(A) The 3D printed smartphone attachment that uses the phone's camera to review the results of the assay. (B–D) The process to administer a blood sample onto the D4 assay. [Daniel Joh/Duke University]
(A) The 3D printed smartphone attachment that uses the phone’s camera to review the results of the assay. (B–D) The process to administer a blood sample onto the D4 assay. [Daniel Joh/Duke University]

Enzyme-linked immunosorbent assays (ELISAs) represent the gold standard for quantitatively detecting protein biomarkers, and are used for applications such as diagnosing or monitoring disease, developing therapeutic approaches, tracking patients’ responses to treatment, and evaluating patients for clinical trials. But ELISAs are not particularly user friendly. The tests are time consuming and resource intensive and require trained personnel or automated liquid-handling robots, together with bulky data analysis equipment. Multiple blocking and washing steps are needed to prevent unwanted binding by nonspecific proteins that otherwise create background noise and reduce sensitivity.

All these factors hold back the use of ELISAs in limited-resource settings and can delay results reaching clinicians who might be waiting to make life-saving decisions on treatment, reports a team of researchers at Duke University, Durham, NC. The Duke team, headed by Ashutosh Chilkoti, Ph.D., Daniel Y. Joh, and Angus M. Hucknall, Ph.D., has now developed a completely self-contained, portable, multiplex immunoassay platform that is as sensitive and accurate as an ELISA, but which can be used in any field or remote setting, without specialist training, to quickly and quantitatively detect biomarkers in a spot of blood.

The team’s D4 lab-on-a-chip is generated by inkjet printing an array of antibodies onto a glass slide that features a non-stick, nanoscale poly(oligo(ethylene glycol)methacrylate) (POEGMA) brush coating that prevents unwanted proteins, cells, and other biomolecules from binding to the chip surface. This dramatically improves the signal-to-noise ratio (SNR), even at low analyte concentrations in complex samples such as blood. It also means that the test doesn’t require any complicated processing steps to prevent nonspecific binding, it just needs a wash in buffer solution.

And unlike ELISAs, which can take up to 24 hours to generate results, the D4 test results are available within 15 minutes, and can be read using a tabletop scanner or using a 3D printed smartphone attachment that has been developed alongside the test.

The D4 assay concept was founded on earlier work by the group, which had created antibody microarrays on the nanoscale brush POEGMA that could detect protein analytes in whole blood with femtomolar sensitivity. “This polymer brush-based platform simplified assay fabrication and workflow – elimination of sample pre-processing and blocking steps, and reducing the number of washes compared to ELISA – but was still far removed from being a point-of-care (POC) test due to the requirement for separate incubation and labelling steps for assay readout,” Dr. Hucknall explained to GEN. “The current work with the D4 assay builds upon this framework to develop a POC test that has performance comparable to laboratory-based ELISA but retains the low-cost, portability, and user-friendliness of existing POC tests such as lateral flow assays.”

Similar to ELISAs, the D4 assay is a fluorescence test that uses a matched pair of antibodies to detect and capture target proteins in a blood sample. “At the core of the D4 is the POEGMA polymer surface coating …” Dr. Hucknall continued. “In the dry state, the coating acts as an ink reservoir that stores and stabilizes reagents, even in unrefrigerated conditions. We took advantage of this feature to store all necessary capture and detection reagents ‘on-chip. In the hydrated state, the coating is non-fouling and virtually eliminates background binding.” This leads to excellent SNR for ELISA-like assay performance, he claims. 

“We fabricate the D4 by inkjet printing two assay reagents directly into the POEGMA brush: immobilized capture antibodies, and ‘soluble,’ fluorescently labeled detection antibodies (co-printed with excipient), Dr. Hucknall further explained.  “Adding blood allows the soluble detection antibodies to dissolve, and subsequently bind to analytes, if present. These detection antibody-analyte complexes can then be captured by the immobilized capture antibodies, effectively forming an antibody ‘sandwich’. Since the detection antibodies are labeled with a reporter, the antibody sandwich complexes, which appear as fluorescent microspots, can be detected using a detector such as a tabletop scanner or the smartphone-based device …”

The Duke team claims that the D4 assay has advantages over other “passive” point-of-care test designs, such as lateral flow immuomassays (LFIAs), paper-based diagnostics (PBDs), and passive microfluidics (PMFs), each of which still exhibits certain drawbacks, for example, lack of sensitivity. Capture and detection antibodies are printed very close together on the D4 chip, which also simplifies the design.

“This design eliminates the need to control fluid movement with fluidics or include sequential liquid transfer steps”, the authors write in their published paper, in Proceedings of the National Academy of Sciences (“Inkjet-Printed Point-of-Care Immunoassay on a Nanoscale Polymer Brush Enables Subpicomolar Detection of Analytes in Blood”). Inkjet printing also allows fine control over assay geometry, spot concentration, and composition, without the need for covalent coupling steps, which further simplifies assay fabrication.

The Duke team validated the D4 assay platform by carrying out a small pilot-scale proof-of-concept study that compared the test with an ELISA for detecting leptin levels in blood samples from lean and obese pediatric patients. The trial confirmed that the D4 results were as good as those of the ELISA test when a tabletop scanner was used to read results from the new assay.

The researchers chose leptin as an analyte because recent work by investigators at Duke University and Mulago Hospital in Uganda indicated that low serum leptin levels represent a major biochemical risk factor for mortality in malnourished children. A portable, rapid POC test for leptin could help to save lives by quickly identifying those children at the highest risk of death, particularly in regions where there isn’t the infrastructure to carry out hormone assays with the same sensitivity and blood tests need to be shipped to centralized laboratories, which delays life-saving treatment. 

The first-generation mobile phone imaging scanner wasn’t as sensitive as the tabletop scanner at detecting blood leptin captured by the D4 assay, but the researchers suggest that this technology will improve in time. “As mobile phone detector technology, computational imaging, and sensing approaches continue to evolve, we expect that the fluorescence collection efficiency and hence sensitivity of these portable, low-cost detectors will rival table-top fluorescence scanners,” they write.

The team hopes that the D4 assay will make possible sensitive, user-friendly quantitative diagnostic immunoassays that can be used in limited resource settings, and widespread point-of-care diagnostics, as well as research applications, for example, in large-scale epidemiological studies.

The underlying technology has been licensed exclusively to Immucor for human in vitro diagnostic applications, while the Duke researchers are exploring opportunities for developing tests for non-human applications, including animal and environmental testing, Dr. Hucknall commented to GEN.  “… we have a robust academic-industrial partnership with Immucor … we are applying this technology to develop POC tests for many different applications and piloting these tests for clinical and/or field studies.  We are currently developing tests for cancer, infectious disease, transfusion medicine, among many other applications.”

*This article was updated on August 11 2017 to include commentary from Dr. Angus M. Hucknall


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