KR102105914B1 - Fluorescent Hydrogel Glucose Droplet Type Biosensors and its Preparing Method - Google Patents

Abstract

The present invention relates to a hydrogel glucose droplet biosensor and a method for manufacturing the same with fluorescence quenching and size reduction, and specifically, carbon dot (CO), glucose oxidase (GOx), and mustard peroxidase (horseradish peroxidase, HRP) immobilized non-enzyme-based fluorescent hydrogel glucose droplet biosensor and a method for manufacturing the same.
Fluorescent hydrogel glucose droplet biosensor has high selectivity for glucose, which is the main component of human serum, because fluorescence quenching and size reduction occur simultaneously in response to glucose, and it can be stored for a long time in a dried state for stability. It has a high, pH-reactive property that swells at a certain pH or higher to have high porosity, so it can be easily applied as a biosensor.

Description

Fluorescent Hydrogel Glucose Droplet Type Biosensors and its Preparing Method

The present invention relates to a hydrogel glucose droplet biosensor and a manufacturing method thereof in which fluorescence quenching and size reduction work simultaneously, specifically carbon dot (CD), glucose oxidase (GOx), and mustard The present invention relates to a non-enzyme-based fluorescent hydrogel glucose droplet biosensor immobilized with horseradish peroxidase (HRP) and a method for manufacturing the same.

Diabetes mellitus is a serious disease associated with an increase in blood sugar due to abnormalities in the production or use of insulin, and various acute and chronic complications. Deficiency or hyperglycemia of insulin causes diabetes, which increases the risk of cardiovascular, nerve, kidney, ocular and peripheral vascular disease. As such, diabetes, which has become a global problem, is expected to increase in invention rate due to an increase in the elderly population and living environment factors, and thus social and economic problems are seriously emerging.

Due to the increase in the number of patients, the demand for an auto-glycemic meter mainly used for the follow-up of blood glucose levels is increasing, and the frequency of use is gradually increasing. Therefore, there is a need to develop a blood glucose sensor that is easy to use with high sensitivity. Proper monitoring of blood sugar levels is important to maintaining a healthy body. Glucose biosensors account for about 85% of the total biosensor market, and research activities on the development of new glucose biosensors that are highly sensitive and easy to use are very active.

Biosensors for glucose detection can be broadly classified into enzyme-based and non-enzyme-based biosensors. Most common biosensors for glucose detection are enzyme-based biosensors that use glucose oxidase (GOx). These enzyme-based biosensors have high sensitivity and can detect sophisticated glucose, but have disadvantages such as inhibiting enzyme activity due to pH, temperature, humidity and the presence of other chemicals, and poor stability and reproducibility. Have

In order to overcome the limitations of the enzyme-based glucose biosensor, studies on the non-enzyme-based glucose biosensor have been actively conducted. Republic of Korea Patent Publication '10 -2011-0057845 'provides a diamond electrode for glucose biosensors and a method of manufacturing the same, but most of the pure metals used therein have low efficiency that does not exhibit satisfactory sensitivity for detecting low concentrations of glucose. , Preparation of the electrode has a problem that the process is complicated, time consuming, sophisticated technology is required.

(Patent Document 1) KR 10-2011-0057845 (Patent Document 2) KR 10-2013-0087239

In order to solve the above problems, the present invention includes a hydrogel droplet, carbon dot (CD), glucose oxidase (GOx), and mustard-free peroxidase (HRP). Fluorescence quenching and size reduction occur simultaneously in response to glucose. It is an object to provide a fluorescent hydrogel glucose droplet type biosensor.

In addition, it is an object of the present invention to provide a method for producing a fluorescent hydrogel glucose droplet type biosensor having the above excellent effect.

In addition, an object of the present invention is to provide a method for producing a hydrogel droplet, which is a component of a fluorescent hydrogel glucose droplet type biosensor.

The present invention to solve the above object,

Hydrogel droplets; And

It provides a fluorescent hydrogel glucose droplet biosensor, characterized in that it comprises; carbon dot (CD), glucose oxidase (GOx), and mustard peroxidase (HRP) immobilized on the hydrogel droplets.

