KR101003534B1 - Quantative detection method of biomolecules using magnetic nano particle and frequency mixing magnetic reader - Google Patents

Abstract

The present invention relates to a method for quantitative detection of biomaterials using magnetic nanoparticles and a frequency mixing magnetic reader. According to the detection method of the present invention, the presence of a biological material can be measured quantitatively as a detection of a signal for the number of magnetic nanoparticles, and is more stable than ELISA (Enzyme-Linked ImmunoSorbent Assay), and has similar sensitivity. Reliable data can be obtained. In addition, according to the detection method of the present invention, since the disposable strip type substrate is used as a sensor, it is very economical in terms of process and cost.

Magnetic Nanoparticles, Frequency Mixed Magnetic Readers, Strips

Description

QUANTATIVE DETECTION METHOD OF BIOMOLECULES USING MAGNETIC NANO PARTICLE AND FREQUENCY MIXING MAGNETIC READER}

The present invention relates to a method for quantitative detection of a biomaterial using magnetic nanoparticles and a frequency mixed magnetic reader. The present invention is derived from research conducted as part of the IT source technology development project of the Ministry of Information and Communication and the Ministry of Information and Communication Research and Development. [Task Management Number: 2006-S-074-02, Title: High-performance biosensor system using nanoparticles ].

Analysis of trace biomaterials is one of the most important challenges in biotechnology, medicine and chemistry. In this respect, much research has been conducted to develop easier analytical tools, and the development of innovative analytical methods is very influential enough to change the academic trends in biotechnology and related fields (eg : MALDI-TOF, HPLC-MS, PCR for macromolecules). In addition to the development of laboratory-level equipment, a lot of research has been conducted in the development of equipment that can be easily measured / analyzed in the field, such as Lab on a Chip (LoC) and System on Chip (SoC). One example is the development of chips capable of array analysis.

Among them, the Giant MagnetoResistance (GMR) developed in the 1970s had a great influence on the development of sensors as well as hard disks. For this reason, magnetic nanoparticles are: 1) much more stable in physical / chemical terms than conventional biosensor labeling materials, and 2) signal generation and capturing of target materials. It was very popular because of its ability to do it at the same time.

Conventional techniques using nanoparticles or beads include a method of immobilizing DNA using particles in which a solid support can be attached to one side of the micro or nanoparticles, and the DNA can be attached to the other side. The immobilization method described above is an immobilization method in which a large amount of DNA is attached to the surface of particles rather than immobilized on a general surface, and a large amount of DNA can be concentrated in a small area. However, in this method the particles were only responsible for the capture of DNA.

In addition, a method of making an array using beads and efficiently analyzing various types of DNA using various enzymes for DNA amplification has been disclosed. However, the method only allowed the DNA amplification reaction to occur on each bead surface.

Meanwhile, conventional techniques using magnetic nanoparticles include 1) a method using a GMR sensor, 2) a method using a spin valve, 3) a method using a Hall probe, and 4) an induction coil. coils) and 5) superconducting quantum interface devices (SQUIDs), which have the highest sensitivity.

Currently, the closest technology to commercialization is GMR, which is very expensive for each sensor, and it is very expensive to make analytical equipment according to the concentration of beads. In other methods, the detection limit itself is very low, but in reality, it is very difficult to commercialize it due to the cost and size / maintenance of equipment.

As a conventional analysis method using such magnetic nanoparticles, an excitation and measurement method for a magnetic biosensor using MRAM in a biosensor system has been disclosed. In this method, at least one digital magnetic sensor element is used, and biomaterials can be analyzed by measuring the degree of magnetization of magnetic beads.

In addition, a method of measuring the binding of antibody-antigen using SQUID and 40 µm beads and reproducing about 20,000 to 50,000 beads has been disclosed. However, SQUID has a disadvantage in that it cannot theoretically make a portable device and in that a sample must be put into the device.

In addition, an analysis method using magnetic beads and a magnetic reader has been disclosed for a bacterium called Yersinia pestis. However, the method is different from the development of a sensor, and uses an antibody immune column made of PE called Antibody Immuno Column for Analytical Processes (ABICAP) for analysis of a sample.

