KR102475238B1 - ELECTROCHEMICALl DETECTION SENSOR OF ETIDRONIC ACID BASED ON NANOCOMPOSITE AND MANUFACTURING METHOD THEREOF - Google Patents

Abstract

The present invention relates to an electrochemical detection sensor based on a reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO2) for detecting etidronic acid, which includes an Au PCB (Au printed circuit board) substrate and the above It includes a layer made of reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO2) formed on the Au PCB substrate.
According to the present invention as described above, by using the electrode modified with the rGO-Ag@SiO ₂ nanocomposite as a detection sensor for etidronic acid, a detection range of a lower concentration and a detection limit of a very small amount are obtained compared to conventional etidronic acid detection sensors. It has excellent stability, repeatability and reproducibility.

Description

Etidronic acid nanocomposite-based electrochemical detection sensor and method for preparing the same. {ELECTROCHEMICALl DETECTION SENSOR OF ETIDRONIC ACID BASED ON NANOCOMPOSITE AND MANUFACTURING METHOD THEREOF}

The present invention relates to a reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ )-based electrochemical detection sensor and a manufacturing method thereof for detecting etidronic acid, and more particularly, to an existing Contains Au PCB (Au printed circuit board) modified with rGO-Ag@SiO ₂ nanocomposite, which has excellent stability, repeatability and reproducibility while having a lower detection range and a very small detection limit than etidronic acid detection sensors It relates to an electrochemical detection sensor that

Bisphosphonate compounds belong to the category of drugs in the field of modern pharmacology that prevent damage to bone density. Etidronic Acid (EA), also known as hydroxyethylidene diphosphonic acid (HEDP), is one of the substances belonging to the bisphosphonate group and is also called etidronate. When etidronic acid is used as a drug to prevent bone density damage, it is mainly used to reduce bone resorption by osteoclasts (Paget's disease and osteoporosis, etc.), and only very low concentrations (200-400 mg) of etidronic acid cause bone damage. is used to treat However, overdose of etidronic acid can cause severe renal failure, joint pain, and low blood calcium levels, and continuous use can cause acute oral and skin damage. Estimated overdose concentrations of etidronic acid have been found to be greater than 500 mg. Therefore, it is important to accurately monitor the concentration of EA in real samples and pharmaceutical formulations.

In the case of etidronic acid, direct detection through photometric and fluorescence measurements is not possible because there is no color-developing and fluorescent substance for detection yet. Existing detection methods for etidronic acid include a direct detection method using evaporative light scattering detection technology and an indirect detection method through chemical derivatization, but the former has a problem of low sensitivity, and the latter takes a long time to detect and the resulting derivative is sometimes There was a problem that it was difficult to perform a complete analysis because it was unstable. Recently, liquid phase chromatography using a conductivity meter has been spotlighted as an effective etidronic acid detection platform, but it is necessary to develop a sensor having higher sensitivity and stability than that.

The present invention uses an electrode modified with a reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) to detect etidronic acid, thereby enabling a lower concentration detection range and a very small amount compared to conventional etidronic acid detection sensors. The purpose is to obtain excellent stability, repeatability, and reproducibility while having a detection limit of .

In order to achieve the above object, the present invention is an Au PCB (Au printed circuit board) substrate; And a layer made of reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO2) formed on the Au PCB substrate; Provides a nanocomposite-based electrochemical detection sensor of etidronic acid including . In the reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ), Ag@SiO ₂ may be adsorbed on reduced graphene oxide (rGO).

In addition, in order to achieve the above object, the present invention provides (a) preparing a silver@silicon dioxide nanocomposite (Ag@SiO2); (b) preparing reduced graphene oxide (rGO); (c) preparing a reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO2) by mixing Ag@SiO2 and rGO prepared in the above step; (d) forming a layer containing the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO2) on an Au printed circuit board (Au PCB) substrate, etidronic acid nano A method for manufacturing a composite-based electrochemical detection sensor is provided.

In step (a), after mixing a solution containing tetraethyl orthosilicate (TEOS) and a solution containing ammonium hydroxide (NHOH), the mixture may be sonicated at 62 to 68 °C.

In step (b), after dispersing graphene oxide in water, hydrazine hydrate may be added while stirring at 76 to 84 °C.

In the step (c), ultrasonic treatment may be performed after mixing Ag@SiO ₂ and rGO.

In step (d), the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) may be drop-casted on an Au printed circuit board (Au PCB) substrate.

According to the present invention as described above, by using the electrode modified with the rGO-Ag@SiO ₂ nanocomposite as a detection sensor for etidronic acid, a detection range of a lower concentration and a detection limit of a very small amount are obtained compared to conventional etidronic acid detection sensors. It has excellent stability, repeatability and reproducibility.

