KR101421887B1 - A method for analyzing nanoparticle using scanning probe microscope(SPM)-impedance analyzer - Google Patents
A method for analyzing nanoparticle using scanning probe microscope(SPM)-impedance analyzer Download PDFInfo
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- KR101421887B1 KR101421887B1 KR1020130018500A KR20130018500A KR101421887B1 KR 101421887 B1 KR101421887 B1 KR 101421887B1 KR 1020130018500 A KR1020130018500 A KR 1020130018500A KR 20130018500 A KR20130018500 A KR 20130018500A KR 101421887 B1 KR101421887 B1 KR 101421887B1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
Abstract
Description
The present invention relates to a method for discriminating protein binding of nanoparticles using a scanning probe microscope-impedance analyzer and a method for detecting proteins bound to nanoparticles.
The Scanning Probe Microscope is a surface measurement instrument with atomic and molecular resolution, and has been widely used for nano-characterization and has contributed greatly to the development of nanotechnology. Unlike optical and electron microscopes, which detect signals at a distance from the sample, the scanning probe microscope is a proximity probe technology that detects near-field of nuclear, tunneling current, and electromagnetic force between the probe and the sample. The scanning probe microscope can detect not only the surface shape but also various physical characteristics by using the near-field interaction felt by the tip of the needle when the probe having a sharp needle at the nano level approaches the sample. Scanning probe microscope refers to a device that measures the amount of physical interaction between a specimen and a probe when a nano-sized sharp needle on a cantilever flies close to the surface of the specimen. The scanning probe microscope consists of a probe, an optical mechanical part for detecting the interaction force, a 3D nano scanner, a digital electronic control device, and software. Scanning probe microscopes are classified into two categories: Scanning Tunneling Microscope (STM), AFM (Atomic Force Microscope), and Magnetic Force Microscope (MFM) ), Lateral Force Microscope (LFM), Force Modulation Microscope (FMM), Electrostatic Force Microscope (EFM), Scanning Capacitance Microscope (SCM) and Electrochemistry scanning probe microscope (ECSPM).
On the other hand, impedance refers to the ratio of the voltage to the current in the AC circuit, and the unit is expressed in ohms and complex numbers. Impedance is a resistor that prevents energy from flowing through a TRANSMISSION LINE (a conductor used to transfer electrical energy from one point to another). It is important to match the impedance data of electronic components for the high speed and accurate processing of electrical signals as communication devices or computers become faster and more versatile. Impedance measurements can be measured with a TIME DOMAIN REFLECTOMETER (TDR). When there is a change in the impedance data (mismatch), a part of the pulse echoes back to the TDR device, and the TDR device measures the impedance by displaying this reflected pulse. Recently, it has been used variously in the field of biotechnology such as measuring specific viruses or bacteria or analyzing body composition using not only electric field but also impedance.
As described above, the scanning probe microscope and impedance are widely used in related fields. However, to date, there has been known a technology for detecting the binding of proteins to nanoparticles or detecting bound proteins by combining a scanning probe microscope-impedance analyzer There is no way.
Techniques for determining the binding of proteins using nanoparticles or for detecting bound proteins can be used in a variety of applications. In the field of medicine or food, nanoparticles can be used to deliver the protein, the active ingredient, to the target site. Before transferring the protein to the target site, it is necessary to analyze whether the active ingredient protein is bound to the nanoparticle, It is necessary to effectively separate protein-bound nanoparticles. Alternatively, if an unspecified protein is bound to the nanoparticle, protein detection techniques can be used to detect what the bound protein is. Therefore, there is a need for an effective and simple process to identify the binding of proteins to nanoparticles or to detect proteins bound to nanoparticles.
Accordingly, the present inventors have studied to develop a new protein detection technique. As a result, they have developed a technique for determining whether or not protein binding of nanoparticles has been established by constructing a scanning probe microscope-impedance analyzer.
Accordingly, the present invention relates to a method for determining the binding of a nanoparticle to a protein, which comprises measuring and analyzing an impedance signal of a nanoparticle sample reacted with a protein and comparing the analyzed impedance data with impedance data of a nanoparticle sample not reacted with the protein ≪ / RTI > In the present invention, a reaction between a protein and a nanoparticle is only a reaction capable of binding a protein and a nanoparticle, and the kind of reaction or binding is not limited. The binding of the protein to the nanoparticles includes both chemical and covalent bonds as well as physical and / or chemical bonds, including when the protein is simply adsorbed on the nanoparticle surface.
