The D4 Assay: A Technology Platform for Point-of-Care Clinical Diagnosis

The D4 assay is a point-of-care diagnostic that we have developed, in which all reagents are printed and stored on a “non-fouling”—protein and cell resistant—polymer brush.  The D4 assay, involves four sequential events: (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse (across surface); and (4) Detect (binding event).  The D4 POCT antibody (Ab) consists of microarrays printed on the polymer brush yields quantitative results, with picomolar sensitivity within 30 minutes.  All reagents are inkjet-printed and stored on D4 POCT cassettes, which do not require refrigeration.  Upon direct application of fingerstick blood onto a cassette, analyte capture and detection occur automatically, generating a quantifiable fluorescence signal obtained by placing the cassette in a small device that magnetically attaches to a smart phone, which images and analyzes microarrays via on-board App. The D4 assay can be used for the diagnosis of all markers for which antibody pairs are available with a speed and sensitivity that is as good or better than commercially available point-of-care tests and is far simpler, cheaper more rugged, and does not require a cold-chain.

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We are currently developing multiplexed POCTs based on the D4 platform for the following diseases:

Global Health: The disease burden in the developing world is still largely dominated by infectious diseases such as malaria, HIV, and re-emerging diseases like Ebola. The availability of rapid and accurate point of care diagnostics is vital in reducing the burden of these diseases. Our lab is developing rapid point of care diagnostic devices targeting the most prevalent diseases in the developing world. By combining three technologies developed in our lab: (1) the D4 assay, (2) self-contained microfluidic platforms and (3) the D4Acope, we are creating point-of-care tests with high (femtomolar) sensitivity, that are low-cost and robust and hence useful for low resource settings for global health applications.

Cancer: We are developing a multiplexed D4 POCT for detection of several serum biomarkers of hepatocellular carcinoma from a single drop of blood. We are currently in the process of validating the test and biomarkers. If successful, this technology will assist in cancer screening programs, particularly in lower-resource settings where incidence and mortality are highest.

Infectious Diseases: We have several active projects that are aiming to developing rapid POCTs for infectious disease (Malaria, Ebola, HIV, Zika, Talaromycosis) screening in low-resource settings.

Publications

Albarghouthi, Faris M., Daria Semeniak, Iman Khanani, James L. Doherty, Brittany N. Smith, Matthew Salfity, Quentin MacFarlane, et al. “Addressing Signal Drift and Screening for Detection of Biomarkers with Carbon Nanotube Transistors.” ACS Nano, February 2024. https://doi.org/10.1021/acsnano.3c11679.

Semeniak, Daria, Daniela F. Cruz, Ashutosh Chilkoti, and Maiken H. Mikkelsen. “Plasmonic Fluorescence Enhancement in Diagnostics for Clinical Tests at Point-of-Care: A Review of Recent Technologies.” Advanced Materials (Deerfield Beach, Fla.) 35, no. 34 (August 2023): e2107986. https://doi.org/10.1002/adma.202107986.

Kinnamon, David S., Jacob T. Heggestad, Jason Liu, Thu Nguyen, Vo Ly, Angus M. Hucknall, Cassio M. Fontes, et al. “Environmentally Resilient Microfluidic Point-of-Care Immunoassay Enables Rapid Diagnosis of Talaromycosis.” ACS Sens 8, no. 6 (June 23, 2023): 2228–36. https://doi.org/10.1021/acssensors.3c00209.

Burrow, Damon T., Jacob T. Heggestad, David S. Kinnamon, and Ashutosh Chilkoti. “Engineering Innovative Interfaces for Point-of-Care Diagnostics.” Current Opinion in Colloid & Interface Science, June 2023, 101718. https://doi.org/10.1016/j.cocis.2023.101718.

Heggestad, Jacob T., Rhett J. Britton, David S. Kinnamon, Jason Liu, Jack G. Anderson, Daniel Y. Joh, Zachary Quinn, et al. “COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.” Anal Chem 95, no. 13 (April 4, 2023): 5610–17. https://doi.org/10.1021/acs.analchem.2c05180.

Kinnamon, David S., Jacob T. Heggestad, Jason Liu, and Ashutosh Chilkoti. “Technologies for Frugal and Sensitive Point-of-Care Immunoassays.” Annual Review of Analytical Chemistry (Palo Alto, Calif.) 15, no. 1 (June 2022): 123–49. https://doi.org/10.1146/annurev-anchem-061020-123817.

