KR101514321B1 - Methods of preparing biosensor for multiplexing detection system by using anisotropic biohybrid microparticles and their applications - Google Patents

Abstract

The present invention relates to a biosensor using anisotropic biohybrid microparticles. More particularly, the present invention relates to a biosensor using an anisotropic microhybrid microparticle, which is physically and chemically compartmentalized, And more particularly, to a biosensor capable of multiple detection and targeting according to the biosensor.

Description

FIELD OF THE INVENTION The present invention relates to a biosensor for multiple detection using anisotropic biohybrid microparticles and a method of manufacturing the biosensor for multiple detection using anisotropic biohybrid microparticles,

The present invention relates to a biosensor using anisotropic biohybrid microparticles, and more particularly, to a biosensor using an anisotropic microparticle structure, which is physically and chemically compartmentalized, to selectively orient an antigen- The present invention relates to a biosensor capable of multiple detection and targeting according to two or more functional groups.

Micro- or nano-sized particles are widely used in biosensors based on physical chemistry. The combination of micro- or nano-sized particles and biomolecules is of interest in biotechnology. Because of its excellent biocompatibility and specific surface area, it plays an important role in improving the biosensor's role, and its smaller surface gravitational surface curvature helps to preserve the original protein structure function compared to larger particles to be.

However, it has not been possible to implement a system that selectively detects multiple substances with conventional isotropic micro or nanoparticle structures.

In other words, in the case of isotropic micro or nanoparticle structures, the detectable moiety is limited to only one, so that multiple bio-sensing, multi-bioimaging, multi-functional drug delivery system) and biomedical devices for multiple detection of biological substances.

Recently, nanostructures with anisotropic segments in the nano-scale have been produced as advanced materials with multi-functional properties possessed by each segment. The two compartments may each be composed of a single polymer, a mixture of two polymers, or an entirely different polymer. A variety of polymers with inherent chemical properties are combined to produce anisotropic nanoparticles and nanofibres for a variety of industrial and biomedical applications. In particular, a variety of drug-therapeutic molecular and inorganic nanoparticles can be introduced into each compartment. Such nano-sized anisotropic structures can potentially be applied to various drug delivery systems, tissue engineering supports, switchable surfaces, and sensors having different drug release mechanisms from each compartment.

Since the anisotropic structure has multiple compartments, it can be used as a material for a switchable image display, a colloidal stabilizer at a contact, a self-propelled motor, an optical sensor, and the like. On the other hand, there have been few studies on anisotropic structures with micro- or nano-scale activation, and such structures can be used as biomedical materials such as tissue engineering, regenerative medicine, and drug delivery.

Accordingly, the present inventors have found that, by using selective orientation properties of an anisotropic biohybrid microparticles structure compartmentalized by physico-chemical compartmentalization, it is possible to selectively detect multiple targets and two or more biomolecules depending on two or more functional groups, A novel concept of a biosensor based on the optical fluorescence capable of being produced by the method of the present invention.

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It is an object of the present invention to provide a biosensor for multiple detection that includes an anisotropic biohybrid microparticles structure that is physically and chemically compartmentalized.

It is another object of the present invention to provide a method for multiplexing biomaterials using the confocal laser scanning microscope (CLSM).

In order to solve the above problems,

An anisotropic biohybrid microparticles structure that is physically and chemically compartmentalized,

Wherein each of the sections of the anisotropic biohybrid microparticles has different types of antibodies introduced therein and has selective orientation according to a different antigen-antibody reaction.

The anisotropic biohybrid microparticles are characterized in that they are composed of copolymer polymers having cross-linking. For example, they form cross-links through chemical cross-linking, UV light cross-linking, thermal cross-linking, can do. In one embodiment of the present invention, thermal crosslinking was used.

At this time, the copolymer polymer may be a poly (acrylamide-co-acrylic acid), a poly (acrylamide-co-acrylic acid), a poly (N-isopropylacrylamide- (N-isopropyl acrylamide-co-stearyl acrylate), poly (NIPAm-co-SA), poly (N-isopropylacrylamide-co and poly (NIPAM-co-AA) having an acrylic moiety, such as poly (NIPAM-co-AA) (Acrylamide-co-acrylic acid, poly (AAm-co-AA)] was used.

The physically and chemically compartmentalized anisotropic biohybrid microparticles of the present invention can be prepared by an electrohydrodynamic co-jetting method.

Particularly, the anisotropic biohybrid microparticles of the present invention are characterized in that, for multiple detection of biomaterials, different functional groups for introducing different kinds of antibodies are introduced into each compartment. That is, the different types of antibodies are introduced into the respective sections of the anisotropic biohybrid microparticles by the different functional groups to be used as a biosensor for multiple detection.

At this time, the different functional groups are composed of an amine, a carboxylic acid, a thiol, a hydroxyl group, acetylene, an azide and other functional groups Lt; / RTI > A homobifunctional crosslinker group or a heterobifunctional crosslinker group may be selected to introduce different kinds of antibodies by the different functional groups. In homobifunctional crosslinkers, 3, 3-dithiobis (sulfosuccinimidylpropionate (DTSSP), bis (sulfosuccinimidyl) suberate (BS), disulfosuccinimidyl tartarate (Sulfo-DST), disuccinimidyl glutarate (DSG), N, N disuccinimidyl carbonate Succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and dimethylsulfoxide (DSC), dimethyl adipimidate dihydrochloride (DMA), and bismaleimidohexane (BMH) N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (Sulfo-NHS-ASA), and the like, but not limited to, 4- (4-N-maleimidophenyl) bytyric acid hydrazide hydrochloride (MPBH)

In the specific examples of the present invention, maleimide and acetylene functional groups were used for the different functional groups, maleimide and acetylene functional groups were substituted for thiol (Polyclonal Antibody) thiol) and azide functional groups to introduce different types of antibodies into each compartment.

Preferably, the anisotropic biohybrid microparticles further include a fluorescence probe so that when two or more biomaterials are multiplexed using the antibodies, the results can be visually confirmed.

For example, the biosensor of the present invention may comprise

Two or more types of monoclonal antibodies immobilized on a substrate; antigen; And an anisotropic biohybrid microparticle structure in which a polyclonal antibody is introduced for each compartment.

In other words, a sandwich-type directional binding of an anisotropic microparticle in which a chip-immobilized monoclonal antibody-antigen-polyclonal antibody is introduced, As shown in FIG.

At this time, it is preferable that the anisotropic biohybrid microparticle structure further contains a fluorescent dye. In an embodiment of the present invention, glass patterned with PDMS (polydimethylsiloxane) is used.

The present invention also relates to a method for detecting a multiplexed biomaterial using the biosensor, characterized by using a method of imaging a fluorescence signal by adjusting a height of a focus using a confocal laser scanning microscope (CLSM) .

In particular, the biosensor of the present invention is advantageous in that it can perform selective multiplex detection and targeting according to two or more functional groups using selective orientation.

The present invention relates to a fluorescence-based biosensor for multi-biomolecule detection using anisotropic biohybrid microparticles and its application, and relates to an anisotropic biohybrid microparticle having two or more functional groups simultaneously , It is possible to introduce two different antibodies into the compartmentalized surface, respectively, so that it is useful for multiplex detection of biomaterials.

FIG. 1 shows a polymer P (AAm-co-AA) structural formula and 1H NMR spectrum of a polymer introduced with maleimide (acetylene) using EDC.
2 is a dual nozzle geometry electrophysiologic co-injector for producing the anisotropic biohybrid microparticles of the present invention; And an anisotropic biohybrid microparticle in which two different polyclonal antibodies were introduced into each compartment.
FIG. 3 is a schematic view showing an image (a) of a chip in which PDMS patterned in a cover glass is coupled by a plasma technique, and an image of a patterned patterned layer after stacking metal and gold layers on a substrate b).
4 is a schematic view illustrating a process of manufacturing the biosensor of the present invention. Based on the characteristics of anisotropic biohybrid microparticles having two or more functional groups simultaneously, two different antibodies are introduced into the compartmentalized surface to detect multiple biomaterials. Specifically, a single clone immobilized on a chip This is a schematic diagram of multiple detection of a biomaterial through a sandwich-type directional binding of an anisotropic microparticle into which a monoclonal antibody-antigen-polyclonal antibody is introduced.
FIG. 5 is a graph showing the relationship between the activity of the isomeric microparticles (AC) and the (GI) azide-introduced particles and the (DF) maleimide and (JL) It is a mimetic diagram and a CLSM and Differential Interference Contrast (DIC) image as to whether a functional group is introduced precisely into a particle using a substance and whether the functional group reacts accurately.
FIG. 6 is a mimetic diagram and a CLSM showing whether an antibody is introduced into an isotropic microparticle and binds to a glass surface depending on whether an antigen is used or not. It is an image demonstrating the selective binding of a sandwich type of anisotropic microparticles in which a monoclonal antibody - an antigen - polyclonal antibody, immobilized on a chip according to presence or absence of an antigen, is introduced.
FIG. 7 is an image showing an anisotropic microparticle prepared by electrohydrodynamic co-injection using a CLSM (AC) dried state and a (DI) hydrated state.
FIG. 8 is a photograph showing an image obtained by observing a directional bond by adjusting a height of a focal point from below by introducing a polyclonal antibody into only one surface of an anisotropic microparticle to the biosensor of the present invention and using CLSM. Specifically, a sandwich-type directional binding of an anisotropic microparticle into which a monoclonal antibody immobilized on a chip-an antigen-polyclonal antibody is introduced, It is an image showing that multiple detection is possible.
FIGS. 9 and 1010 are CLSM images showing in detail the binding selectively by introducing polyclonal antibodies into the green and red portions of the anisotropic microparticles in FIG. From left to right is a photograph showing the channels of fluorescein isothiocyanate (FITC channel), rhodamine B isothiocyanate (RITC channel), and FITC and RITC channels.
FIG. 11 is a photograph showing images observed by adjusting the height of the focal point from the bottom to the top by using CLSM as to whether the biosensor of the present invention can selectively bind polyclonal antibodies that are different on both sides of the anisotropic microparticles. to be.
FIGS. 12 and 13 are images specifically showing whether the anchored microparticles are selectively bound by introducing different polyclonal antibodies on both sides in FIG. From left to right is a photograph showing the channels of fluorescein isothiocyanate (FITC channel), rhodamine B isothiocyanate (RITC channel), and FITC and RITC channels.
14 is a schematic diagram of a method for producing a fluorescent basic biosensor for detecting multiple biomaterials using anisotropic biohybrid microparticles in which different polyclonal antibodies are introduced into each compartment.

