WO2013129710A1 - Label-free aptamer biosensor - Google Patents

Abstract

The present invention relates to a label-free aptamer biosensor, wherein electrochemically active particles generate an electrochemical signal by a redox reaction on the surface of an electrode when an analyte is bound to an aptamer fixed on the electrode so as to specifically bind to the analyte, and the electrochemical signal is reduced due to the screening effect of the electrochemically active particles, thereby enabling measurement of the presence and concentration of the analyte. The aptamer biosensor disclosed in the present invention uses the screening effect of electrochemically active particles on an electrode due to an analyte specifically binding to an aptamer fixed on an electrode, and thus it is possible to improve the sensitivity, reproducibility and reliability of the sensor even without complex pretreatment and analytical steps. Therefore, it is possible to rapidly and economically measure the presence and concentration of an analyte by employing the present invention.

Description

Unlabeled Aptamer Biosensor

The present invention utilizes the screening effect of electrochemically active particles on an electrode by analyte that specifically binds to the aptamer immobilized on the electrode, thereby improving the sensitivity, reproducibility and reliability of the sensor without complex pretreatment and analysis. For improved aptamer biosensors.

Aptamer, a molecular recognition material that specifically binds to various substances (target molecules) such as proteins, enzymes or small drugs, has a high affinity and specificity for target molecules compared to antibodies, and is modified as a small molecule structure. Easy to use, high stability, easy to produce, rarely biomolecular reaction occurs, and easy to transform into a binding material for new target molecules, so it is easy to discover new aptamers. Has been used.

Sensors using aptamers (hereinafter referred to as 'aptamer biosensors', 'aptammer sensors' or simply 'sensors') are labeled and unlabeled.

Referring to FIG. 1, the labeling method is similar to the sandwich assay of the immunoassay. After the first aptamer 13 is immobilized, the analyte according to the concentration is reacted and the labeling material is conjugated again. The method is measured using the secondary aptamer 15 (Zhou, L .; Ou, LJ; Chu, X .; Shen, GL; Yu, RQ Anal. Chem. 2007, 79, 7492-7500). While this labeling method has high sensitivity, it is difficult to find aptamers having two other binding sites of the analyte, and it is also difficult to bond labeling materials 16 such as isotopes and fluorescent materials to the ends of the secondary aptamers. The disadvantage is that the steps are complicated and the measurement takes a long time.

On the other hand, the non-labeling method does not require a complicated labeling system, and has the advantage that it can be analyzed quickly without using a second aptamer. Unlabeled methods, however, require expensive equipment and skilled measurers and have high detection limits (XB Yin et al. Anal. Chem. 2009, 81, 9929-9305 and Rodrluez et al. Talanta 2009, 78). , 212-216).

Therefore, efforts have been made to develop sensors using aptamers that have high sensitivity as a non-labeling method and can obtain reliable results even if they are not expensive equipment or skilled measuring instruments. To this end, research is being actively conducted on biosensors that detect proteins through nano-bio technology that fuses nano technology based on bio technology. In the field of nano-bio, various methods for the detection, analysis and quantification of specific biomaterials have been developed.

Research on introducing the advantages of nanoparticles into immune sensors (Guonan Chen et al. Anal. Chem. 2010, 82, 1527-1534) has been actively conducted. In addition, studies on the synthesis of nanoparticles with electrochemical activity (RW Murray et al. Langmuir 2009, 25, 10370-10375 and S. George et al. ACS Nano 2010, 4, 15-19) and their application to biosensors A study was published (A. Fainstein et al. J. AM. CHEM. SOC. 2008, 130, 12690-12697).

The present invention aims to provide a sensor that can be used in a simple manner by omitting complex pretreatment or analytical processes as an aptamer sensor, having a low detection limit, that is, having high sensitivity and providing reliable results.

In addition, the present invention is to be used in the aptamer sensor, to provide a nanoparticle exhibiting electrochemical activity.

The present invention includes an electrode and an aptamer specifically binding to an analyte immobilized on the electrode, the electrochemically active particles generating an electrochemical signal by a redox reaction on the electrode surface. By measuring the presence and concentration, the electrochemical signal provides an aptamer biosensor, characterized in that it is reduced by the screening effect on the particles when the analyte is bound to the aptamer.

Preferably, the electrochemically active particles are silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), titanium (Ti), tantalum (Ta), niobium (Nb), zirconium (Zr), aluminum (Al), hafnium (Hf), tungsten (W), yttrium (Y), zinc (Zn), 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), rhenium (Re), iridium ( Ir) and at least one selected from the group consisting of oxides thereof.

Preferably, the electrochemically active particles are silica (Si), silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), titanium (Ti), tantalum (Ta), niobium (Nb), zirconium (Zr), aluminum (Al), hafnium (Hf), tungsten (W), yttrium (Y), zinc (Zn), Lanthanum (La), Cesium (Ce), Praseodymium (Pr), Neodymium (Nd), Samarium (Sm), Antimony (Sb), Bismuth (Bi), Lead (Pb), Thallium (Tl), Indium ), Tellurium (Te), chromium (Cr), vanadium (V), manganese (Mn), molybdenum (Mo), cobalt (Co), rhodium (Rh), palladium (Pd), osmium (Os), rhenium ( An electrochemically active compound which redox-reacts at the electrode surface is introduced to at least one surface selected from the group consisting of Re), iridium (Ir) and oxides thereof.

