KR101321082B1

KR101321082B1 - Label-free Electrochemical Aptamer Sensor Using Nanoparticles

Info

Publication number: KR101321082B1
Application number: KR20100085232A
Authority: KR
Inventors: 이지연; 윤종해; 최정연; 한준희; 김석규; 이지훈; 남학현; 차근식; 신재호
Original assignee: 주식회사 아이센스
Priority date: 2010-09-01
Filing date: 2010-09-01
Publication date: 2013-10-23
Also published as: KR20120023208A

Abstract

The present invention is an aptamer biosensor for electrochemically quantitatively analyzing a protein (analyte) that specifically binds to an aptamer using an aptamer, a single-stranded nucleic acid. The present invention relates to an unlabeled aptamer biosensor using nanoparticles having electrochemical activity without labeling of isotope elements.
The aptamer sensor of the present invention is simple and quick to measure in a short time without using two aptamers, unlike the label aptamer sensor in the form of a sandwich immunoassay using two different aptamers previously studied. As a label-type sensor, a methodology is established by quantitatively analyzing proteins (analytes) that specifically bind to aptamers by synthesizing and using electrochemically active nanoparticles to measure electrochemically in a short time. It was.
The aptamer sensor of the present invention drastically improves the problems of low accuracy and reproducibility resulting from the existing complicated experiment process.

Description

Label-free Electrochemical Aptamer Sensor Using Nanoparticles

The present invention relates to a non-labeled aptamer biosensor using nanoparticles, by introducing nanoparticles having electrochemical activity to obtain an electrochemical signal, and adjusting the size of the nanoparticles to a protein-aptamer binding material. The signal according to the shielding effect caused by the was obtained.

By constructing a systematic method of quantifying thrombin in an unlabeled manner using the best known thrombin aptamers, many unknown types of aptamers were sequenced to specific proteins using the methods described herein. Can be predicted.

Recently, aptamers, new molecular recognition materials, have attracted much attention because of their specific binding ability to various target substances, such as proteins, enzymes, and even small drugs, and immune sensors based on these have been developed. Compared with antibodies commonly used in immunosensors, aptamers have a high affinity and specificity and can be mass-produced because they can be synthesized chemically outside the body only by knowing the nucleotide sequence. It has the ability to bind to a wide range of target materials such as ions, small organic molecules, proteins, and cells, making it possible to measure a large number of substances. Its small size also makes it ideal for therapeutic purposes. Sensors using aptamers having many of these advantages include a label-type sensing system and a non-labeled sensing system. Label-based sensing systems have the disadvantage of being able to measure with various techniques, have high sensitivity, take a long time for pretreatment or analysis, and have complex sensing processes (X. Chu et al. , Anal . Chem ., 2007, 79, 7492-7500). The latter has the disadvantage of being simple and short in analysis time, but requiring expensive equipment and high detection limits (XB Yin et al ., Anal . Chem ., 2009, 81, 9929-9305 and MC Rodrluez et. al ., Talanta , 2009, 78, 212-216).

In order to make up for these shortcomings, biosensor research that detects proteins through nano-bio (NT-BT) technology by fusing nanotechnology based on biotechnology (BT) It is actively underway. In the field of nano-bio, various methods for the detection, analysis and quantification of specific biomaterials have been developed.

Among many nanomaterials, nanoparticles (or nanopowders) are fine particles having one or more dimensions of less than 100 nanometers (nm). Nanoparticles are of interest in various fields because they can exhibit essentially new properties when their size becomes smaller than the critical length grade associated with any given property.

Nanoparticles can form stable bonds with various organic molecules, and can be synthesized by controlling various sizes. In addition, since it has a large surface area compared to the volume ratio there is an advantage that can be combined in a larger amount of material even if the same volume.

A study to introduce an immune sensor using the advantages of these nanoparticles (Guonan Chen et al ., Anal . Chem ., 2010, 82, 1527-1534).

A study on the synthesis of nanoparticles having electrochemical activity among the nanoparticles (RW Murray et al . Langmuir , 2009, 25, 10370-10375 and S. George et al . ACS Nano , 2010, 4, 15-19) and its application to biosensors (A. Fainstein et. al ., J. AM. CHEM . SOC . 2008, 130, 12690-12697).

