KR101264204B1 - Sensor based on Redox active gold nanoparticles and the synthesis of the same - Google Patents

Abstract

The present invention relates to an electrochemical sensor which is protected by an organothiol ligand and has an electrode to which gold nanoparticles having redox activity are immobilized, and which do not require addition of a separate oxidation-activating medium or enzyme, Physiological substances such as uric acid, sulfite or dopamine can be detected with high sensitivity.

Description

Sensor based on Redox active gold nanoparticles and the synthesis of the same}

The present invention relates to an electrochemical sensor and a method for manufacturing the same, and more particularly, to an electrochemical sensor comprising a gold nanoparticle having a redox activity and a method for producing the same.

Various kinds of nanoparticles are applied to the field of electrochemical sensors. For example, metal nanoparticles have high electrical conductivity and catalytic properties, oxide nanoparticles have excellent biocompatibility, and semiconductor nanoparticles have been used as labels or tracers. One of the most attracting materials is gold nanoparticles, which are widely used in electrochemical sensors and biosensors due to their excellent biocompatibility, high electrical conductivity, and catalytic activity. However, since the gold nanoparticles that have been studied so far are mainly particles having a large diameter and no oxidation-reduction activity, there is a problem that they must be prepared with molecules or enzymes having electroactivity to be used as electrochemical sensors. Also, in this case, the electrochemical reaction takes place through a very complicated route, which poses a major obstacle to the development of a sensor with a clear response mechanism.

The problem to be solved by the present invention is to provide an electrochemical sensor comprising a nanoparticle having a redox activity that can act as a high-sensitivity electrochemical sensor without binding to a molecule or enzyme having a separate electroactivity and a method of manufacturing the same. .

In order to solve the above technical problem, the present invention provides an electrochemical sensor, which is protected with an organothiol ligand, and comprises an electrode to which gold nanoparticles having redox activity are fixed.

According to one embodiment of the present invention, the ligand-protected gold nanoparticles may be fixed in the sol-gel thin film, wherein the gold nanoparticles are preferably fixed in a trapped state in the sol-gel network.

According to one embodiment of the present invention, the organothiol ligand is an alkanthiol, glutathione, thiophenine, thiolated poly (ethylene glycol), p-mercaptophenol, aromatic alkanethiol, or phenylalkanthiol , (r-mercaptopropyl) -trimethoxysilane), and more preferably, hexane thioleate or glutathione is selected.

In addition, according to one embodiment of the present invention, the diameter of the ligand-protected gold nanoparticles is preferably 0.1 to 5 nm in quantum size, more preferably 3 nm or less. If the diameter of the gold nanoparticles is 5nm or more, it does not exhibit redox activity and thus cannot function as an electrochemical sensor, and the presence or absence of the redox activity can be confirmed by cyclic voltammetry.

According to an embodiment of the present invention, the electrochemical sensor may detect any one material selected from ascorbic acid, uric acid, dopamine or sulfite, and the material may be detected by the electrocatalytic activity of the gold nanoparticles present on the electrode. It is characterized by being.

According to one embodiment of the present invention, the electrode on which the gold nanoparticles having redox activity are fixed is a working electrode, a counter electrode is a platinum (Pt) disk electrode, and a reference electrode is an Ag / AgCl electrode.

In another aspect, the present invention comprises the steps of 1) synthesizing a gold nanoparticle cluster protected with an organothiol ligand; And 2) fixing the gold nanoparticle clusters in the form of a sol-gel thin film on the electrode.

According to an embodiment of the present invention, the step 1) includes: i) mixing and stirring the organic thiol compound solution and a gold (Au) hydrate solution; ii) by adding sodium borohydride to the mixture to perform a reduction reaction,

Step 2) may be performed by drop casting the gold nanoparticle solution synthesized in step 1) on the electrode surface and then drying.

The present invention also provides a sol-gel working electrode comprising a gold nanoparticle protected with an organothiolic ligand, an electrochemical sensor including a counter electrode and a reference electrode.

The sol-gel thin film electrode using gold nanoparticles having redox activity according to the present invention showed excellent electrocatalytic activity, and these were the four main analytes, namely the currents of ascorbic acid, uric acid, sulfite and dopamine. The electron transfer process of the sol-gel thin film as a function of the concentration of gold nanoparticles was used for the detection of the method. In addition, the electrochemical sensor using nanoparticles protected with a quantum size ligand according to the present invention is expected to greatly contribute to the research in nanomaterials and electrochemical fields through the invention of the control of the oxidation-reduction properties and ligand control according to the particle size. .

