HIGH-SENSITIVITY LATERAL FLOW IMMUNOASSAY STRIP BASED ON SURFACE-ENHANCED RAMAN SCATTERING AND DETECTION METHOD USING THE SAME

Abstract

The present disclosure relates to a surface-enhanced Raman scattering (SERS) lateral flow immunoassay strip containing: a sample pad into which a sample containing a target material is introduced; a conjugate pad containing a hollow metal nanoprobe for surface-enhanced Raman scattering, on which an antibody that can be coupled to the target material and a Raman marker are immobilized; and a detection pad including a detection region to which a secondary antibody that can be coupled to the target material coupled to the hollow metal nanoprobe is immobilized. Use of the SERS-based lateral flow immunoassay strip according to the present disclosure enables high-sensitivity quantitative analysis and qualitative analysis of the target material from Raman signal measurement depending on the concentration of the target material.

Claims

1. A method of measuring a concentration of a target material in a sample, comprising: introducing the sample on a surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay strip comprising: a sample pad into which the sample comprising the target material is introduced; a conjugate pad consisting of a hollow metal nanoprobe for surface-enhanced Raman scattering, on which an antibody that is capable of coupling to the target material and a Raman marker are immobilized, wherein only the antibody and the Raman maker are directly immobilized on the hollow metal nanoprobe, wherein only the hollow metal nanoprobe is directly absorbed to the conjugate pad, wherein a primary immune complex of antibody-target material-hollow metal nanoprobe is formed; and a detection pad comprising a test region to which a secondary antibody that is capable of coupling to the target material coupled to the hollow metal nanoprobe is absorbed and a control region to which an another antibody that is capable of coupling to an unbound hollow metal nanoprobe; acquiring a Raman point mapping image of the test region and the control region, respectively, wherein a SERS signal is acquired from 1600 pixels of the test region and the control region, respectively; identifying a color development at the detection pad and measuring the SERS signal to detect the target material; and generating an average SERS intensity for the 1600 pixels of the test region and the control region, respectively, measured by a Raman point mapping.

2. The method of claim 1, wherein the hollow metal nanoprobe is a hollow gold nanoparticle.

3. The method of claim 1, wherein a detection limit of the target material is 0.001 ng/mL or lower.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows a diagnostic strip commonly used in lateral flow immunoassay.

[0049] FIG. 2 compares the existing lateral flow immunoassay (a) with SERS-based high-sensitivity lateral flow immunoassay according to the present disclosure (b).

[0050] FIG. 3 shows the TEM images (a), UV/VIS absorption spectrum (b) and DLS distribution (c) of a hollow gold nanoparticle (HGN).

[0051] FIG. 4 shows a DLS signal from an antibody physically (a) or chemically (b) immobilized on HGN.

[0052] FIG. 5 shows the SEM image of a HGN immune complex when 10 ng/mL SEB is present on a test line (a) and the SEM image of a HGN immune complex when SEB is absent (b).

[0053] FIG. 6 shows the quantitative analysis result of a target material through SERS mapping using a lateral flow immunoassay strip according to an exemplary embodiment of the present disclosure.

[0054] FIG. 7 shows the result of visual detection and detection by enzyme-linked immunosorbent assay (ELISA) of SEB at different concentrations.

[0055] FIG. 8 compares the sensitivity of SERS-based lateral flow immunoassay according to an exemplary embodiment of the present disclosure and the existing technique.

[0056] FIG. 9 shows the images of a SERS-based lateral flow immunoassay strip of the present disclosure and the result of SERS mapping when SEB, SEA (Staphylococcus aureus enterotoxin A), ochratoxin, aflatoxin and fumonisin are present.

[0057] FIG. 10 shows the SERS mapping images of SEB at low concentrations (500, 100, 50, 10 and 1 ng/mL) (a), LFA images (b) and quantitative analysis result obtained from curve fitting of the SERS mapping images (c). The effect of nonspecific conjugation was tested using an antigen cocktail containing five different antigens (SEB, SEA, ochratoxin, aflatoxin and fumonisin).

