rus
Issue #5/2016
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
The continuation of the article about cantilever biosensors considers the integration of cantilever sensors in the bioanalytical devices and the corresponding applications, examples of biosensors for detection of biomolecules, the advantages and disadvantages of biosensors based on microcantilevers, as well as relevant areas of research.
ALTERNATIVES TO INTEGRATION IN BIOANALYTICAL DEVICES
With its small footprint, cantilever sensors can easily be embedded in the bioanalytical devices. Often this integration is carried out in a microfluidic format [1]. Microfluidic chip allows precise control of small-volume liquid sample, consistently providing sample preparation and analysis. Such an integrated system is called the "full-microanalysis system" or "lab-on-a-chip". Cantilever sensors can also be combined with other types of sensors for more complete analysis. In [2] cantilever resonant sensor has been combined with capacitive and calorimetric sensors with the aim of creating a gas sensor for the analysis of airborne volatile organic compounds (Fig.1). Full integration of microelectronic and micromechanical components on the same chip provides control and monitoring of sensor functions, but also increases its overall performance because of signal gain. The circuit also includes an analog-to-digital converters and interface for data transfer to the external writer.
Cantilever sensors can be integrated with systems for wireless data transfer to create a completely autonomous devices. The authors of [3] have designed and built cantilever sensor for determination of C-reactive protein with the integrated wireless module. Such devices can be the prototypes of the implantable sensors (Fig.2), which have been developed relatively recently [4]. Since regeneration of the biosensor is rather difficult, implantable device was used for detection of blood gases such as alcohols. For this purpose on the back side of the cantilever a hydrophobic coating was applied, and the gold side was modified by the polymer thiolised fluorine-siloxane alcohol. The absorption of alcohol by the receptor layer causes bending of the cantilever.
APPLICATIONS OF BIOSENSORS DEPENDING ON TYPES
OF INTEGRATION
The main area of application of biosensors is the diagnosis. Cantilever sensors allow to identify proteins that are markers of various diseases. For example, the possibility of detection of prostate-specific antigen, a marker of prostate cancer, in clinically significant concentrations was shown [5]. In another project [6] the concentration of myoglobin, which is a marker of cardiovascular diseases, was determined. Many tests require considerable time, specially trained staff and a work in the laboratory. Compact and Autonomous sensor devices based on microcantilevers integrated into microfluidic chips have prospects for use as a personalized diagnostic devices [7]. The response time of such devices is tens of minutes. Stationary devices can be used in clinical laboratories as an alternative or supplement to standard diagnostic methods.
However, diagnosis is not the only application of cantilever sensors. "Electronic nose" based on arrays of cantilever sensors can be used for environmental monitoring. Using an eight-cantilever sensor with cantilevers coated with different polymers, the authors of [8] showed the possibility to distinguish in the air "smells" of different alcohols.
Although most studies use the data from one or more sensor cantilevers, the possibility to produce thousands of cantilevers on a single chip of few millimeters is already shown. IBM has demonstrated the location of the 100 cantilevers on 1 mm2. Thus, by packing density of sensor elements, cantilever sensors are close to the DNA microchips. However, these chips do not require fluorescent markers. The main problem is the functionalization of such number of cantilevers with receptor molecules.
EEXAMPLES OF BIOSENSORS
BASED ON MICROCANTILEVERS
FOR DETECTION OF BIOMOLECULES
During the development of cantilever biosensors a large number of applications for detecting various types of biomolecules (proteins [5, 6], nucleic acids, carbohydrates [9]) was demonstrated. The possibility to register single nucleotide polymorphism [10] was shown.
The development of sensors for biological objects that are dangerous for human health is of particular interest. Pathogenic agents can be detected using cantilever sensors, both directly and by analysis of their genome, waste products or toxins.
