Dr. Jongwon Lim

U.S.A.

A biphasic dried blood matrix platform for rapid, extraction-free detection of DNA and RNA pathogens from whole blood

Jongwon Lim1,2,3, Hankeun Lee2,4, Matthew Wester1,2, Katherine Koprowski1,2, An Bao Van1,2,5, Enrique Valera1,2,6, Brian T Cunningham1,2,4,6,7,8,9, Rashid Bashir1,2,4,6,10,11,12,13

1 Department of Bioengineering, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
2 Nick Holonyak Jr. Micro and Nanotechnology Laboratory, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
3 Materials Research Laboratory, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
4 Department of Electrical and Computer Engineering, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
5 VinUni–Illinois Smart Health Center, Building G, VinUni Campus, Vinhomes Ocean Park, Gia Lam District, Hanoi 100000, Vietnam
6 Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
7 Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
8 Center for Genomic Diagnostics, Carl R. Woese Institute for Genomic Biology, Urbana, IL, USA.
9 Gener8 LLC, Wilmington, MA, USA
10 Department of Mechanical Science and Engineering, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
11 Department of Materials Science and Engineering, Grainger College of Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, United States
12 Department of Biomedical and Translational Sciences, Carle Illinois College of Medicine, Urbana, IL 61801, United States
13 Chan Zuckerberg Biohub Chicago, Chicago, IL 60642

Abstract

Background

Blood is one of the most information-rich biofluids for diagnosing infectious diseases, yet it remains one of the most challenging matrices for nucleic acid detection. Conventional workflows require multistep extraction and purification to remove inhibitors such as hemoglobin and immunoglobulins, leading to significant loss of target nucleic acids, increased turnaround time, and reliance on expensive, centralized infrastructure. These limitations are particularly critical in low-resource settings and in diseases with low pathogen concentrations, such as sepsis and viral hepatitis. To address these challenges, we developed a biphasic diagnostic approach that transforms whole blood into a functional dried matrix, enabling direct, highly sensitive pathogen detection without purification.

Methods

Whole blood samples were dried under thermal conditions to form a solid-phase “dried blood matrix” in which inhibitory components are immobilized within a porous fibrin–protein network. A secondary thermal treatment was applied to enhance porosity and facilitate diffusion of amplification reagents. Following this treatment, isothermal amplification reagents including LAMP or RPA master mix were added directly onto the dried matrix, forming a biphasic system consisting of a solid phase containing immobilized DNA and a liquid phase containing primers and enzymes. Structural and material characterization was performed using SEM, BET/BJH analysis, and fluorescence recovery after photobleaching to quantify porosity, surface area, and diffusion properties. The platform was validated using bacterial targets such as MRSA, MSSA, and E. coli, as well as viral DNA including Hepatitis B Virus.

Results

The dried blood matrix inactivated amplification inhibitors while preserving target nucleic acids, and its increased porosity enabled efficient penetration of primers and enzymes. Using this biphasic system, bacterial pathogens were detected directly from whole blood with a sensitivity as low as 1 CFU/mL, and DNA viral detection achieved a limit of detection of 10 IU/mL for Hepatitis B Virus (HBV). In addition, Zika virus was detected with a limit of detection of 10 copies/µL following a controlled reverse transcription step. These results demonstrate that the dried blood matrix enables sensitive, extraction-free detection of both DNA and RNA pathogens from whole blood within 90 minutes using only a simple heater and fluorescence reader.

Conclusions

This work establishes the dried blood matrix as a new functional biomaterial for molecular diagnostics. By eliminating extraction and purification, the biphasic platform simplifies workflows, minimizes target loss, and enables rapid, sensitive detection of pathogens from whole blood. This technology has strong potential for point-of-care diagnostics, outbreak response, and use in resource-limited environments.