KR101598757B1 - Inorganic―organic nanofiber composite substrates for fast and sensitive trace analysis based on surface enhanced raman scattering and the method using the same - Google Patents

Abstract

The present invention relates to an organic / inorganic nanofiber composite substrate for microanalysis of a substance based on Surface Enhanced Raman Scattering (SERS), and more particularly, Nanofiber composite substrates in which aggregates of nanoparticles are uniformly embedded in nanofibers having cross-linkable polymer hydrogel properties at various densities, a method for manufacturing the nanofiber composite substrate, and their applications.

Description

 TECHNICAL FIELD The present invention relates to a nanocomposite substrate for high-speed and high-sensitivity microanalysis based on surface-enhanced Raman scattering, and an analytical method using the same. BACKGROUND ART [0002] In recent years, nanocomposite-

The present invention relates to an organic / inorganic nanofiber composite substrate for microanalysis of a substance based on Surface Enhanced Raman Scattering (SERS) and an analytical method using the same. More particularly, the present invention relates to a variety of Raman dyes, , Nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates, nanofiber composite substrates

As the environmental pollution problem becomes serious today, the need for early detection of environmental pollutants such as various heavy metals and organic phosphorus compounds is increasing. In addition, as the threat of terrorism is rising in the international community, the need for early detection of various bioterror substances such as anthrax and sarin is also increasing. Therefore, it is a very important task to quickly and accurately detect such a dangerous substance in the field and to prevent the diffusion of the dangerous substance. The development of a technique for the analysis of trace amounts of such various chemicals is carried out through environmental monitoring, forensic science, This is a very important issue in the field.

Conventional analysis technology for environmental pollutants requires a complicated pretreatment process and requires a large-scale analysis equipment which is impossible to move or is inconvenient, and it takes a long time and effort to analyze it.

In recent years, Surface Enhanced Raman Scattering (SERS) has attracted attention as a method for detecting and biochemically analyzing chemicals due to its high sensitivity.

In this area, research continues on specific structures that can be used as reliable SERS substrates. In order to make the gold and nanoparticles into a desired shape and shape, attention has been focused on ion beam lithography, electron beam lithography, nanosphere lithography, and vacuum evaporation, and methods of collecting small nanoparticles into nanoclusters have been used. These techniques are used to create nanoclusters of gold and silver nanoparticles with reproducible SERS.

However, these technologies are costly and difficult to mass-produce. Coating silver nanoparticles with a silicon nanowire array to create an economical, stable and reliable SERS substrate [Zhang, M. L., C. Q. Yi, et al. 2008 Applied Physics Letters 92 (19); And Zhang, B. H., H. S. Wang, et al. 2008 Advanced Functional Materials 18 (16): 2348-2355), porous aluminum membranes with gold nanoparticles (Ko, H. and VV Tsukruk 2008 Small 4 (11): 1980-1984.), Hollow gold nanospheres and nanoshells And conjugated antibodies [Lee, S., H. Chon, et al. 2009, Biosensors & Bioelectronics 24 (7): 2260-2263]. However, the increase of the molecular vibrations is high, and the number of the uniaxialized plasmon centers is decreased, so that the reinforcement effect does not effectively take place.

At present, substrates available for SERS have various defects in the enhancement and detection of Raman scattering. Raman scattering signals are generally very weak, especially on flat substrates. A weak Raman signal makes it difficult to detect and measure the Raman scattering signal, thus making it difficult to detect and identify the molecular species. Moreover, even if the usable substrate enhances the Raman scattering signal, the enhanced Raman scattering signal is usually only in the localized area of the substrate and is not uniform across the substrate surface. The area of the local enhancement Raman scattering signal is exponentially smaller than that of the entire area of the substrate surface. Large differences in the area of the Raman scattering signal and the area of the substrate surface make it difficult to locate and locate the signal and thus to detect and identify the molecular species.

Accordingly, the present inventors prepared agglomerates of metal nanoparticles induced by Raman dye, and these agglomerates of metal nanoparticles were subjected to electrohydrodynamic (EHD) jetting to obtain cross-linkable polymer hydrogel Based nanofiber composite substrate prepared by uniformly impregnating nanofibers having various densities at various densities by using an internal standard signal generated by surface-enhanced Raman scattering induced by Raman dyes And the signal intensity of the analyte can be effectively compared and detected. Thus, the present invention has been completed.

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It is an object of the present invention to provide an organic / inorganic nanofiber composite substrate for high-speed and high sensitivity microanalysis based on surface-enhanced Raman scattering and a method for producing the same.

Another object of the present invention is to provide a method of analyzing a material by Surface Enhanced Raman Scattering (SERS) using the nanofiber composite substrate.

In order to solve the above problems,

There is provided a nanofiber composite substrate for surface-enhanced Raman scattering analysis characterized in that agglomerates (clusters) of metal nanoparticles by Raman dye are uniformly distributed in the cross-linkable polymer hydrogel nanofiber.

Crosslinkable polymers include, but are not limited to, poly (acrylamide-co-acrylic acid), poly (AAm-co-AA), poly (N-isopropylacrylamide- Poly (N-isopropylacrylamide-co-stearyl acrylate), poly (NIPAm-co-SA) ), poly (NIPAM-co-AA), and poly (NIPAM-co-AA) having an acrylic moiety. In one embodiment of the present invention, Poly (acrylamide-co-acrylic acid, poly (AAm-co-AA)] was used.

In particular, the cross-linking may be physical, chemical or photo-initiative, preferably ultraviolet (UV) photo-initiatively.

The Raman dyes used in the agglomeration of the metal nanoparticles include rhodamine 6G, rhodamine B isothiocyanate (RBITC), adenine, 4-amino-pyrazole (3,4-d) pyrimidine, Adenine, N6-benzoyladenine, kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza- adenine, 8-azaguanine, 6- Mercapto-pyrazolone (3,4-d) pyrimidine, 8-mercaptoadenine, and 9-amino-acridine. In one embodiment of the present invention, rhodamine B isothiocyanate (RBITC ) Was used.

The metal nanoparticles may be selected from silver, gold, copper, and mixtures thereof, and preferably silver (silver) nanoparticles may be used.

The metal nanoparticles preferably have a diameter of 1 to 00 nm, and the metal nanoparticle aggregate (cluster) preferably has a diameter of 60 to 100 nm.

