KR101586328B1 - 53 Method for Label-Free Electrical Detection of Mutant p53 Using MOSFET Biosensor - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a biosensor device for detecting mutated p53 protein and a method for detecting mutant p53 protein using the same, and more particularly, to a biosensor device using a substrate; A channel doped on the substrate; A source electrode 3 and a drain electrode 4 formed on both sides of the channel 2; An oxide layer (1) in contact with one side of the source electrode (3) and the drain electrode (4) and located above the channel (2); And a gate electrode (7) formed on the oxide layer and having DNA fixed on the surface thereof to specifically bind to p53 protein. The biosensor device for detecting p53 protein and the p53 mutant using the device And a method for detecting a protein.

According to the present invention, the mutated p53 protein can be accurately and sensitively detected, and thus it is highly likely to be used for cancer cancer monitoring.

p53, mutant, field effect transistor, MOSFET, FET, biosensor

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting mutant p53 protein using a field-effect transistor biosensor,

TECHNICAL FIELD The present invention relates to a biosensor device for detecting mutated p53 protein and a method for detecting mutant p53 protein using the same, and more particularly, to a biosensor device using a substrate; A channel doped on the substrate; A source electrode 3 and a drain electrode 4 formed on both sides of the channel 2; An oxide layer (1) in contact with one side of the source electrode (3) and the drain electrode (4) and located above the channel (2); And a gate electrode (7) formed on the oxide layer and having DNA fixed on the surface thereof to specifically bind to p53 protein. The biosensor device for detecting p53 protein and the p53 mutant using the device And a method for detecting a protein.

The p53 gene is a mutation gene that is commonly found in cancer, and it is one of the genes that act as an important factor in cancer development. P53, which is a cancer-suppressing gene, is activated by damage to the intracellular DNA, inducing a base excision repair (BER) mechanism, delaying the cell cycle, inhibiting replication of an incomplete gene And is involved in the repair of damaged DNA. In addition, when p53 is so severe that the damage of the intracellular gene can not be restored, it induces a cell apoptosis mechanism and performs a function of removing a cell which is likely to be transformed into cancer. It is known that p53 prevents the accumulation of damaged genes in the cell and inhibits the mechanism by which the damaged cells develop into cancer.

Mutated p53 protein is found in more than 50% of cancers of the total cancer, and more than 90% of them are mutated in the DNA binding domain. If the p53 protein fails to bind to DNA as a mutation of the DNA binding domain and loses its function as a trans-acting promoter of transcription, it can not inhibit the cell division of the damaged cell, thereby increasing the possibility of cancer cell death of the damaged cell . Therefore, monitoring the DNA binding capacity of normal p53 and mutated p53 proteins is of great value in the field of cancer cell research and diagnosis.

Recently, biosensor technology focuses on the development of non-labeling measurement technology that does not use labeling materials. Representative examples include a method using surface plasmon resonance occurring on a metal surface, a method using a three-dimensional microstructure, A method of measuring the change of the resonance frequency of a quartz crystal according to the change of mass of a biomolecule (Marx, MK, et al. KA., Biomacromolecules, 4 , 1099, 2003), a method for measuring the electric field effect based on a semiconductor process (Yuqing, M. et al., Biotechnol. Adv. , 21; 527, 2003) (Hanahan, G. et al. , J. Environ. Monit. , 6; 657, 2004).

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a biosensor in which a current flowing through a channel between a source and a drain is changed by an electric field effect It is a biosensor using semiconductor characteristics. In general, a field effect transistor used as a biosensor includes source and drain electrodes on both sides of an n-type or p-type semiconductor substrate, and a gate is formed in contact with the source and drain electrodes. The gate generally comprises an insulating layer, an electrode layer and a layer of metal material for attaching probe biomolecules. When the probe reacts with the homologous target on the surface of the gate electrode, a change occurs in the electric field around the gate, and the current value flowing in the channel region between the source electrode and the drain electrode changes due to the change in the electric field around the gate, The target in the sample can be detected. Because field effect transistor sensors have advantages in sensitivity, specificity, agility, portability, and size, they can be used to detect fixed-enzyme field effect transistor sensors that measure H ⁺ ion concentration, DNA-modified field effect transistor sensors that detect DNA hybridization, Cell-based field effect transistor sensors that detect dislocation or cellular metabolism are actively studied. In recent years, nanowire field effect transistor sensors with high sensitivity are becoming popular.