In order to use magnetic nanoparticles as signal materials, 1) very large analysis equipment is required for the analysis of nanoparticles, and 2) GMR, Tunneling MagnetoResistance (TMR), Spin Valve, etc. There is a decisive disadvantage in that the fabrication of the sensor or chip used is very demanding and expensive.

Therefore, the inventors of the present invention are economical in terms of process and cost when using a strip type substrate, magnetic nanoparticles, and frequency-mixing magnetic readers, and are very simple and stable as detection of signals for the number of magnetic nanoparticles. It was found that the presence of a biomaterial can be quantitatively determined and the present invention has been completed.

Accordingly, an object of the present invention is to provide a new method for quantitative detection of a biological material, which can easily and reliably obtain reliable data both indoors and outdoors without requiring an existing expensive sensor or analysis equipment.

In order to achieve the above object, the present invention

Labeling the biomaterial immobilized on the strip-type substrate with magnetic nanoparticles; And

Measuring magnetic nanoparticles labeled with the biomaterial using a magnetic reader of a frequency mixing method;

It provides a quantitative detection method of a biological material comprising a.

According to one embodiment of the invention, the substrate portion is made of a material that is not magnetic.

According to another embodiment of the present invention, the substrate portion is a semiconductor wafer, glass, or quartz.

According to another embodiment of the present invention, the magnetic nanoparticles have a particle diameter of 1nm to 200nm.

According to another embodiment of the present invention, the labeling of the biomaterial immobilized on the substrate with magnetic nanoparticles may be performed on a sandwich immune response using a first capturer and a second capturer for the biomaterial. Performed by

According to another embodiment of the invention, the substrate portion is surface-treated to be fixed to the first trap for the biological material.

According to another embodiment of the invention, the first and second capturers are antigen, antibody, DNA, RNA, PNA, LNA, hapten, aptamer, avidin, biotin, streptavidin, respectively. , At least one selected from the group consisting of neutravidin, lectin, selectin, enzyme, hormone, protein, receptor, ligand, amino acid, oligonucleotide, peptide and oligopeptide, and the same or different from each other.

According to another embodiment of the invention, the biomaterial is selected from the group consisting of DNA, RNA, oligonucleotides, peptides, proteins, antigens, antibodies, viruses, hormones, pathogenic bacteria, viruses, cells, sugars and volatile organics. More than one.

According to another embodiment of the present invention, the detection method of the present invention further comprises removing excess magnetic nanoparticles after the labeling step.

The detection method of the present invention uses stable magnetic nanoparticles instead of HRP (Horse Radish Peroxidase) enzyme as a labeling substance in ELISA sandwich, and uses a frequency-mixing magnetic reader that is portable as a detector. Compared with the analytical method, it is very simple to quantitatively measure the presence of biomaterials as a signal detection for the number of magnetic particles, to obtain more stable data, and to measure biomaterials outside the laboratory. Can be. In addition, as well as obtaining a reliable result of the same sensitivity as in ELISA, unlike the ELISA can be obtained the same result even if the reaction time is different, there is an advantage that can be measured again the same sample later. In addition, since the detection method of the present invention uses a strip type disposable substrate part, it is not necessary to form a complicated pattern on the substrate part like the GMR method, and the material of the substrate part used is very diverse except for magnetic materials. And cost effective in terms of cost.

Hereinafter, with reference to the accompanying drawings of the present invention will be described in detail step by step.

1 is a schematic diagram schematically showing a method for quantitative detection of a biomaterial according to the present invention. As shown in Figure 1, the detection method of the present invention,

Labeling the biomaterial 30 immobilized on the strip-type substrate part 10 with the magnetic nanoparticles 50; And

And measuring the magnetic nanoparticles 50 labeled with the biomaterial 30 using a frequency reader type magnetic reader.

In the present invention, the step of labeling the biomaterial 30 immobilized on the substrate portion 10 with the magnetic nanoparticles 50 is not necessarily limited thereto, but the first capture of the biomaterial 30. It can be carried out by a sandwich immune response using the ruler 20 and the second capturer (40).