In the present invention, the protein and the nanoparticles need only be capable of reacting with each other to bind with each other, and the kind of the protein or the nanoparticles reacted therewith is not limited. Attachable proteins and nanoparticles are suitably selected and are well known in the art. Proteins and nanoparticles can be bound either directly or through a linker. To directly bind a protein and a nanoparticle, a nanoparticle having a functional group capable of reacting with a functional group contained in the protein, such as a carboxyl group or an amine group, may be prepared, or a functional group capable of reacting with the functional group of the protein Can be introduced and used. As a linker for binding protein and nanoparticle, a crosslinking agent and the like can be used. For example, when polystyrene nanoparticles and bovine serum albumin are bound to each other, polystyrene nanoparticles whose surface is substituted with a carboxyl group and N-hydroxysuccinimide as a cross-linking agent are reacted with 1-ethyl-3- (3-dimethylaminopropyl) Polystyrene nanoparticles and bovine serum albumin can be bound by reacting the mixture with bovine serum albumin.
A probe microscope-impedance analyzer is constructed to measure the impedance signal of the nanoparticle sample reacted with the protein. The scanning probe microscope-impedance analyzer of the present invention can be constructed by constituting a scanning probe microscope, an impedance analyzer, and a connection portion. The scanning probe microscope can utilize any known scanning probe microscope without limitation. For example, scanning tunneling microscopy, magnetic force microscopy, electrostatic force microscopy, or atomic force microscopy. In one embodiment of the invention, the scanning probe microscope may comprise a conductive probe. By using a conductive probe, the impedance of the sample can be measured when the probe of the probe microscope is brought into contact with or brought into contact with the sample.
The impedance analyzer can also use any of the analyzers known in the art to measure impedance, without limitations. For example, the Model 1260A Frequency Response Analyzer (Solatron) can be used as an impedance analyzer.
In order to measure the impedance signal of the sample using a scanning probe microscope, a connection is needed to connect the scanning probe microscope and the impedance analyzer. The connection is possible without limitation as long as it can transmit the impedance signal output through the probe microscope to the impedance analyzer while connecting the probe microscope and the impedance analyzer. In one embodiment of the invention, the connection may comprise a conductive substrate and a cable. The conductive substrate may also be fixed to a scanning probe microscope. The impedance signal output through the conductive substrate and the cable can be transmitted to the impedance analyzer.
Scanning probe microscope with scanning probe microscope, impedance analyzer and connection - Measure the impedance signal of the nanoparticle sample reacted with the protein in the impedance analyzer. The measured impedance signal is transmitted to the impedance analyzer through the connection section described above and analyzed by the impedance analyzer.
The analyzed impedance signal is output through software. Software for outputting impedance signals is well known in the industry. For example, in the embodiment of the present invention, LEVMWL software (ver 8.11) developed by MacDonald was used.
Impedance data refers to information about impedances that can analyze and compare impedance signals. In the present invention, the impedance data includes an amount of impedance change according to frequency and / or a circuit element value obtained through data fitting. Since the impedance data varies depending on whether or not the protein is bound, it is possible to determine whether or not the protein is bound by comparing the impedance data. For example, if the impedance data of a nanoparticle reacted with a protein is different from the impedance data of the same nanoparticle that has not reacted with the protein, it can be seen that the protein is bound to the nanoparticle. The shape of the impedance signal according to the frequency of the protein-bound nanoparticles differs from the shape of the impedance signal of the protein-unbound nanoparticles (FIG. 4). After obtaining the impedance signal, circuit element values such as contact resistance (R cont ), capacitance (C), inductance (L p ) and CPE (constant phase element) values are obtained through nonlinear data fitting, can do. Where CPE can be expressed as Z CPE = A / (iAωτ) φ where Z CPE is the impedance due to the constant phase element, A is the resistance, ω is the frequency, i is the imaginary (complex), τ is the capacitance, Indicates the exponent. The capacitance or inductance value varies depending on whether the protein is bound or not, so that it is possible to determine whether the protein is bound or not (FIG. 5).
The present invention also provides a method for detecting proteins bound to nanoparticles, which comprises measuring and analyzing impedance signals of a protein-bound nanoparticle sample and comparing the analyzed impedance data to a database of protein-specific impedance data do. Since each protein shows unique impedance data, it can be used to build a database to detect what proteins are bound to the nanoparticles. For example, a reference nanoparticle can be selected and the protein to be measured can be bound to construct a database of the impedance data inherent to each protein. The number and type of reference proteins are not limited. Specifically, when the polystyrene nanoparticles are selected as the reference nanoparticles, a protein-bound nanoparticle is prepared by binding each protein to construct a database with polystyrene nanoparticles. The binding of the nanoparticles to the protein can be accomplished by direct binding, binding of the appropriate functional groups to the nanoparticles, or binding of the nanoparticles to the protein, or by using a linker capable of binding the nanoparticles to the protein, as described above. Scanning probe microscope - An impedance analyzer is constructed to measure the impedance signal of the sample as described above. The protein-bound polystyrene nanoparticles are separated and the impedance data of each protein-bound nanoparticle is obtained. The database is constructed by analyzing the impedance data of the nanoparticles to which each protein is bound. Impedance data on the database will vary depending on the reference nanoparticle, and one or several reference nanoparticles can be selected to construct the database. For example, in the case of polystyrene nanoparticle-bovine serum albumin, the impedance data may be as shown in Fig. 4 (b), and the polystyrene nanoparticle-fibrinogen may be as shown in Fig. 4 (C). Impedance data for nanoparticle samples with unknown proteins can be compared to impedance data from the database in which they are constructed to detect what proteins are bound to the nanoparticles. For example, impedance data of a sample to which an unknown protein is bound is compared with the database of the impedance data of the unknown protein-bound polystyrene nanoparticle sample and the protein-specific impedance data of the polystyrene nanoparticle- b), it is known that the unknown protein is bovine serum albumin. Also, the database can be constructed by representing the impedance data by the circuit element values through the data fitting of the respective impedance signals (Tables 1 and 2). For example, if the circuit element value of the impedance signal of the unknown protein-bound polystyrene nanoparticle sample corresponds to C = 30.63 ± 9.61, the unknown protein is the bovine serum albumin.