1 is an image of an Au PCB including a working electrode, a counter electrode, and a reference electrode according to an embodiment of the present invention.
2 is (a) bare Au PCB, (b) rGO-modified Au PCB, (c) Ag@SiO ₂ -modified Au PCB, (d) rGO-Ag@SiO ₂ -modified according to an embodiment of the present invention. This is a SEM (scanning electron microscopy) image of the Au PCB surface morphology.
3 shows transmission electron microscopy (TEM) images of high-angle annular (annular) dark regions of (a) Ag@SiO _{2 and} (b) Ag@SiO ₂ according to an embodiment of the present invention, ( c) is an image showing the spatial distribution of Ag, (d) Si, and (e) O measured using EDX spectroscopy (Energy Dispersive X-ray Spectroscopy).
4 is a graph showing EDX spectra of (a) bare Au PCB, (b) rGO-Au PCB, and (c) rGO-Ag@SiO ₂ -Au PCB according to an embodiment of the present invention.
5 shows (a) UV absorption spectra, (b) Fourier transform infrared (FTIR) spectra of rGO, Ag@SiO ₂ and rGO-Ag@SiO ₂ according to an embodiment of the present invention. ) spectra).
Figure 6 shows the X-ray photoelectron spectroscopy (XPS) spectrum of rGO-Ag@SiO ₂ according to an embodiment of the present invention (a) deconvoluted C 1S, (b) deconvoluted O 1S spectrum, (c) extended Ag 3d, (d) extended Si 2p, (e) a graph showing XPS spectrum of overall rGO-Ag@SiO ₂ .
7 (a) and (b) show the CV response of the non-Faraday current response of the bare Au PCB and rGO-Ag@SiO ₂ -Au PCB in 0.1 M KCl, respectively, and FIG. 8 (c) shows the scan rate () 8 (d) and (e) show the relationship between the current density and the CV of bare Au PCB and rGO-Ag@SiO ₂ -Au PCB in 0.1 M KCl containing 5 mM K ₃ Fe(CN) ₆ , respectively. This is the graph showing the response.
8 shows bare Au PCB, rGO-Au-PCB, Ag@SiO ₂ -Au PCB, rGO-Ag@ in 0.1 M KCl containing 1 mM K ₃ Fe(CN) ₆ according to an embodiment of the present invention. It is a graph showing the electrochemical impedance spectroscopy (EIS) Nyquist plot of SiO ₂ -Au PCB.
Figure 9 is (a) in the presence of EA in 0.1 M NaOH according to an embodiment of the present invention at a fixed concentration (1 mg) of Ag@SiO ₂ and various concentration conditions (0, 0.5, 1.0 mg) of rGO Cyclic Voltammetry (CV) response of rGO-Ag@SiO ₂ , (b) is a graph showing a plot of Cr _GO /Cr _GO + C _Ag@SiO2 versus oxidation peak current.
10 is a graph showing Raman spectrum analysis of graphite, GO and rGO.
Figure 11 (a) compares the CV response of the rGO-Ag@SiO ₂ -Au PCB in the presence and absence of 1 mM EA in 0.1 M NaOH according to an embodiment of the present invention, Figure 11 (b) is a graph showing the CV responses of bare Au PCB, rGO-Au PCB, Ag@SiO ₂ -Au PCB, and rGO-Ag@SiO ₂ -Au PCB in the presence of 1 mM EA in 0.1 M NaOH.
12(a) shows the effect of the scan rate (25-200 mV/s) on the CV response of the rGO-Ag@SiO ₂ -Au PCB in the presence of 1 mM EA in 0.1 M NaOH electrolyte at pH 13, (b) is the linear relationship between the oxidation peak current (I _pa ) and the square root of the scan rate ( ^1/2 ) for EA, (c) is the differential pulse voltammetry for 1 mM EA in different pH media. It is a graph showing the differential pulse voltammetric (DPV) response.
Figure 13 is a schematic diagram showing possible steps related to the electrochemical oxidation of etidronic acid (EA).
14 is a schematic diagram showing a mechanism of electrochemical oxidation of EA in an rGO-Ag@SiO ₂ -Au PCB according to an embodiment of the present invention.
Figure 15 (a) shows rGO-Ag for different concentrations of EA (2.0, 5.0, 10.0, 20.0, 50.0, 100.0, 150.0, and 200.0 μM) present in 0.1 M NaOH according to an embodiment of the present invention. The differential pulse voltammetric (DPV) response of the @SiO ₂ -Au PCB, (b) is a graph showing the linear relationship between EA concentration and peak current.
16 is a graph showing the DPV response when EA of 100.0 μM present in 0.1 M NaOH is continuously measured 8 times using an rGO-Ag@SiO ₂ -Au PCB according to an embodiment of the present invention.
17 shows the DPV response of rGO-Ag@SiO ₂ -Au PCB for (a) EA in the presence of different interfering substances according to an embodiment of the present invention, (b) the peak current response for EA It is a graph that represents a bar chart.
18 is a graph showing the DPV response to purification of etidronate at different concentrations in 0.1 M NaOH of an rGO-Ag@SiO ₂ -Au PCB according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention is an Au PCB (Au printed circuit board) substrate; And a reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) formed on the Au PCB substrate; providing a nanocomposite-based electrochemical detection sensor of etidronic acid comprising a layer. do.