According to another embodiment of the present invention, there is provided a method of manufacturing a nanofiber composite substrate for analyzing surface-enhanced Raman scattering comprising:

(i) agglomerating seed metal nanoparticles with a Raman dye to form metal nanoclusters;

(ii) coating the metal nanocluster;

(iii) introducing the metal nanoclusters into the crosslinkable polymer hydrogel nanofibers through an electrohydrodynamic jetting process; And

(iv) cross-linking the cross-linkable polymer hydrogel.

The description of the main constituents used in each step is as described above.

In step (ii), the coating of the metal nanocluster may be carried out by a method selected from the group consisting of avidin, streptavidin, bovine serum albumin (BSA), insulin, soy protein, casein, gelatin and mixtures thereof , Which can stabilize the metal nano-cluster. In one embodiment of the present invention, silver nanoclusters were coated with BSA (bovine serum albumin).

And, in step (iii), when the metal nanoclusters are incorporated into the crosslinkable polymer hydrogel nanofibers, Raman dye type and concentration control; Density control of metal nanoparticle aggregates; Or electrophysiological injection control, it is possible to uniformly distribute aggregates of metal nanoparticles at various densities.

The present invention also provides a surface-enhanced Raman scattering analysis method, characterized in that an analyte is detected in an aqueous solution by using a nanofiber composite substrate for surface-enhanced Raman scattering analysis as another embodiment.

Specifically, the present invention provides the following analytical methods:

Preparing a nanofiber composite substrate for surface-enhanced Raman scattering analysis, wherein aggregates (clusters) of the metal nanoparticles by the Raman dye are uniformly distributed in the cross-linkable polymer hydrogel nanofiber; And

Immersing the nanofiber composite substrate in a solution containing the analyte to detect the analyte;

And a surface-enhanced Raman scattering analysis method comprising the steps of:

The nanofiber composite substrate may be crosslinked between the polymers surrounding the agglomerates of the metal nanoparticles to form a network between the agglomerates of the metal nanoparticles and the polymer.

The method is effective because it can perform comparative analysis based on the intensity of an internal standard signal due to the surface-enhanced Raman scattering induced by the Raman dyes existing in the substrate.

The analytical method of the present invention can be more usefully used when the analyte is present in a concentration of 0.01 to 6 ppm, preferably 0.5 to 6 ppm, that is, even in a trace amount.

The analyte may be a carcinogen, but is not necessarily limited thereto.

When the nanofiber composite substrate is immersed in a solution containing the analyte, the analyte is diffused through the nanofiber composite substrate and adsorbed on the surface of the nanoparticle aggregates, thereby detecting the analyte.

In one embodiment of the present invention, analysis was performed on malachite green (MGITC).

The present invention relates to a nanofiber composite substrate for surface-enhanced Raman scattering analysis and its application, wherein agglomerates (clusters) of metal nanoparticles by Raman dye are uniformly distributed inside the crosslinkable polymer hydrogel nanofiber The distribution density of metal nanoparticles aggregates by Raman dyes can be variously controlled according to an analyte and an internal standard signal by a Raman dye exhibiting a constant intensity by surface-enhanced Raman scattering, , It is useful for effectively detecting Raman signal intensities for analytes by comparative analysis.

1 (A) shows the preparation of agglomerates of metal nanoparticles induced by various Raman dyes for high-speed and high-sensitivity biosensing based on surface-enhanced Raman scattering and for trace analysis of materials, Is a new nanocomposite composite substrate in which nanoparticles of metal nanoparticles are uniformly embedded in nanofibers having polymer hydrogels at various densities using an electrohydrodynamic (EHD) jetting method. FIG. 1 is a schematic view of the present invention on a manufacturing method. 1 (C) is a schematic diagram for quantitatively and qualitatively analyzing MGITC present in an aqueous solution using an organic / inorganic nanofiber composite substrate.
FIG. 2 (A) is a graph showing the UV-Vis spectrum during the formation of silver nanoparticle aggregates by Raman scattering dye (RBITC). FIG. 2 (B) is a graph showing the size of aggregated silver nanoparticles using dynamic light scattering (DLS). 2 (C, D) are transmission electron micrographs of agglomerates of silver nanoparticles and silver nanoparticles. 2 (E) and (F) are graphs showing changes in Raman signal and major Raman peak intensities with respect to agglomeration reaction time during the formation of silver nanoparticle aggregates.
Figure 3 (A) is a cross-sectional view of a UV crosslinkable poly (acrylamide-co-acrylic acid); UV cross-linking using a carbon-carbon double bond (C = C) of UV-P (AAm-co-AA). 3 (B) is a spectrum of UV-P (AAm-co-AA) analyzed by NMR, and FIG. 3 (C) Signal.
Fig. 4 (A) is a graph of a change in the surface-enhanced Raman scattering signal of the nanofiber substrate, which is observed according to the electrospinning time when the concentration of silver nanoparticles in the polymer solution is 0.4 w / v% (1646 cm <" 1 >) for each time. FIG. 4 (C) is a graph showing changes in surface-enhanced Raman scattering signal with increasing concentration of silver nanoparticles in the nanofiber, and FIG. 4 (D) shows the intensity at a main Raman peak (1646 cm -1) .
FIG. 5A is a graph of a change in the surface-enhanced Raman scattering signal of the organic / inorganic nanofiber composite substrate in another solvent (water, ethanol, acetone) and FIG. 5B is a graph showing the Raman scattering signal And the intensity at the main Raman peak (1646 cm < -1 >). Figures 5 (C) and 5 (D) show the Raman scattering signals in various pH 5 - pH 12 and salt concentrations (0.02 M - 0.1 M), followed by the intensity at the main Raman peak (1646 cm -1) Graph.
6 (AD) is an SEM image of polymer nanofibers containing silver nanoparticle aggregate particles at a concentration of 0.4 w / v% at a different magnification, 7.5, 10.0, 12.5 and 15.0 w / v% polymer concentration (AD) FIG. 6 (EF) is a graph showing the distribution of analytes through the swollen nanofibers in an aqueous solution, (E) a dry state for estimating the expansion ratio of the nanofibers, (F) A confocal laser scanning microscope photograph of the composite substrate.
(A, B) 0.2 w / v% (C, D) 0.4 w / v% (E, F) ) 0.8 w / v%].
8 (A) shows a surface-enhanced Raman scattering spectrum according to the concentration of metal nanoparticles aggregated by diffusing through the inside of the organic / inorganic nanofiber composite substrate, which is a swollen hydrogel of malachite green (MGITC) B) is a graph of the relationship between the Raman signal intensity according to the internal standard signal of RBITC and the Raman signal intensity according to the MGITC concentration of carcinogen.

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

The term " metal nanocluster " is a term commonly used in this field as a term for aggregates of aggregated metal particles.