The scanning probe microscope-impedance analyzer according to the present invention is convenient and economical because it can construct a scanning probe microscope-impedance analyzer by a simple connection process using a scanning probe microscope and an impedance analyzer, which can be easily purchased. Through a simple combination of a scanning probe microscope and an impedance analyzer, it is possible to identify the binding of nanoparticles to proteins and to detect proteins bound to nanoparticles by broadening the application field to a previously unexpected field. Therefore, the present invention is an epoch-making technology that can broaden the application field of the scanning probe microscope, which was limited to the analysis of nanoparticles, to protein binding determination or specific protein detection. The scanning probe microscope-impedance analyzer of the present invention can be used for emulsion analysis, or for detecting a medical protein or a specific virus as well as a general core-shell type nanoparticle analysis.
The scanning probe microscope and the impedance analyzer according to the present invention can discriminate the protein binding of the nanoparticles and further build a database of the impedance data specific to the protein to detect the protein bound to the nanoparticles.
Figure 1 shows the preparation of protein-bound nanoparticles.
2 is a schematic diagram of a scanning probe microscope-impedance analyzer of the present invention.
3 is a graph showing impedance signals of nanoparticles and protein-bound nanoparticles.
4 shows the result of data fitting of the impedance signal.
Figure 5 shows the result of measuring the AFM force curve to set the force applied to the conductive tip.
Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention. These embodiments are provided so that the disclosure of the present invention is not limited thereto and that those skilled in the art will fully understand the scope of the present invention and that the present invention is not limited by the scope of the claims Only.
<Examples>
Production Example 1: Production of nanoparticles reacted with protein
A polystyrene latex particle model whose surface was substituted with a carboxyl group as a sample was purchased from Spherotec (2.5% w / v). The average diameter of the polystyrene particles is about 73.7 nm. Model proteins include bovine serum albumin (BSA), fibrinogen (Fib) (Type IS, extracted from the right plasma, protein content 65-68%) and immunoglobulin G (IgG extracted from human plasma, reagent grade
The residue of the polystyrene nanoparticles was washed with distilled water and centrifuged to remove the residue. The above procedure was repeated 2-3 times to obtain clean polystyrene nanoparticles whose surface was modified with a carboxyl group. Then, the protein was immobilized on the surface of the nanoparticles to form a nanoparticle-shell of the protein. To this end, 10 ml of EDC (100 mM) and NHS (100 mM) were added as a crosslinking agent to a vial containing polystyrene nanoparticle solution (2 ml, 2.27% w / v) and reacted at room temperature for 2 to 3 hours. After that, 2 ml of a model protein solution (1.0 ml / ml) was added to the nanoparticle and cross-linking agent mixture, and the mixture was allowed to react at room temperature for 12 hours or longer. After the reaction, the protein-bound nanoparticles were obtained in the form of a core shell of protein-nanoparticles through the distilled water washing-centrifugation process 2 to 3 times as in the previous method. The manufacturing process is shown in Fig.
Manufacturing Example 2. Scanning probe microscope - Impedance analyzer construction
For the impedance analysis of the nanoparticle-protein structure, a scanning probe microscope-impedance analyzer was constructed through the self-isolation of the scanning probe microscope and the connection between the devices.
AFM was used to acquire basic images and to measure the atomic force between the particles and the surface. The conductive probe was a SCM-PIC model coated with Pt from BRUKER and the force constant was 0.2 N / m. The conductive probe was used for the conductive AFM holder (Veeco), and the electrical circuit was constructed by directly connecting the conductive probe, the scanning probe microscope, and the impedance analyzer (Model 1260A Frequency Response Analyzer, Solatron).