In the reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ), Ag@SiO ₂ may be adsorbed on reduced graphene oxide (rGO). The SEM measurement results of the Au PCB electrode modified with rGO-Ag@SiO ₂ show the successful adsorption of Ag@SiO ₂ microspheres on the rGO monolayer. (Fig. 2(d)) If etidronic acid continuously accumulates and is excessively present in the body, serious renal failure, joint pain, etc. may occur. It is important to be able to measure accurately and sensitively even when etidronic acid is present in a very small amount. Referring to Measurement Example 8 and Table 2, in the case of the nanocomposite-based electrochemical detection sensor including the Au PCB modified with rGO-Ag@SiO ₂ of the present invention, it is possible to directly detect etidronic acid, while the previously reported ethide It can be seen that compared to the detection method of Lonic acid, it shows a lower concentration detection range (2 ~ 200 μM) and a very small detection limit (0.68 μM).

Another form according to an embodiment of the present invention includes (a) preparing a silver@silicon dioxide nanocomposite (Ag@SiO ₂ ); (b) preparing reduced graphene oxide (rGO); (c) preparing a reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) by mixing Ag@SiO ₂ and rGO prepared in the above step; (d) forming a layer containing the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) on an Au printed circuit board (Au PCB) substrate of etidronic acid, A method for manufacturing a nanocomposite-based electrochemical detection sensor is provided.

In the step (a), after mixing a solution containing tetraethyl orthosilicate (TEOS) and a solution containing ammonium hydroxide (NH ₄ OH), the mixture may be sonicated at 62 to 68 ° C., the most Preferably, ultrasonication may be performed at 65 °C. When sonication is performed at a temperature outside the range of 62 to 68 °C, Ag may not adhere well to SiO ₂ , and Ag@SiO ₂ nanoparticles may not be properly formed.

In step (b), after dispersing graphene oxide in water, hydrazine hydrate may be added while stirring at 76 to 84 °C. Most preferably, it can be stirred at 80 °C. When stirring at a temperature outside the range of 76 to 84 °C, reduced graphene oxide may not be well formed.

In the step (c), ultrasonic treatment may be performed after mixing Ag@SiO ₂ and rGO. In addition to the ultrasonic treatment, there are methods of synthesizing a nanocomposite (rGO-Ag@SiO ₂ ) through gamma irradiation, spray, and microwave irradiation after mixing Ag@SiO ₂ and rGO, but these methods require a lot of time compared to ultrasonic treatment. required, and the synthesis efficiency is low, so there is a problem that is not suitable for mass production. On the other hand, when ultrasonic treatment is performed, Ag@SiO ₂ nanoparticles and rGO nanosheets may be uniformly present in the nanocomposite, and thus, the nanocomposite (rGO-Ag@SiO ₂ ) prepared through ultrasonic treatment may be applied to the electrode. When used, the reproducibility of the sensor is the highest. More specifically, in relation to sonication, the rGO-modified Ag@SiO ₂ nanocomposite (rGO-Ag@SiO ₂ ) was mixed with Ag@SiO ₂ and rGO, and then the mixture was briefly applied at 45% amplitude for 5 minutes. It can be self-assembled through sonication. Two different absorption peaks for the rGO-Ag@SiO ₂ nanocomposite demonstrate the existence of rGO and Ag@SiO ₂ (FIG. 5(a)), and rGO and Ag@SiO ₂ in rGO-Ag@SiO ₂ The presence of individual bands in a demonstrates that the nanocomposites were successfully formed due to a simple probe sonication procedure. (FIG. 5(b))

In step (d), the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) may be drop-casted on an Au printed circuit board (Au PCB) substrate. Through the drop casting, it is possible to form a layer including a simple reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) on an Au printed circuit board (Au PCB) substrate.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Synthesis of silver@silicon dioxide composite (Ag@SiO _{2 )}

An assisted sonication method was used to prepare the Ag@SiO ₂ nanoparticles. A solution containing 7.5 wt % of tetraethyl orthosilicate (TEOS) in ethanol was injected into a solution containing 4.25 wt % of NH ₄ OH in ethanol through a syringe pump at a flow rate of 0.8 mL/min. The mixture of solutions was then sonicated at 30% amplitude for 30 min at 65 °C, the probe sonication temperature. Thereafter, an aqueous mixture containing 4.75 wt% of AgNO ₃ and 4.25 wt% of NaBH ₄ was injected dropwise into the above solution, and then the solution was sonicated for 30 minutes. The sonicated solution was centrifuged, washed twice with ethanol, and dried at 80 °C for 3 hours.