The present invention to solve the above other object,

Preparing a hydrogel droplet;

Mixing by adding a crosslinking agent to the hydrogel droplets; And

Characterized in that it comprises; a step of adding a carbon dot (CD), glucose oxidase (GOx) and mustard-free peroxidase (HRP) and stirring the mixture, providing a method for manufacturing a fluorescence hydrogel glucose droplet biosensor do.

The present invention to solve the above another object,

In the hydrogel droplet,

Leveling the glass capillaries;

Inserting a dispersant and an initiator into the glass capillary; And

It provides a method for producing a hydrogel droplet, characterized in that it comprises; administering an accelerator and a fluorine-based surfactant from the outside of the glass capillary.

Fluorescent hydrogel glucose droplet type biosensor provided by the present invention includes hydrogel droplets, carbon dot (CD), glucose oxidase (GOx) and mustard peroxidase (HRP) to reduce fluorescence quenching and size in response to glucose Because it occurs at the same time, there is a high selectivity effect on glucose, which is a main component of human serum.

In addition, the fluorescent hydrogel glucose droplet biosensor has a high stability because it can be stored for a long time in a dry state, and has high porosity because it has a pH reactivity and swells at a certain pH or higher, so it can be easily applied as a biosensor.

Figure 1 shows the manufacturing process of a fluorescent hydrogel glucose droplet biosensor according to an embodiment of the present invention.
Figure 2a shows the manufacturing process of a hydrogel droplet using a capillary device according to an embodiment of the present invention.
Figure 2b is a photograph taken with a super-high speed camera manufacturing process of a hydrogel droplet using a capillary device according to an embodiment of the present invention.
Figure 3a is a graph showing the diameter ratio of the fluorescent hydrogel glucose droplet biosensor according to an embodiment of the present invention according to the conditions of pH.
Figure 3b is a graph showing the diameter ratio of the hydrogel (PAAc) droplets according to one embodiment of the present invention according to the conditions of the repeated pH.
4 is a fluorescence photograph according to the presence or absence of glucose in the fluorescent hydrogel glucose droplet type biosensor and PAAc-CD droplets prepared according to an embodiment and a comparative example of the present invention.
5 is a graph showing a sample detection result of a fluorescent hydrogel glucose droplet biosensor according to an embodiment of the present invention.
6 is a fluorescence photograph according to the presence or absence of drying and the presence or absence of glucose of the fluorescent hydrogel glucose droplet biosensor prepared according to an embodiment of the present invention.

The present invention relates to a hydrogel glucose droplet biosensor and a method for manufacturing the same with fluorescence quenching and size reduction, and specifically, carbon dot (CO), glucose oxidase (GOx), and mustard peroxidase (horseradish peroxidase, HRP) immobilized non-enzyme-based fluorescent hydrogel glucose droplet biosensor and a method for manufacturing the same.

Hereinafter, the present invention will be described in detail.

According to one aspect of the invention, the hydrogel droplets; And

It provides a fluorescent hydrogel glucose droplet biosensor, characterized in that it comprises; carbon dot (CD), glucose oxidase (GOx), and mustard peroxidase (HRP) immobilized on the hydrogel droplets.

Hydrogels have high hydrophilicity and, when absorbing large amounts of water, they can react to external stimuli through volumetric changes. Due to these properties, the crosslinked polymer hydrogel has high utilization.

The hydrogel in the present invention can be used as long as it has hydrophilic properties, but polyacrylic acid (poly (acrylic acid) (PAAc)), polyacrylamide (poly- (acrylamide) (PAAm)) and derivatives thereof It is preferred to include.

In addition, hydrogel droplets can be rapidly generated in microfluidic channels with monomer or polymer solutions in the form of water-in-oil (W / O) droplets. The water-in-oil type means that water is the inner phase (dispersion phase) and oil (oil) is the outer phase (dispersion medium). Evaporation of water is small because the trauma is oil.

