KR20160105139A - High-throughput detection method for single-to-multiple nucleotide mutations based on nanoparticle-DNA corona complex via Kelvin probe force microscopy - Google Patents

Abstract

The present invention relates to a high-throughput detection method for single to multiple nucleotide mutations based on a DNA-nanoparticle complex using kelvin probe force microscopy (KPFM) and, more specifically, to a method for identifying the mutation of genes by fixing probe DNA to nanoparticles, spreading a compound hybridized with target DNA on a substrate, and using the surface potential measured by the KPFM.

Description

TECHNICAL FIELD The present invention relates to a DNA-nanoparticle complex-based single-to-multiple nucleotide mutation based on nanoparticle-based DNA-nanoparticle complex-based Kelvin probe force microscopy,

The present invention relates to a DNA-nanoparticle complex-based ultrasensitive, high-speed single to multiple base mutation detection method using a Kelvin probe microscope, and more particularly, to a DNA / Hybridization step of hybridizing a single strand of the target DNA with a single strand of the target DNA by treating a single strand of the target DNA, and a nanoparticle-DNA corona complex having the double strand immobilized thereon, And measuring a surface potential using a Kelvin probe force microscope (KPFM), wherein the measured surface potentials are measured in the same manner The surface potential of the target DNA having a number of base mutations different from the measured normal DNA or the target DNA measured above the present invention relates to a method for detecting the presence or absence of gene mutation on a target DNA in comparison with a surface potential.

Dielectric detection is the most noticeable part of paternity testing, gene expression outlines, drug screening and disease prediction. Particularly, as Korea is progressing into a rapidly aging society, interest in health is gradually increasing. Indeed, the number of hospital users for health care and screening is increasing exponentially every year. Therefore, DNA - specific sequence detection in disease prediction to improve quality of life rather than simple life extension is required as a necessary technique for precise diagnosis.

Previous methods for DNA analysis capable of detecting single nucleotide sequence variants have been based on polymerase chain reaction (PCR) (Mun HY, et al. 2009. Gold Nanoparticle-Based Label-Free Detection of Even if the fluorescent labeling is not carried out by the BRCA1 Mutations Utilizing DNA Ligation on DNA Microarray, Journal of Nanoscience and Nanotechnology, Vol.9, 1019-1024), the polymerase chain reaction must precede and the ligation chain reaction (LCR)), and it is cumbersome and time-consuming because it requires temperature control in each reaction.

Therefore, the technology that is being popularized in recent years is to detect the complementary binding of DNA by an electric or electrochemical method. U.S. Patent No. 2006-0089825 discloses a method for detecting the interaction of biomolecules by using a nucleic acid, a polypeptide, a small molecule, or an antibody antigen binding as a probe and a target. The present invention relates to a method for detecting interaction of biomolecules through a change in contact potential difference image and a contact potential difference image showing surface topography according to intermolecular bonding between molecules, have. However, it is impossible to analyze detailed DNA mutations because it is possible to grasp the degree of the binding according to the interaction between the probe and the target, but not the binding affinity.

 A technique that uses a Kelvin probe microscope to electrically detect the binding between highly sensitive biomolecules is proposed. Using a nanoprobe, it has a resolution of less than 10 nm, a sensitivity of 50 nM and a performance of 1,100 um / s. However, this technique also has the disadvantage that one nucleotide sequence (SEQ ID NO: 1) and one nucleotide sequence (SEQ ID NO: It can not be said that it is a perfect DNA mutation detection method because it can not grasp the mutation.

DNA analysis methods developed by many researchers to date are based on polymerase chain reaction (PCR), which is very complicated and low in reproducibility. In addition, the method of detecting the complementary binding of DNA by an electric or electrochemical method for increasing the efficiency has a problem that the result is obtained with a simple preparation process, but the accuracy is somewhat lower. Therefore, there is a demand for a more precise DNA detection method capable of quickly detecting single nucleotide sequence variation by a simple method overcoming all of the above problems.

