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A capacitive surface stress biosensor for CSFV detection
Microelectronic Engineering 159 (2016) 55–59
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
A capacitive surface stress biosensor for CSFV detection Jianyan Tian a,b, Yang Bai a, Xiaoliang Tang a, Shengbo Sang a,b,c,⁎, Fang Wang a,b a b c
College of Information Engineering, Taiyuan University of Technology, Taiyuan 030024, China Key Laboratory of Advanced Transducers and Intelligent Control System, Ministry of Education, Taiyuan University of Technology, Taiyuan 030024, China MicroNano System Research Center, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 5 February 2016 Accepted 20 February 2016 Available online 24 February 2016 Keywords: Biosensor Capacitive Surface stress Classical swine fever virus (CSFV) Detection
a b s t r a c t Capacitive biosensors are increasingly popular in biomedical analyses. In order to satisfy the requirements in medical diagnostics, such as high sensitivity and quick response, this paper presents a developed capacitive biosensor based on the surface stress to detect classical swine fever virus (CSFV) antigens. The developed biosensor is composed of a substrate layer and a sensitive layer. The sensitive layer is functioned with antibodies for CSFV antigens. Once the CSFV antigens bind to the antibodies, the increased surface stress will result in convex deformation, converting to capacitance changes. Scanning electron microscope (SEM) and atomic force microscope (AFM) are two methods used to monitor the surface changes. The experimental results demonstrate that the sensing electrode modification process is successful, and the surface stress induced by the binding of antibodies and CSFV antigens can cause variations of capacitance. In conclusion, the developed capacitive biosensor can be used to detect the CSFV antigens, and it has the potential to detect other antigens in the future. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are developed as a cross-integration of emerging technologies from a variety of disciplines of biology, physics, medicine and others. It is one of the hottest areas of biotechnology research. Since 1962, Clark and Lyons had presented the basic biosensor concept and made the first biosensor based on the enzyme electrode to measure blood glucose [1]. Many kinds of biosensors are invented for environmental monitoring, food analysis, DNA analysis and other research fields [2–4]. Surface stress-based biosensors, as an important kind of biosensors, attract lots of attention for their advantages, such as short response time, label free, and typical sensitivity at femtojoule, nanogram level. These biosensors use the underlying concept in any binding reaction without energy change, which in result offers a universal platform for chemical and biological sensing. The sensitive elements of these biosensors, composed of a selective layer and the transducer, are widely studied and developed to improve their performances. In addition, most studies of sensitive elements are focused on micro-cantilevers and micro-membranes. However, the cantilever geometry is not best suitable for sensing in the aqueous media, and can lead to the reduction in the signal-to-noise ratio [5]. Then, micro-membranes are studied as sensitive elements to detect surface stress changes. And silicon [6], silicon oxide [7] based materials are replaced by low stiffness materials, such as Parylene, PMMA and PDMS, to solve the problem that ⁎ Corresponding author at: College of Information Engineering, Taiyuan University of Technology, Taiyuan 030024, China. E-mail address: [email protected] (S. Sang).
http://dx.doi.org/10.1016/j.mee.2016.02.037 0167-9317/© 2016 Elsevier B.V. All rights reserved.
membranes are less compliant than cantilevers [8,9]. In general, optical detection and electrical detection are the two main methods in signal detection. The optical detection has some disadvantages compared to the electrical detection. It can cause thermal effects that are troublesome for sensitive elements and are not portable. However, electrical detection method can overcome these problems. And, capacitive detection method, one kind of electrical detection, has been used to biosensors since 1996 [10]. The sensing technique can be found in many applications for the detection of proteins, DNA, antibodies and antigens [11–14]. Recently, capacitive surface stress biosensor is an attractive research area due to the advantages of high-responsive accuracy and simple operation [15,16]. Classical swine fever, which is caused by CSFV, is one of the virulent diseases in domestic pigs. It is characterized by high spreading speed, severe infectiousness and high mortality. It can cause a great economic loss to pig farming industry. Parallelly to the increasing knowledge of CSFV, some diagnostic methods have been developed at the same time. The isolation of CSFV is “gold standard” to detect CSFV [17]. Although this method is still in application, there are drawbacks of this technology such as long processing time, intensive labor force and complex operation [18]. Moreover, the real-time polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RTPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are used for detection of classical swine fever [19–21]. These CSFV detection methods are based on molecular biological diagnostic tools. With rapid development, biosensor is becoming a new method for medical diagnostics [22,23]. Hence, a new poly-dimethylsiloxane (PDMS) micro-membrane capacitive surface stress biosensor is used
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PDMS membrane, which is used to form functional layer to recognize specific targets. The bottom gold electrode is plated on the surface of silicon wafer. These two gold electrodes form a capacitor, and the capacitance can be monitored to reflect the distance changes between the two electrodes. The capacitance can be calculated by the Eq. (1): C ¼ εΑ=d
Fig. 1. Capacitive surface stress biosensor. (a) Top view of a real biosensor; (b) The schematic diagram of A–A'cross-section view.
