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Finnish Researchers Developing Microparticle-Based POC Immunoassay Platform

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NEW YORK (360Dx) – University of Turku researchers said that they have developed a proof-of-concept platform that could be further developed as a diagnostic system for use at the point of care.

The platform, which uses photoluminescent up-converting nanoparticle technology, was described recently in the journal Analytical Biochemistry, and according to the researchers it has limits of detection that are comparable to that of the most sensitive troponin I lab-based assays. They further noted that the technology potentially could detect analytes that require high sensitivity, such as cancer and infectious disease biomarkers in human blood.

The impetus behind the work is a need for sensitive and affordable point-of-care immunoassays, Etvi Juntunen, a developer of the immunoassay who works in the department of biotechnology at the University of Turku, said in an interview, adding such affordable assays could be especially important for applications in low-resource settings where centralized laboratories are not available.

The point-of-care immunoassay platform combines the performance of a microtiter, well-based assay, such as a laboratory ELISA, with the ease-of-use of a rapid assay, the researchers said.

Although the performance of each assay is different depending on the antibodies being used, the platform generally matches the sensitivity and specificity of ELISA technology, Juntunen said.

He noted that the study also demonstrates the feasibility of developing assays for a variety of analytes on the point-of-care platform. In addition to using the platform to measure troponin I, the group has conducted preliminary validation tests for prostate-specific antigen, HIV antigen, and hepatitis B surface antigen.

HIV antibodies are already being detected using conventional lateral flow assays, but the University of Turku researchers are interested in measuring the p24 antigen that "could be more sensitive" and shorten the time to detect a pathogen causing an infection, Juntunen said.

At present, the university does not have specific commercial plans, but the researchers are hopeful that they will generate interest from companies interested in taking the platform forward as a commercial test, he said, and in that context, the university could work as a collaborator with the company.

Among the firms that have developed, and commercialized, POC assays that can be used in decentralized environments are Roche and Abbott Laboratories, which have developed molecular-based POC tests. Assays based on other formats are also in the works, and in general investigators seeking to develop inexpensive assays are leveraging a variety of technologies, such as lateral flow, dipstick, flow-through, and microfluidic approaches, the University of Turku researchers said.

Among point-of-care platforms, lateral flow immunoassays are well-accepted, low-cost, and simple. However, their sensitivity and therefore the usefulness of the approach is limited, the researchers said. 

In lateral flow assays, the reaction time during which an immunocomplex is captured on the solid-phase binder surface is limited by the flow speed of a nitrocellulose membrane, Juntunen noted. By comparison, in the researchers' microparticle-based assay, the reaction time can be selected freely before the microparticles are collected for measurement, and the user has control over sample incubation time.

"It is essential to allow proper time for the Ag-Ag-immunocomplex to form," Juntunen said. "Antibodies with relatively slow on-rate kinetics are unfortunately the reality for many assay developers. By increasing the incubation time more 'sub-optimal' antibodies can be successfully used."

Among their objectives in developing a proof-of-concept test was to retain the ease of use of a lateral flow platform but enable greater flexibility in the sample volume needed for testing. Further, the time to receive a test result for the microparticle platform, up to 20 minutes depending on the analyte being tested, is comparable to that of lateral flow technology, Juntunen noted.

The University of Turku researchers expect that apart from providing sensitivity improvements over lateral flow assays, their platform, if it is eventually commercialized, could be produced at a competitive price to tests that use lateral flow technology. Juntunen noted that the platform and assay can be produced for about $1, partly because the platform is inexpensive to produce from an injection-molded plastic and uses a small amount of reagent.

The researchers said in their paper that their platform consists of separate reaction and detection chambers and microparticles that combine with photoluminescence for detection. A reaction chamber and valve within an injection molded cassette enables control over reaction incubation time. Inside the cassette, microparticles immobilize reagents that capture the analyte of interest. Detection reagents coat fluorescent up-converting nanoparticles that emit light at a shorter wavelength when the analyte is present, and a sensor within a fluorescence reader detects the nanoparticle-emitted light. With a suitable fluorescence reader, the system can conduct analyte quantification, the researchers said.

