Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, 1100 S. Grand Boulevard, St. Louis, MO 63104, USA.
Anal Biochem. 2010 Jan 15;396(2):298-303. doi: 10.1016/j.ab.2009.09.039. Epub 2009 Sep 24.
We developed a straightforward antibody-based assay for rapid homogeneous detection of bacteria. Our sensors utilize antibody recognizing cell-surface epitopes of the target cell. Two samples of the antibody are prepared, each labeled via nanometer size flexible linkers with short complementary oligonucleotides that are modified with fluorochromes that could participate in fluorescence resonance energy transfer (FRET). The length of the complementary oligonucleotide sequences was designed such that very little annealing occurred in the absence of the target cells. In the presence of the target cells the two labeled antibodies bind to the surface of the cell resulting in a large local concentration of the complementary oligonucleotides that are attached to the antibody. This in turn drives the annealing of the complementary oligonucleotides which brings the fluorescence probes to close proximity producing large FRET signals proportional to the amount of target cells. Long flexible linkers used to attach the oligonucleotides to the antibody enable target-induced oligonucleotide annealing even if the density of surface antigens is only modest. We used Escherichia coli 0157:H7 and Salmonella typhimurium to demonstrate that this design produced sensors exhibiting rapid response time, high specificity, and sensitivity in detecting the target bacteria.
我们开发了一种简单的基于抗体的分析方法，用于快速均匀地检测细菌。我们的传感器利用抗体识别靶细胞表面表位。制备了两种抗体样本，每个样本都通过纳米尺寸的柔性接头标记有短的互补寡核苷酸，这些寡核苷酸经过荧光团修饰，可以参与荧光共振能量转移（FRET）。互补寡核苷酸序列的长度设计为在不存在靶细胞的情况下几乎不会退火。在存在靶细胞的情况下，两种标记的抗体结合到细胞表面，导致与抗体相连的互补寡核苷酸的局部浓度非常高。这反过来又促使互补寡核苷酸退火，将荧光探针拉近，产生与靶细胞数量成正比的大 FRET 信号。用于将寡核苷酸连接到抗体上的长柔性接头允许靶标诱导的寡核苷酸退火，即使表面抗原的密度仅适中。我们使用大肠杆菌 0157:H7 和鼠伤寒沙门氏菌证明，这种设计产生的传感器在检测靶细菌时具有快速的响应时间、高特异性和灵敏度。