[0058] FIG. 11 compares the Raman intensity of a test line of a SERS-based LFA strip using HGN (hollow gold nanoparticle) (a) and a SERS-based LFA strip using GNP (gold nanoparticle) depending on the concentration of SEB.

[0059] FIG. 12 shows the calibration curves for quantitative analysis of a SERS-based LFA strip using HGN (a) and a SERS-based LFA strip using GNP depending on the concentration of SEB.

DETAILED DESCRIPTION OF THE INVENTION

[0060] Hereinafter, specific examples are presented to help understanding the present disclosure. However, the following examples are given only as examples of the present disclosure and it will be obvious to those of ordinary skill in the art that various changes and modifications can be made within the scope of the present disclosure. Also, it will be obvious that such changes and modifications belong to the scope of the appended claims.

<Example 1> Materials

[0061] HAuCl.sub.4 (gold(III) chloride trihydrate), Na.sub.3-citrate (trisodium citrate), DHLA (dihydrolipoic acid), EDC (1-ethyl,3-(3-dimethylaminopropyl)carbodiimide), NHS (4-(4-maleimidophenyl)butyric acid N-succinimidyl ester), CoCl.sub.2 (ethanolamine, cobalt (II) chloride), BSA (bovine serum albumin), PVP (polyvinylpyrrolidone), tris-EDTA buffer (TE buffer, pH 8.0), S9008, rabbit anti-SEB (anti-staphylococcal enterotoxin B polyclonal antibody produced in rabbit) and anti-mouse IgG (anti-mouse IgG antibody produced in goat) were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Surfactant G was purchased from Fitzgerald (Concord, Mass., USA). MGITC (malachite green isothiocyanate) was purchased from Invitrogen Corporation (Carlsbad, Calif., USA). S222 and Mouse anti-SEB (anti-staphylococcal enterotoxin B monoclonal antibody produced in mouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). SEB (recombinant enterotoxin type B for Staphylococcus aureus) was purchased from Cusabio (Wuhan, China). A NC (nitrocellulose) membrane-attached backing card (Hi-flow plus HF180) was purchased from Millipore Corporation (Billerica. MA, USA). An absorption pad (CF3) was purchased from Whatman-GE Healthcare (Pittsburgh, Pa., USA).

<Example 2> Preparation of SERS-Based Lateral Flow Immunoassay Kit

[0062] 2-1: Synthesis of Hollow Gold Nanoparticle (HGN) and Immobilization (Conjugation) of Antibody

[0063] A hollow gold nanoparticle (HGN) was synthesized according to methods described in literatures (C. E. Talley, J. B. Jackson, C. Oubre, N. K. Grady, C. W. Hollars, S. M. Lane, T. R. Huser, P. Nordlander and N. J. Halas, Nano Lett., 2005, 5, 1569-1574; A. M. Schwartzberg, T. Y. Oshiro, J. Z. Zhang, T. Huser and C. E. Talley, Anal. Chem., 2006, 78, 4732-4736; H. Chon, S. Lee, S.-Y. Yoon, E. K. Lee, S.-I. Chang and J. Choo, Chem. Commun., 2014, 50, 1058-1060). Briefly, the hollow gold nanoparticle was synthesized by growing a gold nanoshell by reducing gold atoms on the surface of a cobalt nanoparticle used as a support and controlling the same. The cobalt nanoparticle was synthesized by reducing CoCl.sub.2 with NaBH.sub.4 under a N.sub.2 purging condition. After adding a HAuCl.sub.4 solution to the synthesized cobalt nanoparticle to induce nucleation of gold atoms in the solution, a thin shell enclosing the cobalt nanoparticle was grown. Then, the cobalt nanoparticle was completely dissolved to synthesize a hollow gold nanoparticle. The particle size and physical properties of the prepared hollow gold nanoparticle were evaluated by UV/Vis absorption spectroscopy, TEM (transmission electron microscopy) and DLS (dynamic light scattering) (FIG. 3).