U.S. Published Patent Application No. 2006-0089825

Mun, H. Y. & Park H. G. (2009). Gold Nanoparticle-Based Label-Free Detection of BRCA1 Mutations Utilizing DNA Ligation on DNA Microarray, Journal of Nanoscience and Nanotechnology, Vol.9, 1019-1024. Sinensky, A. K. & Belcher, A. M. (2007). Label-free and high-resolution protein / DNA nanoarray analysis using Kelvin probe force microscopy, Nature Nanotechnology 2, 653-659.

In the present invention, the surface potential is measured using a Kelvin probe force microscope (KPFM) based on a DNA-nanoparticle complex in order to detect a gene mutation, and a single target DNA strand or the target A method for detecting the presence or absence of mutation of a gene on a target DNA by measuring and comparing surface potentials with other target DNAs having different numbers of base mutations from DNA.

(A) immobilizing a DNA (deoxyrebonucleic acid) single strand as a probe on the metal nanoparticle; (b) treating the single DNA strand of the target DNA with a single strand of the probe DNA and a single DNA of the target DNA (D) a step of reacting a DNA-nanoparticle complex (nanoparticle-DNA corona complex) immobilized with a double strand through a hybridization step (d) The surface potential was measured using a microscope (Kelvin probe force microscope, KPFM), and the surface potential of the target DNA measured in the same manner or the target DNA having a different number of base mutations from the target DNA measured was measured the present invention provides a method for detecting the presence or absence of a mutation of a gene on a target DNA by comparing the surface potential of a DNA- the sensitivity of the Kelvin probe microscope can be increased by using a nanoparticle-DNA corona complex to detect the presence or absence of a mutation in the target DNA on a single base sequence and further identify the base number of the target DNA.

The gene mutation detection method according to the present invention makes it possible to grasp the degree of binding between DNA-DNA (hybridized double-stranded DNA) by minimizing the binding point at which the probe can be contacted using the metal nanoparticle as a DNA binding surface , A 10 × 10 ㎛ ² Kelvin probe force microscope (KPFM) image, each of which has 2,000 or more DNA immobilized, are measured under the same conditions. Therefore, statistical analysis is possible, Is a high-sensitivity, high-speed gene mutation detection method that can identify a single nucleotide sequence variation with a high confidence level simply and quickly without labeling.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a gene mutation detection method using a Kelvin probe microscope based on a DNA-nanoparticle complex (nanoparticle-DNA corona complex) of the present invention. FIG.
FIG. 2 is a schematic diagram showing a DNA-nanoparticle complex (hereinafter referred to as pDNA coronae) in which DNA nanoparticles immobilized with DNA, a probe DNA single strand immobilized thereon, and a DNA single strand (target cDNA) complementary to a single strand of probe DNA, The DNA-nanoparticle complex (hereinafter referred to as p-cDNA coronae) in which the hybridized double-stranded DNA is immobilized is subjected to a topological image (a to c), a surface potential (surface potential) measured by a Kelvin probe microscope ) Images (d to f), and portions corresponding to each other through the phase height image and the surface potential image are shown by graphs (g to i).
3 is a DNA sequence diagram showing a 3D topology and a surface potential image (10 x 10 탆 ² ) measured by a Kelvin probe microscope and a hybridization state of the probe DNA with the target DNA (a : DNA nanoparticles (100 nm), b: pDNA coronae, c: p-cDNA coronae, d: DNA single strand (target ncDNA) hybridizing with single strand of probe DNA hybridizes (Hereinafter referred to as " p-ncDNA coronae ") in which double-stranded DNA is immobilized, e: double-stranded DNA in which probe DNA is hybridized with target DNA (m ₁ DNA) DNA- nanoparticle complex with immobilized (or less, ₁ pm DNA corona (coronae)), f: 2 of DNA sequence variation in the target DNA (DNA ₂ m) and the probe DNA is hybridized double-stranded DNA is immobilized DNA-nanoparticle complex (hereafter, pm ₂ DNA coronae), g: target DNA having three base sequence mutations ( m ₃ DNA) and a DNA-nanoparticle complex (hereafter, pm ₃ DNA coronae) in which double-stranded DNA hybridizing probe DNA is immobilized, h: a target DNA (m ₄ DNA) and the probe DNA are DNA- nanoparticle complex is the double stranded DNA hybridization is immobilized (hereinafter, DNA ₄ pm coronal (coronae)), i: 5 of sequence variation the target DNA (DNA ₅ m) in the probe DNA is DNA- nanoparticle complexes in the hybridized double-stranded DNA is immobilized (hereinafter, DNA ₅ pm coronal (coronae)).
Figure 4 is a graph illustrating the statistical analysis of the measurements of the DNA- KPFM nanoparticle complexes (DNA-nanoparticle complex corona) for detection of gene mutations (p-cDNA corona (coronae), ₁ pm DNA corona (coronae), pm ₂ histograms of the phase heights and surface potentials of DNA coronae, pm ₃ DNA coronae, pm ₄ DNA coronae, pm ₅ DNA coronae, and p-ncDNA coronae, Gaussian fitting curve (a, b) and box plot and t test (c, d)).
Figure is a graph showing the distribution analysis of the surface potential of the 5 target DNA concentrations (a, d, g: p -cDNA corona (coronae), b, e, h: pm 1 DNA corona (coronae), c, f, i : p-ncDNA corona (coronae), j, kl: two different states quantify the ratio of (single-stranded or double-stranded corona (coronae)) (j: pDNA , k: m 1 DNA, l; ncDNA)).
Fig. 6 is a schematic diagram of a structural change of a DNA molecule by DNA-DNA hybridization. Fig.

