Naked-Eye Enumeration of Single Chlamydia pneumoniae Based on Light Scattering of Gold Nanoparticle Probe

Abstract

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.

Dark-Field Microscopy: Recent Advances in Accurate Analysis and Emerging Applications

“…Similarly, they used antibody-functionalized gold nanoparticles to recognize Chlamydia pneumoniae forming a wreath-like structure (Figure A). The specific optical topography in this counting analysis method could also play important roles in avoiding false-positive results …”

“…Reproduced from Chen, F.; Di, T.; Yang, C.-T.; Zhang, T.; Thierry, B.; Zhou, X. ACS Sens . 2020 , 5 , 1140–1148 (ref ). Copyright 2020 American Chemical Society.…”

“…The specific optical topography in this counting analysis method could also play important roles in avoiding false-positive results. 25 When the morphological characteristics of the targets could be used for an effective distinction, the accuracy of analysis is better. Li and co-workers took advantage of antibodyfunctionalized gold nanoparticles as reporters to count E. coli under DFM with a limit of detection comparable to that of the flow cytometry method.…”

Visual and Ultrasensitive Detection of a Coronavirus Using a Gold Nanorod Probe under Dark Field

“…We have previously shown many DFM counting strategies for pathogens of various sizes, such as Cryptosporidium [ 17 ], Chlamydia pneumoniae [ 18 ], and white spot syndrome virus [ 19 ]. These counting strategies make the best use of the dependence of LSPR on the proximity of other nanoparticles.…”

An Ultrasensitive Virus ELISA Based on a magnetic Mesoporous Silica Nanoprobe

“…Based on our previous experience in in vitro detection using nanoprobe, [ 8–11 ] the design of a nanoprobe for colorimetric ELISA should consider several critical parameters. First, simple design is preferable as probes with complex assembly strategies are faced with reproducibility issues and of questionable translational relevance.…”