[0064] The prepared hollow gold nanoparticle had a size of 45±12 nm and a thickness of 15±5 nm. A SERS nanoprobe was prepared from the hollow gold nanoparticle as follows. 5.0 μL of a MGITC Raman marker at a concentration of 10 μM was added to 1 mL of the hollow gold nanoparticle at a concentration of 0.1 nM and reaction was conducted for 30 minutes. The MGITC-adsorbed hollow gold nanoparticle was reacted for 30 minutes by adding 0.1 μL of 1.0 mM DHLA to substitute the surface of the nanoparticle with carboxyl groups. The carboxyl-substituted hollow gold nanoparticle was reacted for 1 hour by adding 1.0 μL of a 0.1 mM EDC/NHS solution. Then, reaction was conducted for 1 hour by adding 0.1 μL of 1.0 mg/mL mouse anti-SEB. After removing unreacted materials and antibodies through centrifugation, the unreacted portion on the surface of the hollow gold nanoparticle was inactivated by adding 0.5 μL of 1.0 mM ethanolamine. The prepared antibody-immobilized hollow gold nanoparticle was stored at 4° C. In order to improve the efficiency of reaction and diffusion of the nanoparticle in a lateral flow immunoassay (LFA) strip, a mixture of 20 μL of 10× concentration antibody- and MGITC-immobilized hollow gold nanoparticle, 20 μL of surfactant G (10%), 20 μL of PVP (10%) and 40 μL of TET buffer (Tween 20, 0.05 v/v %, pH 8.0) was used for a lateral flow immunoassay strip sensor.

[0065] 2-2: Preparation of Lateral Flow Immunoassay (LFA) Strip

[0066] A lateral flow immunoassay strip consisted of a sample pad for sample injection, a conjugate pad on which the hollow gold nanoparticle is adsorbed, a nitrocellulose (NC) membrane as a detection pad and an absorption pad. In order to prepared the strip, a nitrocellulose membrane with a size of 3-10 μm was attached to a support (plastic backing card) and an absorption pad was attached to the end of the nitrocellulose membrane. A test line and a control line in the nitrocellulose membrane were prepared using 0.5 mg/mL rabbit anti-SEB and 0.1 mg/mL mouse anti-IgG. Each antibody was sprayed onto the nitrocellulose membrane at a concentration of 0.5 μL/cm using a precision line dispensing system (Zeta Corporation, South Korea). The antibody-sprayed nitrocellulose membrane was dried at room temperature for 1 hour. The nitrocellulose membrane having the antibodies adsorbed in the form of lines was cut to a thickness of 3.8 mm using a programmable cutter (Zeta Corporation, South Korea). Immunoassay using the prepared lateral flow immunoassay strip was conducted by dropping a sample onto a 96-well ELISA plate and then immersing the strip for simplification of the analytical procedure.

[0067] 2-3: Methods for Detection and Analysis

[0068] The Raman spectra and SERS mapping images of the test line in the lateral flow immunoassay (LFA) strip were acquired using the inVia Raman microscope system (Renishaw, New Mills, United Kingdom). The inVia Raman microscope system uses a He—Ne laser with a power of 3 mW operating at a wavelength of 633 nm. The Rayleigh line was removed by placing a holographic notch filter in the collection path. Raman scattering was collected using a CCD (charge-coupled device) camera at a spectral resolution of 1 cm.sup.−1. The Raman images were acquired by Raman point mapping using a 50× lens. The detection range was set using a stage that can be translated along x- and y-axes in micrometer scales and Raman signals were acquired from a total of 1600 pixels from a range of 200 μm (x-axis)×800 μm (y-axis) with a step size of 10 μm×10 μm. The SERS images obtained from the strip were corrected using the WiRE software V 4.0 (Renishaw, New Mills, United Kingdom) and the intensity of the Raman signal for each pixel was quantitatively analyzed using the peak of the Raman marker MGITC at 1615 cm.sup.−1. The average spectrum of the SERS image of the lateral flow immunoassay strip at each concentration was obtained from the total pixels and quantitative analysis for different SEB concentrations was conducted based on this.

