A micro-chamber free digital biodetection method via the “sphere-labeled-sphere” strategy

[Display omitted] •“Sphere-labeled-sphere” strategy is first applied to micro-chamber free digital biodetection to completely distinguish “1” and “0” signals.•The feasibility and accuracy of our proposed method conducted on the high-throughput flow cytometry (FCM) are successfully verified.•Size effects of labeled fluorescent particles (FPs) on detection performance are systematically investigated.•FPs display a more objective and accurate result than conventional dyes in digital detection of biomarkers. Digital biodetection is one of the most ultrasensitive methods for quantifying biomarkers and has aroused extensive attention in biomedical fields. However, most of digital biodetections need a large number of isolated micro-chambers (such as microarrays or micro-droplets) at femtoliter or microliter scale to form a single target molecule reactor, which is complicated for practical applications. Here we propose a simple, micro-chamber free digital biodetection method via replacing micro-chambers with microspheres for the first time by employing magnetic spheres (MSs) as carriers and fluorescent particles (FPs) as labels (that is “sphere-labeled-sphere” strategy). An ideal model digital biodetection is first conducted to verify the feasibility and accuracy of our strategy through flow cytometry (FCM). After a systematic investigation of size effects on detection performance, 1.2 μm fluorescent polystyrene spheres (PSs) are determined to be the optimal labels for the carriers of 3.2 μm MSs. Finally, the test of carcinoembryonic antigen (CEA) was performed as a proof of concept, which displays a more objective and accurate result for distinguishing positive signals from negative ones. Therefore, this novel micro-chamber free digital biodetection method via the “sphere-labeled-sphere” strategy paves a promising way as a precise and powerful methodological tool for ultrasensitive biomarkers detection.