A) immobilizing a single strand of DNA (deoxyrebonucleic acid) as a probe in metal nanoparticles; (b) a hybridization step of treating a single strand of the target DNA to induce a single strand of the probe DNA and a single strand of the target DNA; (c) reacting a nanoparticle-DNA corona complex with a double strand immobilized on a metal substrate through a hybridization step; And (d) measuring a surface potential using a Kelvin probe force microscope (KPFM), wherein the measured surface potential is measured in the same manner as the normal DNA Or a surface potential of another target DNA having a different number of base mutations from the measured target DNA, thereby detecting the presence or absence of mutation in the target DNA.

In one aspect of the present invention, the single strand of the probe DNA is a single strand that binds complementarily to a normal DNA single strand.

In one aspect of the invention, the amount of probe DNA single strand or target DNA single strand required for detection may be less than or equal to 100 μl.

In one embodiment of the present invention, the metal nanoparticles may be gold (Au) nanoparticles, and in this case, the size of the gold nanoparticles may be 100 nm or less. If the size of the gold nanoparticles exceeds 100 nm, the nanoparticle characteristics may disappear, making it impossible to prepare particles bound with the bioactive material.

In one embodiment of the present invention, the metal substrate may be a gold (Au) substrate. When gold nanoparticles having a size of 100 nm or less are used, a Kelvin probe microscope (Kelvin probe force microscope (KPFM)) for the measurement of nanoparticle-DNA corona complexes.

In one embodiment of the present invention, in step (a), the single strand of the probe DNA immobilized on the metal nanoparticle may be a thiol-modified probe DNA.

In one embodiment of the present invention, in the step (a), the probe DNA may be DNA for detecting the BRCA1 gene by making a complementary binding with normal BRCA1 .

In one embodiment of the present invention, a single strand of the probe DNA immobilized on the metal nanoparticles in the step (a) is dropped on a metal substrate so that a single strand of the probe DNA is absorbed by electrostatic interaction with the surface of the metal substrate .

In one aspect of the invention, a single point or more mismatched sequence of a single DNA strand of a target DNA can be detected using the method according to the present invention.

In one aspect of the present invention, the surface potential due to hybridization of the target DNA (normal) completely complementary to the probe DNA is about twice the surface potential for the single strand of probe DNA, and the mismatch surface potential decreases with increasing number of mismatched nucleotide sequences and the surface potential value for the target DNA, which has no complementarity with the probe DNA, is similar to the surface potential value for a single strand of probe DNA. That is, when the nucleotide sequence on the target DNA matches with the normal DNA, the surface potential becomes about twice the surface potential of the single strand of the probe DNA. As the number of nucleotide sequences mutated on the target DNA increases, the surface potential becomes smaller. If this mutation is present, it may be similar to the surface potential of a single strand of probe DNA. Here, the surface potential may mean the average value of the surface potential of the DNA-nanoparticle complexes (nanoparticle-DNA corona complexes).

In an embodiment of the present invention, several tens of nanoparticles each having more than 2,200 DNA immobilized on a Kelvin probe force microscope (KPFM) image of 10 × 10 μm ² are measured at the same conditions at a time, A high confidence level statistical analysis can be performed with a trace amount of DNA sample below ㎕.

In one aspect of the present invention, the statistical analysis may use a Gaussian distribution or the like.

In one embodiment of the present invention, the higher the ratio of single-stranded DNA (probe DNA) to double-stranded DNA calculated from the probability distribution of the measured surface potential, the lower the concentration of target DNA have.

In one embodiment of the present invention, the DNA may be a BRCA1 gene.

The present invention can also be used, in one embodiment, to detect super-highly sensitive binding between biomolecules such as RNA, protein, or carbohydrate instead of DNA.

The present invention also relates to a method for detecting the presence or absence of a mutation in a target DNA by detecting the presence or absence of a permanent gene mutation, thereby providing information necessary for cancer diagnosis.

In one aspect of the invention, the cancer is selected from the group consisting of breast cancer, cervical cancer, endometrial cancer, ovarian cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, colon cancer, rectal cancer, colon cancer, prostate cancer, testicular cancer, , Connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma and multiple myeloma blood cancers.

In one embodiment of the present invention, the cancer that can provide information necessary for cancer diagnosis by detecting the presence or absence of gene mutation on the target DNA may be breast cancer.

In the present invention, mismatch of DNA means base mutation or mismatch on a gene, and single mismatch means single base mutation on a gene.

In the present invention, the term "nanoparticle-DNA corona complex" or "DNA corona" means a nanoparticle having a DNA single strand or double strand bound thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail by way of examples according to the present invention, but the scope of the present invention is not limited by the following examples and the like.

EXAMPLES Detection of BRCA1 gene mutations associated with breast cancer

1. Materials and methods of implementation

1.1. Probe DNA fixation on gold nanoparticles

1.1.1. Gold (Au) nanoparticles

100 nm gold (Au) nanoparticles from Sigma Aldrich are suspended in 1 mM PBS at a molecular weight of 196.97 g / mol and are present in a suspension of 3.45 × 10 ⁹ to 4.22 × 10 ⁹ nanoparticles per mL.

1.1.2. DNA solution

Oligonucleotides (Oligonucleotides, Bio Basic Canada, Ontario, Canada) with 1 to 5 base mutations in the BRCA1 gene were artificially prepared with the target DNA as shown in Table 1 below, And purified by high performance liquid chromatography (HPLC). The DNA was dissolved in the same amount of phosphate buffer (pH 7) to prepare a DNA solution of the same concentration.

1.2.3. When the probe DNA is functionalized with gold (Au) nanoparticles

2.5 mL of a probe DNA (base sequence: 5'-HS-CTA CCT TTT TTT TCT G-3 ') with a thiol group (-HS) attached to immobilize probe DNA on gold nanoparticles, 2.5 mL of the particles were mixed at room temperature. The mixture was vortexed vigorously for 16 hours, mixed with 15 mL of phosphate buffer, vortexed mildly for at least 40 hours, and the mixture was centrifuged to remove unreacted reagents 14,000 rpm, 25 min, room temperature), 16 mL of supernatant was loaded, and 1 mL of phosphate buffer was added to prepare gold nanoparticles functionalized with probe DNA.

1.2. Hybridization of probe DNA and target DNA

2.5 mL of the same concentration of target DNA (for example, cDNA, m ₁ DNA, m ₂ DNA, m ₃ DNA, m ₄ DNA, m ₅ DNA, and ncDNA) and ₅ mL of the probe DNA-functionalized gold nanoparticles were mixed The mixture was vortexed vigorously at room temperature for 3 hours to induce hybridization, followed by addition of 12.5 mL of phosphate buffer, followed by centrifugation in the same manner as above to remove unhybridized DNA.

1.3. A nanoparticle-DNA corona complex reaction was performed on a gold (Au) substrate.

1.3.1. Gold (Au) substrate

A silicon dioxide wafer (Silicon Technology, Tokyo, Japan) was rinsed with a piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 1: 1 (v / v)) for at least 10 minutes at room temperature Chromium and gold were sequentially deposited on a silicon substrate using a thermal evaporator and then cut into a 10 × 10 mm ² gold / chromium / silicon substrate (Au (200 Å) / Cr (50 Å) / Si substrates.

1.3.2. Adsorption of nanoparticle-DNA corona complex on gold substrate

1.2. 100 μL of nanoparticle-DNA corona complexes hybridized with probe DNA and target DNA were placed on a gold substrate prepared in 1.3.1, so that the DNA-nanoparticle complex was adsorbed onto the gold substrate for 1 hour. , Rinsed with deionized water, and gently dried with a nitrogen gun to prevent DNA-nanoparticle complexes adsorbed on the gold substrate from clumping together.

1.4. Measurement of surface potential

Measurements of topology and surface potential were performed using commercial AFM (Atomic Force Microscopy, Multimode V, Veeco, CA, USA) and surface potentials were measured in lift mode KPFM. -PIT conductive tips (conducting tips, Bruker, CA, USA) were mounted on a MMEFCH tip holder (Veeco) to control the voltage.

2. Conducted Results

2.1. Properties of nanoparticle-DNA corona complex using KPFM

2.1.1. Phase height measurements of DNA-free, pDNA, and p-cDNA gold nanoparticles using KPFM

(P-cDNA) gold nanoparticles hybridized with target DNA (cDNA) complementarily complementary to probe DNA, and gold nanoparticles without pDNA The topological heights were measured as 89.0 ± 7.3 nm, 107.0 ± 6.6 nm, and 108.5 ± 8.0 nm, respectively. The pDNA and p-cDNA showed relatively higher phase heights than the DNA-free gold nanoparticles, which may be due to the wobbling effect. The gold nanoparticles to which the DNA was bound were strongly adsorbed on the substrate rather than the gold nanoparticles, so that the AFM defects such as the wobbling effect were removed, resulting in a slightly higher height (FIG. 2).

2.1.2. Measurement of surface potential of pDNA gold nanoparticles and p-cDNA gold nanoparticles using KPFM

Surface potentials of (p-cDNA) gold nanoparticles hybridized with probe DNA-functionalized (pDNA) gold nanoparticles and target DNA complementarily complementary to probe DNA (normal) -0.743 V, and -1.455 V, respectively. This means that the surface potential of the p-cDNA is about two times the surface potential of the pDNA, which means that all of the probe DNAs on the gold nanoparticles are completely hybridized complementarily to the target DNA (normal). Therefore, it has been confirmed that the nanoparticle-DNA corona complex for the surface potential measurement of the present invention is sufficiently suitable for measuring the hybridization degree of DNA despite the steric hindrance between DNA-DNA.

2.2. Detection of base mutation of gene mutation based on DNA-nanoparticle complex (nanoparticle-DNA corona complex) using KPFM

2.2.1. Image of three-dimensional (3D) phase and surface potential according to the type of target DNA

(M ₁ DNA, m ₂ DNA, m ₃ DNA, m ₄ DNA, m ₅ DNA) with no more than 1 to 5 nucleotide sequence variations, and no complementary DNA-nanoparticle complexes (nanoparticle-DNA corona complexes) hybridized with target DNA (ncDNA) and probe DNA (pDNA) were hybridized with KPFM at a scanning speed of 10 μm · s ^-1 An image of the dislocation was obtained (Fig. 3).