Accordingly, the present invention introduces the nanoparticles having the electrochemical activity into the unlabeled aptamer biosensor.

In the present invention, a new method of electrochemical aptamer biosensor, which is very simple and has high sensitivity and low detection limit as measured using nanoparticles as an unlabeled method, has been studied.

The principle of protein (analyte) detection of an unlabeled aptamer biosensor using nanoparticles having electrochemical activity is as follows.

In the aptamer biosensor, the aptamer, which binds specifically to a protein (analyte), is fixed on a gold electrode, which is a working electrode. At this time, a thiol group was introduced at 5 ends of the single-stranded nucleic acid aptamer DNA to bind well with the gold electrode, and 6 thymine spacers were also introduced for efficient binding with the protein (analyte). After fixing the aptamer to the gold electrode for about 12 to 14 hours, the aptamer is washed to remove the aptamer not bound to the gold electrode. In addition, 6-mercapto-1hexanol (6-mercapto-1-hexanol) was used to fill the voids on the surface of the gold electrode to facilitate interaction between the aptamer and the protein (analyte). After forming the monolayer, the washing process is performed, and the protein (analyte) is reacted by concentration to induce binding between the aptamer and the protein (analyte). It is also washed once again. Finally, nanoparticles with redox mediators were used to obtain electrochemical signals. These nanoparticles have the advantage of maximizing the screening effect by the protein (analyte). Therefore, in this aptamer biosensor, the protein (analyte) binds to the aptamer at each concentration, so that the higher the concentration of protein (analyte), the more easily the redox mediated nanoparticles with the redox mediator do not reach the electrode. As a result, the electrochemical signal becomes smaller.

The calibration curve of the aptamer biosensor has an inverse curve that increases the electrochemical signal because the lower the protein (analyte), the easier redox-electrode between the electrode and the nanoparticle.

The bound aptamer-protein binding material has a strong negative charge.

In the present invention, by synthesizing the nanoparticles having a strong negatively charged electrochemically active, using them to increase the repulsive force between the nanoparticles and the aptamer-protein binding material to increase the electrochemical signal difference between concentrations to improve the distinction between concentrations I wanted to.

The electrochemical aptamer biosensor is economical without the need for expensive large equipment, and has the advantage that the analysis can be performed in a short time with low detection limit and high sensitivity as other detection equipment.

Known aptamer biosensors have been studied in sandwich immunoassays using two aptamers. Although the sandwich immunoassay is widely used due to its excellent sensitivity, it is difficult to find an aptamer having two other binding sites of an analyte. In addition, it is difficult to conjugate the label of enzyme, isotope, fluorescent material, etc. at the end of the second aptamer, and it has a disadvantage that the experimental step is complicated and the measurement time is long.

There is a high probability that experimental errors will occur in many ways, such as errors resulting from complex experimental stages, and errors from immature parts of handling measurement equipment.

As shown in the existing technology, the conventional methods require a complicated experimental step, a complexity due to the use of a large device, and a problem of requiring an experienced measurer. Therefore, there is a need for an electrochemical aptamer biosensor that is simple but highly sensitive and can be measured without a professionally skilled person.

In order to solve the above problems, the present invention intends to apply nanoparticles having non-labeled electrical activity to an aptamer biosensor so that it can be measured using only one aptamer without using two aptamers.

The aptamer biosensor according to the present invention is a method of measuring a second aptamer without attaching a label to it, and by using a non-labeled model, a nanoparticle having electrochemical activity can be synthesized to obtain an electrochemical signal. Since the experimental procedure is not complicated and can be measured in a short time, the concentration of the analyte can be measured more sensitively and accurately than the conventional sensor.

The sensor according to the present invention measures the electron transfer with the electrode by the electrochemical active material using the interaction between the protein (analyte) specifically binding to the aptamer immobilized on the gold electrode protein (analyte) To quantify. The sensor uses nanoparticles of a certain size as an electrochemical material to further increase the screening effect caused by the protein-aptamer binding material, thereby improving the sensitivity, reproducibility and accuracy of the sensor. Lowering the detection limit to 10 ^-14 M may help the biology field to study the sequence of aptamers that bind specific proteins. Equipment such as quartz crystal microbalance (QCM), surface plasmonresonance (SPR), and atomic force microscopy (AFM), which usually measure the binding of aptamers to specific proteins, Although expensive, and difficult to handle the equipment, there is a problem in the detection, but the measurement using the aptamer biosensor can produce fast, economical and reliable results.