Table 1 The main parameters of the biosensor. Material
Shape
Radius (μm)
Thickness (μm)
Chromium film of top electrode Gold film of top electrode Cavity PDMS membrane Chromium film of bottom electrode Gold film of bottom electrode
Circular Circular
250 250
Circular Circular Circular
250 240 240
0.01 0.04 3 1 0.02 0.04
for the CSFV detection, which is label-free, rapid, high sensitivity and free energy transform. The design and fabrication of the biosensor are presented in our previous work, achieving the integration of PDMS processing with conventional processes [24]. In addition, the bonding technique, which uses uncured PDMS as the intermediate layer, has been proved to be suitable for the binding between the biosensor and the glass microfluidic components. The Finite Element Analysis is used to optimize the sensor geometry parameters. It is found that the circular electrode has smaller fringing effect than the square electrode. Hence, the shape of the proposed biosensor electrodes is designed to be circular. SEM and AFM are used to get information about the biosensor's surface topography and roughness, respectively, verifying the surface changes in the experiment. At the same time, the capacitance measurement method is used to detect the variations of capacitance induced by the CSFV. 2. Capacitive surface stress biosensor The capacitive surface stress biosensors that are designed and fabricated in this paper are illustrated in Fig. 1a. There are two sensitive structures in one biosensor chip. The one is used as the standard signal for reference. The other one is used to detect CSFV antigens. Therefore, compared with the capacitance values of two sensitive structures, it is easier to tell whether CSFV is detected. The sensitive structure is made up of bottom gold electrode, PDMS membrane and top gold electrode as shown in Fig. 1b. The top gold electrode is placed on the surface of
ð1Þ
where, ε is the dielectric constant, A is the area of the electrodes and d is the distance between them. The detailed parameters of the biosensor are extremely important to the sensitivity. Herein, the structure of the developed capacitive surface stress biosensor is studied systematically including the characters of size, shape and depth, which reduces the effects of the external environment and improves sensor sensitivity [15]. The main parameters of the biosensor are shown in Table 1. If biological substances attach on the membrane, deflections of the membrane will be produced by the induced surface stress, and translating distance changes to capacitive signal. The detailed principle on deflections of the membrane induced by surface stress is analyzed in our previous research [16]. 3. Materials and equipment Alkanethiol self-assemed monolayers (HS-(CH2)n-X) are widely studied to construct SAM(self-assemed monolayer). They can be immobilized stably on Au through the S–Au bond and van der Waals force [25], and it is available for their functional groups to bind with analytes. Herein, according to the molecule structure of the CSFV, the HS–(CH2)10–COOH (11-mercaptoundecanonic acid, 1 mM/L) was used to functionalize the biosensor to form a SAM. The biosensor was immersed in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide /Nhydroxysuccinimide (EDC/NHS, 0.3 mM/L) solution for one hour. Antibodies for swine fever could be dropped on the surface of gold electrode. The indirect haemagglutination assay kit and antibodies for swine fever(diluted in 1:30 proportion) were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science. Bovine serum albumin (BSA, 0.1%) was used to block the unreached space, reducing binding chances with nonspecific antibodies. Phosphate-buffered saline (PBS, 0.01 m/L) was used to rinse the excess antibodies. The capacitances of the biosensor were detected by Agilent B1500A (Agilent Technologies, USA). MIRA3 LMH (Tescan, Czech Republic) was used for SEM images. NX-10 (Park Systems, Korea) was used for AFM images. 4. Results and discussion 4.1. Sensing electrode modification process The sensing electrode modification process of capacitive surface stress biosensor can be found in Fig. 2. The initial surface of the biosensor was a naked Au membrane. Then the biosensor was immersed in
Fig. 2. Schematic diagram of the capacitive surface stress biosensor in modification process.