 Therefore, the present inventors have made intensive efforts to develop a more accurate and sensitive method for detecting mutant p53 protein. As a result, it has been found that by using a DNA having a base sequence specific to the p53 DNA binding domain and a field effect transistor, p53 protein can be detected, thereby completing the present invention.

The main object of the present invention is to provide a field effect transistor biosensor device for detecting p53 protein and a method for detecting mutant p53 protein using the same.

According to an aspect of the present invention, A channel doped on the substrate; A source electrode 3 and a drain electrode 4 formed on both sides of the channel 2; An oxide layer (1) in contact with one side of the source electrode (3) and the drain electrode (4) and located above the channel (2); And a gate electrode (7) formed on the oxide layer and having DNA fixed on the surface thereof to specifically bind to p53 protein. The biosensor device for detecting p53 protein and the p53 mutant using the device A method for detecting a protein is provided.

According to the present invention, the mutated p53 protein can be accurately and sensitively detected, and thus it is highly likely to be used for cancer cancer monitoring.

In one aspect, the present invention provides a substrate comprising: a substrate; A channel doped on the substrate; A source electrode 3 and a drain electrode 4 formed on both sides of the channel 2; An oxide layer (1) in contact with one side of the source electrode (3) and the drain electrode (4) and located above the channel (2); And a gate electrode (7) formed on the oxide layer and having a DNA binding specifically to p53 protein immobilized on its surface.

The substrate of the field effect transistor may be formed of a single layer of silicon and silicon dioxide, and the silicon dioxide layer may be formed on the silicon layer as a single layer to serve as an insulating layer.

The gate of the field effect transistor is activated by applying a thin film by an APCVD (Low Pressure Chemical Vapor Deposition) method, an LPCVD (Low Pressure Chemical Vapor Deposition) method and a PECVD (Plasma Enhanced Chemical Vapor Deposition) method. , The LPCVD method is characterized in that it operates at a high temperature, so that a PECVD method which can operate at a low temperature and can perform a rapid deposition is preferable.

A field effect transistor uses a hole as a carrier. When a gate voltage is 0V, a p-type field effect transistor and an electron are used as a carrier for the current to flow in the channel. When the gate voltage is 0.01V to 6V, Type field effect transistor, and both the p-type and n-type transistors can be used as the field effect transistor. However, since the current driving ability of the n-type field effect transistor is two times higher than that of the p-type field effect transistor, The transistor biosensor device preferably uses an n-type field effect transistor to enhance the detection effect.

In the method of immobilizing DNA on the gold thin film electrode, (11-mercaptoundecanoic acid), ion-binding poly L-lysine and 5'-aminoalkylated DNA, and a method of using EDC (1- ( 3-dimethylaminopropyl) -3-ethylcarbodiimidehydrocholide) and NHSS (N-hydroxysulfosuccinimide), and a method using mercaptoalkylated DNA and covalent poly L-lysine and mercaptoalkylated DNA induced by 1-mercaptoundecanoic acid and 1-decanethiol - N-succinimidyl-N, N, N ', N'-tetra methyluronium tetrafluoroborate and N, N-diisopropylethylamine-derived ester groups and 5'-aminoalkylated DNA. Among them, MUA, In the method using poly-L-lysine and 5'-aminoalkylated DNA, since DNA is attached to the gold surface through ionic bonding, DNA can be separated from the gold surface depending on the pH change. However, texture Since it is coupled to receive a relatively less affected by the pH.

In another aspect, the present invention relates to a method for detecting a mutant p53 protein, which uses the biosensor device of the field effect transistor.

According to an embodiment of the present invention, there is provided a method of detecting a p53 protein, comprising: (a) dropping a sample containing p53 protein on the gate electrode of the field effect transistor biosensor device and then reacting; (b) measuring a current value flowing in the channel region between the source electrode and the drain electrode; And (c) confirming whether the p53 protein is mutated using the measured current value.

According to the present invention, the current value between the source and the drain is changed according to the degree of binding of the p53-specific DNA fixed on the surface of the field-effect transistor to the normal p53 protein and the mutant p53 protein. Due to the difference in binding ability to DNA, the current value in the channel region between the source electrode and the drain electrode is steeply increased for the normal p53 protein, but the current value is not changed for the mutated p53 protein.