Determination of the presence of whole pathogenic cells is often used for the detection of bacteria. One of the most popular objects for testing of methods (largely thanks to its presence in various samples of food and water) is E.coli. This bacterium may serve as an indicator of water quality [11]. The detection of individual E. coli cell was demonstrated using the cantilever sensor in a resonant mode [12] (Fig.3). However, in this project, for sensitivity increase measurements were carried out in vacuum and required stage of drying. This limitation can be overcome through the use of cantilevers with an internal channel that can work in a vacuum, wherein analyzing the fluid in the canal [13]. In another study, the piezoelectric cantilever of millimeter size was used to determine E.coli in a buffer solution with a detection limit of 700 cells/ml [14]. It allows to measure directly in the liquid with data receiving in near real time.
The use of static mode showed significantly worse detection limit of about 106 CFU/ml [15]. Apparently the binding of such large objects as the bacteria does not create a uniform surface tension. For the S.Typhimurium bacteria the cantilever in resonance mode showed a detection limit at the level of 5∙103 cells/ml [16], and in the mode of measuring of surface tension – 106 CFU/ml [17]. Campbell and co-authors used a cantilever modified with polyclonal antibodies against B.Anthracis bacteria for measuring in the flow and have shown a sensitivity of 300 spores/ml [18]. In another study [19] with the use of cantilever sensor it has been registered 50 spores in water.
In the last few years the creation of biosensors for the detection of viruses in the environment became popular. Hepatitis is one of the most dangerous diseases in the world. Timurdogan and co-authors [20] used a cantilever biosensor to identify viruses of hepatitis A and C in bovine serum. Antibodies to hepatitis A and C were immobilized on different cantilevers, the detection limit was 0.1 ng/ml (1.6 pmole). Gupta in [21] showed that the sensitivity of the cantilever to measure the mass is enough to weigh single vaccinia virus, but he did not use a specific sensor layer.
In paper [22] it is shown the detection in the static mode of duck influenza A virus with a cantilever modified with synthetically glycopolymer containing residues of sialic acids. The authors suggest that the polymer layer on the surface of the cantilever created a matrix that increased surface tension due to the additional interaction with viral particles. The limit of detection in this system amounted to 106 virions/ml.
The piezoelectric cantilever sensor operating in resonant mode, was used to detect helicase of hepatitis C virus with a concentration of 100 pg/ml [23]. Helicase is the enzyme responsible for the deployment of viral RNA and is specific to this virus. RNA aptamers, a short sequences of nucleotides, which are able to specifically bind the antigen (protein), were used as receptor. They can be easily synthesized, and they are more stable in storage than antibodies.
Another option for the detection of pathogenic agents is the detection of their nucleic acids in the sample. Unlike large objects, DNA and RNA does not have substantial mass, but it has been shown that the binding of nucleic acids with complementary molecules on the surface of the cantilever causes a large surface stress. It was demonstrated detection of not amplified template-RNA in the total mass of RNA of the cell with a detection limit of 10 pmole. With the use of microcantilever sensor and gain by means of silicon nanoparticles it was succeeded to achieve detection of DNA of the hepatitis b virus at femtomolar level [24]. The DNA fragment of the virus with a length of 243 nucleotide was detected using a probe at the level of picomoles. A further increase in sensitivity to femtomoles was achieved by hybridization with a second probe containing a nanoparticle.
Paper [25] describes the detection of toxins producing M.aeruginosa cyanobacteria by the specific section of the 16S gene of ribosomal RNA at the level of a concentration of 50 cells/ml. For this purpose, DNA oligonucleotides containing 27 bases that are complementary to the desired region of the genome, was immobilized on the cantilever. The sensor registered the shift of the resonance frequency of the cantilever caused by the hybridization of complementary DNA molecules that is proportional to the concentration of bacteria in solution. The reliability of the hybridization was confirmed by fluorescence measurements and by secondary hybridization of DNA probes with attached gold nanoparticle.