[0069] The physical properties of the prepared nanoparticle were analyzed using the Cary 100 spectrophotometer (Varian, Salt Lake City, Utah, USA) and the DLS (dynamic light scattering) Nano-ZS90 (Malvern). The shape and size of the prepared nanoparticle were identified from high-magnification TEM (transmission electron microscopy) images. The adsorption of the hollow nanoparticle in the lateral flow immunoassay strip was identified by SEM (scanning electron microscopy). Enzyme-linked immunosorbent assay (ELISA) was conducted for comparison with the immunoassay according to the present disclosure and calibration curves versus SEB concentration were constructed using a microplate reader (Power Wave X340, Bio-Tek, Winooski, Vt., USA). The Raman intensity of the test line in the lateral flow immunoassay strip at different SEB concentrations was identified using the Chemi-Doc imaging system (Bio-Rad, Hercules, Calif., USA).

<Example 3> Detection of Target Material Using SERS-Based LFA Strip

[0070] The operating principle of the surface-enhanced Raman scattering (SERS)-based high-sensitivity lateral flow immunoassay (LFA) strip is sandwich-type immunoassay. FIG. 2 (a) shows the operating principle of a general lateral flow immunoassay strip. An unknown sample containing a target material is dropped onto a sample pad of the LFA strip. The sample passes through a conjugate pad by a capillary phenomenon. An antibody-immobilized nanoparticle physically adsorbed to the conjugate pad undergoes an immune reaction with the target material in the sample. The resulting immune complex (antigen-gold nanoparticle) moves continuously toward a NC membrane by the capillary phenomenon and reaches a test line, where it undergoes a secondary immune reaction with an antibody adsorbed in the test line. A red line develops as the gold nanoparticle is accumulated in the test line. The excess antibody-conjugated gold nanoparticle moves further and is captured by an antibody adsorbed in a control region. Finally, two red lines appear if the target material is present (positive), and only one red line appears if the target material is absent (negative). The red line of the control region shows that the LFA strip works well.

<Example 4> Quantitative Analysis Using SERS-Based LFA Strip

[0071] Although the existing LFA strip is commercialized and used as a POC (point-of-care) detection device, it has the problem that the sensitivity of analysis is low. This is a serious handicap in the early diagnosis of a disease. In addition, the existing lateral flow immunoassay sensor has the problem that quantitative analysis is impossible. The SERS-based LFA strip of the present disclosure overcomes these problems.

[0072] FIG. 2 (b) describes an analysis platform of the SERS-based LFA strip. The analytical method is identical to that of the existing lateral flow immunoassay sensor but there is a difference in the condition of the nanoparticle. The nanoparticle used in the SERS-based LFA strip is a hollow gold nanoparticle (HGN) on which a Raman marker is adsorbed. It is used for quantitative analysis of a target material (antigen) to be detected. In the presence of an antigen, the nanoparticle is accumulated in the test line and the test line develops a red line. Quantitative analysis of the concentration of the antigen is possible using the SERS signal from the Raman marker on the surface of the accumulated nanoparticle.

[0073] The method of immobilizing an antibody on the surface of the hollow gold nanoparticle may affect the flow in the strip because it can induce aggregation and instability of the nanoparticle. Therefore, the stability and flow ability of the nanoparticle in the strip were evaluated for immobilization of the antibody by physical adsorption and immobilization of the antibody by chemical reaction.

