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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. 

Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing E. coli was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments. 

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. 

Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications ‐ bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we usedGaussialuciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV‐1 DNA and IFN‐γ. We demonstrated that the incubation of Gluc conjugated HIV‐1 and IFN‐γ oligonucleotide probes with GO provided for monitoring of probe‐target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN‐γ and HIV‐1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN‐γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter.

 

The ability to monitor types, concentrations, and activities of different biomolecules is essential to obtain information about the molecular processes within cells. Successful monitoring requires a sensitive and selective tool that can respond to these molecular changes. Molecular aptamer beacon (MAB) is a molecular imaging and detection tool that enables visualization of small or large molecules by combining the selectivity and sensitivity of molecular beacon and aptamer technologies. MAB design leverages structure switching and specific recognition to yield an optical on/off switch in the presence of the target. Various donor–quencher pairs such as fluorescent dyes, quantum dots, carbon‐based materials, and metallic nanoparticles have been employed in the design of MABs. In this work, the diverse biomedical applications of MAB technology are focused on. Different conjugation strategies for the energy donor–acceptor pairs are addressed, and the overall sensitivities of each detection system are discussed. The future potential of this technology in the fields of biomedical research and diagnostics is also highlighted.