KR20080104495A - Kits and methods for biological detection using quantum dots - Google Patents

Abstract

A bioconjugate, a sensing membrane where the bioconjugate is fixed on the surface, a biological detection kit containing the sensing membrane, and a biological detection method using the kit are provided to have the energy transfer characteristics between neighbouring molecules by using quantum dot. A bioconjugate comprises a quantum dot which comprises a cadmium selenide (CdSe) core and a zinc sulfide (ZnS) shell coated on the core and is coated with a hydrophilic surfactant and where an oxidase is encapsulated. Preferably the oxidase is selected from the group consisting of glucose oxidase, lactate oxidase, tyramine oxidase, cholesterol oxidase, choline oxidase, alcohol oxidase, ascorbic acid oxidase and xanthine oxidase.

Description

Kits and methods for biological detection using quantum dots}

1 shows a reaction scheme of oxidation of glucose to gluconic acid using GOD / HRP entrapped in CdSe / ZnS QDs;

2A shows absorption spectra of CdSe (550 nm), CZ-QDs (580 nm) and MPA-QDs (580 nm), and (b) shows CdSe (560 nm) and CZ-QDs at an excitation wavelength of 480 nm. (590 nm) and emission spectra of MPA-QDs (590 nm), (c) the overlap of absorption and emission spectra of enzymes (GOD, HRP) and QDs, and (d) CdSe (left) and CdSe under UV light. Is a diagram showing an image of / ZnS (right);

FIG. 3 (a) is a photographic image of gel electrophoresis of Enz-QDs containing different amounts of GOD (GOD = 10, 50, 100, 150, 200, 250 and 300 U) and 0.5 g / l glucose solution. Emission spectra using fixed amounts of QDs (3.145 mg / ml) and HRP (10 U) at excitation wavelength 445 nm, and (b) showed different concentrations of QDs (QDs = 0.4309, 0.8622, 1.7254, 2.5872, 3.445 mg / Photograms of gel electrophoresis of Enz-QDs in ml) and emission spectra using fixed amounts of GOD (200 U) and HRP (10 U) at an excitation wavelength of 445 nm in a 0.5 g / L glucose solution;

4 shows the emission spectrum (445 nm (ex) / 525 nm (em)) of enzyme in the presence and absence of MPA-QDs;

5 shows changes in fluorescence intensity of QD-FRET-based probes at different glucose concentrations and at different volume ratios of QDs / GOD / HRP added to the glucose solution;

6 is a view showing the effect of pH and temperature of the reaction solution during glucose measurement;

7 shows the effect of Fe ³⁺ ions on the fluorescence emission of MPA-QDs during the measurement of 1 g / L glucose solution.

The present invention relates to biological detection using quantum dots (QDs), and more particularly, bioconjugates of QDs and various oxidases coated with a zinc sulfide (ZnS) shell on a cadmium selenide (CdSe) core. The present invention relates to bioconjugates, sensing membranes to which the bioconjugates are immobilized, biological detection kits including the sensing membranes, and biological detection methods using the detection kits.

The invention of semiconductor nanoparticle QDs with various applicability dates back to the 1970's, but only four to five years have been applied to the life sciences. QDs with a ZnS shell on a CdSe core are spherical materials ranging in diameter from several nanometers to several tens of nanometers, and emit fluorescence at different wavelengths depending on the size of the particles. It can also be used in a variety of applications in life sciences such as protein chips and biosensors. Quantum dots can be excited at any wavelength from UV to red and have an adjustable narrow emission spectrum. As it is an inorganic substance, it is stable to chemical reactions and is easily bonded to biological materials by surface treatment. In the experiments of Jaiswal et al. (Jaiswal et al., Nature Biotechnology 21, 47-51, 2003), biotinylated primary antibodies were attached to avidin-embedded quantum dots to observe images of living cells, and negatively charged quantum dots The positively charged protein G-leucine zipper fusion protein was coated and subjected to image analysis by attaching a primary antibody to it. The most significant result of this experiment is that QDs can be used to continuously obtain images of living cells without losing their emission intensity even after long laser irradiation. These features are groundbreaking properties beyond the limits of existing organic fluorophores and are expected to be actively used in the future of cell biology.

QDs are attractive in biosensors because of their excellent light stability and continuous monitoring in real time. However, its application in the field of biosensors has been limited because the photoluminescent quantum yield is significantly reduced when converted from hydrophobic to hydrophilic forms or in contact with other materials. Recently, research has focused on developing sensors for some target analytes, where QDs are used as electron donors for fluorescence resonance energy transfer (FRET) between QDs (donor) and receptor molecules. In addition to FRET, many of the advantages of QDs are being utilized for the development of sensors based on changes in emission wavelength, voltage, or fluorescence intensity.

On the other hand, glucose is an important nutrient for microorganisms in biotechnological processes, and the measurement of glucose concentration is useful for the control of various food and biotechnological processes, as well as for the diagnosis of many metabolic disorders, in particular for the diagnosis and treatment of diabetes. Many methods have been used for the detection of glucose at present, but glucose detection using QDs has not yet been developed.

The present inventors have conducted continuous research to efficiently apply QDs coated with ZnS shells to CdSe cores in the biosensor field, and after coating QDs with hydrophilic surfactants, the bioconjugates coated with various oxidases, including glucose, It has been confirmed that the present invention can be efficiently used for the detection of various biological materials such as monosaccharides, polysaccharides, organic acids, and the like.

Accordingly, a first object of the present invention is to provide a bioconjugate of QDs and oxidases.

A second object of the present invention is to provide a sensing film in which the bioconjugate is immobilized.

A third object of the present invention is to provide a biological detection kit comprising the sensing film.

A fourth object of the present invention is to provide a biological detection method using the detection kit.

The first aspect of the present invention relates to a bioconjugate in which oxidase is encapsulated in QDs coated with a ZnS shell on a CdSe core coated with a hydrophilic surfactant. The oxidase is preferably selected from the group consisting of glucose oxidase (GOD), lactate oxidase (LOD), tyramine oxidase (TOD), cholesterol oxidase, choline oxidase, alcohol oxidase, ascorbic acid oxidase and xanthine oxidase, The hydrophilic surfactant is mercaptopropionic acid (MPA), mercaptoacetic acid (MAA), mercaptosuccinic acid (MSA), dithiothreitol (DTT), glutathione, glutathione, It is preferred that it is histidine or thiol-containing silanes. The bioconjugates of the invention may further be enclosed with peroxidases, in particular horseradish peroxidase (HRP).

The second aspect of the present invention relates to a sensing film in which the bioconjugate is immobilized on a surface thereof. In the present invention, the immobilization can be performed by a sol-gel method.

The third aspect of the present invention relates to a biological detection kit comprising the bioconjugate or the sensing layer. The kit of the present invention may be for detecting a substance selected from the group consisting of monosaccharides, polysaccharides, organic acids, alcohols, cholesterol, choline, xanthine and mixtures thereof. In the kit according to the present invention, a bioconjugate or a sensing film may be formed on a glass plate, a polystyrene plate, or a microtiter plate.

The fourth aspect of the present invention

1) injecting a biological sample into the detection kit to perform the reaction,

2) measuring the fluorescence intensity emitted from the reaction:

It relates to a biological detection method comprising the step. In the process according to the invention, it is preferred to carry out the reaction in the presence of Fe ³⁺ ions. Fluorescence intensities can be measured by optical methods or by changing them into electrical signals.

Hereinafter, the present invention will be described in detail.

The present invention relates to the detection of biological material using CdSe / ZnS core-shell QDs. In the present invention, hydrophilic CdSe / ZnS core-shell QDs are used to detect monosaccharides, polysaccharides or organic acids including glucose, lactate, tyramine, ascorbic acid and the like, alcohols, cholesterol, choline, xanthine and the like. In a representative example of the present invention, fluorescence quenching of QDs is used to determine the concentration of glucose in aqueous solution. The attenuation process is based on the transfer of electrons from QDs to enzymes (glucose oxidase (GOD), horseradish peroxidase (HRP)) that catalyze the oxidation / reduction reaction of glucose. QDs can be used to detect glucose by entrapment with enzymes and then directly added to the glucose solution. 1 shows a reaction scheme of oxidation of glucose to gluconic acid using GOD / HRP contained in CdSe / ZnS quantum dots (QDs). In the present invention, the glucose is detected by measuring the change in the shape strength due to the chemical change, and thus, a detection method commonly known in the art may be used, for example, by using an optical method or converting into an electrical signal. A detection method or the like can be used, but is not particularly limited thereto.

CdSe / ZnS core-shell QDs used in the present invention are synthesized by modifying existing synthetic methods and coated with hydrophilic surfactants (e.g. MPA, MAA, MSA, DTT, glutathione, histidine, thiol-containing silanes, etc.). Can be. The clathrate can be obtained by mixing coated CdSe / ZnS QDs with enzymes such as GOD and HRP. In the present invention, glucose oxidase (GOD), lactate oxidase (LOD), tyramine oxidase (TOD), cholesterol oxidase, choline oxidase, alcohol oxidase, ascorbic acid oxidase, xanthine oxidase and the like can be used. It is not limited to. Furthermore, as disclosed in Korean Application No. 10-2007-0014736, the contents of which are incorporated herein by reference in their entirety, the enzymes and QDs are referred to as 3-glycidoxypropyl-trimethoxysilane (GPTMS ) The sensing film can be prepared by immobilization on a sol-gel alone or a mixture of GPTMS and methyl-triethoxysilane (MTES) or aminopropyl-trimethoxysilane (APTMS). Covalent bonding of the epoxy group of the sol-gel with the amine group of the enzyme prevents the removal of the enzyme during washing and maintains the high sensitivity of the sensing membrane.

The typical properties of sol-gels applied to enzyme immobilization or encapsulation of organic materials and biomass contribute significantly to the sensitivity and stability of sensing membranes for biological material detection. The mixing ratio of silane in the sol-gel leads to different characteristics such as the different response speed of the sensing film to detect the concentration of biological substances. For the immobilization of QDs, the volume ratio of GPTMS and MTES is 1: 1-2, especially 1: 2. It is preferable to include, and for enzyme immobilization, it is preferable to include GPTMS and APTMS in a volume ratio of 2 to 4: 1, especially 4: 1. Thus, the sensing membrane according to the present invention comprises, for example, QDs immobilized in a sol-gel containing GPTMS and MTES in a volume ratio of 1: 1 to 2, especially 1: 2, and 2 to 4: 1, especially 4 to GPTMS and APTMS. It may be to include an enzyme immobilized in a sol-gel containing in a volume ratio of 1: 1.

In the present invention, the bioconjugate or sensing film may be formed on a substrate to prepare a biological detection kit, and the substrate may be selected and used among those commonly known in the art, for example, a glass plate and a polystyrene plate. , Microtiter plates and the like may be used, but are not particularly limited thereto.

Hereinafter, the present invention will be described in detail by way of examples, which are only intended to help understanding of the present invention, but are not intended to limit the scope of the present invention in any way.