A "polymer or polymer" is a compound in which one kind or several kinds of constitutional units are linked to each other by a large number of chemical bonds. The constituent unit is referred to as a monomer, and it is referred to as a dimer, trimer or the like depending on the polymerization degree. A macromolecule having a high degree of polymerization is referred to as a high polymer and is often used in the same meaning as a polymer compound (a compound having a molecular weight of 10,000 or more). In the present specification, the terms polymer and polymer are used in combination.

"Hydrogel" is a water-swelling 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 because of 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.

"Raman scattering" is a phenomenon in which the energy (hv) of an incident photon is inelastic scattered to energy (hv ') at another frequency while changing the vibrational state of the molecule. Such Raman scattering interacts with the photons and exhibits inherent photonic energy changes depending on the molecular structure that induces scattering. Therefore, it is possible to detect, identify and analyze molecules. Surface enhanced Raman scattering or Surface Enhanced Raman spectroscopy is one technique used to enhance the sensitivity of Raman signals. Surface enhanced Raman scattering is a technique for amplifying a Raman signal by locally increasing the electromagnetic field using a very fine structure

The term "analyte or analyte" means any atom, chemical, molecule, compound, composition or aggregate that is to be detected and / or identified. Non-limiting examples of such analytes include, but are not limited to, amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, , Hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals, nutrients, prions, toxins, toxins, Toxicants, radiation isotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagenic factors, anesthetics, amphetamines, barbiturates, hallucinogens, wastes and / or contaminants. In one embodiment of the present invention, malachite green, one of the environmental pollutants, was analyzed.

"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, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are 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 invention relates to a substrate for use in surface enhanced Raman scattering (SERS) analysis.

The surface enhanced Raman scattering (SERS) - based detection method has been spotlighted by high sensitivity analysis method. The analyte-containing solution and the immuno-gold nanoparticles with Raman reporter are added continuously and the specific marker can be quantitatively analyzed by measuring the intensity change of the characteristic SERS peak of the reporter molecule. When the reporter molecule is adsorbed to a rough metal surface and exposed to excitation light (laser light), electromagnetic and chemical enhancement occurs at the SERS active site of the reporter molecule known as the hot spot junction, resulting in a significant increase in the SERS signal Kneipp, J. et al., 2006. Nnao Lett., 6, 2225-2231). This enhancing effect solves the problem of low sensitivity which is a disadvantage of conventional Raman and fluorescence detection methods.

SERS has evolved into a technology capable of measuring biological imaging and biological signals with high sensitivity at a single molecule level. Nanostructures Raman signals of different molecules adsorbed on metal surfaces are very high in the case of silver, gold, copper, and the like. When the Raman active molecules are adsorbed on the surface of the rough metal substrate, their SERS signal increases. This is due to the electromagnetic and chemical enhancement phenomena occurring at the highly flattened plasmon centers of the hot spot junctions. The electronic representation relates to local surface plasmon resonance associated with coarse metal or metal aggregate surfaces. On the other hand, chemical enhancement is associated with direct charge transfer or hole-pair excitation processes.

In order to detect a single molecule level, the SERS signal must be enormously increased. Silver colloidal nanoparticle aggregates were first used for SERS detection of single molecules [Nie, S. M. and S. R. Emery 1997 Science 275 (5303): 1102-1106.]. Subsequent studies have shown that silver nanoclusters are best suited for super sensitivity detection [Kneipp, J., H. Kneipp, et al. 2006, Nano Letters 6 (10): 2225-2231.]. If the Raman reporter molecule is present at the junction of the nanoclusters, the signal intensity increases tremendously [Chen, J. W., Y. Lei, et al. 2008, Analytical and Bioanalytical Chemistry 392 (1-2): 187-193.]. The distance between particles, the intrinsic characteristics of the metal plasmon region, the size and concentration of nanoparticles, and the laser intensity are important parameters controlling signal enhancement.

Studies are underway on specific structures available as SERS substrates or substrates. In order to make the gold and nanoparticles into a desired shape and shape, attention is focused on ion beam lithography, electron beam lithography, nanosphere lithography and vacuum evaporation, and methods of collecting small nanoparticles into nanoclusters have been used, It is difficult to mass-produce them, and the stability of nanoclusters in different chemical and biological environments is very important.

In addition, most of the surface-enhanced Raman scattering substrates conventionally developed as chemical sensors for chemical trace analysis have no internal standard signals due to surface-enhanced Raman scattering induced by Raman dyes, It is difficult to reproducibly perform minute amount analysis.

In the currently developed surface-enhanced Raman scattering substrate, there is a limit to the core technology required for the microanalysis because the detectable part is limited.

However, the nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention can realize a high-speed and high-sensitivity microanalysis of an analyte and realize chemical stability and data reproducibility as a new concept substrate satisfying this demand.

That is, the present inventors produced metal nanoparticles agglomerates (clusters) of metal nanoparticles induced by various Raman dyes for high-speed and high-sensitivity microanalysis and biosensing, (Polymer) hydrogel property by uniformly introducing the nanofiber into the nanofiber at various densities by chemical hydrothermal (electrohydrodynamic (EHD) jetting) method. Through the swollen sturucture of the organic / inorganic nanofiber composite substrate, the analytical substance is diffused to realize a high-speed and high-sensitivity microanalysis capable of detecting a dangerous drug, as well as chemical stability and data reproducibility.

Accordingly, the present invention provides, in one aspect,

A nanofiber composite substrate for surface-enhanced Raman scattering analysis and a method for producing the same, characterized in that agglomerates (clusters) of the metal nanoparticles by the Raman dye are uniformly distributed in the cross-linkable polymer hydrogel nanofiber .

The substrate of the present invention contains nanoclusters, which are agglomerates of metal nanoparticles induced by Raman dye, in uniform density within the nanofibers having cross-linkable polymer hydrogel properties.

Particularly, in order to stabilize the organic / inorganic nanofiber composite substrate in aqueous conditions, the polymer hydrogel nanofibers form crosslinks physically, chemically or photoactively, for example, thermal chemical crosslinking or swollen in an aqueous solution using photo-initiated chemical crosslinking which minimizes the loss of surface-enhanced Raman scattering signals at high temperatures.

Through the inside of the swollen hydrogel substrate, the analytes dissolved in the aqueous solution are diffused and adsorbed on the surface of the metal nanoparticle aggregates, whereby the adsorption of the substances on the surface of the material based on the surface-enhanced Raman scattering signal induced by the Raman dye High-speed and high-sensitivity trace analysis is possible.

