Multidimensional laser induced fluorescent and microfluidic-valve-system-based combinational antibiotics susceptibility screening and sub-5 minute pathogen identification

Overusing broad-spectrum antibiotics, due to the slow process of traditional bulk bacteria identification and antibiotic susceptibility testing (ID & AST), has raised severe public health concerns regarding antibiotic resistance. Despite alternatives such as PCR and MALDI-TOF MS were developed, these methods still face challenges, such as a high risk of contamination, suboptimal specificity, or pre-testing culture required. In response, we herein report the Multi-Dimensional Laser Induced Fluorescent and Microfluidic Valve System as a promising solution for rapid and accurate bacteria ID and AST. The system combines a microfluidic valve-based automated combinational multisample assembling platform, a multi-channel laser-induced fluorescent (LIF) single-cell resolution optical detection system, an innovative thermal permeation-based assay hybridizing fluorescent oligonucleotide probes and 16S ribosomal RNA, and a multidimensional Quadratic Discriminant-based machine learning bacteria ID algorithm. This unique combination allows our system to achieve automated bacteria identification with 98% accuracy among 7 different bacteria species in under 5 minutes and high-throughput combinational AST in under 1 hour with high specificity and sensitivity. Taken together, our system represents a rapid, simple, specific, sensitive, and versatile solution for bacteria ID and AST, and portends an important role in the fight against antibiotic resistance.