equilibrium time of reaction were recorded and analyzed. And linear regression was done between frequency decrease and the target concentration; hence the linear range was determined.Results Any frequency change could not be detected by biosensor when target concentration was 1 pg/L. It enhanced firstly and then tended gently when target concentration varied from 10pg/L to 100μg/L, and 10μg/L was the watershed. And no evident change tendency was observed on equilibrium time for hybridization. The statistic linear regression equation was lgC=-2.745 5+0.069 1×△F among the range of target concentration from 10pg/L to 10μg/L and the correlating coefficient was 0.992 3. Conclusion The frequency change caused by hybridization reaction was a typical saturation curve in accordance with the increase of the target concentration. And the linear range of the target concentration was between 10pg/L to 10μg/L.