For quite a number of illnesses, patients need to have the concentrations of the medications they are taking and their blood metabolites checked on a regular basis, which can make life rather difficult. Researchers at the Max Planck Institute for Medical Research in Heidelberg have now developed a test based on the firefly enzyme luciferase. This enzyme can be used to quickly and cheaply measure parameters of interest in any doctor's surgery or even at home with just one drop of blood on a strip of paper.
The therapeutic outcome of many diseases depends on administering drugs in appropriate doses for individual patients. In addition, the exact concentration of disease-causing metabolites in the body needs to be determined to be able to prescribe the best possible medication. Currently, drug dose and metabolite concentration are determined by taking blood and analysing it in a suitable medical institution using mass spectrometry. Depending on the disease, this type of examination requires patients to see a doctor or visit a hospital on a regular basis, which can be costly and stressful if the visits continue for months or years.
Researchers at the Max Planck Institute (MPI) for Medical Research in Heidelberg are seeking to change this. They have developed a test that makes it possible to measure various parameters directly in patients’ blood quickly and easily, similar to blood glucose measurements done by diabetics. Scientists led by Prof. Dr. Kai Johnsson, director at the MPI for Medical Research, developed the basic test principle back in 2014. The system is called LUCID (luciferase-based indicators of drugs) and is based on the enzyme luciferase, which is also responsible for the bioluminescence of fireflies, making them glow. In the test developed by the Heidelberg researchers, the firefly enzyme also triggers the release of light, which can be measured easily and even seen with the naked eye.
"Our test system is based on a proprietary biosensor that is half protein (i.e. the luciferase) and half synthetic molecule, and can thus emit light," explains Johnsson. "And this combination is what makes the test so powerful. The luciferase is obtained by expressing the luciferase gene in bacteria. We also produce the synthetic molecule in our laboratory."
When analysing the substance in the blood, the biosensor binds to a cofactor, which changes the colour of the light emitted from blue to red. If the substance of interest is either absent or present in a lower quantity, the cofactor either does not bind or only binds in an amount that corresponds to that of the sensor. Consequently, the colour of the emitted light only changes slightly or not at all. The concentration of the metabolite in the blood can now be determined easily from the ratio of blue to red light and measured with a simple camera and a piece of paper. The metabolic assay can be used to determine the concentration of many biomolecules and substances. For example, it can be coupled to glucose, a wide range of amino acids and lipids. In principle, the concentration of any metabolite that can be oxidised by the cofactor nicotinamide adenine dinucleotide phosphate (NADP) can be measured.
"The good thing about the method is that we can do the tests within a few minutes, and they are as reliable as mass spectrometry, which is the method currently used for analysing blood samples," says Johnsson. "This is what makes the novel test so interesting for diagnostic tests and would greatly simplify the lives of many patients." The scientists have already subjected the system to a practical test: blood samples from patients with phenylketonuria were analysed at the Heidelberg Children's Hospital. Phenylketonuria is one of the most common congenital metabolic disorders and can be managed through a low-protein diet. However, the level of the amino acid phenylalanine in the blood has to be monitored effectively. The results of the luciferase test system were very convincing and as accurate as those obtained with mass spectrometry1.
"We are hoping that this test will make life easier for those affected in the future and that they will be able to do the analysis themselves at home," says the biochemist. "However, at the moment, the test is not yet ready and still requires a bit more development work." A functional sensor that can quantify the desired substances in the blood samples exists. What is still lacking is the associated equipment, i.e. a device with a chip to measure the photons. “But I am sure that this can be solved by engineers,” says Johnsson. "You can probably even build a device in the two-digit euro range. In fact, a camera like the ones in standard mobile phones would do the job quite well.”
It has long been possible to determine the concentration of metabolites per se in hospitals. However, direct determination of metabolites in blood has not previously been possible because light of low wavelength is absorbed by the components of the blood and can therefore not be perceived or measured. "The trick with the new test system is that the molecule that emits the light also changes the wavelength," explains the scientist. "And this can be measured, i.e. not the light itself, but the ratio of blue to red light, which must be high enough for us to be able to determine the desired substance directly in the blood. Half a microlitre of blood is more than enough for that, and this is what now makes the biosensor so versatile."
The test system is not only used in medical diagnostics. The biosensor experts from Heidelberg also use the technology to study processes in living cells. "We use the technology to quantify metabolites in different cell compartments, such as the nucleus or the mitochondria," says Johnsson. "For example, we can monitor the changes metabolites undergo when certain processes go awry in the cell.”
The Max Planck researchers intend to spend the next weeks and months further validating and automating the biosensor so that it can be used for patients. "But this is not something that will happen within the next 12 months," says Johnsson. "However, we are convinced that we can overcome the conceptual hurdles that lie ahead - others before us have also managed to achieve similar outcomes. And this is precisely what I find so interesting on a personal level – as a biochemist I now have the opportunity to think about completely new and different things."
(1) Qiuliyang Yu Lin Xue, Julien Hiblot, Rudolf Griss, Sebastian Fabritz, Clothilde Roux, Pierre-Alain Binz, Dorothea Haas, Jürgen G. Okun, Kai Johnsson: "Semisynthetic sensor proteins enable metabolic assays at the point of care." Science, 14 Sep 2018