1 is a view showing a bonding structure of nanoparticles and a sol-gel network in Au ₂₅ SGE.
2 is a MALDI mass spectrum of the synthesized Au ₂₅ nanoparticles.
3, TEM image of synthesized Au ₂₅ nanoparticles, scale bar of TEM image is 20 nm and insert is histogram of core size.
4 is a UV-Vis spectrum of the synthesized Au ₂₅ nanoparticles.
5 is (A) SWV of Au ₂₅ at a Pt working electrode in CH ₂ Cl ₂ comprising 0.1 M Bu ₄ NPF ₆ . (B) Cyclic current voltage graph (CV) of Au ₂₅ SGE for 25 consecutive cycles running at 20 mVs ⁻¹ in 0.1 M KCl.
FIG. 6 shows the CV of Au ₂₅ SGE and GCE (insert of FIG. 6) recorded in the presence or absence of analyte in 0.1 M KCl. Specifically, FIG. 6 shows a voltammetry demonstrating electrocatalytic oxidation of ascorbic acid (A), uric acid (B), sulfite (C) and dopamine (D) at 20 mVs ⁻¹ in 0.1 M KCl, (a) is the CV of Au ₂₅ SGE in the absence of analyte, (b-f) is the CV of Au ₂₅ SGE in the presence of 1, 2, 3, 4 and 5 μM of analyte, and (g) is in the absence of analyte CV of SGE, (h) is CV of SGE in the presence of 5 μM analyte.
7 is a calibration graph for determining the content of ascorbic acid (A), uric acid (B), sulfite (C) and dopamine (D) from a cyclic voltammogram of Au ₂₅ SGE in 0.1 M KCl.
8 is The electron diffusion coefficient ( D _E ), the self-exchange rate constant ( k _EX ), and the detection sensitivity of ascorbic acid ( S _AA ) and the detection sensitivity of uric acid ( S _UA ) according to the concentration of Au ₂₅ are shown.
9 shows a cycle of Au ₂₅ SGE ( C = 15.97 mM) with different scan rates (2, 5, 10, 15, 20, 25, 30, 40, 50, 70, 100 mVs ⁻¹ , respectively, from inside to outside). Voltage and current graph.
10 shows Au ₂₅ Figure showing the change of anode and cathode peak currents with respect to the square root of the scan rate of Au ₂₅ SGE of different concentrations.
FIG. 11 is a diagram illustrating the synthesis of Au ₂₅ nanoparticles (Au ₂₅ ) protected with glutathione.
FIG. 12 is a diagram illustrating a bonding structure of nanoparticles and a sol-gel network in Au ₂₅ SG-SAME.
FIG. 13 shows the CV of Au ₂₅ SG-SAME recorded in the presence or absence of analyte in 0.1 M KCl.
FIG. 14 is a calibration graph for determining the ascorbic acid (A), uric acid (B), sulfite (C) and dopamine (D) contents from the voltammogram of Au ₂₅ SG-SAME in 0.1 M KCl.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Recent advances in the synthesis of ligand-protected gold nanoparticles have made it possible to produce highly uniform particles with the same atomic number. Particularly, in the case of gold nanoparticles composed of tens to hundreds of atoms, the quantum dot shows characteristics of quantum confinement effect as the size decreases without showing bulk metal properties. These quantum-sized metal nanoparticles have intrinsic redox activity, and thus may be used as electrochemical sensors without the addition of molecules or enzymes having electroactivity.

In the present invention, the electrochemically active quantum sized metal nanoparticles were prepared, their electrochemical characteristics were understood, and a series of processes were systematically executed from the basic to the application until the development of the electrochemical sensor using the characteristics. In this process, we developed an electrochemical sensor based on quantum-sized ligand-protected metal clusters (LPCs), which are nanoparticles whose physical and chemical properties are very different from conventional metal nanoparticles. One of the important properties of quantum size LPC is its electrochemical properties. Since quantum-sized LPCs exhibit quantized energy levels and have very small capacitance values (<10 ^-18 F), their oxidation / reduction processes can be observed experimentally. It varies greatly with the size of and shows different redox behavior.

