KR20120088202A - Biosensors for Detecting Microbes - Google Patents

Abstract

PURPOSE: A biosensor containing magnetic nanoparticles and a target-binding moiety for detecting microorganisms is provided to isolate and/or detect various targets with high efficiency. CONSTITUTION: A biosensor for detecting microorganisms contains magnetic nanoparticles and a target-binding moiety conjugated to the mangetic nanoparticles. The magnetic nanoparticles contain an amine group or a carboxylic group on the surface thereof. The target-binding moiety is an antibody to surface protein of the microorganisms. The microorganisms are marine microorganisms. The surface of the magnetic nanoparticles is coated with BSA to suppress non-specific binding of the nanoparticles to a target.

Description

Biosensors for Detecting Microbes

The present invention relates to a biosensor for detecting microorganisms comprising magnetic nanoparticles and target-binding moieties bound thereto, and a method for detecting microorganisms using the same.

In the reverse osmosis membrane process, there is a need to develop a technology for understanding, detecting, and monitoring microorganisms and organic substances that cause biological membrane contamination. If the biofilm is intensified and the operating efficiency is reduced, biofouling may occur. Biofouling can be diagnosed only by operating indicators such as changes in flux or pressure, but diagnosis at the time of biofouling has already occurred. Early warning systems are necessary because they cannot contribute to process efficiency improvements. To date, detection and analysis of biofouling has been performed with water samples, not membrane surfaces. Although the SDI (Slit Density Index) or Modified Fouling Index (MFI) is widely used as an indicator for measuring biofouling potential in water samples, it has limitations in providing accurate information on the biomass of membrane contaminants and Adenosine. Triphosphate) is also difficult to measure precisely because of the high salinity of seawater. In addition, disinfection to kill microorganisms during various pretreatment processes prior to RO membrane inflow is performed. At this time, even though the microorganisms die, the nutrients that the microorganisms can eat and grow increase. Microorganisms survived during sterilization and pretreatment will grow exponentially based on these nutrients and form biofilms on the RO membrane via RO membrane influent, although theoretically impossible. Process efficiency checks and biofouling levels can be indirectly assessed based on the plant's operating performance or the quality of the water produced, but there is no early warning effect and often exceeds the dose of biocides harmful to the organisms in the process. Because it is injected, it can cause water quality problems. Therefore, it is essential to present the target of sensor development based on understanding of biofilm formation and biofouling mechanism of reverse osmosis membrane during RO process and analysis of membrane fouling agents. There is also a need to develop a method for quickly and simply measuring the detection and monitoring of microorganisms associated with membrane fouling in a RO membrane system.

In 2003, when biosensor products began to appear in the domestic market, the Korea Biosystems, established by the Korea Institute of Science and Technology, developed BOD measuring instruments and poison detection devices by applying unique biosensor technology. In addition, iSense, founded by professors in the department of chemistry at Kwangwoon University, has introduced a portable self-glucometer that can measure blood glucose with a very small amount of blood. In May 2005, Professor Park Je-kyun of the Korea Advanced Institute of Science and Technology developed a high-sensitivity nanobiosensor source technology that can detect extremely small amounts of nanograms. In June of the same year, biochip venture venture Genopra developed a biosensor technology that can directly check E. coli contained in drinking water.In July, biosensor company Olmedikus can check blood sugar and cholesterol with a drop of blood. Developed a blood analysis chip. In 2006, the Rural Agricultural Research Institute held a technology transfer signing ceremony with Kmac, a biosensor manufacturer, to promote the commercialization of “residual pesticide detection SPR biosensor”. The developed residual pesticide detection SPR biosensors are carbamate, cabaryl, benpracarb, chiodicarb, etc., which have high detection frequency in Korea, and carbamate-based tetraazines, diazinon, cardusasa, etoproforce, parathion methyl, chlor Nine pesticides can be detected in all five kinds of organophosphates such as pyriphos. In 2007, Professor Yang-Kyu Choi of KAIST developed a biosensor that detects biomolecules using semiconductor transistors. The biosensors form nanometer-sized nanogaps in general transistor structures, It is a source technology for detecting biomolecules using the change of dielectric constant.