FIG. 2 is a schematic view showing a step of labeling the biomaterial 30 immobilized on the substrate portion 10 with the magnetic nanoparticles 50 according to an embodiment of the present invention. Referring to FIG. Immobilizing the first trapper 20 on the surface of the substrate portion 10; Coupling the immobilized first capturer (20) with the biomaterial (30) in a sample; Coupling the combined biomaterial 30 and the second capturer 40 to each other; And labeling the magnetic nanoparticles 50 on the combined second capturer 40.

In the present invention, the substrate portion 10 uses a disposable strip type. When using the strip type as described above, there is no need to form a complex pattern on the substrate portion, such as the GMR method, there is an advantage that the production is simple, easy to handle, and low in cost. At this time, the length and width of the substrate portion 10 is not particularly limited, but may be, for example, length 3cm to 20cm, width 1mm to 15mm.

In addition, the substrate portion 10 is made of a material that is not magnetic so as not to affect the signal to the magnetic nanoparticles. For example, semiconductor wafers, such as a silicon wafer; Glass such as cover glass and slide glass; Alternatively, a quartz plate may be used. In addition, a polymer such as polymethyl methacrylate (PMMA), polypropylene (PP), polystyrene (PS), and pressure-sensitive adhesive (PSA) may be used.

In addition, the substrate unit 10 is used to enable the surface treatment using a common chemical used in the production of the biosensor, so that the first trap 20 for the biological material 30 can be fixed, the surface of the The treatment can be carried out using a method well known to those skilled in the art according to the kind of sample to be measured, the kind of biological material, the kind of the first trap, and the like.

In this regard, Figure 3 shows a schematic diagram of the substrate portion 10 surface treatment according to an embodiment of the present invention. As shown in FIG. 3, in the present invention, a silicon oxide layer (Si—OH _x ) 11 modified in a silanol form is formed on the substrate portion 10, and on the silicon oxide layer 11. After forming the polymer layer 12 having an amine group using polyethyleneimine, the biotin 13 is immobilized through a linker having an amine-reactive group at one end and a biotin at the other end, and again to the biotin 13 The substrate portion 10 can be surface treated by immobilizing the streptavidin 14. However, the present invention is not necessarily limited thereto, and the biotin-streptavidin conjugation may be replaced by another known bioconjugation system such as antigen-antibody, DNA complement, and the like.

Thus, the substrate portion 10 of the present invention, which has been surface-treated, is arrayed to fix one or more first traps 20 of the same or different type on the surface thereof, thereby allowing one or more living bodies in the sample to be fixed. The substance 30 may be measured simultaneously.

The first capturer 20 immobilized on the surface of the substrate 10 and the second capturer 40 coupled to the biomaterial 30 captured by the first capturer 10 are biomaterials. Any biomolecule capable of binding to (30) by antigen-antibody reaction, DNA complementary binding, organic compound specific reactivity, etc. can be used. For example, as the first capturer 20 and the second capturer 40, antigen, antibody, DNA, RNA, PNA, LNA, hapten, aptamer, avidin, biotin, One or more selected from streptavidin, neutravidin, lectin, selectin, enzymes, hormones, proteins, receptors, ligands, amino acids, oligonucleotides, peptides, oligopeptides, etc. may be used, but is not limited thereto. It is not. The first capturer and the second capturer may use the same or different ones.

The biomaterial 30 targeted in the present invention is not limited, but for example, DNA, RNA, oligonucleotides, peptides, proteins, antigens, antibodies, viruses, hormones, pathogenic bacteria, cells, sugars, volatile organic substances, and the like. One or more selected may be used.

In addition, the sample including the biological material 30 is not necessarily limited thereto, but may be a cultured virus, bacteria, cells or tissue-derived extracts, lysates or purified products, blood, plasma, serum and the like.

In the present invention, the magnetic nanoparticles 50 are bound to the second capturer 4 bound to the biological material 30 as a labeling substance, and the magnetic nanoparticles 50 are enzymes which are existing labeling substances. In addition, it is inexpensive and stable compared to fluorescent materials, which enables more reliable analysis, and has the advantage of enabling measurement outdoors.

