KR20200098141A - Multi-layered core-shell particle comprising SERS signal as internal standard and detection method of target analyte using the same - Google Patents

Abstract

The present invention provides a multi-layered core-shell particle comprising a core, an inner layer, and an outer layer, in which a Raman-labeled compound is included in the inner layer as an inner standard material, a method for preparing the same, a composition for detecting a target analyte including the same, a SERS-based chemical sensor, and a method for detecting an internal standard-based target analyte by using the same. According to the present invention, the multi-layered core-shell particle and the method for detecting a target analyte by using the same can be utilized as a technology for rapidly confirming a contamination status and preventing diffusion through quantitative analysis in the field of environmental substance detection as well as major agricultural and aquatic products, and can be also used as a core source technology of biosensor and nanobio technology for early detection and diagnosis of cancer, bio-imaging, customized diagnosis, and diagnostic monitoring in the form of point-of-care testing (POCT).

Description

Multi-layered core-shell particle comprising SERS signal as internal standard and detection method of target analyte using the same}

The present invention relates to a multilayer core-shell particle containing the SERS signal as an internal standard signal and a method of detecting a target analyte using the same, and more particularly, to a multilayer core-shell particle composed of a core, an inner layer, and an outer layer. The present invention relates to a multilayer core-shell particle comprising a Raman label compound (RLC) as a material and a method of detecting a target analyte using the same.

Surface-enhanced Raman scattering (SERS) spectroscopy is recognized as a promising detection technology in a variety of applications because it enables high-sensitivity, label-free, and immediate chemical fingerprint analysis. Accordingly, SERS-based analysis is being developed and studied in a number of research fields related to chemistry, biology and the environment. However, it is difficult to obtain a reproducible signal because the signal of SERS can be influenced by several factors such as the uniformity of the SERS-active substrate and the degree of distribution of analytes adsorbed on the SERS-active substrate.

Recently, several research groups have attempted to overcome this problem and have proposed nanoparticles (NPs) containing internal standards. Chemicals that act as internal standards can be added to the SERS-active substance to plot the ratio of the analyte signal to the internal standard signal and use it to create a calibration curve. For this, a core-internal standard-shell structure was proposed. The internal standard located between the core and the shell is not affected by the detection environment because it exists inside the shell and does not compete with the target analyte on the surface. In addition, signal fluctuations caused by different aggregation states and measurement conditions are corrected. A probe molecule having a vibration band in the Raman-silent region can be used as an internal standard called "AGN" in which gold NPs are decorated in multilayer graphite magnetic nanocapsules. These signals cause relatively low interference with vibration peaks in the surrounding environment. However, immobilizing these probe molecules on a "core-shell" surface is not an easy task. During immobilization, the unstable sol form of the “core-shell” silica substrate may aggregate, resulting in a non-uniform SERS signal. Therefore, there remains room to develop more advanced detection probes using highly sensitive NPs and efficient quantitative detection methods.

On the other hand, linear fitting can improve detection accuracy and the concentration of _analyte can be reliably quantified, so that it has a good linear relationship with I _analyte / I _{internal standard} . Therefore, the ratiometric analysis method is advantageous. However, it is difficult to establish a detection method for a wide dynamic range of NP concentration in an actual detection system.

Korean Patent Publication No. 10-2017-0070351 (June 22, 2017) Korean Patent Publication No. 10-2011-0066881 (June 17, 2011) Korean Patent Publication No. 10-2012-0015227 (February 21, 2012)

The inventors of the present invention study a quantitative analysis method using a surface-enhanced Raman scattering (SERS) spectroscopy that enables a quantitative analysis by showing a wide dynamic range in an actual detection system and capable of stable and reliable field inspection. In the meantime, a multilayer core-shell particle composed of a core, an inner layer and an outer layer, and a multilayer core-shell particle containing a Raman label compound (RLC), an internal standard material in the inner layer, exhibits excellent dispersion stability, and is stable and reproducible. It can be used as a quantitative analysis method capable of stably and reliably on-site inspection by confirming that the concentration of analyte can be obtained by a single measurement regardless of the number of particles by using it to generate a signal to enable accurate quantitative analysis. I found it.