J. Tian et al. / Microelectronic Engineering 159 (2016) 55–59
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Fig. 3. (a) SEM image for the Au layer. (b) SEM image for the (SAM + Au) layer. (c) SEM image for the (CSFV antibodies + SAM + Au) layer.
thiol solution for 24 hours to ensure sufficient reaction between thiol molecules and Au atoms. The Au–S bonds were formed in the reaction, carrying out the aim that thiol molecules could be immobilized on the surface of sensitive membrane layer. SAMs can form on PDMS-Au layer instead of Si–Au layer because there is no solution in the capacitive cavity. These thiol molecules formed SAM are shown in Fig. 2b. After that, EDC/NHS solution was used as catalyst to improve coupling efficiency between antibodies and thiol molecules. In this case, EDC as a coupling agent played the role of activating carboxyl groups, and a stable active ester was provided by NHS. Therefore, the antibodies were cross-linked to the SAM through the amide bond (–CONH–), and the biosensor was modified with classical swine fever antibodies. To avoid the impact of other substances, the unreacted and non-specific sites were filled with BSA as shown in Fig. 2c. To verify the biosensor was modified successfully, SEM analysis, AFM analysis, and capacitance measurements were conducted for the modification process under static conditions, including SEM, AFM, and C–V analyzer.
4.1.1. SEM Analysis SEM images were gained under 10.0 kx to analyze the change in the biosensor surface. As shown in Fig. 3a, a naked surface image was from the biosensor gold surface without modification. During immersion in thiol solution, the formed Au–S bond enabled the thiol molecules to be immobilized on the surface, making the molecular groups larger. As shown in Fig. 3b, the enlarger molecules were reflected as a bunch of spots in the SEM image, indicating that the biosensors were immobilized with thiol molecules successfully. The density of the spot was approximately 0.25/μm2. When the antibodies were linked to the biosensor, larger agglomerations were presented due to the cross-link between the CSFV antibodies and SAMs, as shown in Fig. 3c. Also, the result was similar to reference [26]. In addition, the size of the agglomeration was about 300 μm2. Comparing the three SEM images, it can be inferred that the modification process was successful.
4.1.2. AFM Analysis AFM was used to get three-dimensional topography on the membrane surface of biosensors. In Fig. 4a, homogeneous Au nanoparticles are presented, illustrating the gold membrane surface without modifications. Due to the formation of SAM, small islands appeared on the biosensor membrane surface. Fig. 4b shows the surface change after the formation of SAM. It can be found that the maximum height of islands increased to 120 nm due to the accumulation of thiol molecules. Subsequently, antibodies coupled on the thiol groups significantly amplifies the diameter of islands and the density of the sparser, which were clearly presented in Fig. 4c. Therefore, it is obviously concluded that the immobilization of thiol molecules and antibodies was successful.
4.1.3. Capacity analysis The capacitances of the active biosensor and the reference biosensor were detected during the modification process. Based on the data from six experiments, the results are presented in Fig. 5. The two biosensors' capacitance values are near. The average value represents the capacitance value for the developed biosensor in different stages. The first data represent capacitance values of biosensors with Au layer; The second data represent capacitance values of biosensors with the (SAM + Au) layer; The third data represent capacitance values of biosensors with the (CSFV antibodies + SAM + Au) layer; The top values of error bars represent the highest capacitance in sixth measurements, and the bottom values of error bars represent the lowest capacitance in the six times measurements. After the formation of SAM, the strong covalent bonds (S–Au) were formed by gold and sulfur. As illustrated in reference [27], the force of the covalent bonds is about 185 kJ/mol, resulting in convex deformation of the membrane and decrease in capacitance. As the antibody attaches onto the biosensor membrane surface, the amine group on the antibodies reacted with the –COOH, forming –CONH– bond. Therefore, the deformation of membrane
Fig. 4. (a) AFM image for the Au layer. (b) AFM image for the (SAM + Au) layer. (c) AFM image for the (CSFV antibodies + SAM + Au) layer.