Preferably, the electrochemically active compound is ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives), quinones (quinones), quinone derivatives (quinone derivatives), ruthenium amin complexes, osmium (I), osmium (II) ), Osmium (III) complexes, metallocene, metallocene derivatives, potassium hexacyanoferrate (II), meldola's blue, fr Prussian blue, dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3,4-dihydroxybenzaldehyde (3,4-dihydroxybenzaldehyde (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-meth) ylphenazinium (NMP +)), 3-methyl-2-benzothiozolinone hydrazone, 2-methoxy-4-allylphenol, 4- 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3 ', 5,5'-tetra Methylbenzidine (3,3 ', 5,5'-tetramethylbenzidine (TMB)), 2,2-azino-di- [3-ethyl-benzthiazolinesulfonate] (2,2-azino-di- [3- ethyl-benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4-dichlorophenol, 4-aminophenazone ), And benzidine.

Preferably, the electrochemically active particles are introduced on the surface with a compound derived from at least one selected from the group consisting of sulfonic acid, phosphoric acid, carboxylic acid and acetic acid By doing so, it is negatively charged.

Preferably, the electrochemically active particles are positively charged by the introduction of quaternary amine or organic silane on the surface.

Preferably, the electrode is a gold (Au) or carbon (C) electrode.

Preferably, the gold (Au) electrode is a screen printing electrode using a gold disk electrode or gold sputtering (gold sputtering).

Preferably, the carbon (C) electrode is a screen printing electrode using a carbon disk electrode or carbon paste (carbon paste).

Preferably, the analyte is thrombin, B-type natriuretic peptide (BNP) or Carcinoembryonic antigen (CEA).

Preferably, the electrochemically active particles are ferrocene introduced into the silica nanoparticles.

Preferably, the electrochemically active particles are those in which ferrocene and sulfonic acid groups are introduced into the silica nanoparticles.

Preferably, the electrochemically active particles include silanol groups (Si-OH) and (3-aminopropyl) trimethoxysilane (3-Aminopropyl) triethoxysilane on the surface of silica nanoparticles;

[3- (2-amineethoxyamino) propyl] trimethoxysilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane), and (3-aminopropyl) triethoxysilane triethoxysilane It is prepared by performing a process comprising introducing an amine group on the surface of the silica nanoparticles through a condensation reaction between one or more compounds selected from the group consisting of, and introducing a ferrocene in a coupling reaction with the amine group.

Preferably, the electrochemically active particles further include introducing a sulfonic acid group through a ring opening reaction between silanol groups (Si-OH) and 1,3-propanesultone on the surface of the silica nanoparticles. It is manufactured by.

The present invention provides electrochemically active particles prepared from the above process.

The aptamer biosensor according to the present invention utilizes the screening effect of electrochemically active particles on an electrode by an analyte that specifically binds to an aptamer fixed on the electrode, thereby avoiding a complex pretreatment and analytical processes. Sensitivity, reproducibility and reliability can be improved. The electrochemically active particles have a constant size to control the screening effect by the aptamer-analyte by adjusting the size, and in particular, the detection limit can be lowered by using the repulsive force with the analyte.

Therefore, the present invention has the advantage that it can be analyzed in a short time as an economical, high sensitivity sensor with a low detection limit without the need for expensive equipment. In addition, the sensor of the present invention can lower the detection limit to 10-16 M can be used in the field of studying the nucleotide sequence of the aptamer binding to a specific protein.

Figure 1 shows a comparison between the labeling method and the non-labeling method of the aptamer sensor.

2 is a conceptual diagram of an aptamer sensor of the present invention.

Figure 3 shows the screening curve of the electrochemically active signal according to the screening effect for the electrochemically active particles of the present invention.

4 shows a process for introducing ferrocene into silica nanoparticles of 7 nm and 14 nm.

Figure 5 shows the process of introducing a sulfonic acid group having a negative charge to the silica nanoparticles of 7 nm and 14 nm introduced ferrocene.

6 is a cyclic voltammetry diagram of the electrochemically active particles Fc- [a], Fc- [b], and Fc- [b] -SO3- prepared in Examples 1 and 2. FIG.

7 is a TEM photograph of electrochemically active particles (Fc- [a], Fc- [b], Fc- [a] -SO3-, Fc- [b] -SO3-) prepared in Examples 1 and 2 to be.

8 is a schematic of the masking effect (i.e., almost not shown) when using ferrocenecarboxylic acid as electrochemically active particles.

9 is a cyclic voltammogram measured when using ferrocenecarboxylic acid as the electrochemically active particles.

FIG. 10 shows the square voltammetry and calibration curve measured according to thrombin concentration in the thrombin aptamer sensor manufactured in Example 3 using the electrochemically active particles (Fc- [a]) prepared in Example 1. FIG. will be.

FIG. 11 shows the square voltammetry and calibration curve measured according to thrombin concentration in the thrombin aptamer sensor manufactured in Example 3 using the electrochemically active particles (Fc- [b]) prepared in Example 2. FIG. will be.

12 is a square voltammetry and calibration curve measured according to thrombin concentration in the thrombin aptamer sensor manufactured in Example 3 using the electrochemically active particles (Fc- [b] -SO3-) prepared in Example 2. FIG. It is shown together.