The researchers noted that in developing their proof-of-concept device, they focused on several aspects of the platform's design, including the geometry of the reaction chamber, the need for a suitable optical cap allowing label-specific excitation and emission, the size of the waste chamber, and the need for a practical buffer inlet and suction channel orifice for maintaining undisrupted liquid flow. Initial cassettes were 3-D printed and the finalized prototypes were injection-molded from plastic at Scaletec Oy in Turku, Finland.

The researchers prepared analyte-specific reagents suitable for the detection of cardia troponin I and tested them in five replicate cassettes. During testing, all the reaction materials, except the microparticles entrapped in the detection chamber due to the presence of a filter mesh, flowed from the reaction chamber through a detection chamber and on to a waste container. The investigators used a portable fluorescence reader to measure the up-converting nanoparticle signals being transmitted from the immune complexes bound to the microparticles in the detection chamber.

When they tested their proof-of-concept platform with a cardiac troponin I assay, they achieved a limit of detection of 19.7 ng/L. Current high sensitivity cardiac troponin I assays have a limit of detection of less than 10 ng/L, but point-of-care assays have limits of detection of between 10 and 50 ng/L, the researchers said.

This type of platform permits efficient washing that contributes to better specificity, according to Juntunen. Washing removes residual reporter nanoparticles that generate background signals, and reducing those signals enables more accurate detection of weak signals produced by smaller quantities of specifically bound reporters, he added.

The use of photoluminescent up-converting nanoparticles in the assay in combination with a reader not only provided better sensitivity, but it also eliminated a problem of subjectivity associated with reading the results of lateral flow assays, the researchers said.

New research is overcoming many of the problems of lateral flow assays, primarily by using nanoparticle-reporters, and new designs are providing increased sensitivity and quantification with portable optical or electrochemical reader instrumentation, the researchers said.

Several investigators doing research and development on clinical diagnostic tests are combining nanotechnology and light-based detection in their platforms and they are well aligned with the Finnish team's research, according to Juntunen.

For example, at the center of a test being developed by researchers at Arizona State University, exosome vesicles bound to a sensor chip are exposed to two different antibody-coated nanoparticles: one green nanosphere and one red nanorod.

The close contact of the two particles causes a coupling effect that changes their color and increases the intensity of their refracted light, generating a detectable signal.

They are developing the method for the detection of tumor-derived exosomes in circulation that they said could be turned into a simpler and potentially cheaper platform for cancer diagnostics development than other approaches.

For infectious disease applications, Linear Diagnostics, a recent spinout of the University of Birmingham in the UK, is developing a microfluidic cartridge-based platform to detect multidrug-resistant bacteria in hospitals. 

Its technology is based on the arrangement of long, thin nanometer-sized molecules in a microfluidic chamber inside a test cartridge that holds the reagents needed to perform a test. Using a handheld external optical reader, researchers detect the molecules' alignment in the chamber by shining polarized light from different angles into the cartridge.

A European Union-sponsored research project leverages plasmonics — an optical effect — that combines the transmission of light to gold nanoparticles on a chip, and measures light reflecting from the chip. The system integrates DNA, microRNA, and protein-based tumor autoantibody detection, and it could soon lead to a liquid biopsy test for colorectal cancer, according to its developers.

The system detects protein-based tumor autoantibodies and recognizes specific DNA and microRNA mutations, eliminating the requirement for preliminary amplification of nucleic acid sequences.

In separate nanoplasmonics research, Harvard Medical School and Massachusetts General Hospital researchers are developing a multiplexed assay that relies on nanopores and a shift in light spectra initiated by extracellular vesicles bound to monoclonal antibodies. They're designing it to fit into clinical workflows for high-throughput detection of pancreatic ductal adenocarcinoma, an aggressive and often inoperable form of pancreatic cancer.

Although their platform is still very much in the development phase, the University of Turku researchers noted that the proof-of-concept study demonstrated the performance of microparticle−based platform in "a highly challenging and demanding application." However, the device would need to be further validated in complex sample matrices such as serum, plasma, and whole blood "to truly realize the potential of the developed platform," they added.