rus
Issue #3-4/2020
I.V.Yaminskiy, А.I.Аkhmetova, M.A.Pavlova
Early detection of viral infections using solid-state piezoelectric ceramic biosensors
Early detection of viral infections using solid-state piezoelectric ceramic biosensors
DOI: 10.22184/1993-8578.2020.13.3-4.188.195
The early detection of viral pathogens is a fundamental goal of modern biomedicine. An effective solution to this problem is especially in demand in the context of the current global coronavirus pandemic. At the same time, and upon its completion, the development of new and each time more effective biosensors for viral infections will continue in an intensive manner. Viruses are human companions in the past, present and future.
The early detection of viral pathogens is a fundamental goal of modern biomedicine. An effective solution to this problem is especially in demand in the context of the current global coronavirus pandemic. At the same time, and upon its completion, the development of new and each time more effective biosensors for viral infections will continue in an intensive manner. Viruses are human companions in the past, present and future.
Теги: biochips biosensors influenza a virus sars-cov-2 coronavirus scanning probe microscopy tick-borne encephalitis virus биосенсоры биочипы вирус гриппа а вирус клещевого энцефалита коронавирус sars-cov-2 сканирующая зондовая микроскопия
Early detection of viral infections using solid-state piezoelectric ceramic biosensors
Detecting of viruses has two important aspects. The first one is related to detection of a pathogenic agent in the environment at the moment when viral infection has not yet occurred. It is very particular and vitally important goal of preventive medicine. In this case prevention of the disease is cheaper than its subsequent treatment. The second aspect suggests detection of viral agents after infection contamination.
The approaches to these two cases have their own peculiarities. In the early warning of a viral disease, the main targets of the search are the viral particles themselves in air and / or liquid media. The main tasks are to search, concentrate and identify viruses. Thus, a recording device should be compact, mobile, portable, energy efficient, with high sensitivity and selectivity, simple in operation, use and maintenance, and able to present the analysis result in a short period of time. The price of a biosensor is also not the last criterion. Low cost ensures its availability and mass application.
If it is necessary to identify a cause of the disease, the variants of detectable targets can be different: both the virus particles themselves (similar to the first case), and antibodies to the virus produced by the immune system of an infected person. Detecting damage caused by a virus can also be an alternative way to identify a disease. For example, with coronavirus infections, a reliable diagnostic method is computed tomography, which allows you to see damage to lung tissue or other organs. The presence of a biosensor or device that is capable to record the nature of the effect on human tissue is also a highly demanded need of modern medicine.
Although there are a large number of approaches to creating compact and mobile biosensors without the use of labels, only a few technologies promise to achieve the level of detection of a single virus in a liquid medium. Among these technologies, the following should be mentioned: high-Q optical resonators [1–3], plasmon resonance sensors [4], nanomechanical resonators, and nanowire biosensors [5].
Among electromechanical biosensors, quartz microbalance technology is widely used [6]. In these sensors, the resonance frequency of the quartz plate is measured with a change in the attached mass. One side of a quartz plate faces a liquid and performs shear oscillations. As a result, the sensitivity to changes in the elastic properties of the surface film is insignificant, since the film does not undergo tensile-compression cycles of a sufficient level during measurement. However, it often turns out that it is the change in the elastic properties of the probe-target film that plays a decisive role in the formation of the recorded signal. With all its advantages, the method of quartz microbalance, is not possible to achieve miniaturization of the device.
An increase in sensitivity by six orders of magnitude compared with the best examples of commercial quartz scales is achieved in a cantilever biosensor with an internal channel and a total mass of about 100 ng [7]. Cantilever biosensors [8, 9] present a highly sensitive platform for detecting viruses. In laboratory experiments sensitivity was achieved in detecting a single virus [10] and even a single atom [11]. A disadvantage of cantilever systems is the technological difficulties in the mass production implementation. While preserving high sensitivity the proposed solution involves the use of planar technology for manufacture of a biochip. This yields significant advantages in mass replication. In this case, synthesis of the receptor surface can occur immediately on a large plane of the workpiece followed by its subsequent separation into individual microchips.