Accordingly, the present invention is a multilayer core-shell particle composed of a core, an inner layer and an outer layer, and a multilayer core-shell particle comprising a Raman-labeled compound as an inner standard material in the inner layer, a method for preparing the same, a composition for detecting a target analyte comprising the same, An object of the present invention is to provide a SERS-based chemical sensor including the same and a method for detecting target analytes based on internal standards using the same.

According to an aspect of the present invention, silica core particles; An inner layer including a first Ag particle introduced to the surface of the silica core particle, a Raman label compound (RLC) introduced to the surface of the first Ag particle, and a silica shell surrounding the first Ag particles; And multilayer core-shell particles including an outer layer including the second Ag particles introduced on the surface of the inner layer is provided.

In one embodiment, the silica core particles may have a size of 10 nm to 10 mm.

In one embodiment, the first Ag particles and the second Ag particles may each independently have a size of 1 to 200 nm.

In one embodiment, the silica shell may have a thickness of 1 to 50 nm.

In one embodiment, the multilayer core-shell particle may have a size of 10 nm to 10 mm.

According to another aspect of the present invention, (a) introducing a thiol group to the surface of the silica core particle and then introducing the first Ag particle to prepare SiO ₂ @Ag particles; (b) preparing SiO ₂ @Ag/RLC particles by introducing a Raman label compound (RLC) to the SiO ₂ @Ag particles; (c) preparing SiO ₂ @Ag/RLC@SiO ₂ particles by encapsulating the SiO ₂ @Ag/RLC particles with a silica shell; And (d) introducing a thiol group to the surface of the SiO ₂ @Ag/RLC@SiO ₂ particles and then introducing the second Ag particles to prepare SiO ₂ @Ag/RLC@SiO ₂ @Ag particles. A method for producing core-shell particles is provided.

According to another aspect of the present invention, a composition for detecting a target analyte comprising the multilayer core-shell particles is provided.

According to another aspect of the present invention, a SERS-based chemical sensor comprising the multilayer core-shell particles is provided.

According to another aspect of the present invention, a method for detecting a target analyte based on an internal standard using the multilayer core-shell particles is provided.

In one embodiment, the internal standard-based target analyte detection method may use a ratiometric analysis method.

According to the present invention, silica core particles; An inner layer including a first Ag particle introduced into the surface of the silica core particle, a Raman labeling compound introduced into the surface of the first Ag particle, and a silica shell surrounding them; And a multilayer core-shell particle including an outer layer including the second Ag particles introduced on the inner layer surface as a particle structure that generates a signal that is sufficient and stable for detection, and exhibits a negative zeta potential value in a colloidal state, resulting in excellent dispersion stability. And agglomeration-free properties, and by using a ratiometric analysis method, a stable and reproducible signal is generated for accurate quantitative analysis, and the concentration of analytes can be quantitatively analyzed with a single measurement regardless of the number of particles. It turns out that you can.

Therefore, the multilayer core-shell particle of the present invention and the detection method of the target analyte using the same overcome the limitation of the analysis of quantitative analysis in Raman spectroscopy to enable high-sensitivity and high-speed analysis of harmful substances, thereby enabling quantitative analysis in the field of environmental substance detection. It can be used as a technology to quickly check contamination status and prevent spread, and provides early detection and diagnosis of cancer, bio-imaging, customized diagnosis, and point-of-care testing (POCT) type diagnostic monitoring as well as major agricultural and aquatic products. It can be usefully used as a key source technology for biosensor technology and nanobio technology.