13 is a result of testing the specificity of the thrombin aptamer sensor manufactured in Example 3.

FIG. 14 is a square voltammetry and calibration curve measured according to BNP concentration in a BNP aptamer sensor prepared in Example 4 using the electrochemically active particles (Fc- [b] -SO3-) prepared in Example 2. FIG. It is shown together.

15 is a square voltammetry and calibration curve measured according to the CEA concentration in the CEA aptamer sensor manufactured in Example 5 using the electrochemically active particles (Fc- [b] -SO3-) prepared in Example 2 It is shown together.

Figure 16 shows the calibration curve according to the analyte size in the aptamer sensor of the present invention (BNP (a), thrombin (b) and CEA (c) aptamer sensor).

The aptamer biosensor of the present invention detects an analyte in an unlabeled manner, and specifically includes an electrode and an aptamer specifically binding to an analyte immobilized on the electrode. By measuring the presence and concentration of the analyte by electrochemically active particles which produce an electrochemical signal by means of which the electrochemical signal is “hidden” with respect to the electrode when the analyte is bound to the aptamer Use the principle of "reduction".

The screening effect is to block the access of the electrochemically active particles on the electrode surface and thereby block the electrochemical signal thereby leading to a decrease in the signal. In the present invention, as shown in FIG. 2, the aptamer 15 having the screening effect fixed on the surface of the electrode physically binds to the molecule to which it specifically binds, that is, the analyte 14. 21 and 23 combined state).

The physical blocking means that when the analyte 14 is not bonded to the aptamer 15 fixed to the electrode surface in FIG. 2, the electrochemically active particles 21 and 23 are bonded as shown in the enlarged view on the right. Is generated by the electron transfer reaction or redox reaction on the electrode surface, but it is achieved by the inaccessibility of the electrode surface when the analyte 14 is bound to the aptamer 15). .

The result of the signal change of the sensor of the present invention associated with the screening effect is shown in FIG. 3. That is, when the access of the analyte 14 to the aptamer on the electrode surface is increased, the access of the electrochemically active particles is blocked, thereby reducing the electrochemical signal detected by the sensor. The higher the concentration of the analyte, the more effective the redox reaction because the electrochemically active particles do not reach the electrode due to the screening effect, which results in an inverse calibration curve in which the electrochemical signal decreases depending on the concentration of the analyte.

The analyte may include, but is not limited to, thrombin, B-type natriuretic peptide (BNP), or carcinoembryonic antigen (CEA) as a substance to be detected by the sensor of the present invention. Includes all materials for which you want to develop aptamers.

Therefore, the sensor of the present invention can be used not only for the detection of analytes for which an aptamer has been developed, but also for developing an aptamer for any analyte.

Use as electrochemically active particles in the present invention is to generate an electrochemical signal by a redox reaction on the electrode surface. Specifically, such electrochemically active particles include silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), Titanium (Ti), Tantalum (Ta), Niobium (Nb), Zirconium (Zr), Aluminum (Al), Hafnium (Hf), Tungsten (W), Yttrium (Y), Zinc (Zn), 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), rhenium (Re), iridium Like (Ir) or the like, a conductive metal or an oxide thereof, which itself undergoes a redox reaction at the electrode surface, can be used.

In addition, the present invention can be used as the electrochemically active particles in the form of introducing the electrochemically active compound (23) on the surface of the conductive metal or their oxide or silica (Si) particles 21 as shown in FIG. . Specific examples thereof are described in FIG. 4 and the description of the following examples.

This type of electrochemically active particles can be used as a means of controlling the screening effect by adjusting the particle size according to the selection of conductive metals or their oxides or silica particles and electrochemically active compounds, and thus controlling the sensitivity of the sensor of the present invention. It can be advantageous.

The electrochemically active compounds introduced on the surface of the conductive metal or oxides or silica (Si) particles thereof include ferrocene, ferrocene derivatives, quinones, quinone derivatives, and ruthenium amine complexes. (ruthenium amin complexes), osmium (I), osmium (II), osmium (III) complexes, metallocene, metallocene derivatives, potassium hexacyanoferrate (II) (pottasiumhexacyanoferrate), Meldola's blue, Prussian blue, dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3, 4-dihydroxybenzaldehyde (3,4-dihydroxybenzaldehyde (3,4-DHB)), viologen, 7,7,8,8-tetracyanoquinodimethane (7,7,8,8 -tetracyanoquinodimethane (TCNQ)), tetrathiafulvalene (TTF), N-methylacidinium (NMA +), Tetrathiatetracene (TTT), N-methylphenazinium (NMP +), 3-methyl-2-benzothiozolinone hydrazone, 3-methyl-2-benzothiozolinone hydrazone, 2 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4- 4-methoxynaphthol, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine (TMB)), 2,2-azino-di- [3- Ethyl-benzthiazolinesulfonate] (2,2-azino-di- [3-ethyl-benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4 One or more selected from the group consisting of dichlorophenol (2,4-dichlorophenol), 4-aminophenazone, and benzidine can be used.

In addition, when the electrochemically active particles are in the form of introducing an electrochemically active compound on the surface of a conductive metal or oxide or silica (Si) particles thereof, the conductive metal or oxides or silica (Si) particles thereof are preferably less than 100 nm. Particles having a size of 1 nm or more (hereinafter referred to as 'nano particles') are used.

