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Bioobjects Detection by Scanning Probe

#1734


Development of an Original, High-Sensitive, Express Method for Biological Objects Identification and Control in Water Environment Using Scanning Probe

Tech Area / Field

  • INS-DET/Detection Devices/Instrumentation
  • ENV-WPC/Water Pollution and Control/Environment
  • BIO-MIB/Microbiology/Biotechnology

Status
8 Project completed

Registration date
20.12.1999

Completion date
10.02.2004

Senior Project Manager
Mitina L M

Leading Institute
ITEF (ITEP), Russia, Moscow

Supporting institutes

  • State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk

Collaborators

  • Institute for fundamental research of organic chemistry, Japan, Fukuoka\nBiochemical Institute University of Lisbon Medical Scool, Portugal, Lisbon\nProcter & Gamble Company, USA, OH, Cincinnati

Project summary

This project is devoted to the use of up-to-date ultramicroscopic methods-scanning tunnel and atomic-force microscopy for the study of human viruses and bacterial contaminants in water environments, including drinking water, where the presence of these morbific agents is regularly acknowledged by the investigations, which are using culture of tissue methods. These agents can provoke such dangerous diseases as gastroenteritis, hepatitis A and B, poliomyelitis, serous meningitis, colitis.

Project task: The task of this project is to develop methods of human nosogenic viruses and bacterial contaminants revealing, identification and quantitative calculation in water objects, having different degree of pollution. This express, highsensitive method should allow to reveal single virions and bacterias in large water volumes, which is a typical situation for environmental samples and especially for drinking water.

The proposed method, compared to traditional methods (on a culture of tissue by coliphage content), has the following advantages:


· Analyses time is less (expelling the stage of large water volumes concentration).
· The analyses is highly specific so long as specific antibodies to viruses are used.
· The used equipment is very compact (the sizes of STM/AFM are not greater than those of a light microscope).
· There is a possibility of automatic virus concentration analyses in a sample.

The most suitable application of this method is the use of the method:


· for virusological and bacterialogical water quality control on city water supply stations;
· in sanitary-epidemiological labs;
· for the study of widely spread viruses of different types in lake, pond, river and sea water in different regions.

In order to realize this task, a group of scientists from State Science Center of the Russian Federation-Institute of theoretic and experimental physics,actively using up-to-date ultra microscopic methods (atomic probe microscopy, autoion, scanning tunnel and atomic force microscopy which provide atomic resolution) to investigate radiation effects, proposed to use scanning probe microscopy (tunnel and atomic force) in order to analyze viruses in water. In collaboration with scientists, belonging to a number of other organizations (Lomonosov Moscow State University, Sisin Human Ecology and Environmental Hygiene Institute (RAS), Chumakov poliomyelitis and virus encephalitis Institute).

During the recent years, the basics of original methods have been developed by them as well as the prototypes of experimental plants, realizing these methods and serving for identification and virus presence control in water, with sensitiveness of 0.1 virus per liter, have been created. The difficulty connected with the identification of human viruses is that many of them have close morphological characteristics. The problem of identification is also complicated by the fact that almost every kind of virus has a number of serotypes. The problem connected with the identification of viruses and bacterias which are homogeneous by morphology can be solved by means of their immune fixation by a film of specific antibodies right on the substrate of a microscope. For the deposition of a monolayer film on the substrate Lengmur-Blodget technology, which will allow to get well-ordered monomolecul albumen layers and steady virus distribution on a substrate, will be used. Among the project tasks is the task of experimental determination of the adequate procedure of the immobilization of specific albumens on a substrate in order to get a monolayer of active antibodies, which are able to fix the corresponding viruses 7 types of human viruses and 2 types of bacterias will be used.

Antibodies films on the surface of a water subphase in Lengmur-Blodget trough are supposed to be manufactured in combination with amphiphyl polymers and in pure state.

The problem of experimental search for optimal modes of interaction between viruses and antibodies in reaction antigen-antibody, while depositing viruses or bacterias on a substrate, cultivated by albumens, is directly connected with this task. The investigation of the possibility of virus observation by means of scanning tunnel microscopy, performed before by the authors, showed that because of the considerable force, appearing in tunnel contact, biomolecul deformation is possible. This leads to the image smearing and makes it impossible to determine exact sizes of the biological object, being observed. An optimal mode for the preparation of viruscontaining preparations for observation is the covering of the preparation by a thin (2-5 nm) film of electroconducting material, which gives conductivity and stiffness to the surface of a preparation. The preparations will be also investigated in atomic-force mode.

Since virus identification is performed by means of their immune fixation by a film of specific antibodies, the microscope resolution of 1 nm is allowed, while virus identification program should separate only those particles, found on the microscope substrate which considerably differ from each other by morphology.

Separate problem, whose solution is necessary for project realization is the preparation of viruscontainig sample from the water. Since the content of viruses in lakes and rivers, which are the sources for getting drinking water, and especially in drinking water, is very low, it is necessary to concentrate large water volumes. Up-to-date experimental methods of virus biochemistry will be used in order to realize this task. It will be performed in two stages: ultrafiltration on electropositive adsorbent filters and ultracentrifugation.

Technical approach and methodology: Special attention will be paid to the development and realization of sample preparation technology for the needs of scanning tunnel and atomic-force microscopy. The methods of ultramicroscopic analyses will be essentially updated and adapted for the concrete investigations, being planned.

For the deposition of albumens, which are the antibodies to the analyzed object, lengmur film deposition technology for solid surfaces will be applied. Bacterias and viruses binding to specific antibodies, immobilized in the film on a substrate, is antigen-antibody reaction in solid phase. When viruses will be concentrated in a water sample of a large volume, ultrafiltration and ultracentrifugation methods will be used.

Specialists, who developed biological and nuclear arms, corresponding materials and those who tested special military equipment are invited to take part in this project.

Meeting ISTC Goals and Objectives: as a result of the project scientific and technical personnel, which was occupied by weapon production and testing, will be orientated on a peaceful activity; the method of water environment pollution control (by biological agents: nosogenic viruses and bacterias) will be developed. The use of this method concerns provision of country population (population of Russia) by ecologically safe water.

Partnership of Foreign Collaborators: the role of foreign collaborators in the project-joint forming, discussion and correction of intermediate and final results, some joint investigations and developments, marketing assistance, possible subsequent joint use of developed technologies and of working prototypes of scanning probe microscopes on sanitary-epidemiological stations.


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