Example 2. Preparation of reduced graphene oxide (rGO)

A modified Hummers method was used to prepare rGO. Initially, 1.0 g of graphite powder was dispersed in 40 mL of H ₂ SO ₄ in a round bottom flask, and the solution was stored in an ice bath. Next, 3.5 g of KMnO ₄ was added to the prepared solution and the reaction mixture was stirred for 2 hours, after which 150 mL of Milli-Q water was added. Dropwise addition of hydrogen peroxide (H ₂ O ₂ ) was carried out when the reaction mixture no longer formed bubbles. The prepared solution was then filtered to remove unwanted metal ions, and the filtered solution was washed with 10% hydrochloric acid (HCl) and completely dried to obtain graphene oxide (GO). After dispersing the obtained graphene oxide (GO) in Milli-Q water, GO was reduced to rGO by introducing 1 mL of hydrazine hydrate while continuously stirring at 80° C. for 1 hour. The color of the obtained solution turned black, which confirmed the formation of rGO. The collected precipitate was filtered and then dried in a vacuum oven.

Example 3. Self-assembly of rGO-Ag@SiO ₂

1 mg each of rGO and Ag@SiO ₂ was dispersed in 1 mL of Milli-Q, and the probe was sonicated for 5 min at 45% amplitude.

Example 4. Surface modification of Au PCB

An integrated circuit-based Au Printed Circuit Board (Au PCB) was used as the electrode. The Au PCB contained a working electrode with a diameter of 1.48 mm, a counter electrode with a length of 4.3 mm and a width of 1.6 mm, and a reference electrode with a length of 4.1 mm and a width of 0.8 mm. (FIG. 1) The Au PCB was sequentially washed with acetone, ethanol and Milli-Q water, and after washing and drying the Au PCB, plasma treatment was applied to the Au PCB. Afterwards, about 5L of the prepared rGO-Ag@SiO ₂ nanocomposite was drop-cast onto the clean Au PCB.

Example 5. Preparation of samples

A 200 mg etidronate drug tablet was used to prepare three different concentrations (25.0, 50.0 and 100.0 M) of real samples of etidronic acid (EA) in 0.1 M NaOH diluent. The tablets were ground finely with the help of a mortar and pestle and dispersed in 0.1 M NaOH electrolyte as a diluent. The sample was then filtered to remove undissolved excipients. The filtered sample was diluted 100-fold with 0.1 M NaOH to fit the peak current response of EA to the calibration curve, and the concentration of EA obtained in the final calculation was multiplied by the dilution factor.

Measurement Example 1. SEM (scanning electron microscopy) image

The surface morphology of bare Au PCB, rGO-Au PCB, Ag@SiO ₂ -Au PCB, and rGO-Ag@SiO2- Au PCB was investigated using SEM (scanning electron microscopy). A smooth surface was observed on the bare Au PCB (Fig. 2(a)), which changed to a thin sheet-like monolayer structure due to rGO on the rGO-Au PCB. (FIG. 2(b)) The Ag@SiO ₂ -Au PCB showed uniform and evenly distributed Ag@SiO ₂ microspheres. (FIG. 2(c)) SEM measurement results of the rGO-Ag@SiO ₂ -Au PCB electrode showed successful adsorption of Ag@SiO ₂ microspheres on the rGO monolayer. (Fig. 2(d))

TEM (transmission electron microscope) analysis was performed to recognize Ag-embedded SiO ₂ nanoparticles, and FIG. 3(a) shows the nanostructure of the Ag@SiO ₂ composite. The image confirms the presence of spherical SiO ₂ nanoparticles embedded with silver nanoparticles. Fig. 3(b) shows a high-angle annular (annular) dark field image of Ag@SiO ₂ . Elements of Ag@SiO ₂ were analyzed by energy dispersive X-ray spectroscopy (EDX), and Ag (Fig. 3(c)), Si (Fig. 3(d)) in Ag@SiO ₂ were analyzed. ) and O (FIG. 3(e)) confirmed the presence of spatial distribution of the elements.

The elemental composition of Au PCB, rGO-Au PCB and rGO-Ag@SiO ₂ -Au PCB was analyzed through EDX spectra. The dominant elements observed in the Au PCB were Ni, Au, C, and O, with the corresponding weight percentages of 63.4 wt%, 30.7 wt%, 5.2 wt%, and 0.7 wt%, respectively. (FIG. 4(a)) The rGO-Au PCB contained 57.9 wt% C and 4.9 wt% O. (FIG. 4(b)) EDX analysis of the rGO-Ag@SiO2-Au PCB showed the presence of C, O, Ag and Si with corresponding weight percentages of 16.0 wt%, 8.8 wt%, 48.6 wt% and 1.4 wt%. I checked. (FIG. 4(c))

Absorption spectra of rGO, Ag@SiO ₂ and rGO-Ag@SiO ₂ were analyzed in the wavelength range of 200–800 nm. (FIG. 5(a)) The absorption peak of rGO was observed at 272.7 nm and the very strong absorption peak of Ag@SiO ₂ was observed at 410.2 nm. The rGO-Ag@SiO ₂ nanocomposite showed absorption peaks for rGO and Ag@SiO ₂ at wavelengths of 276.8 and 408.6 nm, respectively. Two different absorption peaks for the rGO-Ag@SiO ₂ nanocomposite demonstrate the presence of rGO and Ag@SiO ₂ .