Piezoresistive cantilever sensor was used [26] for registration of staphylococcal enterotoxin B with a concentration of 1 nmole using thio-modified aptamers as receptors. This toxin can be released by some types of staphylococcus. Ricin in a concentration of 0.14 nmol and 0.28 nmol was also detected. Labeled with biotin polyclonal antibodies were used as the receptor. Ricin is extremely poisonous toxin of plant origin.
ECONOMIC FEASIBILITY
OF USE OF MICROCANTILEVERS
AS BIOSENSOR DEVICES
The basic measuring element in a cantilever sensor is a silicon sensor. The microprocessing of silicon is well studied for its use in microelectronics. This makes it accessible and affordable. According to Yole Development, the cost of microprocessing of 1 cm2 of silicon is $ 0.165. One square centimeter of a silicon substrate can easily accommodate more than 10 cantilever sensors. A number of researches aimed at the development of economical methods of production of cantilever sensors is executed [27, 28]. The results were the techniques allowing to produce cantilever sensors with high quality and minimal costs.
COMMERCIALLY AVAILABLE COMPONENTS AND MODULES
FOR BIOSENSORS BASED
ON MICROCANTILEVERS
Currently, several companies sell commercial cantilevers for the development of sensor devices [29, 30] (Fig.4). Devices that are ready for experiments to study interactions on the surface of the cantilever, which can be used as the basis for sensors, are also presented on the market. Most of the elements of biosensor devices, such as laser sources, photo-detectors, the electronic components are also commercially available.
The following is a list of the major manufacturers of cantilevers:
NanoAndMore (www.nanoandmore.com);
Bruker (brukerafmprobes.com);
Olympus (probe.olympus-global.com);
NanoScience Instruments (www.nanoscience.com).
ADVANTAGES AND DISADVANTAGES OF BIOSENSORS BASED
ON MICROCANTILEVERS
One of the main advantages of cantilever sensors is that they allow direct analysis without use of additional marks. This allows to simplify the procedure of sample preparation and to conduct continuous monitoring in real-time.
Despite the fact that the cantilever sensors can directly measure the interaction between antibodies and antigens or hybridization of DNA, their sensitivity is often insufficient for practical applications [31]. Methods of signal amplification, such as using nanoparticles [24] or the creation of a special matrix on the surface of the cantilever [22] are developed. An important limiting factor for maximum sensitivity is non-specific binding. Cantilever sensors operating in the static mode, have a high degree of protection against non-specific binding, since the energy of nonspecific interaction makes only a small contribution to the bending of the cantilever. Sensors operating in the resonance mode, on the contrary react to any attached mass.
Another disadvantage is the high susceptibility of the cantilever (especially in static mode) to external influences, e.g. temperature variations, fluctuations of the fluid flow, seismic noise, which leads to increased measurement errors. Sensors operating in the resonant mode, are more resistant to such factors, however, in liquids quality factor of the resonant flexural vibrations strongly decreases, which also leads to a reduction of sensitivity. To avoid this, it is possible to use the measurements of the longitudinal vibration modes [32].
CURRENT AREAS OF RESEARCH
Cantilever biosensors are a promising platform for highly sensitive and selective sensor devices. However, there are some critical factors that determine the development of this technology.
The most important area of research to create sensors based on microcantilevers is surface chemistry. The quality of the receptor layer determines the performance and the characteristics of the entire sensor. There are no universal methods of modification of the cantilever. Each specific applications needs in development of a special methodology that provides the best results. Thus, the development of protocols for the creation of receptor layers, and their study is key to the successful creation of the sensor.
Another important area is the search for new materials. As shown, the characteristics of the material significantly affect the sensitivity of the sensor, and using new functional materials, it is possible to significantly increase the sensitivity of the registration system. The use of polymeric materials opens broad prospects. However, the manufacturing of microelectromechanical systems on basis of them is worked out not so well, as for silicon and its compounds.