The terms used in the present invention are defined as follows.

"Nanoparticles are particles in the size range of 10 to 1000 nm, and" microparticles "are particles in the size of 1 to 1000 μm.

"Anisotropic" means that the properties vary depending on the direction, and isotropic means that the properties are the same for all directions.

"Hydrogel" is a water-swellable polymer having a three-dimensional network structure through cross-linking between molecular chains. It is a polymer complex that can contain a large amount of water and is insoluble in water. Hydrogels exhibit excellent absorbency due to the presence of hydrophilic groups in the polymer chain, and through their cross-linking, the absorbed water can be present between the chains. This absorption system is a combination of chemical action and physical action As shown in FIG.

A "biosensor" is a system that uses biologic elements to obtain information from a measurement object or converts them into useful signals that can be recognized by mimicking biological elements. In other words, it is a device that selectively detects a substance to be analyzed by converting it into a recognizable signal which is composed of a bio-receptor and a signal transducer.

Examples of the " biosensor "include enzymes, antibodies, antigens, and hormone receptors that can selectively react with and bind to a specific substance. Examples of the" signal conversion method "include electrochemistry, fluorescence, resonance, field effect transistor (FET), quartz crystal microbalance (QCM), and thermal sensor.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition. Such detection labels may be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes, but is not necessarily limited thereto.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have used anisotropic biohybrid microparticles that are physically and chemically compartmentalized to introduce different antibodies by different functional groups in each compartment so that a selective orientation for antigen- The first multi-detection biosensor was developed.

Accordingly, the present invention provides, in one aspect,

An anisotropic biohybrid microparticles structure that is physically and chemically compartmentalized,

Wherein each of the sections of the anisotropic biohybrid microparticles has different types of antibodies introduced therein and has selective orientation according to different antigen-antibody reactions.

The anisotropic biohybrid particles on which the biosensor of the present invention is based are micro and nano-sized anisotropic polymer structures having multi-functional characteristics possessed by the respective compartments. Each compartment may be composed of a single polymer, a mixture of two polymers, or an entirely different polymer, and may have different physical and chemical properties at the same time in each compartment.

Techniques for the synthesis of anisotropic microstructures or nanostructures in the form of particles can be made using known techniques: electrohydrodynamic co-jetting, microfluidics, differential solvent evaporation, , Flow focusing lithography, spinning disks, selective deposition, partial deformation by masking, picking emulsion using powder particles, and self-assembly. In particular, one embodiment of the present invention produced anisotropic microparticles using electrohydrodynamic co-jetting.

Microparticles or nanoparticles, consisting of two compartments with different optical, electrical or magnetic properties within each compartment, have the potential to dynamically switch in response to light, electricity, and magnetic fields, Can be used.

Since the anisotropic biohybrid particles of the present invention are also physically chemically compartmentalized, different functional groups are introduced into the respective compartments, and different antibodies are bound to the surfaces of the compartments, whereby a biosensor As a base material.

As an example, the anisotropic microparticle structure of the present invention can be prepared by the following method:

(i) introducing a crosslinkable polymer and necessary functional groups;

(ii) preparing an anisotropic structure through an electrohydrodynamic co-jetting method to form a crosslinking; And

(iii) binding the antibody to the anisotropic structure.

The polymer constituting the anisotropic biohybrid particle is a copolymer polymer having thermal crosslinking, for example, poly (acrylamide-co-acrylic acid, poly (AAm -co-AA), poly (N-isopropyl acrylamide-co-stearyl acrylate), poly (NIPAm-co-SA) Poly (NIPAM-co-AA) having poly (N-isopropylacrylamide-co-allylamine), poly (NIPAM-co- AA), and acrylic moiety, But is not limited thereto. In one embodiment of the present invention, poly (acrylamide-co-acrylate) [poly (AAm-co-AA)] was used.

On the other hand, in order to bind an antibody for multiple detection of a biomaterial to the anisotropic biohybrid particle, necessary functional groups are introduced together at the time of producing the anisotropic biohybrid particle.

In one embodiment of the present invention, maleimide or acetylene functional groups are introduced by using [1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide] or EDC in poly (AAm-co-AA).

Two or more types of functional groups are introduced to bind the multiple antigens required for the compartmentalized surface of the anisotropic biohybrid particles of the present invention. Such functional groups can be appropriately selected and used by those skilled in the art depending on the antibody to be introduced later.

That is, the different types of antibodies are introduced into the respective sections of the anisotropic biohybrid microparticles by the different functional groups to be used as a biosensor for multiple detection.

The different functional groups may be, for example, an amine, a carboxylic acid, a thiol, a hydroxyl group, an acetylene, an azide, Functional groups, and the like. A homobifunctional crosslinker group or a heterobifunctional crosslinker group may be selected to introduce different kinds of antibodies by the different functional groups. In homobifunctional crosslinkers, 3, 3-dithiobis (sulfosuccinimidylpropionate (DTSSP), bis (sulfosuccinimidyl) suberate (BS), disulfosuccinimidyl tartarate (Sulfo-DST), disuccinimidyl glutarate (DSG), N, N disuccinimidyl carbonate Succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and dimethylsulfoxide (DSC), dimethyl adipimidate dihydrochloride (DMA), and bismaleimidohexane (BMH) N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (Sulfo-NHS-ASA), and the like, but not limited to, 4- (4-N-maleimidophenyl) bytyric acid hydrazide hydrochloride (MPBH)

Then, an anisotropic microparticle structure physically and chemically compartmentalized by an electrohydrodynamic co-jetting method is formed. A schematic diagram thereof is shown in Fig.

The electrohydrostatic co-injection is used to produce multiple compartments of nanofibers and nanoparticles from various polymer solutions through control of process parameters. Although these multi-compartment nanostructures are used as two mixed polymer solutions, they have different chemical properties within each compartment. Importantly, the viscosity, surface tension and electrical conductivity of other polymer solutions can be matched to each other to maintain a separate contact at the Taylor cone of the electrohydrostatic co-injection, which results in the formation of other multiple compartment anisotropic nanostructures ≪ / RTI > in large quantities.

An anisotropic microparticle is prepared by electrohydrostatic cavity injection as described above, and is crosslinked.

"Cross-linking" of a water-soluble polymer is very important in imparting resistance to being dissolved when exposed to water. The internal molecular cross-linking of the polymer chains helps these nanoparticles maintain their original structure in a substantially solubilized environment, such as aqueous conditions. This also helps the nanoparticles to act as a soft hydrogel containing a significant amount of water. Hydrogels composed of biodegradable polymers are widely used for tissue repair and drug delivery carriers.

Crosslinking of hydrogels can be accomplished through a variety of mechanisms including physical, chemical, and photo initiative cross-linking. Physically or chemically cross-linked nanostructures can be used in stable and aqueous environments. In particular, the production of multiple compartments of hydrogel nanoparticles and nanofibers depends on the rheological compatibility and dynamic order of the other polymer solutions under externally applied electric fields during electrohydrostatic co-injection. The maintenance of the hydrodynamic properties of the polymer solution during electrohydraulic co-injection can be the most critical parameter for producing homogeneous multi-compartment nanoparticles and nanofibers. Chemical cross-linking within the nanofibers requires a cross-linking agent to be added to the polymer solution, which can interfere with rheological properties during the electrohydrostatic co-injection process. However, physical crosslinking based on electrostatic and hydrophobic interactions creates rheological characteristic constants through electrohydraulic co-injection without crosslinking agents.

In particular, one embodiment of the present invention uses thermally crosslinked polymers.

Next, two or more different antibodies are respectively introduced into the segmented surface of the anisotropic biohybrid particle.

At this time, considering the functional group introduced into the anisotropic biohybrid particle, a functional group capable of binding with the required antibody can be introduced into each antibody.

In one embodiment of the present invention, maleimide and acetylene functional groups are introduced into each compartment, and thiol and azide functional groups are introduced into a polyclonal antibody, Two different antibodies were bound to the compartmentalized surface (Figure 2).

That is, in the present invention, the anisotropic biohybrid particles are characterized by having the structure of a structure in which different antibodies are bound to physiochemically partitioned surfaces.

At this time, the size of the anisotropic particles is not particularly limited, but is preferably 0.2 to 20 micrometers. When the particle size is more than 20 micrometers, the surface area of many particles is relatively reduced and the detection efficiency of the biomolecule is low. When the particle size is less than 0.2 micrometer, its production is not easy and there is a limit to the optical- It is because.

In the present invention, it is preferable to further include a fluorescence probe in the anisotropic biohybrid particle.

The fluorescent dyes used as the fluorescent probes are FITC, RITC, IRDye, Cy2 (trade name), Cy3 (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 Alexa Fluor TM 430, Alexa FLOUR 488, Alexa FLOUR 532, Alexa FLOUR 540, and the like. Alexa Floor (TM) 566, Alexa Floor (TM) 594, Alexa Floor (TM) 633, Alexa Floor (TM) 647, Alexa Floor (TM) 660, Alexa Floor (TM) 680 Can

The fluorescence probe is preferably an anisotropic bio-hybrid particle-based biosensor, and may include fluorescence signals of different colors to be emitted for each antibody so as to be identifiable when the biomaterial is detected in multiple.

As such, the present invention relates to a biosensor for multiple detection based on an anisotropic biohybrid microparticles structure,

In one embodiment, the biosensor for multiple detection based on the anisotropic biohybrid microparticle structure

Two or more types of monoclonal antibodies immobilized on a substrate; antigen; And an anisotropic biohybrid microparticle structure in which a polyclonal antibody is introduced for each compartment.

In other words, a sandwich-type directional binding of an anisotropic microparticle in which a chip-immobilized monoclonal antibody-antigen-polyclonal antibody is introduced, As shown in FIG. FIG. 3 and FIG. 4 briefly show a schematic diagram of a process for manufacturing the biosensor of the present invention.

The substrate that can be used in the present invention may be any known support such as glass and the like, and preferably includes patterned wells.

In principle, any substance that can be used as a main component of a well in the present invention can easily produce a desired shape or pattern, and may be any material suitable for binding a biomaterial such as an antigen or an antibody.

For example, polyurethane acrylate (PUA), polydimethylsiloxane (PDMS), polyethyleneglycol (PEG), chitosan, and the like can be used. Polydimethylsiloxane (PDMS) can be used in the examples. In one embodiment of the present invention, a glass substrate patterned with polydimethylsiloxane (PDMS) was used.

"Polydimethylsiloxane (PDMS)" is a silicon oxide polymer, often referred to as silicone, which can vary in its properties depending on its molecular weight. That is, since the gel state changes from a gel state under a molecular weight of 10,000 to a rubber phase of over 300,000, PDMS has an advantage that a desired shape or pattern can be easily manufactured.

In the well preparation method, photo-curing, in particular photo-curing by ultraviolet light, may be used as a method for curing PDMS. Photocuring using ultraviolet (UV), visible ray or electron beam (EB). Generally, free radicals or cations generated from the photoinitiaor by light irradiation. ) Initiates the initiation reaction and the monomer or oligomer having reactivity is cured through the continuous reaction. UV curing is the most widely used industry in the light curing industry. The advantages of UV curing are excellent price performance, low energy consumption, very little emission of VOCs, simple curing facilities, It is possible to make materials having excellent chemical resistance and mechanical properties after curing.

In the present invention, a nano-sized ridge / groove pattern array of 300 nm to 800 nm can be fabricated on a glass cover slip by UV-assisted capillary force lithography using the PDMS. PDMS molds fabricated by photocuring are patterned at a size of 300 nm to 800 nm, adhered to a glass cover slip treated with a PDMS precursor, and then cured by ultraviolet rays to form a nano-sized patterned sheet Can be obtained.

Then, a monoclonal antibody and an antigen are immobilized on a glass patterned with PDMS (polydimethylsiloxane), and an anisotropic (anisotropic) The biohybrid microparticles are immobilized on the substrate.

In one embodiment of the present invention, an amine functional group is introduced into a glass substrate using a solution of APTES [(3-Aminopropyl) triethoxysilane], and a monoclonal antibody is immobilized using glutaraldehyde Respectively. Then, an antigen binding to the monoclonal antibody was bound and immobilized on the substrate through directional binding of an anisotropic biohybrid microparticle into which a polyclonal antibody was introduced.

The above process is briefly shown in the following schematic diagram.

In addition, the present invention relates to a method of detecting a plurality of biomolecules using the biosensor from a different point of view.

In particular, the anisotropic biohybrid microparticles of the present invention can selectively immobilize molecules having target-oriented functionality on each segmented surface, selectively inject different fluorescence signals into the respective compartments, There is a feature that multiple material detection and imaging can be done.

Various known detection methods can be used. Preferably, the fluorescence signal of the cross section is imaged by controlling the height of the focus using a confocal laser scanning microscope (CLSM) can do.

In addition, the biosensor of the present invention can be provided as a conventional kit capable of checking the presence or concentration of a target substance by mixing the target substance with a sample solution to be detected

As described above, the present invention relates to an anisotropic biohybrid microparticle-based biosensor, and more particularly, to an anisotropic biohybrid microparticle-based biosensor, which uses a compartmentalized anisotropic microparticle structure to perform multiple detection and targeting ) And a method of using the same.

Example

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Experimental material

The reaction was carried out using 98% propargylamine, N- (2-aminoethyl) maleimide trifluoroacetate salt, 99% 2-bromoethylamine hydrobromide, sodium azide, sodium hydroxide Sodium hydroxide, Dichloromethane anhydrous and Sodium sulfate anhydrous were purchased from Sigma-Aldrich.

The dialysis tubing (MWCO 3,500, Fisher Scientific, US), deionized water (DIW, 18.2 MΩ) was obtained from a Milli-Q system (Millipore SA, Bedford, US) a dual syringe applicator assembly (Fibrijet SA-0100, Micromedics, MN, US) and a syringe pump (KDS-100, Kd scientific Inc, US). Cysteamine, Fluorescein isothiocyanate-dextran (MW 70,000), Rhodamine B isothiocyanate-dextran (MW 70,000), (3-aminopropyl propyl) human monoclonal anti-human IgG (Fc specific) antibodies (monoclonal anti-human IgG (Fc specific) antibodies), monoclonal antibodies (monoclonal antibodies), triethoxysilane (APTES), glutardialdehyde, polyclonal antibodies ) And antigen (human serum-derived IgG) were purchased from Sigma-Aldrich and used.

