KR100932601B1 - Zinc oxide quantum dot manufacturing method and blood glucose sensor formed using the same - Google Patents

Abstract

The present invention relates to a zinc oxide quantum dot manufacturing method and a blood glucose sensor formed using the same, a zinc oxide quantum dot manufacturing process by adding a mercapto Undecanoic Acid (MUA) acting as a surfactant to the zinc precursor The present invention relates to a method for simplifying blood glucose levels by binding glucose oxidase to zinc oxide quantum dots surface-treated with Mercapto Undecanoic Acid (MUA).

Description

Method of manufacturing zinc oxide quantum dots and blood glucose sensor formed using the same {Method of Fabricating ZnO quantum dot and Glucose Sensor Using The Same}

The present invention is a non-toxic and biochemically stable material, which is surface treated with a mercapto Undecanoic Acid (MUA) material on zinc oxide, which has high value for use in biolabel and electrochemical applications. The present invention relates to a technology for easily preparing a zinc oxide quantum dot and easily forming a blood glucose sensor using the same.

Recently, the use of electrochemical biosensors for analyzing biological samples including blood in the pharmaceutical field is increasing.

A biosensor is a system that uses biological elements or mimics biological elements to obtain information from an object to be converted into useful signals that can be recognized, such as color, fluorescence, and electrical signals.

These biosensors focused mainly on blood glucose sensors with high clinical demand until just a few years ago. However, recent advances in molecular biology, nanotechnology (NT) and information and communication technology (IT) have led to the development of multi-sensor characteristics. The development of various sensors incorporating the PSA has been attempted, attracting much attention in terms of mass retrieval, multi-measurement or multi-diagnosis in addition to the purpose of simple biochemical aspects.

The most demanded fields for biosensors are known as the medical sector, which allows for free movement and immediate detection, which facilitates the use of high-risk drugs in the medical field and enables rapid medical care for critically ill patients. At the same time, demand is expected to continue to grow in the medical sector.

As applied to the medical field as described above may include a biosensor capable of selectively quantitative analysis of a specific substance in a biological sample, such as blood glucose, cholesterol, etc., and improves performance and new technologies around the world's manufacturers. Various studies are underway for development.

The most commonly used biosensors to date include a glucose sensor for measuring blood glucose, which is described as an example of a glucose sensor.

Blood glucose sensors have been constantly developed to date based on the first sensor made by incorporating glucose oxidase, which oxidizes glucose, onto a polyacrylamide gel membrane and attaching it to the diaphragm sensor electrode.

Glucose oxidant, an enzyme used in blood glucose sensors, can be obtained easily and cheaply, and is more stable against pH, ionic strength, and temperature than other enzymes, and the optimal condition for oxidizing glucose to match the concentration of glucose in human blood. It is widely used.

The analysis method of the above blood glucose sensor can be largely divided into photometeric method and electrochemical method. Both photometric and electrochemical methods basically use an oxidase which can react with glucose to oxidize glucose. do.

Photometric method quantifies the degree of color change by measuring the reflectance or transmittance of light using a photometer using a dye source that produces a color change when glucose is oxidized.

In contrast, electrochemistry measures glucose by measuring the electrons generated when oxygen or oxidized mediators are converted to hydrogen peroxide or reduced mediators when glucose is oxidized and then oxidized and returned to their original oxidized form in the form of current flowing through the electrodes. Quantify

Photometric methods generally require a longer measurement time, require a relatively large amount of blood, and are difficult to analyze important biological materials due to measurement errors due to turbidity of biological samples.

Therefore, recently, after forming an electrode, a screen reagent, a film adhesive method, or the like is used to expose an analyte to which only the analyte is fixed, and the remaining reagent is blocked to react with the measurement component in the sample. Electrochemical methods for quantitatively measuring a specific substance in a biological sample by applying and applying a constant potential after coating and fixing the exposed part and introducing a biological sample such as blood have been widely applied to a biosensor.

In the electrochemical method, a blood glucose sensor having a rectangular working electrode and a reference electrode formed on the insulating substrate and a recognition electrode formed on the other side of the insulating substrate is used.

Here, the recognition electrode is formed at a predetermined position on the test strip determined according to what substance is fixed by the reagent fixed across the working electrode and the reference electrode, and when the test strip is inserted into the biosensor measuring device, the biosensor measuring device By identifying the position of the recognition electrode on the test strip, it is possible to identify which material the test strip is intended to analyze.