The ultimate goal of improving the technology of cantilever sensors would be the achievement of sensitivity, allowing to detect a single molecule of a substance while maintaining selectivity. ■
The study was performed with the financial support of the Ministry of education and science of Russian Federation (project 02.G25.31.0135).
With its small footprint, cantilever sensors can easily be embedded in the bioanalytical devices. Often this integration is carried out in a microfluidic format [1]. Microfluidic chip allows precise control of small-volume liquid sample, consistently providing sample preparation and analysis. Such an integrated system is called the "full-microanalysis system" or "lab-on-a-chip". Cantilever sensors can also be combined with other types of sensors for more complete analysis. In [2] cantilever resonant sensor has been combined with capacitive and calorimetric sensors with the aim of creating a gas sensor for the analysis of airborne volatile organic compounds (Fig.1). Full integration of microelectronic and micromechanical components on the same chip provides control and monitoring of sensor functions, but also increases its overall performance because of signal gain. The circuit also includes an analog-to-digital converters and interface for data transfer to the external writer.
Cantilever sensors can be integrated with systems for wireless data transfer to create a completely autonomous devices. The authors of [3] have designed and built cantilever sensor for determination of C-reactive protein with the integrated wireless module. Such devices can be the prototypes of the implantable sensors (Fig.2), which have been developed relatively recently [4]. Since regeneration of the biosensor is rather difficult, implantable device was used for detection of blood gases such as alcohols. For this purpose on the back side of the cantilever a hydrophobic coating was applied, and the gold side was modified by the polymer thiolised fluorine-siloxane alcohol. The absorption of alcohol by the receptor layer causes bending of the cantilever.
APPLICATIONS OF BIOSENSORS DEPENDING ON TYPES
OF INTEGRATION
The main area of application of biosensors is the diagnosis. Cantilever sensors allow to identify proteins that are markers of various diseases. For example, the possibility of detection of prostate-specific antigen, a marker of prostate cancer, in clinically significant concentrations was shown [5]. In another project [6] the concentration of myoglobin, which is a marker of cardiovascular diseases, was determined. Many tests require considerable time, specially trained staff and a work in the laboratory. Compact and Autonomous sensor devices based on microcantilevers integrated into microfluidic chips have prospects for use as a personalized diagnostic devices [7]. The response time of such devices is tens of minutes. Stationary devices can be used in clinical laboratories as an alternative or supplement to standard diagnostic methods.
However, diagnosis is not the only application of cantilever sensors. "Electronic nose" based on arrays of cantilever sensors can be used for environmental monitoring. Using an eight-cantilever sensor with cantilevers coated with different polymers, the authors of [8] showed the possibility to distinguish in the air "smells" of different alcohols.
Although most studies use the data from one or more sensor cantilevers, the possibility to produce thousands of cantilevers on a single chip of few millimeters is already shown. IBM has demonstrated the location of the 100 cantilevers on 1 mm2. Thus, by packing density of sensor elements, cantilever sensors are close to the DNA microchips. However, these chips do not require fluorescent markers. The main problem is the functionalization of such number of cantilevers with receptor molecules.
EEXAMPLES OF BIOSENSORS
BASED ON MICROCANTILEVERS
FOR DETECTION OF BIOMOLECULES
During the development of cantilever biosensors a large number of applications for detecting various types of biomolecules (proteins [5, 6], nucleic acids, carbohydrates [9]) was demonstrated. The possibility to register single nucleotide polymorphism [10] was shown.
The development of sensors for biological objects that are dangerous for human health is of particular interest. Pathogenic agents can be detected using cantilever sensors, both directly and by analysis of their genome, waste products or toxins.