In particular, silica nanoparticles can form stable bonds with various organic molecules, and have the advantage of being synthesized by controlling various sizes (see Beesley, CA; Murray, RW Langmuir 2009, 25, 10370-10375). . It also has the advantage of being able to combine with larger amounts of material even when using the same volume of particles because of its large surface area relative to the volume. Accordingly, the present invention utilizes silica nanoparticles in one embodiment for the preparation of electrochemically active particles, and functionalizes the surface by partial chemical reaction with inorganic materials and organic molecules to improve functionality. Impart desirable properties.

Such silica nanoparticles may be of various sizes synthesized from tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) using the Stover method, or of various sizes available commercially. Fumed silica nanoparticles can also be used.

In the case of using the silica nanoparticles for the production of electrochemically active particles, as an example, silica nanoparticles selected from various sizes, such as, but not limited to, 7 having a number of hydroxyl groups introduced on the surface 7 nm or 14 nm, condensation-polymerization reaction of an organic silane having a primary amine or a secondary amine under anhydrous toluene under amine to nanoparticles After the introduction, a method of introducing a ferrocene, which is a ferrocene having a carboxyl group, through a coupling reaction between ferrocenecarboxylic acid and the amine may be used. The organic silane is (3-aminopropyl) trimethoxy silane (3-Aminopropyl) triethoxysilane,

[3- (2-amineethoxyamino) propyl] trimethoxysilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane), and (3-aminopropyl) triethoxysilane triethoxysilane It may be one or more compounds selected from the group consisting of.

As described above, in the sensor of the present invention, when the aptamer and the analyte are combined at the electrode surface, the presence of the analyte is prevented from reducing the redox reaction by blocking the access of the electrochemically active particles to the electrode surface by the screening effect. And since the concentration is detected, controlling the size of the electrochemically active particles is a means of controlling the sensitivity of the sensor. In the present invention, the size of the whole particle is controlled by controlling the kind, size and amount of the conductive metal or their oxide or silica (Si) and the electrochemically active compound introduced to the surface thereof.

The analyte to be detected in the present invention is mostly negatively charged as a protein or glycoprotein. Therefore, if the electrochemically active particles have a negative charge, the covering effect may be further improved by the electric repulsive force. In the present invention, a compound derived from at least one selected from the group consisting of sulfonic acid, phosphoric acid, carboxylic acid and acetic acid is introduced on the surface of the electrochemically active particles. To make a negative charge. Specific examples thereof are described in FIG. 5 and the description of the following examples.

The negatively charged electrochemically active particles also have the advantage of being kept very stable in solution by lowering the zeta potential. As an example, the zeta potential is -38.5 ± 0.2 mV for 14 nm size silica nanoparticles, and the zeta potential rapidly increases to -5.0 ± 0.9 mV when ferrocene is introduced thereto. In this case, the repulsive force of the particles is weakened in the solution phase, causing agglomeration. However, when a sulfonic acid group is introduced to make a negative charge, the zeta potential decreases rapidly to -52.5 ± 2.1 mV, thus maintaining a very stable state in solution.

In the present invention, in order to produce the negatively charged electrochemically active particles, as an example, silanol groups and 1,3- groups which do not react to the surface of the silica nanoparticles exhibited electrochemically by the method as described above. A method of introducing a sulfonic acid group through a ring opening reaction between propane sultones is used.

Although the above example shows the introduction of negative charges on the surface of the electrochemically active particles, as an alternative, the surface of the electrochemically active particles is a substituent for introducing a positive charge, for example quaternary amine or organic silane. You can also introduce a positive charge. At this time, the signal of the sensor is changed by the attraction force with the analyte.

As the electrode system used in the sensor of the present invention, a two-electrode system including an electrode having the aptamer fixed as a working electrode and a reference electrode therefor may be used. It is also possible to use a three-electrode system further comprising an auxiliary electrode. Both the two-electrode system and the three-electrode system can be clearly understood by those skilled in the art.

In the present invention, the working electrode to which the aptamer is fixed may be a gold (Au) or carbon (C) electrode. The gold electrode may be a gold disk electrode or a type including a gold surface deposited using gold sputtering. The carbon electrode may be a carbon disk electrode or an electrode manufactured by screen printing using carbon paste.

Although described above with respect to the aptamer biosensor, it should be understood that the technology related to the electrochemically active particles included in the sensor is included in the scope of rights. That is, in the present invention, the particles include silanol groups (Si-OH) and (3-aminopropyl) trimethoxysilane (3-Aminopropyl) triethoxysilane on the surface of silica nanoparticles;

[3- (2-amineethoxyamino) propyl] trimethoxysilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane), and (3-aminopropyl) triethoxysilane triethoxysilane Introducing an amine group to the surface of the silica nanoparticles through a condensation reaction between one or more compounds selected from the group consisting of; And it provides an electrochemically active particles, characterized in that prepared by performing a process comprising the step of introducing a ferrocene coupling reaction with the amine group. In addition, the electrochemically active particles are negatively charged by introducing sulfonic acid groups through a ring opening reaction between silanol groups (Si-OH) and 1,3-propanesultone on the surface of silica nanoparticles. Can be.

