Fluorescent probe for rapid bacterial detection in aqueous solutions

Overview

This technology addresses the critical need for rapid, sensitive detection of live bacteria in aqueous solutions, including clinically relevant fluids such as urine and blood. The approach leverages a fluorescent probe called beta-LEAF (beta-lactamase enzyme activated fluorophore) that identifies bacteria by detecting the activity of enzymes unique to bacterial cells, specifically penicillin-binding proteins (PBPs), which are universally involved in bacterial cell wall synthesis, and beta-lactamases, which are increasingly prevalent enzymes that degrade beta-lactam antibiotics. This dual-enzyme targeting strategy enables detection of both antibiotic-resistant and non-resistant bacteria, offering a versatile diagnostic platform.

The technology is designed for applications in clinical diagnostics, water quality monitoring, pharmaceutical manufacturing quality control, and environmental testing, where timely identification of bacterial contamination is essential.

Technical specifications

How it works:

• The beta-LEAF probe contains a cleavable beta-lactam core conjugated to two fluorophores positioned in close proximity, creating a quenched (non-fluorescent) state
• When the probe encounters bacterial enzymes such as PBPs or beta-lactamases, the beta-lactam core is cleaved
• Cleavage separates the two fluorophores, restoring their fluorescent properties and producing a detectable signal
• The presence of fluorescence indicates bacterial contamination

Key features:

• Single-mixing-step detection completed within approximately 10 minutes
• Validated in multiple fluid types including PBS buffer, human urine, and human blood
• Designed to detect bacteria regardless of beta-lactamase expression status
• A portable cell phone-based system coupled with a customized microfluidic device has been developed
• The portable platform aims to concentrate bacteria from large sample volumes, enabling detection at extremely low concentrations (potentially as low as 0.1 CFU/ml)
• Automated fluorescent result collection and interpretation capabilities

Technology readiness level

The beta-LEAF probe has undergone initial laboratory validation, demonstrating successful detection of bacterial beta-lactamase activity in PBS, human urine, and human blood samples through a straightforward mixing protocol. A portable cell phone-coupled microfluidic platform has been prototyped to enable field-ready detection. Ongoing and planned validation efforts include testing across diverse aqueous solution types using controlled bacterial contamination at concentrations ranging from 0.001 to 100 CFU/ml, encompassing both beta-lactamase-producing and non-producing strains. Assay parameters, including sample volume, bacterial concentration methods, and probe concentration, are being optimized to enhance sensitivity and reliability. The technology is currently at an early-to-mid stage of development, progressing toward broader validation and potential commercialization.