In order to manufacture the test strip as described above, the electrode material is formed by sputtering electrode material on a predetermined region on the insulating substrate, and the reaction reacts with the measurement component in the sample while exposing only the part where the analysis reagent is fixed and blocking the remaining part. The reagents are applied and fixed to the exposed part.

Meanwhile, in the biosensor in which electrons are transferred through the working electrode as described above, current is generated for a predetermined time while electrons are generated through the reaction appearing at the working electrode after the sample is inputted, and the current signal is read by the measuring device to correspond to the sample. The components will be quantitatively analyzed.

In this case, when the same reaction occurs in the working electrode, the same current signal should be generated.

That is, even if the measurement using a plurality of test strips for the same sample of the same component (the same component to be measured) the reaction of the component and the reagent in the sample at the working electrode is the same, the same current signal should be output.

However, this should be the same for all the test strips manufactured, the area of the working electrode part (reaction part) to which the reagent is fixed, that is, the area of the working electrode to which the reagent is fixed (reaction part area of the working electrode) and the test strips. On the premise that

If the test strip is manufactured to have a difference in the area of the working electrode part (reaction part) in the reagent fixing part in the manufacturing process, the area of the reaction part between the sample and the reagent in the working electrode is different. Inevitably, a difference occurs in the current signal.

Therefore, in order to reduce the measurement error and increase the reliability, it is natural that the reaction area of the working electrode, that is, the area where the reagent is fixed on the working electrode must be the same area in all test strips without a slight error.

However, in the actual manufacturing process, it is difficult to produce the same area of the reaction part (fixed part) of the working electrode for each test strip without a minute error, thus reducing the process error as much as possible, thereby reducing the area of the reaction part in the working electrode. The focus is on reducing measurement errors as much as possible.

To this end, the area of the reagent fixing part exposed during screening or film-gluing for each test strip during the manufacturing process, especially the area where the reagent is fixed on the working electrode, is reduced to the same as possible by reducing the process error. However, there is still insufficient research or efforts to improve.

The present invention synthesizes zinc oxide quantum dots that can be easily combined with biomaterials at one time by using mercapto undecanoic acid (MUA) as a surfactant, and immobilizes glucose oxidase on its surface without going through several steps. By doing so, it is an object of the present invention to provide a zinc oxide quantum dot manufacturing method and a blood glucose sensor formed using the same to provide a blood glucose sensor more efficient than the conventional method for detecting the concentration of glucose by electrochemistry.

The zinc oxide quantum dot manufacturing method according to the present invention comprises the steps of forming a first solution by adding a zinc precursor and mercapto undecanoic acid (MUA) to the first ethanol, and a second solution in which sodium hydroxide is dissolved in the second ethanol Adding to the first solution and stirring to form a third solution in which zinc oxide quantum dots are dissolved in ethanol, washing the zinc oxide quantum dots of the third solution, dispersing them in a buffer solution and the zinc oxide quantum dots N-hydroxysulfo-succinimide (Sulfo-NHS), 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and glucose oxidase were added to the buffer solution containing the mixture, followed by stirring. Forming a quantum dot.

Here, the addition ratio of the first ethanol and the mercapto undecanoic acid (MUA) is 100: 20 ~ 100: 25, the first ethanol is characterized in that 15 to 25ml is added, the third solution In the forming step, the stirring process is performed at a temperature of 40 to 60 ° C. for 11 to 13 hours, and the washing of the third solution is performed 4 to 6 times, and the washing of the third solution is performed. The step of adding a deionized water (Deionized Water) to the third solution, characterized in that performed using a centrifuge, the ratio of ethanol to deionized water (Deionized Water) contained in the third solution Silver 100: 80 to 100: 100, characterized in that for washing the third solution is performed using chloroform and methanol, the buffer solution using ethanol or chloroform Thing The gong, and characterized by further performing the steps of dispersing the wash solution and the buffer after the formation of the glucose oxidase is immobilized with zinc oxide quantum dots.

In addition, the blood glucose sensor according to the present invention is manufactured by the above-described method, characterized in that formed using zinc oxide quantum dots immobilized glucose oxidase.

The present invention provides a method for preparing zinc oxide synthesis, which is surface-treated with mercapto undecanoic acid (MUA) and can be combined with a biological material without being replaced with a bio-friendly surfactant, and immobilizes glucose oxidase on the zinc oxide. By providing a measurement of the sugar value according to the photoluminescence intensity after the step, it solves a number of problems in the existing electrochemical method to provide the effect of the detection of other bio-materials other than sugar.