Hydrogels are biologically similar to soft bio-tissues, and can be widely used in 3D cell culture, tissue engineering, and regenerative medicine. Synthetic hydrogels can be used as biosensors because of the ease of network formation and functionalization during synthesis compared to natural hydrogels of proteins and polysaccharides.

The stimulation-reactive hydrogel can be used as a valve to regulate the flow of flow in the microfluidic channel without external control, and the hydrogel microdroplets of a porous structure immobilized with receptor molecules can be combined with a molecule to be specifically and efficiently analyzed or It can be used to capture cell secretions.

Carbon dot (CD) is a carbon nanoparticle that has excellent photophysical properties of quantum dots, but is excellent in biocompatibility, non-toxicity, manufacturability and stability. It has the characteristics of excellent solubility in water, strong luminescence, high compatibility with a living body, easy functionalization of a surface, preventing long-term light discoloration, and high economic efficiency.

In general, it receives ultraviolet rays and emits blue to green light, and in this luminescence phenomenon, various chemical functional groups present on the orbital and surface due to internal carbon double bonds play an important role.

Since the maximum quenching effect of carbon dot (CD) occurs in a water-soluble state, a hydrogel in which water is absorbed can be suitably used as a solid matrix of a biosensor.

In addition to its high fluorescence properties, carbon dot (CD) can bind therapeutic molecules in the carbon skeleton due to its unique chemical structure, and molecules such as biocompatible ligands can be additionally bound to its surface. Due to these characteristics, carbon dot (CD) is a nanoparticle capable of realizing personalized medicine as an ideal material in the field of diagnostics where diagnosis and treatment are possible simultaneously.

Glucose oxidase (GOx) is an enzyme that oxidizes glucose to oxygen to produce gluconic acid (D-gluconic acid), which is not particularly limited as long as it can be usually sold or synthesized, but is preferably phenicylium May include those found in mold and honey, such as Penitacillium notatum.

When glucose is oxidized by an enzymatic reaction of glucose and glucose oxidase (GOx), gluconic acid (D-gluconic acid) and H ₂ O ₂ may be produced.

Horseradish peroxidase (HRP) is a type of peroxidase found in horseradish. It has a molecular weight of about 44,000 and contains one functional group protohemin per molecule. The absorption spectrum of this enzyme has an absorption maximum at 640, 500, and 402 nm, but H ₂ O ₂ or CH ₃ OOH is added because hydrogen peroxide is combined with Fe ³⁺ of the enzyme protohematine to form an enzyme-substrate bond. If you do, the absorption spectrum may change.

H ₂ O ₂ produced by the enzymatic reaction of glucose and glucose oxidase (GOx) reacts with HRP It is decomposed into -OH radical, and fluorescence quenching of carbon dot (CD) may be caused by the decomposed -OH radical.

In addition, gluconic acid produced by the enzymatic reaction of glucose and glucose oxidase (GOx) reduces the pH, thereby reducing the size of the fluorescent hydrogel glucose droplet biosensor.

Therefore, the fluorescent hydrogel glucose droplet type biosensor can simultaneously exhibit the degree of fluorescence quenching and the reduction in the size of the droplets depending on the amount of glucose.

In the present invention, the fluorescent hydrogel glucose droplet biosensor has a diameter of 360 µm to 570 µm. Small sized FHGB droplets can be useful as small samples and can be used to detect glucose in narrow, confined areas, providing local site information.

In addition, the fluorescent hydrogel glucose droplet biosensor can be stored for a long period of time in a dry solid state and can be used with water whenever necessary.

According to another aspect of the invention, the step of preparing a hydrogel droplets; Mixing by adding a crosslinking agent to the hydrogel droplets; And adding and agitating the mixture by adding carbon dot (CD), glucose oxidase (GOx) and mustard-free peroxidase (HRP); and providing a method for producing a fluorescent hydrogel glucose droplet biosensor. do.

The hydrogel in the present invention can be used as long as it has hydrophilic properties, but polyacrylic acid (poly (acrylic acid) (PAAc)), polyacrylamide (poly- (acrylamide) (PAAm)) and derivatives thereof It is preferred to include.

N- (3- (dimethylamino) propyl-N-ethylcarbodiimide hydrochloride (EDCHCl), N-hydroxysuccinimide (N-hydroxysuccinimide, NHS), and, N, N-methylenebis (acrylamide) (N, N-methylenebis (acrylamide), MBAm).

Carbon dot (CD), glucose oxidase (GOx), and horseradish peroxidase (HRP) included in the fluorescent hydrogel glucose droplet biosensor are immobilized on the hydrogel droplet.

According to another aspect of the invention, the leveling of the glass capillaries; Inserting a dispersant and an initiator into the glass capillary; And administering an accelerator and a fluorine-based surfactant from the outside of the glass capillary to the inside.

Dispersant may be at least one selected from acrylamide (AAm), and N, N-methylenebis (acrylamide), MBAm, the initiator is ammonium persulfate, APS), and tetramethylethylenediamine (tetramethylethylenediamine, TEMED).

In addition, hydrogel droplets can be rapidly generated in microfluidic channels with monomer or polymer solutions in the form of water-in-oil (W / O) droplets. The water-in-oil type means that water is the inner phase (dispersion phase) and oil (oil) is the outer phase (dispersion medium). Evaporation of water is small because the trauma is oil. Hydrogel droplets can be applied to encapsulation of single molecules or cells, as they allow precise control of some environments and high permeation analysis.

As an embodiment of the present invention, a hydrogel droplet converts PAAm into an anionic polymer electrolyte, poly (acrylic acid) (PAAc), and then a microfluidics method having a glass capillary having a geometric structure flowing in a horizontal direction. ).

Since PAAc has excellent pH reactivity, it swells under high pH conditions by deprotonation. These PAAc droplets also have a high porosity characteristic because they can release internal substances when the pH is changed in a contracted state (acidic condition).

 PAAc has a carboxyl group that can crosslink with amine-containing substances, and N- (3- (dimethylamino) propyl-N-ethylcarbodiimide hydrochloride (N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride , EDC · HCl) can crosslink with amine-containing substances through crosslinking agents, which causes carbon dots (CD) to be immobilized on PAAc droplets to contain a number of functional groups including amine groups, including amine-containing substances such as ethylene diamine It can be made of carbon dot (CD) having a group and immobilized on PAAc.

Fluorescent hydrogel glucose droplet biosensors can be prepared by fixing glucose oxidase (GOx) and HRP in addition to carbon dot (CD) to PAAc droplets. These fluorescent hydrogel glucose droplet biosensors may show a new way to detect glucose in small samples.

In addition, due to its high biocompatibility, it can also be applied to an implantable continuous detection droplet type biosensor.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.

<Example>

Example 1. Preparation of carbon dot (CD)

Carbon dot (CD) was synthesized using a mini bench-top reactor (Parr series 4560, Parr Instrument Co., USA). 0.4 g citric acid and 270 μl of ethylenediamine were dissolved in 80 ml of distilled water. When the solution was uniformly mixed, 200 ml of the mixture was transferred to a reactor and sealed to heat at 200 ° C. for 4 hours. Thereafter, the reactor was synthesized using air. 0.4 g citric acid and 270 μl ethylenediamine were cooled to distilled room temperature (20 ° C.). Finally, a transparent and brownish pure carbon dot (CD) solution was obtained.

Example 2. Preparation of PAAm hydrogel droplets-Microfluidics method

Square glass capillaries (Vitrocom, USA, 0.9 mm × 0.9 mm) were used without any surface treatment, round glass capillaries (Vitrocom, USA, 0.7 mm inner diameter (ID) × 0.87 mm outer diameter (OD) × 100 mm length) ) Was tapered in half using a Micropipette Puller (P-97, Sutter Instrument, USA).

