Digital Bio-detection Based On Ultra-bright Conjugated Polymer Nanoparticles

Digital bio-detection is a major breakthrough in in vitro diagnosis,because it can achieve the absolute counting of target molecules and posseses ultra-high sensitivity.Current digital bio-detection methods including digital PCR and digital ELISA,mainly achieve single-molecular detection through physical isolation to limit the target molecules to a small space so as to increase the concentration of the target molecules.However,physical isolation methods such as microarray chips and micro-droplets not only increase the complexity of the detection system wich leads to higher costs,but also make it more difficult to achieve high reproducibility.Therefore,a digital bio-detection method without additional physical separation was established in this study,adopting ultra-bright conjugated polymer nanoparticles(CPNs)as fluorescent labelling material.First,ultra-bright conjugated polymer nanoparticles as fluorescent labelling particles were prepared.A series of CPNs with different particle sizes were synthesized by co-precipitation of PFBT with a comb-like amphiphilic polymer PS-PEG-COOH and their optical properties were investigated.With the increase of particle size,CPNs could maintain stable and excellent optical properties.And it was revealed that the single-particle brightness of CPNs increased quadratically with the increase of diameter.CPNs with average diameter of 140 nm were adopted as fluorescent labelling material for the following digital bio-detection since a single particle could be detected by laser confocal scanning microscopy and had a relatively small particle size.Then,digital bio-detection platform based on microspheres was established.Microspheres and CPNs were modified with streptavidin and biotin respectively.And CPNs were added to the microsphere reaction system at a certain ratio to perform the simulated biological reaction.The feeding amount of CPNs-biotin were much less than microbead-SA,which insured that there was at most one CPNs-biotin conjugated with every microbead-SA.Then,the fluorescence of each microsphere was detected by laser confocal scanning microscopy.The microspheres combining fluorescent nanoparticles served as signal “1”,and the microspheres without fluorescent nanoparticles served as signal “0”.The number ratio of fluorescent microspheres in total microspheres were worked out to calculate the concentration of fluorescent nanoparticles according to Poisson distribution,thus achieving digital bio-detection.It showed that the number of conjugated polymer nanoparticles bounding to the microspheres obeyed the poisson distribution,and the LOD of simulated bio-detection reached 18 aM,which verified the feasibility of the constructed digital bio-detection method.Finally,human chorionic gonadotropin(hCG)was chosen as target molecule in the digital bio-detection method.Capture antibody and labelling antibody were conjugated with microbeads and CPNs respectively.Choosing microbeads as carrier material and CPNs as labelling material,the beads-Digital ELISA was constructed and its feasibility was explored in this study.In summary,the constructed beads-Digital ELISA shows ultra-high sensitivity and possesses huge application potential in the field of in vitro diagnosis.