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Fig. 5. Capacitances for the active biosensor and the reference biosensor with Au layer, the (SAM + Au) layer, and the (CSFV antibodies + SAM + Au) layer.
caused by the –CONH– bonds was increased again, and the capacitance decreased corresponding.
4.2 CSFV detection After the sensing electrode modification process, the capacitive surface stress biosensor, which has the ability to recognize its associated target with an immobilized antibody, was used for the detection of CSFV. If the capacitance of the tested biosensor is lower than that of the reference biosensor, CSFV antigens can be considered to be detected by the biosensor. CSFV is a RNA virus, which the genome is characterized by positive polarity and polyprotein [28]. Multiple non-covalent bonds are produced during the binding of CSFV antigen and antibody, forming an antigen–antibody complex [29]. In addition, the binding forces come from these non-covalent bonds, such as hydrogen bonds, hydrophobic force, salt bridges and Van-der Waals forces [30]. Fig. 6a presents a schematic diagram of the interaction between antibody and CSFV. After the reaction, SEM and AFM were used to characterize the biosensor membrane surface, as shown in Fig. 6b and c. Because the CSFV was been added to the membrane surface of the biosensor, the molecular groups are larger than these that are shown in Fig. 4. For the capacity measurement, the binding force increased the surface stress, resulting in a longer distance between two gold electrodes. Therefore, the capacitance continued to decrease as shown in Fig. 7. However, the capacitance of the reference biosensor without CSFV
Fig. 7. Capacitances for the active biosensor with Au layer, the (SAM + Au) layer, the (CSFV antibodies + SAM + Au) layer, and the (CSFV antigens + CSFV antibodies + SAM + Au) layer; Capacitances for the reference biosensor with Au layer, the (SAM + Au) layer, the (CSFV antibodies + SAM + Au) layer, and the (CSFV antibodies + SAM + Au) layer.
antigens changed little. The results demonstrated that the new capacitive surface stress biosensor could detect CSFV successfully. The experiment based on white light interferometry was conducted to test the deformations of the capacitive surface stress biosensor. In the experiment, two white light interferometry tests are adopted to compare the variations of the biosensor: one test for the membrane without CSFV; the other for the membrane with CSFV. As shown in Fig. 8, it is visible that the Z-range value becomes higher, which means the distance between two electrodes becomes higher. According to Eq. (1), it is easy to know that the raised height of d will lead to the decrease of the biosensor's capacitance. The membrane upon CSFV binding will generate force, causing the deflection of the membrane. Therefore, it can verified that the successful binding between the biosensor and CSFV antigen with the combination of the variations in Z-range and the decrease in capacitance. 5. Conclusions In this paper, a new capacitive surface stress biosensor was used to detect CSFV. The modification and detection process were successful based on the experimental tests. The results from SEM and AFM demonstrated the surface features changed in the sensing electrode
Fig. 6. (a) The schematic diagram of the biosensor surface structure. (b) SEM images of the detection process in 5.00 kx. (c) AFM image after the interaction between antibody and virus for classical swine fever.
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References [1] [2] [3] [4] [5] [6]
[7] [8] [9] Fig. 8. (a) The white light interferometry test for the membrane without CSFV; (b) The white light interferometry test for the membrane with CSFV.
[10] [11] [12] [13]
modification process and detection process. Moreover, the semiconductor parameter analyzer was used to monitor the capacitance changes in the process. From the reaction principle and capacitance change trend, it was found that when the CSFV was detected, the capacitance would decrease because the surface stress from the binding of antibodies and CSFV antigens increased the distance between two gold electrodes. The results from the biosensor's surface feature and capacitance demonstrated that the biosensor could detect the CSFV antigens through the capacitance changes. This study provides a new convenient method for CSFV detection.
Acknowledgments This study was supported by the National High Technology Research and Development Program of China (863 Program) (2013AA102306, 2013AA041109), the National Natural Science Foundation of China (No. 61471255, 61474079), the National Research Foundation for the Doctoral Program of Higher Education of China (No. 20131402110013).
