rus
Issue #4/2015
G.Kiselev, P.Gorelkin, A.Erofeev, D.Kolesov, I.Yaminsky
Rapid direct analysis of influenza A virus in a liquid medium may be performed using a piezoelectric cantilever with a glycopolymer-modified receptor containing sialic groups that are specific to the protein of the viral envelope.
Rapid direct analysis of influenza A virus in a liquid medium may be performed using a piezoelectric cantilever with a glycopolymer-modified receptor containing sialic groups that are specific to the protein of the viral envelope.
Rapid direct analysis of influenza A virus in a liquid medium may be performed using a piezoelectric cantilever with a glycopolymer-modified receptor containing sialic groups that are specific to the protein of the viral envelope.
Modern sensor systems must meet extremely high requirements. Their critical characteristics include such parameters as a detection threshold, linear size of the sensor and speed of assays. In all the variety of sensors, we make a special note of the rapidly growing subclass of microelectromechanical systems (MEMS) with different kinds of resonators, in which nano-amplitude vibrations occur due to the alternating electromagnetic field. The resonance in such systems directly depends on their geometric topology. Essentially, MEMS are pendulums, springs, strings or membranes with their specific frequencies at different harmonics.
A good example of special application of the quartz resonator fork, which is well-known from the clock mechanisms, to obtain a scientific result is the experiment carried out under the guidance of Franz Giessibl [1]. The nano-vibrations occurring in a quartz fork, which were used for setting a reference frequency, appeared very useful for the employees of the University of Augsburg. They proposed using the fork as a tunneling microscope by attaching a piece of platinum wire to its end. The needle made from the wire served as a probe of the surface of the mono-crystal silicon (fig.1). Due to the high quality factor of the quartz resonator, the atomic structure of the silicon was determined with a precision of a few fractions of an angstrom.
MEMS in the control
of bacteria and viruses
Increasingly, attempts are being made to use MEMS as sensors for bacteria and viruses. The latter ones are quite well characterized by various methods of microscopy, PCR, and Raman spectrometers. However, applying these methods in everyday life and integrating them into small appliances is practically impossible. MEMS in this regard have unsurpassed advantages: they have a small size and a simple system of reading an electrical signal from the sensor with ordinary electronic components.
An extremely relevant use of micromechanical sensors could be controlling bacteria and viruses that cause common diseases, such as influenza and pneumonia.
The current technologies allow the creation of MEMS that can measure individual viral particles with a sensitivity of 10–19 g/Hz [2] (fig.2a). The study [3] experimentally demonstrated the measurement of a virus mass of the vaccinia virus with a weight of 9.5 femtograms using a cantilever type resonator with a 1.8 μm width and a 4 μm length (fig.2b).
In the past decade, a critical breakthrough was made in the design of micromechanical sensor systems with the highest levels of sensitivity by the mass [2] and amount of the attached analyte [4]. These systems use a fundamentally new method of converting biochemical reactions into analytical signals through a static deformation resulting from thermal, electrostatic and entropic effects inside the receptor layer and the MEMS structures. With a wide range of operating modes (static, dynamic, control of the Q-factor and amplitude of the analytical signal), MEMS are an excellent complement for electrochemical, optical and acoustic sensors. Besides, the combinations of various schemes for recording the analytical signal help to optimize the performance of MEMS in practically any environment [5]: vacuum, gas and liquid phases of a substance. The microscopic size of sensing devices allows lowering the threshold of their sensitivity to the size of an individual bacteria [6] and a viral particle [2, 3].
Numerous publications describe the use of various resonant sensors for virus detection. The most common sensors are quartz crystal based microbalances [7, 8, 9] and sensors based on the effect of surface acoustic waves in a piezoelectric crystal [10]. In new developments, it is also proposed to use capacitive micromechanical ultrasonic transducers [11] and membrane piezo-acoustic resonators [12]. All these methods are effective and, as a rule, are related to controlling the resonant frequency of MEMS. In the simplest case, MEMS follow the harmonic oscillator formula, in which the reduction of the system’s own frequency is proportional to the square root of the attached mass. However, in some cases, the sensitivity of the MEMS depends not only on the effect of the attached mass, but also on the strain occurring in the receptor of the sensor with the attachment of the analyte. Strains result in increasing stiffness of the system and can be used alternatively to attaching masses for recording resonant frequency changes [13].
Analysis of viral particles using a piezoelectric cantilever
The joint laboratory of LG Electronics and Lomonosov Moscow State University carried out experiments for analysis of viral particles in a liquid medium by means of a piezoelectric cantilever (fig.3).