Biosensors / biochips are paying more attention to simplify measurement, improve real-time measurement sensitivity, improve reliability, and prolong life. In addition, problems such as weak adhesion of the thin film, severe change in sensitivity, lack of components, noise interference, specificity of analyte detection, and difficulty in miniaturization should be calculated. Therefore, there is a demand for the development of biosensors with high test stability, excellent data processing capability, various analysis capabilities, small size, easy to use, low cost, and long life. In Korea, despite the enormous growth potential of biosensors, social awareness and support are still lacking, and many parts are still imported and supplied. Therefore, it is necessary to develop technology for import substitution and foster support of domestic companies. It is understood. In particular, biochip technology is important on its own, but synergies can only be achieved when the research subjects using it, ie biological information, are fused. Already, advanced countries of biochip technology are accumulating large amount of biological information, and applying biochip technology to this accumulated information is showing rapid growth in bio industry and research. Therefore, the focus of the biochip development technology so far is mainly focused on the microarray manufacturing technology, but in the future, it is expected to be further affected by the development of peripheral technologies such as MEMS, robotics, CAD, data mining, and printing technology. do. Accordingly, active mutual exchanges between technologies are also required.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have sought to develop a method that can detect microorganisms in a sample in a relatively simple manner with higher accuracy. As a result, the inventors have found that the use of magnetic nanoparticles and target-binding moieties bound thereto, in particular magnetic nanoparticles bound to bacterial antibodies, allows detection of microorganisms (preferably marine microorganisms). By quantitatively measuring the number of the magnetic nanoparticles using a particle number measuring device, the present invention has been completed by discovering a method for detecting microorganisms more quickly and simply.

Accordingly, an object of the present invention is to provide a biosensor for detecting microorganisms.

Another object of the present invention is to provide a method for detecting microorganisms in a sample.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a biosensor for detecting microorganisms comprising magnetic nanoparticles and a target-binding moiety coupled to the magnetic nanoparticles.

The inventors have sought to develop a method for detecting microorganisms in a sample in a relatively simple manner with higher accuracy. As a result, the inventors have found that the use of magnetic nanoparticles and target-binding moieties bound thereto, in particular magnetic nanoparticles bound to bacterial antibodies, allows detection of microorganisms (preferably marine microorganisms). It has been found that the method can be carried out specifically and by detecting the microorganisms more quickly and simply by quantitatively measuring the number of the magnetic nanoparticles using a particle number measuring device.

Biofouling or biological fouling means undesirable accumulation of microorganisms, plants, algae and / or animals on highly humid structures. In particular, biofouling by microorganisms causes a great industrial problem. Therefore, there is an urgent need to develop an efficient and economical method for detecting microorganisms (eg, marine microorganisms) that cause biofouling.

According to the present invention, the biosensor for detecting a microorganism of the present invention includes magnetic nanoparticles and a target-binding moiety coupled to the magnetic nanoparticles.

According to a preferred embodiment of the present invention, a target-binding moiety having a binding affinity to a target is bound to the surface of the magnetic nanoparticles used in the present invention. The target-binding moiety is bound to the surface of the magnetic nanoparticles directly or indirectly, in a covalent or non-covalent manner.

For example, the target-binding moiety may be covalently or non-covalently directly to the surface of the magnetic nanoparticles such as ionic bonds, electrostatic bonds, hydrophobic bonds, hydrogen bonds, covalent bonds, hydrophilic bonds, or van der Waals bonds. Can be combined. In addition, the target-binding moiety may be bound to the surface of the magnetic nanoparticles indirectly through an intervening agent.

As used herein, a "target" is a substance in a sample to be separated (or separated and detected, or separated, detected and quantified), and is a bacterium, virus, cell, nucleic acid molecule (DNA or RNA), protein, peptide, antigen. , Sugars, lipids, organic compounds, inorganic compounds, metals and inorganic ions, but are not limited thereto. In addition, the sample includes, but is not limited to, biological samples, chemical samples and environmental samples. The biological sample is for example blood, plasma, serum, virus, bacteria, tissue, cells, lymph, bone marrow, saliva, milk, urine, feces, ocular fluid, semen, brain extract, spinal fluid, joint fluid, thymus, ascites, amniotic membrane Fluids, cell tissue fluids, and cell culture fluids, including but not limited to.

As used herein, the term "target-binding moiety" is a material having a specific binding ability to the target material to be separated, any material having a binding affinity to the material to be separated. It is possible. For example, the target-binding moiety may comprise antibodies, antibody fragments, antigens, peptides, aptamers (RNA, DNA and peptide aptamers), proteins, receptors, drugs, nutraceuticals, streptavidin, avidin, biotin, Lectins, ligands, agonists, antagonists, enzymes, coenzymes, inorganic ions, enzyme cofactors, carbohydrates, sugars, lipids, enzyme substrates, hapten, neutravidin, protein A, protein G, selectin Or calcium sulfate, gas binders (eg, Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru). Preferably, the target-binding moiety of the present invention may be bonded to the surface of the magnetic nanoparticles directly (eg, covalently) or indirectly (eg, ionic, coordinating, and non-covalently) to the target (preferably). Preferably, a substance having a specific affinity for the surface protein of the microorganism).

According to a preferred embodiment of the invention, the target-binding moiety of the invention is an antibody molecule, more preferably a bacterial antibody, even more preferably an E. coli antibody, most preferably an E. coli surface protein. Is an antibody against.

According to a preferred embodiment of the present invention, the surface protein of the microorganism that can be bound by the target-binding moiety of the present invention is OmpA, OmpC, OmpF, OmpG, OmpN, OmpR, OmpW, OmpX, FecA, FhuA, FhuE, HlpA , HtrE, InvE, LamB, MltA, MltB, MltC, MltD, PgpB, PhoE, PhoP, RfaY, YbfM, YbiL or YeaF proteins.

According to a preferred embodiment of the present invention, the magnetic nanoparticles used in the present invention include an amine group or a carboxyl group on the surface thereof, and more preferably include a carboxyl group.

According to a preferred embodiment of the present invention, the magnetic nanoparticles used in the present invention are directly bonded to an affinity material for the target-binding moiety on its surface. The affinity material includes various linkers and nanoparticle surface coating materials used in the art, preferably an affinity material for antibodies, more preferably protein G.

In addition, the target-binding moiety can be modified with a reactor suitable for the terminal portion of the protein to directly bind to the nanoparticle surface. For example, the magnetic nanoparticles of the present invention include an activated carboxyl group through which the direct binding to the E. coli antibody is possible.

According to a preferred embodiment of the present invention, the surface of the magnetic nanoparticles used in the present invention is coated with bovine serum albumin (BSA) to inhibit nonspecific binding of the nanoparticles to the target.

In the biosensor of the present invention, the target-binding moiety bound to the magnetic nanoparticles has a specificity for binding to microorganisms, and the magnetic nanoparticle-microbe complex formed through the binding has a change in the size of the microorganism. Induces a change in the electrical signal in the microfluidic channel through which the presence of the target microorganism can be detected. In addition, the number (or amount) of microorganisms in the sample may be quantitatively analyzed by separating and quantifying the magnetic nanoparticles bound to the microorganisms.

According to the present invention, the biosensor of the present invention exhibited a binding capacity and particle number linearly proportional to the number (or amount) of microorganisms present in the sample (see FIG. 6), and its microbial detection limit was 10 ⁵ CFU. (colony forming unit) / number of microorganisms more than mL was shown (see Fig. 7). That is, the biosensor of the present invention has specificity for a target, preferably a microorganism (more preferably a marine microorganism) via a target-binding moiety, and counts the number (or amount) of the microorganism by counting the number of magnetic nanoparticles. ) Can be analyzed quantitatively.

According to a preferred embodiment of the present invention, the magnetic nanoparticles of the present invention exhibit aggregation of the nanoparticles through binding to microorganisms. Accordingly, the complex formation between the magnetic nanoparticles and the microorganism of the present invention causes a change in the electrical signal in the microfluidic channel, thereby detecting the presence of the microorganism in the sample (see FIG. 7). Therefore, the biosensor of the present invention is a very good and effective biosensor for the detection of microorganisms.

According to another aspect of the invention, the invention provides a method for detecting microorganisms in a sample comprising the following steps:

(a) contacting the sample with the biosensor described above;

(b) separating the magnetic nanoparticle-microbial complex in the sample using magnetic; And

(c) measuring the number of magnetic nanoparticles.

The method of the present invention is basically carried out using the biosensor of the present invention described above, and the common content between the two is omitted in order to avoid excessive complexity of the present specification.

Applying a magnetic field to the sample separates the magnetic nanoparticle-target (preferably microorganism) complex from other components in the sample. The complex thus separated can be removed from other components in the sample in a variety of ways (e.g., pipetting, draining, skimming, pouring, etc.), thereby obtaining a sample concentrate consisting solely of magnetic nanoparticle-target complexes. . For example, the magnetic nanoparticles-target material complex is separated into one portion of the reaction vessel by adding and mixing the magnetic nanoparticles to the reaction vessel containing the sample and reacting the magnetic field. Subsequently, when the solution is removed by pipetting the sample while the magnetic field is applied, only the magnetic nanoparticle-target material complex remains in the reaction vessel. Then, the magnetic nanoparticle-target complex is washed and suspended in a suitable solvent to obtain a sample concentrate consisting only of the magnetic nanoparticle-target complex.

According to a preferred embodiment of the present invention, the separation method of the present invention is combined with a detection or quantification process.

Detecting the formation of magnetic nanoparticle-target complexes in a sample can be carried out according to conventional methods known in the art.

For example, in a sample with magnetic nanoparticle-target complexes, the number of magnetic nanoparticles can be measured with a particle counter to quantitatively determine the number (or quantity) of the target (preferably, microorganism). have.

In addition, when a signal generating label is bound to the magnetic nanoparticles, complex formation can be quantitatively detected by detecting a signal from the magnetic nanoparticle.

The signal generating label may be a chemical label (eg biotin), an enzyme label (eg alkaline phosphatase, peroxidase, β-galactosidase and β-glucosidase), radiolabels (eg I ¹²⁵ and C). ¹⁴ ), but not limited to fluorescent labels, luminescent labels, chemiluminescent labels, fluorescence resonance energy transfer (FRET) labels and heavy metal labels (eg, gold).

Non-limiting examples of such fluorescent labels are fluorosane, rhodamine, lucifer yellow, B-phytoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio -Cyanatostilben-2,2'-disulfonic acid, 7-diethylamino-3- (4'-isothiocyatophenyl) -4-methylcoumarin, succinimidyl-pyrenebutyrate, 4-acet Amido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives, LC ™ -Red 640, LC ™ -Red 705, Cy5, Cy5.5, lysamine, isothiocyanate, ery Throcin isothiocyanate, diethylenetriamine pentaacetate, 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidiyl-6-naphthalene Sulfonate, 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacridine, acridine orange, N- (p- (2-benzoxazoylyl) phenyl) melimide, benzoxa Diazole, stilbene, wave Ylenes and their derivatives.

Detection of the formation of the label-bound magnetic nanoparticle-target complex may be performed by a fluorometer, spectrophotometer, colorimetric detection, or radioactivity detection.

The present invention can be used for the separation, concentration and detection of specific target substances in a sample. In the present invention, by manufacturing the magnetic nanoparticles by varying the kind of target-binding moiety that binds to the target material, it is possible to design an economical system capable of separating and / or detecting various targets with high efficiency. In addition, according to the present invention, there is no need for complicated pre-enrichment, purification or processing required in the general detection process.

Because the nanoparticles of the present invention are small in size and have a very high saturation susceptibility, they are rapidly distributed in biological samples (generally aqueous solution samples) (brown diffusion increases the chance of contact with the target material in the aqueous sample). In the case of applying a magnetic field, there is an advantage of being quickly collected and separated.

Thus, the present invention has a variety of applications: First, the present invention can be used for diagnosis. Magnetic nanoparticles of the invention can be applied to biological samples to isolate / detect disease-related substances (eg, bacteria, viruses, proteins, antigens, nucleic acid molecules, carbohydrates, and lipids) in the sample. Second, the present invention can be applied to the compound production process and used for the separation of the final product. Third, the present invention can be used for water desalination. For example, magnetic nanoparticles modified with a chelating agent / complexing agent to selectively remove specific salts or ions or metals can be used in water desalination or desalination processes.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a biosensor for detecting a microorganism comprising magnetic nanoparticles and a target-binding moiety bound thereto, and a method for detecting microorganisms using the same.

(b) In the biosensor of the present invention, the production of magnetic nanoparticles with different types of target-binding moieties that bind to a target may design an economical system capable of separating and / or detecting various targets with high efficiency. Can be.

(c) In addition, according to the present invention, there is no need for complicated pre-enrichment, purification or processing procedures required for general detection procedures.

(d) Thus, the biosensors of the present invention can be effectively applied to a variety of applications, such as diagnosis of microbial-induced diseases, separation of final products, or water desalination or desalination processes.

1 is a schematic diagram showing the immobilization of E. coli antibodies on the surface of carboxyl magnetic particles and the aggregation of magnetic particles in the presence of E. coli bacteria.
2 is a result of analyzing the magnetic particle state according to each step using an optical microscope (magnification: 400X). From the first on the left, the magnetic particles before the antibody treatment group (Fig. 2a; only MBs), the non-sonication group after the antibody treatment (Fig. 2b; MB-Ab conjugation, No sonic), after the antibody treatment group (Fig. 2c) MB-Ab conjugation, sonic) and BSA blocking treatment group after antibody treatment (FIG. 2D; BSA blocking).
3 is a result of investigating the selectivity according to the type of bacteria using an optical microscope (magnification: 400X). From the first photo on the left, no bacteria treated group (Fig. 3a; No bacteria), BL21 treated group (Fig. 3b; BL21_1 × 10 ⁵ / ml), DH5A treated group (Fig. 3c; DH5A_1 × 10 ⁵ / ml) and SA treated group (FIG. 3D; SA_1 × 10 ⁵ / ml).
4 is a result of observing the selectivity according to the type of bacteria through fluorescence using a confocal microscope (magnification: 600X). In the group treated with DH5A (FIG. 4A; DH5A_1 × 10 ⁵ / ml) and the group treated with SA (FIG. 4B; SA_1 × 10 ⁵ / ml), fluorescence (left panel) and optical microscopy (by confocal microscopy) Right panel). It has been found that a technique for surface modification of magnetic nanoparticles has been developed that can minimize nonspecific reactions and induce effective conjugate-bacterial responses.
FIG. 5 is a schematic diagram illustrating a bacterial detection method using magnetic nanoparticles immobilized with protein G and a particle counter. FIG.
Figure 6 is a graph showing the linearity test results according to the bacterial concentration using the particle count meter.
7 is a result of measuring the linearity and the detection limit according to the concentration of bacteria using a particle count meter.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

Capboxyl group magnetic nanoparticles were purchased from invitrogen (Carlsbad, CA, USA). E. coli antibodies were purchased from Fitzgerald (Concord, MA, USA), micromagnetic particles were purchased from invitrogen (Carlsbad, CA, USA), and fluorescent magnetic nanoparticle- or protein G-labeled magnetic nanoparticles were obtained from Chemicell ( Berlin, Germany). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and bovine serum albumin (BSA) were purchased from sigma-aldrich (CA, USA).

Example 1 Preparation of Antibody Immobilized Magnetic Nanoparticles That Minimize Nonspecific Responses and Induce Effective Bacterial Responses

a. Immobilization of Antibodies on Carboxyl Magnetic Nanoparticles

The next method uses carboxyl-activated magnetic nanoparticles on the surface to induce aggregation in the presence of specific bacteria. More preferably, the BSA is blocked by immobilizing specific antibodies on the surface of the magnetic nanoparticles and preventing non-specific reactions so as to induce an effective magnetic nanoparticle-bacterial reaction. The specific experimental sequence is as follows (FIG. 1). First, 1 μm-sized magnetic beads (80 μl) having 10 mg / mL carboxyl group activated were prepared and washed three times using deionized water (DI water). In the method of washing magnetic nanoparticles, first, deionized water was added and then mixed evenly using voltex, and reacted with a magnet for 3 minutes. Then, the magnetic nanoparticles can be observed to be attracted in the direction of the magnet, at which time the supernatant was carefully removed while leaving the pellet using a pipette. After washing three times in the same manner, unnecessary materials contained in the magnetic particle solution were removed as much as possible. Next, the inventors activated the carboxyl group of the magnetic beads in the previous step for immobilizing the antibody. The carboxyl activation method using EDC is as follows. 200 μl of EDC solution (20 mg / ml, DI water) was reacted with carboxyl magnetic nanoparticles for 30 minutes at 4 ° C. using a rotator. After the reaction, the supernatant was removed by placing in a magnet for 5 minutes. After washing with 300 μl of cold deionized water as described above, 25 mM MES at pH 6 was rapidly treated to prevent hydrolysis of the activated carboxyl groups.

We then immobilized E. coli bacterial antibodies reactive to all antigenic serotypes of O and K of E. coli to detect specific bacteria on activated carboxyl magnetic nanoparticles using EDC. The present inventors conducted the experiment as follows to immobilize the E. coli antibody. First, the antibody was dissolved in 60 μl MES and mixed with the activated magnetic beads to bind the carboxyl-activated magnetic beads and the antibody, and then 40 μl of MES was added to adjust the final dose to 100 μl. After reacting at 4 ° C. for 2 hours, antibodies and residues that did not react with the magnetic particles were removed using a magnet. In the case of the amine group present in the antibody, it reacts with the activated carboxyl group to form an amide bond. Since such amide bond does not easily fall into a very strong bond, the magnetic nanoparticle and the antibody may be strongly bound. Next, a blocking operation was performed to prevent nonspecific adsorption of bacteria. First, PBS was used as a blocking buffer, and 0.5% BSA was added as a protein for blocking to react for 12 hours at 4 ° C. In the case of BSA, antibodies are immobilized by nonspecific binding to some surfaces of unsecured magnetic nanoparticles. As described above, in the case of magnetic nanoparticles to which E. coli antibodies are immobilized, the size of aggregates increases due to the binding between the E. coli bacteria and the magnetic nanoparticles of the present invention, and other bacteria other than E. coli. It did not increase in size because there was no reaction in presence.