The present inventors manufactured a new type of electrode based on the electroactive LPC, and understood the catalytic characteristics, and developed an electrochemical sensor using the characteristics. As described above, in the case of the conventional electrochemical sensor using gold nanoparticles, the gold nanoparticles themselves have no electrical activity and are mainly used as conductive materials to improve electrical conductivity. Therefore, in order to act as an electrochemical sensor, the redox mediator or enzyme sensitive to the analyte must be used together, so the configuration of the sensor is very complicated and the response mechanism is very complicated. There was a problem.

However, in the case of the electrochemical sensor based on LPC as in the present invention, since the LPC itself has oxidation / reduction activity, a redox mediator or enzyme is not required. In addition, since LPC itself can act as a small conductive material, it can be expected to exhibit excellent conductivity without the addition of other conductive materials. In other words, the quantum size LPC plays a dual role not only as a redox mediator but also as a conductive material, thus making it possible to manufacture a sensor having a much simpler structure than a conventional sensor.

Ligand for protecting the gold nanoparticles in the present invention can be selected from organothiol-based compounds, alkanedylate or glutathione is preferred. Specific examples are as follows.

Arkansiuol

Glutathione

Thiopronin

Thiolated Poly (Ethylene Glycol)

p-mercaptophenol,

Aromatic alkanthiols,

Phenyl alkanthiol

(r-mercaptopropyl) -trimethoxysilane

Hereinafter, the present invention will be described in detail through examples. However, the following examples are intended to help the understanding of the present invention, and the scope of the present invention should not be construed as being limited thereto.

Examples 1-1: hexane synthesis of Au ₂₅ nanoparticles protected with a thiol-rate

In view of the above advantages of an LPC-based electrochemical sensor, the embodiment of the present invention synthesizes highly monodisperse, hexanethiolate protected Au ₂₅ nanoparticles (Au ₂₅ ), [Au ₂₅ (SC It was identified as ^- ₆ H _{13) 18].} Au ₂₅ nanoparticles were captured in the sol-gel network using ethyltrimethoxysilane precursor to form Au ₂₅ SGE. As a typical example, 10 μM of a sol-gel mixture comprising Au ₂₅ was dropped on the surface of a glassy carbon electrode (GCE) and then dried overnight at room temperature.

And Figure 1 is Au ₂₅ SGE, sol-as shown by the reaction of the Au ₂₅ trapped in the gel network schematically, Au ₂₅ SGE by in Au _25, illustrates the electrocatalytic oxidation and mechanism of the delivery process by the parameters to be.

The specific synthesis process is as follows. Gold tetrachloride trihydrate (HAuCl ₄ · 3H ₂ O, reagent grade), 1-hexanethiol (98%), tetraoctylammonium bromide (Oct ₄ NBr, 98%), sodium borohydride (NaBH ₄ , 99%) , Ethyltrimethoxysilane (ETMOS, 97%), glutaraldehyde (25%), L-ascorbic acid (AA, reagent grade), uric acid (UA, 98%), sodium sulfite (reagent grade), dopamine hydrochloride, Potassium chloride (KCl) and tetrabutylammonium perchlorate (Bu ₄ NClO ₄ ) were purchased from Aldrich. Ultrapure grade toluene, acetone, anhydrous ethanol, acetonitrile, dichloromethane and dimethyl sulfoxide (DMSO) were used. Water was purified using Millipore Milli-Q system (18.2 MΩcm). All chemicals were used as purchased without further purification.

Au ₂₅ nanoparticles (Au ₂₅ ) was synthesized by a previously reported Brust two-phase process. The molar ratio of thiol / gold was 5: 1, and was synthesized by performing NaBH ₄ reduction at 0 ° C. for 30 minutes. Briefly, 3.3 mmol of Oct ₄ NBr dissolved in 120 mL of toluene was stirred vigorously for 30 minutes, followed by the addition of 2.5 mmol of HAuCl ₄ · 3H ₂ O dissolved in 20 mL of water. The aqueous layer turned colorless, while the toluene layer turned dark red with the introduction of AuCl ₄ ⁻ . 12.8 mmol of 1-hexanethiol was added to the organic layer and stirred at 0 ° C. until the solution turned colorless, ie, until an Au ^I -thiol polymer formed. A 25.6 mmol NaBH ₄ solution dissolved in 20 mL of water, cooled with ice, was added quickly to this mixture with vigorous stirring. The solution immediately turned black, indicating that a cluster protected by thiolate was formed. Then stirred at 0 ° C. for 30 min. After removing the aqueous layer, the organic layer was washed with water and rotary evaporated to give a black product. The resulting black product was left overnight in DMSO (200 mL). DMSO was removed and the black product was washed with acetonitrile and anhydrous ethanol and evaporated to dryness. Au ₂₅ was repeatedly extracted from the dried product using a 1: 1 acetonitrile / acetone mixture. Overall yield was 80-100 mg. It was confirmed that the ^anion-mass spectrometry, a Au ₂₅ nanoparticles synthesized by voltammetry and the open circuit potential measurements _{[Au 25 (SC 6 H 13} ) 18]. _{[Au 25 (SCH 2 CH 2} Ph) 18] - , as in the case identified in the crystal structure of the cluster, the counter ion is assumed to be Oct ₄ N ^+.