1 is a comparison between the sensing method of the label and non-label method.
Figure 2 is a conceptual diagram of a non-labeled aptamer biosensor presented in the present invention.
3 is a calibration curve in the aptamer biosensor using nanoparticles to act as a redox mediator according to the present invention.
Figure 4 is a synthesis of ferrocene-incorporated silica nanoparticles having a size of 7 nm and 14 nm.
5 is a synthesis process of the negatively charged silica nanoparticles introduced ferrocene having a size of 7 nm and 14 nm.
6 is a cyclic voltammogram according to the change in scanning speed of ferrocene-introduced silica nanoparticles.
7 is a TEM image of nanoparticles.
8 is a conceptual diagram of an aptamer biosensor using a single molecule having electrochemical activity.
9 is a cyclic voltammogram measured using a single molecule having electrochemical activity.
10 shows square wave voltages of thrombin measured using 7 nm silica nanoparticles containing ferrocene.
11 is a square wave voltammetry and calibration curve of thrombin measured using 14 nm silica nanoparticles in which ferrocene is introduced.
12 is a square wave voltammogram and calibration curve of thrombin measured using 14 nm negatively charged silica nanoparticles in which ferrocene is introduced.

In order to achieve the above object, the non-labeled electrochemical aptamer biosensor using nanoparticles includes an electrochemical pressure including a working electrode to which an aptamer-protein (analyte) binding material is fixed and a reference electrode and an auxiliary electrode. As a timer biosensor, redox currents are obtained according to protein (analyte) concentration using nanoparticles having electrochemical activity.

Each reaction reagent layer formed on the working electrode according to the present invention is formed by applying about 40 [mu] l of reagent solution. This reagent layer contains aptamer, blocking solution and protein (analyte) to form a monolayer.

Single-stranded nucleic acid aptamer having a sequence of 5'-GGT TGG TGT GGT TGG-3 'according to the present invention introduced a thiol group at the 5' end for binding to the gold electrode, In order to maintain a three-dimensional structure for the thymine (cymine), cytosine (cytosine), it is preferable to introduce a space, such as a methyl group (methyl group).

The electrochemically active nanoparticles used in the present invention are characterized by the use of conductive nanoparticles whose intrinsic properties serve as oxidation-reduction mediators, as well as materials having electrochemical activity on the surface or inside the nanoparticles. It can be divided into ways of introduction.

As the nanoparticles whose original properties act as redox mediators, silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), Ruthenium (Ru), Titanium (Ti), Tantalum (Ta), Niobium (Nb), Hafnium (Hf), Tungsten (W), Yttrium (Y), Zinc (Zn), Zirconium (Zr), Aluminum (Al ), Lanthanum (La), Cesium (Ce), Praseodymium (Pr), Neodymium (Nd), Samarium (Sm), Antimony (Sb), Bismuth (Bi), Lead (Pb), Thallium (Tl), Indium (In), tellurium (Te), chromium (Cr), vanadium (V), manganese (Mn), molybdenum (Mo), cobalt (Co), rhodium (Rh), palladium (Pd), osmium (Os) ), Nanoparticles are selected from the group in which conductive metal elements such as rheium (Re), iridium (Ir), or metal oxides thereof are synthesized in one or more combinations.

As nanoparticles into which the electron transfer redox mediator can be introduced, silica (Si), silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), Nickel (Ni), Ruthenium (Ru), Titanium (Ti), Tantalum (Ta), Niobium (Nb), Hafnium (Hf), Tungsten (W), Yttrium (Y), Zinc (Zn), Zirconium (Zr ), Aluminum (Al), lanthanum (La), cesium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), antimony (Sb), bismuth (Bi), lead (Pb), thallium (Tl), indium (In), tellurium (Te), chromium (Cr), vanadium (V), manganese (Mn), molybdenum (Mo), cobalt (Co), rhodium (Rh), palladium (Pd) ), Nanoparticles synthesized from one or more combinations of elements such as osmium (Os), rheumium (Re), iridium (Ir), or metal oxides or inorganic oxides.

