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Max Planck Team Develops Paper-Based Metabolite Test

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NEW YORK (360Dx) – A team led by researchers at the Max Planck Institute for Medical Research has developed a semisynthetic protein biosensor that could enable point-of-care metabolite testing.

In a study published last week in Science, the researchers used the sensor to detect the metabolite phenylalanine, measuring it in a matter of minutes from less than a microliter of blood. According to Kai Johnsson, a Max Planck professor and senior author on the paper, the results point to the possibility of POC testing of clinically relevant metabolites.

The test adapts an existing clinical approach to measuring metabolites that is based on measuring the reduced form of the molecule nicotinamide adenine dinucleotide (NAD) or its phosphorylated form (NADP). Both molecules are cofactors that oxidize a variety of metabolites, and through this oxidation reaction they are converted to their reduced forms (NADH and NADPH). These reduced forms can then be measured and the amount of the metabolite they oxidized can be calculated from that measurement.

In a typical assay, a clinician adds an enzyme specific to the analyte of interest to a patient blood sample and then quantifies the production of NADH or NADPH. Accurately measuring the levels of these co-factors in whole blood, however, requires a level of performance that is beyond typical POC technologies, Johnsson said, and so such testing is generally done in a clinical chemistry lab.

There are a number of metabolites used for regular patient monitoring of different conditions for which a POC test could be useful, though, he noted. For instance, people with phenylketonuria — pediatric and pregnant patients — must carefully monitor their phenylalanine levels, but existing assays for that metabolite require sending out a blood sample to a lab.

"The turnaround time is, in the best case, around two days," Johnsson said. "But depending on how well the postal service in your country works, it could be longer."

The test developed by Johnsson and his colleagues, on the other hand, could in theory return a result in 15 minutes and without the need for any equipment more advanced than a digital camera, he said.

The paper-based test uses a semisynthetic sensor protein that changes color upon binding to NAD+ or NADP+. The sensor consists of the luciferase NanoLuc linked to an NADPH-dependent receptor protein and a fluorescently labeled ligand that has an NADPH-dependent affinity for the receptor protein. When NADPH is present, the fluorescently labeled ligand binds to the receptor protein, which brings the fluorophore close to the NanoLuc luciferase, changing the color of the fluorescence emitted. This color change can be measured with a digital camera and used to calculate the quantity of NADPH present and, from that, the level of the target metabolite.

The major advantage of the approach is its simplicity, Johnsson said. "The recognition happens in solution. No washing is required."

Additionally, he said, because the assay is essentially a version of the NADH and NADPH metabolite assays currently used in clinical chemistry, it can, in principle, be adapted to a wide range of metabolites. That fact could help drive development of the assay as a POC test, Johnsson said, "because once you have that, then you can use this [approach] for a variety of different assays."

In the Science paper, the researchers included a list of 42 clinically significant metabolites that they said could be measured by the assay, including galactose, ethanol, triglycerides, cholesterol, and branched-chain amino acids. They also demonstrated the assay's suitability to a 96-well format, measuring phenylalanine levels in 96 whole blood samples in parallel, a capability they suggested could allow for applications like "PKU screening in resource-limited settings."

They compared the performance of their phenylalanine assay to that of existing clinical assays including LC-MS/MS, finding that it offered a comparable level of performance. In a series of experiments testing whole blood spiked with phenylalanine, measurements from the paper-based test achieved a correlation of .999 with measurement by mass spec. In measurements made in 40 PKU patient plasma samples, the paper-based assay also showed good correlation with mass spec, achieving an r of .992. The test also showed good reproducibility, with an average coefficient of variation across triplicate measurements of 7 percent.

Additional development will be needed before the assay can be run in a POC format, Johnsson said, noting that it will have to be packaged in an integrated and automated device that will draw patient blood in a simple and reproducible fashion and run the test.

That "is not impossible," he added. "There are devices and chips that can run the kind of steps [involved in the assay] in an automated fashion. There are hurdles, but from our point of view, we should be able to overcome them. It's an engineering problem now."

He said his lab is currently looking into collaborations with other labs that have expertise in automating such tests.

Among the challenges will be developing a system capable of diluting patient blood to a level appropriate for running the assay, which, in the study, used samples diluted by a factor of 50. The authors noted this could likely be handled by a microfluidic system.

Additionally, awareness of potential interferences will be important. The authors noted, for instance, that patients taking the antibiotic trimethoprim (TMP) would not be able to use the published version of the phenylalanine test as TMP binds to the receptor protein used in the assay.

That said, a POC version of the assay could be highly useful to patients with PKU and other conditions that require regular monitoring of metabolites, Johnsson suggested.

"It would just make the whole process much easier," he said. "It puts patients in control of their disease, if you will."