rus
Issue #4/2016
D.Kolesov, I.Yaminsky, A.Ahmetova, O.Sinitsyna, G.Meshkov
Cantilever biosensors for detection of viruses and bacteria
Cantilever biosensors for detection of viruses and bacteria
Cantilever biosensors are a promising platform for development of highly sensitive and selective sensors. The first part of the paper describes the background of the problem, properties of microcantilevers, technologies of their production and biofunctionalisation, and also detection methods.
Теги: biosensor microcantilever scanning probe microscope биосенсор микрокантилевер сканирующий зондовый микроскоп
The cantilever is the basic measuring element of atomic force microscopy [1]. It is a thin long plate, fixed at one end to a massive chip with a needle at the opposite end. Thomas Albrecht in 1989 has first used photolithography to create microcantilever of silicon nitride. Now the most commonly used materials for the production of cantilevers are silicon, silicon oxide and silicon nitride. Early researches on the use of microcantilevers for chemical detection was carried out about 20 years ago in IBM research laboratory (Zurich, Switzerland) and in the University of Basel [2]. The authors demonstrated static bending of the cantilever as a result of chemical reactions of catalytic conversion of hydrogen and oxygen into water. The first application of cantilever sensors for biological systems was introduced in 1996 on the example of nonspecific adsorption of the protein of bovine serum albumin [3]. Since then a lot of projects have demonstrated the potential of micromechanical cantilevers to create a highly sensitive chemical and biological sensors.
There are two basic modes of operation of cantilever sensors: static and dynamic (Fig.1). Due to the low thickness, the console of the cantilever is very sensitive to the lateral stresses on its surface. Biospecific binding of analyte molecules to the receptor layer on one surface of the cantilever leads to lateral stresses in the layer, which causes bending of the cantilever. Stress can be both a tightening and stretching. Thus the deviation of the free end of the console of the cantilever from its equilibrium position in the simplest case is connected with the lateral voltage via Stoney's formula:
,
where ∆σ is the change in surface stress, ∆Z is the relative deflection of the cantilever, υ is Poisson's ratio, E is young's modulus of the cantilever material, T and L are its thickness and length. Thus, by measuring the deflection of the cantilever as the result of biospecific binding, we can calculate the surface tension, which depends on the concentration of the target substance in the environment.
Dynamic mode of the cantilever sensors is based on the change in resonance frequency of the cantilever upon binding of analyte from a solution. The cantilever is a high-q resonator whose resonant frequency depends on the effective mass and stiffness of the console. As a result of interaction of analyte molecules with the receptor layer, one or both of these characteristics can change, which can be registered in a shift of the resonance frequency of the cantilever [4]:
,
where Δf, Δk, Δm are changes of the resonance frequency, stiffness and mass of the cantilever and fn, k and m are the initial values of these parameters.
Variety of microcantilevers and their properties
that determine possibility
of their use as biosensors
The first experiments in the field of creation of micromechanical sensors have used standard commercial cantilevers for scanning probe microscopy. Due to the development of this area the cantilevers without a needle for scanning of a surface, who are specially adapted for use in biosensors began to be developed. The cantilevers for static modes have a long and thin console with low rigidity. The cantilever for resonance mode are characterized by high stiffness and, respectively, by a higher resonant frequency. Usually the cantilevers have a gold coating on one side that serves two purposes: enhances the reflective properties and provides selective modification on only one side of the cantilever. Placement of several consoles on one chip for multivariate analysis is possible.
Thanks to the development of photolithography many scientific groups create their own configurations of cantilevers of different shapes and sizes for specific tasks. The traditional form of the cantilever is rectangular. It is possible to vary the ratio of length, width and thickness. In the static mode, the lateral dimensions do not directly affect the sensitivity of the sensor, because the surface tension is isotropic and uniformly over the entire active surface [5]. However, they can affect the sensitivity of the detection system, for example, of laser-optical system, in which the displacement of the free end of the cantilever, which quadratically depends on its length, is measured. At the same time the thickness of the cantilever should be minimal. For a resonant cantilever has been shown that the decrease of the ratio of the length and width at a constant thickness leads to the increased sensitivity of molecular binding of angiopoietin-1 in the fluid [6]. This result is logical, as the resonant frequency and q-factor depend on the specified ratio.
The cantilevers may have not a rectangular shape. So, for example, triangular cantilevers of silicon nitride have lower stiffness and therefore are more sensitive in static mode. Changing the shape of the cantilever for resonance mode can have the following background: change the vibration modes, selective excitation of vibrations, the increase of q-factor and decrease of damping in the fluid and, as a result, increase of sensitivity. For example, a T-shaped piezoelectric resonator showed a sensitivity of the order of femtograms [7].