Zinc oxide quantum dots have a wide band spacing of 3.26 eV and a large exciton energy of 60 mV. Due to this property of zinc oxide, it is applicable to the field of laser diode (LD), light emitting diode (LED) and the like. In addition, it is a material that is well received in the biological field because it reacts well electrically and optically from the electron, and is biochemically stable, and unlike other compounds, it is not toxic. Combining a biomaterial called glucose oxidase with zinc oxide can produce a biosensor that can measure glucose by varying the amount of electrons generated depending on the amount of glucose.

Specific details of the embodiments are included in the detailed description and the accompanying drawings.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, only the present embodiments to make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to fully inform the person having the scope of the invention, which is defined only by the scope of the claims. Like reference numerals refer to like elements throughout.

1A to 1C are schematic diagrams illustrating a method for preparing zinc oxide quantum dots according to the present invention, illustrating a process of immobilizing glucose oxidase on zinc oxide quantum dots surface-treated with mercapto undecanoic acid (MUA). .

Referring to FIG. 1A, a first solution 120 is placed in a flask 100, and a zinc precursor (Zn precursor) and a mercapto undecanoic acid (MUA) are added to the first solution. In this case, the first solution 120 uses 15 to 25 ml of an ethanol solution, and the molar ratio of zinc precursor to mercapto undecanoic acid (MUA) is preferably 100: 20 to 100: 25. .

Next, in a vial, 1M sodium hydroxide and 20 ml of ethanol were added to prepare a second solution in which sodium hydroxide was completely desorbed.

Then, the second solution is put in the first solution 120 and stirred for 11 to 13 hours at a temperature of 40 ~ 60 ℃ zinc oxide quantum dots in the first solution 120 is prepared.

1B illustrates a shape of mercapto undecanoic acid (MUA) bonded to the zinc oxide quantum dot 150.

The reason why mercapto undecanoic acid (MUA) is bound to the surface of zinc oxide is explained by two mechanisms.

One is a dehydration reaction between the hydroxyl group (-OH) on the zinc oxide surface and the carboxyl group (-COOH) in the mercapto undecanoic acid (MUA) bond, and the other on the zinc oxide surface. It is a bond by substitution reaction between oxygen atom and zinc sulfide in thiloate (SH) group in mercapto undecanoic acid (MUA).

For these two reasons, the zinc oxide quantum dot 150 is formed to wrap around the surface of the mercapto-undecanoic acid (MUA) zinc oxide.

1C shows that zinc oxide quantum dots 150 are dispersed in ethanol, and quantum dots 150 dispersed in ethanol are washed again using a buffer solution and ethanol, and then dispersed in a buffer solution.

Here, the buffer solution uses phosphate saline (PHOSPHATE BUFFER SOLUTION) and serves to adjust the pH of the solution to neutral (pH7.4). In addition, the same washing and dispersing buffer solutions are used.

Next, in a solution containing zinc oxide quantum dots 150 substituted with mercapto undecanoic acid (MUA), mercapto undecanoic acid (MUA) and n -hydroxysulfo-succinimide (Sulfo-NHS), 1-Ethyl 3 (3-dimethylaminopropyl) carbodiimide (EDC) and glucose oxidase are mixed to activate the carboxyl group on the surface of zinc oxide quantum dots 150, and the coupling reaction with the amine group on the surface of glucose oxidase By inducing glucose oxidase immobilized on the surface of the zinc oxide quantum dot (150). At this time, n-hydroxysulfo-succinimide (Sulfo-NHS) 40 ~ 60mM, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 10 ~ 20mM and glucose oxidase 0.6mg / ㎖ ~ 1mg / ml is added, It is desirable to stir the solution slowly at 150 to 250 rpm.

Finally, the cleaning process is performed. Here, in the washing step, the zinc oxide quantum dot powder is obtained by turning the buffer solution into a centrifuge, and washing the obtained zinc oxide quantum dot powder again and dispersing it in the buffer solution is preferably performed 4 to 6 times. At this time, the washed zinc oxide quantum dot 150 is preferably dispersed in 4 ~ 5mL buffer solution.

As described above, when the sugar is added to the solution in a buffer solution containing zinc oxide quantum dots to which glucose oxidase is immobilized, the sugar concentration is increased by a certain concentration, and the optical change occurs in the zinc oxide quantum dots according to the sugar concentration. This optical change is called photoluminescence, and by measuring the change in the photoluminescence, the sugar level can be accurately measured.