Example  1-2: Au ₂₅ in Reformed  Sol-gel thin film electrode Au ₂₅ SGE ) Production

Au ₂₅ nanoparticles were fixed in the form of a sol-gel thin film on a glassy carbon electrode (GCE, 3 mm in diameter). GCE was continuously polished with 1, 0.3 and 0.05 μm of Al ₂ O ₃ and then rinsed thoroughly with water. The electrode was sonicated successively in 0.1 M HNO ₃ , 0.1 MH ₂ SO ₄ , CH ₂ Cl ₂ and water and dried at room temperature. Subsequently, a slight modification of the method reported in Thenmozhi, K .; Narayanan, SS Electroanalysis 2007, 19 , 2362-2368, hydrolyzed ETMOS to trap Au ₂₅ nanoparticles in the sol-gel network. As a representative example, Au ₂₅ solution (10 mg / 0.2 mL CH ₂ Cl ₂ ) is mixed with 200 μM of water containing 25% (v / v) glutaraldehyde and 0.3 mL of ETMOS and then the mixture is mixed for 30 minutes. Ultrasonicated. The homogeneous solution obtained was stored at room temperature for 30 minutes. Au ₂₅ SGE was prepared by drop casting 10 μL of this mixture onto the GCE surface and drying at room temperature overnight. The prepared Au ₂₅ SGE was washed with water and used as a working electrode.

Through the above process, Au ₂₅ SGE was estimated to have an Au ₂₅ concentration of 15.97 mM. Au ₂₅ concentration of Au is ₂₅ SGE Au sol _{25 (25} to know the content of Au) were estimated by measuring the final volume and then drying the resultant gel mixture at 100μL capillary. Au ₂₅ SGE with various Au ₂₅ concentrations can be made by varying the weight of Au _{25 in} 0.5 mL of the original sol-gel mixture (before drying) to 2.0, 2.5, 3.0, 4.0, 5.0 and 7.5 mg. Were obtained ₂₅ Au concentration is 3.94, 4.87, 5.78, 7.54, 9.23 and 12.84 mM of Au ₂₅ SGE.

Example  1-3: Electrochemical Characterization

Electrochemical measurements were performed using an electrochemical analysis workstation (Model 660 B, CH Instruments). Square wave voltammetry (SWV) measurement of Au ₂₅ was carried out in CH ₂ Cl ₂ containing 0.1 M Bu ₄ NClO ₄ . Ar gas was used to remove oxygen in the solution. The experiment was performed in CH ₂ Cl ₂ using a Pt disk (0.4 mm diameter) as the working electrode, a Pt disk (0.4 mm diameter) as the counter electrode, and an Ag pseudo reference electrode as the reference electrode. Electrochemical properties and electrocatalytic activity of Au ₂₅ SGE were investigated by cyclic voltammetry (CV) in aqueous solution containing 0.1 M KCl. A three-electrode system was used with Au ₂₅ SGE as the working electrode, Pt disk as the counter electrode, and Ag / AgCl (3 M NaCl) as the reference electrode.

Transmission electron microscope (TEM) images of Au ₂₅ were recorded using JEOL transmission electron microscope (JEOL 2100F). TEM samples were prepared by dropcasting a 1 mg / mL Au ₂₅ solution dissolved in CH ₂ Cl ₂ onto a 400 mesh foam bar / carbon coated copper grid and drying at room temperature for 1 hour. Matrix assisted laser desorption ionization (MALDI) mass spectra were obtained using a MALDI-TOF Microflex mass spectrometer (Bruker Daltonics) equipped with a standard UV nitrogen laser (337 nm). The acceleration voltage was maintained at 15 kV and the spectrum was obtained in the linear cation detection mode. A CH ₂ Cl ₂ solution of Au ₂₅ was dissolved (saturated in CH ₂ Cl ₂ ) trans-2- [3- (4-t-butylphenyl) -2-methyl-2-propenylidene] malononitrile (DCTB) After mixing with the matrix it was applied to the sample plate and air dried. UV-Vis spectra of the prepared Au ₂₅ is Shimadzu UV-Vis NIR spectrometer to obtain a (UV 3600) using the new CH ₂ Cl ₂ solution of the prepared Au _25.