In addition to different shapes, the cantilever may include multiple layers of different materials. Additional layers are often used to integrate in the sensor of system for registration or excitation of oscillations [8]. An active layers for excitation of oscillations are often made of piezoelectric or magnetoelastic material. The cantilever may contain also passive layers that separate the active or change its properties, e.g., stiffness. The simplest option are bimorphs, consisting of two layers of equal length. The cantilevers with layers of different length and geometry are more complex systems. Such systems have internal heterogeneity, leading to a unique resonance properties [9].
The sensor can contain not only the cantilever, but can be combined with other elements of the sensor, e.g. measuring chamber. So, in [10], the authors used cantilevers combined with a microfluidic channel through which the sample flows (Fig.3). Using the channel in the cantilever it was succeeded to achieve high sensitivity in the measurement of mass in the resonance mode [11].
Also the sensors are developed that operate based on the measurement of lateral stresses, in which instead of the cantilevers the membranes that are fixed on perimeter [12] or in several points are used. Such sensors have several advantages. For example, the reverse side of the membrane can be isolated from contact with the sample which reduces the effect of nonspecific binding. It was shown that the membrane sensor, fixed by the four small "bridges" (Fig.4), has a high sensitivity for the measurement of the lateral stress compared with conventional cantilevers [13].
Technologies for production
of microcantilevers
Photolithography is the main technology for the production of microcantilevers. Commercial cantilevers of silicon, silicon nitride or silicon oxide in a variety of shapes and sizes of length from 10 to 500 µm and thickness up to 12 nm are available. However, for specific tasks, including achievement of the maximum sensitivity, the cantilever should be specially designed and produced to meet all requirements. The cantilevers are manufactured using well-established thin-film technology providing low cost, good production yield and reproducibility. It comprises applying a thin film of material, structuring via photolithography and etching, and subsequent micromachining. Usually a protective layer is applied on a substrate before the main material to facilitate the subsequent separation of the product.
The sensitivity of cantilever sensors in the static mode depends on the young's modulus of the material of the cantilever. Silicon has a large young's modulus, therefore, the cantilever made of soft materials will be more sensitive. Polymeric materials such as SU-8 and polymethylmethacrylate (PMMA) are widely used. Since they are photoresists, the direct photolithography is suitable, but other technologies, for example, molding or screen printing can also be applied [14]. In [15], the authors fabricated the cantilevers using 6-µm film of polyethylene terephthalate. Plates of the required forms were cut with a UV laser. The result is an array of three cantilevers that are 600 µm long with a width of 250 µm and a thickness of 6 µm. On the basis of such cantilevers the biosensor for registration of single-stranded DNA has been created: one side of a cantilever was coated with a 50 nm thick layer of gold on which DNA, complementary to a detected was attached. In the experiment were able to detect concentrations of DNA to 0.01 µM in a volume of 0.2 ml.
Technologies for biofunctionalization
of microcantilevers
The sensor layer is a key element of cantilever sensors. Its characteristics determine the sensitivity and selectivity of the biosensor. This layer usually consists of receptor molecules, having the property of biospecifically binding, and accessory molecules that provide a secure fit and correct location of the receptors. Antibodies and nucleic acids (DNA probes) are the most commonly used receptor molecules for the detection of pathogenic agents.
Physical adsorption is the simplest method of immobilization of antibodies on the surface of the cantilever, however, their activity may decline as a result of denaturation or the wrong orientation. Regeneration of such sensitive layers is also difficult, since physically adsorbed molecules can be easily washed away. Covalent attachment of antibodies, which uses a helper molecule that performs the role of linkers, is more preferred.
Self-organizing monolayers (SOM) based on thiols [16] for a gold surface or on silatranes [17] for silicon, are often used to create a sensor layer on the surface of the cantilever. Compounds on the basis of organosilanes create a developed surface. For achievement of the best activity only Fab-fragments of antibodies can be used as receptors [18]. The oligonucleotides can be easily modified by thiol group and attached directly on the gold surface of the cantilever [19].
The application of a sensor layer may be performed by full immersion of the cantilever in the modifier solution [20], however, this method does not apply if the cantilever has a few consoles. Modification of each console by different substances is a difficult task. One of the possible solutions is the use of a set of capillaries of a suitable size for each console [21]. Micro-printing technology that is similar to used in the creation of DNA chips, is also suitable for deposition of sensor layer [22].