Here, photoluminescence is also called photoluminescence, and a luminescence material absorbs energy such as light, electricity, and radiation into an excited state, and when it returns to the ground state, it absorbs energy. It refers to the phenomenon of emitting.

Photoluminescence is a luminescence generated by excitation by light, and generally emits light having a wavelength equal to or longer than that of irradiated light (irradiation light). In some cases, the light emitting center absorbs light and is excited and emits light. In some cases, a carrier generated by the absorption of light is captured by the light emitting center and emits light. Therefore, blood glucose can be measured using the photoluminescence spectrum which measures such photoluminescence.

Hereinafter, specific examples of a method for preparing zinc oxide quantum dots and a method for fixing glucose oxidase according to embodiments of the present invention will be described. However, the content not described herein is omitted because it can be inferred technically sufficient by those skilled in the art.

EXAMPLE

First, the zinc precursor and acetate and mercapto undecanoic acid (MUA) are dissolved in ethanol well and then stirred to form a first solution.

Next, a second solution in which sodium hydroxide is dissolved in ethanol is added to the first solution, followed by stirring while heating at a temperature of 50 ° C. for about 12 hours to form a third solution in which zinc oxide quantum dots are dissolved in ethanol.

Next, deionized water was mixed in a third solution at a ratio of 100: 80 to 100: 100, put into a centrifuge and rotated to wash the zinc oxide quantum dots to obtain a powder, and the zinc oxide quantum dot powder was returned to ethanol. Disperse Such a washing process is preferably carried out 4 to 6 times.

Afterwards, the zinc oxide quantum dots are washed again with a buffer solution and adjusted to pH 7.

Next, add n-hydroxysulfo-succinimide (Sulfo-NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and glucose oxidase in a buffer solution containing zinc oxide quantum dots. The reaction allows the glucose oxidase to be immobilized on the zinc oxide quantum dots.

Finally, the washing is performed once more, and the sugar is added to the zinc oxide quantum dot solution in which glucose oxidase is immobilized to check the increase in photoluminescence.

[Comparative Example]

In the above embodiment, zinc nitrate (Zn (NO ₃ )) is synthesized without adding zinc acetate in the preparation of the first solution, and then zinc oxide quantum dots are prepared in the same manner as in the above example.

[Characteristic Analysis]

The zinc oxide quantum dots immobilized with glucose oxidase prepared as described above are subjected to UV-visible, XRD, PL or FT-IR analysis.

[Feature Analysis Results]

FIG. 2 is a graph of XRD analysis results of zinc oxide quantum dots prepared using Examples and Comparative Examples according to the present invention.

Referring to FIG. 2, it can be seen that the size of the prepared zinc oxide quantum dot has a hexagonal wurtzite phase having a high crystallinity of about 3 nm.

Figure 3 is a graph of the results of UV-Visable spectrum analysis of zinc oxide quantum dots prepared using the Examples and Comparative Examples according to the present invention.

Referring to FIG. 3, the produced zinc oxide quantum dots exhibit intrinsic optical properties at the zinc oxide at 374 and a wide pick at about 550 degrees due to trap sites on the zinc oxide surface. Another reason is the recombination of holes and holes ionized into one in zinc oxide. This green emission increases the intensity of the pick as the powder size decreases.

4 is a graph of a PL analysis result of zinc oxide quantum dots prepared using the Examples and Comparative Examples according to the present invention.

Referring to FIG. 4, due to stokes shift, it comes out of a smaller wavelength band than the pick shown in PL. The blue shift occurred in both the UV-Visable spectrum and PL results. This is due to the quantum confinement effect.

The pick in FIG. 3 appears on the left side (347 nm) rather than the position (383 nm) of the pick in FIG. 4 that appears when in bulk. It is an expression that the material synthesized in the present invention is showing its characteristics as a nanomaterial.

5 is a surface analysis result of a Fourier change infrared ray detector (FTIR spectrum) of the zinc oxide quantum dots prepared by the embodiment according to the present invention.

Referring to Figure 5, surface analysis of zinc oxide quantum dots surrounded by mercapto undecanoic acid (MUA) 2928 and 2842 cm ^-1 is- CH ₂ is shown and the picks shown at 3418 represent the -OH group. The peaks of 1378 and 1565 represent C-OH and -CH bonds and 468 represent zinc and sulfur bonds. These results show that mercapto undecanoic acid (MUA) surrounds the surface of zinc oxide quantum dots well.