Preparation Example 1 Synthesis of CdSe / ZnS Core-Shell Quantum Dots (QDs)

CdSe / ZnS core-shell QDs were synthesized by modifying existing synthesis methods. First, CdSe nanoparticles were prepared by Qu and Peng (L. Qu, X. Peng, J. Am. Chem. Soc. 124 (2002) 2049-2051), and Gaunt et al. (J. Gaunt et al., J.). Coll. Interf. Sci. 290 (2005)] was synthesized by modifying the method of cadmium acetate dehydrate (0.6 mM, 147 mg) and stearic acid (2.13 mM, 607 mg) in a 50 ml three-necked flask. Heated under vacuum at 150 ° C. until a colorless liquid was obtained, after cooling to room temperature hexadecylamine (1.94 g) and trioctylphosphine oxide (TOPO) (2.2 g) were added to the flask. The mixture was then degassed using a pump and heated under vacuum to 120-150 [deg.] C. The reaction vessel was then charged with nitrogen gas and heated to 310-320 [deg.] C. at this point, trioctylphosphine (TOP) A solution in which selenium (211 g) was dissolved in (2.5 mL) was injected rapidly with vigorous stirring. After removing the flask from the heating mantle, it was heated for 25 seconds and allowed to cool to room temperature The reaction mixture was dissolved in chloroform and then precipitated with an equal volume of methanol to purify the resulting CdSe nanoparticles. CdSe particles were used to synthesize CdSe / ZnS core-shell QDs (CZ-QDs) A mixture of hexadecylamine (2 g) and TOPO (2.5 g) was loaded into a 50 ml three-necked flask and degassed to 180 ° C. The purified CdSe particles dispersed in 2 ml of chloroform were added to this solution at 180 ° C. The chloroform was completely removed using a pump and the flask was filled with nitrogen gas, and the reaction temperature was raised to 180-185 ° C. A mixture of zinc acetate (54 mg) and hexamethyldisilathiane (TMS) ₂ S (0.05 mL) dissolved in 1 mL TOP was added dropwise for 5 to 10 minutes. After the dropwise addition, the mixture was stirred at 180-185 ° C for 1 hour.

Preparation Example 2 Synthesis of Hydrophilic Surfactant Coated CdSe / ZnS Core-Shell QDs

CZ-QDs synthesized in Preparation Example 1 were coated with mercaptopropionic acid (MPA) with a slight modification of the existing method. 200 mg of CZ-QDs in TOP-TOPO-hexadecylamine were purified by dissolving and precipitation in anhydrous chloroform and ethanol, respectively. The wet precipitate was dispersed in a mixture of 2 mL N, N-dimethylformamide (DMF) and 0.25 mL 3-mercaptopropionic acid. It was sonicated for about 30 minutes until the mixture became clear and stored for 1 week at room temperature. In the next step, 0.5-0.7 ml 4-dimethylaminopyridine (DMAP) dissolved in DMF was added (50 mg DMAP / 1.0 ml DMF) and the solution was centrifuged at 5000 rpm for 30 minutes. The supernatant was removed and the precipitate was dried in a desiccator and dissolved in 10 mM phosphate buffered saline (PBS). The resulting MPA-coated CdSe / ZnS core-shell QDs (MPA-QDs) were used for glucose detection.

Example 1 Bioconjugate Synthesis of MPA-Coated CdSe / ZnS Core-shell QDs with Oxidase

A fixed amount of enzyme was mixed with different volumes of MPA-QDs or a constant volume of MPA-QDs was added to various amounts of enzyme. Evaluation of their inclusion and interaction was performed after 3 to 24 hours of incubation and inclusion of MPA-QDs and enzymes was measured by electrophoresis (2% agarose). A voltage of 100 V was applied to the gel for 1 hour. Enzymatic activity of GOD- or non-inclusion MPA-QDs was determined by diammonium 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a peroxidase substrate in the presence of 0.5 g / l glucose. ) Was measured. This substrate is green and produces an aqueous final product that can be spectroscopically read at 405 nm (see Scheme 1). Microtiter plate reader (Wallac Victor 2, Perkin-Elmer Co. USA) was used for absorbance measurements.

Scheme 1

H ₂ O ₂ + reduced ABTS --- (HRP) ---> H ₂ O + 0.5O ₂ + oxidized ABTS

Example 2 Measurement of Optical Properties of QDs

Absorption and emission spectra of CdSe nanoparticles, MPA-QDs and enzyme-enclosed MPA-QDs were analyzed using the Multiskan spectrum (Thermo electron corporation, Finland) and the fluorescence spectrophotometer (Model: F-4500, Hitachi Co., Ltd.). , Japan).