Optical biosensors represent a widespread type of biosensors. An optical signal of high sensitivity, resistance to external noise, stability and low noise present advantages over other physical signals. However, presence of the diffraction limit does not allow the optical method to record a signal from the nanometer region to detect single proteins or antibodies. Recently, a group of American scientists developed an original femtosecond adaptive spectroscopic method with improved resolution due to coherent anti-Stokes Raman spectroscopy (FASTER CARS), using signal amplification with the aid of a probe (tip enhanced Raman spectroscopy). As a result, single viral particles can be detected [12].
Transmission electron microscopy (TEM) and atomic force microscopy make it possible to successfully visualize single viral particles. Moreover, various methods of contrasting make it possible to see the structural elements of the virus envelope in the TEM. With all the advantages, these methods are unlikely to be compact, mobile and inexpensive. Although in the field of atomic force microscopy there are options for devices (without taking into account the dimensions of the electronics) of a millimeter size, they remain complicated and expensive as before.
Electrochemical biosensors are based on the entry into the electrochemical reaction of a biological material with an analyte [13]. The main categories of electrochemical biosensors are potentiometric, amperometric and impedimetric converters. Electrochemical biosensors have several advantages in terms of sensitivity and selectivity. They can be mobile and inexpensive.
There are, for example, many variations of commercial amperometric biosensors. Glucose biosensors are best known and widely available, for example: SIRE P201 (Chemel AB, Lund, Sweden), the blood glucose monitoring system FreeStyle Freedom, Precision Xtra (Abbot Diabetes Care, Alameda, California, USA) and GlucoWatch Biographer (Cygnus, Redwood City, California, USA). The Midas Pro device (Biosensori SpA, Milan, Italy) is widely used for surface water analysis.
Currently, reverse transcription PCR analysis on SARS-CoV-2 can only be done in the laboratory. It requires a set of reagents and laboratory equipment. A test of the best systems is able to detect 0.3–3 copies of viral RNA in a microliter (0.001 ml). Testing time is a few hours. Now they are striving to further reduce the analysis time.
Piezoceramic biosensors appeared not so long ago, but they have already shown high sensitivity when detecting the epidermal growth factor receptor (Her2) [14], the virus (WSSV) of plants [15], bacterial spores (Bacillus anthracis) [16], DNA [17] and influenza A virus [18].
In our work on the development of a piezoceramic biosensor for detecting viral infections, we record the probe-target binding by changing characteristics of the biochip oscillations. This solution does not involve the use of additional labels and chemicals. The selective sensor system is completely located on the surface of the biochip, therefore, the need to use additional chemical and biological reagents: DNA primers, TAG polymerase, various labels, etc., disappears, in contrast to PCR diagnostics and enzyme immunoassay.
We selected influenza A virus (Fig.1), tick-borne encephalitis virus (Fig.2), and coronavirus as targets for testing the biosensor. The choice of targets is due primarily to the urgent need for active opposition to these viral infections. Influenza A virus is a constant seasonal disease, tick-borne encephalitis virus has become widely spread over the territory of the Russian Federation over the last short period of time, SARC-CoV-2 coronavirus is the cause of a modern pandemic.
The proposed biosensor solution consists of a piezoceramic biochip in the form of a 0.1–3 mm dia. disk. The symmetric three-electrode design eliminates the influence of a double electric layer and parasitic diffusion processes on the surface of the biochip (Patent for invention No. 2636048, Biosensor device for detecting biological micro- and nano-objects, 2017). In this biochip design, the potential is supplied to the central electrode, while the external electrodes facing the solution can be grounded or kept at the potential of the solution. In this case, unlike the known structures, there is no need for an additional layer insulating the external electrodes. The use of periodic and pulsed modes of excitation of oscillations allows, due to the use of original algorithms, to achieve high sensitivity when detecting the influenza A virus [18].
The measuring electronic system is implemented using FPGA (Field-Programmable Gate Array) technologies or its further development in the form of ASIC (Application Specific Integrated Circuit). It is planned to adapt the existing electronic system for collecting information from a FemtoScan scanning probe microscope for conducting both resonant measurements and processing of electrochemical signals. The signal from the piezoceramic biosenosor arrives at the frequency of the mechanical resonance of the biochip, as a rule, this is the high-frequency range from hundreds of kHz to hundreds of MHz. The signal from the electrochemical biosensor is located in the range of significantly lower frequencies. This allows you to synchronously register signals from electrochemical and piezoelectric ceramic biosensors using just electronic means. Moreover, it is possible to combine two biosensors – piezoceramic and electrochemical – in one biosensor, while two signals will come together from the same biochip.
