de la Rica Roberto, Baldi Antonio, Fernández-Sánchez César, Matsui Hiroshi
Department of Chemistry and Biochemistry, City University of New York-Hunter College, 695 Park Avenue, New York, New York 10065, USA.
Anal Chem. 2009 Sep 15;81(18):7732-6. doi: 10.1021/ac901210f.
The risk of infectious diseases has compelled some industries to establish a zero-tolerance standard for the presence of microorganisms in a given sample. Here, we address this issue with a novel reverse-phase immunoassay on impedimetric transducers for the specific detection of extremely low numbers of pathogens (less than 10 cells). After simply spotting the sample onto the electrodes, physisorbed analytes were targeted with urease-labeled antibodies, and the urease on the pathogens hydrolyzed urea to ionic species with a concomitant decrease of the resistivity of the solution. By this methodology, the limit of detection (LOD) based on the 3sigma criterion was 1 Escherichia coli cell with an assay time under 1 h. However, the precise number of cells present in highly diluted samples is uncertain, making it difficult to assess the final LOD of the sensor. We overcome this problem by using an atomic force microscope to deposit and image in situ the exact number cells on the transducer. After performing the immunoassay, a single E. coli cell was successfully detected without ambiguity in the number of cells even in the presence of a 10(4) excess of a competing microorganism, thus demonstrating the outstanding LOD and selectivity of the proposed reverse-phase immunoassay.
传染病风险促使一些行业针对给定样本中的微生物存在情况制定零容忍标准。在此，我们利用一种新型的基于阻抗传感器的反相免疫分析法来解决这一问题，以特异性检测极少量病原体（少于10个细胞）。将样本简单点样到电极上后，用脲酶标记的抗体靶向物理吸附的分析物，病原体上的脲酶将尿素水解为离子物质，同时溶液的电阻率降低。通过这种方法，基于3σ标准的检测限为1个大肠杆菌细胞，检测时间不到1小时。然而，高度稀释样本中存在的细胞精确数量不确定，这使得难以评估传感器的最终检测限。我们通过使用原子力显微镜在传感器上原位沉积并成像细胞的确切数量来克服这个问题。进行免疫分析后，即使存在10⁴倍过量的竞争微生物，也能成功检测到单个大肠杆菌细胞，且细胞数量明确无误，从而证明了所提出的反相免疫分析法具有出色的检测限和选择性。