CdSe / ZnS core-shell QDs had the highest fluorescence intensity when the quantum yield (QY) was 64% and the particle size was 2 to 4.5 nm. 2 (a) shows absorption spectra of CdSe particles, CZ-QDs and MPA-QDs. Their broad spectrum of absorption allows for efficient excitation of several QD-based phosphors with a single light source. That is, easy control using the emission filter of the microplate reader and excitation at short wavelengths without directly exciting the receptor are possible. In Figure 2 (b), the emission wavelengths of the QDs (e.g. MPA-QDs) are 590 nm at 40 nm FWHM (full width at half of the maximum emission spectrum), which corresponds to the absorption bands of GOD and HRP. It overlaps (FIG. 2 (c)). This property was used for the use of QDs for glucose detection through electron transfer from QDs to enzymes. After covering the surface with a carboxyl group (MPA), the quantum yield of QDs was reduced to about 50% of the initial value.

Two-dimensional fluorescence spectra of MPA-QDs and enzymes (HRP and GOD) observed changes in the fluorescence intensity of the enzyme at excitation wavelengths of 260-320 nm / 445 nm and emission wavelengths of 280-450 nm / 525 nm (results). Not shown). On the other hand, the fluorescence intensity of QDs varied between 280-550 nm excitation and 580-610 nm emission wavelengths. That is, the fluorescence intensities of QDs and enzymes vary with their inclusion and interaction, activity, and the amount of each component involved.

Clear binding of QDs and enzymes was observed in the photographic image of FIG. 3. The amine group of the enzyme easily binds to the carboxyl group on the surface of the QDs. After inclusion with the enzyme, the size of the QDs increased, and as the amount of GOD increased, the amount of enzyme bound to the binding site of the QD surface increased (see FIG. 3 (a)). The enzyme-bound QDs in the gel plate migrated more slowly than the enzyme-inclusion MPA-QDs (Enz-QDs) containing smaller amounts of MPA-QDs. Therefore, the distance traveled in the gel was slightly shorter than that of MPA-QDs containing small amounts of GOD. The same tendency appeared when the QD concentration increased with a certain amount of enzyme, resulting in more quantum dots being entrapped with the enzyme and their migration slower. In the picture, the bands of QDs were not clear because QDs aggregated under basic conditions and only a small amount of QDs migrated to the anode, and changes in enzyme or QDs amounts resulted in changes in fluorescence intensity of enzymes and QDs. 3 (a) shows the fluorescence attenuation of QDs at an emission wavelength of 590 nm as the amount of GOD increases. In addition, enzymes can receive energy from excited QDs and increase their fluorescence intensity. An increase in fluorescence intensity of the enzyme was also observed at an emission wavelength of 525 nm. This phenomenon is associated with increased amounts of enzymes and fluorescence resonance energy transfer (FRET) from QDs to enzymes. The use of a constant amount of GOD entrapped with different concentrations of QDs increases the fluorescence intensity of the enzyme and decreases the fluorescence intensity of the QDs compared to enzyme entrapped QDs, so FRET is also shown in FIG. 3 (b).

Example 3: Evaluation of the Effect of MPA-QDs on Enzyme Activity

GOD, a structurally robust glycoprotein of 160,000 Da, consists of two identical polypeptide chains with a hydrodynamic radius of 43 mm 3. The robust nature of GODs derives from polysaccharides that form hydrophilic envelopes. When the electrons migrate between the redox enzyme and H ₂ O ₂ in the electrochemically reduced form produced during the O ₂ -biocatalyzed oxidation of glucose, the electrons move to the substrate (e.g. glucose, H ₂ O ₂ , O ₂ ). The turnover rate of electron exchange between QDs and enzymes, as well as catalysts (GOD and HRP), is rapidly increased. The delivered physical energy of the QDs associated with the attenuated phosphor thus reflects the concentration of the substrate in the system.

