Quantitative DNA Amplification on PCB (qDAMP): Low-cost diagnostic technology
Lead Research Organisation:
University of York
Department Name: Electronics
Abstract
Context: Diarrhoeal diseases result in approximately 1.8 million deaths each year worldwide, in many cases due to a lack of access to clean water. Current analytical technologies to assess water quality in low and middle income countries are limited in terms of technical performance (e.g. sensitivity, speed and specificity) and suitability (e.g. cost, sustainability, and integration with local and national context). We have recently worked with the Department of Water Resources (DWR) in Vanuatu to co-develop the specifications of next generation water quality monitoring technology that addresses these limitations. This studentship will investigate such analytical technology based on real-time detection of DNA amplification using low-cost field effect transistor sensors.
Aims and Objectives: This project will demonstrate an electronic water quality sensor array that can be readily manufactured and deployed in Vanuatu and that is able to rapidly detect and quantify a panel of molecular targets that are indicators of faecal contamination.
1. Understand relationship between the structure and composition of IrOx electrodeposited on a printed circuit board (PCB) electrode and its pH sensitivity and stability
2. Demonstrate a PCB ion sensitive field effect transistor (PCB- ISFET) optimised for stability, reproducibility and pH sensitivity (aiming for better than 0.1 pH).
3. Fabricate PCB-ISFET for label-free detection and quantification of DNA amplification and evaluate in-lab aiming for <10 CFU/100mL in < 2 hours.
Research Methodology: Research will focus on quantification of E. coli. We will use known primers for E. coli and, using a laboratory PCR system, optimise a loop-mediated isothermal amplification (LAMP) protocol focusing on sensitivity to buffer and contaminants, temperature and assay speed.
In parallel, we will investigate the fabrication of PCB-ISFETs and associated drive circuitry. We will use standard PCB fabrication of copper-clad FR4, which can be replicated or made available in Vanuatu and develop the analogue front-end, including ISFET bias circuitry and signal conditioning. The performance of the ISFET is dependent on the pH sensitivity, stability and reproducibility of the IrOx layer. We will apply a range of analytical tools (electron and atomic force microscopy and x-ray photoelectron spectroscopy) to characterise and optimise IrOx deposition for use in extended gate, PCB-ISFETs. The pH sensitivity will be assessed electrochemically through measurements of the open circuit potential and CV. The optimised deposition conditions will be used to fabricate an extended gate PCB-ISFET and we will characterise the performance aiming for a sensitivity of > 70mV /pH in the pH range 4-10.
We will demonstrate the complete system for real-time monitoring and quantification of E. coli. Measurements will be performed in a fluid cell consisting of a 3D printed holder and PDMS gaskets using E. coli spiked into pure and environmental water with a known background of other bacteria. We will assess quantification of E coli via time-dependent, ISFET measurements of pH change and quantify the limit of detection for the qDAMP prototype.
Alignment to EPSRC strategy: Aligned with the principles of ODA, the primary goal of this project is the promotion of the economic development and welfare in developing countries and is thus aligned with the EPSRC's role in the GCRF. We also anticipate benefits to the UK. In particular, this proposal will develop a novel, low-cost analytical technology that aligns with the aims of the EPSRC Physical Science theme, Analytical science and extends the position of the UK in analytical science and technology. Our low-cost diagnostic technology will also contribute directly to the EPSRC Healthcare Technologies strategy and has potential beyond healthcare, for example as an analytical tool for food security, environmental monitoring and bioterrorism.
Aims and Objectives: This project will demonstrate an electronic water quality sensor array that can be readily manufactured and deployed in Vanuatu and that is able to rapidly detect and quantify a panel of molecular targets that are indicators of faecal contamination.
1. Understand relationship between the structure and composition of IrOx electrodeposited on a printed circuit board (PCB) electrode and its pH sensitivity and stability
2. Demonstrate a PCB ion sensitive field effect transistor (PCB- ISFET) optimised for stability, reproducibility and pH sensitivity (aiming for better than 0.1 pH).
3. Fabricate PCB-ISFET for label-free detection and quantification of DNA amplification and evaluate in-lab aiming for <10 CFU/100mL in < 2 hours.
Research Methodology: Research will focus on quantification of E. coli. We will use known primers for E. coli and, using a laboratory PCR system, optimise a loop-mediated isothermal amplification (LAMP) protocol focusing on sensitivity to buffer and contaminants, temperature and assay speed.
In parallel, we will investigate the fabrication of PCB-ISFETs and associated drive circuitry. We will use standard PCB fabrication of copper-clad FR4, which can be replicated or made available in Vanuatu and develop the analogue front-end, including ISFET bias circuitry and signal conditioning. The performance of the ISFET is dependent on the pH sensitivity, stability and reproducibility of the IrOx layer. We will apply a range of analytical tools (electron and atomic force microscopy and x-ray photoelectron spectroscopy) to characterise and optimise IrOx deposition for use in extended gate, PCB-ISFETs. The pH sensitivity will be assessed electrochemically through measurements of the open circuit potential and CV. The optimised deposition conditions will be used to fabricate an extended gate PCB-ISFET and we will characterise the performance aiming for a sensitivity of > 70mV /pH in the pH range 4-10.
We will demonstrate the complete system for real-time monitoring and quantification of E. coli. Measurements will be performed in a fluid cell consisting of a 3D printed holder and PDMS gaskets using E. coli spiked into pure and environmental water with a known background of other bacteria. We will assess quantification of E coli via time-dependent, ISFET measurements of pH change and quantify the limit of detection for the qDAMP prototype.
Alignment to EPSRC strategy: Aligned with the principles of ODA, the primary goal of this project is the promotion of the economic development and welfare in developing countries and is thus aligned with the EPSRC's role in the GCRF. We also anticipate benefits to the UK. In particular, this proposal will develop a novel, low-cost analytical technology that aligns with the aims of the EPSRC Physical Science theme, Analytical science and extends the position of the UK in analytical science and technology. Our low-cost diagnostic technology will also contribute directly to the EPSRC Healthcare Technologies strategy and has potential beyond healthcare, for example as an analytical tool for food security, environmental monitoring and bioterrorism.
Organisations
People |
ORCID iD |
| Steven Johnson (Primary Supervisor) | |
| Rhys Ashton (Student) |
| Description | This work utilised a range of complimentary analysis techniques to better understand issues with low sensitivity and poor repeatability of pH sensitive devices. This understanding and the addition of a simple treatment step in the fabrication process have now enabled highly pH sensitive and repeatable devices, based on easy to manufacture and widely available printed circuit board (PCB) technology. Finally, we demonstrate the application of the PCB-based sensors through detection and quantification of enzymatic activity via turnover of ß-lactamase antibiotics. |
| Exploitation Route | These low-cost pH sensitive devices are based on widely available PCB substrates and have been demonstrated towards an assay of relevance in the anti microbial resistance space. Future work can apply these sensors to a range of other low cost applications due to the high pH sensitivity and repeatability of manufacture. We see applications across a range of other biochemical assays, as well as environmental monitoring and the food industry. The benefit of electrochemical polishing on biosensors may also be applied across other materials and substrates to improve performance and repeatability. |
| Sectors | Agriculture, Food and Drink,Energy,Environment,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |