KR100561911B1 - Apparatus for analyzing of characteristics of biomolecules - Google Patents

Abstract

The present invention includes a biomolecule having a charge placed between the first electrode and the second electrode, and measures the change in capacitance measured as the difference in the amount of charge between the two electrodes changes, thereby measuring the chemical bonding between the biomolecules. It provides an analysis device for analyzing the. The distance between the two electrodes can be adjusted using the micrometer principle, providing the advantage of convenience.

Capacitance, Biomolecule, DNA, SNP

Description

Apparatus for analyzing of characteristics of biomolecules}

1 is a schematic configuration diagram of an apparatus for characterizing biomolecules according to a preferred embodiment of the present invention.

FIG. 2 is a schematic diagram for explaining an application example for DNA analysis (eg, genotyping) in a device for characterizing biomolecules according to an embodiment of the present invention.

3 is a conceptual diagram illustrating a basic principle in the C-V measurement according to an embodiment of the present invention.

4A, 4B, and 4C are conceptual views for conceptually illustrating that a biomolecule characterization apparatus may be manufactured using a MOS structure.

5 to 7 are capacitance-voltage curves for explaining the principle of measuring a characteristic of a biomolecule by observing a change in capacitance by changing a voltage on an electrode.

The present invention includes biomolecules having electrical properties between the first electrode and the second electrode, and analyzes the characteristics of the biomolecules by measuring capacitance values measured as the difference in the amount of charge between the two electrodes changes. It provides a biomolecule characterization device.

The interaction between biomolecules is basically the reaction between protein, DNA and RNA. DNA, RNA, and protein interactions are based on southern analysis (DNA to DNA), western analysis (protein to protein), and northern analysis (DNA, RNA to RNA) methods. Methods, other detection methods, and giving methods have been mainly used.

In this way, not only gene function studies, mutation studies, cell signaling, and other molecular biological studies but also diseases diagnosis can be performed.

By analyzing DNA and its interactions, gene detection and disease prediction can be performed. After the reaction between the DNA detector and the target DNA, the results are analyzed before and after the reaction. When the DNA to be detected (Genomic DNA, phage library, metagenome ....) is fixed on a solid surface and the labeled detector is reacted, the affinity is different depending on the sequence complementarity. Diagnosis such as detection and disease prediction becomes possible. The detection method may vary depending on the type of label. In this way, a commercialized DNA chip has been developed and sold by many companies, and various content development will be the main flow of future research.

 The interaction between protein and protein, protein and DNA can also be applied to the above principles, such as pregnancy diagnosis, hepatitis diagnosis and various diagnostic kits have already been applied. Analytical detection methods include various methods such as fluorescence, gel mobility shift, precipitation, and chromatography.

There are a variety of methods for measuring the interaction of biomolecules and each has advantages and disadvantages. The advantage has been developed and used in the past, so that the desired measurement can be made easily by the optimal protocol, but the disadvantage is that the whole analysis takes a long time and depending on the method, the reagent cost is not small.

In order to solve the above problems, it is an object of the present invention to provide a new device for measuring the properties of biological molecules.

Another object of the present invention is to fix the same molecule to the thin film in advance and to change the reaction sample so that the operation of comparing the amount of specific molecules in the two samples, comparing the properties of the specific molecules.

Another object of the present invention is to use the capacitance value and the change of the value in the measurement to examine the interaction of the fixed molecule and the target molecule in the fluid sample.

It is still another object of the present invention to provide an apparatus for detecting an intermolecular reaction by measuring the capacitance of the remaining reactant on the metal thin film before and after the reaction by interaction between charged biomolecules.

As a technical means for achieving the above object, one side of the present invention is fixed to at least one of the first electrode, the second electrode formed to be spaced apart from the first electrode a predetermined distance, the first electrode and the second electrode Capacitance measured as a change in the amount of charge between the first electrode and the second electrode by the reaction of the first biomolecule, including the first biomolecule that is charged, the sample containing the second biomolecule to be analyzed A biomolecule characterization apparatus for analyzing the degree of interaction between the first biomolecule and the second biomolecule using the value of is provided.

Preferably, the spaced distance between the first electrode and the second electrode is configurable to be variable, and a semiconductor may be attached to the first electrode. The distance between two electrodes can be finely adjusted like the principle of micrometer, and the desired pattern of biomolecules can be obtained through the change pattern of capacitance.

The biomolecule may be composed of charged components such as charged DNA, RNA, protein, or charged synthetic compounds.

Preferably, the reaction sample to be submitted is compared to compare the amount or nature of the specific molecule in the two samples. For example, anchoring one of the two interacting biomolecules to an electrode surface and exposing it to a sample containing the other biomolecule creates a bond between the two affinity biomolecules, Detection can be made by measuring the capacitance. The greater the affinity, the greater the amount of specific molecule binding to the detector, resulting in greater capacitance.

Hereinafter, an apparatus for characterizing a biomolecule according to a preferred embodiment of the present invention will be described. The present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, only the embodiments to complete the disclosure of the present invention and to those skilled in the art to fully understand the scope of the invention It is provided to inform you.

1 is a schematic configuration diagram of an apparatus for characterizing biomolecules according to a preferred embodiment of the present invention.

Referring to FIG. 1, the apparatus for characterizing a biomolecule according to an exemplary embodiment of the present invention may include a first electrode 10, a second electrode 20 formed to be spaced apart from the first electrode 10 by a predetermined distance, and a first electrode. And a biomolecule 40 having a charge placed between the electrode 10 and the second electrode 20. The characteristics of the biomolecules may be analyzed by measuring capacitance values measured as the difference in the amount of charge between the first electrode 10 and the second electrode 20 is changed. The electrodes are preferably made of metal.

Meanwhile, the semiconductor layer 30 may be fixed to the surface of the first electrode 10 in advance, and the amount or properties of specific molecules in the two samples may be compared by varying the reaction samples. A semiconductor may or may not be attached to the first electrode 10. The distance between the electrodes has a micrometer structure that is usually used for thickness measurement so that the distance can be finely adjusted.

Figure 2 is a schematic diagram illustrating an application example for DNA analysis in the apparatus for characterizing biomolecules according to an embodiment of the present invention.

Referring to FIG. 2, the apparatus for analyzing the characteristics of the biomolecule includes a first electrode 100, a second electrode 110, insulating layers 120 and 130, detector molecules 150 and 160, and a detection target 170.

First, the probe molecules 150 and 160 are fixed to the surfaces of the insulating layers 120 and 130. For example, in case of DNA, thiol, amine, carbosyl group, biotin, etc. are attached to fix the DNA, and the protein can be fixed using its own functional group or immobilization antibody. ) Can be used.

Next, after passing the fluid including the detection target 170 to the thin film on which the detector molecules are attached, the non-specific reactants are removed using a salt-free washing solution. After drying to remove moisture from the fluid, the backside of the silicon semiconductor can be attached to one electrode of the micrometer.

Then, after measuring the capacitance-voltage characteristic, the amount of DNA is measured. In addition, the capacitance according to the distance can be measured by moving the electrode that can adjust the left and right fine displacement. The more interaction between the detector and the target in the thin film, the more molecules to be detected and the shorter the distance between the two electrodes, the higher the capacitance.

Hereinafter, the operation principle of the device for analyzing the characteristics of biomolecules according to the present invention will be analyzed in detail. 3 is a conceptual diagram illustrating a basic principle in C-V measurement.

If the space between the two plates 210,220 of the capacitor is empty or full of air, the capacitor has about the same capacitance. This is because the dielectric constants of vacuum (dielectric constant 1) and air (dielectric constant (1.0006) are similar. Therefore, you can use air as the reference value of the capacitance. Now if you put a substance between the two plates of the capacitor, the capacitance of the capacitor Increasingly, these materials are called genomes, and DNA, in a general sense, belongs to genetic material.

The charged capacitor is connected to a voltmeter capable of measuring the potential difference between the two plates 210 and 220. Inserting dielectric material 230 here reduces the potential difference. In addition, the value of C (capacitance) is increased by decreasing V (voltage) while keeping Q (charge amount) constant in the definition of C = Q / V. The charge Q is invariant. The voltage will return to its original position when the dielectric material is removed again. At this point, the voltmeter has returned to its original position, so V or C will return to its original value.

The reason why the dielectric increases the capacitance of the capacitor is due to the polarization of the dielectric. The molecules are normally neutral. When placed in an electric field, electrons are charged toward the positive electrode and the nucleus toward the negative electrode. The electrons in the insulator cannot move freely, but may have some displacement in parallel, so that the local electric field generated by the bimolecules is created in the opposite direction to the original electric field. As a result, the effective charge of the capacitor is reduced without changing the original charge. This results in a reduction of the effective electric field. If we consider DNA as a kind of genome, we can observe the increase of capacitance C due to the polarization of DNA in the electrode and analyze the change in capacitance C value according to the amount of DNA in the electrode.

4A, 4B, and 4C are conceptual views for conceptually illustrating that a biomolecule characterization apparatus may be manufactured using a metal oxide semiconductor (MOS) structure. That is, in FIG. 4, MOS, metal air semiconductor (MAS), and metal DNA semiconductor (MDS) structures are compared, and negative charges in the oxide film, air, and DNA are shown between the electrodes.

Referring to FIG. 4A, the MOS device includes a silicon substrate 310, an oxide film 320, and a metal film 330. As a method of analyzing the charge component in the oxide film 320, a voltage is applied to the electrodes of the metal film 330 and the silicon substrate 310 to observe a change in capacitance caused by the amount of charge in the oxide film 320. By measuring the change in capacitance according to the change in voltage, the flat band voltage Vfb of the silicon substrate, the charge density in the oxide film 20, and the like can be calculated.

Referring to FIG. 4B, the oxide film 320 is replaced with an air layer having a predetermined thickness among the components of the MOS device. This is called a MAS structure because it is similar to the MOS structure. A is the initial character of AIR. The dielectric constant of the oxide film 320 is about 10 and the dielectric constant of air is approximately 1. The difference between the two dielectrics is that a large capacitance is measured in the oxide film having a high dielectric constant with respect to the same voltage across the electrode, and 1/10 capacitance is generated in the air compared with the oxide film.

Referring to FIG. 4C, if DNA is used as a dielectric instead of air in the MAS structure of FIG. 4B, a metal / DNA / semiconductor (MDS) structure may be obtained. In this case, knowing the dielectric constant of the DNA, the thickness of the dielectric, the area of the electrode, and the applied voltage, the capacitance of the DNA can be known. According to the qualitative analysis method of the charge amount in the MOS structure and the MDS structure, it is possible to obtain the inverse capacitance-voltage curve which observes the change in capacitance by changing the voltage.

5 is a typical capacitance-voltage curve that observes a change in capacitance by varying the voltage across the MOS structure.

The semiconductor layer used is a P-type silicon semiconductor with a majority carrier of holes. Thus, the minority carrier becomes an electron. The solid line is the C-V (Capacitance-Voltage) curve of a typical thermal oxide film. If there is a negative charge in the oxide film, the curve moves to the right. This is because negative charges in the oxide film attract holes in the semiconductor-side interface, which are majority carriers, which are evenly distributed in the bulk of the semiconductor layer. Similarly, the more negatively charged DNA, the more the C-V curve shifts to the right.

In general, examples of measurement voltages used for C-V measurement include a DC signal as a large signal, an AC voltage as a small signal, a DC only, or a method of sending AC to DC.

Meanwhile, referring to FIG. 6, since the degree of movement of the curve to the right varies depending on the amount of negative charge, the amount of negative charge can be calculated by changing the flat band voltage Vfb. In this case, however, the semiconductor is a P type. This principle is known in the art.