The n-type field effect transistor biosensor device of the present invention does not operate when the gate voltage is less than 0.01 V. When the voltage exceeds 6 V, the current flowing in the channel is shifted from the linear region to the saturation region, The gate voltage applied to the transistor biosensor device is preferably 0.01V to 6V.

In one embodiment, the core domain of the mutated p53 protein (R248W) lacking the DNA binding ability with the core domain of the normal p53 protein is prepared and the surface of the gate electrode of the field effect transistor biosensor is specifically bound to the normal p53 protein And the change of the current value between the source and the drain of the normal p53 protein or the mutant p53 protein in the sample was analyzed. As a result, a significant change in the current value was measured in response to the normal p53 protein, but the change in the current value was not observed for the mutant p53 protein. These results confirmed that DNA sequence-specific DNA-protein interactions can be monitored by field effect transistor biosensors. The above results are consistent with the results of a surface plasmon resonance biosensor currently used as a biosensor. Using the biosensor of the field effect transistor type of the present invention, It was confirmed that the interaction between DNA and protein can be monitored electronically.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Preparation of p53 Protein Binding DNA and Production of Strain Transformed with p53 Mutant Gene

(SEQ ID NO: 1) and a negative control DNA sequence (SEQ ID NO: 2) of the GADD45 promoter known to bind to p53 protein, a DNA sequence complementary to the GADD45 promoter DNA sequence (SEQ ID NO: 3) DNA sequence (SEQ ID NO: 4) complementary to the DNA sequence was prepared.

SEQ ID NO: 1: 5'-GTA CAG AAC ATG TCT AAG CAT GCT GGG GAC-3 '

SEQ ID NO: 2: 5'-TTT TCG AGA GCT AGC AAG ACT GAG GAA GCC-3 '

SEQ ID NO: 3: 5'-GTC CCC AGC ATG CTT AGA CAT GTT CTG TAC-3 '

SEQ ID NO: 4: 5'-GGC TTC CTC AGT CTT GCT AGC TCT CGA AAA-3 '

In order to replace the 248th arginine codon UGG in the p53 protein amino acid sequence with tryptophan codon CGG, PCR was performed to cause point mutation with the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6.

SEQ ID NO: 5: 5'-GGG CGG CAT GAA CTG GAG GCC CAT CCT CA-3 '

SEQ ID NO: 6: 5'-TGA GGA TGG GCC TCC AGT TCA TGC CGC CC-3 '

The p53 gene was subjected to PCR using the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 to separate the core domains containing NdeI and BamHI restriction enzyme sites.

SEQ ID NO: 7: 5'-GGA ATT CCA TAT GGC GGC CCC TGC ACC AG-3 '

SEQ ID NO: 8: 5'-CGG GAT CCA ACC CTT TCT TGC GG-3 '

PCR amplification was performed using Pfu DNA polymerase (Bioprogen, Korea). The amplified DNA was cut into fragments using NdeI and BamHI restriction enzymes and inserted into the pER21a plasmid (Novagen, Germany) Expression vectors were constructed. Plasmid purification and gel extraction of DNA fragments were performed with DNA purification kit (Bioneer, Korea). The prepared expression vector was transformed into Escherichia coli Strain Rosetta BL21 (DE3) strain (Novagen, Germany) to produce a strain producing p53 mutant protein.

Example 2: Expression and purification of mutant p53 protein

The transformed strain in Example 1 was cultured in LB medium containing 100 μg / mL of ampicillin at 37 ° C and 200 rpm. The concentration of the cell culture solution was determined by measuring the absorbance at a wavelength of 600 nm using a spectrophotometer. When the absorbance of the cell culture solution was 0.6-0.8, 1 mM IPTG and 0.1 mM zinc acetate were added to induce expression at 15 ° C for 12 hours to 16 hours. The cultured strain was centrifuged at 6000 rpm for 8 minutes, washed twice with a column-binding buffer solution (50 mM Tris-HCl, 500 mM NaCl, pH 8.0), and then disrupted with an ultrasonic disintegrator. To obtain crude cell lysate. To purify normal p53 protein and mutant p53 protein, crude cell lysate was eluted with Ni-NTA column and elution buffer (50 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, pH 8.0). Purified proteins were identified by performing 10% SDS-PAGE and dialyzed sequentially with 1X PBS (GIBCO, BRL, USA) and 0.1X PBS at 4 ° C for 12 to 16 hours to remove salts and concentrate. Concentration of protein was measured by Bradford method using BSA standard reagent.