Figure 2 is a MALDI mass spectrum of the synthesized Au ₂₅ nanoparticles, Figure 3 TEM image of synthesized Au ₂₅ nanoparticles, scale bar of TEM image is 20 nm and insert is histogram of core size. 4 is a UV-Vis spectrum of the synthesized Au ₂₅ nanoparticles.

5 is (A) SWV of Au ₂₅ at a Pt working electrode in CH ₂ Cl ₂ comprising 0.1 M Bu ₄ NPF ₆ . (B) CV of Au ₂₅ SGE for 25 consecutive cycles run at 20 mVs ⁻¹ in 0.1 M KCl. 5A is a square wave voltammogram (SWV) of Au ₂₅ in CH ₂ Cl ₂ , illustrating redox characteristics of Au ₂₅ . Three distinct oxidation-reduction potential in the form of 0.62, 0.31 and -1.33 V for Ag pseudo-reference electrode (AgQRE) peaks, respectively ^{_{Au 25 1 + / 0, Au}} 25 0 / 1- and Au ₂₅ ^{1 - / 2-} redox By pairs. The cyclic voltammogram (CV) of Au ₂₅ SGE in 0.1 M KCl (FIG. 5B) also shows a distinct and reversible redox peak at the 0.34 V formal potential for Ag / AgCl, which corresponds to the Au ₂₅ ^{0 / 1-} pair. Is due. The redox peaks of the Au ₂₅ ^{1 + / 0} pairs are indistinguishable and appear as small shoulders around 0.43 V. The reason is not yet clear. The high dielectric constant of the solution may be due to the narrow peak spacing of the Au ₂₅ ^{1 + / 0} pair and the Au ₂₅ ^{0 / 1-} pair. In addition, as observed in Langmuir monolayers of similar particles, the first oxidation reaction of Au ₂₅ ^{1 + / 0} (Au ₂₅ ^{0 / 1-} ) compensates the charge in the sol-gel network. This may be due to the lack of counterion. However, the first oxidation reaction (Au ₂₅ ^{0 / 1-} ) appears to be very stable and reproducible. The peak potential and peak current of Au ₂₅ SGE remained unchanged for 25 consecutive cycles (FIG. 5B), indicating that Au ₂₅ was stably fixed in the sol-gel network.

On the other hand, the electrocatalytic activity of Au ₂₅ SGE on the oxidation of ascorbic acid (AA), uric acid (UA), sulfite and dopamine, four biologically or industrially important analytes, was investigated using the electrode according to the present invention. . FIG. 6 shows the CV of Au ₂₅ SGE and GCE (insert of FIG. 6) recorded in the presence or absence of analyte in 0.1 M KCl. Specifically, FIG. 6 shows a voltammetry demonstrating electrocatalytic oxidation of ascorbic acid (A), uric acid (B), sulfite (C) and dopamine (D) at 20 mVs ⁻¹ in 0.1 M KCl, (a) is the CV of Au ₂₅ SGE in the absence of analyte, (b-f) is the CV of Au ₂₅ SGE in the presence of 1, 2, 3, 4 and 5 μM of analyte, and (g) is in the absence of analyte CV of SGE, (h) is CV of SGE in the presence of 5 μM analyte.

As shown in FIG. 6, when the analyte was added in 1 μM, the anode peak current of Au ₂₅ SGE rapidly increased (curves b to f), whereas in the case of GCE, the anode current increased only slightly after 5 μM of the analyte was added ( Curve h).

In addition, as can be seen in Table 1 and FIG. 6, in the case of Au ₂₅ SGE, all analytes were found to be oxidized at a significantly lower potential (about 70 to 260 mV) compared to GCE. This reduction in oxidation potential, with the increase of the anode current, demonstrates that the electrocatalytic activity by the mediation of fixed Au ₂₅ is due to the following reaction.

