rus
Issue #8/2017
A.Ahmetova, I.Nazarov, G.Presnova, M.Rubtsova, A.Egorov, I.Yaminsky
Detection of protein biomacromolecules using piezoceramic biochip
Detection of protein biomacromolecules using piezoceramic biochip
The design of the piezoceramic cantilever biochip and methods for detecting protein macromolecules for use in medicine are considered.
Теги: biochip biosensor cantilever microalbumin probe microscopy protein macromolecules белковые макромолекулы биосенсоры биочип зондовая микроскопия кантилевер микроальбумин
The use of probe microscopy to detect biological macromolecules is a relatively new area. Earlier we presented methods for detecting viruses [1] and DNA [2, 3] using piezoceramic sensors. In particular, to detect the influenza A virus, a biosensor is proposed in which sialic acids that are capable of binding to the hemagglutinin of the virus envelope are used as biospecific recognition reagents. For the detection of DNA, piezoceramic biochips can be used, on the surface of which oligonucleotide probes of known sequence are immobilized. The principle of detection is based on the hybridization of these probes with complementary fragments of DNA molecules from the analyzed sample.
The aim of this work is to check the activity of antibodies on microalbumin for further development of piezoceramic biochip, which detects microalbumin using specific antibodies. Microalbuminuria is considered a harbinger of diseases of internal organs, in particular kidneys, and can indicate the development of heart failure and vascular damage. Thus, the index of microalbumin in the urine is an important factor determining the general state of the patient's body [4].
For the study, two types of piezoceramic discs were used (Fig.1): round single piezoceramic plates with a diameter of 16 mm and a thickness of 0.1 mm and twin discs consisting of two piezoceramic plates with a diameter of 4 mm and a thickness of 0.1 mm each. Plates with a diameter of 4 mm were cut mechanically from plates of a larger size and then glued in such a way that their polarization directions were different. Connecting wires were soldered to the electrodes of the plates: two wires for disks of the first type and three for disks of the second type.
In the experiment, we used six disks with a diameter of 16 mm and three twin discs with a diameter of 4 mm. All the disks were coated with a 40 nm thick gold layer using the Q300T D (Quorum Technologies Ltd., UK) deposition system. Immediately after the deposition of gold, three twin discs with a diameter of 4 mm and three single 16 mm discs (set No.1) were placed into a solution of 4-aminothiophenol in ethyl alcohol with a concentration of 0.16 g/l, and then three 16 mm discs (set No.2) were placed into ethyl alcohol for six days at room temperature.
Mouse monoclonal antibodies (Immunotek, Russia) against human serum albumin (H-C15, Bialexa, Russia) were used. On the set of disks No.1, after modification of the surface with aminothiophenol, antibodies were applied in a solution of EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) for the covalent binding of the carboxyl group of immunoglobulins to the amino group on the carrier surface. Antibodies with EDC in an amount of 29 μl with concentrations of 5, 50 and 100 μg/ml were applied to 16 mm discs, and 5 μl of the same concentration were placed on twin 4 mm discs, after which they were left for 12 hours at –4⁰С.
The second set of disks was placed for 12 hours into a solution of cysteamine (mercaptoethylamine) at a concentration of 0.05 M. Then, a solution of antibodies with EDC in a volume of 29 μl with concentrations of 5, 50 and 100 μg/ml was applied to each piezoceramic disk.
16 mm disks from the first set were combined with a second set, after which immobilized antibodies were detected using a conjugate of goat antibodies against mouse antibodies marked by horseradish peroxidase. The incubation was carried out in PBST buffer (phosphate buffer solution with Tween-20), the substrate of tetramethylbenzidine (TMB) was used to detect the enzymatic activity of peroxidase.
Based on the results of the immunoenzyme analysis, it was shown that aminothiophenol is better suited for modifying the carrier surface than cysteamine (Fig.2).
In parallel with immunoenzyme analysis, an experiment was performed with 4 mm piezoceramic discs (set No.1). Twin discs with an antibody concentration of 50 μg/ml were placed in the biosensor flow system [5]. To measure the noise in the PBST buffer, the dependence of the oscillation amplitude of the disk on frequency in the region of the resonance peak was determined. The measurements were repeated at intervals of 1 second. After that, for each measurement, the resonant frequency was calculated by the method of determining the center of mass. According to the measurements, a graph of the resonance frequency of the piezoceramic biochip was plotted against time (measurement number – frequency scan) (Fig.3).
In the next step, a conjugate of goat antibodies against mouse antibodies marked with peroxidase was added to the buffer solution. The ratio of the conjugate to the buffer was 1 : 4,000,000. The experiments were carried out under the same conditions. Fig.4 shows the results for a 4 mm piezoceramic disc with an antibody concentration of 50 μg/ml.
Comparing the results of measurements of different concentrations of specific antibodies to albumin (50 μg/ml and 5 μg/ml) it was found that when the concentration of antibodies immobilized on a piezoceramic disk decreases, the range of the resonance frequency changes decreases, which confirms the results obtained by immunoenzyme analysis (Fig.2). The lack of result at excess concentration of specific antibodies (100 μg/ml) deposited on piezoceramic discs is explained by the fact that several monolayers are formed on the surface, which bind to each other by free fragments. Because of this, the conjugate added to the system practically does not bind to the antibodies and does not contribute to the change in the resonant frequency. The same conclusion was confirmed by the immunoenzyme analysis.