Individual functional groups of rGO, Ag@SiO ₂ and rGO-Ag@SiO ₂ were studied by Fourier transform infrared (FTIR) spectroscopy.

Analysis was performed using wave numbers from 500 to 4,000 cm ^-1 . (FIG. 5(b)) C-OH (1190 cm ^-1 ), C = C (1556 cm ^-1 ) and OH (3695 cm ^-1 ) vibrations were identified in rGO. The bands of Ag@SiO ₂ recorded at wavenumbers of 831, 978, 2896 and 2981 cm ^-1 corresponded to Si-O, Si-OH, -CH ₂ and -CH ₂ , respectively. After the rGO-Ag@SiO ₂ nanocomposite was formed, the individual vibrations present in rGO and Ag@SiO ₂ were C-OH (1212 cm ^-1 ), C=C (1553 cm ^-1 ), and OH (3675 cm ^-1 ). , and the individual vibrations present in Ag@SiO ₂ are Si-O (827 cm ^-1 ), Si-OH (983 cm ^-1 ), -CH ₂ (2900 cm ^-1 ), and -CH ₂ (2985 cm ⁻¹ ). The presence of individual bands of rGO and Ag@SiO ₂ in rGO-Ag@SiO ₂ demonstrates that the nanocomposite was successfully formed due to a simple probe sonication process.

X-ray photoelectron spectroscopy was used to analyze the elemental and electronic states in the rGO-Ag@SiO ₂ nanocomposite. The analysis results showed the presence of C, O, Ag and Si in the rGO-Ag@SiO ₂ nanocomposite. (FIG. 6(e)) In the extended C 1S spectrum (FIG. 6(a)), the deconvoluted peak (separating a peak in the spectrum into several different peaks) is attributed to the CC, CO, and C = O spectra, respectively. were 284.4, 286.8 and 288.9 eV. The extended O 1s spectrum (Fig. 6(b)) showed deconvoluted peaks at 534.3, 532.5 and 530.8 eV corresponding to the CO, Si-O and C = O spectra. The extended Ag 3d spectrum shown in Fig. 6(c) contains two orbitals (orbitals) attributed to Ag 3d _5/2 (368.5 eV) and Ag 3d _3/2 (374.4 eV). An extended Si 2p spectrum (FIG. 6(d)) was observed at 103.2 eV.

Measurement Example 2. Electrochemical characteristics of rGO-Ag@SiO2-Au PCB

Two methods were used to estimate the electrochemical active surface area (ECASA). The first method involves the estimation of the roughness factor and the second method involves the evaluation of ECASA in the presence of the redox probe [(K3Fe(CN) ₆ ].

The roughness factor (Rf) of the electrode is calculated from the double layer capacitance (Cdl). Non-Faraday currents were captured over a voltage range of −0.7 to −0.2 V and at different scan rates (25 to 200 mV/s). The current density at −0.45 V was considered to constitute Cdl. 7(a) and (b) show the cyclic voltammetry (CV) responses of bare Au PCB and rGO-Ag@SiO ₂ -Au PCB in 0.1M KCl, respectively. The calculated Cdl values of bare Au and rGO-Ag@SiO ₂ -Au PCBs were 1.58 and 2.20 mF/mm ² , respectively. (FIG. 7(c)) This shows that the roughness factor of the Au PCB increased by 1.4 times by modifying the Au PCB with the rGO-Ag @ SiO2 nanocomposite.

ECASA evaluation of bare Au PCB and rGO-Ag@SiO2-Au PCB was performed in the presence of 5mM K ₃ Fe(CN) ₆ in 0.1 M KCl. 7(d) and (e) show the CV responses of a bare Au PCB and an Au PCB modified with rGO-Ag@SiO ₂ , respectively. The Randles-Sevcik equation was applied to estimate ECASA values.

I _pa = 2.69 × 105 n ^3/2 AD ^1/2 C ^1/2

where I _pa = anodic peak current, n = number of electrons transferred, A = ECASA, D = diffusion coefficient (D = 7.6 × 10-6 cm ² / s), C is potassium ferricyanide concentration (mol / cm ³ ) , = scan rate (mV/s).

The calculated ECASA values of the Au PCB and the Au PCB modified with rGO-Ag@SiO ₂ (rGO-Ag@SiO ₂ -Au PCB) were found to be 2.210 and 13.812 mm ² , respectively. (Table 1) The actual percentage value (%) in relation to the surface area is the ratio between the ECASA and the geometric surface area of the Au PCB, which is found to be 122.3%.