Example 3: Detection using mutant p53 protein fluorescence assay

The gold thin film was washed with piranha solution (70% H ₂ SO ₄ , 30% H ₂ O ₂ ), washed with distilled water and ethanol, and the single-stranded thiol-modified GADD45 promoter DNA The nucleotide sequence (SEQ ID NO: 1) and the thiol-modified control nucleotide sequence (SEQ ID NO: 2) were respectively dissolved in 0.1X PBS at a concentration of 10 μM and spotted on the surface of the gold chip with BioOdyssey Calligrapher Mini Arrayer (Bio-Rad, USA) After standing at room temperature for 90 minutes, it was washed twice with 0.1X PBS for 5 minutes.

(SEQ ID NO: 3) complementary to the single-stranded thiol-modified GADD45 DNA sequence (SEQ ID NO: 1) and complementary DNA nucleotide sequence (SEQ ID NO: 2) complementary to Cy5-labeled DNA (SEQ ID NO: 4) was prepared and the single-stranded DNA immobilized on the surface of the gold chip was subjected to a cover slip reaction for 60 minutes and then washed.

The fluorescence image of the DNA chip was analyzed with a fluorescence scanner, GenPix 4200 (Axon, USA). DNA chip fluorescence image analysis showed that single-stranded thiol-modified DNA was efficiently immobilized on the gold thin film surface when tested under the same conditions as the buffer solution used for the field effect transistor biosensor measurement, At the same time.

Example 4: Detection of mutant p53 protein by SPR analysis

SPR analysis was performed using BIACORE 3000 (BIAcore AB, UK) and carboxymethylated dextran CM5 chip (BIAcore AB, UK). The standards of the sensorgram were set using PBS as a developing solvent. Neutravidin (Pierce, USA) was immersed in acetic acid (pH 4.5) at a concentration of 50 μg / ml to fix the carbodiimide-N-hydroxyl succinimide coupling reaction with the dextran carboxyl group on the chip surface. Unreacted carboxyl groups were inactivated using ethanolamine. 500 nM of DNA (SEQ ID NO: 1) whose 5 'end was labeled with biotin was flowed on the surface of the chip on which neutravidin was immobilized at a flow rate of 5 μl / min for 6 minutes and hybridization with the complementary base sequence DNA shown in SEQ ID NO: Double stranded DNA was immobilized on the chip. SPR standards were set with 0.1X PBS, and the purified normal p53 protein and the mutant p53 protein were flowed at a flow rate of 5 μl / min for 6 minutes at a concentration of 1 μM, and the SPR was measured. As a result of SPR analysis, mutant p53 protein showed about 5,500 RU (Resonance Unit) lower than that of normal p53 protein, indicating that the binding ability to DNA was extinguished when compared with normal p53 protein.

Example 5: Detection of a mutant p53 protein using a field effect transistor biosensor

An n-type field effect transistor sensor was fabricated using CMOS (complementary metal oxide semiconductor). After the gate electrode was made of gold thin films for thiol modification, each wash for 5 minutes ultrasonic the gate electrode surface with acetone, methanol and distilled water, and treated O ₂ plasma for 120 seconds to 120W enable the gate electrode, 5 ' (SEQ ID NO: 1) whose terminal was labeled with thiol was prepared at a concentration of 100 nM in 0.1X PBS and allowed to react with the surface of the gold thin film for 90 minutes to fix. The surface of the gold thin film was treated with 10 μM 6-mercapto-1-hexanol (Sigma Aldrich) dissolved in 0.1 × PBS for 60 minutes to stop the reaction and serve as a spacer. Single-stranded DNA (SEQ ID NO: 3) prepared in a concentration of 100 nM in 0.1X PBS was treated on the gate electrode for 60 minutes to allow double-stranded DNA to be fixed on the surface of the gate electrode through a hybridization reaction. The p53 protein sample was dissolved in 0.1 X PBS at a concentration of 100 nM and the solution was applied to the gate electrode. The current flowing through the channel region between the source electrode and the drain electrode was measured for 30 minutes. The measurement was performed by washing the gate electrode three times with 0.1X PBS and measuring the current value after washing the probe station of the shield box at a humidity of 50%.