Production Example 1: Conjugation of acetylene and maleimide-introduced P (AAm-co-AA)

P (AAm-co-AA) (MW 200,000; 10% acrylic acids; Polysciences, Warrington, Pa., US), acetylene and maleimide- co-AA) s and propargylamine or N- (2-aminoacetyl) maleimide triflouroacetate salt respectively.

 (EDC, Thermo SCIENTIFIC, US) and 0.7 mmol of propargylamine < RTI ID = 0.0 > propargylamine was reacted with 10 ml of DIW at room temperature (RT) overnight, and the reaction mixture was dialyzed with distilled water at room temperature for 48 hours. The solution was dried in a freezing dryer (MCFD 8508, illShinBioBase, KR) for 2 days.

5 umol P (AAm-co-AA) and 1.4 mmol EDC were dissolved in 9 ml DIW. 0.7 mmol of N- (2-Aminoethyl) maleimide trifluoroacetate salt was dissolved in 1 ml DIW. Then, N- (2-Aminoethyl) maleimide trifluoroacetate salt solution was added dropwise to the polymer solution, and reacted overnight. The reaction mixture was then dialyzed using distilled water at room temperature for 48 hours. This solution was dried in a freeze dryer for 2 days.

In FIG. 1, functional groups (maleimide, acetylene) using EDC were introduced into the carboxyl groups of poly (acrylamide-co-acrylic acid) and P (AAm-co-AA) (MW 200 kDa; A structural formula of a polymer is shown.

Then, by dissolving each polymer in D ₂ O the data analyzed using the ¹ H NMR, (a) and maleimide was introduced the P (AAm-co-AA) (b) acetylene is introduced into the P (AAm- It is shown with the ¹ H NMR chart of the co-AA) (Fig. 1).

Production Example 2: Synthesis of 2-azaidoethylamine (azidoethylamine)

24 mmol of 2-bromoethylamine hydrobromide were dissolved in 30 ml DIW. 55 mmol sodium azide was added to the solution. The resulting solution was stirred overnight at 75 < 0 > C. After cooling to RT, 24 mmol sodium hydroxide was added to the reacted compound. The reaction was extracted three times with dichloromethane (50 mL). The organic phase was dried over anhydrous sodium sulfate. After filtration, the organic solvent was removed using a rotary evaporator (N-1110S, EYELA, JP).

Example 1: Production of anisotropic biohybrid microparticles-based detection device

1-1. Mono & biphasic particles by electrohydrodynamic co-jetting

Two polymer solutions were prepared separately in DIW.

(a) One solution contained P (AAm-co-AA) (7.0%, w / v) and maleimide modified P (AAm-co-AA) (0.1%, w / v) of P (AAm-co-AA) (7.0%, w / v) and acetylene modified P (AAm-co-AA) in DIW. The two solutions were loaded into 1 ml syringes (BD, Franklin Lakes, NJ, US) and the same mixture was loaded onto a dual channel tip (Fibrijet) with two 26- and 3.25- SA-3610, Micromedics, MN, US).

 (b) One solution contained P (AAm-co-AA) (7.0%, w / v), maleimide modified P (AAm-co-AA) (0.1%, w / v) and FITC-dextran %, w / v). The other solutions were P (AAm-co-AA) (7.0%, w / v), acetylene modified P (AAm-co- AA) (0.1% w / v) and RITC-dextran v). One set was loaded in a 1 ml syringe and the other set was loaded in two syringes separately as a mixture and prepared side by side by a dual channel tip.

The cathode was connected to the needle and the opposite electrode was connected to an aluminum foil (0.018 mm thick, Fisherbrand, US). The distance between two kinds of electrodes was kept in the range of 28 ~ 30 cm vertically. During the EHD co-injection, the high-voltage power supply (ESN-HV30, Nano NC, KR) was supplied with a current of 15 to 18 kV in a current of 0.20 ml / h and the substrate was collected and thermally crosslinked overnight at 175 ° C.

Figure 2 shows a side-by-side dual-nozzle geometry electrophysiologic co-injector for making anisotropic particles. The apparatus allows the maleimide and acetylene-introduced polymers to be ejected in equal amounts using a micro-syringe pump [taylor cone, jet stream, jet break up]

1-2. Conjugation of fluorescein and FITC-BSA

Thermally cross-linked monophasic particles (14 μmol, 10 mg) were dispersed in DIW (1 ml) with a tip sonicator (VC-505, Sonics & Materials, Inc., US).

First, the fluorescein sodium salt (14 mu mol) was dissolved in DIW (1 ml) and coupled with cysteamine (14 mu mol) by EDC (28 mu mol). The thiol-introduced fluororesin was then combined with the maleimide-introduced particles for 2 hours at room temperature. Second, FITC-BSA (1.4 mu mol) was dissolved in DIW (1 ml) and coupled with 2-azaidoethylamine (14 mu mol) by EDC (28 mu mol) Dialyzed in DIW. The azide-introduced FITC-BSA was bound to the acetylene-introduced particles at room temperature for 2 hours with a 10 mM copper (II) sulfate solution (140 mu mol) and a 50 mM sodium ascorbate solution (140 mu mol). The centrifugation was repeated to purify the bound particles. All particles were centrifuged to remove unreacted fluorescein dye.