The liquid system can be made either with microfluidic technology or with the use of a millimeter-sized tank. Antibodies, aptamers and / or synthetic receptors can be used as probes. In the case of the SARC-CoV-2 coronavirus, its virus can attach to ACE2 protein of the epithelial cells upon infection. Moreover, the binding constant has an unusually high value. When testing, it is possible to use the recombinant water-soluble protein ACE2, available on the market of bioreagents.
The preliminary calculated value of the biosensor sensitivity to SARC-CoV-2 is 1–100 viral particles depending on the size of the biochip area (0.1–3 mm). For influenza A virus and tick-borne encephalitis virus, the sensitivity is slightly worse, since the binding constant of the currently available receptors is 3–10 times less.
Using the biosensor of the proposed design, it is possible to record not only the act of binding, but also the moment of detachment of a viral particle with an increase in the amplitude of mechanical vibrations of the biochip to threshold values when the mechanical energy of the vibrating viral particle becomes greater than the binding energy. At the moment of separation, a separation signal is given and the electronic measuring system records it. Thus, the biosensor will be able to detect both binding of the virus and its detachment.
It should be noted that nonspecific binding in liquid in the presence of various impurities deposited on the surface of the biochip does not lead to a significant change in the mechanical rigidity of the surface layer, and does not affect the separation signal of the particle at large vibration amplitude.
Subsequent characterization of the virus in a sufficiently large quantity can be carried out by chromatography, transmission electron microscopy and atomic force microscopy. For data processing, it is advisable to use the FemtoScan Online software developed by us [19].
In contrast to quartz microbalance, longitudinal vibrations are excited in the biochip, which lead to stretching / compression of the sensor film with attached viral particles or biomacromolecules.
The measurement of film stiffness is 300–1000 times more sensitive than simply determining the attached mass (as in the quartz microbalance method). Curiously, this experimental fact was noticed in the 18th century by Benjamin Franklin. Traveling from the New to the Old World on a whaling ship, Benjamin Franklin noticed that during the storm the captain ordered that a barrel of whale oil be poured into the sea. After that, the waves near the ship calmed down. The modern explanation of this effect is quite simple: a monolayer film of fat is formed on the surface of water. A lot of mechanical energy is expended on its compression and expansion during a storm; as a result, the waves attenuate. A film of fat converts mechanical energy into thermal energy transmitting it to the sea wave. Surprisingly, the calculation shows that a film with a thickness of nanometers calms meter high waves on the water.
Creating an "ideal" biosensor for viral infections is a noble task though a difficult one. On this path, new ideas and solutions will lead to continuous progress. The combined efforts of specialists from different fields of knowledge will undoubtedly contribute to moving forward. On our way, we will be glad to have new companions. ■
Detecting of viruses has two important aspects. The first one is related to detection of a pathogenic agent in the environment at the moment when viral infection has not yet occurred. It is very particular and vitally important goal of preventive medicine. In this case prevention of the disease is cheaper than its subsequent treatment. The second aspect suggests detection of viral agents after infection contamination.
The approaches to these two cases have their own peculiarities. In the early warning of a viral disease, the main targets of the search are the viral particles themselves in air and / or liquid media. The main tasks are to search, concentrate and identify viruses. Thus, a recording device should be compact, mobile, portable, energy efficient, with high sensitivity and selectivity, simple in operation, use and maintenance, and able to present the analysis result in a short period of time. The price of a biosensor is also not the last criterion. Low cost ensures its availability and mass application.
If it is necessary to identify a cause of the disease, the variants of detectable targets can be different: both the virus particles themselves (similar to the first case), and antibodies to the virus produced by the immune system of an infected person. Detecting damage caused by a virus can also be an alternative way to identify a disease. For example, with coronavirus infections, a reliable diagnostic method is computed tomography, which allows you to see damage to lung tissue or other organs. The presence of a biosensor or device that is capable to record the nature of the effect on human tissue is also a highly demanded need of modern medicine.
Although there are a large number of approaches to creating compact and mobile biosensors without the use of labels, only a few technologies promise to achieve the level of detection of a single virus in a liquid medium. Among these technologies, the following should be mentioned: high-Q optical resonators [1–3], plasmon resonance sensors [4], nanomechanical resonators, and nanowire biosensors [5].
