Biochips
A new method in the diagnosis of diseases
Dmitry Grydunov, Doctor of Biological Sciences, Chief Researcher, Deputy Director for Scientific Work of the V.A. Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (IMB RAS).
The main task of modern personalized medicine is to identify the risks of the disease in a particular person. This will help prevent diseases in advance and prescribe adequate treatment. You can examine the entire human body in detail to understand exactly what is affected in it, but this is an expensive process that takes a lot of time. That is why scientists have developed biochips that identify hundreds and thousands of biomarkers in one analysis – compounds indicating the presence of any disorder or susceptibility to the disease.
The history of the creation of biological microchips
The idea of developing biochips appeared in the late 1980s and belonged to several groups of scientists, including from the USSR, Yugoslavia and Great Britain. In the Soviet Union, Academician Andrey Darievich Mirzabekov, director of the V. A. Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, proposed the idea of biochips as an alternative to the then complex method of decoding DNA sequences. Mirzabekov postulated the possibility of placing short fragments of DNA probes on the flat surface of micro–matrices in three-dimensional hemispherical hydrogel cells. The cells with probes were placed on the substrates in a strictly ordered form. Since the localization of the probes in the cells is known in advance, the interaction of the probes with the analyzed DNA molecules made it possible to establish the structure of extended DNA sections. Thus, the theory of sequencing on DNA biochips was proposed and substantiated for the first time in the world.
How does the biochip work?
A biological microchip is a matrix of cells, each of which contains a specific probe (DNA molecule, protein, glycan, low molecular weight ligand, and so on) that recognizes its target in the analyzed sample, for example, in a drop of blood. Pretreated and labeled with a fluorescent dye, the molecules of the analyzed sample introduced into the biochip chamber are able to penetrate into the cells and interact with specific probes. When irradiated with light of a certain wavelength of the surface of the biochip, the cells in which a specific interaction occurred fluoresce. This fluorescence can be registered using a special analyzer-microscope. Then calculate the intensity of the signals in each cell and, knowing the signal processing algorithm, establish the presence of a specific target in the test sample – a microorganism, virus, mutation, chromosomal rearrangement, cancer marker, allergen, and so on. This process allows you to quickly choose an adequate treatment strategy for the patient.
The matrix of the biochip cells is made by transferring drops of a mixture of probes and hydrogel with a volume of 0.1 nanoliters from the wells of the microplate to a plastic substrate. This is done with the help of ultra-thin metal rods of a mechanical robot. Such droplets can move and merge on the hydrophobic surface of the substrate, so it is very important to fix them through a photopolymerization reaction with the formation of covalent bonds both between the droplet and the substrate with the active groups of the substrate, and between the probe molecules and hydrogel monomers. To do this, the substrates are irradiated with light with a certain wavelength under strict conditions: temperature, humidity, irradiation time, the presence of an inert gas displacing oxygen molecules. Thus, under the influence of ultraviolet radiation, the joint polymerization of molecular probes (DNA fragments, proteins, and so on) occurs with the main components of the hydrogel. During the reaction, the molecular probes covalently attach to the growing polymer chains and are evenly distributed throughout the gel volume, thereby immobilizing the probes.
Substrates and hydrogel for microchips
A matrix of biochip cells is formed on a glass coated with a monomolecular layer of a substance containing active chemical groups interacting with the applied probes. The technology of hydrogel biochips of IMB RAS uses plastic substrates, which significantly simplifies and reduces the cost of manufacturing matrices. Russian engineers have created molds for the production of substrates and reaction chambers of a biochip by injection molding, which ensures our technological independence in this area.
A key aspect of the domestic biochip technology is the use of a special hydrogel in which molecular probes are placed. The immobilization capacity of the probes in the hydrogel cells of the biochip increases by several orders of magnitude. This makes it possible to register signals in the chip elements that are 10-100 times higher than those during immobilization on flat glass matrices, and, accordingly, to create and use inexpensive biochip analyzers. The three-dimensional hydrogel cell provides a much greater specificity of the interaction between the analyzed molecule and the probe. This helps to detect clinically significant markers with very high specificity, giving the result in the "yes – no" format.