Determination of the presence of whole pathogenic cells is often used for the detection of bacteria. One of the most popular objects for testing of methods (largely thanks to its presence in various samples of food and water) is E.coli. This bacterium may serve as an indicator of water quality [11]. The detection of individual E. coli cell was demonstrated using the cantilever sensor in a resonant mode [12] (Fig.3). However, in this project, for sensitivity increase measurements were carried out in vacuum and required stage of drying. This limitation can be overcome through the use of cantilevers with an internal channel that can work in a vacuum, wherein analyzing the fluid in the canal [13]. In another study, the piezoelectric cantilever of millimeter size was used to determine E.coli in a buffer solution with a detection limit of 700 cells/ml [14]. It allows to measure directly in the liquid with data receiving in near real time.
The use of static mode showed significantly worse detection limit of about 106 CFU/ml [15]. Apparently the binding of such large objects as the bacteria does not create a uniform surface tension. For the S.Typhimurium bacteria the cantilever in resonance mode showed a detection limit at the level of 5∙103 cells/ml [16], and in the mode of measuring of surface tension – 106 CFU/ml [17]. Campbell and co-authors used a cantilever modified with polyclonal antibodies against B.Anthracis bacteria for measuring in the flow and have shown a sensitivity of 300 spores/ml [18]. In another study [19] with the use of cantilever sensor it has been registered 50 spores in water.
In the last few years the creation of biosensors for the detection of viruses in the environment became popular. Hepatitis is one of the most dangerous diseases in the world. Timurdogan and co-authors [20] used a cantilever biosensor to identify viruses of hepatitis A and C in bovine serum. Antibodies to hepatitis A and C were immobilized on different cantilevers, the detection limit was 0.1 ng/ml (1.6 pmole). Gupta in [21] showed that the sensitivity of the cantilever to measure the mass is enough to weigh single vaccinia virus, but he did not use a specific sensor layer.
In paper [22] it is shown the detection in the static mode of duck influenza A virus with a cantilever modified with synthetically glycopolymer containing residues of sialic acids. The authors suggest that the polymer layer on the surface of the cantilever created a matrix that increased surface tension due to the additional interaction with viral particles. The limit of detection in this system amounted to 106 virions/ml.
The piezoelectric cantilever sensor operating in resonant mode, was used to detect helicase of hepatitis C virus with a concentration of 100 pg/ml [23]. Helicase is the enzyme responsible for the deployment of viral RNA and is specific to this virus. RNA aptamers, a short sequences of nucleotides, which are able to specifically bind the antigen (protein), were used as receptor. They can be easily synthesized, and they are more stable in storage than antibodies.
Another option for the detection of pathogenic agents is the detection of their nucleic acids in the sample. Unlike large objects, DNA and RNA does not have substantial mass, but it has been shown that the binding of nucleic acids with complementary molecules on the surface of the cantilever causes a large surface stress. It was demonstrated detection of not amplified template-RNA in the total mass of RNA of the cell with a detection limit of 10 pmole. With the use of microcantilever sensor and gain by means of silicon nanoparticles it was succeeded to achieve detection of DNA of the hepatitis b virus at femtomolar level [24]. The DNA fragment of the virus with a length of 243 nucleotide was detected using a probe at the level of picomoles. A further increase in sensitivity to femtomoles was achieved by hybridization with a second probe containing a nanoparticle.
Paper [25] describes the detection of toxins producing M.aeruginosa cyanobacteria by the specific section of the 16S gene of ribosomal RNA at the level of a concentration of 50 cells/ml. For this purpose, DNA oligonucleotides containing 27 bases that are complementary to the desired region of the genome, was immobilized on the cantilever. The sensor registered the shift of the resonance frequency of the cantilever caused by the hybridization of complementary DNA molecules that is proportional to the concentration of bacteria in solution. The reliability of the hybridization was confirmed by fluorescence measurements and by secondary hybridization of DNA probes with attached gold nanoparticle.
Piezoresistive cantilever sensor was used [26] for registration of staphylococcal enterotoxin B with a concentration of 1 nmole using thio-modified aptamers as receptors. This toxin can be released by some types of staphylococcus. Ricin in a concentration of 0.14 nmol and 0.28 nmol was also detected. Labeled with biotin polyclonal antibodies were used as the receptor. Ricin is extremely poisonous toxin of plant origin.
