rus
Issue #8/2016
A.Akhmetova, N.Gutnik, G.Meshkov, I.Nazarov, O.Sinitsyna, I.Yaminsky
Biosensor for detection of viruses and bacteria in liquids
Biosensor for detection of viruses and bacteria in liquids
This paper describes the development of a compact and inexpensive biosensor intended for use in medical diagnostics.
Теги: biochip flow-through liquid cell scanning probe microscopy биочип проточная жидкостная ячейка сканирующий зондовый микроскоп
A block diagram of the biosensor is shown in Fig.1. The biochip represents a miniature piezoceramic disk with sensory layers on the opposite sides. The attaching of the biological agent to sensor surface of the biochip changes the resonant frequency of mechanical oscillations of the disk.
The interaction of pathogens with the receptor layer leads to a change in the effective mass and stiffness of a biochip that can be registered by shift of the resonance frequency of the cantilever:
, (1)
where Δf, Δk, Δm are changes of the resonance frequency, stiffness and mass of the biochip, and fn, k and m are the initial values of these parameters.
Let’s consider the detection of bacterial cell of Escherichia coli. Length of bacteria is of about 2–6 µm and a diameter – of about 1 µm. With an average length of 4 µm the mass of bacteria is equal to about 3.1 ∙ 10–12 g. The biochip with a diameter of 3 mm and a thickness of 0.1 mm is made of piezoelectric ceramics ЦТС-19. It has a volume of 0.7 10–3 cm3 and weight of 5.3 ∙ 10–3 g (the density of material is 7.5 g/cm3). Attachment of a single bacterial cell leads to a relative change in the resonant frequency by the value:
(2)
According to relation (2), the relative frequency stability of the drive-pulse generator should be at the level of 10–10.
General view of a flow-through cell with the piezoceramic biochip placed in it is shown in Fig.2. Peristaltic pump and connecting tubes are used for circulation of the fluid containing the detected pathogens. Flow-through liquid cell and the connecting tubes form a hermetically sealed chamber with the circulating biological fluid. Connecting tubes and flow-through liquid cell are made of cheap and inert materials (silicone rubber and polyethylene, respectively) and can be a consumable one-time use items.
The frequency measurement error Δf of the piezoelectric biochip, which is caused by electronic noise of the measuring circuit, instability of piezoceramics and other factors, limits the sensitivity of the method. The ZTB piezoelectric ceramic resonator with a resonant frequency of 1 MHz was used to assess the self-noise level of the measuring system of the biosensor.
Determining the resonant frequency of oscillation of the biochip was conducted in the following way. Using a digital frequency synthesizer with AD7008 chip (Analog Devices) and precision input amplifier, the amplitude-frequency characteristic of the piezoelectric resonator was registered (Fig.3). For determination of the amplitude-frequency characteristic 512 measurements in the selected frequency range were carried out. To improve accuracy it is possible to consecutively measure several characteristics. The resonant frequency was calculated according to the method of determining the "center of mass":
.
This method provides much greater accuracy in the presence of real noises than the simple search for the maximum on the curve.
Fig.4 shows the results of processing of 512 resonance curves, each of which was drawn using 512 points. Thus, the total number of individual measurements is 262, 144.
Mean square error in determining the resonant frequency of the biochip in case of linear averaging of five consecutive measurements was 1.8 Hz, which corresponds to the standard deviation of experimental curve (black line in Fig.4) from the average value. In our case, the error in determining the mass of a biochip, calculated by the formula 2 is Δm = 6 ∙ 10–10 g, which is approximately equal to the mass of two hundred E. coli bacteria.
The control unit of the biosensor consists of electronic boards of digital frequency synthesizer, precision input amplifier, interface for communication with computer, thermostat of flow-through liquid cell, DAC-ADC, voltage stabilizer. Fig.5 shows the boards of the prototype of biosensor and its case.
The symmetry of the design of the biosensor is a significant factor. The plate of the biochip has the geometric symmetry, however, the voltage supply circuit is asymmetric, because voltage of different polarity is supplied to the opposite sides. As a result, the structure of the surface dual conductive layer in close proximity to opposite sides of the biochip is substantially different, which may affect both the receptor layer and the properties of adsorbed films of biological agents. To achieve the full symmetry of the electric circuit an original solution [5] using the composite piezoceramic biochip is proposed (Fig.6).
In the specified design of the biochip, voltage can be fed only to the inner electrodes, and outer electrodes are grounded or have a potential of solution. In this case, the symmetry is achieved in the geometry of the biochip and in the voltages supply. As a result, the near-surface dual conductive layers are fully symmetric.
The calculations and estimates show that the technical parameters of the digital frequency synthesizer (accuracy of frequency setting is 0.001 Hz thanks to use of a 32-bit control) allow to reach the sensitivity required to register by a precision input amplifier at the following minimum concentrations of biological agents:
• 104 virus/ml for influenza
A viruses in a fluid;
• 102 bacteria/ml for E. coli bacteria in a fluid.