As a result, the DNA-nanoparticle complexes (nanoparticle-DNA corona complexes) were protruded with a positive value in the phase, while the surface potential image showed a negative conical shape with a negative value. (Fig. 3). ≪ tb >< TABLE >

2.2.2. Negative Control Experiment: Surface potential for target DNA (ncDNA) with no complementarity

For accurate gene mutation analysis, KPFM analysis was performed on DNA-nanoparticle complexes (nanoparticle-DNA corona complexes) hybridized with target DNA (ncDNA) and probe DNA (pDNA) without negative complement control.

As a result, the surface potential distribution of p-ncDNA was similar to that of pDNA when only a single strand of probe DNA was bound to gold nanoparticles. Specifically, the average of p-ncDNA surface potentials was about 0.747 V, and the average of pDNA surface potentials was about 0.743 V. In other words, it was confirmed that the surface potential for p-ncDNA was hardly changed from the surface potential for pDNA because the target DNA did not interact with the probe DNA at all if it had no complementarity.

2.2.3. Surface potential according to the number of mutated bases

A DNA-nanoparticle complex (hybridization) in which target DNA (m ₁ DNA, m ₂ DNA, m ₃ DNA, m ₄ DNA, m ₅ DNA) and probe DNA (pDNA) hybridized with 1 to 5 base mutations (nanoparticle-DNA corona complexes) were analyzed by KPFM.

As a _{result, p-cDNA, pm 1 DNA} , pm 2 DNA, pm 3 DNA, pm 4 DNA, pm 5 DNA, and the average of the surface potential of the p-ncDNA are each 1.455 V, 1.410 V, 1.334 V , 1.156 V , 0.912 V, 0.748 V, and 0.747 V, the surface potential decreased with increasing number of bases.

2.2.4. Statistical analysis by KPFM measurement

All types of the target DNA (cDNA, DNA m _1, m ₂ DNA, DNA m _3, m ₄ DNA, DNA ₅ m, ncDNA) with probe DNA (pDNA) is (nanoparticle- respective DNA- nanoparticle complexes each hybridization 30 to 55 DNA corona complexes were randomly selected, and the phase height and surface potential measured by KPFM were represented by a Gaussian fitting curve suitable for a histogram and a histogram, and a box plot was used for statistical analysis Respectively.

As a _{result, p-cDNA, pm 1 DNA} , pm 2 DNA, pm 3 DNA, pm 4 DNA, pm 5 DNA, and 107.0 ± 6.6 nm, respectively to increase the phase of the p-ncDNA, 103.8 ± 5.3 nm , 104.4 ± 4.5 nm , 105.3 ± 6.5 nm, 105.2 ± 3.9 nm, 106.3 ± 7.7 nm, and 103.5 ± 6.1 nm, respectively, indicating that the probe DNA does not significantly affect the phase height regardless of the interaction with any target DNA (FIG.

On the other hand, the surface potential of the nanoparticle-DNA corona complex hybridized with the target DNA (tDNA) and probe DNA (pDNA) was found to be highly dependent on the number of mutated bases on the target DNA. Specifically looking in, represented by p-cDNA, pm ₁ DNA, pm ₂ DNA, pm ₃ DNA, pm ₄ DNA, pm ₅ DNA, and the mean and standard deviation of a suitable Gaussian curve the surface potential in the histogram for the p-ncDNA statistical The values were -1.455 0.026 V (p-cDNA), -1.379 0.036 V (pm ₁ DNA), -1.334 0.041 V (pm ₂ DNA), -1.156 0.044 V (pm ₃ DNA), -0.912 0.055 V (pm ₄ DNA), -0.748 ± 0.034 V (pm ₅ DNA), and -0.747 ± 0.041 V (p-ncDNA), indicating that the absolute value of the surface potential decreases as the number of bases displaced on the target DNA increases (Fig. 4B).