b. Magnetic Nanoparticle Observation Using Optical Microscope

Figure 2 shows an optical microscope image according to the manufacturing step of the magnetic nanoparticles described above. Figure 2a is a magnetic nanoparticle image washed using only distilled water (DW) without processing anything, wherein it was confirmed that the magnetic nanoparticles are well dispersed alone without aggregation (aggregation). Figure 2b is an image when the immobilization of the E. coli antibody to the magnetic nanoparticles was not subjected to sonication, it can be seen that the magnetic particles remain in the aggregate form after the reaction with the antibody. Nonspecific binding between magnetic nanoparticles after antibody reaction is a form of aggregation that we do not want. Thus, we implemented sonication to block aggregation between beads through unwanted nonspecific binding. The Sony process is as follows. First, put the reaction tube into a sponge and put it in an ultrasonic crusher (sonicator, HWASHIN TECH.-power sonic 505) and set it to 'M' in the ultrasonic intensity controller (divided by L, M or H), and then for 5 minutes. Sony was allowed. Figure 2c is a result of the sonication after the reaction of the E. coli antibody to the magnetic nanoparticles, showing the dispersed form of the magnetic nanoparticles were aggregated. 2D is a group subjected to BSA blocking after antibody immobilization, and it was confirmed that nonspecific binding between the magnetic nanoparticles does not occur even without the sonication process.

Example 2: Selectivity of Antibody-Immobilized Magnetic Nanoparticle Conjugates Against Bacteria

a. Bacterial Preparation and Antibody-Immobilized Magnetic Nanoparticle Reactions

As can be seen in the above-described examples, the inventors of the ball confirmed that the antibody can be immobilized on the surface of the magnetic nanoparticles, and when BSA blocking is performed, it is possible to inhibit nonspecific binding between the magnetic nanoparticles. Next, the inventors examined whether the magnetic nanoparticles to which the E. coli antibody was immobilized selectively bind to E. coli bacteria to form aggregates. Two different bacterial species, E. coli and Staphylococcus aureus , were used as targets. More specifically, bacterial samples of various concentrations were prepared. Bacterial samples were prepared by the following procedure. BL21, DH5α and S. aureus , stored frozen at −80 ° C., were inoculated in 10 mL LB medium in 15 mL tubes using pipettes at cell stocks. Inoculated LB medium was incubated for 12 hours at a speed of 200 rpm in a 37 ° C incubator (HANBAEK SCIENTIFIC CO.-HB 201SF). The next day, E. coli in cultured LB medium was grown to OD = 0.6-0.8 to confirm that the total number of bacteria was about 6.0 × 10 ⁸ cells / mL. Centrifugation at 4,000 g for 20 minutes to remove all supernatants except precipitated cells, and suspended the cells in PBS buffer. Suspended 10 ⁵ CFU / mL (PBS) bacteria were prepared in 1.5 mL E-tubes, and the magnetic nanoparticle-antibody conjugate was added, followed by shaking voltex (JIOO TECH.-VM 96B) at room temperature. The reaction was carried out for 1 hour. Then, in order to separate the reacted conjugate, the sample is placed on the magnet, and the reacted particles are collected in the magnet, and the pipette removes the unreacted particles, thereby removing the bacteria that do not react with the magnetic nanoparticles. gave. Finally, the presence or absence of aggregation was confirmed by using an optical microscope.

 b. Confirmation of Cohesion Using Optical Microscope

3 is a result of observing particles using an optical microscope. As shown in FIG. 3, in the group not treated with bacteria (FIG. 3A), it was confirmed that the magnetic nanoparticles were not dispersed but were evenly dispersed. However, in the group treated with BL21 (FIG. 3B), one of E. coli species, significant aggregation of magnetic nanoparticles was observed. Similarly, magnetic nanoparticles were agglomerated even when DH5a, another E. coli species, was treated (FIG. 3C). This aggregation is predicted to be caused by specific binding between E. coli antibodies located on the magnetic nanoparticle surface and receptors on the E. coli surface. Figure 3d is a group treated with bacteria of Staphylococcus aureus species but not E. coli as a control, the phenomenon of agglomeration of magnetic nanoparticles was not observed. As expected, since the E. coli antibody located on the surface of the magnetic nanoparticles does not specifically bind to Staphylococcus aureus, the magnetic nanoparticles are not aggregated but remain dispersed.