The round glass capillaries washed with ethanol and distilled water were dried at 85 ° C. for 10 minutes, and subjected to oxygen plasma treatment for 5 minutes using a plasma system (FC-10005, Femto Science, South Korea). The treated round glass capillary tube was immersed in a glass bottle containing 5 wt% tetraethyl orthosilicate (TEOS) in ethanol for 30 minutes, washed with ethanol and distilled water, and dried in a dry oven at 70 ° C for 15 minutes. Thereafter, the round glass capillaries were inserted into the square glass capillaries and leveled by tight coupling.

To prepare the hydrogel droplets, water-soluble acrylamide (AAm) monomer (5 M) and cross-linked N, N-methylenebis (acrylamide) (MBAm) (0.15 M) and ammonium persulfate (APS) (0.05 M) as dispersants It flowed through a centrally thin round capillary tube. In addition, at the same time, fluorocarbon oil (Novec 7500) containing tetramethylethy-lenediamine (TEMED, 0.5 wt%) and fluorine-based surfactant (Krytox 157FSH, 2 wt%) from the outside to the inside of the capillary tube is squared in the same direction as the solution flowing inside the capillary. Allowed to flow in the capillaries.

This is a continuous process for generating a single W / O droplet in a horizontally flowing geometric structure, in which fluorocarbon oil compresses the aqueous AAm monomer solution into simple dispersed droplets, and TEMED performs a continuous process. Through, it was used and dissolved as an accelerator for polymerization of AAm, and APS was used as an initiator for polymerization of AAm.

After the continuous process, poly (acrylamide) (PAAm) droplets were finally prepared by performing a post-heat treatment at 100 ° C. in a 50 ml glass bottle.

In this embodiment, the flow rate was controlled using a pneumatic microfluidic flow control system (OB1 pressure controller, Elveflow, France) that can send two fluids at two different speeds, the system using a shrinking connecting tube ( Tommyheco, Korea, 2.38 mm × 1.59 mm × 2.38 mm) and flexible plastic tube (Norton, 0.51 mm ID, 1.52 mm OD).

In addition, the speed of the air gas was finely adjusted and the tube was pressurized by applying it into the tube containing the liquid, thereby allowing the liquid to flow through the tube to the fine capillary device. The pressures of the internal and external fluids were 10 and 130 mbar, respectively.

Example 3.Fluorescent hydrogel glucose biosensor (FHGB) droplet preparation

In a 20 ml glass bottle, 1 g of the prepared PAAm hydrogel droplet was immersed in 13.5 mL of a NaOH aqueous solution (3 M) in the presence of TEMED (10 vol%) 1.5 m, stirred for 12 hours, and then washed with distilled water to remove poly ( acrylic acid) PAAc droplets were prepared.

Activated by adding 2 ml of EDC / NHS (0.2 M / 0.2 M) mixed with N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) to PAAc droplets . Thereafter, 5 ml of CD (0.58 mg / mL), GOx (45 μM) and HRP (15 μM) were stirred for 12 hours to prepare FHGB droplets, and the process is shown in FIG. 1.

Example 4. Measurement of the diameter of FHGB droplets according to pH

PAAc is typically a weak polymer electrolyte with pH reactivity, so the size of the FHGB droplets made with PAAc will have reactivity to pH.

The diameter of the FHGB droplets prepared according to the Examples was measured by inserting them into water having a different pH, and expressed as a ratio. In the ratio of the droplet diameter (D / D ₀ ), D is the diameter at a specific pH, and D ₀ is the maximum diameter of the droplet.

Example 5. Glucose detection test of FHGB droplets

The glucose detection ability of the FHGB droplets prepared according to the above example was tested. In each case, fluorescence pictures of FHGB droplets were taken using a fluorescence microscope (E600POL, Nikon eclipse, Japan).

Example 6. Glucose detection selectivity test of FHGB droplets

After adding 2 ml of 30 mM urea, ascorbic acid, dopamine, lactose, NaCl and glucose aqueous solution to the cells, the Q _f value was checked to confirm the detection degree of FHGB droplets (0.05 g) prepared according to the above example. Compared.

