Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

Abstract

BackgroundUrinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.Methodology/Principal FindingsThe integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).Conclusion/SignificanceWe successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.

New and developing diagnostic technologies for urinary tract infections

“…This sandwich hybridization electrochemical biosensor platform consists of an array of individually addressable sensors that can be used to detect different analytes. Development of a panel of probes enables identification of a wide variety of uropathogens in a 1 hour assay 122 . Validation of this biosensor with patient-derived samples has demonstrated robust uropathogen detection and speciation from single-species and polymicrobial samples.…”

A Universal Electrode Approach for Automated Electrochemical Molecular Analyses

“…The sequences of oligonucleotide probes used are summarized in Table 1. The specificity of the probes has been verified in our previous studies [19-21]. The sensor surface is first immobilized with the biotinylated capture probes through streptavidin-biotin binding.…”

“…Among different transduction mechanisms, electrochemical biosensing is sensitive, specific, and rapid, and represents an attractive strategy for point-of-care diagnostics of infectious diseases, such as urinary tract infection (UTI), one of the most common bacterial infections [17, 18]. For instance, we have demonstrated a self-assembled monolayer (SAM)-based electrochemical biosensor for detecting bacterial 16S rRNA and protein biomarkers [19-21]. We have also shown the implementation of in situ AC electrothermal flow (ACEF) induced mixing and on-chip heating on the electrochemical sensor [16].…”

Electrochemical Detection of RNA

“…Using electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60 % and allowed single-base mismatch discrimination (Liu et al 2014a). Additionally, the biosensor cassette was validated using patient-derived clinical urine samples (Bercovici et al 2012;Mohan et al 2011;Ouyang et al 2013). Liu et al (2014b) applied the electrokinetics-enhanced biosensor platform for rapid antimicrobial susceptibility testing.…”