First-passage time analysis of diffusion-controlled reactions in single-molecule detection.

Single-molecule detection (SMD) aims to achieve the ultimate limit-of-detection (LOD) in biosensing. This method detects a countable number of targeted analyte molecules in solution, where the dynamics of molecule diffusion, capturing, identification and delivery greatly impact the SMD's efficiency and accuracy. In this study, we adopt the first-passage time method to investigate the diffusion-controlled reaction process in SMD. We analyze the influence of detection conditions on incubation time and the expected coefficient of variation (CV) under three SMD molecule capturing strategies, including solid-phase capturing (one-dimensional solid-liquid interface fixation), liquid-phase magnetic bead (MB) capturing, and liquid-phase direct fluorescence pair labeling. We find that inside a finite-sized reaction chamber, a finite average reaction time exists in all three capturing strategies, while the liquid-phase strategies are in general more efficient than the solid-phase approaches. CV can be estimated by averaging first-passage time solely in all three strategies, and the CV reduction is achievable given an extended reaction time. To further enable zeptomolar detection, extra treatments, such as adopting liquid-phase fluorescence pairs with high diffusion rates to label the molecule, or designing specific sensing devices with large effective sensing areas would be required. This framework provides solid theoretical support to guide the design of SMD sensing strategies and sensor structures to achieve desired measurement time and CV.

Automated Raman based cell sorting with 3D microfluidics.

Raman activated cell sorting has emerged as a label-free technology that can link phenotypic function with genotypic properties of cells. However, its broad implementation is limited by challenges associated with throughput and the complexity of biological systems. Here, we describe a three-dimensional hydrodynamic focusing microfluidic system for a fully automated, continuous Raman activated cell sorting (3D-RACS). The system consists of a 3D printed detection chamber (1 mm3) that is integrated with a PDMS based sorting unit, optical sensors and an in-line collection module. It has the ability to precisely position cells in the detection chamber for Raman measurements, effectively eliminating spectroscopic interference from the device materials. This enables the sorting of a range of cell sizes (from 1 µm bacteria to 10's µm mammalian cells) with stable operation over >8 hours and high throughput. As a proof-of-concept demonstration, Raman-activated sorting of mixtures of Chlorella vulgaris and E. coli has demonstrated a purity level of 92.0% at a throughput of 310 cells per min. The platform employed in this demonstration features a simple "Raman window" detection system, enabling it to be built on a standard, inverted microscope. Together with its facile and robust operation, it provides a versatile tool for function-based flow cytometry and sorting applications in the fields of microbiology, biotechnology, life science and diagnostics.