Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies—tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)—were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens—namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 10⁵ CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 10⁵–10⁶ CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.

Enzyme linked immunosorbent assay (ELISA) is one of the most utilized serological methods to diagnose and identify etiologic agents of many infectious diseases and other physiologically important analytes. ELISA can be used either alone or adjunct to other diagnostic methods such as molecular arrays, and other serological techniques. Most ELISA assays utilize reagents that are proteinaceous in nature, which are not very stable and require cold-chain transport systems. Development of a desirable immunoassay requires stability of reagents used and its ability to be stored at room temperature without sacrificing the activity of the reagents or the protein of interest. Metal organic frameworks (MOFs) are a rapidly emerging and evolving class of porous polymeric materials used in a variety of biosensor applications. In this study, we introduce the use of MOFs to stabilize a universal reporter fusion protein, specifically, avidin-like protein (Tam-avidin2) and the small bioluminescent protein Gaussia luciferase (Gluc) forming the fusion reporter, tamavidin2-Gluc (TA2-Gluc). This fusion protein serves as a universal reporter for any assays that utilize biotin–avidin binding strategy. Using SARS-CoV2 S1 spike antigen as the model target antigen, we demonstrated that encapsulation of TA2-Gluc fusion protein using a nano-porous material, zeolitic imidazolate framework-8 (ZIF-8), allows us to store and preserve this reporter protein at room temperature for over 6 months and use it as a reporter for an ELISA assay. Our optimized assay was validated demonstrating a 0.26 μg mL⁻¹ limit of detection, high reproducibility of assay over days, detection of spiked non-virulent SARS-COV2 pseudovirus in real sample matrix, and detection in real COVID-19 infected individuals. This result can lead to the utilization of our TA2-Gluc fusion protein reporter with other assays and potentially in diagnostic technologies in a point-of-care setting.

Point-of-care testing (POCT) offers a transformative approach to diagnostics by enabling rapid and accurate results at or near the site of patient care. This is especially valuable in critical care, emergency settings, and resource-limited areas. However, one major limitation of POCT remains its analytical sensitivity, particularly in detecting low concentrations of analytes. To address this, various innovations are being explored, including advanced sensors, signal amplification, and sensitive labels. Among these, bioluminescent proteins have gained attention for their high sensitivity, fast readout, minimal background interference, and simplified instrumentation. Bioluminescence—light emission from biochemical reactions—presents an ideal platform for enhancing POCT sensitivity. In parallel, metal–organic frameworks (MOFs), especially structures like ZIF-8, are emerging as valuable materials in biosensing. Their high porosity, tunable surface properties, and ability to host biomolecules make them excellent candidates for improving analyte capture and signal transduction. When integrated with bioluminescent systems, MOFs can stabilize proteins, concentrate targets, and enhance overall assay performance. This review highlights the role of bioluminescent proteins in medical diagnostics and their application in POCT platforms. We also discuss the potential synergy between MOFs and bioluminescence to overcome current sensitivity limitations. Finally, we examine existing challenges and strategies to optimize these technologies for robust, field-deployable diagnostic tools. By leveraging both the natural sensitivity of bioluminescence and the structural advantages of MOFs, next-generation POCT systems can achieve superior performance, driving forward diagnostic accessibility and patient care outcomes.

A high-throughput assay system is developed for measuring communication in microbial biofilms in a 96-well microtiter plate format. In this assay, bioluminescent microbial whole cell biosensor systems (MWCBs) for quorum-sensing molecules (QSMs) are embedded into biofilms, and their response to chemical cues relevant for bacterial communication is assessed. For measuring the response to stress, a sigma factor 54 (σ⁵⁴, RpoN)-dependent MWCB was developed. Biofilms generated in this platform were exposed to gradients of communication signals (QSMs such as N-acetyl-homoserine lactones (AHLs), 3,5- dimethylpyrazin-2-ol (DPO), or phytochemicals that can act as natural quorum-sensing inhibitors (QSIs) such as curcumin or 3,3′-diindolylmethane (DIM)), and the response pattern was monitored. Further, the regulatory role of stressors such as oxidants (H₂O₂) or antibiotics (ciprofloxacin, trimethoprim/sulfamethoxazole) on the communication response is assessed. QSMs induced the MWCBs at 1 h and 4 h in biofilms, but high concentrations inhibited them at 24 h. Curcumin and DIM at higher concentrations lead to inhibition of quorum sensing in biofilms after 4 h and 24 h, but this is not followed by biofilm disintegration. H₂O₂ above 0.002% efficiently inhibited the MWCB activities and led to biofilm disintegration. At lower concentrations of H₂O₂, we observed induction of MWCBs. The antibiotics inhibited the MWCB activity at concentrations above their minimal inhibitory concentration (MIC), but this did not necessarily lead to disintegration of the biofilm. Like low concentrations of H₂O₂, the antibiotics activated the MWCBs at concentrations close to their MIC, possibly as a result of H₂O₂ generated during their bactericidal action. Interestingly, the induction of communication in response to antibiotics can be quenched by iron chelators, suggesting involvement of H₂O₂ and free radicals generated by the Fenton reaction. We hypothesize that the observed response to these stressors reflects increased communication in the biofilm, possibly enhancing tolerance and increasing survival.