Q _f represents the degree of quenching and is (F-F ₀ ) / F ₀ . In the present invention, F ₀ is PL intensity measured at 450 nm when glucose is not excited when excited at 360 nm, and F means PL intensity measured at 450 nm when glucose is excited when excited at 360 nm.

Photoluminescence (PL) was measured using a UV-vis spectrophotometer (UV-2401 PC, Shimadzu, Japan) or spectrofluorophotometer (RF-5301PC, Shimadzu, Japan), and finally the Q _f value was determined according to the type of the substance to be detected. Graphed.

Example 7. Stability test of FHGB droplets

After the FHGB droplets prepared according to the above examples were dried in a dry oven and stored for a long time, a glucose detection test was conducted to confirm the stability of the FHGB droplets.

After 4 weeks of drying of the droplets, fluorescence photographs were taken in a dry state, a state added to an aqueous solution of 30 mM glucose and a state added to an aqueous solution without glucose.

Example 8. Glucose detection test of FHGB droplets in human serum

The ability to detect glucose in human serum was tested using FHGB droplets prepared according to the above example.

0.05 g of FHGB droplets were added to 2 ml of serum diluted 10-fold with distilled water, and the amount of glucose in the original serum and the amount of glucose to be added to the serum and FHGB droplets were used using high-performance liquid chromatography (HPLC). The detected amount of glucose in the serum was measured.

After measuring the glucose in the original serum, the experiment was performed by adding the measured amount of glucose to the centrifuged serum sample to remove the large molecule.

Comparative Example 1. PAAc-CD droplet preparation and glucose detection test

A PAAc-CD droplet immobilized only with CD without GOx and HRP was prepared according to the above example, and the FHGB droplet and glucose detection ability of the present invention were compared.

<Evaluation and Results>

Results 1. Preparation of PAAm hydrogel droplets-Microfluidics method

According to Example 2, PAAm hydrogel droplets were prepared, and the process and results are shown in FIGS. 2A and 2B.

Figure 2a shows a fine capillary device for producing PAAm hydrogel droplets. The round glass capillaries were inserted into the inside of the square glass capillaries, making them horizontal and tightly adhered, and it was confirmed that droplets were formed at the inlet tapered in half. The solution flowing inside (AAm + MBAm + APS + distilled water) and the solution flowing from the outside (fluorocarbon oil, fluorine-based surfactant and TEMED) flowed horizontally and formed droplets.

2B is a photograph taken in the process of Example 2 with an ultra-high speed camera. In the bio-fluidic device for producing PAAm hydrogel droplets, the actual droplets are formed and the reference size is 200 µm. It was confirmed that droplets were formed at the inlet of the thin round glass capillary tube as shown in FIG. 2A.

Result 2. Measurement of the diameter of the FHGB droplet according to the pH

The diameter of the FHGB droplet according to pH was measured, and the ratio and pH are shown in FIG. 3A. It was found that the ratio of the diameter of the carboxyl group (pH 2-4), which was less than 0.6 at pK _a , was gradually increased from pH 5 to about 0.9.

However, the diameter ratio was repeatedly raised and lowered at pH 6-12, and the diameter ratio reached 1 at pH 12. Accordingly, after pK _a , the carboxyl group of PAAc is deprotonated and ionized to exhibit repulsive force and expand, and the volume of the droplet increases by 5 times or more.

pK _a is It is about the intensity of the acid defined by -log [K _a] . The stronger the acid, the smaller the value, and easily loses H ^+, thereby increasing the tendency to deprotonation and ionization.

Figure 3b shows the change in the ratio of the diameter of the PAAc droplets appearing as the pH value is repeated from 2 to 12. As for the diameter ratio, when the pH value was changed from 2 to 12 and 10 times, it was repeated 0.55 to 1, and it was confirmed that the value changed.

Accordingly, it was confirmed that the diameter ratio was almost constant for each changed ash, and that the expansion and contraction of the FHGB droplets were stable.

