Quantitative Detection Of Chlamydia Pneumoniae Using Gold Nanoparticle Probe Based Dark-field Microscopy

Chlamydia pneumoniae(C.pneumoniae,C.pn)is a member of the Chlamydiae,an obligate intracellular bacterium which causes infections not only inhuman,but also in a wide variety of animal species,such as koalas,horses,amphibians and reptiles.Recent research suggests that the persistent infections of C.pn also association with a diverse range of chronic diseases,including lung cancer,asthma,Alzheimer's disease and many serious cardiovascular diseases like coronary heart disease.Due to the lack of characteristic clinical symptoms,further identification of C.pn often needs to be further determined using a certain detection technique in the laboratoryAt present,the diagnosis of C.pn in clinic still relies on a few traditional methods such as culturing,enzyme-linked immunosorbent assay(ELISA),micro-immunofluorescence(MIF)and polymerase chain reaction(PCR),among which MIF is considered as the gold standard for qualitative detection of C.pn.However,the isolation and culture of C.pn from host issues or cells is laborious and time-consuming,which has little significance for early diagnosis.Serological tests may delay detection time because a large number of antibodies usually appear after C.pn infect host for more than a week.PCR assay requires specially trained technician and considerable laboratory resources while they are usually not available in the vast majority of underdeveloped regions of developing countries,although it is sensitive.In this study,to address the problem for on-site detection of C.pn,using a time-saving,simple and convenient method,we constructed a quantitative detection technology by employing gold nanoparticle(GNP)probes and dark-field microscope,which can meet the needs of detection in a rapid,low cost,high sensitivity and specificity,easy to operate and on-site wayThe application of nanomaterial-based biosensor has been extensively studied in many fields with unique characteristics.A variety of nanomaterial-based detection methods for small molecules and microorganisms have been successfully reported.Herein,we offer a gold nanoparticle probe based dark-field counting strategy,which relies on a gold nanoparticle(GNP)modified with C.pn that can bind well to C.pn and a common optical microscope with a dark-field lens.After mixing GNP probes with sample containing C.pn,hundreds of GNP probes attach to each C.pn to form a bright golden garland-like structure under dark-field microscopy,which is easy to count with naked eye because the reunited gold nanoparticles attaching C.pn can emit strong scattering lights in dark-field.After the construction of GNP probes,we used SDS-PAGE,transmission electron microscope(TEM)and the established RT-qPCR to verify the specificity and the accuracy of our GNP-based dark-field counting strategy and used it to detect pure C.pn samples.The results show that GNP probes have been constructed and our counting method worked well for detection of pure C.pn samples,which only costs about 30 min for assay of each sample.The pure-culture sample detection data show that limit of detection(LOD)of this counting strategy is down to single pathogen per ?L,which is about 10 times more sensitive than real-time fluorescence quantitative PCR(RT-qPCR)in this work.Furthermore,we collected the pathogens from lung tissues of C57BL/6J mice infected with C.pn,and detected the concentration of these real samples by the established GNP-based dark-field counting strategy and verified by digital PCR as well.The data shows that our counting strategy is valid for detection of real samples.In the future,our GNP-based dark-field counting strategy can be further developed and is potential to be used for clinically quantitative on-site detection of C.pn as well as other pathogens with similar size.In summary,this study used protein A modified GNP and C.pn antibody to construct GNP probes,which can effectively capture C.pn and form a cake-like structure in which hundreds of GNP probes attach to single C.pn surface and exhibits a bright golden garland-like structure under the dark field of a general optical microscope,therefore offering a direct counting by the naked eye.Detection result for real sample also agreed with that LOD of this method is down to single C.pn cell per ?L.Our counting method is ultrasensitive and easy-to-operation and is suitable for quickly qualitative on-site detection of C.pn and other pathogens with the similar size in real samples.