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Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
A capacitive surface stress biosensor for CSFV detection Jianyan Tian a,b, Yang Bai a, Xiaoliang Tang a, Shengbo Sang a,b,c,⁎, Fang Wang a,b a b c
College of Information Engineering, Taiyuan University of Technology, Taiyuan 030024, China Key Laboratory of Advanced Transducers and Intelligent Control System, Ministry of Education, Taiyuan University of Technology, Taiyuan 030024, China MicroNano System Research Center, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 3 November 2015 Received in revised form 5 February 2016 Accepted 20 February 2016 Available online 24 February 2016 Keywords: Biosensor Capacitive Surface stress Classical swine fever virus (CSFV) Detection
a b s t r a c t Capacitive biosensors are increasingly popular in biomedical analyses. In order to satisfy the requirements in medical diagnostics, such as high sensitivity and quick response, this paper presents a developed capacitive biosensor based on the surface stress to detect classical swine fever virus (CSFV) antigens. The developed biosensor is composed of a substrate layer and a sensitive layer. The sensitive layer is functioned with antibodies for CSFV antigens. Once the CSFV antigens bind to the antibodies, the increased surface stress will result in convex deformation, converting to capacitance changes. Scanning electron microscope (SEM) and atomic force microscope (AFM) are two methods used to monitor the surface changes. The experimental results demonstrate that the sensing electrode modification process is successful, and the surface stress induced by the binding of antibodies and CSFV antigens can cause variations of capacitance. In conclusion, the developed capacitive biosensor can be used to detect the CSFV antigens, and it has the potential to detect other antigens in the future. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are developed as a cross-integration of emerging technologies from a variety of disciplines of biology, physics, medicine and others. It is one of the hottest areas of biotechnology research. Since 1962, Clark and Lyons had presented the basic biosensor concept and made the first biosensor based on the enzyme electrode to measure blood glucose [1]. Many kinds of biosensors are invented for environmental monitoring, food analysis, DNA analysis and other research fields [2–4]. Surface stress-based biosensors, as an important kind of biosensors, attract lots of attention for their advantages, such as short response time, label free, and typical sensitivity at femtojoule, nanogram level. These biosensors use the underlying concept in any binding reaction without energy change, which in result offers a universal platform for chemical and biological sensing. The sensitive elements of these biosensors, composed of a selective layer and the transducer, are widely studied and developed to improve their performances. In addition, most studies of sensitive elements are focused on micro-cantilevers and micro-membranes. However, the cantilever geometry is not best suitable for sensing in the aqueous media, and can lead to the reduction in the signal-to-noise ratio [5]. Then, micro-membranes are studied as sensitive elements to detect surface stress changes. And silicon [6], silicon oxide [7] based materials are replaced by low stiffness materials, such as Parylene, PMMA and PDMS, to solve the problem that ⁎ Corresponding author at: College of Information Engineering, Taiyuan University of Technology, Taiyuan 030024, China. E-mail address: [email protected] (S. Sang).
http://dx.doi.org/10.1016/j.mee.2016.02.037 0167-9317/© 2016 Elsevier B.V. All rights reserved.
membranes are less compliant than cantilevers [8,9]. In general, optical detection and electrical detection are the two main methods in signal detection. The optical detection has some disadvantages compared to the electrical detection. It can cause thermal effects that are troublesome for sensitive elements and are not portable. However, electrical detection method can overcome these problems. And, capacitive detection method, one kind of electrical detection, has been used to biosensors since 1996 [10]. The sensing technique can be found in many applications for the detection of proteins, DNA, antibodies and antigens [11–14]. Recently, capacitive surface stress biosensor is an attractive research area due to the advantages of high-responsive accuracy and simple operation [15,16]. Classical swine fever, which is caused by CSFV, is one of the virulent diseases in domestic pigs. It is characterized by high spreading speed, severe infectiousness and high mortality. It can cause a great economic loss to pig farming industry. Parallelly to the increasing knowledge of CSFV, some diagnostic methods have been developed at the same time. The isolation of CSFV is “gold standard” to detect CSFV [17]. Although this method is still in application, there are drawbacks of this technology such as long processing time, intensive labor force and complex operation [18]. Moreover, the real-time polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RTPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are used for detection of classical swine fever [19–21]. These CSFV detection methods are based on molecular biological diagnostic tools. With rapid development, biosensor is becoming a new method for medical diagnostics [22,23]. Hence, a new poly-dimethylsiloxane (PDMS) micro-membrane capacitive surface stress biosensor is used
56
J. Tian et al. / Microelectronic Engineering 159 (2016) 55–59
PDMS membrane, which is used to form functional layer to recognize specific targets. The bottom gold electrode is plated on the surface of silicon wafer. These two gold electrodes form a capacitor, and the capacitance can be monitored to reflect the distance changes between the two electrodes. The capacitance can be calculated by the Eq. (1): C ¼ εΑ=d
Fig. 1. Capacitive surface stress biosensor. (a) Top view of a real biosensor; (b) The schematic diagram of A–A'cross-section view.