On the other hand, referring to Figure 7, if the MDS structure instead of the oxide film can be formed, the charge density in the DNA layer can be calculated. Since DNA has a negative charge in the phosphate structure, the amount of negative charge is determined according to the amount of DNA so that the amount can be easily determined in the capacitance-voltage measurement. 7 may be interpreted by inferring the case of FIG. 6 as the amount of negatively charged DNA increases. On the other hand, if the dielectric has a positive charge, the C-V curve will shift to the left, but DNA is generally known to have a negative charge, so it shifts to the right.

On the other hand, the present invention is developed by dividing the main body and the cartridge, the measurement related device is mounted on the main body, it will be able to be configured as a chip in the fluidic control (fluidic control) in the cartridge.

Since multiple electrodes / probes / electrode structures can be manufactured in a single cartridge using a semiconductor process, the interaction between various molecules can be measured simultaneously. On the other hand, the use of disposable cartridges can minimize the cost of testing and measurement, and it can be considered to have the advantage of saving time.

 Biomolecules such as DNA, RNA, and proteins interact with high specificity for complementary sequences or specific molecules. When one of the two interacting biomolecules is immobilized on a metal thin film surface and exposed to a sample containing other biomolecules, a bond is formed between the two molecules with mutual affinity, which is measured by measuring the capacitance under the voltage applied to both sides. Detection is possible.

 For example, genotyping such as SNP analysis can be performed by using the hybridization of DNA probe and target DNA, and the DNA probe and sequence specific DNA binding protein can be searched and qualitatively analyzed. .

Efforts to easily detect, miniaturize and enable mass analysis of these reactions are growing rapidly in the development of diagnostic and research equipment, and are expected to grow even more in the post-genome era.

As one technique for analyzing the interactions between biomolecules, the present invention is expected to be manufactured in a chip form using a silicon process, and thus, it is expected to have advantages of low cost and short analysis time through mass production.

Claims (11)

A first electrode; A second electrode formed to be spaced apart from the first electrode by a predetermined distance; And A first biomolecule having a charge fixed to at least one of the first electrode and the second electrode, The first biomolecule and the first biomolecule are reacted by the reaction between the sample containing the second biomolecule to be analyzed and the first biomolecule, and the capacitance is measured according to the change in the amount of charge between the first electrode and the second electrode. Characterization apparatus of the biomolecule, characterized in that for analyzing the degree of interaction of the second biomolecule. The method of claim 1, Characterized in that the distance between the first electrode and the second electrode is a biomolecule characterized in that the variable. The method according to claim 1 or 2, The first electrode is a characteristic analysis device of the biomolecule characterized in that the semiconductor is attached. The method according to claim 1 or 2, The first biomolecule and the second biomolecule may be composed of charged components such as charged DNA, RNA, protein, or charged synthetic compounds. The method of claim 4, wherein When the biomolecule is negatively charged, the semiconductor is a P-type semiconductor, when the positive charge is used, the semiconductor is an N-type semiconductor. The method of claim 4, wherein And wherein the semiconductor is a P-type semiconductor when the second biomolecule is a negatively charged DNA. The method according to claim 1 or 2, Characterization of the biomolecule is characterized in that the analysis of the characteristics of the biomolecule, characterized in that using the difference in capacitance with the control group. The method according to claim 1 or 2, Characterizing apparatus for biomolecules, characterized in that the amount or properties of the specific molecules in the first biomolecule and the second biomolecule are compared. The method of claim 8, The molecular weight of the specific molecule is characterized in that the biomolecule characterization device characterized in that the affinity for the first biomolecule after the reaction. The method according to claim 1 or 2, The device of claim 1, wherein an insulator is further added to the first electrode and / or the second electrode. The method of claim 10, The insulator is a device for characterizing the biomolecule, characterized in that the material which enables the fixation of the first biomolecule.

Images