OIST Researchers Create 3D Printed Modular Microfluidic Prototype for Point-of-Care Testing

Common on-site water quality test kits and at-home infection disease tests typically require the user to do all of the work – collect fluid samples, measure and mix reagents, perform assays, etc. Even though these kits decrease how long you have to wait for results, the risk of false positives, biocontamination, and inaccurate analyses increases. But, longer wait times and higher costs can occur if you choose more precise results by shipping pre-processed fluid samples to a lab. It seems like a no-win situation, right?

Researchers Shivani Sathish, Kazumi Toda‐Peters, and Amy Q. Shen with the Okinawa Institute of Science and Technology (OIST) in Japan recently published a paper, titled “Proof-of-concept modular fluid handling prototype integrated with microfluidic biochemical assay modules for point-of-care testing,” about their work creating a modular prototype system for accurate, rapid, and reproducible diagnosis that have a decreased risk of contamination.

“Large populations around the world suffer from numerous but treatable health issues, caused by either lifestyle choices or environmental factors. Over the past decades, point‐of‐care testing kits have been developed to circumvent the reliance on laboratories, by allowing users to perform preliminary health or environmental testing from the privacy of their homes. However, these kits heavily rely on the precision of the user to perform the procedures, leading to increased variability in final assessments,” the trio wrote. “To eliminate user‐induced errors, we present an integrated, completely sealed, and disposable point‐of‐care testing prototype that exploits the benefits of microfluidics and 3D‐printing fabrication techniques. The palm‐sized modular prototype consists of a manually operated fluid handling device that allows precise mixing, filtration, and delivery of fluids to an on‐board microfluidic assay unit for subsequent detection of specific biochemical analytes, with a minimized risk of contamination.”

A fluid handling device (FHD) module is the first component in their system, and makes it possible to collect and mix a fluid sample with pre-loaded reagents, then filter it, collect fluid waste, and bring the processed sample to the second component, an on-board microfluidic assay device. Contamination caused by the user is reduced in this sealed system, and makes it possible to get accurate results in just a few minutes.

“The FHD is the base module for our point‐of‐care testing system, where it serves as a platform to secure different analysis/detection modules for subsequent biochemical tests. The reagent storage cartridge is capable of housing 1‐ 3 mL of primary reagents, used for sample dilution and priming for the biochemical assay,” they wrote.

Here’s how it works: the bottom parts of the FHD are locked with the upper portions – made of secondary reagents employed during the assay – once a fluid sample is deposited. The user then pulls down on the buffer cartridge tabs to dilute and mix the solution. This causes a vacuum pressure, which forces the primary reagents to flow into the mixing chamber to mix and dilute the sample. Then, the sample goes through the upper part of the FHD for filtration and assay in the microfluidic assay module; simultaneously, the waste fluid is collected in a separate unit. A final compression step delivers the secondary reagents to the microfluidic assay module – this finishes the assay and lets the user see the results.

The researchers used a Form 2 3D printer to create the body of the FHD out of white V4 photocurable resin. To ensure fluid delivery with no leaks, the custom components slide concentrically within each other, thanks to a piston/cylinder system.

“As a case study, we carried out biochemical assays to detect antibodies specific to bacterial Chlamydia trachomatis infections using our integrated prototype,” they explained. “Specifically, buffer samples doped with C. trachomatis specific immunoglobulin G (IgG) antibodies3 were mixed with on‐board fluorescently labeled antibodies in the FHD and assayed in poly(methyl methacrylate) (PMMA) microfluidic assay devices, patterned with antigenic major outer membrane proteins (MOMP) of C. trachomatis.4 Varying concentrations (125 ng/mL ‐ 2 µg/mL) of anti‐MOMP IgGs could be detected and reproducibly differentiated from negative samples using our manually powered prototype within 15 min.”

They used PBS as the negative control. Six individual FHD sets were soaked in an N101 blocking buffer at 4°C for 15 hours for the team’s “non-specific protein adsorption studies.”

“The samples were loaded onto the lower portion of the FHD and mixed with the primary reagent buffer (2 mL) comprising fluorescently‐labeled detection antibodies,” the team wrote.

(A) FHD components, (B) fabrication strategy of microfluidic assay module, (C) quantification of non‐specific protein adsorption in FHD, (D) processing steps for an anti‐MOMP IgG fluorescence assay. The graph and fluorescence images depict the linear range of detectable anti‐MOMP IgG concentrations with the integrated prototype.

An air-plasma enhanced bio-functionalization strategy was used to fabricate a microfluidic chlamydia assay module, and microchannels were formed with a carbon dioxide laser cutter. MOMP was bonded to PMMA strips on one side, and IPA-cleaned PMMA strips on the other. A 1% weight/volume of bovine serum albumin was used to prevent non-specific adsorption of proteins and block the bare surfaces of the PMMA microchannels, which were attached to the top part of the FHD.

The mixed samples, after being filtered through the FHD’s upper portion, were delivered to the MOMP-patterned microfluidic assay modules. After washing the assay to get rid of any unbound biomolecules, the team used a spectrophotometer to quantify the “concentrations of antibodies in the eluates.”

“The inner layers of the FHD body showed minimal non‐specific protein adsorption, with an insignificant difference between the blocked and pristine units (Figure 1C),” they explained. “As seen in Figure 1D, the integrated prototype enabled the detection of anti‐MOMP IgGs with a linear concentration range of 125 ng/mL ‐ 2 µg/mL, and a limit of detection8 of 1.05 µg/mL.”

The researchers were consistently able to tell the difference between the assay and the negative controls, which provides that their prototype has high potential for applications in point-of-care testing.

“In the future, we envision the inclusion of multiplexed microfluidic assay chambers within one single device, to allow detection of multiple bioanalytes from one single sample, thereby reducing both the testing time (<30 min) and the cost (< US$25 for multiple tests), for discrete at‐home testing,” the researchers concluded.

This 3D printed prototype joins many other microfluidic, water-testing, and medical testing devices that are making this type of research less expensive and time-consuming.

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