Methods of detection
using microcantilevers
The main method of detection of the deviation of the cantilever from the equilibrium position is the optical method (Fig.5), which, as the cantilever came from the AFM. The laser beam is directed to the console tip of the cantilever and reflected from it, falls on the position detector. Sensitive photodiode [23] or CCD camera [24] can be used as a detector. When the cantilever is deflected from the initial position, the reflected beam moves across the detector, and its movement is enhanced by the "optical leverage". This method of registration is extremely precise, allowing the detection of even subnanometre move, but has a number of disadvantages, for example, relatively large geometric size of the system, as the need to strengthen the movement of the beam on the photodiode leads to an increase in the distance from the cantilever to the detector. In addition, focused laser radiation can cause local heating of the sample and the sensor. To achieve maximum sensitivity it is necessary to use a quality radiation source and detector, which makes the whole system quite expensive.
An alternative method of detection that has been widely used in the last decade due to the high level of development of the lithographic techniques is the piezoresistive system. It is based on the change in resistance of the sensor as a result of surface stresses during the bending of the cantilever. A plain gold electrode of nanometer thickness deposited on the surface of the cantilever (Fig.6) can be used as such a sensor. The electric circuit based on the Wheatstone bridge is also often used for measurements using piezoresistive cantilever [25]. This method has great potential for the creation of compact sensor devices with a high degree of integration, however, typically has an order of magnitude lower sensitivity. In addition, the production of cantilevers with integrated piezoresistive sensor requires special equipment that is reasonable only in case of large volume of production.
Other detection methods have not received wide use. For example, a capacitive method, based on measuring capacitance of a plane capacitor, one of electrodes of which is microcantilever sensor [26], allows high accuracy of registration of the bending of the cantilever, however, is not applicable to solutions of electrolytes. There is also an optical technique based on interference of reflected from a cantilever beam with a reference laser beam [27; 28]. The cleaved end of the optical fiber is placed near the surface of the cantilever, and one part of radiation is reflected from the boundary of the fiber and the environment, and the second part – from the surface of the cantilever. These two radiation interfere inside the fiber and the interference signal can be measured by a photodetector. This method is extremely sensitive, allowing to measure deviation of 0.1 Å, but is not suitable for large displacements and is complex because of the need very precisely position the optical fiber.
Optical detection methods differ by the big variety. The cantilever itself can act as a waveguide [29]. Its deviation can be registered by using a diffraction grating or by changing the interference pattern on its surface.
An interesting approach to the measurement of surface tension by embedding a MOSFET transistor in the base of the cantilever is shown in [30]. The shutter of the field transistor increases with increasing of surface tension, causing a decrease of drain current upon deviation. The authors declare low noise and high sensitivity of this method. ■
To be continued in the next issue
Our sincere gratitude to the RFBR (project 15-04-07678) and the Ministry of education and science (project 02.G25.31.0135) for financial support
There are two basic modes of operation of cantilever sensors: static and dynamic (Fig.1). Due to the low thickness, the console of the cantilever is very sensitive to the lateral stresses on its surface. Biospecific binding of analyte molecules to the receptor layer on one surface of the cantilever leads to lateral stresses in the layer, which causes bending of the cantilever. Stress can be both a tightening and stretching. Thus the deviation of the free end of the console of the cantilever from its equilibrium position in the simplest case is connected with the lateral voltage via Stoney's formula:
,
where ∆σ is the change in surface stress, ∆Z is the relative deflection of the cantilever, υ is Poisson's ratio, E is young's modulus of the cantilever material, T and L are its thickness and length. Thus, by measuring the deflection of the cantilever as the result of biospecific binding, we can calculate the surface tension, which depends on the concentration of the target substance in the environment.
Dynamic mode of the cantilever sensors is based on the change in resonance frequency of the cantilever upon binding of analyte from a solution. The cantilever is a high-q resonator whose resonant frequency depends on the effective mass and stiffness of the console. As a result of interaction of analyte molecules with the receptor layer, one or both of these characteristics can change, which can be registered in a shift of the resonance frequency of the cantilever [4]:
,
where Δf, Δk, Δm are changes of the resonance frequency, stiffness and mass of the cantilever and fn, k and m are the initial values of these parameters.