The present invention will be described through the following examples. However, this is to facilitate the understanding of the invention, the present invention should not be considered to be limited thereto.

Example 1

Preparation of Electrochemically Active Particles (Fc- [a])

The procedure as shown in FIG. 4 was carried out to produce electrochemically active particles. That is, 2 g of fumed silica nanoparticles (6.02 mmol, Aldrich) having a size of 7 nm were refluxed in 55 mL of toluene (Carlo Erbr, Milano, Italy). After 5 minutes, a solution of 5 mL of 3-aminopropyltrimethoxysilane (APTMS) (Aldrich) was added dropwise very slowly, dropwise, dropwise to 5 mL of toluene for 24 hours. Reacted for a while. After separating the nanoparticles and the solution at 5000 rpm using a centrifuge (1580-MGR model, GYROZEN, Daejeon, Korea), toluene was added again and dispersed and washed. After the same process three more times, the nanoparticles were separated by centrifugation and dried in vacuo to complete drying.

Next, 2.08 g of ferrocenecarboxilic acid (Fluka, Buchs, Swizerland) and 1.22 g of hydroxybenzotiazole (HOBt) (Fluka, Buchs, Swizerland) were added dimethylformamide (dimethylformamide ( DMF)) (Daejeongsa, Gyeonggi-do, Korea), and added the silica nanoparticles into which the amine group obtained by the above method was introduced. After completely dispersing the solution through an ultrasonic mill, 1.4 mL of N, N'-diisopropylcarbodiimide (DIC) (Aldrich), a reaction derivative of an amine and a carboxylic acid, was used. 9 mmoL) was added and reacted vigorously at room temperature for 24 hours. The nanoparticles and the solution were separated using a centrifuge at 9000 rpm, after which dimethylformamide was added again and the nanoparticles were dispersed and washed with an ultrasonic mill for 15 minutes. Washed four times with dimethylformamide three times with methanol in the same manner. The fully washed synthesized nanoparticles were completely dispersed in a round bottom flask for vacuum drying and then dried under vacuum for 12 hours.

<Preparation of Electrochemically Active Particles (Fc- [a] -SO3-) Having Negative Charge>

A process as shown in FIG. 5 was performed to prepare electrochemically active particles having negative charge. That is, an ultrasonic crusher was added to 30 mL of purified tetrahydrofuran after adding 0.6 g of 1 mmol potassium tert-butoxide (Acros, Geel, Belgium) and 0.3 g of the electrochemically active particles prepared above. It was dispersed completely using. After diluting 0.263 mL of 1,3-propanesultone (Aldrich) in 5 mL of tetrahydrofuran, the mixture was added dropwise using micropipet and reacted at 80 ° C. for 12 hours. The nanoparticles and the solution were separated at 9000 rpm using a centrifuge. After that, tetrahydrofuran was added and the nanoparticles were dispersed and washed by using an ultrasonic mill for 15 minutes. Tetrahydrofuran was washed three times with methanol three times in the same manner. The fully washed synthesized nanoparticles were completely dispersed in a round bottom flask for vacuum drying and then dried under vacuum for 12 hours.

Example 2

By electrochemically active particles (Fc- [b]) from 2 g (3.98 mmoL) of 14 nm silica nanoparticles by the same procedure as in Example 1 except for using the reagent in the same amount as in Table 1 Electrochemically active particles (Fc- [b] -SO3-) with negative charge were prepared.

Table 1

reagent	APTMS	Ferrocene carboxylic acid	HOBt	DIC		1,3-propane sultone	Potassium tert-butoxide
usage
	2 mL	1.38 g	0.82 g	0.93 mL	0.526 mL	0.22 g

Example 3

<Production of Thrombin Aptamer Biosensor>

In order to manufacture the working electrode of the sensor, a planar-type electrode was manufactured by using ion deposition (working electrode: gold electrode) and screen printing (reference electrode: silver electrode). Polyester (PE) was purchased from 3M (St. Paul, MN, USA) in the United States, silver chloride (Ag / AgCl) dough was used by Japan ASAHI, and insulator dough was used by Jujo, Japan. . Plasma evaporator used NCVS-580S model of Nuricell of Korea and LS-150 product of Newlong Seimitsu Kogyo of Japan. Gold was deposited on polyethylene terephthalate (PE) by ion deposition, and then screen printing was used to sequentially pattern the silver chloride dough and the insulation layer dough.

Next, thiol groups were introduced at the five ends of the aptamer DNA to bind well with the gold electrode, and six thymine spacers were introduced for efficient binding with thrombin. Aptamer (5'-SH-TTT TTT GGT TGG TGT GGT TGG-3 ') (Cosmo Genetech, Seoul, South Korea) that binds specifically to thrombin on a working electrode, 0.1 M PBS After dilution with pH 7.4 (10 mM NaCl, 5 mM KCl, 1 mM MgCl2), the aptamer was soaked in water at 90 ° C. for 5 minutes in order to transform the aptamer into a better binding structure for thrombin, followed by 5 minutes in an ice bath. After soaking, it was fixed to the electrode. The aptamer was reacted for 1 hour, and then washed three times using an electric pipette with 0.1 M PBS pH 7.4 (10 mM NaCl, 5 mM KCl, 1 mM MgCl 2) in order to remove the aptamer not bound to the electrode. Then, a 1 mM 6-mercapto-1-hexanol of blocking molecule was filled to fill the empty space with no aptamer bound on the electrode surface to facilitate the interaction between the aptamer and the analyte. ) (Aldrich Chemical, Milwaukee, WI) was diluted with ethanol and fixed for 20 minutes.