A 2 × 3 mm cantilever was cut out with the help of a diamond disc of a composite membrane consisting of a 60-micron layer of brass and 50-micron layer of PZT-ceramics covered with silver foil. The cantilever was placed for two minutes in a solution of sulfuric acid in a 37% solution of hydrogen peroxide 1 : 1, washed in 98% ethanol and was incubated for 16 hours in a solution of 4-aminothiophenol 10-3 M in ethanol. After the incubation and sequential rinsing in ethanol and in water, it was placed into an aqueous solution of sialic glycopolymer for 12 hours. Then, the surface of the receptor developed a film containing glycopolymers groups that are specific to the hemagglutinin, i.e. the protein of the influenza A viral envelope A/Duck/Moscow/4182/2008 (Chumakov Institute of Poliomyelitis and Viral Encephalitides of RAMS, Moscow), which was used in this work.
The influenza A virus was obtained by a 10-day infection of a chicken embryo in the egg with further extraction of allantoic fluid from the egg. The initial concentration of the virus in the allantoic fluid was about 1•108 virions/ml. The test solution with a concentration of 1•106 virions/ml was prepared by 100-fold diluting of the original solution in an uninfected allantoic fluid.
The measurements were carried out in a longitudinal vibration mode of the cantilever in allantoic fluid. The longitudinal mode, in contrast to the bending mode, has a high quality factor in a liquid due to the minimal friction of the cantilever in the medium. The frequency of the longitudinal mode (fig.4) was determined using an electronic unit of the FemtoScan atomic force microscope (Advanced Technologies Center, Moscow, www.nanoscopy.ru).
Before measurement, the cantilever was incubated in a flowing virus free allantoic fluid. After the mean drift (Δf/f) was set at 0.5⋅10-3, the system was injected a solution of viral particles with a concentration of 1⋅106 virions/ml.
It is known that the interaction between the virus and the receptor layer on the sensor surface causes additional strain in the film [14] or an increased effective stiffness of the whole system [13]. This results in an increased resonance frequency. By the moment, we have received a positive change in the resonant frequency of the cantilever during the sorption of viral particles on its surface from the solution with a concentration of 106 virion/ml (fig.5). The signal/noise ratio was 5:1, which means high prospects of using piezoelectric cantilevers in such applications.
Thus, we can conclude that the future development of nanotechnological biosensors measuring masses of individual virus particles and the small stress changes in molecular films can be based on microelectromechanical systems that already demonstrate good sensitivity, compactness and simplicity of the direct analysis. ■
The study was conducted in the joint laboratory of LG Electronics and Lomonosov Moscow State University in the framework of agreements No. JM-02/2014 and No. JY-01/2014. The authors thank the employees of the Chumakov Institute of Poliomyelitis and Viral Encephalitides of RAMS, A.Ghambaryan and A.Tuzikov, as well as N.Bovin (IBCh of RAS), for the preparation of the virus and the synthesis of glycopolymer. Additionally, the authors thank K.Kvak and I.Borodina (LG Electronics).
A good example of special application of the quartz resonator fork, which is well-known from the clock mechanisms, to obtain a scientific result is the experiment carried out under the guidance of Franz Giessibl [1]. The nano-vibrations occurring in a quartz fork, which were used for setting a reference frequency, appeared very useful for the employees of the University of Augsburg. They proposed using the fork as a tunneling microscope by attaching a piece of platinum wire to its end. The needle made from the wire served as a probe of the surface of the mono-crystal silicon (fig.1). Due to the high quality factor of the quartz resonator, the atomic structure of the silicon was determined with a precision of a few fractions of an angstrom.
MEMS in the control
of bacteria and viruses
Increasingly, attempts are being made to use MEMS as sensors for bacteria and viruses. The latter ones are quite well characterized by various methods of microscopy, PCR, and Raman spectrometers. However, applying these methods in everyday life and integrating them into small appliances is practically impossible. MEMS in this regard have unsurpassed advantages: they have a small size and a simple system of reading an electrical signal from the sensor with ordinary electronic components.
An extremely relevant use of micromechanical sensors could be controlling bacteria and viruses that cause common diseases, such as influenza and pneumonia.
The current technologies allow the creation of MEMS that can measure individual viral particles with a sensitivity of 10–19 g/Hz [2] (fig.2a). The study [3] experimentally demonstrated the measurement of a virus mass of the vaccinia virus with a weight of 9.5 femtograms using a cantilever type resonator with a 1.8 μm width and a 4 μm length (fig.2b).