Variety of microcantilevers and their properties
that determine possibility
of their use as biosensors
The first experiments in the field of creation of micromechanical sensors have used standard commercial cantilevers for scanning probe microscopy. Due to the development of this area the cantilevers without a needle for scanning of a surface, who are specially adapted for use in biosensors began to be developed. The cantilevers for static modes have a long and thin console with low rigidity. The cantilever for resonance mode are characterized by high stiffness and, respectively, by a higher resonant frequency. Usually the cantilevers have a gold coating on one side that serves two purposes: enhances the reflective properties and provides selective modification on only one side of the cantilever. Placement of several consoles on one chip for multivariate analysis is possible.
Thanks to the development of photolithography many scientific groups create their own configurations of cantilevers of different shapes and sizes for specific tasks. The traditional form of the cantilever is rectangular. It is possible to vary the ratio of length, width and thickness. In the static mode, the lateral dimensions do not directly affect the sensitivity of the sensor, because the surface tension is isotropic and uniformly over the entire active surface [5]. However, they can affect the sensitivity of the detection system, for example, of laser-optical system, in which the displacement of the free end of the cantilever, which quadratically depends on its length, is measured. At the same time the thickness of the cantilever should be minimal. For a resonant cantilever has been shown that the decrease of the ratio of the length and width at a constant thickness leads to the increased sensitivity of molecular binding of angiopoietin-1 in the fluid [6]. This result is logical, as the resonant frequency and q-factor depend on the specified ratio.
The cantilevers may have not a rectangular shape. So, for example, triangular cantilevers of silicon nitride have lower stiffness and therefore are more sensitive in static mode. Changing the shape of the cantilever for resonance mode can have the following background: change the vibration modes, selective excitation of vibrations, the increase of q-factor and decrease of damping in the fluid and, as a result, increase of sensitivity. For example, a T-shaped piezoelectric resonator showed a sensitivity of the order of femtograms [7].
In addition to different shapes, the cantilever may include multiple layers of different materials. Additional layers are often used to integrate in the sensor of system for registration or excitation of oscillations [8]. An active layers for excitation of oscillations are often made of piezoelectric or magnetoelastic material. The cantilever may contain also passive layers that separate the active or change its properties, e.g., stiffness. The simplest option are bimorphs, consisting of two layers of equal length. The cantilevers with layers of different length and geometry are more complex systems. Such systems have internal heterogeneity, leading to a unique resonance properties [9].
The sensor can contain not only the cantilever, but can be combined with other elements of the sensor, e.g. measuring chamber. So, in [10], the authors used cantilevers combined with a microfluidic channel through which the sample flows (Fig.3). Using the channel in the cantilever it was succeeded to achieve high sensitivity in the measurement of mass in the resonance mode [11].
Also the sensors are developed that operate based on the measurement of lateral stresses, in which instead of the cantilevers the membranes that are fixed on perimeter [12] or in several points are used. Such sensors have several advantages. For example, the reverse side of the membrane can be isolated from contact with the sample which reduces the effect of nonspecific binding. It was shown that the membrane sensor, fixed by the four small "bridges" (Fig.4), has a high sensitivity for the measurement of the lateral stress compared with conventional cantilevers [13].
Technologies for production
of microcantilevers
Photolithography is the main technology for the production of microcantilevers. Commercial cantilevers of silicon, silicon nitride or silicon oxide in a variety of shapes and sizes of length from 10 to 500 µm and thickness up to 12 nm are available. However, for specific tasks, including achievement of the maximum sensitivity, the cantilever should be specially designed and produced to meet all requirements. The cantilevers are manufactured using well-established thin-film technology providing low cost, good production yield and reproducibility. It comprises applying a thin film of material, structuring via photolithography and etching, and subsequent micromachining. Usually a protective layer is applied on a substrate before the main material to facilitate the subsequent separation of the product.
The sensitivity of cantilever sensors in the static mode depends on the young's modulus of the material of the cantilever. Silicon has a large young's modulus, therefore, the cantilever made of soft materials will be more sensitive. Polymeric materials such as SU-8 and polymethylmethacrylate (PMMA) are widely used. Since they are photoresists, the direct photolithography is suitable, but other technologies, for example, molding or screen printing can also be applied [14]. In [15], the authors fabricated the cantilevers using 6-µm film of polyethylene terephthalate. Plates of the required forms were cut with a UV laser. The result is an array of three cantilevers that are 600 µm long with a width of 250 µm and a thickness of 6 µm. On the basis of such cantilevers the biosensor for registration of single-stranded DNA has been created: one side of a cantilever was coated with a 50 nm thick layer of gold on which DNA, complementary to a detected was attached. In the experiment were able to detect concentrations of DNA to 0.01 µM in a volume of 0.2 ml.