<Scheme 1>

Au ⁰ + ₂₅ analytes (reduced state) → Au ₂₅ ^- + analyte (oxidation state)

Also, as shown in the calibration graph (FIG. 7), the anode peak current increase of all analytes was linear with respect to the concentration of the analyte added. Table 1 shows the electrocatalytic activity and amphoteric detection performance of Au ₂₅ SGE. As can be seen from this table, Au ₂₅ SGE was used to detect these analytes in amperometric range, with low detection limits and high sensitivity, in a linear range. At this time, the linear range, detection limits and sensitivity were equal or better than the recently reported electrochemical sensors for the detection of these analytes. This is the first application of redox-activated gold nanoparticles to current detection.

Oxidation of ascorbic acid (AA) and uric acid (UA) at Au ₂₅ SGE (FIGS. 6A and 6B) shows only one peak closer to the peak of oxidation of the medium (Au ₂₅ ), which is modified with all AA and UA modified. It means that the electrode has undergone mediated electrocatalytic oxidation. In comparison, sulfite and dopamine (FIGS. 6C and 6D) show two oxide peaks. The first peak is close to the oxidation potential of the mediator (~ 370 mV), and the second peak is close to the oxidation potential (~ 600 mV) of the bare electrode (SGE electrode not modified with Au). This may be due to the slow electrocatalytic reaction between Au ₂₅ 0 and the analyte. In other words, when these two analytes, only some of which have been oxidized in the vicinity of the oxidation potential of Au ₂₅ electrochemically catalytic rest will be oxidized at the higher potential to the Au ₂₅ nanoparticles act as an electrode. In the case of sulfite and dopamine, the detection performance (detection limit and sensitivity) was lower than that of AA and UA due to the low catalytic activity (Table 1). The reason for this is not clear. However, in the case of sulfite and dopamine, the electrocatalyst reaction may not be effective because the potential difference between E 1 and E 2 is large (Table 1).

8 is The electron diffusion coefficient ( D _E ), the self-exchange rate constant ( k _EX ), and the detection sensitivity of ascorbic acid ( S _AA ) and the detection sensitivity of uric acid ( S _UA ) according to the concentration of Au ₂₅ are shown.

As mentioned above, Au ₂₅ can act as an electron conductor and a redox mediator. In order to further investigate the role of Au _{25 as} a conductor, electron transfer of Au ₂₅ SGE according to Au ₂₅ concentration ( C ) was investigated. Stable voltammetric responses were observed at all concentrations ranging from 3.94 to 15.97 mM. The voltammetric response of Au ₂₅ SGE ( C = 15.97 mM) at various scan rates is shown in FIG. 9. In all modified electrodes fabricated with different Au ₂₅ concentrations, both the anode and cathode peak currents showed a change proportional to the square root of the scan rate in the scan rate range of 2 to 100 mVs ^-1 (Fig. 10). It means that the electron transfer within the diffusion control process. The apparent diffusion coefficient ( D _APP ) was calculated from the peak current. This diffusion process may include physical diffusion ( D _PHYS ) in the thin film, electron transfer diffusion ( D _E ) between the Au ₂₅ cores (ie, self-exchange of electrons) or both.

The electrocatalytic reaction between the Au ₂₅ 0 and the analyte in oxidation state and the electron transfer through the Au ₂₅ thin film are shown in FIG. 1. In order to calculate the travel speed from the _E-E D, it is assumed that D _E D >> _PHYS. Given the structure of Au ₂₅ SGE in which Au ₂₅ nanoparticles are trapped in the polymer network (Scheme 1), this assumption is very reasonable. Thus, in the ^{23 and 24} a thin film using the following equation Au ₂₅ 0 / - it can be calculated for a pair of self-exchange rate constant (k _EX) from the D _E.

<Reaction Scheme 2>

Where δ is the spacing between the centers of the Au ₂₅ cores. The calculated D _E and k _{EX are shown} for Au ₂₅ concentration (FIG. 8) and the values are shown in the following [Table 2].

