Chlamydia 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 rapidity, ultrasensitivity, 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) under dark-field microscopy (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 microscopy. Hundreds of GNP probes can bind to the surface of C. pneumoniae due to the high stability and specificity of the nucleic acid immuno-GNP probes, which generates by the hybridization of DNA-modified GNP with DNA-functionalized antibodies. The limit of detection (LOD) of the GNP-labeled darkfield counting strategy for C. pneumoniae detection in spiked samples or real samples is down to four C. pneumoniae per microliter, which is about 4 times more sensitive than that of quantitative polymerase chain reaction (qPCR). Together with the advantages of the strong light scattering characteristic of aggregated GNPs under dark-field microscopy and the specific identification of functionalized GNP probes, 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 demands 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.

Abstract. In recent years, nanomaterials have attracted great interest from industrial community and researchers due to many reports showing that nanomaterials can be promising vehicles for in vitro diagnosis, in vivo tumor imaging and targeted therapy of many diseases in addition to being wide-spectrum antibacterial agents. Since nanomaterial can readily diffuse into aquatic environments and further enter human body, which probably pose a threat to human health thus limit the real application of nanomaterials in organism. To this end, it is very important to investigate the toxicity of nanomaterial to organisms. Herein, we evaluated the toxicity of two representative silver nanoparticles (AgNPs@PVP, AgNPs@citrate) on the development of zebrafish (a classic model animal) embryos by incubating these silver agents and silver ions (as a positive control) with early embryos at various concentrations for 24, 48, 72 and 96 hours, respectively. The results showed that after 24 hours incubation, Ag + of 0.2 μg/mL led to up to 80 % of mortality of embryos and AgNPs@citrate (~15 nm diameter) can result in death of a small percentage of embryos (mortality of 20 %). However, AgNPs@PVP (~15 nm diameter) did not cause any death of embryos. As the concentration went up to 0.5 μg/mL, Ag + caused 100 % mortality of embryos with 48 hours incubation, and AgNPs@citrate was also highly toxic (mortality of 96 %), but the mortality rate induced by AgNPs@PVP was only 40 %. This data clearly indicate that the AgNPs@PVP is less toxic than AgNPs@citrate and the toxicity of AgNPs@citrate to embryos is comparable to silver ion.