AMS Spring 2023

Researchers Use Inkjet 3D Printing to Create Fast, Accurate, Inexpensive Diagnostic Tool

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From whirligigs and models to printers and smart bathrooms, 3D printing technology has definitely made an impact on diagnostics. Healthcare workers use diagnostic tools to detect and determine the severity of a disease, as well as plan treatment and track the patient’s response. There aren’t many diagnostic tests that are portable and easy to use, and the ones that are often don’t have enough sensitivity to offer any information other than if a biomarker is there or not. The optimal quantitative diagnostic test is the enzyme-linked immunosorbent assay, or ELISA, which is sensitive enough to identify how many specific antigens (protein markers) are in a particular biological sample. But researchers at Duke University have used 3D inkjet printing to create a diagnostic tool that has the potential to be better than ELISA.

The ELISA platform is one of the most sensitive immunoassay tools available for detecting diseases and tracking hormone levels in blood, but it’s not without its flaws: it can take up to 24 hours to show test results, requires trained researchers or liquid handling robotic devices to follow multiple steps so that no unwanted proteins mess up the results, and, due to its necessary laboratory instruments, is not good in environments with limited space and resources.

[Image: Digital Journal]

The 3D printed biomedical tool created by Duke University scientists can be used in point-of-care settings to screen patients for certain disease markers. In addition to high accuracy, the tool can help reduce the time patients have to wait for test results from days to just 15 minutes.

It’s called the D4 assay, a self-contained test in the vein of a lab-on-a-chip that can detect low levels of antigens from a single drop of blood. The scientists used inkjet technology to print an array of antibodies onto a glass slide with a nonstick polymer coating. Two types of antibodies are in the printed array – soluble detection antibodies and immobilized capture antibodies – and tagged with fluorescent markers show physicians can see how much of a particular antigen is present. The D4 assay is just as sensitive as the ELISA, but much quicker and less bulky.

“The real significance of the assay is the polymer brush coating,” explained Ashutosh Chilkoti, one of the lead researchers and the chair of the Department of Biomedical Engineering at Duke. “The polymer brush allowed us to store all of the tools we need on the chip while maintaining a simple design.”

The research team published a paper on their findings, titled “Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood,” in the journal Proceedings of the National Academy of Sciences (PNAS). In addition to Chilkoti, co-authors include Daniel Y. Joh, Angus M. Hucknall, Qingshan Wei, Kelly A. Mason, Margaret L. Lund, Cassio M. Fontes, Ryan T. Hill, Rebecca Blair, Zackary Zimmers, Rohan K. Achar, Derek Tseng, Raluca Gordan, Michael Freemark, and Aydogan Ozcan.

D4 assay prototype

The abstract reads, “Here, we describe a self-contained immunoassayplatform (the ‘D4 assay’) that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are ‘on-chip,’ and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.”

The results from the D4 assay can be read with a tabletop scanner, or a 3D printed smartphone attachment that uses the camera to read results. This allows the portable test kit to be brought closer to the patient.

Once a drop of blood is placed on the D4 assay slide, detection antibodies dissolve and separate from the array, then bind to the target proteins in the blood. Then, the “fluorescing antibody-protein pairs” attach to the capture antibodies on the slide. The antibody array is 3D printed on a novel polymer brush coating, which keeps unwanted proteins from binding to the rest of the assay.

Joh said, “What’s cool is that our assay can achieve comparable sensitivity to the ELISA within 15 minutes, and if further sensitivity is needed, longer incubation times can be used. This device can also be compared to a lateral flow test, which is quite fast as it takes less than five minutes to get a reading, but that test isn’t as sensitive. This is really the best of both worlds.”

Unlike the ELISA, which has a complicated workflow for researchers to follow, once a test with the D4 assay is complete, the slide simply needs to be washed in a buffer solution to get rid of any extraneous particles. Another way the 3D printed Duke diagnostic tool differs from the ELISA is its portability – the dry slides don’t need refrigeration during travel, and the design is cost-efficient. The research team believes that the D4 chips will cost less than one dollar, and the 3D printed smartphone attachment, which was developed at UCLA will cost less than $30 once it’s mass-produced.

Image A shows the smartphone attachment to read results. Images B-D show how to add a sample to the D4 assay.

The researchers plan to make rapid diagnostic testing accessible to areas that may not have access to lab-based diagnostic technologies.

Hucknall said, “The D4 assay enables us to conduct high-performance diagnostic testing with minimal resources, making it a promising platform for increasing access to sensitive and quantitative diagnostic tools.”

The researchers plan to study how they can use the 3D printed tool to perform even more diagnostic tests, and Joh and Hucknall will be conducting a field test of the prototype to screen participants’ serum leptin levels in Liberia to determine how diagnostic results from the D4 assay can help monitor and plan malnutrition treatment strategies.

[Sources: Duke University, Digital Journal / Images: Joh et al.]

 

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