As the redox mediator to be introduced into the nanoparticles, ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives), quinones (quinones), quinone derivatives (ruthenium ammine complexes), osmium (II), Osmium (III), osmium (IV) complex, metallocene, metallocene derivatives, potassium hexa-cyanoferrate (II), Mel aldolase blue (Melola's blue), prussian blue (prussian blue) dichlorophenol in flight play (dichlorophenolindophenol (DCPIP)), o - phenylenediamine (o- phenylenediamine (o -PDA), 3,4- dihydroxy- Benzaldehyde (3,4-hydroxybenzaldehyde (3,4-DHB)), viologen, 7,7,8,8-tetracyanoquinodimethane (7,7,8,8-tetracyanoquinodimethane (TCNQ) )), Tetrathiafulvalene (TTF), N-methylacidinium (NMA +), tetrathiatetracene (TTT), N- Methylphenazinium (N-methylphenazinium (NMP +)), 3-methyl-2-benzothiozolinonehydrazone (3-methyl-2-benzothiozolinonehydrazone), 2-methoxy-4-arylphenol (2-methoxy-4 -allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 4-methylnaphthol, 3,3 ', 5,5'-tetramethylbenzidine (3,3 ', 5,5-tetramethylbenzidine (TMB)), 2,2-azino-di- [3-ethyl-benzthiazolinesulfonate] (2,2-azinodi- [3-ethylbenzthiazolinesulfonate]), o - Dia Genie Dean (o -dianisidine), o - toluidine (o -toluidine), 2,4- dichloro-phenol (2,4-dichlorophenol), 4- amino-Pena zone (4-aminophenazone ), One or more of the group consisting of benzidine is introduced into the nanoparticles and synthesized.

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

Figure 4 is a process diagram for a method for producing silica nanoparticles introduced redox mediator according to the present invention. With reference to this, a process for producing silica nanoparticles into which an oxidation-reduction medium is introduced will be described in detail.

Example 1. Having electrochemical activity Silica nanoparticles synthesis

<Step 1> Synthesis of Silica Nanoparticles with an Amine Group

2 g of commercially available fumed silica nanoparticles of 7 nm or 14 nm (3.01 mmol for 7 nm nanoparticles and 1.99 mmol for 14 nm nanoparticles per 1 g silica nanoparticles) Add 55 mL of toluene and start reflux. After 5 minutes, 5 ml of toluene was mixed with an appropriate amount of 3-aminopropyltrimethoxysilane (APTMS), 6 mL (7 nm nanoparticles) or 3 mL (14 nm nanoparticles). Dropwise dropwise very slowly and react for 24 hours while maintaining reflux. The nanoparticles and the solution were separated using a centrifuge at 4000 rpm. Then, the toluene was added again, dispersed and washed. After the same procedure three more times, the nanoparticles were separated by centrifugation and dried in an oven at 130 ° C. for 24 hours.

<Step 2> Synthesis of Silica Nanoparticles with Ferrocene

2.06 g (9 mmol, 7 nm nanoparticles) or 1.4 g (9 mmol, 14 nm nanoparticles) and 1.3 g (hydroxybenzo-triazole (HOBt)) of ferrocenecarboxylic acid 9.6 mmol, 7 nm nanoparticles) or 0.86 g (6.2 mmol) is dissolved in dimethylformamide (DMF). To this solution is added silica nanoparticles having an amine group obtained through synthesis (about 6.02 mmol of amine group at 7 nm and about 3.98 mmol of amine group at 14 nm). After the solution was completely dispersed by an ultrasonic grinder, amine and carboxylic acid derivatives the reaction of N, N in - diisopropyl amide carbonyl di (N, N -diisopropylcarbodiimide) nanoparticles of 1.5 mL (9.6 mmol, 7 nm ) Or 1 mL (3.2 mmol, 14 nm nanoparticles) was added slowly and then reacted vigorously at room temperature for 24 hours. The nanoparticles and the solution are separated by using a centrifuge at 9000 rpm, and then dimethylformamide is added again, and the nanoparticles are dispersed and washed by using an ultrasonic grinder (ultrasonicator) for 5 minutes. After the same procedure three more times, two washes with a buffer solution (pH 7.0 PBS 0.1M (NaCl 10 mM)) to be used for electrochemical measurement, followed by two washes with ethanol. The fully washed synthesized nanoparticles were completely dispersed in a round bottom flask for vacuum drying and then dried under vacuum for 24 hours.