The clear structure of Au ₂₅ SGE makes it possible to understand the effect of thin film electron conductivity on electrochemical detection. As shown in FIG. 8, D _E and k _EX increase rapidly at low Au ₂₅ concentrations and slowly reach a maximum at concentrations of 9.23 mM or more. This behavior is reflected in the detection sensitivity of ascorbic acid ( S _AA ) and uric acid ( S _UA ). S _AA and S _UA showed a more than 25-fold increase when the concentration increased 2.3 times from 3.94 mM to 9.23 mM, but almost linearly at concentrations above 9.23 mM. These results clearly show that the detection sensitivity is determined by the electron transfer process in the thin film, mainly at low Au ₂₅ concentrations. In addition, the linear increase in sensitivity with high concentrations at high concentrations means that all Au ₂₅ nanoparticles in the thin film are in an electrically activated state and are electrically connected to one another. Finally, the dependence of k _EX on the Au ₂₅ concentration provides new information on the electron transport mechanism in the thin film. When the Au ₂₅ concentration is above 9.23 mM, k _EX reaches maximum velocity, which appears to be limited by the tunneling rate through the hexanethiolate ligands between the Au ₂₅ cores. The maximum value of k _EX is 2.27 x10 ⁸ M ^-1 s ^{- 1} (Table 2), are compared with the thin film mesh of gold nanoparticles. The electron transfer rate at low concentrations ( C <9.23 mM) appears to be limited by the rate at which Au ₂₅ migrates to form precursor complexes for electron transfer. This interpretation is supported by the structural data of Au ₂₅ SGE, described in Table 2. Since the average distance between the centers of Au ₂₅ is too long, it is thought that electron transfer does not occur at this equilibrium distance.

In short, it was confirmed that the redox activity of the Au ₂₅ nanoparticles having excellent electrocatalytic activity can be used for the development of the amperometric sensor. Studies on the electron transfer process of Au ₂₅ modified electrodes confirmed that Au ₂₅ nanoparticles act as electron conductors and redox mediators. In addition, the effect of the electron transfer process on the detection sensitivity in the nanoparticle thin film was analyzed for the first time. A better understanding of the role of gold nanoparticles and the electron transfer process between gold nanoparticles will provide valuable information for the development of nanoparticle-based electrochemical sensors.

Example  2-1: With glutathione  Protected Au ₂₅ Synthesis of nanoparticles

Glutathione protected Au ₂₅ nanoparticles (Au ₂₅ ) were synthesized by the previously reported Brust method. The molar ratio of thiol / gold was 3: 1 and synthesized by performing NaBH ₄ reduction at room temperature for 90 minutes. A specific synthesis process of Au ₂₅ nanoparticles (Au ₂₅ ) protected with glutathione is shown in FIG. 11.

Briefly, 3.0 mmol of glutathione dissolved in 40 mL of water was stirred vigorously for 15 minutes, followed by addition of 1.0 mmol of HAuCl ₄ .3H ₂ O dissolved in 80 mL of methanol. It was then stirred at room temperature until the solution turned colorless, ie, until an Au ^I -thiol polymer was formed. Thereafter, a solution of 10.0 mmol of NaBH ₄ dissolved in 10 ml of water was added rapidly, and vigorously stirred for 1 hour and 30 minutes to form a cluster. The solution immediately turned black, indicating that a cluster protected by thiolate was formed. After the reaction was completed by rotary evaporation to remove the solvent to give a black product. The obtained black product was dissolved with a small amount of water, and then recrystallized with methanol, and separated by size. At this time, since the size is large, the methanol sinks, and additionally, methanol can be added to the liquid that is not settled to obtain a small size nanoparticle. The yield of the smallest nanoparticles separated last was 80-100 mg. The obtained nanoparticles are more soluble in water than in organic solvents.

Example  2-2: Au ₂₅ in Reformed  Sol-gel Single Layer Electrode (Au) ₂₅ SG-SAME  making

Au ₂₅ nanoparticles protected with glutathione were immobilized in the form of a sol-gel monolayer on a gold electrode (AuE, 3 mm in diameter). AuE was continuously polished with 1, 0.3 and 0.05 μm of Al ₂ O ₃ and then rinsed thoroughly with water. Electrodes were prepared using 0.1 M HNO ₃ , 0.1 MH ₂ SO ₄ , CH ₂ Cl _2. And continuously sonicated in water and dried at room temperature. In addition, 0.1 M sulfuric acid solution was electrochemically washed in the range of -0.2 V to 1.5 V. MPS was then hydrolyzed to capture Au ₂₅ nanoparticles in the sol-gel network.

In the sol-gel thin film electrode (Au ₂₅ SGE) modified with Au ₂₅ , the electrode is formed only through physical immobilization without chemical bonding between the nanoparticles and the sol-gel network. On the other hand, in Example 2, the chemical bond between the nanoparticles and the sol-gel network's thiol functional period makes it possible to manufacture a more stable electrode, which is illustrated in FIG. 12. Chemical bonds between nanoparticles and thiols and physical immobilization to porous sol-gel networks can be used to produce more stable quartz electrodes. Also in this implementation, it is possible to uniformly mix the nanoparticles and the sol-gel precursor and to condense / polymerize the mixture to form a stable sol-gel film.