Among electromechanical biosensors, quartz microbalance technology is widely used [6]. In these sensors, the resonance frequency of the quartz plate is measured with a change in the attached mass. One side of a quartz plate faces a liquid and performs shear oscillations. As a result, the sensitivity to changes in the elastic properties of the surface film is insignificant, since the film does not undergo tensile-compression cycles of a sufficient level during measurement. However, it often turns out that it is the change in the elastic properties of the probe-target film that plays a decisive role in the formation of the recorded signal. With all its advantages, the method of quartz microbalance, is not possible to achieve miniaturization of the device.
An increase in sensitivity by six orders of magnitude compared with the best examples of commercial quartz scales is achieved in a cantilever biosensor with an internal channel and a total mass of about 100 ng [7]. Cantilever biosensors [8, 9] present a highly sensitive platform for detecting viruses. In laboratory experiments sensitivity was achieved in detecting a single virus [10] and even a single atom [11]. A disadvantage of cantilever systems is the technological difficulties in the mass production implementation. While preserving high sensitivity the proposed solution involves the use of planar technology for manufacture of a biochip. This yields significant advantages in mass replication. In this case, synthesis of the receptor surface can occur immediately on a large plane of the workpiece followed by its subsequent separation into individual microchips.
Optical biosensors represent a widespread type of biosensors. An optical signal of high sensitivity, resistance to external noise, stability and low noise present advantages over other physical signals. However, presence of the diffraction limit does not allow the optical method to record a signal from the nanometer region to detect single proteins or antibodies. Recently, a group of American scientists developed an original femtosecond adaptive spectroscopic method with improved resolution due to coherent anti-Stokes Raman spectroscopy (FASTER CARS), using signal amplification with the aid of a probe (tip enhanced Raman spectroscopy). As a result, single viral particles can be detected [12].
Transmission electron microscopy (TEM) and atomic force microscopy make it possible to successfully visualize single viral particles. Moreover, various methods of contrasting make it possible to see the structural elements of the virus envelope in the TEM. With all the advantages, these methods are unlikely to be compact, mobile and inexpensive. Although in the field of atomic force microscopy there are options for devices (without taking into account the dimensions of the electronics) of a millimeter size, they remain complicated and expensive as before.
Electrochemical biosensors are based on the entry into the electrochemical reaction of a biological material with an analyte [13]. The main categories of electrochemical biosensors are potentiometric, amperometric and impedimetric converters. Electrochemical biosensors have several advantages in terms of sensitivity and selectivity. They can be mobile and inexpensive.
There are, for example, many variations of commercial amperometric biosensors. Glucose biosensors are best known and widely available, for example: SIRE P201 (Chemel AB, Lund, Sweden), the blood glucose monitoring system FreeStyle Freedom, Precision Xtra (Abbot Diabetes Care, Alameda, California, USA) and GlucoWatch Biographer (Cygnus, Redwood City, California, USA). The Midas Pro device (Biosensori SpA, Milan, Italy) is widely used for surface water analysis.
Currently, reverse transcription PCR analysis on SARS-CoV-2 can only be done in the laboratory. It requires a set of reagents and laboratory equipment. A test of the best systems is able to detect 0.3–3 copies of viral RNA in a microliter (0.001 ml). Testing time is a few hours. Now they are striving to further reduce the analysis time.
Piezoceramic biosensors appeared not so long ago, but they have already shown high sensitivity when detecting the epidermal growth factor receptor (Her2) [14], the virus (WSSV) of plants [15], bacterial spores (Bacillus anthracis) [16], DNA [17] and influenza A virus [18].
In our work on the development of a piezoceramic biosensor for detecting viral infections, we record the probe-target binding by changing characteristics of the biochip oscillations. This solution does not involve the use of additional labels and chemicals. The selective sensor system is completely located on the surface of the biochip, therefore, the need to use additional chemical and biological reagents: DNA primers, TAG polymerase, various labels, etc., disappears, in contrast to PCR diagnostics and enzyme immunoassay.
We selected influenza A virus (Fig.1), tick-borne encephalitis virus (Fig.2), and coronavirus as targets for testing the biosensor. The choice of targets is due primarily to the urgent need for active opposition to these viral infections. Influenza A virus is a constant seasonal disease, tick-borne encephalitis virus has become widely spread over the territory of the Russian Federation over the last short period of time, SARC-CoV-2 coronavirus is the cause of a modern pandemic.