Figure 5 is a procedure for the manufacturing method of the negatively charged silica nanoparticles having electrochemical activity according to the present invention. With reference to this it will be described in detail for the process of producing negatively charged silica nanoparticles having electrochemical activity.

Example 2. Having electrochemical activity Negative charge Silica Nanoparticle Synthesis

<Step 1> Ferrocene-introduced silica nanoparticle synthesis

The procedure of Example 1 was carried out to synthesize silica nanoparticles into which a molecule having electrochemical activity, ferrocene was introduced.

<Step 2> Synthesis of negatively charged silica nanoparticles with ferrocene

100 mL of purified tetrahydrofuran (THF) was added to the synthesized ferrocene-incorporated silica nanoparticles of 0.2 M potassium tert - butoxide. The synthesized ferrocene-incorporated silica nanoparticles were added to the solution and completely dispersed using an ultrasonic crusher, followed by 2.22 g (18 mmol, 7 nm nanoparticles) of 1,3-propanesultone. ) Or 1.46 g (12 mmol, 14 nm nanoparticles) was diluted in 20 mL of tetrahydrofuran, and then added dropwise using an additional funnel and reacted at 50 ° C. for 24 hours. Separate the nanoparticles and solution at 9000 rpm using a centrifuge. Tetrahydrofuran was put back and the nanoparticles were dispersed and washed using an ultrasonic mill for 5 minutes. After washing the same process two more times, three more washes with a buffer solution (pH 7.0 PBS 0.1 M (NaCl 10mM)), three times with ethanol (ethanol) to completely dry the nanoparticles in vacuum drying.

Example 3. Cyclic voltammetry Measurement of electrochemical activity using

Silica nanoparticles having a certain amount of electrochemical activity are completely dispersed in a buffer solution (pH 7.0 PBS 0.1M (NaCl 10mM)) using an ultrasonic mill. The dispersed solution was measured using an electrochemical analyzer workstation 760D instrument of CH Instrument, an electrochemical measuring apparatus. The scanning speed of cyclic voltammetry is 100 mV / sec. to be. The measurement results can be seen through the sensitivity curve of FIG. 9, and FIG. 6 shows a graph of changes in electrochemical signals with increasing scanning speed. 100 to 500 mV / sec. Scanning speed was increased to 2 cycles and cyclic voltammetry was performed in the potential range of 100 mV to 700 mV, respectively. It was found that the electrochemical signal increased with increasing scanning speed, and that the increase was constant through the response curve of the current value at the applied potential of 450 mV. 6, it was confirmed that the electrochemical signal of the ferrocene formed in the nanoparticles was reversible, and it was confirmed that there was no problem in the electrochemical signal.

Example 4. Having electrochemical activity Single molecule Used Thrombin Measure

Experimental method was used the method and principle presented above, the experimental conditions are as follows. As a buffer solution, pH 7.0, 0.1 M phosphate buffer was used as a base solution, and a single molecule having electrochemical activity was ferrocenecarboxylic acid, and 1 mM was used. The scanning speed of cyclic voltammetry is 100 mV / sec. to be.

The measurement result can be seen through the sensitivity curve of FIG. 9, and the signal according to the concentration of the sample can be measured by measuring the oxidation-reduction current value according to each potential. 9 is a measurement of cyclic voltammetry by applying a single molecule having electrochemical activity to the present invention, and the signal difference according to the thrombin concentration could not be seen. It can be seen that the shielding effect presented in the present invention is not exhibited by small molecules. In the case of small molecules, it can be seen that the electrochemically active material reaches the electrode and transmits electrons because the thrombin has no masking effect of the thrombin regardless of the concentration. Therefore, it can be seen that the screening effect is applied only when the particle size is large.

Example 5. 7 nm Of silica nanoparticles Thrombin Measure

Since it was confirmed that the small molecule is not applied to the screening effect through Example 4, a nanoparticle having a predetermined size or more was synthesized and a signal according to the concentration of thrombin was measured.