Ready-made biochip // Daniil Gurbanov for post-science
Specialization of biochips
The history of biochips began with the analysis of DNA sequences. Simultaneous reactions of interaction of the analyzed DNA molecules with immobilized DNA probes carried out in each cell provide parallel identification of multiple genomic targets. This makes it possible to use DNA biochips as an effective molecular tool for identifying clinically significant markers of pathogens and causes of socially significant diseases, monitoring food products, plant raw materials, possible agro- and natural biocenoses.
Biochips can also simultaneously contain cells with immobilized proteins or oligosaccharides. Moreover, both an individual molecular probe and their combinations can be immobilized in each individual cell, depending on the objectives of the experiment. Interaction between different classes of molecules can occur in the form of "receptor – ligand", "antigen – antibody", "enzyme – substrate" and so on. The immobilized ligand of protein or non-protein nature located in the cell forms a specific complex when interacting with a sample containing the analyzed compounds. At this stage, the analyzed compounds are separated from the mixture according to their ability to bind specifically to immobilized ligands. This makes it possible to perform simultaneous analysis of several biological objects on a single biochip, implementing the principle of multiplex immunoassay, based on the conduct of multiple reactions of the interaction "antigen – antibody".
Such a test is necessary for research in the field of proteomics, for the diagnosis of all types of diseases that are characterized by changes in a large number of indicators in the patient's blood serum. Personalized medicine implies a comprehensive examination of the patient, that is, the analysis of his biological fluids according to the maximum range of parameters.
Test systems based on biochips
30 years have passed since the first publication on the theory of the interaction of DNA molecules with probes mounted on a biochip substrate. During this period, the staff of the Laboratory of Biological microchips of the IMB RAS built a universal methodology for the analysis of protein and DNA markers on biochips. A production line for the production of hydrogel biochips with a capacity of up to 1 million per year was created and certified according to the international standard ISO 13485.
The developed diagnostic test systems based on biochips cover a wide range of applications - from identification of markers of drug resistance of microorganisms and viruses, identification of targets in the human genome associated with the risk and development of malignant tumors and the effectiveness of therapy, to multiplex immunoassay of protein markers in the patient's blood serum.
The first technique in the world developed for the needs of practical medicine and approved by the Federal Service for Supervision of Healthcare of the Russian Federation (Roszdravnadzor) for use in clinical practice was a set of reagents "TB-Biochip-1". It was created to identify the genetic markers of the causative agent of tuberculosis responsible for resistance to the first–line drugs of therapy - rifampicin and isoniazid.
Such biochips for the analysis of drug resistance of the causative agent of tuberculosis have been used since 2005 in more than 30 institutions of the anti-tuberculosis service of the Russian Federation and CIS countries, bacteriological laboratories of the Federal Penitentiary Service. During this period, more than 500 thousand analyses were performed and the diagnostic, economic and clinical effectiveness of biochips was proved, which allowed at least 3 times to increase the number of cured patients with drug-resistant forms of tuberculosis.
The latest generation of TB-biochips in the form of a set of "TB-TEST" provides simultaneous detection of more than a hundred markers of resistance of the causative agent of tuberculosis to topical drugs of the first and second series. This makes it possible to differentially prescribe different doses of chemotherapy drugs and transfer patients to treatment with the latest drugs. This is extremely important with the current extremely limited range of anti-tuberculosis drugs.
Biochips for the detection of hepatitis C and gonococcal infection
Another development – a unique way to identify varieties of hepatitis C virus – was created in joint research with the Laboratory of Virology at the University Hospital of Toulouse (France). The biochip allows you to choose the mode of antiviral therapy with direct-acting drugs, depending on the established type of virus.
Also, scientists of the IMB RAS created a biochip for the analysis of genetic markers of antibiotic resistance of microorganisms – pathogens of infections of human reproductive organs. This biochip has been an effective tool for monitoring the drug resistance of the causative agent of gonococcal infection since 2016. Its application made it possible for the first time to describe the molecular evolution of drug resistance in the modern gonococcus population in Russia and to fix the level of resistance at a record low level, unlike in the EU and the USA.
Determination of DNA mutations and somatic mutations
An important area of application of hydrogel biochip technology was the analysis of mutations and polymorphisms of human DNA associated with various diseases to determine the treatment strategy and the choice of therapy.