Table 1 The main parameters of the biosensor. Material
Shape
Radius (μm)
Thickness (μm)
Chromium film of top electrode Gold film of top electrode Cavity PDMS membrane Chromium film of bottom electrode Gold film of bottom electrode
Circular Circular
250 250
Circular Circular Circular
250 240 240
0.01 0.04 3 1 0.02 0.04
for the CSFV detection, which is label-free, rapid, high sensitivity and free energy transform. The design and fabrication of the biosensor are presented in our previous work, achieving the integration of PDMS processing with conventional processes [24]. In addition, the bonding technique, which uses uncured PDMS as the intermediate layer, has been proved to be suitable for the binding between the biosensor and the glass microfluidic components. The Finite Element Analysis is used to optimize the sensor geometry parameters. It is found that the circular electrode has smaller fringing effect than the square electrode. Hence, the shape of the proposed biosensor electrodes is designed to be circular. SEM and AFM are used to get information about the biosensor's surface topography and roughness, respectively, verifying the surface changes in the experiment. At the same time, the capacitance measurement method is used to detect the variations of capacitance induced by the CSFV. 2. Capacitive surface stress biosensor The capacitive surface stress biosensors that are designed and fabricated in this paper are illustrated in Fig. 1a. There are two sensitive structures in one biosensor chip. The one is used as the standard signal for reference. The other one is used to detect CSFV antigens. Therefore, compared with the capacitance values of two sensitive structures, it is easier to tell whether CSFV is detected. The sensitive structure is made up of bottom gold electrode, PDMS membrane and top gold electrode as shown in Fig. 1b. The top gold electrode is placed on the surface of
ð1Þ
where, ε is the dielectric constant, A is the area of the electrodes and d is the distance between them. The detailed parameters of the biosensor are extremely important to the sensitivity. Herein, the structure of the developed capacitive surface stress biosensor is studied systematically including the characters of size, shape and depth, which reduces the effects of the external environment and improves sensor sensitivity [15]. The main parameters of the biosensor are shown in Table 1. If biological substances attach on the membrane, deflections of the membrane will be produced by the induced surface stress, and translating distance changes to capacitive signal. The detailed principle on deflections of the membrane induced by surface stress is analyzed in our previous research [16]. 3. Materials and equipment Alkanethiol self-assemed monolayers (HS-(CH2)n-X) are widely studied to construct SAM(self-assemed monolayer). They can be immobilized stably on Au through the S–Au bond and van der Waals force [25], and it is available for their functional groups to bind with analytes. Herein, according to the molecule structure of the CSFV, the HS–(CH2)10–COOH (11-mercaptoundecanonic acid, 1 mM/L) was used to functionalize the biosensor to form a SAM. The biosensor was immersed in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide /Nhydroxysuccinimide (EDC/NHS, 0.3 mM/L) solution for one hour. Antibodies for swine fever could be dropped on the surface of gold electrode. The indirect haemagglutination assay kit and antibodies for swine fever(diluted in 1:30 proportion) were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science. Bovine serum albumin (BSA, 0.1%) was used to block the unreached space, reducing binding chances with nonspecific antibodies. Phosphate-buffered saline (PBS, 0.01 m/L) was used to rinse the excess antibodies. The capacitances of the biosensor were detected by Agilent B1500A (Agilent Technologies, USA). MIRA3 LMH (Tescan, Czech Republic) was used for SEM images. NX-10 (Park Systems, Korea) was used for AFM images. 4. Results and discussion 4.1. Sensing electrode modification process The sensing electrode modification process of capacitive surface stress biosensor can be found in Fig. 2. The initial surface of the biosensor was a naked Au membrane. Then the biosensor was immersed in
Fig. 2. Schematic diagram of the capacitive surface stress biosensor in modification process.
