Watts H J, Yeung D, Parkes H
Laboratory of the Government Chemist, Teddington, Middlesex, U.K.
Anal Chem. 1995 Dec 1;67(23):4283-9. doi: 10.1021/ac00119a013.
The use of an optical biosensor, the resonant mirror, for direct and rapid detection of DNA-DNA hybridization has been demonstrated. Biotinylated oligonucleotide probes were immobilized on the sensor surface, via streptavidin, and the hybridization of a complementary target oligonucleotide (40-mer) was monitored in real time. The interaction at the sensor surface was shown to be sequence specific under conditions of low stringency. Regeneration of the surface-immobilized probe was possible, allowing reuse without a significant loss of hybridization activity. A comparison of probes indicated that the relative position of complementary sequence and the length of probe affected the hybridization response obtained. The potential of the sensor for quantitation of a hybridized DNA target was investigated. From radiolabeling data, the lowest amount of hybridized target sequence which could be determined directly was 19.9 fmol/mm2 (263 pg/mm2) of sensor surface. The dependence of the sensor response on the concentration of probe and target oligonucleotide was established. Utilizing the assay as an end-point determination method, the lowest detectable concentration of target oligonucleotide (40-mer) was 9.2 nM. This compares favorably to other sensor methods described previously without the requirement for labels.
已证明使用光学生物传感器——共振镜可直接快速检测DNA-DNA杂交。生物素化的寡核苷酸探针通过链霉亲和素固定在传感器表面，并实时监测互补靶寡核苷酸（40聚体）的杂交情况。在低严格度条件下，传感器表面的相互作用显示出序列特异性。表面固定的探针可以再生，可重复使用且杂交活性不会显著损失。对探针的比较表明，互补序列的相对位置和探针长度会影响获得的杂交反应。研究了该传感器对杂交DNA靶标进行定量的潜力。根据放射性标记数据，可直接测定出的最低杂交靶序列量为传感器表面19.9飞摩尔/平方毫米（263皮克/平方毫米）。确定了传感器响应与探针和靶寡核苷酸浓度的关系。将该检测方法用作终点测定法时，靶寡核苷酸（40聚体）的最低可检测浓度为9.2纳摩尔。与之前描述的其他无需标记的传感器方法相比，这一结果很有优势。