Example 4

<Manufacture of BNP Aptamer Biosensor>

In place of the thrombin aptamer in Example 3, a sensor was prepared by introducing an aptamer of B-type Natriuretic Peptide (BNP) (molecular weight of 3 kDa), which is a cardiovascular disease marker. For this purpose, BNP aptamer (Cosmo Genetech, Seoul, Korea) having the sequence of 5'-SH-TTT TTT TAA ACG CTC AAA GGA CAG AGG GTG CGT AGG AAG GGT ATT CGA CAG GAG GCT CAC A-3 ' The sensor was manufactured in the same manner as in Example 3, except that it was fixed to.

Example 5

<Manufacture of CEA Aptamer Biosensor>

Instead of the thrombin aptamer in Example 3, a sensor was prepared by introducing an aptamer of a colon cancer marker, Carcinoembryonic antigen (CEA), a glycoprotein having a molecular weight of 150 kDa to 200 kDa. For this purpose, the BNP aptamer (Cosmo Genetech, Seoul, Korea) having the sequence of 3'-SH-TTT TTT ATG ATT TCT GGT GGC TGT GCG TAG CTG TGG ATG TGG-5 'is fixed on the electrode surface. The sensor was manufactured by the same procedure as in Example 3.

Experimental Example 1

<Measurement of electrochemical activity of electrochemically active particles>

The electrochemically active particles (Fc- [a], Fc- [b], and Fc- [b] -SO3-) prepared in Examples 1 and 2 were buffered (0.1 M PBS pH 7.0 (NaCl 10 mM)). ) Was completely dispersed using an ultrasonic grinder. The solution was measured by cyclic voltammetry using an electrochemical analyzer workstaion 760D instrument from CH Instrument, an electrochemical measuring instrument.

All experiments were conducted at room temperature, gold disk electrode (CH Instruments, CHI 101, 2 mm diameter), vycor Ag / AgCl reference electrode (CH Instruments, MF-2052), platinum wire auxiliary electrode (BAS Inc., MW-1033) ) 3 electrode system was used.

The scanning speed of the cyclic voltammetry was 0.2 V / s, and two cycles were performed in the potential region of 0 V to 0.7 V. The results are shown in FIG. Both Fc- [a] and Fc- [b] show good electrochemical signals, but the negatively charged Fc- [b] -SO3- shows an increased signal due to the improved dispersion effect.

<TEM image measurement of electrochemically active particles>

Electrochemically active particles (Fc- [a], Fc- [b], Fc- [b] -SO3-) prepared in Examples 1 and 2 using Model Tecnai 20 having an acceleration voltage of 200 kV and TEM images of bare silica nanoparticles (ie, 7 nm silica nanoparticles [a] and 14 nm silica nanoparticles [b]) were taken for comparison. TEM images of Fc- [a] and Fc- [b] into which particles [a], [b] and their ferrocenes were introduced are shown in FIG. 7. In each case it can be seen that the size of the particles increased.

Specifically, it was confirmed that particles Fc- [a] had a size of 16.8 ± 3.0, Fc- [b] had a size of 20.0 ± 2.2, and Fc- [b] -SO3- had a size of 20.6 ± 2.5. . This is larger than the silica nanoparticles of 7 nm and 14 nm, respectively.

Observation of zeta potential and cohesiveness of electrochemically active particles

Zeta Potential of Electrochemically Active Particles Fc- [a], Fc- [b], and Fc- [b] -SO3- Prepared in Examples 1 and 2 Using Dynamic Light Scattering Was measured. Zeta potentials of 7 nm and 14 nm silica nanoparticles (ie, [a] and [b]) were measured together for comparison. Zeta potential measurements are made by Microtrac Inc. A model Zetatrac zeta potential analyzer was used (Montgomeryville, PA, USA). The results are shown in Table 2 below.

TABLE 2

Sample	TEM average diameter	Zeta potential (mV)
[a]	10.5 ± 2.7	-39.4 ± 0.1
[b]	13.6 ± 0.7	-38.5 ± 0.2
Fc- [a]	16.8 ± 3.0	-0.6 ± 1.2
Fc- [b]	20.0 ± 2.2	-0.5 ± 0.9
Fc- [b] -SO 3 -	20.6 ± 2.5	-52.5 ± 2.1

From the table, it can be seen that after the introduction of ferrocene and negative charge to the silica nanoparticles the size of the particles is somewhat larger, and also after the introduction, the properties as particles are maintained.

In addition, although the 7 nm silica nanoparticles ([a]) and 14 nm silica nanoparticles ([b]) have negative charge values of -39.4 ± 0.1 and -38.5 ± 0.2 mV, respectively, an amine group and ferrocene are introduced into the surface. The zeta potential of the surface was reduced to -0.6 ± 1.2 and -0.5 ± 0.9 mV, respectively. This results in the phenomenon of aggregation in aqueous solution.

On the other hand, when negatively charged (Fc- [b] -SO3-) zeta potential has a large value of -52.5 ± 2.1 mV, which is expected to remain stable in aqueous solution.