J. Tian et al. / Microelectronic Engineering 159 (2016) 55–59
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Fig. 3. (a) SEM image for the Au layer. (b) SEM image for the (SAM + Au) layer. (c) SEM image for the (CSFV antibodies + SAM + Au) layer.
thiol solution for 24 hours to ensure sufficient reaction between thiol molecules and Au atoms. The Au–S bonds were formed in the reaction, carrying out the aim that thiol molecules could be immobilized on the surface of sensitive membrane layer. SAMs can form on PDMS-Au layer instead of Si–Au layer because there is no solution in the capacitive cavity. These thiol molecules formed SAM are shown in Fig. 2b. After that, EDC/NHS solution was used as catalyst to improve coupling efficiency between antibodies and thiol molecules. In this case, EDC as a coupling agent played the role of activating carboxyl groups, and a stable active ester was provided by NHS. Therefore, the antibodies were cross-linked to the SAM through the amide bond (–CONH–), and the biosensor was modified with classical swine fever antibodies. To avoid the impact of other substances, the unreacted and non-specific sites were filled with BSA as shown in Fig. 2c. To verify the biosensor was modified successfully, SEM analysis, AFM analysis, and capacitance measurements were conducted for the modification process under static conditions, including SEM, AFM, and C–V analyzer.
4.1.1. SEM Analysis SEM images were gained under 10.0 kx to analyze the change in the biosensor surface. As shown in Fig. 3a, a naked surface image was from the biosensor gold surface without modification. During immersion in thiol solution, the formed Au–S bond enabled the thiol molecules to be immobilized on the surface, making the molecular groups larger. As shown in Fig. 3b, the enlarger molecules were reflected as a bunch of spots in the SEM image, indicating that the biosensors were immobilized with thiol molecules successfully. The density of the spot was approximately 0.25/μm2. When the antibodies were linked to the biosensor, larger agglomerations were presented due to the cross-link between the CSFV antibodies and SAMs, as shown in Fig. 3c. Also, the result was similar to reference [26]. In addition, the size of the agglomeration was about 300 μm2. Comparing the three SEM images, it can be inferred that the modification process was successful.
4.1.2. AFM Analysis AFM was used to get three-dimensional topography on the membrane surface of biosensors. In Fig. 4a, homogeneous Au nanoparticles are presented, illustrating the gold membrane surface without modifications. Due to the formation of SAM, small islands appeared on the biosensor membrane surface. Fig. 4b shows the surface change after the formation of SAM. It can be found that the maximum height of islands increased to 120 nm due to the accumulation of thiol molecules. Subsequently, antibodies coupled on the thiol groups significantly amplifies the diameter of islands and the density of the sparser, which were clearly presented in Fig. 4c. Therefore, it is obviously concluded that the immobilization of thiol molecules and antibodies was successful.
4.1.3. Capacity analysis The capacitances of the active biosensor and the reference biosensor were detected during the modification process. Based on the data from six experiments, the results are presented in Fig. 5. The two biosensors' capacitance values are near. The average value represents the capacitance value for the developed biosensor in different stages. The first data represent capacitance values of biosensors with Au layer; The second data represent capacitance values of biosensors with the (SAM + Au) layer; The third data represent capacitance values of biosensors with the (CSFV antibodies + SAM + Au) layer; The top values of error bars represent the highest capacitance in sixth measurements, and the bottom values of error bars represent the lowest capacitance in the six times measurements. After the formation of SAM, the strong covalent bonds (S–Au) were formed by gold and sulfur. As illustrated in reference [27], the force of the covalent bonds is about 185 kJ/mol, resulting in convex deformation of the membrane and decrease in capacitance. As the antibody attaches onto the biosensor membrane surface, the amine group on the antibodies reacted with the –COOH, forming –CONH– bond. Therefore, the deformation of membrane
Fig. 4. (a) AFM image for the Au layer. (b) AFM image for the (SAM + Au) layer. (c) AFM image for the (CSFV antibodies + SAM + Au) layer.