1-3. Combination of thiol group and azide-introduced antibody

25 μ㏖ cysteamine, was dissolved in a 25 μ㏖ EDC and 2.5x10 ^-2 μ㏖ polyclonal antibody in PBS, respectively (100 ㎕. 100 ㎕ and 400 ㎕). Mixed cysteamine, EDC solution and 400 [mu] l PBS were then added, then the polyclonal antibody solution was added dropwise to the mixture. After the reaction was carried out at room temperature for 4 hours, the reaction mixture was dialyzed against PBS at 4 ° C for 48 hours.

The synthesized 25 mu mol ^2- azaidoethylamine, 25 mu mol EDC and 2.5x10 ^-2 mu mol polyclonal antibody were separately dissolved in PBS (100 mu l, 100 mu l, and 400 mu l, respectively). Mixed 2-azaidoethylamine, EDC solution, and 300 [mu] l PBS were then added, then the polyclonal antibody solution was added dropwise to the mixture. After the reaction was carried out at room temperature for 4 hours, the reaction mixture was dialyzed against PBS at 4 ° C for 48 hours.

1-4. Combination of Particles and Antibodies

At 13 nM concentration, single & abnormal particles in PBS (1 ml) were suspended by tip sonicator. The maleimide-introduced monoclinic particles were added to the thiol-introduced antibody solution at room temperature for 4 hours. The acetylenically introduced single phase particles were added to a 10 mM CuSO ₄ solution (140 μl) and a 50 mM sodium ascorbate solution (140 μl) and the mixture was added to the suspension one drop. The reaction was carried out at room temperature for 4 hours. The bound particles were purified by repeated centrifugation. Unreacted antibodies were removed using centrifugation with all particles and antibody purification method.

1-5. PDMS ( Polydimethylsiloxane ) ≪ / RTI >

A cover glass mixed with sulfuric acid and hydrogen peroxide in a ratio of 3: 1 was immersed in the liquid as a whole to remove organic substances. A 5 mm diameter hole was made in the cured PDMS using a curing agent.

PDMS and glass were treated with plasmons (PDC-32G, Harrick Plasma, US) to couple the two materials. APTES was dissolved in ethanol. 50 [mu] l APTES solution (10 w / v% in ethanol) was incubated on the substrate at room temperature for 0.5 hour. After incubation, unreacted APTES was removed by washing with ethanol and distilled water. 50 [mu] l glutardialdehyde solution (5.0%, w / v) was incubated at room temperature for 45 minutes. 3 (a) is an image of a chip combining a cover glass and a PDMS, and (b) is an image of a chip patterned with gold using a gold array.

An anisotropic biohybrid microparticle-based detection device manufacturing process is shown in FIGS. 2, 3, and 4. FIG.

In the figure below, two cytoamine and 2-azidoethylamines were used for the two polyclonal antibodies, and thiol and azide were introduced. Maleimide-thiol coupling between maleimide and thiol and 1,3- schematic diagram of the reaction mechanism of dipolar cycloaddition; And two antibodies were bound to the anisotropic microparticle compartment.

FIG. 3 (a) shows images of a chip in which PDMS patterned in a cover glass is combined by a plasma technique, FIG. 3 (b) shows a pattern in which a layer of metal and gold is stacked on a substrate, "He said.

4, a sandwich immunocomplex pattern diagram on a glass substrate patterned with PDMS (Polydimethylsiloxane) is shown. Each process is described as follows: (1) Introducing amine functional groups using a solution of APTES [(3-Aminopropyl) triethoxysilane]. (2) Immobilization of monoclonal antibodies using glutaraldehyde. (3) Antigen binding. (4) A directional bond of an anisotropic microparticle into which a polyclonal antibody is introduced.

On the other hand, mimetic diagrams and CLSM and DIC images of each particle introduced with each functional group were confirmed.

Figures 5 and 6 relate to maleimide-introduced particles,

5 (AF) shows a mimetic diagram of a particle (A) in which maleimide is introduced and bonded to fluorescein-SH and a pure particle (D) in which fluorescein-SH is not bonded, and CLSM DIC image, and in FIG. 6 (GL), fluorescence microscopic images and CLSM images of the maleimide-introduced particles in combination with antibody are shown. FIG. 5 (GL) shows a mimetic diagram and CLSM and DIC images of acetylene-introduced and FITC-BSA-N3 bonded particles (G) and pure FITC-BSA-N3 bonded particles (J) And FIGS. 6 (HI) and (KL)) show fluorescent microscope images and CLSM images of acetylene-introduced particles combined with antibody.

FIG. 7 shows an image of anisotropic microparticles using CLSM using electrohydrodynamic co-injection.

From left to right is a photograph showing the channels of fluorescein isothiocyanate (FITC channel), rhodamine B isothiocyanate (RITC channel), and FITC and RITC channels.

