Sensor technology of molecular diagnostics for personalized medicine
Joint efforts of physicians from many countries have been helped to achieve impressive results. The number of deaths from measles worldwide has decreased from 2.5 million in 1983 to 1.1 million in 1992, and from polio during the same period – from 360 thousand to 140 thousand. It has been expected that polio will eradicated from most countries by 1995, however, due to a substantial increase of immunization cost in South-East Asia this infection is not yet eliminated. Malaria continues to cause significant damage to the mankind, taking of life from 1 million to 2 million people every year.
Despite the improvement of living conditions in economically developed countries, widespread vaccination and effective antibiotics, infectious diseases occupy a significant share of morbidity and mortality, second only to diseases of the cardiovascular system and malignant cancer. In tropical developing countries due to poor sanitary conditions and malnutrition infectious diseases kill over 10 million people per year. The majority of deaths among children is caused by infectious diseases of the respiratory system and intestines. High immigration of people from third world countries has led in industrialized states to a sharp increase in the number of persons suffering from infectious diseases. Thus, new diseases are periodically identified.
The modern medical practitioner must be able to correctly determine the tactics of diagnosis, treatment and epidemic control measures. In this regard, the developments providing simplification and acceleration of virus diagnostics are topical, which will allow promptly to diagnose the patient and start the treatment.
The Advanced Technologies Center and Lomonosov Moscow State University implement a project, aimed at further development of highly sensitive physical methods of detection of virus particles in air and in liquids. The developed methods are based on the detection of viruses without the use of additional marks that can reduce the number of stages of sample preparation and total time for detection of infection .
Creation of high-tech production of biosensors for the early diagnosis will help to identify infection at an early stage in 5 minutes. The mobility of such sensors facilitates diagnostics and provides the development of self-diagnostic. In the future it will be possible using a high speed scanning probe microscope, which will adapted for different tasks, to identify a small gas leak, purity of drinking water, a mass of virus or bacterial cells, or even of a single atom, as well as to provide control of drugs, and to identify doping and antibodies.
Currently, laboratory diagnosis of viral infections is developing. The following methods are used for virus detection:
electron microscopy (fig.1), which provides a sensitivity about 1 million particles per ml., not always allows to type viruses, and also involves the use of expensive equipment;
reaction of immunofluorescence that can be successfully applied for direct detection of viruses only if clinical material contains a sufficiently large number of infected cells, and in the case of slight contamination by microorganisms that cause non-specific luminescence;
enzyme-linked immunosorbent assay, involving several stages of sample preparation;
radioimmunoassay analysis, requiring the use of radioactive substances and expensive gamma counters;
in real-time PCR, which provides high specificity of reactions due to the use of highly specific fluorescent probes and allows the quantitative analysis;
hemagglutination assay, which allows to evaluate the activity of the virus, but requires cell culture and erythrocytes.
These methods are developed, but their application in portable systems is difficult because they all require not compact laboratory equipment.
A large number of universities, research centers and commercial companies explores the problem of early detection of viral infection, because it is extremely important. The results of the systematic analysis of activity of the world centers in the field of diagnostics of viruses are presented on the website http://www.virology.net/garryfavweb.html featuring the list of more than 60 centers in the US, the UK, Germany, Russia and other countries. Following the motto "Turn a competitor into an effective partner", the authors have established scientific cooperation with the following domestic and international organizations: Virology Department at the Scottish Crop Research Institute, Dundee (Prof. M.Taliansky); Imperial Colledge London (Prof Yu. Korchev); University of Nebraska Medical Center (Prof. Yu. Lyubchenko); Center of Nanotechnology, Muenster (Prof. L. Heinrich); Institute of Pharmaceutical and Medicinal Chemistry, Muenster (Prof. Martina Duefer); Institute of Poliomyelitis and Viral Encephalitides, Moscow (Prof. A.Gambaryan); Research Institute of Influenza, St. Petersburg (Prof. A.Somina); Institute of Virology, Moscow (prof. Yu.Smirnov); the Department of Virology of Lomonosov Moscow State University (Academician I.Atabekov, Prof. O.Karpova); Department of Enzymology of Lomonosov Moscow State University (Academician A.Egorov); Institute of Biomedical Chemistry, Moscow (Academician A.Archakov, Prof. Yu.Ivanov); SRI of Physical-Chemical Medicine, Moscow (leading researcher D.Klinov); LG Electonics, Seoul, South Korea (Dr. K.Kwak); Korean Institute of Science and Technology, Seoul, South Korea (Dr. Sang Kyung Kim).
The authors develop the following methods of virus detection in air atmosphere, the aquatic environment and biological media:
scanning probe microscopy;
high-Q mechanical resonators;
modern high-sensitivity camera.
Scanning probe microscopy as a promising tool for direct visualization of virus sorption on sensor surfaces (fig.2). Besides, the measurement modes can be improved to reduce the impact of the probe on the investigated objects. Ultrafast scanning microscopy using video mode at the sampling rate of 1 MHz can significantly reduce measurement time. Probe microscopy is a promising metrological tool to control all stages of manufacturing of biosensor element, and also to evaluate the operability of this element in experimental measurements. An additional quality control of biosensor element is carried out using scanning and electron microscopy.
High-Q mechanical resonators are promising systems for the measurement of attached mass by virus adsorption, and for registration of changes in the surface hardness of the receptor layer during biospecific interaction between the virus hemagglutinin and sialic acids located on the surface of the biosensor element . The atomic scale is already developed, which allows in model experiments the registration of the attached mass in air (or vacuum) at the level of 10 ag. The mass of influenza A virus is about 500-100 times greater. However, the detection of viral particles must be carried out in liquids, which significantly (several orders of magnitude) reduces the sensitivity of the measurement of attached mass. As a result, required sensitivity is not achieved by existing geometry of the mechanical microresonator. To increase the sensitivity of the detection of viruses in liquid media, we have developed a solution for miniaturization of the microresonator with the excitation of longitudinal and/or surface modes of oscillations, which damping in liquid media is insignificantly .
Modern high-sensitivity cameras allow to record the Rayleigh scattering caused by single virus particles, which allows to monitor their position in space and to record the trajectory of their movement.
The authors have created a new laboratory device for medical diagnostics, which combines all three methods (scanning probe microscopy, microresonators registration and Rayleigh scattering from virus particles) in a single measurement system with the unique capability of multiparameter study of sorption kinetics of virus. ■
The authors thank the Russian Foundation for basic research for support (project 15-4-07678).