At this time, based on the constant intensity of the internal standard signal by the surface-enhanced Raman scattering induced by the Raman dye present in the surface-enhanced Raman scattering substrate of the present invention, The analyte can be effectively detected by comparing and analyzing the signal intensity.

In order to produce the nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention, it can be produced by the following method as one embodiment:

(i) agglomerating seed metal nanoparticles with a Raman dye to form metal nanoclusters;

(ii) coating the metal nanocluster;

(iii) introducing the metal nanoclusters into the crosslinkable polymer hydrogel nanofibers through an electrohydrodynamic jetting process; And

(iv) cross-linking the cross-linkable polymer hydrogel

The manufacturing method is schematically shown in Fig.

Hereinafter, the present invention will be described in more detail with reference to the above method in more detail.

The term "seed metal nanoparticle" refers to a metal particle that becomes a seed for making a metal nanocluster, and the metal may be selected from the group consisting of silver, gold, copper, and mixtures thereof . In one embodiment of the present invention, silver (silver) nanoparticles were used.

In the present invention, the aging of the seed metal nanoparticles is carried out by Raman dye. That is, agglomerates of seed metal nanoparticles, that is, nanoclusters, are produced by Raman dye. In the specification of the present invention, the term aggregate or cluster is used in combination.

The Raman dye means a Raman active organic compound, and any of them can be used without limitation as long as it is widely used in this technical field. In the present invention, the Raman dye may be variously included. Specific examples thereof include rhodamine 6G, rhodamine B isothiocyanate (RBITC), adenine, 4-amino-pyrazole (3,4-d) pyrimidine, 2-fluoroadenine, N6-benzoyladenine, , Dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine, 6-mercaptopurine, -d) pyrimindine, 8-mercaptoadenine, and 9-amino-acridine, and the like. In one embodiment of the present invention, rhodamine B isothiocyanate (RBITC) was used.

The size of the metal nanoclusters agglomerated by the Raman dye may be 10-1000 nm, more preferably 60-100 nm.

For example, when the size of each of the seed nanoparticles is 1 to 100 nm and the nanocluster size is 10 to 1000 nm, the Raman signal is maximized.

Next, the metal nanocluster is stabilized by coating the nanocluster.

There are no limitations on the type of protein coating the metal nanoclusters, and any of the widely used in the art can be used. For example, it can be selected from the group consisting of avidin, streptavidin, bovine serum albumin (BSA), insulin, soy protein, casein, gelatin and mixtures thereof. In one embodiment of the present invention, nanoclusters were coated and stabilized with BSA (bovine serum albumin).

The following is a process schematic diagram of steps (i) and (ii) according to an embodiment of the present invention.

The figure shows the mechanism of silver nanocluster, silver nanoparticle aggregates (AgNCs) formation according to an embodiment of the present invention. Initial seed particles having a diameter in the range of 6 to 13 nm aggregated by introducing different organic dye agents as flocculants and Raman reporters. The particles were grown simultaneously from silver ions reduced by sodium citrate tribasic dihydrate at the surface of the preexisting seed. Stabilizers were added from BSA to stabilize the clusters.

Subsequently, the coated metal nanocluster is homogeneously embedded in the crosslinkable polymer hydrogel nanofibers through an electrohydrodynamic co-jetting method, and the polymers are crosslinked.

A crosslinkable polymer hydrogel is encapsulated encapsulating the metal nanocluster by spraying by electrohydraulic (electrohydrodynamic) spraying.

Electrohydrodynamic jetting (EHD jetting) is commonly used as an easy technique for the production of polymeric nanofibers and nanoparticles having different shapes and sizes. The manipulation of mainly adjustable parameters depends on several factors, including 1) the viscosity, electrical conductivity and surface tension of the polymer solution, 2) the voltage and distance applied between the two electrodes, and 3) the various polymeric nano- Lt; RTI ID = 0.0 > a < / RTI > structure. Among them, electrohydrodynamic co-jetting (EHD co-jetting) is a method in which an equilibrated laminar flow of a polymer solution far from the vertex of a Taylor cone having two phases - to be jetted in a thin jet stream form by charge to charge repulsion.

On the other hand, the crosslinkable polymer (polymer) may be any hydrophilic polymer without limitation.

The polymer of the present invention is a copolymer polymer capable of 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 (N-isopropyl acrylamide) (NIPAM-co-AA) with poly (N-isopropylacrylamide-co-allylamine), poly (NIPAM-co-AA) and acrylic moiety But not limited thereto. In one embodiment of the present invention, poly (acrylamide-co-acrylate) [poly (AAm-co-AA)] was used.

"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 nanofibers to maintain their original structure in a substantially solubilized environment, such as water-soluble conditions, which also allows the nanofibers to act as soft hydrogels containing a significant amount of water Help. 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.

Therefore, when heating the metal nanoclusters encapsulated with the crosslinkable polymers (polymers) of the present invention or treating ultraviolet rays, crosslinking is formed between the polymers surrounding the metal nanoclusters, A network is formed. And is not soluble in water due to the formation of such a network through crosslinking.

The following is a schematic diagram of steps (iii) and (iv) according to an embodiment of the present invention

The figure shows a typical electrospinning device encapsulating silver nanoparticle aggregates in polymer nanofibers according to one embodiment of the present invention. In a monodisperse suspension of silver nanoparticle aggregates in aqueous solution of hydrophilic, biocompatible and UV crosslinkable polymer UV-poly (acrylamide-co-acrylic acid), UV-P (AAm- -co-AA) is filled with a plastic syringe fitted with a stainless steel capillary attached to a plastic syringe pump. Digital photographs show the formation of taylor cone, jet stream, and jet breakup during electrospraying experiments.

The nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention produced by the above method is characterized in that aggregates of metal nanoparticles formed by the Raman dye are uniformly distributed in the polymer hydrogel nanofiber.

At this time, the agglomerates of the metal nanoparticles can be evenly distributed at various densities by (i) controlling the type and concentration of the Raman dye, (ii) controlling the density of the metal nanoparticle agglomerates, and (iii) controlling the electrohydrodynamic injection.

In addition, the nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention is not difficult to manufacture and is economical to manufacture.

(Raman dyes) induced in metal cluster aggregates of the desired cluster size, stabilization by protein coating, and subsequent encapsulation with polymer (polymer).