As the magnetic nanoparticles 50, any particles having magnetic size and nano size may be used without limitation, and for example, metals, magnetic materials, magnetic alloys or iron oxides therein may be magnetized to an external magnetic field. Polymers and the like can be used. In addition, the particle diameter of the magnetic nanoparticles may be in the range of 1nm to 200nm, preferably used in the range of 20nm to 100nm. This is because the smaller the particle size, the larger the number of bonds to the relatively narrow substrate surface and the more the analytical range is expanded.

Meanwhile, in the present invention, the immobilization of the first capturer 20 to the substrate portion 10 and the coupling between the second capturer 40 and the magnetic nanoparticles 50 may be coupled to the binding material immobilization means and to each other. It can be achieved by chemical bonding between the binding materials.

Accordingly, the first capturer 20 and the second capturer 40 may be ones previously conjugated with a binder (eg, biotin), and the magnetic nanoparticles 50 may be used to bind the binder (eg, a binder). Surface coated with streptavidin) is used. As described above, the substrate unit 10 may be surface treated so that the streptavidin, which is a binder immobilization means, is immobilized on the surface.

In the present invention, the above-mentioned streptavidin may be used as the binding means for immobilizing, in addition to biotin, avidin, aptamer, lectin, DNA, RNA, ligand, coenzyme, inorganic Any one of an ion, an enzyme cofactor, a sugar, a lipid, and a substrate may be used, but is not limited thereto. In addition, the binder may be chemically bonded to the binder immobilization means, and may be bonded to a functional group possessed by the first trapper and the second trap, such as a primary amine group, a sulfhydryl group, a carbonyl group, a carboxyl group, and the like. It will not be limited if it is a substance which can be used.

At this time, some examples of the material that can bind the binding material by reacting with the functional group are as follows. That is, a substance reacting with an amine group may include an imidoester compound, N-hydroxysuccinimide, and the like, and the substance reacting with a sulfhydryl group may include maleimide and haloacetyls. And pyridyl disulfide, and the like. Examples of the material that reacts with the carbonyl group include carbodiimides. Examples of the material that react with the carboxyl group include primary amines and hydrazides. Can be mentioned.

In one embodiment of the present invention, by using streptavidin as a binding material immobilization means and biotin as a binding material, N-hydroxysuccinimide was used as a material for binding the biotin to the trapper. It is not necessarily limited thereto.

Meanwhile, in the present invention, the labeling of the biological material 30 immobilized on the substrate part 10 with the magnetic nanoparticles 50 may be performed by first placing the biological material 30 in the sample as the second capturer 40. After capturing, the biomaterial 30 to which the second capturer 40 is coupled is bonded to the first capturer 20 immobilized on the substrate 10, and the magnetic nanoparticles 50 are labeled. It may be performed in a step. The surface treatment of the board | substrate part 10 at this time includes the step of fixing the 1st trap 20 to the board | substrate part 10. FIG.

Subsequently, the magnetic nanoparticles 50 labeled with the immobilized biological material 30 are quantitatively measured using a magnetic reader of a frequency mixing method.

The frequency-mixing magnetic reader detects a small fringe field generated by repeatedly applying two different wavelengths, that is, low frequency (high energy) and high frequency (low energy) to a metal object or magnetic particle. It is a detector of the principle of measuring the presence or absence of metal or magnetic particles.

The contents of the frequency-mixing magnetic reader are disclosed in US Patent Application Publication No. 2007 / 0155024A1, which includes a low frequency portion, a high frequency portion, and a detection portion (detection portion), respectively, to form a conventional magnetic Unlike readers (eg GMR, TMR, SQUID, etc.), there is no need for devices such as a cooling device or an amplifier for amplification, so it can be miniaturized and portable.

In the present invention, by using such a frequency-mixed magnetic reader as a detector and using a strip-type substrate as a sensor, the present invention provides an effect of simply quantitatively measuring the presence of a biomaterial both indoors and outdoors.