Experimental method was used the method and principle presented above, the experimental conditions are as follows. As a buffer solution, pH 7.0 and 0.1 M phosphate buffer were used as the base solution, and silica nanoparticles having a size of 7 nm in which ferrocene was introduced were measured at a concentration of 1.5 mg / mL. In the analysis, the oxidation current was measured by using square wave voltammtetry, which has a Faraday current due to the oxidation / reduction reaction rather than the charging current.

As can be seen in Figure 10, in Example 5, unlike the signal difference according to the concentration of thrombin, it can be seen that the signal difference according to the concentration of thrombin is apparent. That is, it can be seen that the screening effect of the nanoparticles by thrombin, it can be seen that the thrombin can be effectively detected.

Example 6. 14 nm of Silica nanoparticles Used Thrombin Measure

Through Example 5, it was confirmed that the 7 nm silica nanoparticles showed a clear signal according to the thrombin concentration. Thus, the nano nanoparticles having a larger size were synthesized and the signal according to the thrombin concentration was measured. Experimental methods and measurement techniques are the same as in Example 5.

As can be seen in FIG. 11, it was found that the signal was larger and the signal gap between the thrombin concentrations was larger than that measured using 7 nm silica nanoparticles. It was also confirmed that the detection limit was 10 ^-12 M.

Example 7. Having electrochemical activity Negative charge Using silica nanoparticles Thrombin Measure

Using the characteristics of thrombin, which is generally negatively charged, silica nanoparticles having a size of 14 nm in which ferrocene was introduced at the same time as negative charges were further synthesized to further improve the screening effect, and current values according to thrombin concentrations were measured. Experimental methods and measurement techniques are the same as in Examples 5 and 6 above.

As can be seen in Figure 12, a lower detection limit of 10 ^-14 M than the normal nanoparticles ferrocene introduced. As a result, a more efficient screening effect could be seen.

11: electrode 12: blocking solution
13: primary aptamer 14: protein
15: secondary aptamer 16: enzyme
21: nanoparticle 22: electron
23: ferrocene 24: ferrocene
25: calibration curve 31: ferrocenecarboxylic acid

Claims

By immobilizing the aptamer on the working electrode and using a nanoparticle having electrochemical activity for the concentration of a protein (analyte) specifically binding thereto, the difference in accessibility of the nanoparticle to the working electrode according to the concentration of the protein or Obtained electrochemical signal by the screening effect according to the protein concentration, quantify the protein without using a label, by controlling the size of the nanoparticles having the electrochemical activity specifically bound to the aptamer immobilized on the working electrode Aptamer biosensor, characterized in that to amplify the signal difference between the concentration intervals of the protein by different access to the nanoparticles.

The method of claim 1, wherein the electrochemical signal is obtained by cyclic voltammetry, square wave voltammetry, normal pulse voltammetry, pulse difference voltammetry, Aptamer biosensor characterized by using differential pulse voltammetry) or impedance.

The electrode system used in the method of obtaining an electrochemical signal is characterized in that the third electrode system consisting of the working electrode, the auxiliary electrode and the reference electrode or the second electrode system consisting of the working electrode and the reference electrode. Aptamer biosensor.

The aptamer biosensor according to claim 1, wherein the nanoparticles having electrochemical activity are nanoparticles which cause oxidation or reduction by electrons.

The aptamer biosensor according to claim 1, wherein the nanoparticles having electrochemical activity are nanoparticles having inherent properties of electrochemical activity or nanoparticles having molecules having electrochemical activity.

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According to claim 1, Signal difference between the concentration of the protein through the repulsive force of the nanoparticles and aptamer-coupled protein using the negatively charged nanoparticles and the electrochemical activity of the protein bound to the aptamer Aptamer biosensor, characterized in that for amplifying.

According to claim 1, Signal difference between the concentration interval of the protein through the attraction of the nanoparticles and the protein bound to the aptamer using the negatively charged nanoparticles having an electrochemical activity and negative charge properties of the protein bound to the aptamer Aptamer biosensor, characterized in that for amplifying.

The aptamer biosensor according to claim 3, wherein gold (Au) or carbon (C) is used as the working electrode.