As described above, polymeric polymers are biohybrid, non-toxic and hydrogel polymers, and encapsulation utilizes electrohydrodynamic injection technology. This macromolecule polymer is well swollen in a water-soluble environment and is in a soft matrix state, which serves to hold the metal nanoclusters well. Electrohydrodynamic jetting (EHD jetting) is a well-known technique for encapsulating many inorganic nanoparticles within various polymers [Yoshida, M., KH Roh, et al. 2009, Advanced Materials 21 (48): 4920]. After EHD injection, the polymer polymer is cured thermally or light to stabilize nanoprobes placed in aqueous media. Crosslinked polymers with a Raman center in the interior can be used as sensitive SERS substrates for biosensing and bioimaging.

On the other hand, the present invention relates to a material analysis method using nanofiber composite substrates for surface-enhanced Raman scattering analysis from a different point of view.

There are no restrictions on the material to be analyzed. For example, dipicolinic acid (DPA), malachite green (MG) and the like can be usefully detected and analyzed as a risk substance such as a carcinogen. In one embodiment of the present invention, malachite green (MG) was used as an analyte.

Malachite green is a green industrial pigment and antibacterial agent that has been used in aquaculture to protect against parasites, fungi and bacteria, but since it was found to be highly toxic to fish in the 1950s, In 2002, Europe and China also regulated the use of malachite green for edible fish, and Korea is also classified as toxic chemicals.

In particular, since the nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention has Raman dyes used in the production of metal nanoclusters, it is possible to obtain an internal standard signal indicative of a constant intensity by surface-enhanced Raman scattering signal is used as a reference, the Raman signal intensity of the analyte can be effectively detected by comparative analysis.

Since the surface-enhanced Raman scattering substrate itself, which has been developed as a chemical sensor for most chemical substance trace analysis, does not have an internal standard signal due to surface-enhanced Raman scattering induced by Raman dye, However, the present invention solves this problem due to the existence of an internal standard signal.

In one embodiment of the present invention, malachite green (MGITC), which is a carcinogenic substance dissolved in a small amount of a solution, is diffused through the interior of the substrate of the organic / inorganic nanofiber composite substrate, which is a swollen hydrogel, And the surface - enhanced Raman scattering spectrum according to its concentration, which is observed by adsorption on the surface - enhanced Raman scattering spectrum of the malachite green.

In particular, the intensity of the Raman signal increases with increasing concentration of malachite green, compared with a certain internal standard signal intensity due to surface-enhanced Raman scattering induced by the RBITC Raman dye present in the surface-enhanced Raman scattering substrate itself And quantitative evaluation was carried out by observing the change in the amount of the water.

The figure shows a schematic diagram of trace analysis of MGITC according to the SERS technique, according to one embodiment of the present invention. The crosslinked polymeric nanofibers include hydrogel properties, allowing dye molecules to permeate the matrix, and allowing silver nanoclusters to be absorbed. The incoming laser beam interacted with the stained molecules absorbed on the silver nanoparticle aggregates to generate respective Raman signals from the substrate before and after immersing the MGITC solution.

Meanwhile, according to the method of the present invention, the MGITC concentration range of 0.5-6 ppm and the SERS signal have a first-order linear relationship, and the limit concentration of the detectable analyte is defined as the limit of detection (LOD) Up to now, LOD can be up to about 0.5 ppm (part per million) according to the embodiment, and can be usefully used for the detection of a wide range of target substances present in trace amounts. Liquid chromatographic mass spectroscopy (LC-MS) is typically used as a representative microanalysis method. The LOD of SERS-based MGITC analytes has been reported up to about 1-2 ppb (parts per billion) , A detection limit corresponding to the LOD of the LC-MS is possible.

In the present invention, the aggregation of metal nanoparticles is uniformly distributed at various densities through the control of (i) the type and concentration of Raman dyes, (ii) the density control of metal nanoparticle aggregates, and (iii) the electrohydrodynamic injection control, It is possible to overcome the detection limit corresponding to the LOD of the LC-MS of FIG.

Thus, the present invention provides a nanofiber composite substrate for analyzing surface-enhanced Raman scattering, which is characterized in that aggregates of metal nanoparticles formed by the Raman dye are uniformly distributed in the cross-linkable polymer hydrogel nanofiber To a method for effectively detecting and comparing and analyzing a trace amount of a substance.

All of the features described above can be useful features for use in the biosensing and bioimaging tool of the nanofiber composite substrate for surface-enhanced Raman scattering analysis of the present invention.

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

(Bovine serum albumin), rhodamine B isothiocyanate-linked dextran RITC-dextran (MW 70 kDa), rhodamine (sodium nitrate), sodium borohydride, sodium citrate tribasic dihydrate, BSA B isothiocyanate (RBITC mixed isomer), 2-hydroxy-2-methylpropenone, acetone, albumin-fluororesin isothiocyanate bonded (FITC-BSA), ethylene glycol anhydride, crotononyl Crotonoyl chloride, Malachite green isothiocyanate and sodium hydroxide (= 98%) were purchased from Sigma Aldrich. Poly (acrylamide-co-acrylic acid, sodium salts) and P (AAm-co AA) (MW 200 kDa; 10% acrylic acids) were purchased from Polysciences Inc. and all reagents were used.

Experimental Method

One. UV - Vis  And Raman measurement

All UV-Vis absorbance spectra were measured at wavelengths of 300-800 nm using UV-1800 (Shimadzu, Japan) at room temperature.

The maximum absorbance was labeled to analyze the characteristics of AgNPs and AgNCs (silver nanoparticle aggregates). A small sample of the sample was taken at the specified time interval and a Raman spectrum was obtained under a He-Ne laser beam excited at a wavelength of 633 nm on a Reinshaw 2000 microscope integrated system. A He-Ne laser with a wavelength of 633 nm was used to excite the Raman reporter molecule with 10 mW laser power. Rayleigh lines were removed from the collected spectra using a holographic notch filter.

2. SEM , TEM Imaging

To characterize the shape of the polymer nanofibers by scanning electron microscopy, small square pieces of unstained steel sheets (5 x 5 mm) were placed on the aluminum foil surface during electrospinning.

These fibers were then coated with Pt sputtering using a K575X Turbo Sputter Coater and imaged using SEM VEGA (TESCAN, USA) operating at an accelerating voltage of 0.5 to 30 kV. Images were taken at different magnifications of 5.0KX, 8KX and 10KX. Single silver nanoparticles (AgNPs) and silver nanoparticle aggregates (AgNCs) were characterized for particle size and size distribution analysis using transmission electron microscopy (TEM). As the prepared silver seed, the suspension was filtered through a 0.2 μm Dismic 13 Jp (ADVANTEC, Japan) disposable PTFE hydrophobic syringe filter and diluted 5 times before sample preparation for TEM. A small amount of ethanol can be mixed for rapid evaporation.

