Naked-eye enumeration of single chlamydia pneumoniae based on light scattering of gold nanoparticle probe.

Chlamydia pneumoniae (C. pneumoniae) is a spherical zoonotic pathogen with a diameter of ~200 nm, which can lead to a wide range of acute and chronic diseases in human body. Early and reliable on-site detection of C. pneumoniae is the key step to control the spread of the pathogen. However, the lack of a current technology with advantages of rapid-ity, ultra-sensitivity, and convenience limits the implementation of traditional techniques for on-site detection of C. pneumoniae. Herein, we developed a naked-eye counting of C. pneumoniae based on the light scattering properties of gold nanoparticle (GNP) with a dark-field microscope (termed "GNP-labeled dark-field counting strategy"). The recognition of single C. pneumoniae by anti-C. pneumoniae antibodies functionalized GNP probes with size of 15 nm leads to the formation of wreath-like structure due to the strong scattered light resulted from hundreds of GNP probes binding on one C. pneumoniae under dark-field. Hundreds of GNP probes can bind to the surface of a C. pneumoniae due to the high stability and specificity of the nucleic acid immuno-GNP probe, which generates by the hybridization of DNA modified GNP with DNA-functionalized antibodies. The limit of detection (LOD) of GNP-labeled dark-field counting strategy for the detection of C. pneumoniae in spiked samples or real samples is down to 4 C. pneumoniae per microL, which is about 4 times more sensitive than that of qPCR. And the GNP probes. Together with the advantages of the strong light-scattering characteristic of aggregated GNPs under dark-field microscope and the specific identification of functionalized GNP probe, we can detect C. pneumoniae in less than 30 min using a cheap and portable microscope even if the sample contains only a few targets of interest and other species at high concentration. The GNP-labeled dark-field counting strategy meets the demand of rapid detection, low cost, easy to operate and on-site detection, which paves the way for early and on-site detection of infectious pathogens.