Materials & Tools for Developing POC & Rapid Dx 2023 Agenda

Point of Care Diagnostics – The Opportunity For Real-Time POC Using Microfluidic Sensors and Biosensors
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

The concentration of biomarker molecules can give important information about the health of a person as they are dynamically challenged by acute illness or for example during clinical treatment. Our view is that to do such monitoring effectively ideally requires moment-by-moment measurement of blood or tissue concentrations as this allows treatments to be guided in a personalized way by such data. We have been developing a range of sensing and biosensing solutions for the invasive, minimally invasive, and non-invasive monitoring of people in health care situations. Microfluidics provide a valuable means of clinical sampling and robust quantification of measured signals. I will describe the key challenges in the development of such integrated sensing devices and  present our recent data obtained during models of cardiac arrest and from the neonatal intensive care unit.

Digital Resolution Detection Without Droplets or Enzymes through Photonic Crystal Amplification
Brian Cunningham, Professor and Intel Alumni Endowed Chair, University of Illinois at Urbana-Champaign, United States of America

Droplet digital biomarker detection technologies achieve ultrasensitive detection limits by partitioning the test sample into thousands of small volumes that confine the fluorescent products of an enzymatic amplification reaction, followed by counting bright droplets.  Using photonic crystal surfaces to amplify the fluorescence of light-emitting tags such as quantum dots and plasmonic fluors, individual nucleic acid biomarkers, proteins, and virus particles can be digitally counted without sample partitioning or enzymatic reactions, resulting in simple workflow, rapid sample-to-answer time, and ultrasensitivity using simple inexpensive instruments.  We have also developed digital alternatives to PCR that offer room temperature, isothermal biochemistry methods based upon nucleic acid strand displacement reactions and CRISPR/Cas technology which rapidly amplify single target nucleic acid biomarkers (miRNA and ctDNA) into large numbers of gold nanoparticles that are digitally counted by Photonic Resonator Absorption Microscopy (PRAM).  We show that an “amplify-then-digitize” strategy results in detection limits below PCR while utilizing a simple protocol and inexpensive instrument.  Overall, we envision application of the biodetection technologies and assay methods towards ultrasensitive and multiplexed point of care diagnostics.

Chemically Programmable Isothermal PCR Enables Rapid Nucleic Acid-based Bioanalysis at the Point of Need
Victor Ugaz, Professor & Interim Department Head, Texas A&M University, United States of America

The polymerase chain reaction (PCR) and its variants are analytical gold standards for nucleic acid-based testing that play a critical role in diagnosing and monitoring infectious diseases. But robust portable PCR-based testing at the point of need (PON) remains elusive, especially in resource-limited settings, because the required thermocycling instrumentation is neither amenable to nor validated for operation in a portable format. This presentation describes a microfluidic platform that overcomes these barriers by exploiting the ability to perform isothermal PCR via natural convection. We apply “chemical programming” to manipulate the interplay between the PCR biochemistry and the microscale convective flow field, enabling 100% repeatability to be achieved in a format that can be easily and cost-effectively manufactured, dramatically increasing simplicity, portability, and affordability. We demonstrate rapid replication of targets associated with multiple pathogens, including SARS-CoV-2, attaining performance rivaling ultra-fast PCR instruments while using lower reagent concentrations than conventional protocols. This discovery paves the way for widespread adoption of PCR-based analysis at the PON, making it possible to bring lab-quality diagnostics to decentralized settings.

Detection of Cancer Related Protease Biomarkers from Blood Spot Cards – Transition to Viable POC Diagnostics for Cancer
Michael Heller, Professor, Dept Bioengineering, University of California-San Diego, United States of America

Important cancer related protease biomarkers can be detected rapidly from blood spot card samples using simple electrophoretic and lateral flow devices. The combination of blood spot card samples and simple detection devices represents an ideal approach for new POC and rapid molecular diagnostics. Proteases represent a class of enzymes that degrade proteins and have been associated with a range of diseases, including cardiovascular; coagulation disorders; inflammatory diseases, diabetes, sepsis; infectious diseases and cancer. The protease assays utilize fluorescent charge-changing peptide substrates and can be carried out using small volumes (5ul-10ul) of whole blood, plasma or serum. No sample preparation is required, and the fluorescent peptide products can be detected in about 30 minutes using simple electrophoretic and lateral flow formats We now have results showing the elevation of specific proteases (trypsin, chymotrypsin, MMPs and Cathepsins) in pancreatic (PDAC) and other cancers. We also have preliminary results showing protease biomarkers can be detected from samples applied to “Blood Spot Cards.” Use of blood spot cards represents a paradigm change with advantages including they require only a small blood sample (5-10ul), are cost $$$ effective (compared to a blood draw tube) and would allow viable time course studies and companion diagnostics to be carried out (hours, days, weeks).