Similarly, the prepared silver nanoparticle aggregates were filtered with a 0.2 μm filter, diluted 4 times with DI water, and placed on a TEM grid (Ted Pella Inc. USA). These samples were imaged using a JEM-2100F (FEG) Field Emission Scanning Transmission Electron Microscope (JEOL, Germany) operating at an acceleration voltage of 80-200 kV.

3. Confocal  Laser scanning microscope

Polymeric nanofibers were imaged directly from the cover glass using a Leica TCS SP2 (Leica, Germany) confocal laser scanning microscope with a 100X objective (oil-immersed lens). The suspended fibers of the small specimens were placed on a cover glass and the component properties and coupling efficiency were investigated.

Example  One : Silver nano  Particle aggregates (silver nanoclusters, AgNCs )

1-1. Synthesis of silver seed particles

Seed particles were prepared according to the method described in the paper (J Colloid Interface Sci. 211, 122-129 (1999)). A stock solution of 0.5 M nitrate, sodium citrate and 0.05 M sodium borohydride in stock solution was prepared in DI water. Both solutions were prepared separately. Solution A contained 4 mM silver nitrate in 50 ml of DI water while Solution B contained 0.6 mM sodium borohydride, 8 mM sodium citrate and 2 mM sodium hydroxide in 50 ml of DI water. Both solutions were stirred for 5 minutes before mixing to homogenize the concentration. By adding Solution A to Solution B, silver seed particles were prepared. The reaction was completed by performing for 10 hours under dark conditions.

The seed particles were filtered through a 0.2 [mu] m filter to remove any silver nanorods or large particles. The silver seeds produced in this way were in the range of 6-13 nm in diameter. These particles were stored at 8 ~ 10 ℃ under dark conditions.

1-2. Silver nano  Particle aggregates ( AgNCs ) Synthesis of

For synthesis of silver nanoclusters (AgNCs) with RBITC as Raman receptor and flocculant, 50 ml of silver seed particles were placed in glass vials and mixed with 0.01 M RBITC, 0.5 M AgNO ₃ and 0.5 M sodium citrate solution to give final concentration To be 7.5 mu M, 0.85 mM and 0.5 mM, respectively. The mixture was gently agitated for at least 5 minutes and then placed in an electrically heated oven at 95 < 0 > C for a fixed period of time.

Small samples of samples (300 μl each) were taken at regular time intervals to optimize cluster size and maximum Raman intensity and were tested for SERS signal measurements. After diluting the samples 5 times with 1 mM sodium citrate aqueous solution, UV-Vis absorbance spectroscopy. When maximum Raman intensity was obtained, the reaction was stopped. The mixture containing AgNCs was quickly cooled to room temperature and mixed with 0.5% w / v BSA aqueous solution to a final concentration of 0.0018 5 w / v to stabilize AgNCs. The suspension was filtered through a 0.2 [mu] m PTFE hydrophobic syringe filter and stored at room temperature for further analysis

Continuous experiments were performed to optimize cluster size and individual particle size and achieve maximum Raman signal.

Example  2 : AgNCs  Encapsulation ( Encapsulation ) ≪ / RTI >

2-1. UV - crosslinking Poly ( acrylamide - co - acrylic acid ; UV- P ( AAm - co-AA)

The photo-crosslinkable polymer was prepared from the reaction of P (AAm-co-AA) and Crotonoyl chloride.

The carboxy group of acrylic acid can be partially changed into a double bond containing a residue. Crotonyl chloride was reacted with the carboxy group and a double bond was introduced at this position.

In a typical reaction, 2.0x10 < -6 > M P (AAm-co-AA) was present in 50 ml DI at room temperature. The crotonyl chloride was added very slowly to the vigorously stirred polymer solution to avoid a temperature rise to a final concentration of 0.0208M. After 5.0 hours of reaction, the mixture was poured into 150 ml of dry acetone and precipitated. The precipitate was washed three times with acetone and stored under dark conditions.

2-2. Polymer  Within nanofiber AgNCs  Encapsulation ( Encapsulation )

Prepared AgNCs were repeatedly centrifuged at 7000 rpm for 8 minutes and washed 3 times with 1 mM sodium citrate solution to remove any excess BSA and other reagents.

AgNCs were then resuspended in DI water containing a small amount of citrate ions. A 15.0 w / v% solution of UV-P (AAm-co-AA) was prepared in DI water. RBITC-Dextran was added at 0.5 w / v% of the fluorescence staining reagent of the polymer. 2-Hydroxy-2-methylpropiophenone (photoinitiator) was added at a concentration of 0.0024 w / w% polymer.

Various amounts of AgNCs were suspended in the polymer solution. The well dispersed suspension was loaded onto a 1 ml syringe (Becton-Dickinson, New Jersey, US) equipped with a 21 gauge (NanoNC, Korea) metal capillary. A continuous syringe pump (KdScientific) was used to maintain a continuous flow rate of 0.2 ml per hour. The anode of the high voltage supply (NNC HV 30) was connected to a capillary and the cathode was contacted to a collected substrate made of aluminum foil (Fisherbrand, US). The interelectrode separation varied from 10 to 15 cm.

The voltage was varied from 14 to 17 kV and the flow rate was maintained in the range of 0.20 to 0.30 ml / hr. Electrospinning experiments were performed in ambient conditions. For nanofibers for use in confocal laser scanning microscopy in the solid state, a small cover glass was placed on an aluminum foil. The aluminum foil was exposed to UV light for 30 seconds using Omnicure-1500A (Lumen Dynamics Group Inc. Canada) at 60 mW / cm ² power in the 320-500 nm wavelength range to crosslink the polymer chains.

Example  3: silver nanoparticle size and nanocluster size

UV-Vis absorbance spectra were obtained during AgNCs formation, confirming the ideal individual particle size and cluster size.

The results are shown in Fig.

Figure 2 (A) shows the absorbance spectra collected at different time intervals during the formation of silver nanoparticle aggregates. Individual particle sizes increased with increasing incubation time, resulting in large, narrow absorbance peaks at 400 nm. At 400 nm, the major absorbance peak shifted to a long wavelength band of 429 nm in 60 minutes. An additional peak observed at 649 nm at 50 min indicates cluster formation. RBITC had a characteristic peak at 555 nm.

Fig. 2 (A) shows the start of incubation, where the increase in size occurred more rapidly due to the reduction of sufficient silver ions, and the curve became more flat over time of 40 minutes.

