Half a needle in a haystack
The new express method will find ultra-low concentrations of low molecular weight substances
Phys Tech blog, Naked Science
Russian researchers from the A. M. Prokhorov Institute of General Physics of the Russian Academy of Sciences and the Moscow Institute of Physics and Technology have developed the world's first ultra-sensitive method for rapid detection of low-molecular compounds. It can be used to detect trace amounts of toxins, hormones, vitamins and biologically active small molecules important for modern medical diagnostics, food safety control and many other purposes. The study is published in the journal Analytica Chimica Acta.
The immunochemical analysis developed by scientists based on magnetic tags takes less than 30 minutes, but at the same time it is 100 times more sensitive than enzyme immunoassay (ELISA) used in standard laboratory tests. The effectiveness of the approach was demonstrated by measuring the concentration of thyroxine in human blood plasma, one of the main hormones used to monitor the state of the thyroid gland. The accessibility and simplicity of the procedure of the developed test system allow diagnostics to be carried out directly at the site of biomaterial sampling.
High sensitivity in the detection of small molecules is necessary precisely because low molecular weight substances often have a strong effect on the body, even in small concentrations. Today, enzyme immunoassay is widely used to measure concentrations of such compounds. It is characterized by high labor intensity and can only be carried out by qualified personnel in specially equipped laboratories. To increase sensitivity in the detection of thyroid hormones, radioimmune analysis is still used, which is based on the use of radioactive tags with a short half-life and therefore has significant limitations and is potentially unsafe. In addition, traditional methods require a long time of sample preparation and the availability of specialized equipment.
There is also an immunochromatographic analysis (IHA), used, for example, in standard pregnancy tests. It is less time-consuming than the ELISA. However, this method is approved in many countries only for obtaining threshold results and cases that do not require high sensitivity analysis. Thus, one of the primary tasks in the field of medical diagnostics is the development of a fast and simple highly sensitive method for detecting small molecules.
The new analytical system, created jointly by scientists of the IOF RAS and MIPT, is a modification of immunochromatographic analysis using magnetic nanometers and bifunctional ligands. As a model for studying its capabilities, the definition of the main thyroid hormone – thyroxine was chosen, which informed the work and clinical significance.
The developed magnetic immunoassay is implemented differently than traditional IHA formats. Antibodies labeled with magnetic nanoparticles and a bifunctional thyroxine ligand are added to the patient's blood serum containing the studied free thyroxine. A bifunctional ligand is a thyroxine molecule covalently bound to biotin by means of a "bridge" separating them in space. Antibodies on magnetic nanoparticles, therefore, have the ability to bind both thyroxine from blood serum and a bifunctional ligand.
Scheme of the proposed method of thyroxine detection. Designations: T4 – thyroxine, T4-bt – thyroxine bifunctional ligand with biotin, MR-Ab – biotin antibodies labeled with magnetic nanoparticles (from an article in Analytica Chimica Acta).
After some time, after giving the molecules in the solution the opportunity to contact each other, it is applied to the membrane. In the case of interaction with a bifunctional ligand, the particles are retained on a test line of streptavidin, a protein with a strong affinity for biotin. The high affinity factor, as well as the highly sensitive counting of magnetic nanometers bound to the membrane as a result of the reaction, provide unprecedented sensitivity of the analysis. The achieved limit of hormone detection was 16 fg/ml (or 20 femtomol/liter, which corresponds to a million molecules in one milliliter of solution) in a dynamic range of 3 orders of magnitude.
The original electronic devices used to read the results of the analysis are based on the ultra-sensitive method of detecting magnetic nanoparticles MPQ (English – magnetic particle quantification). It is based on the phenomenon of nonlinear remagnetization of particles by an alternating magnetic field at two frequencies and registration of the induction response at combinatorial frequencies.
Explains the head of research and Head of the laboratory of IOF RAS Pyotr Nikitin (1979 graduate of MIPT): "The developed methods for measuring concentrations of low molecular weight substances use universal immunochromatographic strips with streptavidin on the recognition line, so they are not difficult to replicate. The variety of possible tests for other analytes requires standard antibodies and special bifunctional ligands, the synthesis of which for a variety of small molecules is a very difficult task. Fortunately, we have previously developed original interferometric methods and instruments in our arsenal that allow us to register the dynamics of molecular interactions in real time. With the help of such devices, optimal immunoreagents and thyroxine-biotin bifunctional ligands were selected, which, due to special spatially separating molecular "bridges", ensure good accessibility of both small molecules for effective interaction with two large molecules simultaneously: with a recognizing antibody and with streptavidin. This saved a lot of time when developing tests and greatly contributed to the success of the work."
The lead author of the study, Alexey Orlov, a researcher at IOF RAS and the Laboratory of Nanobiotechnology at MIPT, says: "We use magnetic particles as nanometers of immunochemical reactions, which are recorded quantitatively with record high sensitivity from the entire volume of the three-dimensional reaction zone on the test strip using a portable device, and not only from the membrane surface, as it happens for optical labels. This circumstance, among other factors mentioned, also ensures the simplicity of measuring ultra-low concentrations of low molecular weight substances in media of complex composition, which eliminates the need for complex sample preparation of samples and specially trained specialists to conduct unique sensitivity analyses."
According to the first author of the article Sergey Znoko: "The developed analysis is an express test with characteristics significantly exceeding the characteristics of existing laboratory diagnostic systems. Further expansion of the spectrum of biological molecules detected in this way will make it possible to implement multiparametric analysis of complex media with a significant reduction in its cost."
Graduate student of MIPT Natalia Guteneva, co-author of the article, states: "This approach is simple, affordable, adaptable for the detection of other small molecules, and we hope that it can be actively used to search for new markers of diseases, in medical diagnostics, environmental monitoring, food industry control, biosafety and other vital industries."
The work was supported by the Russian Science Foundation.
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