Programmable receptors enable bacterial biosensors to detect pathological biomarkers in clinical samples

Early detection and monitoring of chronic pathologies are crucial for reducing mortality and improving patient quality of life. In vitro diagnostic technologies play a vital role, but many require expensive equipment and trained personnel, limiting accessibility. Point-of-care diagnostic devices, like those used for glucose monitoring, offer a significant advancement. Bacteria, capable of sensing and responding to various signals, are ideal candidates for engineering biosensors. Whole-cell biosensors (WCBs) use genetically modified cells to detect target molecules, activating a reporter gene. Recent advancements in synthetic biology have enhanced WCBs' robustness, sensitivity, and capabilities, enabling their use in complex media. Liver disease is a major global health concern, causing millions of deaths annually. Current diagnostic methods are centralized, often leading to late-stage diagnoses. Bile salts, key components of bile crucial for fat absorption, have emerged as promising biomarkers for various liver diseases, offering a specific and dynamic assessment of liver function. This study aims to engineer an *E. coli* based biosensor to detect bile salts in clinical samples, leveraging the natural bile salt-sensing capabilities of enteropathogenic bacteria.

Existing literature highlights the potential of bacterial biosensors in medical and environmental diagnostics. However, challenges remain in creating scalable and versatile systems for detecting various ligands. The use of whole-cell biosensors, particularly those engineered from bacteria, offers advantages such as miniaturization, field-deployability, and cost-effectiveness. Previous studies have shown the potential of bile salts as sensitive and specific biomarkers for liver dysfunction. However, current methods for bile salt detection often require complex laboratory equipment and centralized testing facilities, limiting their widespread use. This study builds upon these previous studies by introducing a novel, modular platform for engineering bacterial biosensors capable of detecting bile salts with improved sensitivity and point-of-care capabilities.

The researchers utilized a novel synthetic receptor platform termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization). EMeRALD allows for the modular assembly of various sensing modules onto a high-performance signaling scaffold in *E. coli*. They repurposed bile salt-sensing modules from enteropathogenic *Vibrio* species (*V. cholerae* and *V. parahaemolyticus*), specifically the TcpP-TcpH and VtrA-VtrC systems, respectively. The TcpP-TcpH system was chosen due to its high specificity for primary conjugated bile salts. The EMeRALD system used the CadC DNA-binding domain as a transcriptional regulator, controlling the expression of a reporter gene (initially sfGFP, later LacZ). The sensitivity and limit of detection (LOD) of the TcpP-sensing module were improved via directed evolution, using comprehensive mutagenesis coupled with functional screening and next-generation sequencing (NGS). This allowed for identification of key amino acid residues in the TcpP protein that affect sensitivity. A colorimetric output was developed using the LacZ reporter gene and the substrate CPRG, providing a visual readout. The optimized biosensor, TcpP18-LacZ, was tested on serum samples from liver transplant patients, comparing the results to a standard bile salt assay kit. The study also included various control experiments, such as testing the biosensor's specificity towards different types of bile salts.

The EMeRALD platform successfully integrated bile salt-sensing modules from *Vibrio* species into *E. coli*. Both TcpP-TcpH and VtrA-VtrC modules functioned within the EMeRALD scaffold, demonstrating the platform's modularity. Directed evolution of the TcpP module significantly improved the biosensor's sensitivity and lowered the LOD. Analysis of the evolved TcpP variants revealed specific amino acid sequence features influencing the sensor's responsiveness to bile salts. A colorimetric biosensor based on TcpP18-LacZ was successfully developed and validated. This colorimetric biosensor showed a linear response to GDCA within the concentration range of 0 to 40 µM. Testing on serum samples from liver transplant patients showed good correlation between the biosensor's output and the results from a standard bile salt assay kit. This validated its potential for clinical applications.

This study demonstrates a significant advancement in bacterial biosensor technology. The EMeRALD platform provides a robust and versatile tool for engineering biosensors with customizable ligand specificity. The successful detection of clinically relevant bile salt levels in patient serum highlights the potential of this technology for point-of-care diagnostics. The directed evolution strategy effectively improved the sensitivity of the biosensor, demonstrating the power of this approach for optimizing biosensor performance. The findings pave the way for developing more sophisticated whole-cell biosensors for various applications, including the development of novel point of care diagnostics. The insights gained into the sequence-function relationships of bacterial sensing modules will be valuable for future engineering efforts. The colorimetric output of the biosensor is particularly significant for its potential in creating easy-to-use and field-deployable diagnostic tools.

This research successfully engineered a bacterial biosensor for detecting bile salts, a critical biomarker for liver disease. The EMeRALD platform and the directed evolution strategy demonstrated efficacy in creating a sensitive and specific biosensor. Future work could focus on optimizing the biosensor for use in other biological samples, developing a fully integrated point-of-care device, and exploring the application of this technology to detect other biomarkers.

The study's sample size of liver transplant patients is relatively small, limiting the generalizability of the findings. The biosensor's performance in other biological fluids besides serum requires further investigation. While the colorimetric assay provides a user-friendly readout, further optimization may be needed for improved sensitivity and robustness in real-world settings. The study focused on detecting bile salts, and future research should explore the applicability of the EMeRALD platform for detecting other clinically relevant biomarkers.