In the past decade, a critical breakthrough was made in the design of micromechanical sensor systems with the highest levels of sensitivity by the mass [2] and amount of the attached analyte [4]. These systems use a fundamentally new method of converting biochemical reactions into analytical signals through a static deformation resulting from thermal, electrostatic and entropic effects inside the receptor layer and the MEMS structures. With a wide range of operating modes (static, dynamic, control of the Q-factor and amplitude of the analytical signal), MEMS are an excellent complement for electrochemical, optical and acoustic sensors. Besides, the combinations of various schemes for recording the analytical signal help to optimize the performance of MEMS in practically any environment [5]: vacuum, gas and liquid phases of a substance. The microscopic size of sensing devices allows lowering the threshold of their sensitivity to the size of an individual bacteria [6] and a viral particle [2, 3].
Numerous publications describe the use of various resonant sensors for virus detection. The most common sensors are quartz crystal based microbalances [7, 8, 9] and sensors based on the effect of surface acoustic waves in a piezoelectric crystal [10]. In new developments, it is also proposed to use capacitive micromechanical ultrasonic transducers [11] and membrane piezo-acoustic resonators [12]. All these methods are effective and, as a rule, are related to controlling the resonant frequency of MEMS. In the simplest case, MEMS follow the harmonic oscillator formula, in which the reduction of the system’s own frequency is proportional to the square root of the attached mass. However, in some cases, the sensitivity of the MEMS depends not only on the effect of the attached mass, but also on the strain occurring in the receptor of the sensor with the attachment of the analyte. Strains result in increasing stiffness of the system and can be used alternatively to attaching masses for recording resonant frequency changes [13].
Analysis of viral particles using a piezoelectric cantilever
The joint laboratory of LG Electronics and Lomonosov Moscow State University carried out experiments for analysis of viral particles in a liquid medium by means of a piezoelectric cantilever (fig.3).
A 2 × 3 mm cantilever was cut out with the help of a diamond disc of a composite membrane consisting of a 60-micron layer of brass and 50-micron layer of PZT-ceramics covered with silver foil. The cantilever was placed for two minutes in a solution of sulfuric acid in a 37% solution of hydrogen peroxide 1 : 1, washed in 98% ethanol and was incubated for 16 hours in a solution of 4-aminothiophenol 10-3 M in ethanol. After the incubation and sequential rinsing in ethanol and in water, it was placed into an aqueous solution of sialic glycopolymer for 12 hours. Then, the surface of the receptor developed a film containing glycopolymers groups that are specific to the hemagglutinin, i.e. the protein of the influenza A viral envelope A/Duck/Moscow/4182/2008 (Chumakov Institute of Poliomyelitis and Viral Encephalitides of RAMS, Moscow), which was used in this work.
The influenza A virus was obtained by a 10-day infection of a chicken embryo in the egg with further extraction of allantoic fluid from the egg. The initial concentration of the virus in the allantoic fluid was about 1•108 virions/ml. The test solution with a concentration of 1•106 virions/ml was prepared by 100-fold diluting of the original solution in an uninfected allantoic fluid.
The measurements were carried out in a longitudinal vibration mode of the cantilever in allantoic fluid. The longitudinal mode, in contrast to the bending mode, has a high quality factor in a liquid due to the minimal friction of the cantilever in the medium. The frequency of the longitudinal mode (fig.4) was determined using an electronic unit of the FemtoScan atomic force microscope (Advanced Technologies Center, Moscow, www.nanoscopy.ru).
Before measurement, the cantilever was incubated in a flowing virus free allantoic fluid. After the mean drift (Δf/f) was set at 0.5⋅10-3, the system was injected a solution of viral particles with a concentration of 1⋅106 virions/ml.
It is known that the interaction between the virus and the receptor layer on the sensor surface causes additional strain in the film [14] or an increased effective stiffness of the whole system [13]. This results in an increased resonance frequency. By the moment, we have received a positive change in the resonant frequency of the cantilever during the sorption of viral particles on its surface from the solution with a concentration of 106 virion/ml (fig.5). The signal/noise ratio was 5:1, which means high prospects of using piezoelectric cantilevers in such applications.
Thus, we can conclude that the future development of nanotechnological biosensors measuring masses of individual virus particles and the small stress changes in molecular films can be based on microelectromechanical systems that already demonstrate good sensitivity, compactness and simplicity of the direct analysis. ■
The study was conducted in the joint laboratory of LG Electronics and Lomonosov Moscow State University in the framework of agreements No. JM-02/2014 and No. JY-01/2014. The authors thank the employees of the Chumakov Institute of Poliomyelitis and Viral Encephalitides of RAMS, A.Ghambaryan and A.Tuzikov, as well as N.Bovin (IBCh of RAS), for the preparation of the virus and the synthesis of glycopolymer. Additionally, the authors thank K.Kvak and I.Borodina (LG Electronics).
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