2.2.5. The significance of the distinction between the two groups with a difference of 1 in the number of mutated nucleotides through box plot and t-test

The surface potential data according to the present invention was used to perform box plot and t-test using the measured surface potential data in order to confirm whether the two groups having a difference of one base can be distinguished through the surface potential data Respectively.

As a result, since the median of the surface potentials according to the type of target DNA in the box plot showed a clear distinction from each other, it is easy to distinguish all two groups in which the number of mutated bases is 1 (Fig. 4D).

In addition, the significance (P-value) of the mean surface potential between all two groups with a single base difference of 1 in the t-test was less than 5% The t-test confirmed that there was a significant difference between the mean values of the group surface potentials (Fig. 4d).

Therefore, it has been confirmed through the box plot and the t-test that it is possible to distinguish between two groups in which the number of bases shifted through the surface potential data according to the present invention is one difference, Surface potential measurements on nanoparticle-DNA corona complexes in which target DNA (tDNA) and probe DNA (pDNA) are hybridized can be used to distinguish only one base level mutation from a specific gene, It is possible to accurately distinguish between single and / or multiple mutations of the sequence.

2.2.6. The surface potential distribution of the DNA-nanoparticle complex (nanoparticle-DNA corona complex) according to the target DNA concentration

For further evaluation of the detection method of the present invention, three different concentrations of 768 pM, 7.68 nM, and 76.8 nM were used as the target DNA (cDNA), single base Detection was carried out using a DNA-nanoparticle complex (nanoparticle-DNA corona complex) -based KPFM of the present invention in the case of the target DNA (m ₁ DNA) having mutated sequence and the target DNA (ncDNA) having no complementarity.

As a result, the distribution of surface potentials for DNA-nanoparticle-DNA corona complexes hybridized with probe DNA (pDNA) at low concentrations (7.68 nM and 768 pM) of target DNA (cDNA or m1 DNA) In contrast, when the target DNA was hybridized to a sufficient concentration (76.8 nM), it showed a Gaussian peak. In the case of the target DNA (ncDNA) having no complementarity, There was no change in surface potential (Fig. 5). This means that if the target DNA to be hybridized is not present at a sufficient concentration, DNA is present in the DNA-nanoparticle complex in two states, a single strand and a double strand.

On the other hand, there was no intermediate surface potential distribution between single-stranded DNA and double-stranded DNA distribution in the surface potential distribution. This is because the hybridization on the nanoparticle-DNA corona complex takes place randomly but occurs in a cascade manner, and the onset of DNA hybridization is caused by the conformational change of the DNA molecule, steric hinderance. As a result, the nanoparticle-DNA corona complex is an all or nothing hybridization model in which the target DNA is fully or completely hybridized. It was confirmed that the reaction occurred.

In addition, there was a small size difference between the distributions of surface potentials depending on the type of target DNA (cDNA and m ₁ DNA) or concentration. The difference according to the type of target DNA (cDNA and m ₁ DNA) is the numerical difference of the surface potential. The distribution of the target DNA (m ₁ DNA) having a single base sequence mutation than the target DNA (cDNA) Showed a low surface potential. This is because the binding affinity of the target DNA (m ₁ DNA) having a single base sequence mutation to the probe DNA is lower than that of the target DNA (cDNA) which makes perfect complementary hybridization as described above, The interaction between the target DNA (m ₁ DNA) is weaker and the surface potential becomes smaller.

In order to quantify the difference depending on the concentration of the target DNA, the ratio of the single strand DNA to the double strand DNA is shown by a ratio table of the probability distribution (Fig. 5j-i).

As a result, the ratio of single-stranded DNA to double-stranded DNA increased as the concentration of probe DNA decreased.

Therefore, in the detection method according to the present invention, as the number of mutated bases on the target DNA hybridized with the probe DNA decreases, the absolute value of the surface potential becomes smaller, thereby making it possible to clearly grasp the number of bases mutated on the gene, It was confirmed that the concentration of target DNA can be estimated through the ratio of single stranded DNA to double stranded DNA depending on the concentration of DNA.