As the frequency-mixed magnetic reader, any magnetic reader having the above-described measuring principle may be used, and the present invention is not limited thereto, but the frequency range is 50 to 100 Hz at the low frequency and 10 to 100 KHz at the high frequency. Frequency in the range can be used. For example, a magnetic reader of Senova, Germany may be used as the magnetic reader, and FIG. 4 shows a picture of the frequency mixed magnetic reader used in this embodiment of the present invention.

On the other hand, in the detection method of the present invention, after labeling the biological material 30 immobilized on the substrate portion 10 with the magnetic nanoparticles 50, the step of removing the extra magnetic nanoparticles that did not react It may further comprise. In this way, when the extra magnetic nanoparticles are removed, more accurate measurement results can be obtained. Removal of the excess magnetic nanoparticles can be carried out using a method generally known in the art, for example using a PBS buffer.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed.

Example 1

In order to obtain information about whether the magnetic nanoparticles of continuous concentration can be measured by the detection method of the present invention, it was confirmed whether the magnetic nanoparticles were applied on the substrate without surface treatment and measured by concentration. .

The magnetic nanoparticles used were 50 nm beads purchased from Chemicell, Germany, and 100 μl samples were applied at various concentrations (117, 234, 351, 468, 585, 936, 1053 pcs / ml). It was measured by.

The results are shown in Fig. From FIG. 5, it can be seen that magnetic nanoparticles can be quantitatively measured for each concentration in the case of the detection method of the present invention.

[Example 2]

(1) Preparation of substrate part-surface treatment

A silicon wafer (Nano fab center, charging) having a silicon oxide layer having a thickness of about 10 nm was cut to a width of 6 mm and a length of 8 cm. The organic material was removed from the wafer by using an RCA cleaning method or a plasma cleaning method. The wafer surface was transformed into silanol form using a MeOH: HCl (1: 1) solution. PEI 10% solution (in 50 mM, calcium carbonate, pH 8.0) was treated on the treated surface as described above to allow free amine to appear on the surface. Using NHS-sulfo-biotin purchased from Pierce, the substrate side allowed N-hydroxysuccinimide (NHS) to bind free amines, while the rest allowed biotin to emerge. After applying the PBS (pH 7.1) solution in which streptavidin is dissolved on the substrate prepared as described above, the streptavidin was immobilized by standing at room temperature for about 30 minutes to 1 hour.

(2) Labeling and Measuring Biological Substances

1) DCR-3 antibody as a first capture was purchased from Santa Cruz, DCR-3 standard protein used as a biomaterial (antigen), and a biotin conjugated secondary antibody used as a second capture was Austrian non It was purchased from Bender Medsystems of Vienna. In addition, magnetic nanoparticles were used by diluting Fluidmag-BC-Biotin (size: 100 nm, concentration: 10.0 mg / ml) purchased from Chemicell, Germany, in PBS solution 100 times.

2) Biotin was conjugated to the DCR-3 antibody using a DCR-3 antibody purchased from Santa Cruz, and NHS-sulfo-biotin purchased from Pierce. The biotin-conjugated DCR-3 antibody was dissolved in PBS solution and applied to the surface-treated substrate prepared in (1), followed by standing at room temperature for about 30 minutes. Materials that did not react after standing were removed using a PBS (PBS solution containing 1% Tween 20) solution.

3) Next, DCR-3 antigen was dissolved to 1000 pg / 50 µl using ultrapure water, and then the prepared solution was applied to the substrate prepared in 2), and then incubated at room temperature for about 2 hours.

4) Subsequently, biotin conjugated secondary antibody (in PBS) was applied to the substrate prepared in 3) to complete the sandwich structure of ELISA.

5) The streptavidin-coated beads (magnetic nanoparticles) were frozen on the substrate prepared in 4) to confirm whether the beads were selectively attached. At this time, the beads play the same role as the HRP enzyme used in ELISA.

6) The filtrates that did not participate in the reaction were then removed using PBS buffer.

7) The signal for labeled beads (magnetic nanoparticles) was measured using a frequency-mixed magnetic reader (SENOVA, Germany) with a low frequency of 61 Hz and a high frequency of 49 KHz.