As shown in FIG. 4, the fluorescence emission and activity of the enzyme is significantly changed after inclusion with MPA-QDs. After 3 or 12 hours of incubation of GOD and HRP with MPA-QDs, the fluorescence intensity at ex: 445 nm / em: 525 nm increased by about 30% or 43%, respectively. When only GOD was incubated for 3 or 12 hours with MPA-QDs, the fluorescence intensity of GOD rapidly decreased to 20% or 41% of the original value, respectively. This means that enzymes that retain activity receive large amounts of energy from excited QDs, while their fluorescence emission is stronger than that of the original enzyme, while functional groups on the surface of MPA-QDs inhibit enzyme activity. In addition, fluorescence emission of MPA-QDs was reduced after prolonged incubation (results not shown).

Example 4: Glucose Concentration Measurement

Glucose was measured using MPA-QDs, GOD and HRP. Enzymes (GOD, HRP) and MPA-QDs were added to microtiter plate wells dispensed with 100 μl of varying concentrations of glucose solution. Mixtures of MPA-QDs, GOD and HRP at 10:10:10, 10: 5: 5 and 10: 5: 0 (μl / well) ratios were used. The microtiter plate was then immediately inserted into the measurement chamber of the plate reader, and the fluorescence intensity was measured 40 times at 3 minute intervals at an excitation / emission wavelength of 485/525 nm.

As mentioned above, direct electrical activation of enzymes, in particular redox enzymes, is a general approach for the stimulation of bioelectrocatalytic oxidation (or reduction) of enzyme substrates. 5 shows the effect of volume ratios of MPA-QDs, GOD and HRP in the glucose solution and the ratio between enzymes and MPA-QDs on the output signal of QD-FRET-based probes after exposure to various concentrations of glucose. When the volume ratio of QDs, GOD and HRP was 10/10/10, the fluorescence intensity changed significantly at low glucose concentrations, and the sensitivity (tilt) was very high in the linear concentration range of 0 to 1.0 g / l with an R value of 0.990 ( S = -11360). As the volume ratio of QDs, GOD and HRP decreased in the amount of HRP used at 10/10/5, 10/5/3 or 10/5/0, the sensitivity or reaction efficiency of QD-FRET-based probes decreased. In addition, at low glucose concentrations (0.2-1.0 g / l), the fluorescence intensity decreased with decreasing HRP content. That is, the linear detection interval of glucose is 0 to 5.0 g / l (R = 0.992) when the volume ratio of QDs / GOD / HRP is 10/5/5, and 0.2 to 5.0 g / l when the volume ratio is 10/5/3 ( R = 0.985) and 0 to 5.0 g / L (R = 0.982) at a volume ratio of 10/5/0. The path of electron transfer from excited QDs to hydrogen peroxide (H ₂ O ₂ ) reduction reaction was inhibited or prevented. Thus, a decrease in the number of electrons results in a decrease in the conversion rate of the electron exchange, resulting in low photoluminescence attenuation of QDs and low sensitivity to glucose.

The response time of the QD-FRET-based probe to obtain signal stability was 30 minutes. Although the response time is slower than other glucose detection systems developed in previous studies, this system is difficult to compare because it is manufactured and operated differently from the existing system. The late response time of the probe may be due to the low diffusion rate of molecules in the free environment, resulting in component heterogeneity in the reaction. However, this demonstrates the potential of QDs for use as QD-FRET-based probes in detecting glucose in solution. The slow response time and the stability of QDs and enzyme mixtures in glucose detection could be improved by immobilizing enzymes and MPA-QDs with sol-gel layers of GPTMS and APTMS.