10. The aptamer biosensor according to claim 9, wherein the working electrode is a screen printing electrode including a gold disk electrode or a working electrode using a gold sputtering method.

10. The aptamer biosensor of claim 9, wherein the working electrode is a screen printing electrode using a carbon disk electrode or a carbon paste.

The nanoparticles of claim 5, wherein the nanoparticles to which the molecules having electrochemical activity are introduced are silica nanoparticles synthesized by a tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) using the Stover method. Or fumed silica nanoparticles.

The method of claim 12, wherein the silica nanoparticles are introduced by condensation polymerization of an organosilane having a primary amine or a secondary amine under toluene under anhydrous conditions. , Aptamer biosensor, characterized in that a molecule having electrochemical activity is introduced through a coupling reaction between ferrocene phosphorus or ferrocenecarboxylic acid having a carboxyl group and a ferrocenecarboxylic acid and an amine period.

The sulfonic acid synthesized according to claim 13, wherein the silica nanoparticles into which the electrochemically active molecule is introduced are synthesized through a ring opening reaction between unreacted silanol and 1,3-propane sultone. Aptamer biosensor, characterized in that it has a negative charge nanoparticles having electrochemical activity by having a sulfonic acid.

The nanoparticles of claim 5, wherein the nanoparticles having electrochemical activity of inherent properties include silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), and nickel. (Ni), ruthenium (Ru), titanium (Ti), tantalum (Ta), niobium (Nb), zirconium (Zr), aluminum (Al), and metal oxides thereof. Aptamer biosensor.

6. The molecule of claim 5, wherein the molecules with electrochemical activity to be introduced into the nanoparticles are ferrocene, ferrocene derivatives, quinones, quinone derivatives, ruthenium ammine complexes. ), Osmium (II), osmium (III), osmium (IV) complex, metallocene, metallocene derivatives, potassium hexacyanoferrate (potassiumhexacyanoferrate) , Melola's blue, Prussian blue dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3,4-di Hydroxybenzaldehyde (3,4-hydroxybenzaldehyde (3,4-DHB)), viologen, 7,7,8,8-tetracyanoquinodimethane (7,7,8,8-tetra cyanoquino-dimethane (TCNQ)), tetrathiafulvalene (TTF), N-methylacidinium (NMA +), tetrathiatetracene (tetrath) iatetracene (TTT)), N-methylphenazinium (NMP +), 3-methyl-2-benzothiozolinone hydrazone (3-methyl-2-benzo-thiozolinone hydrazone), 2-methoxy 4-arylphenol (2-methoxy-4-allylphenol), 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol (4-methylnaphthol), 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine (TMB)), 2,2-azino-di- [3-ethyl-benz Thiazolinsulfonate] 2,2-azino-di- [3-ethyl-benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4-dichlorophenol ( Aptamer biosensor, characterized in that selected from the group consisting of 2,4-dichlorophenol), 4-aminophenazone (4-aminophenazone) and benzidine.

The method of claim 5, wherein the nanoparticles to introduce a molecule having an electrochemical activity is silica (Si), silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron ( Fe), nickel (Ni), ruthenium (Ru), titanium (Ti), tantalum (Ta), niobium (Nb), hafnium (Hf), tungsten (W), yttrium (Y), zinc (Zn), Zirconium (Zr), Aluminum (Al), Lanthanum (La), Cesium (Ce), Praseodymium (Pr), Neodymium (Nd), Samarium (Sm), Antimony (Sb), Bismuth (Bi), Lead (Pb) ), Thallium (Tl), indium (In), tellurium (Te), chromium (Cr), vanadium (V), manganese (Mn), molybdenum (Mo), cobalt (Co), rhodium (Rh), An aptamer biosensor, characterized in that selected from the group consisting of palladium (Pd), osmium (Os), rhenium (Re), iridium (Ir), metal oxides and inorganic oxides thereof.

The method of claim 7, wherein the negatively charged nanoparticles having electrochemical activity is characterized in that an organosilane or functional group containing phosphoric acid, carboxylic acid, acetoxy acid is introduced. Aptamer biosensor.

9. The aptamer biosensor according to claim 8, wherein the positively charged nanoparticles having electrochemical activity are introduced with organosilanes or functional groups containing quaternary ammonium.