As a result of measurement of the field effect transistor current value, the current value of the mutated p53 protein showed 13% of the value measured in the normal p53 protein because the mutated p53 protein can not bind to the DNA, On the other hand, in the case of normal p53 protein, p53 protein having positive charge binds to DNA to offset the negative charge effect of DNA, and at the same time, the p53 protein exhibits (+) charge effect, ≪ / RTI >

FIG. 1 shows a schematic view of a field effect transistor biosensor, wherein (A) shows a photo of a field effect transistor biosensor, and (B) shows a cross-sectional view of a field effect transistor in which a mutant p53 protein is detected.

2 shows a schematic diagram of a pET21a-p53 expression vector expressing a normal p53 protein or a mutant p53 protein [R248W], wherein (A) shows a gene map of a pET21a-p53 plasmid vector expressing p53 protein, and ) Shows SDS-PAGE results of normal p53 protein or mutated p53 protein (R248W).

 FIG. 3 is a conceptual diagram of a principle of a field-effect transistor biosensor for detecting mutant p53 protein.

Description of the Related Art

1 oxide layer

2 channels

3 source electrode

4 drain electrode

5 Silicon dioxide layer (substrate)

6 silicon layer (substrate)

7 DNA-immobilized gold thin-film gate electrode

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Label-free electrical detection of mutant p53 using          MOSFET biosensor <130> P09-B019 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> p53 binding DNA sequence <400> 1 gtacagaaca tgtctaagca tgctggggac 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> control DNA sequence <400> 2 ttttcgagag ctagcaagac tgaggaagcc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> p53 binding DNA sequence <400> 3 gtccccagca tgcttagaca tgttctgtac 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> control DNA sequence <400> 4 ggcttcctca gtcttgctag ctctcgaaaa 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer sequence <400> 5 gggcggcatg aactggaggc ccatcctca 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer sequence <400> 6 tgaggatggg cctccagttc atgccgccc 29 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer sequence <400> 7 ggaattccat atggcggccc ctgcaccag 29 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer sequence <400> 8 cgggatccaa ccctttcttg cgg 23

Claims (11)

Board; A channel doped on the substrate; A source electrode 3 and a drain electrode 4 formed on both sides of the doped channel 2; An oxide layer (1) in contact with one side of the source electrode (3) and the drain electrode (4) and located above the channel (2); And a gate electrode (7) formed on the oxide layer and having a DNA sequence having a nucleotide sequence of SEQ ID NO: 1, which specifically binds to the p53 protein, is immobilized on the surface thereof. Biosensor device. 2. The biosensor device of claim 1, wherein the substrate is composed of a single layer of silicon (6) and silicon dioxide (5). 2. The field effect transistor biosensor device of claim 1, wherein the field effect transistor is an n-type field effect transistor. The field effect transistor biosensor device according to claim 1, wherein the gate electrode is a gold thin film electrode. The method according to claim 1 or 4, wherein the gate electrode is processed by a method selected from the group consisting of APCVD (Low Pressure Chemical Vapor Deposition), LPCVD (Low Pressure Chemical Vapor Deposition) and PECVD (Plasma Enhanced Chemical Vapor Deposition) Wherein the biosensor is activated. The biosensor device according to claim 1, wherein the DNA does not bind mutated p53 protein. delete The DNA of claim 1 or 6, wherein the DNA specifically binding to the p53 protein is selected from the group consisting of a thiol group, an amine group, 11-mercaptoundecanoic acid (MUA), poly L-lysine, a mercaptoalkyl group, Wherein the biosensor is modified with a substance and fixed. A method for detecting a mutant p53 protein, comprising using the field effect transistor biosensor device of claim 1. 10. The method for detecting mutant p53 protein according to claim 9, which comprises the step of: (a) dropping a sample containing p53 protein on the gate electrode of the field effect transistor biosensor device of claim 1, and then reacting; (b) measuring a current value flowing in a channel region between the source electrode and the drain electrode; And (c) confirming whether the p53 protein is mutated using the measured current value. 11. The method according to claim 10, wherein the voltage applied to the gate electrode is 0.01 V to 0.6 V.

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