As described above, qualitative analysis was possible to determine the presence or absence of E. coli , and further experiments were performed for quantitative analysis of the amount of E. coli .

c. Selective Aggregation Between Fluorescent Magnetic Nanoparticles and Bacteria Using Confocal Microscopy

To more accurately identify the phenomenon of aggregation between the beads and certain bacteria, the inventors used fluorescent magnetic nanoparticles. First, 10 μl of 1 μm fluorescent magnetic nanoparticles having 50 mg / mL carboxyl group activated was prepared, washed twice with 300 μl of 0.1 M carbonate buffer, and finally, 200 μl of deionized water was used. Additional washing. The washing procedure was the same as described above. Next, the inventors activated the carboxyl group of the magnetic beads in the previous step for immobilizing the antibody. 100 μl of 400 mM EDC and 100 mM NHS were treated to the prepared fluorescent magnetic nanoparticles, respectively, and then reacted with a rotator at room temperature for 30 minutes. After the incubation process, the supernatant was removed by leaving it in the magnet for 5 minutes. 300 μl of cold deionized water was washed in the same manner, followed by 20 μl of PBS. Using this method, we converted the carboxyl group to the NHS ester form, which is a highly reactive form of the amine group. Next, the E. coli antibody was immobilized on the surface of the carboxyl fluorescent magnetic nanoparticles. 20 μl of E. coli antibody (5 mg / mL) was treated to the activated carboxyl fluorescent magnetic nanoparticles, and then reacted at 4 ° C. for at least 8 hours in a shaking incubator. After the reaction, the mixture was washed three times with 200 µl PBS and blocked with 0.1% BSA. Then, washed twice with 200 μl of PBS buffer and finally stored at 4 ° C.

Next, an experiment was conducted to investigate whether the magnetic nanoparticles to which the E. coli antibody was immobilized selectively bind and aggregate only to E. coli bacteria. At this time, Staphylococcus aureus was used as a control group DH5a and one of E. coli species. As described above, the concentration of bacteria was prepared at 10 ⁵ CFU / mL (PBS). Bacteria were prepared in 1.5 mL E-tubes and the magnetic nanoparticle-antibody conjugates prepared above were reacted for one hour using a vortex (shaking voltex) at room temperature. After the reaction, when the sample is placed on the magnet for particle separation, the reacted nanoparticles are collected on the magnet and the pipette is used to remove the unreacted nanoparticles. It was. Finally, it was observed by confocal microscopy to confirm the presence or absence of aggregation. As can be seen in Figure 4, one of the E. coli species DH5a (Fig. 4a) was able to observe the aggregation phenomenon and the fluorescence was detected in the aggregated nanoparticles. This means that specific binding between the magnetic nanoparticles and E. coli occurred, not the nonspecific binding between the magnetic nanoparticles. On the contrary, in the case of Staphylococcus aureus, the aggregation phenomenon could not be observed, and since there was no specific reaction as in the optical microscope result of FIG. 3d, it was confirmed that the magnetic nanoparticles were observed in the dispersed form ( 4b).

Example 3: Bacteria Detection Using Antibody-Immobilized Magnetic Nanoparticles

In order to induce aggregation in the presence of certain bacteria, magnetic nanoparticles immobilized with protein G were used. In the case of protein G, the specific binding to the Fc region of the antibody serves to induce the proper introduction of the immobilized antibody. More precisely, in order to induce an effective magnetic nanoparticle conjugate-bacterial reaction, the inventors blocked the antibody to immobilize specific antibodies on the surface of the magnetic nanoparticles and prevent nonspecific reactions. The specific experimental sequence is as follows. According to FIG. 5, 40 μl of magnetic nanoparticles immobilized with 10 mg / mL Protein G were washed twice with 200 μl sodium phosphate buffer. The washing method of the magnetic nanoparticles was mixed with the buffer and then evenly mixed with vortex. After that, it was left on the magnet for 5 minutes. As a result, the magnetic nanoparticles were attracted in the direction of the magnet, and the supernatant except the pellets formed around the magnet was carefully removed with a pipette. The same procedure was repeated twice to remove unnecessary materials in the magnetic nanoparticle solution. To detect specific bacteria, E. coli bacterial antibodies were immobilized on magnetic nanoparticles. The present inventors performed the immobilization of E. coli antibody as follows. First, 20 μl of a 5 mg / mL E. coli antibody sample was treated to bind the antibody onto the magnetic nanoparticle surface to which Protein G was immobilized, and reacted in sodium phosphate buffer using vortex at room temperature for 30 minutes. Then, washed with PBS and finally stored at 4 ℃.

