WO2008146966A1

WO2008146966A1 - Kits and methods for biological detection using quantum dots

Info

Publication number: WO2008146966A1
Application number: PCT/KR2007/002748
Authority: WO
Inventors: Jong Il Rhee
Original assignee: Industry Foundation of Chonnam National University
Current assignee: Industry Foundation of Chonnam National University
Priority date: 2007-05-28
Filing date: 2007-06-07
Publication date: 2008-12-04
Anticipated expiration: 2009-11-28
Also published as: KR20080104495A; KR100914455B1

Abstract

Disclosed are bioconjυgates of quantum dots (QDs) having CdSe core and ZnS shell and various oxidases, sensing membranes onto which said bioconjugates are immobilized, biological detection kits comprising said sensing membranes, and methods for biological detection using said detection kits. QDs having various advantages over organic fluorophores can be used for the detection of monosaccharides, polysaccharides, organic acids, etc., and particularly for detecting glucose over a broad range of concentration.

Description

KITS AND METHODS FOR BIOLOGICAL DETECTION USING QUANTUM DOTS

[Technical Field]

The present invention relates to the biological detection

5 using quantum dots (QDs) . More specifically, the present invention relates to bioconjugates of QDs having CdSe core and

ZnS shell and various oxidases, sensing membranes onto which said bioconjugates are immobilized, biological detection kits comprising said sensing membranes, and to methods for

10 biological detection using said detection kits.

[Background Art]

Semiconductor nanoparticle QDs having various applicability were first invented in 1970s. However, it has

!.^") been only four or five years since QDs were applied in the field of biological sciences. QDs consisting of CdSe core and ZnS shell are spherical materials having a diameter of several to tens of nanometers . QDs have a property of emitting fluorescence at different wavelengths depending on their 0 particle sizes, and thus, they can be used not only for basic biology such as cell biology but also for applied biology including various protein chips and biosensors, etc. QDs can be excited by light of any wavelength ranging from UV to red and have a controllable narrow emission spectrum. They are inorganic, and so stable against chemical reactions and can be easily linked with biological materials through a surface treatment. Jaiswal et al . reported that avidin-conjugated QDs were linked with a biotinylated primary antibody and the resulting QD bioconjugates were used to image live cells

[Jaiswal et al . , Nature Biotechnology 21, 47-51, 2003]. In addition, they reported that negatively-charged QDs were coated with positively-charged protein G-leucine zipper fusion protein and the coated QDs were conjugated with a primary antibody to get images. It is the most meaningful result obtained from the above studies that images of live cells can be continuously obtained without any decline in the emission intensity even under the irradiation of laser light over a long period of time. This is a remarkable property which can overcome limitations of organic fluorophores of the prior art. Thus, QDs are expected to be widely used in the field of cell biology in the future.

QDs have a high photo-stability and allow for continuous real-time monitoring, and thus, are attractive materials in the field of biosensors. However, the application of QDs in this field is still limited because the photoluminescence quantum yield of the QDs decreases significantly when they are transformed from the hydrophobic to hydrophilic form or when they are conjugated with other materials. Recently, researches have been focused on the development of sensors for some target analytes, wherein QDs are used as electron donors for fluorescence resonance energy transfer (FRET) between the QDs (donor) and an acceptor molecule. Besides FRET, many advantageous properties of QDs have been exploited for the development of sensors based on the change in the emission wavelength, voltage or fluorescence intensity.

Glucose is an important nutrient source for microorganisms in biotechnological processes. Thus, the measurement of glucose concentration is always useful in controlling various food and biotechnological processes, as well as in diagnosing many metabolic disorders, especially in the diagnosis and therapy of diabetes. Many methods have been used for the detection of glucose, but none of the methods developed so far have used QDs for glucose detection.

[Disclosure] [Technical Problem]

The present inventors have performed extensive studies for efficient application of QDs consisting of CdSe core and ZnS shell to biosensors. As a result, the inventors found that bioconjugates of QDs and various oxidases, which are obtained by coating QDs with a hydrophilic surfactant, and then, conjugating the coated QDs with the oxidases, can be efficiently used for the detection of many biological materials including monosaccharides, polysaccharides, organic acids, etc., and therefore, completed the present invention.

Accordingly, the first object of the present invention is to provide QDs-oxidase bioconjugates .

The second object of the present invention is to provide sensing membranes onto which said bioconjugates are immobilized .

The third object of the present invention is to provide biological detection kits comprising said membranes.

The fourth object of the present invention is to provide methods for biological detection using said detection kits.

[Technical Solution] The first aspect of the present invention relates to a bioconjugate, in which a QD having CdSe core and ZnS shell is coated with a hydrophilic surfactant, and the coated QD is conjugated with an oxidase. In a preferable embodiment, said oxidase is selected from the group consisting of glucose oxidase (GOD) , lactate oxidase (LOD) , tyramine oxiade (TOD) , cholesterol oxidase, choline oxidase, alcohol oxidase, ascorbic acid oxidase, and xanthine oxidase. Said hydrophilic surfactant is preferably mercaptopropionic acid (MPA) , mercaptoacetic acid (MAA) , mercaptosuccinic acid (MSA) , dithiothreitol (DTT) , glutathione, histidine or thiol- containing silane. Bioconjugate of the present invention can be further conjugated to peroxidase, particularly to horseradish peroxidase (HRP) .

5 The second aspect of the present invention relates to a sensing membrane onto which said bioconjugate is immobilized. The immobilization may be performed by a sol-gel method.

The third aspect of the present invention relates to a biological detection kit comprising said bioconjugate or said

10 membrane. The kit of the present invention may be used for detection of a substance selected from the group consisting of monosaccharides, polysaccharides, organic acids, alcohols, cholesterol, choline, xanthine, and mixtures thereof. The kit of the present invention may have the bioconjugate or the

To sensing membrane formed on a glass plate, a polystyrene plate, or a microtiter plate.

The fourth object of the present invention relates to a method for biological detection comprising the steps of;

1) injecting a biological sample to said detection kit to ^!).{) carry out a reaction; and

2) measuring fluorescence intensity emitted from said reaction.

In the method of the present invention, said reaction is preferably carried out in the presence of Fe³⁺ ions . Fluorescence intensity may be measured by an optical method or by converting it to an electrical signal.

Hereinafter, the present invention will be described in

.^") detail.

The present invention relates to the detection of biological materials using CdSe/ZnS core-shell QDs. According to the present invention, hydrophilic CdSe/ZnS core- shell QDs

10 are used for sensing monosaccharides, polysaccharides, organic acids, alcohols, cholesterol, choline, or xanthine, including glucose, lactate, tyramine, ascorbic acids, etc. In one representative example of the present invention, the fluorescence quenching of the QDs is used to measure the

IT) concentrations of glucose in aqueous solution. The quenching process is based on the transfer of electrons from the QDs to enzymes including glucose oxidase (GOD) and horseradish peroxidase (HRP) , which catalyze the oxidation/reduction reactions of glucose. The QDs are able to be used to detect

'-}() glucose by introducing them directly into the glucose solution after their conjugation with the enzymes. Figure 1 depicts the reaction scheme of glucose oxidation to gluconic acid using GOD/HRP conjugated to CdSe/ZnS QDs. In the present invention, glucose is detected by measuring a change in fluorescence intensity by the above chemical modification. Thus, any detection method known in the art may be used, and examples thereof include, but are not limited to, optical methods and other methods involving conversion into electrical signals. 5 CdSe/ZnS core-shell QDs that are used in the present invention are synthesized using a modified version of the conventional synthetic method, and they can be coated with a hydrophilic surfactant (for example, MPA, MAA, MSA, DTT, glutathione, histidine, thiol-comprising silane, etc.). The

10 coated CdSe/ZnS QDs are mixed with enzymes (for example, GOD and HRP) to obtain a conjugate. According to the present invention, examples of the enzyme include, but are not limited to, glucose oxidase (GOD) , lactate oxidase (LOD) , tyramine oxiade (TOD), cholesterol oxidase, choline oxidase, alcohol

IT) oxidase, ascorbic acid oxidase, xanthine oxidase, etc. As set forth in the co-pending Korean Patent Application No. 10-2007- 0050538, the entire disclosures of which are incorporated herein by reference, said enzyme-QDs can be immobilized onto a sol-gel layer containing 3-glicydoxypropyl-trimethoxysilane

'/.() (GPTMS) alone or a mixture of GPTMS and methyl-triethoxysilane (MTES) or aminopropyl-trimethoxysilane (APTMS) to prepare sensing membranes . Covalent bond between an epoxy group of sol-gel and an amine group of the enzyme prevents the loss of the enzyme upon washing of the membrane, and further, contributes to maintaining a high sensitivity of sensing membranes .

Typical characteristics of sol-gel, which is used for immobilization of enzymes or encapsulation of organic T) materials and biomaterials , contribute significantly to the stability and sensitivity of the membranes for detection of the biological materials. Mixing ratio of silane in sol-gel results in different properties including different response rates of sensing membranes for measurement of biological

K) materials concentration. Thus, for the immobilization of QDs, GPTMS and MTES are used preferably in the volume ratio of 1:1-2, particularly of 1:2. For the immobilization of enzymes, GPTMS and APTMS are used preferably in the volume ratio of 2-4:1, particularly of 4:1. Therefore, for example, the

IT) sensing membranes according to the present invention comprise QDs, which are immobilized onto sol-gel comprising GPTMS and MTES in the volume ratio of 1:1-2, particularly of 1:2, and the enzymes, which are immobilized onto sol-gel comprising GPTMS and GPTMS in the volume ratio of 2-4:1, particularly of

20 4:1.

According to the present invention, said bioconjugates or sensing membranes can be formed on a substrate to manufacture a biological detection kit. As the substrate, any ones known in the art can be chosen and used. Examples thereof include, but are not specially limited to, a glass plate, a polystyrene plate, a microtiter plate, etc.

[Description of Drawings]

.^'") Figure 1 depicts the reaction scheme of glucose oxidation to gluconic acid using GOD/HRP conjugated to CdSe/ZnS QDs;

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

Figure 3 (a) is a photo image of gel electrophoresis of

!f> Enzyme-QDs containing different amounts of GOD (GOD = 10, 50, 100, 150, 200, 250 or 300 U) and their emission spectra with a fixed amount of QDs (3.145 mg/mi) and HRP (10 U) at an excitation wavelength of 445 nm in 0.5 g/ i glucose solution, and Figure 3 (b) is a photo image of gel electrophoresis of 0 Enzyme-QDs at different concentrations of QDs (QDs = 0.4309, 0.8622, 1.7254, 2.5872 or 3.445 nig/iuC) and their emission spectra with a fixed amount of GOD (200 U) and HRP (10 U) at an excitation wavelength of 445 nm in 0.5 g/C glucose solution; Figure 4 shows emission spectra of enzymes in the presence and absence of MPA-QDs (excitation; 445 nm/ emission,- 525 nni) ;

Figure 5 shows change in fluorescence intensities of the QD-FRET-based probe at different glucose concentrations and various volume ratios of QDs/GOD/HRP added to glucose solution;

Figure 6 shows effect of pH and temperature of reaction solution during glucose measurements; and, Figure 7 shows effect of Fe³⁺ ions on the fluorescence emission of MPA-QDs during the measurement of glucose solution

[Best Mode] The present invention will be specifically explained with reference to the following examples, which are only for the better understanding of the present invention but should not be construed to limit the scope of the present invention in any manner .

Preparation Example 1: Synthesis of CdSe/ZnS core-shell QDs

The synthesis of the CdSe/ZnS core-shell QDs were based on a modified version of the existing method. Firstly, CdSe nanoparticles were synthesized using a modified version of the methods m L. Qu, X. Peng, J. Am. Chem. Soc . 124 (2002) 2049- 2051, and J. A. Gaunt et al . , J. Coll. Interf . Sci. 290 (2005). Cadmium acetate dehydrate (0.6 mM, 147 mg) and stearic acid (2.13 mM, 607 mg) were loaded into a 50 mi three-neck flask, and the mixture was heated to 150 ^°C under vacuum conditions until a colorless liquid was obtained. After cooling to room temperature, hexadecylamme (1.94 g) and trioctylphosphme oxide (TOPO; 2.2 g) were added to the flask. The mixture was then degassed using a pump and heated at 120-150 ^°C under

K) vacuum. The reaction vessel was then filled with nitrogen gas and heated to 310-320 ^°C , and at this point, a solution of selenium (211 g) m trioctylphosphme (TOP; 2.5 ml-) was rapidly injected into the vigorously stirred reaction mixture. The solution was heated for 25 seconds before removing the flask i ^r) from the heating mantle and then allowing it to cool to room temperature. The resulting CdSe nanoparticles were purified by dissolving the reaction mixture in chloroform, followed by precipitation with an equal volume of methanol. In the next step, these purified CdSe particles were used to synthesize 0 the CdSe/ZnS core-shell QDs (CZ-QDs) . A mixture of hexadecylamme (2 g) and TOPO (2.5 g) was loaded into a 50 mf three-neck flask. The mixture was degassed and heated to 180 V. At 180 ^°C , the purified CdSe particles dispersed m chloroform (2 ml) were added to this solution. After chloroform was completely pumped out, the flask was filled with nitrogen gas. The temperature of the reaction then increased to 180-185 ^°C . A mixture of zinc acetate (54 mg) and hexamethyldisilathiane (TMS)₂S; 0.05 ml) dissolved in 1 ml TOP o was then injected dropwise for 5 to 10 min. After the injection, the mixture was stirred for an hour at 180-185 ^°C .

Preparation Example 2: Synthesis of hydrophilic surfactant coated CdSe/ZnS core-shell QDs

U) The CZ-QDs synthesized in the above Preparation Example 1 were coated with mercaptopropionic acid (MPA) using a slightly modified version of the protocols reported in the literature. 200 mg of CZ-QDs in TOP-TOPO-hexadecylamine were purified by dissolution and precipitation in anhydrous chloroform and

' .^") ethanol, respectively. The wet precipitates were then dispersed in a mixture of 2 ml of N, N-dimethylformamide (DMF) and 0.25 ml of 3 -mercaptopropionic acid. The mixture was sonicated for about 30 minutes until it became transparent, and then, stored for one week at room temperature. In the next

'■O step, 0.5-0.7 ml of 4 -dimetnylaminopyridine (DMAP) dissolved in DMF was added (50 mg DMAP/1.0 ml DMF) and the solution centrifuged for 30 minutes at 5000 rpm. The supernatant was discarded, while the precipitate was dried in a desiccator and then dissolved in 10 mM phosphate buffered saline (PBS) . The resultant MPA-coated CdSe/ZnS core-shell QDs (MPA-QDs) were used for glucose sensing.

Example 1: Synthesis of bioconjugates of MPA-coated CdSe/ZnS 5 core- shell QDs and oxidases

A fixed amount of enzymes was mixed with different volumes of MPA-QDs or a given volume of the MPA-QDs was added to various amounts of the enzymes. The evaluation for their conjugation and interactions was carried out after 3-24 hour

H) incubation. The conjugation of MPA-QDs and enzymes was also determined by gel electrophoresis (2% agarose) . A voltage of 100 V was applied along the gel for 1 hour. The enzyme activity of the GOD- conjugated or -unconjugated MPA-QDs was determined using diammonium 2 , 2 ' -azino-bis (3-

15 ethylbenzothiazoline-6-sulfonate) (ABTS) as a peroxidase substrate in the presence of 0.5 g/ I glucose. This substrate produces a soluble end product that is green in color and can be read spectrophotometrically at 405 nm (see Reaction Scheme 1 below) . A microtiter plate reader (Wallac Victor 2, Perkin- '--!O Elmer Co. USA) was used for measuring the absorbance. [Reaction Scheme 1] H_?O_?. + reduced ABTS (HRP) > H₂O + 0.5 O₂ + oxidized ABTS

Example 2: Optical characterization of QDs The absorption and emission spectra of CdSe nanoparticles, MPA-QDs and enzyme-conjugated MPA-QDs were determined using a Multiskan spectrum (Thermo electron corporation, Finland) and Fluorescence spectrophotometer (Model: F-4500, Hitachi Co., r> Japan), respectively.

The synthesized CdSe/ZnS core-shell QDs had a high fluorescence intensity with a quantum yield (QY) of 64% and their particle size ranged from 2 nm to 4.5 lira. Figure 2 (a) shows the absorption spectra of the CdSe particles, CZ-QDs and

10 MPA-QDs. Their broad absorption spectra allows for the efficient excitation of multiple QD-based fluorophores with a single light source. That is, easy tuning with the excitation filter of a microplate reader or excitation at a short wavelength that does not directly excite the acceptor is

If) possible. In Figure 2 (b) , the emission wavelength of the QDs

(e.g., MPA-QDs) was 590 nm with a FWHM (full width at half maximum for emission spectrum) of 40 nm, which overlapped with the absorption band of GOD and HRP (see Figure 2(c)). This property was exploited in order to utilize the QDs for glucose 0 sensing via the electron transfer from the QDs to the enzymes. The quantum yield of the QDs after converting the carboxyl groups (MPA) on their surface was decreased to about 50% of its initial value.

In the two-dimensional fluorescence spectra of the mixture of MPA-QDs and enzymes (HRP and GOD) , it was observed that the fluorescence intensity of the enzymes changed in the excitation wavelength range of 260-320 nm/445 ran and in the emission wavelength range of 280-450 nm/525 ran (data not shown) , T) while the QDs changed in the fluorescence intensity range of 280-550 ran for excitation and 580-610 nπi for emission. That is, the fluorescence intensities of the QDs and enzymes are changed by their conjugation and interaction, their activities and the amount of each component involved. iϋ The clear binding of the QDs and the enzymes was observed in the photo images of Figure 3. The amine groups of the enzymes are easily bound to the carboxyl groups located on the surface of QDs. The sizes of the QD increased after their conjugation with the enzymes, and increasing the amount of GOD i .1 resulted in an increase in the amount of the enzymes bound to the binding sites of the QD surface (see Figure 3 (a) ) . Their movement in the gel plate was slower than that of the enzyme- conjugated MPA-QDs (Enz-QDs) containing a larger amount of enzymes and, therefore, their migration distances in the gel 0 were slightly shorter than those of the MPA-QDs comprising a smaller amount of GOD. The same tendency was observed when increasing the QD concentrations, which were mixed with a given amount of enzymes, in which more quantum dots were conjugated with the enzymes and their movement was slower. In the photos, the bands of the QD were not clear because the aggregation of QDs under basic conditions leads to only a small amount of the QDs moving to the positive pole. The change in the amounts of enzymes or QDs leads to a change in

7> the fluorescence intensity of the enzymes and QDs. Figure 3 (a) shows the fluorophore quenching of the QDs at an emission wavelength of 590 nm when the amount of GOD is increased. The enzymes were able to receive energy from the excited QDs, resulting in an increase in their fluorescence intensity. An

10 increase in the fluorescence intensity of the enzymes was also observed at an emission wavelength of 525 nm. This is associated with the increase in the amount of enzymes and the fluorescence resonance energy transfer (FRET) from the QDs to the enzymes. The use of a given amount of GOD conjugated with

': .^') different concentrations of QDs resulted in an increase in the fluorescence intensity of the enzymes and a decrease in the fluorescence intensity of the QDs, as compared to the enzyme- conjugated QDs. The FRET is also shown in Figure 3 (b) .

:0 Example 3 : Evaluation of effects of MPA-QDs on enzyme activity

GOD, a structurally rigid glycoprotein of 160,000 Da, has a hydrodynamic radius of 43 A and consists of two identical polypeptide chains. The rigidity and ruggedness of GOD are derived from the polysaccharide that forms its outer hydrophilic envelope. Provided the electrons are transferred between the redox enzyme and the electrochemically reduced form of H₂O₂, which is generated upon the 0₂-biocatlayzed oxidation of glucose, the turnover rate of the electron exchange between the substrates (e.g., glucose, H₂O₂ or O₂) and the biocatalysts (including GOD and HRP) , as well as between the QDs and enzymes, is rapidly increased. Hence, the transduced physical energy of the QDs associated with the quenched fluorophores reflects the substrate concentration in the system.

As shown in Figure 4, the fluorescence emission of the enzymes and their activities were considerably changed after their conjugation with the MPA-QDs. After the incubation of GOD and HRP with the MPA-QDs for 3 or 12 hours, the fluorescence intensity (Excitation: 445 ran/ Emission: 525 ran) increased to about 30% or 43% of its original value, respectively. In the case where only GOD was incubated with the MPA-QDs for 3 or 12 hours, the fluorescence intensity of GOD decreased rapidly to about 20% or 41% of its original value, respectively. This means that the functional groups on the MPA-QDs surface inhibited the enzyme activities, whereas those enzymes which retained their activity received a large amount of energy from the excited QDs, leading to their fluorescence emission being stronger than that of the intact enzymes. In addition, the fluorescence emission of the MPA-QDs was decreased after a long period of incubation (data not shown) .

7) Example 4 : Measurement of glucose concentration

Glucose was measured using a mixture of MPA-QDs, GOD and HRP. The enzymes (GOD, HRP) and MPA-QDs were added to the wells of a microtiter plate which included 100 jΛ. of various concentrations of glucose solution. Mixtures of the MPA-QDs,

!0 GOD and HRP at ratios of 10:10:10, 10:5:5, and 10:5:0 (/λ£/well) were used. The microtiter plate was then immediately inserted into the measurement chamber of the plate reader, and the fluorescence intensity at an excitation/emission wavelength of

485/525 nni was measured 40 times at 3 minute intervals. i.) As described above, the direct electrical activation of enzymes, particularly redox enzymes, represents a general approach to the stimulation of the bioelectrocatalyzed oxidation (or reduction) of the enzyme substrates. Figure 5 shows the effect of the volume ratios of the MPA-QDs, GOD and

'■^>.') HRP in the glucose solutions and ratio between the enzymes and MPA-QDs on the output signals of the QD-FRET-based probes, after exposing them to various concentrations of glucose.

With the volume ratios of QDs, GOD and HRP set to 10/10/10, the fluorescence intensity changed significantly in the low range of glucose concentrations. The sensitivity

(slope value) was very high (S=-11360) in the linear concentration range of 0-1.0 g/ I with R = 0.990. With the volume ratios of QDs, GOD and HRP set to 10/10/5, 10/5/3 or

F) 10/5/0, the sensitivity or reaction efficiency of the QD-FRET- based probe decreased with decreasing amount of HRP used.

Furthermore, the fluorescence intensities in the low range of glucose concentration (0.2-1.0 g/ I ) became smaller with decreasing amount of HRP used. That is, the linear detection

10 ranges of glucose concentrations were 0-5.0 g/ I (R=O.992) for the volume ratio of 10/5/5, 0.2-5.0 g/ i (R=O.985) for the volume ratio of 10/5/3, and 1.0-5.0 g/ i (R=O.982) for the volume ratio of 10/5/0. The pathway of electron transfer from the excited QDs to the H₂O₂ reduction reaction was depressed or i F) prevented. Therefore, a decrease of the electron numbers resulted in a decrease of the turnover rate of the electron exchange, reflecting the lower photoluminescence quenching of the QDs and lower sensitivity to glucose.

The reaction time of the QD-FRET-based probes to obtain

!^0 signal stability was 30 min. This is slower than that of the other glucose sensing systems reported in the previous studies, however, it is difficult to compare them, since their fabrication and operation method are totally different. The low response time of the present probes could be due to the low diffusion of the molecules in the free environment, leading to inhomogeneity of the running of the components in the reactions. However, this application demonstrates the potential of the QDs for use as QD-FRET-based probes in the detection of glucose in solution. The low response time and stability of the mixture solution of QDs and enzymes in glucose sensing could be improved by immobilizing the QDs and enzymes in a sol-gel layer of GPTMS and APTMS.

'0 Example 5: Preparation of sensing membranes using a sol-gel method

Sensing membranes were prepared using a sol-gel method, m accordance with the method described in Korean Patent Application No. 10-2007-0050538. Specifically, a mixture (GM2)

IT) comprising GPTMS and MTES in the volume ratio of 1:2 and a mixture (GA2) comprising GPTMS and APTMS in the volume ratio of 4:1 were mixed in 99% ethanol, respectively, to give sol- gel. To the mixture solution, 35% HCl was added at the volume of 40 μt/ml. After the addition of HCl, the resulting sol-gel 0 was stored at room temperature for at least two hours prior to use in the next step.

The MPA-coated QDs of 50 jΛ synthesized in Preparation Example 2 was added to the sol-gel GM2 of 200 [Λ to give a transducer. After completely mixing the MPA-coated QDs and the sol-gel by mechanical stirring, the mixture was stored at room temperature for two hours. 5 μi of the MPA-coated QDs mixture was injected into the bottom of a 96-well microtiter plate, and then, dried at 95 ^°C for 18 hours. After the heat 5 treatment, the sol-gel GA2 was added onto the transducer, on which 40 fd of the enzyme solution (GOD: 100 unit, LOD: 1 unit, or TOD: 0.005 unit) was added to the well of 96-well microtiter plate. The enzymes were immobilized at room temperature for 18 hours.

10

Example 6: Evaluation of effects of pH and temperature

The effects of pH and temperature on the glucose sensing were investigated by using a mixture of GOD, HRP and MPA-QDs. The universal buffer used in this example contained 0.1 M i.) Na₂SO_4, 0.04 M NaOAc, 0.04 M H₃BO₃ and 0.04 M NaH₂PO₄, whose pH was adjusted with 3 N NaOH and 3 M HCl, and measured using a pH meter (Metrohm Co., Switzerland) to obtain the pH range of 4-11. 100 βl of glucose solution (1 g/ I ) prepared in universal buffer at a given pH and 20 μjt of the mixture of GOD, HRP and O MPA-QDs were introduced into a well of a microtiter plate and then the fluorescence intensity was measured. In order to investigate the interference effect of temperature on the measurements, 120 j^ή, of the reaction mixture solution at a glucose concentration of 1.0 g/ I in the well was incubated at temperatures between 23 ^°C and 37 ^°C , and the fluorescence intensity was then measured at an excitation/emission wavelength of 485/525 ran .

As shown in Figure 6, the temperature and pH had only a r> slight effect on the glucose measurement of the present invention. The change in the fluorescence intensity was different at low (i.e., 4-5) or high (i.e., 11) pH . Although the fluorescence intensity at low or high pH seems to be higher than that at neutral pH, in practice the QD

!0 nanoparticles could not be used under strong acidic or basic conditions since they became aggregated and precipitated. Based on another published paper, in which the photoluminescence intensity increased with increasing pH from 8-11.5, this pH range can be taken as the region of stability

15 of the MPA-QDs toward changes in the environment. The pH range of 6-10 is a stable environment for measurement. It is well known that increasing the temperature results in the decrease of the reaction time. Indeed, the reaction time was decreased by 60% at temperatures above 26 ^°C . However, the fluorescence 0 intensity decreased rapidly at these temperatures, due to the self-fluorescence quenching of the QDs and no significant difference in the fluorescence intensity was observed over the temperature range of 29-37 ^°C . Example 7 : Interference study- In this example, effects of various ions including NH₄ ⁺, NOj , Na^H, Cl^", COj^2" and Fe³⁺ on the glucose sensing according to the present invention were investigated. The concentration

Ti range of the ions was from 0.01 to 200 mM. 100 βl of the ion solution was added to a well of a microtiter plate and then mixed with 10 g/ i glucose to obtain a final glucose concentration of 1 g/ t . Each solution of ions at the different concentrations was prepared on a microtiter plate in

K) the form of a duplicate and control sample (without any ions) as well. Thereafter, 20 μl of the MPA-QDs were introduced into each well containing 1 g/ U glucose solution at different ion concentrations and their fluorescence intensity was then measured. Afterwards, the enzyme mixture comprising GOD and

IT) HRP (30 U and 3 U, respectively) was added to these samples and their fluorescence intensity was measured for the second time. The difference in the fluorescence intensity of the samples and control sample before and after adding the enzymes was statistically assessed by ANOVA using Instat software.

Λ) The various ions that were tested, viz. NH₄ ⁺, NO₃ ^", Na⁺, Cl^", in the concentration range of 0.01-200 mM had no significant effect on the fluorescence intensity of the MPA-QDs which were taking part in the glucose reaction. All of their p values were larger than 0.05 when comparing their fluorescence intensities with the fluorescence intensity of the control sample before and after adding enzymes to the glucose solution. For the CO₃ ^2" ions, only the concentration of 200 mM had a weak effect on the fluorescence intensity of the MPA-QDs before adding the enzymes to the glucose solution. As shown in Table 1 below, the Fe³⁺ ion had a strong effect on the fluorescence emission of the MPA-QDs in the glucose oxidation.

[Table l]

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

Before adding the enzymes to the glucose solution, the fluorescence emission of the MPA-QDs was quenched at an Fe^Jf ion concentration of 0.1 mM and absolutely quenched at higher concentrations (>0.5 mM) (see Figure 7). After adding the enzymes, the fluorescence intensity of the MPA-QDs decreased at low concentrations of Fe³⁺ (0.5 mM and 1.0 mM) . In theory, the Fe³⁺ ion acts as a mediator, which has appropriate oxidation potentials, to replace oxygen in the glucose T) oxidation. Therefore, the response of the signal after adding the enzymes was too fast and tenfold higher fluorescence intensity was observed at high concentrations of Fe³⁺.

[industrial Applicability]

H) According to the present invention, CdSe/ZnS core-shell QDs that have been coated with a hydrophilic surfactant can be conjugated with oxidases. The bioconjugates of the present invention have the property of energy transfer between neighboring molecules, and thus, can be widely used in the

\7> field of biosensors, particularly for detecting glucose over a broad range of concentration.

Claims

[CLAIMS]

[Claim l]

A bioconjugate, in which quantum dot (QD) having cadmium selenide (CdSe) core and zinc sulfide (ZnS) shell is coated T) with a hydrophilic surfactant and the coated QD is conjugated with an oxidase.

[Claim 2]

The bioconjugate of claim 1, wherein said oxidase is

K) 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.

}:^■> [Claim 3]

The bioconjugate of claim 1, wherein said bioconjugate is further conjugated with a peroxidase.

[Claim 4] iO The bioconjugate of claim 3, wherein the volume ratio of quantum dot, oxidase and peroxidase is 1:1:1.

[Claim 5]

The bioconjugate of claim 1, wherein said hydrophilic surfactant is mercaptopropionic acid (MPA) , mercaptoacetic acid (MAA) , mercaptosuccinic acid (MSA) , dithiothreitol (DTT) , glutathione, histidine or thiol-containing silane.

^'>^■ [claim 6]

A sensing membrane, onto which said bioconjugate of any one of claims 1 to 5 is immobilized.

[Claim 7]

¹O The sensing membrane of claim 6, wherein the immobilization is performed by a sol-gel method.

[Claim 8]

A biological detection kit, comprising said bioconjugate > , of claim 1 or said sensing membrane of claim 6.

[Claim 9]

The biological detection kit of claim 8, which is used for detection of a substance selected from the group ^■O consisting of monosaccharides, polysaccharides, organic acids, alcohols, cholesterol, choline, xanthine, and mixtures thereof.

[Claim lθ]

The biological detection kit of claim 8, wherein the bioconjugate or the sensing membrane is formed on a glass plate, a polystyrene plate, or a microtiter plate.

[Claim ll]

A method for biological detection, comprising the steps of;

1) injecting a biological sample to said detection kit of claim 8 to carry out a reaction; and

2) measuring fluorescence intensity emitted from said 10 reaction.

[Claim 12]

The method for biological detection of claim 11, wherein said reaction is carried out in the presence of Fe³⁺ ions . i^r>

[Claim 13]

The method for biological detection of claim 11, wherein said fluorescence intensity in step 2) is measured by an optical method or by converting it to an electrical signal. O