Paper-Based Point-of-Care Diagnostic Tools: Electrokinetic Nucleic Acid Extraction and Saliva-Based Glucose Detection for Diabetes Monitoring

Abstract

Currently, we face a global challenge posed by the dual burden of chronic diseases such as diabetes and infectious diseases such as tuberculosis (TB). Low-and middle-income countries are disproportionately overburdened by these crises due to a lack of well-developed healthcare infrastructure. In order to prevent the spread of infection and for effective treatment, the development of point-of-care (POC) diagnostic solutions has recently garnered attention and has elicited enormous interest in developing rapid, affordable, accessible, and accurate POC tools for disease diagnostics and monitoring. In recent years, there has been a surge in the use of paper substrates for developing POC medical diagnostic tests. In this work, we investigate the potential of inexpensive paper substrates to develop diagnostic solutions for three applications– i) sample preparation for nucleic acid amplification tests (NAATs), ii) storage and transportation of sputum samples for TB diagnostics, and iii) glucose detection for diabetes monitoring.

Bacterial infection remains a global threat, and POC-NAATs have shown great promise in addressing this challenge. However, their dependence on complex traditional sample preparation methods remains a significant barrier to their widespread adoption. To overcome this limitation, we present a paper-based sample preparation module that automates bacterial cell lysis, genomic DNA purification, and concentration from complex biological samples (like saliva, blood serum, and urine) using an electrokinetic technique called isotachophoresis (ITP). We demonstrate 12X reconcentration of purified Mycobacterium tuberculosis genomic DNA using a paper-based ITP sample preparation device (p-ITPrep). From complex biological matrices – pooled human saliva, human blood serum, and artificial urine, our device extracted bacterial (Mycobacterium smegmatis, Msm) genomic DNA from samples containing 10^2 CFU Msm/mL saliva or artificial urine and 10^3 CFU Msm/mL serum within 20 minutes. This low-voltage standalone nucleic acid sample preparation device requires minimal user intervention and has no dependence on expensive ancillary equipment. It can potentially expand the accessibility of DNA purification and concentration in resource-limited settings and replace conventional multi step nucleic acid extraction protocols in any laboratory conducting nucleic acid analyses.

Another way to approach disease diagnosis in low-resource settings is to maximize the use of nearby well-equipped centralized diagnostic facilities by sample transportation. However, the absence of efficient cold chains for sample transportation leads to poor specimen quality. To address this problem, we developed a paper-based specimen dry storage technology, SPECTRA-tube (SPECimen TRAnsportation-tube), that allows collection, storage, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 ml) specimens, such as sputum samples. This novel device enables molecular and culture-based diagnosis of tuberculosis (or any other infectious disease) without any cold chains, solving the problem of existing technologies. SPECTRA-tube, therefore, holds the potential to improve diagnostic testing in the developing world with better quality specimens to central labs and enables patient-centric sampling for at-home collection in the developed world.

In addition to addressing the problem with sample preparation associated with infectious disease diagnostics, we also provide a solution to manage chronic metabolic diseases like diabetes. Regular glucose monitoring is critical to managing diabetes. However, the current method involves an invasive and painful way of monitoring blood glucose levels. We developed a non-invasive and user-friendly paper-based glucose-sensing test strip based on bi-enzymatic colorimetric detection, sensitive enough to detect low salivary glucose levels (SGL) within 3 minutes. The finalized bienzymatic chromogen chemistry demonstrated a detection limit of 0.58 mg/dL. The self-testing strips were designed to work with a smartphone camera, enabling the capture of the generated colorimetric signal. Our preliminary clinical study demonstrates that this saliva-based glucose testing kit could be used as an effective tool for detecting increased glucose levels. Larger clinical trials are currently in progress.

In conclusion, these innovative technologies utilizing paper substrates have immense potential to address challenging problems in disease diagnosis and monitoring. They offer promising solutions for improving healthcare outcomes in low-resource settings and beyond. Moreover, their commercial viability presents opportunities for widespread adoption, positively impacting global healthcare systems and benefiting a wide range of individuals.