Routes of biomedical scanning probe microscopy
• early detection of biological agents (viruses and bacteria) and various biological targets in air and biological fluids;
• determination of morphology and quality of human cells, development of a quantitative method for rapid cell analysis;
• creation of a bacterial cell atlas for diagnostic purposes with the use of scanning probe microscopy data;
• development of methods for detecting DNA of viruses and pathogenic bacteria by direct observation of hybridization on the surface of biochips using scanning probe microscopy;
• creation of methods for detection of viruses and pathogenic cells using affine surfaces.
Over the last decade, there has been a qualitative leap in the development of a number of methods of scanning probe microscopy, including scanning capillary microscopy [1, 2]. It is noted that capillary microscopy allows obtaining more accurate images of living cells, since there is no force interaction between the probe (capillary) and the cell membrane . At the same time, high-speed atomic force microscopy makes it possible to observe biological processes at the level of single biomacromolecules with high resolution and speed . In general, we can confidently state that modern models of scanning capillary and atomic force microscopes in the able hands are extremely useful tools for solving the problems of biology and medicine. For biomedical purposes, we developed the FemtoScan X high-speed scanning probe microscope, in which the feedback operates at a frequency of 1 MHz .
Scanning ion-conductance microscopy or scanning capillary microscopy opens new possibilities for medicine. In addition to observing high-resolution topography, scanning capillary microscopy can perform multifunctional analysis of living cells, including the observation of morphological transformations caused by physiological effects, the identification of intracellular signaling pathways and the characterization of mechanical responses, which demonstrates the versatility of the method. The use of multichannel nano-capillaries allows local delivery of chemicals (medical preparations) prior to direct contact with the cell. Such a nano-capillary can also be an electrochemical probe for determining various important parameters, for example, the concentration of reactive oxygen species near the surface of a living cell, and also inside it .
EARLY DETECTION OF INFLUENZA VIRUS BY SCANNING PROBE MICROSCOPY
Early detection of viruses and bacteria in the fluid can significantly reduce the risk of infectious diseases. This urgent and socially significant task is quite complex and requires innovative solutions.
We have developed a compact and inexpensive biosensor for detecting various biological targets – influenza A virus, microalbumin, prostate-specific antigen, etc. The key element of the biosensor is the cantilever biochip – piezoceramic disk, the surface of which is covered with biospecific sensory layers. The binding of the target to the surface of the biochip is determined from the change in the resonance frequency of the piezoceramic disk. As a result of the interaction of pathogens with the biochip receptor layer, the effective mass and rigidity of the latter will vary, which can be estimated from the shift of the cantilever resonant frequency:
where Δf, Δk, Δm are the changes in the resonance frequency, rigidity and mass of the biochip, and fn, k and m are the initial values of these parameters.
In the biosensor control unit, the electronic boards of the scanning probe microscope are used, including a digital frequency synthesizer, a precision amplifier of the input signal, an interface for communication with a computer unit, a thermostat of the flowing liquid cell, a DAC-ADC, a stabilized power supply, and a digital signal processor. A number of technical solutions including the symmetry of the design of the biochip and of the electronic circuit for supplying and removing the signal have made it possible to significantly improve the accuracy and reliability of measurements .
In the conducted experiments the sensitivity for detection of the influenza A virus at the level of 105 units in ml. was achieved . The possibility of detecting low concentrations of microalbumin in biological fluids using a piezoceramic biochip with sensory layers based on monoclonal antibodies was demonstrated .
The use of the original design of a flowing liquid cell and a piezoceramic biochip as a substrate in the scanning probe microscope allows real-time observation of adsorption of individual virus particles with simultaneous registration of the shift of the resonant frequency of mechanical oscillations of the biochip. With the help of such measurements it is possible to calibrate the biochip, which is necessary for practical applications in medicine. Accurate measurement of the size of virus particles can be performed using the previously developed nanometer gauge .
Since its development, atomic force microscopy has been used to implement various modes of nanolithography with the help of power, heat, or electrical impact. Scanning capillary microscopy has become an effective tool for directed mass transfer of individual biomacromolecules, nanoparticles, etc. . Multi-channel probes can be used for 3D printing of specified molecular configurations. The supply of macromolecules through the capillary is carried out in various ways, for example, by electrophoresis or by creating excess pressure in the capillary.
Two-dimensional lithography with fluorescent proteins was successfully performed on a scanning ion-conductance microscope with the use of a two-channel capillary in . In this way, a color miniature copy of the Degas "Dancers" was created. The size of the miniature corresponded to the diameter of the hair (about 50 microns).
Scanning capillary stereolithography has certain advantages over laser stereolithography, the spatial resolution of which is limited by the diffraction limit that is about half the wavelength of the laser radiation. Capillary microscopy achieves nanometer and subnanometer positioning accuracy. The diameter of the output capillary can vary from a few to hundreds of nanometers, which opens the possibility of creating a molecular 3D printer.
When manufacturing single-channel or multichannel nano-capillaries for stereolithography, glass microtubules made of quartz or borosilicate glass are used, which are widely used to solve electrophysiological problems.
It is possible to predict further expansion of the use of a scanning capillary microscope in biomedical applications and testing of drugs using only one cell, rather than their culture . Practical applications of scanning probe microscopy for molecular medicine problems, in particular, early detection of viruses in air and liquid media, are also rapidly and successfully developed. ■
The study was carried out with the financial support of the Russian Foundation for Basic Research (Projects 17-52-560001 and 16-29-06290).