Comparative Example 1 and Comparative Example 2

For comparison, the case where only the silicon wafer was inserted in Example 2 was tested as a non-treated group (Comparative Example 1), and the same procedure as in (2) -2) but using only distilled water instead of DCR-3 antigen was performed. The experiment was performed as a group (negative reference, Comparative Example 2).

The results obtained in Example 2 and Comparative Examples 1 and 2 are shown in FIG. 6. From the results of FIG. 6, it can be seen that the detection method of the present invention can quantitatively measure the presence of the actual biomaterial very stably.

Example 3

In addition, the experiment was performed in the same manner as in Example 2 except that the magnetic nanoparticles were treated at a continuous concentration.

Comparative Example 3

ELISA (Human) using the same biomaterial (DCR-3 antigen) and the same first and second trappers (DCR-3 antibody, secondary antibody) as in Example 3, and using HRP enzyme as a label instead of magnetic nanoparticles DcR-3 BMS2031, Bender Med system) was analyzed and absorbance was measured at 450 nm.

The results (quantitative analysis graphs) obtained in Example 3 and Comparative Example 3 are shown in FIGS. 7A and 7B, respectively. From the results of FIGS. 7A and 7B, it can be confirmed that the biomaterial can be measured more stably with a similar sensitivity to ELISA in the case of the detection method of the present invention.

1 is a schematic diagram schematically showing a method for quantitative detection of a biomaterial according to the present invention.

Figure 2 is a schematic diagram showing the step of labeling the biological material immobilized on the substrate portion with magnetic nanoparticles according to an embodiment of the present invention.

Figure 3 is a schematic diagram showing a surface treated substrate portion according to an embodiment of the present invention.

4 is a photograph of a frequency mixed magnetic reader used in one embodiment of the present invention.

5 is a basic statistical data of the quantitation curve using 50nm magnetic nanoparticles of different concentrations obtained in Example 1.

6 is a graph showing the measurement results obtained in Example 2, Comparative Example 1 and Comparative Example 2. FIG.

7A is a quantitative graph obtained in Example 3, and FIG. 7B is a quantitative graph obtained in Comparative Example 3. FIG.

Explanation of symbols for main parts of the drawings

10: substrate portion 11: silicon oxide layer modified to silanol form

12: polymer layer having an amine group 13: biotin

14: streptavidin 20: first captor

30: Biomaterial 40: Second Capturer

50: magnetic nanoparticles

Claims (9)

Labeling the biomaterial immobilized on a strip type substrate with magnetic nanoparticles; And Measuring magnetic nanoparticles labeled with the biomaterial using a frequency mixing magnetic reader; Quantitative detection method of a biological material comprising a. The method of claim 1, wherein the substrate portion is made of a non-magnetic material. The method of claim 2, wherein the substrate portion is a semiconductor wafer, glass, or quartz. The method of claim 1, wherein the magnetic nanoparticles have a particle diameter of 1 nm to 200 nm. The method of claim 1, wherein the labeling of the biomaterial immobilized on the substrate with magnetic nanoparticles is performed by a sandwich immune reaction using a first capturer and a second capturer for the biomaterial. A quantitative detection method of a biological substance. The method of claim 5, wherein the substrate portion is surface-treated to be fixable to a first trapper with respect to the biomaterial. 6. The method of claim 5, wherein the first and second capturers are antigen, antibody, DNA, RNA, PNA, LNA, hapten, aptamer, avidin, biotin, streptavidin, neutravidin, respectively. (neturavidin), lectins, selectins, enzymes, hormones, proteins, receptors, ligands, amino acids, oligonucleotides, peptides and oligopeptides, one or more selected from the group consisting of the same or different from each other, Quantitative Detection of Substances. The method of claim 1, wherein the biomaterial is at least one selected from the group consisting of DNA, RNA, oligonucleotides, peptides, proteins, antigens, antibodies, viruses, hormones, pathogenic bacteria, viruses, cells, sugars and volatile organic substances. The quantitative detection method of a biological material, characterized in that. The method of claim 1, further comprising removing excess magnetic nanoparticles after labeling the biomaterial immobilized on the substrate with magnetic nanoparticles.

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