Thus, a technique has been developed for the covalent immobilization of specific antibodies on the surface of piezoceramic biochips. In the future, biosensors can be used to identify microalbumin. The concentration of microalbumin in the sample will be determined by the shift of the resonant frequency. The obtained data testify to the possibility of applying a piezoceramic biosensor for the detection of protein biomacromolecules.
It should be noted that a significant increase in the sensitivity of the piezoceramic cantilever biosensor can be obtained by choosing the optimal geometry of the biochip. Reducing the geometric dimensions of the biochip to micron sizes will greatly improve the sensitivity of the method. The following dimensions of the biochip are technologically feasible: diameter of 50–100 microns, thickness of 5–10 microns.
The aim of this work is to check the activity of antibodies on microalbumin for further development of piezoceramic biochip, which detects microalbumin using specific antibodies. Microalbuminuria is considered a harbinger of diseases of internal organs, in particular kidneys, and can indicate the development of heart failure and vascular damage. Thus, the index of microalbumin in the urine is an important factor determining the general state of the patient's body [4].
For the study, two types of piezoceramic discs were used (Fig.1): round single piezoceramic plates with a diameter of 16 mm and a thickness of 0.1 mm and twin discs consisting of two piezoceramic plates with a diameter of 4 mm and a thickness of 0.1 mm each. Plates with a diameter of 4 mm were cut mechanically from plates of a larger size and then glued in such a way that their polarization directions were different. Connecting wires were soldered to the electrodes of the plates: two wires for disks of the first type and three for disks of the second type.
In the experiment, we used six disks with a diameter of 16 mm and three twin discs with a diameter of 4 mm. All the disks were coated with a 40 nm thick gold layer using the Q300T D (Quorum Technologies Ltd., UK) deposition system. Immediately after the deposition of gold, three twin discs with a diameter of 4 mm and three single 16 mm discs (set No.1) were placed into a solution of 4-aminothiophenol in ethyl alcohol with a concentration of 0.16 g/l, and then three 16 mm discs (set No.2) were placed into ethyl alcohol for six days at room temperature.
Mouse monoclonal antibodies (Immunotek, Russia) against human serum albumin (H-C15, Bialexa, Russia) were used. On the set of disks No.1, after modification of the surface with aminothiophenol, antibodies were applied in a solution of EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) for the covalent binding of the carboxyl group of immunoglobulins to the amino group on the carrier surface. Antibodies with EDC in an amount of 29 μl with concentrations of 5, 50 and 100 μg/ml were applied to 16 mm discs, and 5 μl of the same concentration were placed on twin 4 mm discs, after which they were left for 12 hours at –4⁰С.
The second set of disks was placed for 12 hours into a solution of cysteamine (mercaptoethylamine) at a concentration of 0.05 M. Then, a solution of antibodies with EDC in a volume of 29 μl with concentrations of 5, 50 and 100 μg/ml was applied to each piezoceramic disk.
16 mm disks from the first set were combined with a second set, after which immobilized antibodies were detected using a conjugate of goat antibodies against mouse antibodies marked by horseradish peroxidase. The incubation was carried out in PBST buffer (phosphate buffer solution with Tween-20), the substrate of tetramethylbenzidine (TMB) was used to detect the enzymatic activity of peroxidase.
Based on the results of the immunoenzyme analysis, it was shown that aminothiophenol is better suited for modifying the carrier surface than cysteamine (Fig.2).
In parallel with immunoenzyme analysis, an experiment was performed with 4 mm piezoceramic discs (set No.1). Twin discs with an antibody concentration of 50 μg/ml were placed in the biosensor flow system [5]. To measure the noise in the PBST buffer, the dependence of the oscillation amplitude of the disk on frequency in the region of the resonance peak was determined. The measurements were repeated at intervals of 1 second. After that, for each measurement, the resonant frequency was calculated by the method of determining the center of mass. According to the measurements, a graph of the resonance frequency of the piezoceramic biochip was plotted against time (measurement number – frequency scan) (Fig.3).
In the next step, a conjugate of goat antibodies against mouse antibodies marked with peroxidase was added to the buffer solution. The ratio of the conjugate to the buffer was 1 : 4,000,000. The experiments were carried out under the same conditions. Fig.4 shows the results for a 4 mm piezoceramic disc with an antibody concentration of 50 μg/ml.
Comparing the results of measurements of different concentrations of specific antibodies to albumin (50 μg/ml and 5 μg/ml) it was found that when the concentration of antibodies immobilized on a piezoceramic disk decreases, the range of the resonance frequency changes decreases, which confirms the results obtained by immunoenzyme analysis (Fig.2). The lack of result at excess concentration of specific antibodies (100 μg/ml) deposited on piezoceramic discs is explained by the fact that several monolayers are formed on the surface, which bind to each other by free fragments. Because of this, the conjugate added to the system practically does not bind to the antibodies and does not contribute to the change in the resonant frequency. The same conclusion was confirmed by the immunoenzyme analysis.
Thus, a technique has been developed for the covalent immobilization of specific antibodies on the surface of piezoceramic biochips. In the future, biosensors can be used to identify microalbumin. The concentration of microalbumin in the sample will be determined by the shift of the resonant frequency. The obtained data testify to the possibility of applying a piezoceramic biosensor for the detection of protein biomacromolecules.
It should be noted that a significant increase in the sensitivity of the piezoceramic cantilever biosensor can be obtained by choosing the optimal geometry of the biochip. Reducing the geometric dimensions of the biochip to micron sizes will greatly improve the sensitivity of the method. The following dimensions of the biochip are technologically feasible: diameter of 50–100 microns, thickness of 5–10 microns.
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