As a result of observing the aggregation phenomenon in the actual aqueous solution, it was confirmed that the aggregation phenomenon hardly occurred even after 48 hours in the case of Fc- [b] -SO3-.

Experimental Example 2

<Sensibility Evaluation of Aptamer Biosensor 1>

In order to measure the sensitivity of the aptamer biosensor prepared in Example 3, after applying thrombin to the electrode fixed to the aptamer on the surface in Example 3 by concentration, the aptamer and thrombin were reacted for 30 minutes (Sigma, St. Louis). , MO, USA). The thrombin that did not bind to the aptamer was then removed by washing.

Next, in order to obtain an electrochemical signal, electrochemically active particles (Fc- [a], Fc- [) prepared in Examples 1 and 2 using buffer 0.1 M PBS pH 7.0 (NaCl 10 mM) as a base solution. b], Fc- [b] -SO3-) was used at a concentration of 2 mg / mL. The oxidation current was measured by square wave voltammetry using an electrochemical analyzer workstaion 760D instrument from CH Instrument (using an electrochemical analyzer workstaion 760D instrument from CH Instrument). The results are shown in Figs. 10 (Fc- [a]), 11 (Fc- [a]) and 12 (Fc- [b] -SO3-), respectively.

10 and 11 both graphs were able to confirm that the signal difference according to the concentration of thrombin is apparent, it was confirmed that the detection limit is 10-12 M. In addition, the electrochemical signal is larger in FIG. 11 than in FIG. 10, and the signal gap between the thrombin concentrations is larger. Therefore, it was confirmed that the screening effect is different depending on the size of the prepared silica nanoparticles of the electrochemically active particles.

Meanwhile, in FIG. 12, the screening effect was improved by the repulsive force between the electrochemically active particles having a negative charge and thrombin, and the detection limit was further improved to 10-16 M. FIG.

Also for comparison, ferrocene, a single molecule having electrochemical activity instead of the electrochemically active particles (Fc- [a], Fc- [b], Fc- [b] -SO3-) prepared in Examples 1 and 2 The sensitivity of the sensor according to the thrombin concentration was measured using a concentration of 1 mM carboxylic acid (ferrocenecarboxylic acid). CH Instrument's electrochemical analyzer workstaion 760D was used for cyclic voltammetry at a scanning rate of 100 mV / s. The results are shown in FIG.

As a result, no signal difference was observed according to the thrombin concentration. It can be seen that the hiding effect presented in the present invention is not exhibited by small molecules. That is, in the case of a small molecule as shown in Figure 8, regardless of the thrombin concentration, thrombin has no shielding effect, the electrochemically active particles reach the electrode and the redox reaction occurs. Therefore, the size of the electrochemically active particles is a means of controlling the screening effect of the present invention.

 <Specific evaluation of aptamer biosensor 1>

Cross-reactivity tests were performed to evaluate the specificity of the sensor, which is the core of the immune sensor technology. That is, it evaluated what kind of reaction result the sensor of this invention shows with respect to the other substance which exists simultaneously in blood. For this purpose, the lysozyme of the interfering species 5.40x10-7 M (Sigma, St. Louis, Mo., USA), 2.19x10-3 M, using electrochemically active particles (Fc- [b] -SO3-) measuring the sensor's response to hemoglobin (Sigma, St. Louis, MO, USA) and 5.97 × 10-4 M human serum albumin (HSA) (Sigma, St. Louis, MO, USA) It was. The results are shown in FIG.

As shown in (a) to (c) in FIG. 13, it is observed that the signal of the sensor changes according to the change in concentration of thrombin (10-12 M, 10-15 M, 10-16 M, respectively). However, in (d) to (f) measured by mixing with 10-16 M thrombin, only the response by 10-16 M thrombin was confirmed. Therefore, it can be seen that it is not sensitive to the interfering species.

Experimental Example 3

<Evaluation of Sensor Sensitivity According to Analyte Size>

In order to evaluate how the sensitivity of the sensor varies depending on the size of the analyte, the electrochemically active particles ((Fc- [b] -SO3-) prepared in Example 2 were prepared for the sensors manufactured in Examples 4 and 5. The signal of the sensor according to the concentration of each analyte (ie, BNP and CEA) was measured using the same technique as that of the thrombin aptamer sensor in Experimental Example 2. The results are shown in FIG. In each case, the concentrations within the clinical ranges (BNP: 1x10-14 to 1x10-10 M and CEA: 1x10-13 to 1x10-9 M) were distinguished from each other to confirm detection.

 Next, the results were combined to compare the sensitivity in thrombin, BNP and CEA aptamer sensors. The calibration curve of each sensor is shown in FIG. As a result, BNP had the smallest molecular weight of 3 kDa and the slope value of the sensor was 0.249 μA / M. Thrombin was about 30 kDa with a slope value of 0.342 μA / M. Among the three analytes, CEA with the highest molecular weight of 200 kDa showed the highest with a slope value of 0.422 μA / M. As the molecular weight of the analyte increases by about 10 times, the slope value, that is, the sensitivity of the sensor may be improved.