Results 3. Comparison of glucose detection of FHGB droplets and PAAc-CD droplets

The results of comparing the glucose detection ability of the FHGB droplets and PAAc-CD droplets prepared according to the examples and comparisons are shown in FIG. 4.

Figure 4 (i) is a PAAc-CD droplet (PAAc- 0.05 g, CD (0.58 mg / ml)-5 ml) before adding an aqueous glucose solution to the cell, (ii) after adding the aqueous glucose solution to the cell PAAc-CD droplets (PAAc- 0.05 g, CD (0.58 mg / ml)-5 ml), (i) FHGB droplets before adding an aqueous glucose solution to the cells, and (ⅳ) adding an aqueous glucose solution to the cells. The FHGB droplets are then shown.

As a result, it was confirmed that the PAAc-CD droplets showed a similar fluorescence degree with or without the addition of an aqueous glucose solution, but the FHGB droplets exhibited a significant fluorescence quenching as well as a reduction in size after the glucose aqueous solution was added.

When glucose is oxidized by an enzymatic reaction of glucose and glucose oxidase (GOx), gluconic acid (D-gluconic acid) and H ₂ O ₂ may be produced. At this time, the generated H ₂ O ₂ can be decomposed into -OH radicals by HRP, and this -OH radicals can cause fluorescence quenching of CD.

Therefore, it was confirmed that the reduction in the size of the droplets is caused by the production of gluconic acid by the reaction of GOx and glucose, and the bioenzyme reaction with glucose changes not only the size of the droplets but also the fluorescence of the CD.

Result 4. FHGB droplet glucose detection selectivity test

The Q _f values of the FHGB droplets according to 30 mM urea, ascorbic acid, dopamine, lactose, NaCl and glucose aqueous solutions are compared and shown in FIG. 5.

As a result, the Q _f value of all analytes except glucose showed a low value of less than 0.03, so that fluorescence quenching did not occur except when exposed to glucose, and the dopamine and glucose mixture had a Q _f value similar to glucose. Shown.

Accordingly, it was found that other substances did not interfere with glucose detection due to the strong selectivity of the GOx enzyme, and it was confirmed that the FHGB droplet was very selective for glucose.

Results 5. Stability test of FHGB droplets

After 4 weeks of drying of the FHGB droplets, a fluorescence photograph was taken in a dry state, a state added to an aqueous solution of 30 mM glucose and a state added to an aqueous solution without glucose, and shown in FIG. 6.

As a result, the dried FHGB water droplets (i) showed high fluorescence intensity, and after 4 weeks after drying, droplets in the expanded state in water without glucose (ii) also showed weaker but relatively strong fluorescence than (i). Floated.

However, it was confirmed that the FHGB droplets in the expanded state in the water containing glucose extinguish at almost the same level as the fluorescence quenching of the FHGB immediately after drying.

Therefore, it was confirmed that the FHGB droplets can be dried and stored in a solidified state if necessary, and the stability is also high.

Results 6. Glucose detection test of FHGB droplets in human serum

The amount of glucose in the serum detected using the original amount of glucose in the serum, the amount of glucose to be added to the serum, and FHGB droplets is shown in Table 1 below.

glucose (mM) sample original added measured recovery (%) RSD (n = 5,%) One 0.53 0 0.49 93.6 3.91 2 0.53 0.47 1.10 110.4 3.76 3 0.53 1.47 1.92 96.6 6.07 4 0.53 9.47 10.3 100.3 4.61

In Table 1, original represents the original glucose amount of human serum, added represents the amount of added glucose, and measured represents the amount of glucose in serum measured using FHGB droplets.

In addition, recovery means the recovery rate of glucose, and was calculated using the amount of glucose in Table 1. [recovery = (measured) / (original + added) × 100%]

As a result, depending on the amount of glucose added, a high glucose recovery rate of 95.6 to 110.4% was confirmed, and thus it was confirmed that FHGB droplets can be applied to and used in actual human serum.

However, in Table 1, when the recovery rate is more than 100%, interference by other biological substances in human serum is reflected, but the deviation is insignificant.