When the blood glucose sensor is manufactured using the zinc oxide quantum dots formed as described above, it is as follows.

First, in order to measure photoluminescence, a quartz cell (1 × 1 cm) made of quartz is prepared on a slope, and 1.5-2.5 ml of the solution prepared according to the embodiment is added to the cell.

Next, the intensity of the pick is measured while adding 0.01 to 0.02 ml of a small amount of glucose to the solution.

6 is a graph showing photoluminescence according to glucose concentration change.

Referring to FIG. 6, it can be seen that as the amount of blood sugar increases, the intensity of the pick in the ultraviolet region increases. Above 11.1mM the pick strength no longer increases.

7 is a graph showing the change in photoluminescence intensity according to the glucose concentration change.

Referring to FIG. 7, the PL pick increases linearly from 11.1 mM. This shows that glucose can be measured using zinc oxide fixed with glucose oxidase.

Here, the reason why the strength of PL increases with the amount of glucose is as follows.

When glucose (D-glucose) enters a solvent containing a zinc oxide quantum dot to which glucose oxidase is immobilized, two protons and electrons are generated and transferred to riboflavin adenine dinucleotide (FAD), a coenzyme of glucose oxidant. React as follows.

The two electrons are transferred to the zinc oxide quantum dots via riboflavin adenine dinucleotide (FAD), which further increases the photoluminescence as the electrons move from the conduction band to the valence band.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above embodiments but may be manufactured in various forms, and having ordinary skill in the art to which the present invention pertains. It will be understood by those skilled in the art that the present invention may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting.

1A to 1C are schematic views showing a method for manufacturing zinc oxide quantum dots according to the present invention.

Figure 2 is a graph of the results of XRD analysis of zinc oxide quantum dots prepared using the Examples and Comparative Examples according to the present invention.

Figure 3 is a graph of the results of UV-Visable spectrum analysis of zinc oxide quantum dots prepared using the Examples and Comparative Examples according to the present invention.

Figure 4 is a graph of the results of PL analysis of zinc oxide quantum dots prepared using the Examples and Comparative Examples according to the present invention.

5 is a surface analysis result of the Fourier change infrared detector (FTIR spectrum) of the zinc oxide quantum dots produced by the embodiment according to the present invention.

Figure 6 is a graph showing the photoluminescence according to the glucose concentration change.

7 is a graph showing the change in photoluminescence intensity according to the glucose concentration change.

Claims (10)

Adding a zinc precursor and mercapto undecanoic acid (MUA) to the first ethanol to form a first solution; Adding a second solution of sodium hydroxide dissolved in a second ethanol into the first solution and stirring to form a third solution having zinc oxide quantum dots dissolved in ethanol; Washing the zinc oxide quantum dots of the third solution and dispersing them in a buffer solution; And To the buffer solution containing the zinc oxide quantum dots, n-hydroxysulfo-succinimide (Sulfo-NHS), 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and glucose oxidase were added to the solution, followed by stirring. A method of manufacturing a zinc oxide quantum dot comprising the step of forming an immobilized zinc oxide quantum dot. The method of claim 1, The addition ratio of the first ethanol and the mercapto undecanoic acid (MUA) is 100: 20 ~ 100: 25, the first ethanol is a zinc oxide quantum dot manufacturing method characterized in that the addition of 15 to 25ml. The method of claim 1, In the third solution forming step, the stirring process is performed for 11 to 13 hours at a temperature of 40 to 60 ℃ zinc oxide quantum dot manufacturing method. The method of claim 1, Washing the third solution is a zinc oxide quantum dot production method characterized in that it is performed 4 to 6 times. The method of claim 1, The washing of the third solution is performed by using a centrifuge after adding deionized water (Deionized Water) to the third solution. The method of claim 5, wherein Zinc oxide quantum dot manufacturing method characterized in that the ratio of ethanol to deionized water (Deionized Water) contained in the third solution is 100: 80 ~ 100: 100. The method of claim 1, Washing the zinc oxide quantum dots in the third solution is a method of producing zinc oxide quantum dots, characterized in that performed using chloroform and methanol. The method of claim 1, The buffer solution is zinc oxide quantum dot production method characterized in that using ethanol or chloroform. The method of claim 1, The method for producing zinc oxide quantum dots characterized in that the step of forming the glucose oxidase immobilized zinc oxide quantum dots and then further dispersed in a buffer solution. The glucose sensor according to claim 1, wherein the glucose oxidase is formed using an immobilized zinc oxide quantum dot.

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