[0074] In Example 2-1, the antibody was immobilized by a chemical method. It is contrasted with physical adsorption. In the physical adsorption, the antibody is adsorbed on the surface of the hollow gold nanoparticle using electrostatic attraction. Details are as follows. After adding 1 μL of 1 mg/mL mouse anti-SEB to 1 mL of the prepared hollow gold nanoparticle, reaction was conducted for 1 hour. The surface of the hollow gold nanoparticle interacts with the antibody through electrostatic attraction. Unreacted residues were removed by centrifugation.

[0075] FIG. 4 shows DLS signals for the two types of antibody immobilization method (physical and chemical immobilization). The hollow gold nanoparticle on which the antibody is immobilized by physical adsorption (a) exhibits increased nonspecific aggregation of the nanoparticle and decreased efficiency of antibody reaction as compared to chemical immobilization. From comparison of FIGS. 4 (a) and (b), it was confirmed that the antibody immobilization by chemical substitution results in a narrower nanoparticle size distribution as well as a superior reaction efficiency without nonspecific aggregation or disturbed flow in the strip.

[0076] FIG. 5 shows the SEM image of the HGN immune complex formed in the test line depending on the presence or absence of the antigen (SEB). From FIG. 5 (a), it can be seen that the HGN immune complex forms clusters between the pores of the NC membrane when the antigen (SEB) is present (10 ng/mL). Due to these clusters, the test line turns red (“On”) and the SERS activity is exhibited with high sensitivity. In the absence of the antigen (SEB), as seen from FIG. 5 (b), no color change (“Off”) or SERS activity is observed because the immune complex is not formed in the test line. This is consistent with the color development of the test line depending on the antigen concentration. To conclude, when the SERS-based LFA strip of the present disclosure is used, the presence or absence of the target material can be detected visually as in the existing LFA strip. However, the SERS-based LFA strip of the present disclosure also enables quantitative analysis of the target material because the characteristic Raman signals of the SERS nanoprobe can be acquired depending on the concentration of the target material.

[0077] FIG. 6 (a) shows the SERS mapping result for the peak of the lateral flow immunoassay strip at 1615 cm.sup.−1 for SEB at different concentrations (0-1000 ng/mL). Images were acquired from 80×20 pixels (1 pixel=10 μm×10 μm) at each concentration from 0 to 1000 ng/mL. The detection range of Raman point mapping image was set using a stage that can be translated along x- and y-axes in micrometer scales and Raman signals were acquired from a total of 1600 pixels from a range of 200 μm (x-axis)×800 μm (y-axis) with a step size of 10 μm×10 μm. The scale bar at the bottom left represents the SERS intensity, which is determined by the intensity of the peak at 1615 cm.sup.−1. To conclude, it was confirmed that the amount of the immune complex formed in the test line increases as the SEB concentration changes from 0.1 pg/mL to 1000 ng/mL and this leads to increase in the SERS intensity.

[0078] However, the intensity was not uniform in the same region of the 1600 pixels of the mapping images due to the difference in nanoparticle aggregation and surface morphology depending on pixels. The average SERS intensity for the 1600 pixels of the strip was determined to solve this problem. FIG. 6 (b) shows the average spectrum of the 1600 pixels measured at different SEB concentrations. It can be seen that the intensity of the spectral signal (1615 cm.sup.−1) increases with the concentration of the target material (antigen). The SERS mapping image of the control region was constant regardless of the SEB concentration.

<Comparative Example 1> Detection of Target Material Using POC-Based LFA and ELISA

[0079] POC-based LFA and enzyme-linked immunosorbent assay (ELISA) were conducted to evaluate the detection sensitivity of the SERS-based LFA strip. The POC-based LFA refers to detecting the target material without conducting SERS measurement. 20 μL of a SEB solution was loaded on the LFA strip and passed through the absorption pad. Then, an antibody-conjugated HGN and a running buffer were loaded. The antibody-conjugated HGN formed a sandwich immune complex in the detection region by reacting with the SEB antigen. The remaining antibody-conjugated HGN reacted with a secondary antibody adsorbed at the control region. FIG. 7 (a) shows the change of the LFA strip at the SEB concentrations from 1 to 20,000 ng/mL. A red line was observed up to the SEB concentration of 10 ng/mL. Contrast images were measured using the Chemi-Doc imaging system in order to evaluate the optical density of the test line depending on the change in SEB concentration. The detection limit was found to be 10 ng/mL.