3. Conclusion

Through the above example, a DNA-nanoparticle-DNA corona complex and a Kelvin probe force microscopy (KPFM) bond can be used to accurately identify one to five base mutations on the same gene It is confirmed that it is a useful technique. Nanoparticles immobilized with probe DNA were used as a nanoscale platform for detecting target DNA. In addition, the phase height according to the number of mutated bases of the target DNA does not show any appreciable change through the measurement data of the DNA-nanoparticle complex (nanoparticle-DNA corona complex) using KPFM, but the surface potential is changed by the sugar-phosphate backbone It was confirmed that as the number of mutated bases increased due to negative charge DNA, it decreased steadily. Thus, it was used as a very useful practical physical term that allows precise detection of the surface potential of a DNA-nanoparticle complex to a single base level variation. The detection method according to the present invention is advantageous in that it is quick and reproducible in a simple preparation process and statistical analysis of several tens DNA-nanoparticle complexes is realized by one KPFM imaging And suggests ultra-high sensitivity and fast detection for specific mutations of the nucleotide sequence on the gene. Furthermore, the detection method according to the present invention can not only detect the relationship between disease expression and base mutation, but also detect other biomolecules that could not be detected by conventional methods.

Claims (15)

(a) immobilizing a single strand of DNA (deoxyrebonucleic acid) as a probe in metal nanoparticles;
(b) a hybridization step of treating a single strand of the target DNA to induce a single strand of the probe DNA and a single strand of the target DNA;
(c) reacting a nanoparticle-DNA corona complex with a double strand immobilized on a metal substrate through a hybridization step; And
(d) measuring the surface potential using a Kelvin probe force microscope (KPFM)
The surface potential of the target DNA is compared with the normal DNA measured in the same manner or the surface potential of the target DNA having a different number of base mutations from the measured target DNA, / RTI >
The method according to claim 1,
Wherein the probe DNA single strand is a single strand that binds complementarily to a normal DNA single strand.
The method according to claim 1,
Wherein the amount of probe DNA single strand or target DNA single strand required for detection is not more than 100 占 퐇.
The method according to claim 1,
Wherein the metal nanoparticles are gold (Au) nanoparticles.
5. The method of claim 4,
Wherein the gold (Au) nanoparticles have a particle size of 90 to 100 nm or less.
The method according to claim 1,
Wherein the metal substrate is a gold (Au) substrate.
The method according to claim 6,
Wherein the gold (Au) substrate has a thickness of 18 to 20 nm or less.
Wherein the single strand of the probe DNA immobilized on the metal nanoparticle in the step (a) is a thiol-modified probe DNA.
The method according to claim 1,
Characterized in that in step (a), a single strand of probe DNA immobilized on the metal nanoparticle is dropped on a metal substrate and the single strand of probe DNA is absorbed by electrostatic interaction with the surface of the metal substrate. A method for detecting the presence or absence of an upper gene mutation.
The method according to claim 1,
And detecting a mismatch of a single point or more of a single strand of the target DNA.
The method according to claim 1,
Wherein the surface potential is decreased as the number of mismatched base sequences increases.
The method according to claim 1,
Characterized in that the higher the ratio of single-stranded DNA (probe DNA) to the double-stranded DNA calculated from the probability distribution of the measured surface potential is the lower concentration of the target DNA, A method for detecting the presence or absence of a mutation.
A method for providing information necessary for cancer diagnosis by detecting the presence or absence of gene mutation on a target DNA by the method according to claim 1.
14. The method of claim 13,
Wherein said cancer is selected from the group consisting of breast cancer, cervical cancer, endometrial cancer, ovarian cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, colon cancer, rectal cancer, colon cancer, prostate cancer, testicular cancer, bladder cancer, Wherein the cancer is selected from the group consisting of brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma and multiple myeloma blood cancers.
14. The method of claim 13,
Wherein the cancer is breast cancer.

Images