[Table 1]

In the presence of 5mM K ₃ Fe(CN) ₆ , as a redox probe, electrochemical impedance spectroscopy (EIS) was performed on an rGO-Ag@SiO ₂ -modified Au PCB in the frequency range between 0.1 mHz and 1 MHz ( rGO-Ag@SiO ₂ -Au PCB) electrode. Diffusion was promoted at low frequencies of the electrodes, while high frequencies could create resistance. The bare Au PCB showed a higher charge transfer resistance (Rct) of 8.62 kΩ. (Fig. 8)

In this study, the rGO-modified Au PCB (rGO-Au PCB) showed the lowest Rct (5.53KΩ) compared to other modified PCBs. The fast electron transport of rGO causes fast charge transfer at the rGO-Au PCB electrode interface, which can be attributed to the low Rct of the rGO-modified Au PCB. The Au PCB modified with Ag@SiO ₂ (Ag@SiO ₂ -Au PCB) showed a high Rct (7.73KΩ) due to the formation of an electron blocking layer on the Ag@SiO2-Au PCB electrode. The formation of an electronegative charge on the Ag@SiO ₂ -Au PCB caused an electrostatic repulsion between the Ag@SiO ₂ -Au PCB and ferricyanide ions, which also created an electron blocking layer.

The Au PCB modified with rGO-Ag@SiO ₂ (rGO-Ag@SiO ₂ -Au PCB) produced a low Rct (6.60 KΩ). The presence of rGO in the rGO-Ag@SiO ₂ can enhance the charge transfer of the rGO-Ag@SiO ₂ -Au PCB. Through this EIS (electrochemical impedance spectroscopy) study, it was confirmed that the importance of rGO in the Au PCB modified with rGO-Ag@SiO ₂ and diffusion occurred in the rGO-Ag@SiO ₂ -Au PCB.

Measurement Example 3. Optimization of the rGO and Ag@SiO2 quantities

Optimization of rGO and Ag@SiO ₂ was performed by increasing the concentration of rGO from 0 to 1 mg/mL (0, 0.5 and 1.0 mg/mL), while holding the concentration of Ag@SiO ₂ at 1 mg/mL. . Optimization of rGO and Ag@SiO ₂ loading was investigated through cyclic voltammetry (CV) in 0.1M NaOH containing 1mM EA. The CV response to different concentrations of rGO-Ag@SiO ₂ showed that the anode peak current improved to 19.4 μA as the concentration of rGO increased from 0 to 1 mg/mL. (Fig. 9) This shows the importance of rGO in enhancing the electrocatalytic activity in the electrochemical detection of EA.

Measurement Example 4. Raman spectrum analysis of rGO

Raman spectral analysis revealed two characteristic peaks for graphite, GO and rGO: D (defects in graphene) and G (symmetric peaks) bands and a weak 2D band. 10 shows Raman spectral analysis of graphite, GO and rGO. I _D / I _G values provide information about the disorder of graphene and can be used to distinguish GO from rGO. As GO is converted to rGO, I _D / I _G may increase or decrease. The intensities of the corresponding Raman shift values for graphite, GO and rGO are shown in Table 2. In graphite, weak D (1330.5 cm ^-1 ), intense G (1560.1 cm ^-1 ) and 2D (2679.3 cm ^-1 ) peaks were observed. GO exhibited intense D and G peaks and weak 2D peaks at 1340.9, 1573.5 and 2654.3 cm ⁻¹ , respectively. Finally, high-intensity D (1348.4 cm ^-1 ) and G (1585.4 cm ^-1 ) peaks were observed in rGO along with a very weak 2D peak (2670.9 cm ^-1 ). The increase of I _D /I _G strongly suggests the high defect density of GO and rGO. rGO has a higher I _D / I _G value compared to graphite and GO, which means a higher number of nanocrystalline aromatic carbons.

[Table 2]

Measurement Example 5. Electrochemical oxidation of etidronic acid on Au PCB modified with rGO-Ag@SiO ₂

To study the effect of etidronic acid (EA) on Au PCB modified with rGO-Ag @ SiO ₂ (rGO-Ag @ SiO ₂ -Au PCB), 1 mM EA in 0.1 M NaOH was used. CV responses were obtained in the presence and absence of EA to investigate the oxidation potential of EA and the electrocatalytic activity of the rGO-Ag@SiO ₂ -Au PCB on EA. (FIG. 11(a)) After adding 1 mM EA, the current response increased to 19.4 μA at 0.67 V. It showed enhanced electrocatalytic ability and ECASA of Au PCB modified with rGO-Ag@SiO ₂ .