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Fig. 5. Capacitances for the active biosensor and the reference biosensor with Au layer, the (SAM + Au) layer, and the (CSFV antibodies + SAM + Au) layer.
caused by the –CONH– bonds was increased again, and the capacitance decreased corresponding.
4.2 CSFV detection After the sensing electrode modification process, the capacitive surface stress biosensor, which has the ability to recognize its associated target with an immobilized antibody, was used for the detection of CSFV. If the capacitance of the tested biosensor is lower than that of the reference biosensor, CSFV antigens can be considered to be detected by the biosensor. CSFV is a RNA virus, which the genome is characterized by positive polarity and polyprotein [28]. Multiple non-covalent bonds are produced during the binding of CSFV antigen and antibody, forming an antigen–antibody complex [29]. In addition, the binding forces come from these non-covalent bonds, such as hydrogen bonds, hydrophobic force, salt bridges and Van-der Waals forces [30]. Fig. 6a presents a schematic diagram of the interaction between antibody and CSFV. After the reaction, SEM and AFM were used to characterize the biosensor membrane surface, as shown in Fig. 6b and c. Because the CSFV was been added to the membrane surface of the biosensor, the molecular groups are larger than these that are shown in Fig. 4. For the capacity measurement, the binding force increased the surface stress, resulting in a longer distance between two gold electrodes. Therefore, the capacitance continued to decrease as shown in Fig. 7. However, the capacitance of the reference biosensor without CSFV
Fig. 7. Capacitances for the active biosensor with Au layer, the (SAM + Au) layer, the (CSFV antibodies + SAM + Au) layer, and the (CSFV antigens + CSFV antibodies + SAM + Au) layer; Capacitances for the reference biosensor with Au layer, the (SAM + Au) layer, the (CSFV antibodies + SAM + Au) layer, and the (CSFV antibodies + SAM + Au) layer.
antigens changed little. The results demonstrated that the new capacitive surface stress biosensor could detect CSFV successfully. The experiment based on white light interferometry was conducted to test the deformations of the capacitive surface stress biosensor. In the experiment, two white light interferometry tests are adopted to compare the variations of the biosensor: one test for the membrane without CSFV; the other for the membrane with CSFV. As shown in Fig. 8, it is visible that the Z-range value becomes higher, which means the distance between two electrodes becomes higher. According to Eq. (1), it is easy to know that the raised height of d will lead to the decrease of the biosensor's capacitance. The membrane upon CSFV binding will generate force, causing the deflection of the membrane. Therefore, it can verified that the successful binding between the biosensor and CSFV antigen with the combination of the variations in Z-range and the decrease in capacitance. 5. Conclusions In this paper, a new capacitive surface stress biosensor was used to detect CSFV. The modification and detection process were successful based on the experimental tests. The results from SEM and AFM demonstrated the surface features changed in the sensing electrode
Fig. 6. (a) The schematic diagram of the biosensor surface structure. (b) SEM images of the detection process in 5.00 kx. (c) AFM image after the interaction between antibody and virus for classical swine fever.
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[7] [8] [9] Fig. 8. (a) The white light interferometry test for the membrane without CSFV; (b) The white light interferometry test for the membrane with CSFV.
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modification process and detection process. Moreover, the semiconductor parameter analyzer was used to monitor the capacitance changes in the process. From the reaction principle and capacitance change trend, it was found that when the CSFV was detected, the capacitance would decrease because the surface stress from the binding of antibodies and CSFV antigens increased the distance between two gold electrodes. The results from the biosensor's surface feature and capacitance demonstrated that the biosensor could detect the CSFV antigens through the capacitance changes. This study provides a new convenient method for CSFV detection.
Acknowledgments This study was supported by the National High Technology Research and Development Program of China (863 Program) (2013AA102306, 2013AA041109), the National Natural Science Foundation of China (No. 61471255, 61474079), the National Research Foundation for the Doctoral Program of Higher Education of China (No. 20131402110013).
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