In the next step, bacterial samples of various concentrations were prepared. The bacterial sample preparation procedure was as follows. DH5a lyophilized at −80 ° C. was inoculated into 10 mL LB medium in a 15 mL tube using a pipette in a cell stock. Inoculated LB medium was incubated overnight at a speed of 200 rpm in a 37 ℃ incubator. The next day, E. coli in cultured LB medium was grown to OD = 0.6-0.8 to confirm that the total number of bacteria was about 6.0 × 10 ⁸ cells / mL. Centrifugation at 4,000 g for 20 minutes to remove all supernatants except precipitated cells, and suspended the cells in PBS buffer. Bacteria at various concentrations (10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ CFU / mL in PBS) were prepared in 1.5 mL E-tubes and the magnetic nanoparticle-antibody conjugate was added to room temperature. Vortex was used for 1 hour to induce an antibody-antigen reaction between the magnetic nanoparticle-antibody conjugate and bacteria. After an hour, the reaction is placed in a magnet for 5 minutes, and bacteria (1-3 μm) reacted with the magnetic nanoparticles are attracted to the magnet. In order to isolate the reacted bacteria, the pipette was carefully removed twice with the supernatant containing the unreacted bacteria. Residual particles were diluted in sodium phosphate buffer at a rate and measured quantitatively using a particle counter (BECKMAN COULTER ^™ -Z2).

FIG. 6 shows the results of quantitative analysis of DH5a, which is a type of E. coli , by using a particle size meter using the above-described method. The particle size meter is filled with 10 mL of an electrolyte solution in a cuvette to which the sensor is attached using an electrical resistance method, and the sensor analyzes 100 μl of a diluted solution containing magnetic nanoparticles. In addition, particle size analysis is possible by three times the specified minimum magnification. In other words, for a minimum magnification of 1 μm, the maximum possible magnification is 1-3 μm. As can be seen in Figure 6, the concentration of DH5a after the reaction was terminated at 10 ⁶ , 10 ⁵ , 10 ⁴ CFU / mL, respectively, it was confirmed that the number of magnetic nanoparticles decreased linearly with the decrease in bacterial concentration.

7 is a result of measuring the linearity and the detection limit according to the bacterial concentration using a particle count meter. As described above, bacteria at various concentrations (10 ⁸ CFU / mL-10 ⁵ CFU / mL) were reacted with the magnetic nanoparticle-antibody conjugates. As a result, as the concentration of bacteria decreased, the number of magnetic nanoparticles also decreased linearly, and the size of the magnetic nanoparticles was about 1-2 μm. In addition, considering the number of magnetic nanoparticles (about 7420) measured in the group not treated with bacteria, the detection limit for bacteria could be determined to be about 10 ⁵ CFU / mL.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A biosensor for detecting microorganisms comprising magnetic nanoparticles and a target-binding moiety coupled to the magnetic nanoparticles.
The biosensor of claim 1, wherein the magnetic nanoparticles include an amine group or a carboxyl group on a surface thereof.
The biosensor of claim 2, wherein the magnetic nanoparticles include a carboxyl group on a surface thereof.
The biosensor of claim 1, wherein the magnetic nanoparticles are bound to an affinity material for the target-binding moiety.
5. The biosensor of claim 4, wherein the affinity material is Protein G. 6.
The biosensor of claim 1, wherein the target-binding moiety is an antibody against a surface protein of a microorganism.
The biosensor of claim 1, wherein the microorganism is a marine microorganism.
The biosensor of claim 1, wherein the biosensor forms a magnetic nanoparticle-microbe complex by combining with the microorganism.
The biosensor of claim 1, wherein the limit of detection of the microorganisms of the biosensor is 10 ⁵ CFU (colony forming unit) / mL or more.
The biosensor of claim 1, wherein the surface of the magnetic nanoparticles is coated with bovine serum albumin (BSA) to inhibit nonspecific binding of the nanoparticles to a target.
(a) contacting the biosensor of any one of claims 1 to 10 to a sample; (b) separating the magnetic nanoparticle-microbial complex in the sample using magnetic; And (c) measuring the number of magnetic nanoparticles.
The method of claim 11, wherein the sample is a biological sample, a chemical sample, or an environmental sample.
12. The method of claim 11, wherein the number of magnetic nanoparticles is measured using a particle counter.

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