Transmission electron microscopy confirmed the diameter and shape of single silver nanoparticles and silver nanoclusters (AgNCs).

The results are shown in Figs. 2 (C) and 2 (D).

FIG. 2 (C) shows that the cluster has a size in the range of 60 to 100 nm after a 60-minute incubation time in the TEM image, and FIG. 2 (D) shows that the single seed particle has a size in the range of 6 to 13 nm. Individual particle sizes increased with increasing incubation time, and also the aggregation of single particles resulted in the formation of aggregates of nano-particles, which are uniquely observed in the UV-Vis spectrum.

Example  3: AgNCs Raman signal measurement of

In addition, Raman signals were measured during manufacture of AgNCs.

FIG. 2 (E) shows the increasing Raman signal of the aggregate of the silver nanoparticles over the agglomeration reaction time. The maximum Raman signal was obtained after 60 minutes incubation time. Very long incubation times had adverse effects on signal intensity, presumably due to larger cluster formation.

Figure 2 (F) shows two major Raman peak intensity flats versus RBITC spectra versus incubation time. No noticeable difference in the position of the peak among all the spectra collected at different time intervals appeared.

The RBITC concentration was optimized so that the cluster had a maximum Raman signal after growing to 60 ~ 100 nm. Isothiocyanate dyes such as RBITC and MGITC have stronger agglomeration tendencies compared to other dyes. It was found that the optimal dye concentration of 7.5 μM RBITC according to the incubation time of 60 minutes produced the maximum SERS signal.

The colloidal stability of silver nanoparticles is confirmed by the capping of the negatively charged citrate ions around the silver nanoparticles. This is dependent on the principle that the coalescence mechanism of silver nanoparticles by Raman dyes decreases as the electrostatic repulsion between the nanoparticles by the anions decreases as the Raman dye is absorbed from the surface of the metal nanoparticles. The size of the individual particles increases due to the additional deposition of silver, and the dye molecules are captured with high probability at the junction of the merging particles.

This type of structure is associated with high Raman signals due to the high electromagnetic fields present in the spots. The small silver seed nanoparticles had very low Raman intensity as evidenced by the increase in nanoparticles until the individual nanoparticles had a maximum particle size of 25-30 nm and an average aggregate (cluster) size of 76 nm. After a long incubation time of 80 to 90 minutes, aggregate (cluster) size was quite large and precipitated quickly, resulting in a decrease in signal intensity.

The useful meaning of aggregate (cluster) formation is to increase the turbidity of the mixture, the appearance of added peaks in the UV-Vis spectrum, and the maximum Raman signal. During electrospinning, AgNCs in the polymer nanofibers are encapsulated and the polymer crosslinks by exposure to UV light.

To confirm this, the fibers obtained were exposed to water and observed by CLSM imaging in the swollen state.

As a result, as shown in Fig. 3, the Raman signals were very stable and retained their original characteristic peaks before and after the photo-crosslinking.

There was no significant decrease in the signal after UV cross-linking compared to the signal decreased in thermal cross-linking, which resulted in a significant reduction of the Raman signal by other cross-linking, e. G. Desorption of the dye at high temperatures.

Example  5: Various parameters and Rahman signal  Relationship

Based on the results of previous experiments on incubation time, cross-linking type, temperature, etc., the Raman signal can be easily quantified by varying the number of parameters that can be readily met by controlling the number of AgNCs in the polymer nanofiber or the number of hot spots And the like.

Therefore, SERS signal characteristics according to various parameters were investigated.

Another mechanism that occurs simultaneously in nanofibers is the potential fusion of the SERS signal because of the local fusion of the dye molecules with the single nanoparticles at the binding site where the dye molecules are located in the electromagnetic field at the binding site. Such dye aggregation is useful for achieving high signal intensification. Therefore, the experiment was used for temperature, incubation time, dye concentration, temperature and amount of silver ion added.

5-1 Relationship with Temperature

First, it can be seen that the SERS signal exhibits adverse effects at high temperatures.

Although other dyes have different stability as signals at high temperatures, in the case of dyeings containing isothiocyanate groups, as compared to other dye agents such as R6G and CV, the isothiocyanate group and the metal-sulfur bond between the metal particles And is stabilized by bond formation. The lower signal is thought to result from the desorption of dye from silver nanoparticles, which reduces the SERS effect. Most organic dyes have a stable chemical structure at 175 ° C (thermal cross-linking temperature), eliminating the possibility of dye degradation.

However, the photocrosslinking of nanofibers with Raman active centers inside was done under moderate conditions to prevent significant degradation of the SERS signal.

Successful polymerisation of P (AAm-co-AA) and crotonyl chloride was confirmed from the H ¹ NMR spectrum.

The pure P (AAm-co-AA) showed no characteristic peaks of double bonds in the crotonyl chloride at 6.640, 6.957, 6.974, 6.977, 6.996, 7.013 and 7.030, which was also seen in the modified polymer as shown in Figure 3 .

5-2. AgNCs  Relationship with quantity

In addition, the Raman signal intensity varied depending on the amount of AgNCs in the polymer nanofibers, while retaining other unchanging parameters such as spinning time, dye concentration, cluster size, and individual silver particle size.

In particular, as shown in FIG. 4, the relationship between the Raman intensity and the amount of AgNCs was shown as a linear graph. Therefore, it can be predicted that the SERS signal can be controlled by the introduction of various amounts of AgNCs.

A plot of the Raman intensity of the specific peak in the RBITC spectrum versus the amount of encapsulated AgNCs (nanoparticle aggregates) in nanofibres is shown in Figures 4 (C, D).

* 1645.9 cm < -1 > was selected as the reference peak from the RBITC spectrum and signals at different AgNCs concentrations were compared. It was also found that the introduction of different amounts of AgNCs in the polymer nanofibers facilitates the control of the desired Raman signal.

5-3. Relationship with Solvent

The Raman signal stability of the encapsulated AgNCs in nanofibers was analyzed by immersing these fiber mats in other solvents.

The change in the Raman signal intensity in other solvents, i.e. DI water, acetone and ethanol, is shown in Fig. 5 (A).

The signal was stable and no significant degradation due to exposure to this solvent was seen. It is believed that the crosslinking of the polymer shell by UV irradiation prevents degradation of the silver nanoclusters that stabilize the SERS substrate.

5-4. Reproducibility of signal

On the other hand, one of the most important forms of the SERS substrate is the reproducibility of the SERS signal, especially for quantification purposes. Raman spectra were collected from 14 randomly selected spots on the surface of the nanofiber substrate to confirm the reproducibility of the signal.