To further increase the sensitivity of the biosensor up to the level of single pathogens, it is necessary to use the biochip of substantially smaller size: with thickness and diameter of micron size. The manufacturing of such a biochip is challenging, but with adequate production base, technically implementable process. ■
R&D is performed in the Lomonosov Moscow State University with financial support of the Ministry of education and science of the Russian Federation (project 02.G25.31.0135).
The interaction of pathogens with the receptor layer leads to a change in the effective mass and stiffness of a biochip that can be registered by shift of the resonance frequency of the cantilever:
, (1)
where Δf, Δk, Δm are changes of the resonance frequency, stiffness and mass of the biochip, and fn, k and m are the initial values of these parameters.
Let’s consider the detection of bacterial cell of Escherichia coli. Length of bacteria is of about 2–6 µm and a diameter – of about 1 µm. With an average length of 4 µm the mass of bacteria is equal to about 3.1 ∙ 10–12 g. The biochip with a diameter of 3 mm and a thickness of 0.1 mm is made of piezoelectric ceramics ЦТС-19. It has a volume of 0.7 10–3 cm3 and weight of 5.3 ∙ 10–3 g (the density of material is 7.5 g/cm3). Attachment of a single bacterial cell leads to a relative change in the resonant frequency by the value:
(2)
According to relation (2), the relative frequency stability of the drive-pulse generator should be at the level of 10–10.
General view of a flow-through cell with the piezoceramic biochip placed in it is shown in Fig.2. Peristaltic pump and connecting tubes are used for circulation of the fluid containing the detected pathogens. Flow-through liquid cell and the connecting tubes form a hermetically sealed chamber with the circulating biological fluid. Connecting tubes and flow-through liquid cell are made of cheap and inert materials (silicone rubber and polyethylene, respectively) and can be a consumable one-time use items.
The frequency measurement error Δf of the piezoelectric biochip, which is caused by electronic noise of the measuring circuit, instability of piezoceramics and other factors, limits the sensitivity of the method. The ZTB piezoelectric ceramic resonator with a resonant frequency of 1 MHz was used to assess the self-noise level of the measuring system of the biosensor.
Determining the resonant frequency of oscillation of the biochip was conducted in the following way. Using a digital frequency synthesizer with AD7008 chip (Analog Devices) and precision input amplifier, the amplitude-frequency characteristic of the piezoelectric resonator was registered (Fig.3). For determination of the amplitude-frequency characteristic 512 measurements in the selected frequency range were carried out. To improve accuracy it is possible to consecutively measure several characteristics. The resonant frequency was calculated according to the method of determining the "center of mass":
.
This method provides much greater accuracy in the presence of real noises than the simple search for the maximum on the curve.
Fig.4 shows the results of processing of 512 resonance curves, each of which was drawn using 512 points. Thus, the total number of individual measurements is 262, 144.
Mean square error in determining the resonant frequency of the biochip in case of linear averaging of five consecutive measurements was 1.8 Hz, which corresponds to the standard deviation of experimental curve (black line in Fig.4) from the average value. In our case, the error in determining the mass of a biochip, calculated by the formula 2 is Δm = 6 ∙ 10–10 g, which is approximately equal to the mass of two hundred E. coli bacteria.
The control unit of the biosensor consists of electronic boards of digital frequency synthesizer, precision input amplifier, interface for communication with computer, thermostat of flow-through liquid cell, DAC-ADC, voltage stabilizer. Fig.5 shows the boards of the prototype of biosensor and its case.
The symmetry of the design of the biosensor is a significant factor. The plate of the biochip has the geometric symmetry, however, the voltage supply circuit is asymmetric, because voltage of different polarity is supplied to the opposite sides. As a result, the structure of the surface dual conductive layer in close proximity to opposite sides of the biochip is substantially different, which may affect both the receptor layer and the properties of adsorbed films of biological agents. To achieve the full symmetry of the electric circuit an original solution [5] using the composite piezoceramic biochip is proposed (Fig.6).
In the specified design of the biochip, voltage can be fed only to the inner electrodes, and outer electrodes are grounded or have a potential of solution. In this case, the symmetry is achieved in the geometry of the biochip and in the voltages supply. As a result, the near-surface dual conductive layers are fully symmetric.
The calculations and estimates show that the technical parameters of the digital frequency synthesizer (accuracy of frequency setting is 0.001 Hz thanks to use of a 32-bit control) allow to reach the sensitivity required to register by a precision input amplifier at the following minimum concentrations of biological agents:
• 104 virus/ml for influenza
A viruses in a fluid;
• 102 bacteria/ml for E. coli bacteria in a fluid.
To further increase the sensitivity of the biosensor up to the level of single pathogens, it is necessary to use the biochip of substantially smaller size: with thickness and diameter of micron size. The manufacturing of such a biochip is challenging, but with adequate production base, technically implementable process. ■
R&D is performed in the Lomonosov Moscow State University with financial support of the Ministry of education and science of the Russian Federation (project 02.G25.31.0135).
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