The proposed biosensor solution consists of a piezoceramic biochip in the form of a 0.1–3 mm dia. disk. The symmetric three-electrode design eliminates the influence of a double electric layer and parasitic diffusion processes on the surface of the biochip (Patent for invention No. 2636048, Biosensor device for detecting biological micro- and nano-objects, 2017). In this biochip design, the potential is supplied to the central electrode, while the external electrodes facing the solution can be grounded or kept at the potential of the solution. In this case, unlike the known structures, there is no need for an additional layer insulating the external electrodes. The use of periodic and pulsed modes of excitation of oscillations allows, due to the use of original algorithms, to achieve high sensitivity when detecting the influenza A virus [18].
The measuring electronic system is implemented using FPGA (Field-Programmable Gate Array) technologies or its further development in the form of ASIC (Application Specific Integrated Circuit). It is planned to adapt the existing electronic system for collecting information from a FemtoScan scanning probe microscope for conducting both resonant measurements and processing of electrochemical signals. The signal from the piezoceramic biosenosor arrives at the frequency of the mechanical resonance of the biochip, as a rule, this is the high-frequency range from hundreds of kHz to hundreds of MHz. The signal from the electrochemical biosensor is located in the range of significantly lower frequencies. This allows you to synchronously register signals from electrochemical and piezoelectric ceramic biosensors using just electronic means. Moreover, it is possible to combine two biosensors – piezoceramic and electrochemical – in one biosensor, while two signals will come together from the same biochip.
The liquid system can be made either with microfluidic technology or with the use of a millimeter-sized tank. Antibodies, aptamers and / or synthetic receptors can be used as probes. In the case of the SARC-CoV-2 coronavirus, its virus can attach to ACE2 protein of the epithelial cells upon infection. Moreover, the binding constant has an unusually high value. When testing, it is possible to use the recombinant water-soluble protein ACE2, available on the market of bioreagents.
The preliminary calculated value of the biosensor sensitivity to SARC-CoV-2 is 1–100 viral particles depending on the size of the biochip area (0.1–3 mm). For influenza A virus and tick-borne encephalitis virus, the sensitivity is slightly worse, since the binding constant of the currently available receptors is 3–10 times less.
Using the biosensor of the proposed design, it is possible to record not only the act of binding, but also the moment of detachment of a viral particle with an increase in the amplitude of mechanical vibrations of the biochip to threshold values when the mechanical energy of the vibrating viral particle becomes greater than the binding energy. At the moment of separation, a separation signal is given and the electronic measuring system records it. Thus, the biosensor will be able to detect both binding of the virus and its detachment.
It should be noted that nonspecific binding in liquid in the presence of various impurities deposited on the surface of the biochip does not lead to a significant change in the mechanical rigidity of the surface layer, and does not affect the separation signal of the particle at large vibration amplitude.
Subsequent characterization of the virus in a sufficiently large quantity can be carried out by chromatography, transmission electron microscopy and atomic force microscopy. For data processing, it is advisable to use the FemtoScan Online software developed by us [19].
In contrast to quartz microbalance, longitudinal vibrations are excited in the biochip, which lead to stretching / compression of the sensor film with attached viral particles or biomacromolecules.
The measurement of film stiffness is 300–1000 times more sensitive than simply determining the attached mass (as in the quartz microbalance method). Curiously, this experimental fact was noticed in the 18th century by Benjamin Franklin. Traveling from the New to the Old World on a whaling ship, Benjamin Franklin noticed that during the storm the captain ordered that a barrel of whale oil be poured into the sea. After that, the waves near the ship calmed down. The modern explanation of this effect is quite simple: a monolayer film of fat is formed on the surface of water. A lot of mechanical energy is expended on its compression and expansion during a storm; as a result, the waves attenuate. A film of fat converts mechanical energy into thermal energy transmitting it to the sea wave. Surprisingly, the calculation shows that a film with a thickness of nanometers calms meter high waves on the water.
Creating an "ideal" biosensor for viral infections is a noble task though a difficult one. On this path, new ideas and solutions will lead to continuous progress. The combined efforts of specialists from different fields of knowledge will undoubtedly contribute to moving forward. On our way, we will be glad to have new companions. ■
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