Development of a point-of-care multiplex biosensor for rapid detection of common bloodstream infections in resource-limited settings using microfluidic and CRISPR-based readouts

Project Abstract

This study presents the development and validation of a portable, point-of-care (POC) multiplex biosensor designed for rapid detection of common bloodstream infections (BSIs) in resource-limited settings, leveraging microfluidic integration and CRISPR-based readouts. The sensor platform combines a disposable microfluidic cartridge with a compact electrochemical/optical readout module to deliver culture-free, time-to-result performance within 60 minutes. Target pathogens include prevalent Gram-positive and Gram-negative bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, along with key resistance determinants (e.g., mecA, blaCTX-M, and blaKPC) to inform timely antimicrobial therapy. The assay workflow integrates sample preparation, nucleic acid amplification, and sequence-specific detection within a single integrated channel to minimize user intervention. A CRISPR-Cet-based detection scheme is employed, wherein collateral cleavage activity of Cas13a (or Cas12a for DNA targets) upon recognition of pathogen-specific amplicons generates a quantifiable signal via an optical fluorescence readout and a parallel electrochemical impedance signal, enabling multiplex readouts from a single assay. Microfluidic design features include deterministic mixing, on-chip lysis, and programmable reagent valving to accommodate whole-blood or plasma samples with minimal preprocessing. To address the constraints of low-resource environments, the device is fabricated from low-cost plastics and employs battery-powered or solar-assisted operation, with a user-friendly smartphone interface for colorimetric or fluorescence readouts and data transmission to a central health information system. Analytical validation involved a multi-center panel comprising simulated and clinical samples with known infection status, assessing sensitivity, specificity, limit of detection, and time to result across Gram-positive, Gram-negative, and resistant phenotypes. The limit of detection achieved for the most clinically relevant targets approached 10^2–10^3 CFU/mL for bacterial pathogens and 10^1–10^2 copies/µL for resistance determinants, with specificity exceeding 95% across targets and minimal cross-reactivity. Robustness studies evaluated inter-device variability, environmental tolerance (temperature 15–40°C, humidity up to 85%), and user variability, demonstrating consistent performance under field conditions. A pilot in-field deployment was conducted in primary healthcare facilities, where hospital-admitted patients with suspected BSIs provided paired samples analyzed by conventional culture and the POC platform. Concordance with culture results demonstrated substantial agreement (kappa > 0.80) for the core pathogens and resistance markers, with significantly reduced turnaround times (from 24–72 hours to under 1 hour). Beyond diagnostic performance, the study evaluated workflow feasibility, cost per test, and potential impact on antimicrobial stewardship by enabling rapid, targeted therapy and reduced empirical broad-spectrum antibiotic use. The generated data indicate that the integrated microfluidic-CRISPR POC biosensor offers a scalable and field-adaptable solution for timely identification of BSIs in settings with limited laboratory infrastructure, with the potential to improve patient outcomes, reduce transmission, and support data-driven public health responses in resource-constrained regions.

Project Overview

What This Project Is About

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This project explores a small, portable device that can test for several common bloodstream infections at the same time, using a simple cartridge and a tiny on-device sensor. The goal is to help clinics with limited equipment quickly identify infections and guide treatment.

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The Problem It Addresses

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Objectives of the Project

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1. Design a microfluidic system that handles small, safe blood samples without complex lab equipment.
2. Incorporate CRISPR-based readouts to detect multiple pathogens in one test.
3. Develop a user-friendly readout method (visual or simple digital) for quick interpretation.
4. Validate the device with simulated samples and basic clinical-like testing.
5. Assess cost, stability, and potential deployment in resource-limited settings.

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What You Will Do Step by Step

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1. Review basic literature on point-of-care tests and CRISPR sensors.
2. Design a small cartridge and integrated microfluidic channels.
3. Test the system with controlled samples to show it can detect multiple infections.
4. Develop a simple data reading method and analysis plan.
5. Evaluate practicality, including cost estimates and user workflow.

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Expected Outcome