[0080] The enzyme-linked immunosorbent assay was conducted using the same antigen and antibody as those used for the SERS-based LFA strip. The capture antibody was immobilized on the surface of a 96-well plate and the remaining sites were treated with BSA to prevent nonspecific conjugation. Then, the SEB antigen was added for conjugation with the capture antibody. After washing 3 times with a micropipette, a detection antibody was added. For conjugation with the detection antibody, an enzyme-conjugated secondary antibody was added. Finally, a substrate was added to enable detection by an enzyme. FIG. 7 (b) shows the color change (from yellow to dark yellow) depending on the change in SEB concentration. The detection limit of the enzyme-linked immunosorbent assay using SEB was found to be 1.0 ng/mL.

[0081] FIG. 8 compares a result of normalizing the result of the SERS-based LFA, POC-based LFA and ELISA for SEB concentrations changing from 10-4 to 103 ng/mL. The SERS intensity was determined by the peak intensity of the Raman marker MGITC at 1615 cm.sup.−1.

[0082] At large, the SERS-based LFA strip showed higher sensitivity of quantitative analysis as compared to other analytical methods. In particular, quantitative analysis was possible at concentrations of 1 ng/mL or lower unlike other analytical methods. This confirms higher sensitivity as compared to the existing POC-based LFA or ELISA. From the normalized curves depending on SEB concentration, the detection limits of the POC-based LFA strip (optical density), ELISA and SERS-based LFA strip were found to be 10, 1.0 and 0.001 ng/mL, respectively.

<Example 4> Evaluation of Selectivity and Quantitative Analysis of SERS-Based LFA Strip

[0083] The selectivity of the SERS-based LFA strip according to the present disclosure was evaluated using five different toxoproteins at 1,000 ng/mL. FIG. 9 shows the immunoassay result for SEB, SEA (Staphylococcus aureus enterotoxin A) (Cusabio (Wuhan, China)), ochratoxin (Sigma-Aldrich (St. Louis, Mo., USA)), aflatoxin (Sigma-Aldrich (St. Louis, Mo., USA)) and fumonisin (Abcam (Cambridge, United Kingdom)) conducted using the SERS-based LFA strip. The detection region developed a red line in the presence of SEB only and the SERS mapping image was observed for SEB only. No positive response was detected for the toxoproteins other than SEB and the positive response was detected in the presence of SEB. That is to say, it was confirmed that the SERS-based LFA strip of the present disclosure shows high analytical selectivity.

[0084] Selectivity and quantitative analysis tests were conducted with SEB at low concentrations (500, 100, 50, 10 and 1 ng/mL). The effect of nonspecific conjugation was tested using an antigen cocktail solution containing five different antigens (SEB, SEA, ochratoxin, aflatoxin and fumonisin at the same concentration of 100 ng/mL). The result is shown in FIG. 10. As seen from FIGS. 10 (a) and (b), the SERS-based LFA strip showed high selectivity only for SEB. The concentration of SEB in the antigen cocktail solution mixed with SEB at different concentrations was quantitated as shown in FIG. 10 (c).