ECONOMIC FEASIBILITY
OF USE OF MICROCANTILEVERS
AS BIOSENSOR DEVICES
The basic measuring element in a cantilever sensor is a silicon sensor. The microprocessing of silicon is well studied for its use in microelectronics. This makes it accessible and affordable. According to Yole Development, the cost of microprocessing of 1 cm2 of silicon is $ 0.165. One square centimeter of a silicon substrate can easily accommodate more than 10 cantilever sensors. A number of researches aimed at the development of economical methods of production of cantilever sensors is executed [27, 28]. The results were the techniques allowing to produce cantilever sensors with high quality and minimal costs.
COMMERCIALLY AVAILABLE COMPONENTS AND MODULES
FOR BIOSENSORS BASED
ON MICROCANTILEVERS
Currently, several companies sell commercial cantilevers for the development of sensor devices [29, 30] (Fig.4). Devices that are ready for experiments to study interactions on the surface of the cantilever, which can be used as the basis for sensors, are also presented on the market. Most of the elements of biosensor devices, such as laser sources, photo-detectors, the electronic components are also commercially available.
The following is a list of the major manufacturers of cantilevers:
NanoAndMore (www.nanoandmore.com);
Bruker (brukerafmprobes.com);
Olympus (probe.olympus-global.com);
NanoScience Instruments (www.nanoscience.com).
ADVANTAGES AND DISADVANTAGES OF BIOSENSORS BASED
ON MICROCANTILEVERS
One of the main advantages of cantilever sensors is that they allow direct analysis without use of additional marks. This allows to simplify the procedure of sample preparation and to conduct continuous monitoring in real-time.
Despite the fact that the cantilever sensors can directly measure the interaction between antibodies and antigens or hybridization of DNA, their sensitivity is often insufficient for practical applications [31]. Methods of signal amplification, such as using nanoparticles [24] or the creation of a special matrix on the surface of the cantilever [22] are developed. An important limiting factor for maximum sensitivity is non-specific binding. Cantilever sensors operating in the static mode, have a high degree of protection against non-specific binding, since the energy of nonspecific interaction makes only a small contribution to the bending of the cantilever. Sensors operating in the resonance mode, on the contrary react to any attached mass.
Another disadvantage is the high susceptibility of the cantilever (especially in static mode) to external influences, e.g. temperature variations, fluctuations of the fluid flow, seismic noise, which leads to increased measurement errors. Sensors operating in the resonant mode, are more resistant to such factors, however, in liquids quality factor of the resonant flexural vibrations strongly decreases, which also leads to a reduction of sensitivity. To avoid this, it is possible to use the measurements of the longitudinal vibration modes [32].
CURRENT AREAS OF RESEARCH
Cantilever biosensors are a promising platform for highly sensitive and selective sensor devices. However, there are some critical factors that determine the development of this technology.
The most important area of research to create sensors based on microcantilevers is surface chemistry. The quality of the receptor layer determines the performance and the characteristics of the entire sensor. There are no universal methods of modification of the cantilever. Each specific applications needs in development of a special methodology that provides the best results. Thus, the development of protocols for the creation of receptor layers, and their study is key to the successful creation of the sensor.
Another important area is the search for new materials. As shown, the characteristics of the material significantly affect the sensitivity of the sensor, and using new functional materials, it is possible to significantly increase the sensitivity of the registration system. The use of polymeric materials opens broad prospects. However, the manufacturing of microelectromechanical systems on basis of them is worked out not so well, as for silicon and its compounds.
The ultimate goal of improving the technology of cantilever sensors would be the achievement of sensitivity, allowing to detect a single molecule of a substance while maintaining selectivity. ■
The study was performed with the financial support of the Ministry of education and science of Russian Federation (project 02.G25.31.0135).
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