The gold-coated conductive silicon wafer is about 1 cm
Experimental Example 1. Impedance Signal Measurement and Data Analysis
Scanning probe microscope - The impedance analyzer consists of an impedance analyzer, a scanning probe microscope, a cable and a substrate as a connection. Each component has circuit components such as resistance, capacitance, and parasitic inductance. The factors other than the resistance and capacitance value of the nanoparticles are determined at the time of equipment configuration, and these values are known through measurement. A conductive scanning probe microscope tip was placed over a gold coated silicon wafer with nanoparticles distributed and contacted with a constant force of about 200 nN or more. This was obtained by AFM Force curve measurement and the related graph is shown in FIG. Through this, a circuit was created and an alternating current was applied to obtain an impedance signal according to the frequency change.
In order to analyze the obtained signal, the electric circuit configuration and circuit model for the impedance signal were constructed. We considered resistance, capacitance, and inductance for circuit model analysis. Also, the existence of unknown parasitic capacitors and parasitic inductance values are not considered to exist in actual equipment when constructing a circuit, but they are considered because they may affect the analysis of electrical circuit signals. The impedance signal obtained from the impedance analyzer is represented by a nonlinear function according to the circuit model configuration, and LEVMWL software (ver. 8.11) developed by MacDonald is used to obtain the optimized value.
Electrochemical analysis of protein-polystyrene nanoparticle surfaces was performed using an impedance-scanning probe microscope. Impedance was measured by varying the frequency of AC current and the values were compared. The impedance signals of the protein-polystyrene nanoparticles are shown in Fig. In each experiment, the mean value was measured after 5 or more measurements. The impedance signals of the polystyrene nanoparticles immobilized with different kinds of proteins were found to vary depending on the protein in the high frequency region of 10 5 Hz or more. The capacitor effect of nanoparticles began to be characteristic when compared with the impedance signal without the nanoparticles between the conductive tip and the substrate. It was concluded that the change in the impedance signal at such a high frequency was attributed to the characteristics of the protein. Thus confirming its usefulness as a protein marker. Also, a high contact resistance of about 200 OMEGA was generated in each sample.
Experimental Example 2: Circuit element value through data fitting
In order to analyze each protein-polystyrene nanoparticle, an AC circuit model was constructed by the above method and an impedance signal was obtained. Then, a contact resistance (R cont ), a capacitance (C), an inductance (L p ) (constant phase element) value. Here, the CPE can be expressed as Z CPE = A / (iAωτ) φ , and the impedance data fitting can be optimized. Where Z CPE is the impedance due to the constant phase element, A is the resistance, ω is the frequency, i is the imaginary (complex number), τ is the capacitance, and φ is the exponent. In this experiment, CPE values were connected in series to set the AC circuit model and data fitting was performed. The fitting results are shown in FIG. 5, and the values of the respective circuit elements are shown in Tables 1 and 2. The capacitance values for each protein were different and thus the capacitor effect was observed. It is considered that this is due to the influence of the protein layer formed on the polystyrene nanoparticles and is due to the layer thickness and the inherent properties of each protein. Also, the parasitic inductance value showed a large difference according to the samples. Experimental results show that nonlinear data fittings for impedance signals in the high frequency domain are reliable markers for protein identification.
Claims (7)
A conductive substrate disposed between the conductive probe and the conductive substrate,
A method of determining whether a nanoparticle is bound to a protein, comprising measuring and analyzing the impedance signal of the nanoparticle sample reacted with the protein, and comparing the impedance data with the impedance data of the nanoparticle sample not reacted with the protein.
A conductive substrate disposed between the conductive probe and the conductive substrate,
A method of detecting a protein bound to a nanoparticle comprising measuring, analyzing and analyzing the impedance signal of a protein-bound nanoparticle sample and comparing the analyzed impedance data to a database of protein-specific impedance data.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20100073636A (en) * | 2008-12-23 | 2010-07-01 | 연세대학교 산학협력단 | Bioprobes, preparation method thereof, analysis apparatus and method using the same |
| KR20110047823A (en) * | 2009-10-30 | 2011-05-09 | 배재대학교 산학협력단 | Methods for Detection and Isolation of DNAs Using Nanoparticles |
| KR20130005849A (en) * | 2011-07-07 | 2013-01-16 | 전자부품연구원 | Hand-held diagnostic system using electrical parameter analysis |
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| KR20100073636A (en) * | 2008-12-23 | 2010-07-01 | 연세대학교 산학협력단 | Bioprobes, preparation method thereof, analysis apparatus and method using the same |
| KR20110047823A (en) * | 2009-10-30 | 2011-05-09 | 배재대학교 산학협력단 | Methods for Detection and Isolation of DNAs Using Nanoparticles |
| KR20130005849A (en) * | 2011-07-07 | 2013-01-16 | 전자부품연구원 | Hand-held diagnostic system using electrical parameter analysis |
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