These results are shown in Fig. 5 (B).

The intensity of the main peak is indicated in the other spot. Significantly reproducible signals showed small deviations from the mean.

5-5. pH  And salt concentration

In addition, the signal stability at different pH values was analyzed in the range of 1.5 to 11.0 by varying the pH value using 1.0 M NaOH and 0.5 M HCl at room temperature.

As a result, as shown in Fig. 5 (C), the Raman signal intensity of the encapsulated Raman active nanoparticle aggregates was stable over a wide range of pH. A crosslinked polymer network covering silver nanoparticle aggregates protects the unique properties of these substrates.

Then, the signal intensity of the substrate was examined over the salt concentration. A 1.0 M aqueous NaCl solution was used as the source of the chloride ion.

* Raman spectral results are shown in Fig. 5 (D). The nanofiber matte encapsulating AgNCs showed a very stable signal over a wide range of salt concentrations.

Example  6: Characterization of nanocluster and nanofiber mat

SEM imaging was used to determine the morphological characteristics and size of the nanofiber mat that encapsulated the AgNCs.

The results are shown in Fig.

6-1. Nanofiber size

The nanofibers were prepared by electrospinning directly on an unstained steel substrate for 5 minutes and the diameters of the nanofibers ranged from 220 to 260 nm.

Other polymer concentrations of 7.5% w / v, 10.0% w / v, 12.5 w / v% and 15.0 w / v% were used to improve fiber size and uniformity.

The lower polymer concentration showed a mixture of nanofibers and nanoparticles along the bead along the string structure, and as the polymer concentration increased, the relative amount of fibers increased.

In addition, in experiments using polymer concentrations of at least 12.5 w / v%, the particles and beads in the string structure were removed. It was found that the average diameter of the nanofibers was increased by using a high polymer concentration, decreasing the distance on the electrode and increasing the flow rate.

6-2. zeta Potential

Also, the adsorption of dye on the surface of the nanoparticles resulted in a significant reduction in the zeta potential of the silver colloid, which leads to cluster formation (data not shown).

6-3. Crosslinking

The crosslinked polymer nanofiber mat was also confirmed in a dry state and in a swollen state using a confocal laser scanning microscope.

Figure 6 (E) shows the polymer nanofibers encapsulating AgNCs in the dry state directly imaged under a microscope.

Figure 6 (F) is an image of fibers swollen in aqueous solution after successful cross-linking of the polymer. The nanofibers were photocrosslinked, ultrasonicated for 1 minute and resuspended in DI water. The fibers were swollen into a soft matrix after immersion in water to hold AgNCs. Such hydrogel properties will be very useful for many biological devices.

6-4. Varying amounts of AgNCs  Introduction

In addition, successful encapsulation of RBITC-based AgNCs in nanofibers at various densities was confirmed using field emission scanning transmission electron microscopy (SEM).

The increase in Raman signal with increasing AgNCs in the nanofibers was consistent with the increased density of AgNCs in the polymeric nanofibers identified by the TEM image as shown in FIG. In other words, it can be seen that the SERS signal can be controlled by introducing various amounts of AgNCs in the nanofiber.

Example  7: Malachite  Green isothiocyanate trail detection

Another solution of malachite green isothiocyanate with various concentrations ranging from 0.5 ppm to 6.0 ppm was prepared in a 3: 1 by volume ratio water and ethanol mixture. The nanofiber mat placed on an aluminum foil was cut to an appropriate size and immersed in the MGITC solution for 30 minutes. The mat was thoroughly washed three times with DI water and used for Raman spectral analysis.

The Raman spectra were collected for an exposure time of 5 seconds at 633 nm with He / Ne laser illumination. All samples were analyzed under the same conditions of a Rahman scope. The captured spectra were modified and used for quantitative analysis. Before use for analyte detection, a uniform SERS signal was observed at various spots of the substrate.

The SERS spectra collected after immersion of the nanofiber mat in solutions of various concentrations of the analyte were compared to the original spectra of the nanofiber mat. Each sample was tested for at least 5 scan cycles and the average of the resulting SERS spectra was determined. Raman intensity at 1621 cm-1, the inner Raman dye, 1646cm generated from RBITC ^- consistent with peak for analysis in the ^first water. The ratio of the internal analyte peak intensity to the intensity of the internal peak was displayed for increasing concentrations of the analyte solution.

In the ethanol-water mixture, MGITC, which is water-soluble, readily adsorbs to the surface of nanoclusters that diffuse into nanofibers and produce high SERS signals. Raman signals generated from MGITC after diffusion into polymer nanofibers were measured and shown for various concentrations. It was sensitive to 0.5 ppm of MGITC in aqueous solution. The RBITC peak was used as an internal standard.

As a result, as shown in FIG. 8, the MGITC peak intensity increased with concentration, confirming that there was a linear relation between the concentration and the Raman intensity.

The RBITC characteristic peak area is kept constant through analysis, which means that it exhibits excellent stability without severe diffusion of RBITC in solution. The RBITC peak-constant region was used as an internal standard to eliminate errors caused by the intensities of the different peaks in the MGITC spectrum under different conditions, which can be compared to the gradually increasing MGITC peak-under regions. In order to obtain highly reproducible quantitative results, it was found that the ratio of the increasing SERS signal to the constant internal signal is important, instead of using only one analyte as the quantitative analysis tool.

Claims (6)

Preparing a nanofiber composite substrate for surface-enhanced Raman scattering analysis, wherein aggregates (clusters) of the metal nanoparticles by the Raman dye are uniformly distributed in the cross-linkable polymer hydrogel nanofiber; And
Detecting the analyte by immersing the nanofiber composite substrate in a solution containing the analyte to cause the analyte to diffuse through the nanofiber composite substrate to be adsorbed on the surface of the nanoparticle aggregates;
A surface-enhanced Raman scattering analysis method comprising the steps of:
(Raman scattering) induced by the Raman dyes existing in the substrate, the intensity of the internal standard signal can be compared and analyzed.
Wherein the nanofiber composite substrate is formed by cross-linking between the polymers surrounding the agglomerates of the metal nanoparticles by treating the nanofibers with ultraviolet light, forming a network between the aggregates of the metal nanoparticles and the polymer, Wherein the agglomerates of the metal nanoparticles are fixed to the nanofibers to form a nanofiber substrate.
delete delete The method according to claim 1,
The method
Wherein the analyte is detectable when the analyte is present in a concentration of 0.01 to 6 ppm.
The method according to claim 1,
Wherein the analyte is a carcinogenic substance.
delete

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