Example 5 Preparation of Sensing Film by Sol-Gel Method

According to the method described in Korean Patent Application No. 10-2007-0014736, a sensing film was prepared using the sol-gel method. That is, a sol-gel was prepared by mixing GPTMS and MTES in a volume ratio of 1: 2 (GM2) and GPTMS and APTMS in a ratio of 4: 1 by volume (GA2), respectively, in 99% ethanol. 35% HCl was added to the mixed solution in a volume of 40 μl / ml. After HCl addition, the sol-gel was stored at room temperature for at least 2 hours before being used in the next step.

A transducer was prepared by adding 50 μl of MPA-coated QDs synthesized in Preparation Example 2 to 200 μl of sol-gel GM2. The mixture of MPA-coated QDs and sol-gel was thoroughly mixed by mechanical stirring and then stored at room temperature for 2 hours. 5 μL MPA-coated QDs mixture was dispensed to the bottom of a 96 well microtiter plate and dried at 95 ° C. for 18 hours. After the heat treatment, sol-gel GA2 was added on the transducer and 40 μl of enzyme solution (GOD: 100 units, LOD: 1 unit, or TOD: 0.005 units) was added to the wells of a 96 well microtiter plate. The enzyme was immobilized at room temperature for 18 hours.

Example 6: Evaluation of the Effect of pH and Temperature

The effect of pH and temperature on glucose detection was investigated using a mixture of GOD, HRP and MPA-QDs. The universal buffer used in this example contains 0.1 M Na ₂ SO ₄ , 0.04 M NaOAc, 0.04 MH ₃ BO ₃ and 0.04 M NaH ₂ PO _4, and the pH of the buffer solution is measured using a pH meter (Metrohm Co , Switzerland) was adjusted to pH 4-11 with 3 N NaOH and 3 M HCl. At a given pH, 100 μl of glucose solution (1 g / L) and 20 μl of GOD, HRP, and MPA-QDs mixed solution prepared in general buffer solution were dispensed into the wells of a microtiter plate and the fluorescence intensity was measured. To investigate the effect of temperature interference on the measurement, 120 µl of the reaction mixture solution was incubated at a glucose concentration of 1.0 g / l at 23-37 ° C, followed by fluorescence intensity at an excitation / emission wavelength of 485/525 nm. Measured.

As shown in FIG. 6, temperature and pH only slightly influenced the glucose measurement. The change in fluorescence intensity was different at low pH (4-5) or high pH (11). Although the fluorescence intensity at low or high pH is higher than at neutral pH, QD nanoparticles cannot be used because they aggregate and precipitate under strong acid or base conditions. Based on the results of previous papers dealing with the effect of pH on the fluorescence intensity of QD, the fluorescence intensity increased with increasing pH in the range of pH 8 ~ 11.5. Can be used as a stability zone for QDs. A pH range of 6 to 10 is a stable environment for the measurement. The increase in temperature also results in a decrease in reaction time. In fact, the reaction time was reduced by more than 60% at temperatures above 26 ℃. However, due to the autofluorescence attenuation of QDs at temperatures above 26 ℃, the fluorescence intensity decreased rapidly.

Example 7: Interference Studies

In this embodiment, a variety of ions was performed to evaluate the effect of _{^{(NH 4 +, NO 3 -}} -, Na +, Cl, CO 3 2-, Fe 3+), the concentration range of the used ion was 0.01 ~ 200 mM. 100 μl ionic solution was added to the microtiter well and mixed with 10 g / l glucose to obtain a final glucose concentration of 1 g / l. Different ionic solutions of different concentrations were prepared on microtiter plates in plural measurement form with control samples (without ions). The fluorescence intensity was then measured by adding 20 μl MPA-QDs to each well containing 1 g / L glucose solution at different ion concentrations. An enzyme mixture (GOD / HRP: 30 U / 3 U each) was added to these samples and secondly the fluorescence intensity was measured. Fluorescence differences between samples before and after enzyme addition and control samples were statistically processed by ANOVA using Instat software.