Heggestad, Jacob T., David S. Kinnamon, Jason Liu, Daniel Y. Joh, Cassio M. Fontes, Qingshan Wei, Aydogan Ozcan, Angus M. Hucknall, and Ashutosh Chilkoti. “Smartphone Enabled Point-of-Care Detection of Serum Biomarkers.” Methods in Molecular Biology (Clifton, N.J.) 2393 (January 2022): 343–65. https://doi.org/10.1007/978-1-0716-1803-5_19.

Heggestad, Jacob T., Rhett J. Britton, David S. Kinnamon, Simone A. Wall, Daniel Y. Joh, Angus M. Hucknall, Lyra B. Olson, et al. “Rapid test to assess the escape of SARS-CoV-2 variants of concern.” Sci Adv 7, no. 49 (December 3, 2021): eabl7682. https://doi.org/10.1126/sciadv.abl7682.

J. T. Heggestad, Kinnamon, D. S. , Liu, J. , Joh, D. Y. , Fontes, C. M. , Wei, Q. , Ozcan, A. , Hucknall, A. M. , and Chilkoti, A. , Methods in Molecular BiologyBiomedical Engineering TechnologiesSmartphone Enabled Point-of-Care Detection of Serum Biomarkers, vol. 2393. New York, NY: Springer US, 2021, pp. 343 - 365.

Joh, Daniel Y., Jacob T. Heggestad, Shengwei Zhang, Gray R. Anderson, Jayanta Bhattacharyya, Suzanne E. Wardell, Simone A. Wall, et al. “Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.” NPJ Breast Cancer 7, no. 1 (July 2, 2021): 85. https://doi.org/10.1038/s41523-021-00290-0.

Heggestad, Jacob T., David S. Kinnamon, Lyra B. Olson, Jason Liu, Garrett Kelly, Simone A. Wall, Solomon Oshabaheebwa, et al. “Multiplexed, quantitative serological profiling of COVID-19 from blood by a point-of-care test.” Sci Adv 7, no. 26 (June 2021). https://doi.org/10.1126/sciadv.abg4901.

Fontes, Cassio M., Barbara D. Lipes, Jason Liu, Krystle N. Agans, Aiwei Yan, Patricia Shi, Daniela F. Cruz, et al. “Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR.” Sci Transl Med 13, no. 588 (April 7, 2021). https://doi.org/10.1126/scitranslmed.abd9696.

Joh, Daniel Y., Angus M. Hucknall, Qingshan Wei, Kelly A. Mason, Margaret L. Lund, Cassio M. Fontes, Ryan T. Hill, et al. “Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood.” Proc Natl Acad Sci U S A 114, no. 34 (August 22, 2017): E7054–62. https://doi.org/10.1073/pnas.1703200114.

Patents

A. Chilkoti, Franklin, A. , Yellen, B. , Hucknall, A. , Joh, D. , Abedini-Nassab, R. , and Andrews, J. , “Nonfouling biosensors”, 2022.

“CHIPS, DETECTORS, AND METHODS OF MAKING AND USING THE SAME”, U.S. Patent 17/521,3572022.

A. Chilkoti and Hucknall, A. , “Detection devices and related methods of use”, 2022.

A. Chilkoti and Hucknall, A. , “Chips, detectors, and methods of making and using the same”, 2021.

A. Chilkoti, “Non-fouling polymeric surface modification and signal amplification method for biomolecular detection”, 2021.

A. Chilkoti, Rangarajan, S. , Amiram, M. , and Hucknall, A. , “Direct detection of RNA by surface initiated enzymatic polymerization”, 2021.

Chilkoti, A., Heggestad, J., Kinnamon, D., and Hucknall, A., "Methods for Detecting Neutralizing Antibodies." U.S. Patent Application US20230098149A1, Filed September 30, 2022, Published March 30, 2023. https://patents.google.com/patent/US20230098149A1

Chilkoti, A., Fontes, C., Hucknall, A., "Semi-quantitative lateral flow devices", US Patent Application US20230314434A1, Filed March 3, 2023, Published October 10, 2023. https://patents.google.com/patent/US20230314434A1