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Point-of-care testing (POCT) for uropathogen detection and chemical screening has great benefits for the diagnosis of urinary tract infections (UTIs). The goal of this study was to develop a portable and inexpensive paper-based analytical device (PAD) for cultivating bacteria in situ and rapidly testing for nitrite on the same device. The PAD was fabricated using a wax printing technique to create a pattern on Whatman No. 1 filter paper, which was then combined with a cotton sheet to support bacterial growth. Nitrite detection was based on the principle of the Griess reaction, and a linear detection range of 0–1.6 mg/dL (R2 = 0.989) was obtained. Scanning electron microscopy (SEM) analysis demonstrated that the bacteria were able to grow and formed a cluster on the cellulose fibres within 2 hours. The enzyme β-glucuronidase, which is specifically produced by Escherichia coli, was able to convert the pre-immobilized 5-bromo-4-chloro-3-indolyl-β-D-glucuronide sodium salt (X-GlcA), a colourless substrate, generating a blue colour. Under optimum conditions, the proposed device allowed bacterial concentrations in the range of 104–107 colony forming units (CFU)/mL to be quantified within 6 hours. Moreover, the use of this device enables the identification of E. coli pathogens with selectivity in real urine samples. In conclusion, the PAD developed in this study for UTI screening provides a rapid, cost-effective diagnostic method for use in remote areas.

Rationale: Saliva as a sample matrix is rapidly gaining interest for disease diagnosis and point-of-care assays because it is easy to collect (non-invasive) and contains many health-related biomarkers. However, saliva poses particular problems relative to more common urine and blood matrices, which includes low analyte concentrations, lack of understanding of biomolecule transportation and inherent viscosity variability in human samples. While several studies have sought to improve assay sensitivity, few have addressed sample viscosity specifically. The goal of this study is to minimize the effect of sample viscosity on paper-based analytical devices (PADs) for the measurement of pH and nitrite in human saliva.Methods: PADs were used to measure salivary pH from 5.0 to 10.0 with a universal indicator consisting of chlorophenol red, phenol red and phenolphthalein. Nitrite determination was performed using the Griess reaction. Artificial saliva with viscosity values between 1.54 and 5.10 mPa∙s was tested on the proposed PAD. To ensure the proposed PADs can be tailored for use in-field analysis, the devices were shipped to Australia and tested with human specimens.Results: Initial experiments showed that viscosity had a significant impact on the calibration curve for nitrite; however, a more consistent curve could be generated when buffer was added after the sample, irrespective of sample viscosity. The linear range for nitrite detection was 0.1 to 2.4 mg/dL using the improved method. The nitrite measurement in artificial saliva also showed a good correlation with the standard spectrophotometry method (p=0.8484, paired sample t-test, n=20). Measured pH values from samples with varying viscosities correlated well with the results from our pH meter.Conclusions: The inherent variation of salivary viscosity that impacts nitrite and pH results can be addressed using a simple washing step on the PAD without the need for complex procedures.

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