Of the tested concentration range of 0.01 ~ 200 mM _{^{NH 4 +, NO 3 -,}} Na +, Cl - various ions such as, CO ₃ ^2-, Fe ³⁺ have a significant effect on the fluorescence intensity of MPA-QDs involved in the reaction of glucose It was not crazy. However, when these ions were compared with the fluorescence intensities of glucose and control samples before and after enzyme addition, the p-value was measured to be greater than 0.05. 200 mM CO ₃ ^2- ions had a slight effect on the fluorescence intensity of MPA-QDs before glucose addition, and as shown in Table 1, Fe ³⁺ ions had a significant effect on the fluorescence emission of MPA-QDs in glucose oxidation. I could see the crazy.

Ions (mM) CO ₃ ^{2 -} Fe ^{3 +} Enzyme-free enzyme Enzyme-free enzyme 0.01 ns ns ns ns 0.1 ns ns * ns 0.5 ns ns *** *** 1.0 ns ns *** *** 10 ns ns *** ns 50 ns ns *** ** 100 ns ns *** *** 200 * ns *** ***

(ns: no significance, * P <0.05: ** P <0.01: *** P <0.001)

Prior to the addition of enzymes to the glucose solution, the fluorescence emission of MPA-QDs was attenuated at Fe ³⁺ ion concentrations of 0.1 mM and strong fluorescence attenuation occurred at concentrations above 0.5 mM (FIG. 7). After enzyme addition, the fluorescence intensity decreased at low concentrations (0.5 mM and 1.0 mM) of Fe ³⁺ ions. In theory, Fe ³⁺ ions act as mediators with adequate oxidation potential, replacing oxygen in glucose oxidation. Therefore, the response signal was very fast after the enzyme addition, and the fluorescence intensity was measured up to 10 times before the enzyme addition at the high Fe ³⁺ ion concentration.

According to the present invention, CdSe / ZnS core-shell QDs coated with hydrophilic surfactants can be encapsulated with oxidase and have neighboring intermolecular energy transfer properties, so that they can be widely applied in the field of biosensors, especially glucose in a wide concentration. It has been shown that it can be efficiently detected in the range.

Claims (13)

Bioconjugates in which oxidases are enclosed in quantum dots (QDs) coated with a zinc sulfide (ZnS) shell on a cadmium selenide (CdSe) core coated with a hydrophilic surfactant. The bioconjugate of claim 1, wherein the oxidase is selected from the group consisting of glucose oxidase, lactate oxidase, tyramine oxidase, cholesterol oxidase, choline oxidase, alcohol oxidase, ascorbic acid oxidase and xanthine oxidase. The bioconjugate of claim 1, further comprising a peroxidase. The bioconjugate of claim 3, wherein the volume ratio of the quantum dots, the oxidase, and the peroxidase is 1: 1: 1. The bioconjugate of claim 1, wherein the hydrophilic surfactant is mercaptopropionic acid, mercaptoacetic acid, mercaptosuccinic acid, dithiothreitol, glutathione, histidine, or thiol-containing silane. A sensing membrane in which the bioconjugate according to any one of claims 1 to 5 is immobilized on a surface thereof. The sensing film of claim 6 which is immobilized by a sol-gel method. A biological detection kit comprising a bioconjugate according to claim 1 or a sensing film according to claim 6. The kit of claim 8 for detecting a substance selected from the group consisting of monosaccharides, polysaccharides, organic acids, alcohols, cholesterol, choline, xanthine and mixtures thereof. The kit according to claim 8, wherein the bioconjugate or sensing film is formed on a glass plate, polystyrene plate or microtiter plate. 1) injecting a biological sample into the detection kit according to claim 8 to perform the reaction, 2) measuring the fluorescence intensity emitted from the reaction: Comprising the steps of: biological detection method. The method of claim 11, wherein the reaction is carried out in the presence of Fe ³⁺ ions. 12. The method according to claim 11, wherein in step 2) the fluorescence intensity is measured by an optical method or by converting it into an electrical signal.

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