Claims (12)

Neutralized porous hydrogel droplets comprising a carboxyl group;
A carbon dot (CD) immobilized by covalently bonding to the outer surface of the hydrogel droplet; And
Includes; glucose oxidase (GOx) and mustard-free peroxidase (HRP) immobilized on at least one selected from the outer surface of the hydrogel droplet and the carbon dot; and
The hydrogel droplet and the carbon dot form an amide bond,
Fluorescent hydrogel droplet type glucose biosensor, characterized in that the ratio of the diameter at pH 2 to pH 12 is 0.55 to 1.00 based on the maximum diameter of the droplet.
According to claim 1,
The hydrogel droplet is characterized in that it contains any one or more selected from the group consisting of polyacrylic acid (poly (acrylic acid) (PAAc)), polyacrylamide (poly- (acrylamide) (PAAm)), and derivatives thereof Fluorescent hydrogel droplet type glucose biosensor.
According to claim 1,
The hydrogel droplet is characterized in that the water-in-oil (W / O) droplet form, a fluorescent hydrogel droplet type glucose biosensor.
The method according to any one of claims 1 to 3,
Fluorescent hydrogel droplet type glucose biosensor, characterized in that the diameter is 360 μm to 570 μm.
Preparing a neutralized porous hydrogel droplet containing a carboxyl group;
Adding a crosslinking agent to the hydrogel droplets and mixing to activate the crosslinking agent; And
Including the reaction by adding carbon dot (CD), glucose oxidase (GOx), and mustard-free peroxidase (HRP) to the mixture;
The carbon dot is immobilized on the outer surface of the hydrogel droplet by forming an amide bond with the hydrogel droplet through a crosslinking agent,
The glucose oxidase and mustard-free peroxidase are characterized in that they are immobilized on at least one selected from the outer surface of the hydrogel droplets and carbon dots, a method for manufacturing a fluorescent hydrogel droplet-type glucose biosensor.
The method of claim 5,
The hydrogel droplet is characterized in that it contains any one or more selected from the group consisting of polyacrylic acid (poly (acrylic acid) (PAAc)), polyacrylamide (poly- (acrylamide) (PAAm)), and derivatives thereof A method of manufacturing a fluorescent hydrogel droplet type glucose biosensor.
The method of claim 5,
N- (3- (dimethylamino) propyl-N-ethylcarbodiimide hydrochloride (EDCHCl), N-hydroxysuccinimide Fluorescent hydro, characterized in that it contains at least one selected from the group consisting of (N-hydroxysuccinimide, NHS), and, N, N-methylenebis (acrylamide) (N, N-methylenebis (acrylamide), MBAm) Method for manufacturing a gel droplet type biosensor.
The method of claim 5,
The carbon dot (CD), glucose oxidase (GOx), and mustard-free peroxidase (HRP), characterized in that immobilized on the hydrogel droplets, fluorescent hydrogel droplet type glucose biosensor manufacturing method.
A first step of fitting a round glass capillary inside a square glass capillary and leveling to form a fine capillary device;
A second step of administering a dispersant, an initiator, an accelerator, and a fluorine-based surfactant from outside the inside of the microcapillary device; And
And a third step of heat-treating the fine capillary device of the second step,
The method for producing a hydrogel droplet according to claim 1, wherein the accelerator and the fluorine-based surfactant form droplets while flowing outside the round glass capillary and inside the square glass capillary.
The method of claim 9,
The dispersant is at least one selected from acrylamide (acrylamide, AAm), and N, N-methylenebis (acrylamide), MBAm, hydro according to claim 1 Method of manufacturing a gel droplet.
The method of claim 9,
The initiator is characterized in that at least one selected from ammonium persulfate (ammonium persulfate, APS), and tetramethylethylenediamine (TEMED), hydrogel droplet production method according to claim 1.
The method of claim 9,
The method of claim 1, wherein the hydrogel droplet is in the form of a water-in-oil (W / O) droplet.

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