Since EA contains numerous hydroxyl groups in its structure, the strengthening of hydrogen bonds between SiO ₂ and -OH groups of EA may be responsible for the high selectivity toward EA. The electrochemical oxidation of EA on individual layers of an rGO-Ag@SiO ₂ -Au PCB electrode was investigated. Bare Au PCB, rGO-Au PCB, Ag @ SiO ₂ -Au PCB and rGO-Ag @ SiO ₂ -Au PCB electrodes were studied in the presence of 1 mM EA in 0.1 M NaOH electrolyte (pH = 13). Fig. 11(b) shows the CV responses of individual layers of the rGO-Ag@SiO ₂ -Au PCB electrode. The bare Au PCB did not show a significant current response to EA and obtained a very negligible current (0.25 μA) across EA at 0.61V. A significant increase in current response (3.45 μA) was observed at 0.67 V for the rGO-Au PCB. The Ag@SiO ₂ -Au PCB produced an improved current response (5.99 μA) at 0.68 V. Although the Au PCB modified with individual layers as above did not show an elevated current response, the rGO-Ag@SiO ₂ -Au PCB produced a current response of 19.4 μA at 0.67 V, which was 77.6 μA higher than the current response of the bare Au PCB. times higher. The higher current response to EA on the rGO-Ag@SiO ₂ -Au PCB confirmed the enhanced electrocatalytic activity and electron transfer.

Measurement Example 6. Effect of pH and scan rate

To evaluate the effect of scan rate on the electrochemical oxidation of 1 mM EA in 0.1 M NaOH, CV responses were recorded at scan rates of 25 to 200 mV/s and the results are shown in Fig. 12(a). At 0.67 V, the anode peak current corresponding to oxidized EA increases with increasing scan rate. The Randles-Sevcik equation was applied to study the electrochemical oxidation process of EA on rGO-Ag@SiO ₂ -Au PCB.

A linear graph between the square root of the scan rate ( ^1/2 ) and the corresponding anodic peak current of EA (FIG. 12(b)) provides specific confirmation that the electrochemical oxidation of EA is a diffusion process.

To improve the stability and performance of the electrochemical sensor, pH studies were performed in three different pH media containing 1 mM EA. : 0.1M acetate buffer (pH = 4.4), 10mM PBS (pH = 7.4) and 0.1M NaOH (pH = 13)

A negative shift of the oxidation potential (Ep) was observed with increasing pH of the electrolyte (Fig. 12(c)) and a low current response (3.29 μA) was observed in acidic medium. An enhanced peak current (10.3 μA) was observed at neutral pH compared to the acid medium. The highest peak current (18.4 μA) of EA was observed at 0.68 V in 0.1 M NaOH and pH = 13.

Measurement Example 7. EA Detection Mechanism

EA follows an oxidation procedure similar to that of tertiary alcohols. (FIG. 13) In the case of oxidation of EA, the CP bond is initially broken due to oxidation, resulting in the formation of phosphate ions. The oxidation process is driven by the participation of 1.5 times more protons than electrons. The second step involves removing H ₂ O from the intermediate product. The final step deals with the cleavage of the CP bond, with the participation of 3 protons and 2 electrons, creating a phosphate ion.

14 shows how the Au PCB modified with rGO-Ag@SiO ₂ (rGO-Ag@SiO ₂ -Au PCB) promotes enhanced electron transport in the electrochemical detection of EA . The improved electrochemical detection of EA is attributed to the enhanced ECASA of the Au PCB modified with rGO-Ag@SiO ₂ in addition to the many active sites possessed by the rGO-Ag@SiO ₂ composite.

Measurement Example 8. DPV Study

The feasibility verification of the Au PCB electrode (rGO-Ag@SiO ₂ -Au PCB) modified with rGO-Ag@SiO ₂ at various concentrations of EA was performed using the Differential Pulse Voltammetric (DPV) technique. . As the concentration of EA increased, the oxidation peak current of EA increased. Figure 15(a) shows DPV curves obtained at various concentrations of EA. The verified Ep of EA was obtained at 0.68V. The Ep of EA was kept constant and the Au PCB electrode modified with rGO-Ag@SiO ₂ showed considerable stability. The EA concentration versus oxidation peak current plot (FIG. 15(b)) shows that the oxidation peak current of EA increases linearly as the EA concentration increases. A linear expression of EA concentration versus EA peak current can be written as Y = 0.0497X + 1.0293 (R ² = 0.9890). The error bar represents the standard deviation of the five determinants.

To calculate the limit of detection (LOD) for the electrochemical detection of EA, the IUPAC method was used. Following this method, 10 empty injections of 0.1 M NaOH were added and the DPV response was recorded.

LOD = 3S _B /S (S _B : standard deviation of 10 blank samples, S : slope of calibration curve)

For the Au PCB modified with rGO-Ag@SiO ₂ , the LOD of electrochemical detection of EA was calculated to be 0.68 μM. The constructed linear equation can be used to determine the unknown concentration of EA in real sample analysis. The effectiveness of the EA sensor based on the rGO-Ag@SiO ₂ -Au PCB was compared with previously evaluated sensors, and the results are shown in Table 3.

[Table 3]

Measurement Example 9. Stability, reproducibility, reusability

The operational stability of the rGO-Ag@ SiO ₂ -Au PCB electrochemical sensor was evaluated via CV in the presence of 1 mM EA in 0.1 M NaOH at pH = 13.

