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Cell-Free Platform Developed to Aid in Detecting Nutritional Deficiency in Children

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NEW YORK – Researchers at Georgia Institute of Technology and Northwestern University have developed a cell-free expression proof-of-concept platform that detected clinically relevant concentrations of zinc, a biomarker for a nutritional deficiency that kills 100,000 children under the age of 5 worldwide each year.

The platform, using minimal equipment, may one day be used to develop a range of diagnostic assays, including those used at the point of care by organizations seeking to prioritize outreach and treatments for children with nutritional deficiencies, Mark Styczynski, one of the platform's developers and an associate professor of chemical and biomolecular engineering at Georgia Tech, said in an interview.

On Wednesday, Styczynski and his colleagues published a study in Science Advances describing the development of their platform, which used freeze-dried bacterial extracts mixed with synthetic DNA within plastic tubes. 

In the current version of the testing platform, the researchers ran the zinc assay by adding serum to plastic tubes that consisted of the relevant reaction components to obtain color changes associated with quantities of zinc in the serum. The assay had a clinically relevant range of response for detection of zinc ions between 5 microns and 20 microns, Styczynski said.

They obtained results in less than 60 minutes, running reactions in 8 microliter samples. For blood tests, the samples were 25 percent serum when they were not freeze-dried and 100 percent serum when they were freeze-dried. 

In their study, the Georgia Tech-Northwestern researchers designed DNA that they then added to the test platform to enable key functions. To begin, they used "a small amount" of chemically synthesized DNA and then deployed bacteria to manufacture the DNA for use in the test, Styczynski said.

DNA extracted from bacteria triggered an enzyme switch, enabling it to be turned on or off depending on the concentration of zinc ions encountered in blood samples. The activity of the enzyme, in turn, controlled a reaction that changed one color to another in the test, enabling an equipment-free color readout.

Such a design solves challenges associated with matrix effects — interference by components of a sample other than the analyte of interest — that can hamper the accuracy of laboratory tests, according to developers of cell-free expression systems.

The work by Styczynski and his colleagues "reflects an important trend in the field of synthetic biology, which is to develop technologies in the context of real-world practice," Keith Pardee, Canada research chair in synthetic biology and human health at the University of Toronto, said in an interview. "Through a clever molecular solution to creating standard curves, [the researchers] were able to generate high fidelity measurements for the micronutrient zinc from patient serum samples, which is clinically relevant for global health applications."

Importantly, the team showed that its approach was generalizable to a wide range of analytes, including nucleic acids, said Pardee who was not involved in the work described in Science Advances. "I anticipate seeing this work incorporated into many cell-free sensor-based diagnostics in the coming years." 

The work demonstrated by Styczynski and his colleagues "provides a generalizable solution to the problem of matrix effects that hamper a lot of…tests that work in idealized samples but fail in the real world," Khalid Alam, cofounder of Evanston, Illinois-based Stemloop, a biological sensor developer, said in an interview. "I suspect their approach will not only be used by others but will inspire additional strategies for dealing with matrix effects," said Alam, who is a former postdoctoral fellow of Northwestern but is not involved in the current research and development work.

Styczynski said that the calibration strategy that he and his colleagues employed to cope with matrix effects involves use of a test reaction site and "carefully designed" standard reaction sites. A sample undergoing analysis is added to both types of sites, ensuring that all test reactions are run in parallel using the same sample matrix. After a preset time for incubation, the color of the test reaction site can be matched to the color of the standard reaction sites to determine the biomarker concentration in the test reaction.

The research team developed the platform with the objective of retaining a minimal amount of equipment to operate it. The system uses only plastic tubes and mixtures to generate reactions reflective of test results, reducing its complexity and cost, Styczynski said.

For future versions of the test, the group plans to provide an analytical component as an option. The team is engaging with a high school synthetic biology research team to develop a smartphone application that could enable a quantitative readout based on an image of the colors produced by the test results.

The results could be readily incorporated into nutritional surveys currently being conducted by epidemiological researchers using tablets and smartphones. In low-resource settings, these researchers conduct the surveys in a bid to estimate the nutritional consumption of children and then allocate resources for treatment.

The platform developers are also experimenting with using paper to implement its diagnostic approach. Other groups developing cell-free expression diagnostics have shown that the use of paper is possible, Styczynski noted.

A future format for implementing the approach may involve integrating the reaction components within a combined lateral flow and microfluidic structure.

Styczynski said that the team has been focused on the specific zinc ion because of its importance within nutritional epidemiology, but the group has also moved beyond ions in its development work to include nucleic acid detection on its platform.

The researchers are currently developing the platform to detect levels of vitamin B12, an important dietary health ingredient. Further, as a next step to support validating the platform, they are seeking to attract interest from a partner that would collaborate in deploying the system to conduct field testing of zinc levels in children living in low-resource settings.

The team is also in discussions with "well-funded startups," that may be interested in integrating the platform into their diagnostic test development pipelines, Styczynski said.

Julius Beau Lucks, who heads the Lucks Laboratory for RNA engineering at Northwestern University, said that he believes the work described in the Science Advances paper tackles an important challenge to make these platforms quantitative, while retaining ease of use. "This and other recent work are marking a turning point for synthetic biology to deliver impactful technologies for addressing important global health needs," said Lucks, who is not involved the work of Styczynski and his colleagues.

Nonetheless, getting a diagnostic approved by the US Food and Drug Administration or endorsed by the World Health Organization will not be easy, Styczynski said. "The path to commercialization is always bumpy, and this is targeting the resource-constrained developing world," he said. Launch of a platform that's available for purchase may be possible within a few years, but that estimate could also be too optimistic, he said.