<Comparative Example 2> Comparative Analysis of SERS-Based LFA Strip Using Hollow Gold Nanoparticle (HGN) and SERS-Based LFA Strip Using Gold Nanoparticle (GNP)

[0085] The SERS-based LFA strip according to the present disclosure used HGN for SERS measurement. There are several other metal nanoparticles used as metal nanoprobes for SERS measurement in addition to the HGN. Among them, the gold nanoparticle (GNP) was selected for comparison of sensitivity with the SERS-based LFA strip HGN. Unlike HGN, GNP does not have a hollow cavity. The GNP was synthesized using a HAuCl.sub.4 solution and trisodium citrate according to the method described in the literature (Frens, G. et al., 1973. Nat. Phys. Sci. 241, pp. 20-22). Briefly, a nanoparticle with a size of about 40 nm was synthesized by adding 500 μL of 1% trisodium citrate (Sigma-Aldrich) as a reducing agent to 50 mL of a boiling 0.01% HAuCl.sub.4 solution (Sigma-Aldrich) and conduction reaction for 20 minutes.

[0086] A SERS nanoprobe and a SERS-based LFA strip using GNP were prepared under the same condition as the SERS-based LFA using HGN (see Example 2-2).

[0087] After preparing SERS-based LFA strips using HGN and GNP SERS nanoprobes, quantitative analysis was conducted using SEB as a target material at different SEB concentrations. Details are described in Table 1.

TABLE-US-00001 TABLE 1 SERS-based LFA using SERS-based LFA using HGN GNP Detection Hollow gold nanoparticle Gold nanoparticle probe (SERS Raman marker: MGITC Raman marker: MGITC probe) Anti-SEB antibody Anti-SEB antibody immobilized immobilized Preparation Prepared in the same manner as in Example 2-2 of LFA Flow condition in LFA is identical Comparative Quantitative analysis: comparison of Raman intensity analysis of test line at different SEB concentrations Result LOD: 0.001 ng/mL LOD: 0.1 ng/mL (1 pg/mL) (100 pg/mL)

[0088] FIG. 11 compares the Raman intensity of the test line of the SERS-based LFA strip using HGN (a) and the SERS-based LFA strip using GNP (b) depending on the concentration of SEB. It can be seen that the two strips show decrease in Raman intensity s the SEB concentration is decreased. However, the LFA using HGN and the LFA using GNP show difference in Raman intensity at the same SEB concentration. At each concentration, the HGN-based LFA exhibits about 8-10 times higher Raman intensity than the GNP-based LFA. Accordingly, it was confirmed that the LFA using HGN has higher sensitivity than the GNP-based LFA.

[0089] FIG. 12 shows the calibration curves for quantitative analysis of the SERS-based LFA strip using HGN (a) and the SERS-based LFA strip using GNP (b) depending on the concentration of SEB. The calibration curves of FIG. 12 were obtained by comparing the intensity of Raman signals depending on SEB concentration (FIG. 11). It was confirmed that the LFA using HGN shows change in Raman intensity as the SEB concentration changes from 1,000 to 0.001 ng/mL. The GNP-based LFA showed change in Raman intensity as the SEB concentration from 1,000 to 0.1 ng/mL. From the calibration curves, it was confirmed that the LFA using HGN has higher sensitivity than the GNP-based LFA.

[0090] In the present disclosure, the hollow metal nanoprobe with the Raman marker adsorbed was introduced to a lateral flow immunoassay sensor to overcome the low sensitivity of the existing lateral flow immunoassay sensor. As a result, high-sensitivity quantitative analysis was made possible through Raman mapping and imaging. For quantitative analysis and evaluation of sensitivity, the SEB toxoprotein was used as the target material and POC-based LFA and ELISA were selected as control groups for comparison. AS a result, it was confirmed that SEB could be detected with a high sensitivity of 0.001 ng/mL regardless of other toxoproteins. The result was 1,000-10,000 times superior as compared to the POC-based LFA or ELISA. In addition, it was confirmed that particularly the hollow metal nanoparticle from among the metal nanoprobes for SERS has high sensitivity. Accordingly, it is expected that the present disclosure is applicable to early diagnosis, environmental sensor, etc., to which the existing lateral flow immunoassay sensor is inapplicable.