20 CV cycles were recorded, and 92.7% of the initial peak current was maintained at the end of the 20th cycle. To evaluate the long-term stability of the rGO-Ag@SiO ₂ -Au PCB in EA detection, the rGO-Ag@SiO ₂ -Au PCB was stored at room temperature (25 °C), and storage stability studies were conducted at 7-day intervals for 4 weeks. has been carried out At the end of the fourth week, 93.1% of the DPV peak current response of the initial peak current was maintained.

The evaluation of the reusability of the rGO-Ag@SiO ₂ -Au PCB in the electrochemical detection of 100.0 μM EA was performed using the DPV technique. A single rGO-Ag@SiO ₂ -Au PCB was used continuously for 8 runs. At the end of each run, the rGO-Ag@SiO ₂ -Au PCB was washed with 0.1M NaOH buffer to remove the oxidized form of EA adsorbed on the Au PCB. Observed DPV current responses were plotted in a bar chart (FIG. 16) for comparison across 8 runs. The relative standard deviation (% RSD) evaluated between the 8 runs was calculated to be 3.51 %.

The reproducibility of the rGO-Ag@SiO ₂ -Au PCB was investigated using four independent rGO-Ag @ SiO ₂ -Au PCBs in the presence of 100.0 μM EA in 0.1 M NaOH. The % RSD between DPV current responses of EA was estimated at 2.88%.

Table 4 shows the performance of sensor parameters of rGO-Ag @ SiO ₂ in EA detection.

[Table 4]

Measurement Example 10 Interference Study

The selectivity of EA detection on the rGO-Ag@SiO ₂ -Au PCB was evaluated in the presence of different excipients, coexisting and ion-interfering substances. To determine the effect of selectivity and interferers on the peak current of EA and Ep, 50 μM of EA was added to 5 mM of each other interferer. : Glucose (Glu), uric acid (UA), ascorbic acid (AA), potassium hydroxide (KOH), starch, nitric acid (HNO ₃ ), sodium chloride (NaCl), magnesium stearate (MS) and cetyl trimethylammonium bromide (CTAB) . The recorded DPV response is shown in Fig. 17(a).

A comparison of the effect of different interfering substances on the EA peak current is shown in Fig. 17(b). Table 5 shows the Ep values and current response of EA after addition of different interfering substances. No significant changes were observed in Ep of EA. The change in EA peak current due to the addition of interfering substances was less than 5%. This demonstrates that the developed sensor is highly selective for EA.

[Table 5]

Measurement Example 11. Analysis of real samples

To confirm the real-time application of the proposed sensor, the Au PCB modified with rGO-Ag@SiO ₂ (rGO-Ag@ An electrochemical sensor based on a SiO ₂ -Au PCB) was used. The obtained DPV current response (FIG. 18) was fitted with a calibration curve (Y = 0.0497X + 1.0293) to calculate the concentration of EA present in the actual sample. Recovery of EA from pharmaceutical samples was calculated to be between 98.3% and 102.9%. (Table 6)

[Table 6]

Claims (7)

Au printed circuit board (Au PCB) substrate; and
A nanocomposite-based electrochemical detection sensor of etidronic acid comprising a layer made of reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) formed on the Au PCB substrate.
According to claim 1,
The reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) is an electrochemical based nanocomposite of etidronic acid, characterized in that Ag@SiO ₂ is adsorbed on rGO (reduced graphene oxide) detection sensor.
(a) preparing a silver@silicon dioxide nanocomposite (Ag@SiO _{2 )} ;
(b) preparing reduced graphene oxide (rGO);
(c) preparing a reduced graphene oxide-silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) by mixing Ag@SiO ₂ and rGO prepared in the above step;
(d) forming a layer containing the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) on an Au printed circuit board (Au PCB) substrate of etidronic acid, Manufacturing method of nanocomposite-based electrochemical detection sensor.
According to claim 3,
In the step (a), a solution containing tetraethyl orthosilicate (TEOS) and a solution containing ammonium hydroxide (NH ₄ OH) are mixed and the mixture is sonicated at 62 to 68 ° C. Characterized in that Manufacturing method of nanocomposite-based electrochemical detection sensor of etidronic acid.
According to claim 3,
The step (b) is a nanocomposite-based electrochemical detection sensor of etidronic acid, characterized in that after dispersing graphene oxide in water, adding hydrazine hydrate while stirring at 76 to 84 ° C. Manufacturing method of.
According to claim 3,
The step (c) is a method for producing a nanocomposite-based electrochemical detection sensor of etidronic acid, characterized in that Ag@SiO ₂ and rGO are mixed and then ultrasonicated.
According to claim 3,
In the step (d), the reduced graphene oxide-silver@silicon dioxide nanocomposite (rGO-Ag@SiO ₂ ) is drop-casted on an Au printed circuit board (Au PCB) substrate. Manufacturing method of nanocomposite-based electrochemical detection sensor of tidronic acid.

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