As a representative example, a solution of Au ₂₅ (7.5 mg / 0.6 mL water) protected with glutathione is mixed with 1.8 mL of MPS, 0.1 mL of hydrochloric acid (0.1M), and 0.3 mL of ethanol, followed by mixing the mixture with 1 mL. Ultrasonicated for hours. The resulting homogeneous solution was stored at room temperature for 30 minutes. The mixture was then immersed in cleanly washed AuE overnight to form a sol-gel monolayer on the electrode surface. The prepared Au ₂₅ SG-SAME was washed with water, dried and used as a working electrode.

`

Example  2-3: Electrochemical Characterization

The redox activity of Au ₂₅ SG-SAME prepared in Example 2-2 was confirmed by cyclic voltammogram. FIG. 13 shows the CV of Au ₂₅ SG-SAME recorded in the presence or absence of analyte in 0.1 M KCl. Specifically shows a voltammetric curve demonstrating the electrocatalytic oxidation of ascorbic acid (A), dopamine (B) and sulfite (C) at 20 mVs ^-1 in 0.1 M KCl, (a) in the absence of analyte CV of Au ₂₅ SG-SAME, (b-f) is the CV of Au ₂₅ SG-SAME in the presence of 5, 10, 15, and 25 μM analyte.

FIG. 14 is a calibration graph for determining ascorbic acid (A), uric acid (B), sulfite (C) and dopamine (D) contents from a voltammogram of Au ₂₅ SG-SAME in 0.1 M KCl. As shown in the graph of FIG. 14, the anode peak current increase of all analytes was linear with respect to the concentration of analyte added.

Oxidation of ascorbic acid (AA) with dopamine and sulfide in Au ₂₅ SG-SAME (FIG. 13) shows only one peak closer to the oxide peak of the medium (Au ₂₅ ). This shows that all ascorbic acid, dopamine, and all of the sulfide involved in the measurement are electrocatalytically oxidized near the oxidation potential of Au ₂₅ . In sum, it has been confirmed that the redox activity of gold nanoparticles protected with glutathione ligands as well as Au ₂₅ nanoparticles protected with alkanthiolate ligands can be used in the development of amperometric sensors. In addition, depending on the ligand of the gold nanoparticles will be able to adjust the detection sensitivity of the electrochemical sensor.

Claims (12)

An electrochemical sensor, protected with an organothiol-based ligand, in which gold nanoparticles having redox activity are fixed in a sol-gel thin film. delete The method of claim 1,
The gold nanoparticles are electrochemical sensor, characterized in that fixed in the trapped state in the sol-gel network. The method of claim 1,
The organothiol ligands include alkanethiol, glutathione, thiophenine, thiolated poly (ethylene glycol), p-mercaptophenol, aromatic alkanethiol, phenylalkanethiol, (r-mercaptopropyl ) -Trimethoxysilane) electrochemical sensor. The method of claim 1,
The diameter of the gold nanoparticles protected with the ligand is an electrochemical sensor, characterized in that the quantum size of 0.1 to 5 nm. The method of claim 5,
The diameter of the ligand-protected gold nanoparticles is characterized in that the quantum size of 0.1 to 3 nm. The method of claim 1,
Electrochemical sensor, characterized in that for detecting any one selected from ascorbic acid, uric acid, dopamine or sulfite. The method of claim 1,
Electrochemical sensor, characterized in that for detecting the material by the electrocatalytic activity of the gold nanoparticles present on the electrode. The method of claim 1,
An electrochemical sensor comprising an Ag / AgCl electrode as a reference electrode and a platinum (Pt) disk electrode as a counter electrode. 1) synthesizing a gold nanoparticle cluster protected with an organothiol ligand; And
2) A method of manufacturing a sol-gel electrode comprising gold nanoparticles, comprising the step of fixing the gold nanoparticle cluster in the form of a sol-gel thin film on the electrode. The method of claim 10,
Step 1) comprises: i) mixing and stirring the organothiol compound solution and the gold (Au) hydrate solution; ii) a method for producing a sol-gel electrode comprising gold nanoparticles, comprising the step of performing a reduction reaction by adding sodium borohydride to the mixture. The method of claim 10,
Step 2) is a method of manufacturing a sol-gel electrode comprising gold nanoparticles, characterized in that the method is carried out by drop casting the gold nanoparticle solution synthesized in step 1) on the electrode surface and dried.

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