Particularly, an anisotropic microparticle was directly collected in a cover glass using electrohydrodynamic co-injection and observed, and an image was observed in Figure 7 (AC). The collected anisotropic microparticles were suspended in phosphate buffered saline, PBS (pH 7.4) An observed image is shown in Fig. 7 (DI).

Example  2 : Anisotropy  Particle detection

Monoclonal antibodies (50 l, 2.05 mg / ml, 0.7 mu mol) were incubated in aldehyde-introduced glass substrate wells at room temperature for 2 hours. As above, 50 [mu] l BSA (1.0%, w / v) was cultured in the wells for 2 hours.

One well was incubated with antigen (50 [mu] l, 5 mg / ml) and the other well with no antigen and only with PBS. Finally, polyclonal antibodies labeled with anisotropic particles were incubated in two wells at room temperature for 2 hours. At all final stages of incubation the wells were washed with PBS and treated with nitrogen gas.

Confocal microscopy experiments were performed using a scanning microscope using Ar, He-Ne and Gre-Ne laser sources on a Leica (Germany) TCS SL confocal laser scanning microscope.

The mono and biphasic particles were imaged in x-y-z mode with 100 planapochromatic oil immersion objective variable PMT gain, digital zoom 4 a line frequency of 200 Hz, and 8-bit acquisition mode.

The bound particles were observed using an IX81 inverted microscope (Olympus, JP) equipped with DIC slides with 20 x objective. The light source was a 100W HBO lamp housing and an HBO transformer.

A CEA or IgG monoclonal antibody and an antigen were introduced into a patterned glass substrate, sandwich immobilization was performed by adding an anisotropic microparticle into which polyclonal antibodies were introduced, and CLSM was used to adjust the height of the focus from below to above 8, 9, 10, 11, 12 and 13 show the images observed.

A method of directional binding using the selective directionality of the anisotropic microparticle structure is to adjust the height of the focus using a confocal laser scanning microscope (CLSM) The fluorescence signal of the cross section was imaged.

As a result, it was confirmed that the positions of the anisotropic microparticles were varied depending on the type of the monoclonal antibody immobilized on the glass substrate (Figs. 8, 9, 10, 11, 12 and 13).

Claims (14)

Claim 1 has been abandoned due to the setting registration fee. An anisotropic biohybrid microparticles structure that is physically and chemically compartmentalized,
Wherein a different kind of antibody is introduced into each of the sections of the anisotropic biohybrid microparticles and has selective orientation according to a different antigen-antibody reaction. Claim 2 has been abandoned due to the setting registration fee. The biosensor for multiple detection according to claim 1, wherein the anisotropic biohybrid microparticles are composed of a copolymer polymer having crosslinking. The biosensor for multiple detection according to claim 2, wherein the cross-linking is performed by thermal crosslinking. The method of claim 2, wherein the copolymer polymer is selected from the group consisting of poly (acrylamide-co-acrylic acid, poly (AAm-co-AAc) (N-isopropyl acrylamide-co-stearyl acrylate), poly (NIPAm-co-SA) and poly (N-isopropylacrylamide- co-allylamine) isopropylacrylamide-co-allylamine, poly (NIPAM-co-AA)]. The multi-detection biosensor according to claim 4, wherein the copolymer polymer is poly (acrylamide-co-acrylic acid, poly (AAm-co-AAc) . Claim 6 has been abandoned due to the setting registration fee. The biosensor for multiple detection according to claim 1, wherein the anisotropic bio-hybrid microparticles are produced by an electrohydrodynamic co-jetting method. Claim 7 has been abandoned due to the setting registration fee. The method of claim 1, wherein the different types of antibodies are introduced by different functional groups, wherein the different functional groups are selected from the group consisting of an amine, a carboxylic acid, a thiol, a hydroxyl group, Wherein the biosensor is selected from the group consisting of acetylene and azide. 8. The biosensor for multiple detection according to claim 7, wherein the different functional groups are maleimide and acetylene functional groups, respectively. The method according to claim 8, wherein the Maleimide and Acetylene functional groups and the thiol introduced into the polyclonal antibody are combined with the azide functional groups so that different sections of the anisotropic biohybrid microparticles Type antibody is introduced in the biosensor for multiple detection. Claim 10 has been abandoned due to the setting registration fee. The biosensor for multiple detection according to claim 1, wherein the anisotropic biohybrid microparticle further comprises a fluorescence probe. Claim 11 has been abandoned due to the set registration fee. 2. The biosensor according to claim 1, wherein the biosensor
Two or more types of monoclonal antibodies immobilized on a substrate; antigen; And an anisotropic biohybrid microparticle structure in which a polyclonal antibody is introduced for each of the compartments. 12. The biosensor for multiple detection according to claim 11, wherein the anisotropic bio-hybrid microparticle structure further comprises a fluorescent dye. The biosensor for multiple detection according to claim 11, wherein the substrate is glass patterned with PDMS (Polydimethylsiloxane). Claim 14 has been abandoned due to the setting registration fee. The multiple detection method of biomaterial using the biosensor according to any one of claims 1 to 11, characterized by using a method of imaging a fluorescence signal by adjusting the height of the focus using a confocal laser scanning microscope (CLSM) .

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