Technologies for biofunctionalization
of microcantilevers
The sensor layer is a key element of cantilever sensors. Its characteristics determine the sensitivity and selectivity of the biosensor. This layer usually consists of receptor molecules, having the property of biospecifically binding, and accessory molecules that provide a secure fit and correct location of the receptors. Antibodies and nucleic acids (DNA probes) are the most commonly used receptor molecules for the detection of pathogenic agents.
Physical adsorption is the simplest method of immobilization of antibodies on the surface of the cantilever, however, their activity may decline as a result of denaturation or the wrong orientation. Regeneration of such sensitive layers is also difficult, since physically adsorbed molecules can be easily washed away. Covalent attachment of antibodies, which uses a helper molecule that performs the role of linkers, is more preferred.
Self-organizing monolayers (SOM) based on thiols [16] for a gold surface or on silatranes [17] for silicon, are often used to create a sensor layer on the surface of the cantilever. Compounds on the basis of organosilanes create a developed surface. For achievement of the best activity only Fab-fragments of antibodies can be used as receptors [18]. The oligonucleotides can be easily modified by thiol group and attached directly on the gold surface of the cantilever [19].
The application of a sensor layer may be performed by full immersion of the cantilever in the modifier solution [20], however, this method does not apply if the cantilever has a few consoles. Modification of each console by different substances is a difficult task. One of the possible solutions is the use of a set of capillaries of a suitable size for each console [21]. Micro-printing technology that is similar to used in the creation of DNA chips, is also suitable for deposition of sensor layer [22].
Methods of detection
using microcantilevers
The main method of detection of the deviation of the cantilever from the equilibrium position is the optical method (Fig.5), which, as the cantilever came from the AFM. The laser beam is directed to the console tip of the cantilever and reflected from it, falls on the position detector. Sensitive photodiode [23] or CCD camera [24] can be used as a detector. When the cantilever is deflected from the initial position, the reflected beam moves across the detector, and its movement is enhanced by the "optical leverage". This method of registration is extremely precise, allowing the detection of even subnanometre move, but has a number of disadvantages, for example, relatively large geometric size of the system, as the need to strengthen the movement of the beam on the photodiode leads to an increase in the distance from the cantilever to the detector. In addition, focused laser radiation can cause local heating of the sample and the sensor. To achieve maximum sensitivity it is necessary to use a quality radiation source and detector, which makes the whole system quite expensive.
An alternative method of detection that has been widely used in the last decade due to the high level of development of the lithographic techniques is the piezoresistive system. It is based on the change in resistance of the sensor as a result of surface stresses during the bending of the cantilever. A plain gold electrode of nanometer thickness deposited on the surface of the cantilever (Fig.6) can be used as such a sensor. The electric circuit based on the Wheatstone bridge is also often used for measurements using piezoresistive cantilever [25]. This method has great potential for the creation of compact sensor devices with a high degree of integration, however, typically has an order of magnitude lower sensitivity. In addition, the production of cantilevers with integrated piezoresistive sensor requires special equipment that is reasonable only in case of large volume of production.
Other detection methods have not received wide use. For example, a capacitive method, based on measuring capacitance of a plane capacitor, one of electrodes of which is microcantilever sensor [26], allows high accuracy of registration of the bending of the cantilever, however, is not applicable to solutions of electrolytes. There is also an optical technique based on interference of reflected from a cantilever beam with a reference laser beam [27; 28]. The cleaved end of the optical fiber is placed near the surface of the cantilever, and one part of radiation is reflected from the boundary of the fiber and the environment, and the second part – from the surface of the cantilever. These two radiation interfere inside the fiber and the interference signal can be measured by a photodetector. This method is extremely sensitive, allowing to measure deviation of 0.1 Å, but is not suitable for large displacements and is complex because of the need very precisely position the optical fiber.
Optical detection methods differ by the big variety. The cantilever itself can act as a waveguide [29]. Its deviation can be registered by using a diffraction grating or by changing the interference pattern on its surface.
An interesting approach to the measurement of surface tension by embedding a MOSFET transistor in the base of the cantilever is shown in [30]. The shutter of the field transistor increases with increasing of surface tension, causing a decrease of drain current upon deviation. The authors declare low noise and high sensitivity of this method. ■
To be continued in the next issue
Our sincere gratitude to the RFBR (project 15-04-07678) and the Ministry of education and science (project 02.G25.31.0135) for financial support
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