Reagentless fluorescent protein paper sensor for ultrasensitive endotoxin detection

Overview

This solution introduces a novel biosensor platform for detecting endotoxins, the dangerous contaminants that trigger severe immune responses and must be rigorously monitored in pharmaceuticals, medical devices, and biotechnology products. Current industry-standard methods such as the LAL (Limulus Amebocyte Lysate) assay require multiple reagents, trained operators, and specialized instrumentation, creating bottlenecks in quality control workflows.

The proposed approach eliminates these limitations by combining engineered fluorescent proteins with high-affinity endotoxin-binding domains and self-assembling peptides. The result is a simple paper strip that produces a measurable fluorescent signal upon endotoxin binding, with no reagents, no complex sample preparation, and no expensive readers required. This makes rapid endotoxin testing accessible in settings ranging from pharmaceutical manufacturing floors to field-based bioprocess monitoring.

Technical specifications

Core technology:

• Single fluorescent protein (FP) biosensor genetically fused to endotoxin-binding proteins such as BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), which exhibit higher binding affinities than recombinant Factor C used in the LAL assay
• Self-assembling peptide tags enable high-density deposition of FP biosensors directly onto paper substrates, amplifying fluorescence signal intensity
• Structure-guided linker design and computational screening to optimize biosensor performance
• Expression in LPS-eliminated E. coli hosts to avoid contamination during production

Key advantages:

• Reagentless operation removes the need for multi-step assays and cold-chain reagent storage
• Target detection limit of 0.03–0.25 EU/ml, comparable to regulatory thresholds for many pharmaceutical and medical device applications
• Readout achievable with an inexpensive transilluminator rather than specialized plate readers
• Potential for multiplexed sensing by combining different FP variants on a single strip

Technology readiness level

The underlying engineering strategy has been validated through prior demonstrations: structure-guided fusion of fluorescent proteins with diverse sensing domains has successfully produced FP biosensors for metal ions, small molecules, sugars, and lipids. The principal investigator and collaborators have also previously demonstrated FP biosensor self-assembly for enhanced sensitivity and multiplexed detection, along with a protein assembly platform that fabricates high-density FP nanostructures on paper and other substrates.

Future work will proceed in defined stages: (1) genetic construction and computational screening of GFP-LBP and GFP-BPI fusion candidates with optimized linkers, (2) expression and functional testing in endotoxin-free E. coli, (3) purification of lead candidates, and (4) fabrication and optimization of self-assembled paper sensor strips to meet the target detection sensitivity. The technology is currently at an early-to-mid stage of development, with strong preliminary validation of the core platform components and ongoing work toward endotoxin-specific performance targets.