Thus, in order to detect genetic changes in blood cells that lead to the occurrence of leukemia, and to identify chromosomal rearrangements associated with certain types of acute and chronic leukemia (the most common childhood oncological diseases), a test system "LC-BIOCHIP" was developed. This test system has a high prognostic value and was used at the Dmitry Rogachev National Research and Practice Center for Pediatric Hematology, Oncology and Immunology in Moscow. Samples from 18 regional hematology centers of the Russian Federation were analyzed there.
Another test system, PF-BIOCHIP, created to determine DNA markers correlating with a predisposition to the development of cardiovascular pathologies during pregnancy, is used at the D. O. Ott Research Institute of Obstetrics and Gynecology in St. Petersburg.
Variants of test systems based on biochips are being developed to determine the sensitivity of malignant cells to antitumor therapy. For the individual selection of drugs acting on molecular targets in melanoma tumor cells, a biochip has been created that allows detecting mutations in the genes BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2, in which the use of targeted therapy drugs such as trametinib (protein kinase inhibitor MEK1/2) is indicated (or, conversely, useless). or MAP2K1/2), imatinib (KIT protein inhibitor) and vemurafenib (BRAF protein kinase inhibitor). This approach makes it possible to effectively identify clinically significant somatic mutations and make a choice in favor of a certain targeted drug in 70% of melanoma patients.
Biochips that detect the occurrence of allergies
Scientists from Germany have created a test system "ALLERGO-BIOCHIP" for parallel analysis of panels of specific immunoglobulins of classes E and G4 (SiGe and sIgG4) to allergens of the following classes: pollen (trees and shrubs), pollen (weeds and flowers), pollen (herbs and cereals), allergens of house dust and animals, epidermal, insect poisons, tick, food, fungal. Such a biochip quickly and effectively identifies the causes of allergies.
An epidemiological study was conducted, which included 800 patients of Filatov DSCB aged 0 to 16 years and 50 healthy donors as a model population of children in central Russia, in order to assess the frequency of occurrence of various causes of allergies, depending on age. It was found that among the inhaled allergens, birch pollen and cat epithelium most often cause sensitization, while among food allergens, the SiGe response is most often caused by egg and milk allergens. sIgG4 production is mainly caused by food allergens, especially egg white. With age, the proportion of patients with elevated levels of SiGe to inhaled allergens increases, while most food allergens are characterized by a decrease in the proportion of sensitized patients, with the exception of carrot, apple and peach allergens.
Prospects for the development of biochip technology
Multiplex immunoassay on hydrogel biochips makes it possible to cover almost all areas of medical diagnostics. A promising direction for the development of protein biochips is the study of markers of inflammatory reactions for the differential diagnosis of rheumatological, endocrine diseases and other disorders in the immune system.
Currently, more than two thousand patients of the Filatov DGKB have been examined using allergobiochips. In relation to pediatrics, in addition to the pronounced economic effect due to the "one sample – one analysis on a chip" format, this approach makes it possible to use only 100 ml of blood serum to detect an allergen that causes a pronounced reaction in a child. When examining young children, the use of such a small amount of sample is a significant advantage. Another promising direction is the expansion of the biochip for the analysis of predictive markers of oncological diseases. It is expected that the development of a new approach based on a comprehensive analysis of combinations of biomarkers – signatures – will solve this difficult task.
Due to the rapid development of polymerase chain reaction technologies and next-generation sequencing platforms, DNA biochips have faced serious competition. Today, they occupy an intermediate niche in the field of nucleic acid analysis techniques, supported by a variety of tests based on polymerase chain reaction, and are under increasing pressure from high-performance sequencing technologies. In the case of the IMB RAS technology, the immobilization of any types of biomolecules in a hydrogel and the possibility of carrying out various biochemical reactions in it opens up prospects for the creation of a new generation of biosensors. Hydrogel elements will become a platform for the immobilization of enzymes – genomic editors – nucleases together with guiding and detecting DNA or RNA molecules. This will make it possible to create highly sensitive biosensors, the use of which will be possible in the field. Such complex autonomous systems on the platform of hydrogel biochips will allow obtaining results faster, more informative and more accurate than now, and will play a key role in personalized medicine of the future.
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