Claims (16)

1. Presence and concentration of the analyte by electrochemically active particles comprising an electrode and an aptamer specifically binding to the analyte immobilized on the electrode and generating an electrochemical signal by a redox reaction at the electrode surface By measuring, wherein the electrochemical signal is reduced by the screening effect on the particles when the analyte is bound to the aptamer.
2. In claim 1,

   The electrochemically active particles are silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), titanium (Ti) , Tantalum (Ta), Niobium (Nb), Zirconium (Zr), Aluminum (Al), Hafnium (Hf), Tungsten (W), Yttrium (Y), Zinc (Zn), 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), rhenium (Re), iridium (Ir) and Aptamer biosensor, characterized in that at least one selected from the group consisting of these oxides.
3. In claim 1,

   The electrochemically active particles are silica (Si), silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru) , Titanium (Ti), tantalum (Ta), niobium (Nb), zirconium (Zr), aluminum (Al), hafnium (Hf), tungsten (W), yttrium (Y), zinc (Zn), lanthanum ( La, cesium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), antimony (Sb), bismuth (Bi), lead (Pb), thallium (Tl), indium (In), tel Rulium (Te), Chromium (Cr), Vanadium (V), Manganese (Mn), Molybdenum (Mo), Cobalt (Co), Rhodium (Rh), Palladium (Pd), Osmium (Os), Lelium (Re), An aptamer biosensor characterized in that an electrochemically active compound which redox-reacts at an electrode surface is introduced on at least one surface selected from the group consisting of iridium (Ir) and oxides thereof.
4. In claim 1,

   The electrochemically active particles are (3-aminopropyl) trimethoxysilane (3-Aminopropyl) triethoxysilane on the surface,

   [3- (2-amineethoxyamino) propyl] trimethoxysilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane), and (3-aminopropyl) triethoxysilane triethoxysilane Aptamer biosensor, characterized in that the amine is introduced into a substituent derived from one or more selected from the group consisting of.
5. In claim 1,

   The electrochemically active compounds are ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives), quinones (quinones), quinone derivatives (quinone derivatives), ruthenium amin complexes (osthenium (I), osmium (II), osmium (III) Osmium complexes, metallocenes, metallocene derivatives, potassium hexacyanoferrate (II), Meldola's blue, Prussian blue Prussian blue), dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3,4-dihydroxybenzaldehyde (3,4-dihydroxybenzaldehyde DHB)), viologen, 7,7,8,8-tetracyanoquinodimethane (7,7,8,8-tetracyanoquinodimethane (TCNQ)), tetrathiafulvalene (TTF) , N-methylacidinium (NMA +), tetrathiatetracene (TTT), N-methylphenazinium (NMP +)), 3-methyl-2-benzothiozolinone hydrazone (3-methyl-2-benzothiozolinone hydrazone), 2-methoxy-4-allylphenol, 4-aminoantipyrine (4-aminoantipyrin (AAP)), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3 ', 5,5'-tetramethylbenzidine (3,3 ', 5,5'-tetramethylbenzidine (TMB)), 2,2-azino-di- [3-ethyl-benzthiazolinesulfonate] (2,2-azino-di- [3-ethyl- benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4-dichlorophenol, 4-aminophenazone, And aptamer biosensor, characterized in that at least one selected from the group consisting of benzidine.
6. In claim 1,

   The electrochemically active particles have a negative charge on the surface by introducing a compound derived from at least one selected from the group consisting of sulfonic acid, phosphoric acid, carboxylic acid and acetic acid. Aptamer biosensor, characterized in that prominent.
7. In claim 1,

   The electrochemically active particles are aptamer biosensor, characterized in that by the introduction of quaternary amine (organic silane) on the surface of the positive charge.
8. In claim 1,

   The electrode is an aptamer biosensor, characterized in that the gold (Au) or carbon (C) electrode.
9. In claim 1,

   The gold (Au) electrode is an aptamer biosensor, characterized in that the screen printing electrode using a gold disk electrode or gold sputtering (gold sputtering).
10. In claim 1,

    The carbon (C) electrode is an aptamer biosensor, characterized in that the screen printing electrode using a carbon disk electrode or carbon paste (carbon paste).
11. In claim 1,

    The analyte is thrombin, B-type natriuretic peptide (BNP) or Carcinoembryonic antigen (CEA), characterized in that the aptamer biosensor.
12. In claim 1,

    The electrochemically active particles are aptamer biosensors characterized in that ferrocene is introduced into the silica nanoparticles.
13. In claim 1,

    The electrochemically active particles are aptamer biosensors characterized in that ferrocene and sulfonic acid groups are introduced into the silica nanoparticles.
14. In claim 1,

    The electrochemically active particles include silanol groups (Si-OH) and (3-aminopropyl) trimethoxysilane (3-Aminopropyl) triethoxysilane on the surface of silica nanoparticles,

    [3- (2-amineethoxyamino) propyl] trimethoxysilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane), and (3-aminopropyl) triethoxysilane triethoxysilane Introducing an amine group to the surface of the silica nanoparticles through a condensation reaction between one or more compounds selected from the group consisting of Aptamer biosensor.
15. The method of claim 14,

    The electrochemically active particles are prepared by additionally introducing a sulfonic acid group through a ring opening reaction between silanol groups (Si-OH) and 1,3-propanesultone on the surface of silica nanoparticles. Aptamer biosensor, characterized in that.
16. An electrochemically active particle produced from a process comprising the step as claimed in claim 14.

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