1 is a schematic diagram of a synthesis and detection principle of a multilayer SERS NP, (a) a synthesis process of SiO ₂ @Ag/RLC@SiO ₂ @Ag (ML _RLC dots), and (b) an ML _RLC dot using an internal standard Detection concept and structure (A: internal standard, B: analyte).
2 is a TEM image and composition analysis result of NP at each step of the synthesis, (a) SiO ₂ NP, (b) SiO ₂ @Ag, (c) SiO ₂ @Ag/4-BBT@SiO ₂ , (d ) ML _4-BBT dot, (e) ML _4-BBT dot at low magnification and (f) the thickness of the silica shell at SiO ₂ @Ag/4-BBT@SiO ₂ , and (g) ML _4-BBT dot and (h) TEM-EDX image of three components (Ag, Si and Br from above) of ML _4-BBT dot, and sample [(i) SiO ₂ @Ag, (ii) SiO ₂ @Ag@SiO ₂ , (Iii) ML dot] is a UV-visible ray absorption spectrum.
3 is a TEM image of SiO ₂ NP and a histogram of the size distribution.
4 is an SEM image of an ML _4-BBT dot colloid.
5 is the zeta potential of the ML _4-BBT dot colloid.
6 is an EDX analysis result of ML _4-BBT dots.
7 is a characteristic of the internal standard embedded ML _4-BBT dot, (a) SERS spectrum of the ML _4-BBT dot in each synthesis step [(i) SiO ₂ @Ag, (ii) SiO ₂ @Ag/ 4-BBT, (iii) SiO ₂ @Ag/4-BBT@SiO ₂ , and (iv) ML _4-BBT dots], (b) SERS intensity at 491 cm ^-1 of the nanostructure synthesized in each step , (c) SERS spectra of ML _4-BBT dots reacted with 4-CBT and 4-FBT, respectively.
Figure 8 is (a) SERS intensity according to the amount of ML _4-BBT dots, (b) SERS intensity ratio of the ML _4-BBT dots reacted with various concentrations of Tiram (10 ^-3 ~ 10 ^-6 M) (I _4-FBT /I _4-BBT ).
9 is an internal standard SERS intensity according to the amount of ML _4-BBT dots.
Figure 10 is (a) a three-dimensional SERS intensity map of the ML _4-BBT dot in the NPs introduced with different concentrations of 4-FBT and 4-BBT, the internal standard 4-BBT concentration ranging from 0.1 mg to 1.2 mg Was included in NP, and 4-FBT (1×10 ⁻³ M to 1×10 ⁻⁶ M) was used as the target analyte. (b) It is the percentage of the experimental and calculated concentration of the analyte.
11 is a photograph of (a) ML _4-BBT dots after mixing with an analyte for 5 hours, (i) ML _4-BBT dots, (ii) ML _4-BBT dots mixed with Tiram, and (iii) 4-FBT Mixed with ML _4-BBT dots. (b) UV-Vis spectrum of ML _4-BBT dots mixed with various concentrations of analytes. (c) The z potential of the ML _4-BBT dot mixed with 4-FBT.
12 is the SERS intensity ratio (I _Thiram /I _4-BBT , I _1397cm-1 /I _491cm-1 ) of various concentrations of Tiram (5×10 ^-5 ~ 10 ^-7 M) for ML _4-BBT dots. .
13 shows the process of detecting Tiram on the surface of an apple peel using (a) ML _4-BBT dots, and (b) ML _4-BBT reacted with various amounts of Tiram (10 ^-8 mol ~ 10 ^-11 mol) It is the SERS spectrum of the dot.

In the present specification, the term "Raman label compound (RLC)" refers to any substance that exhibits a Raman signal of a substance's vibrational, rotational, and other low-frequency modes. The Raman signal is recognized as a structural fingerprint of the molecule.

The present invention is a silica core particle; An inner layer including a first Ag particle introduced into the surface of the silica core particle, a Raman labeling compound introduced into the surface of the first Ag particle, and a silica shell surrounding them; And it provides a multi-layered core-shell particles comprising an outer layer including the second Ag particles introduced on the surface of the inner layer.

NP as a SERS substrate uses a variety of metals such as precious metals and transition metals, controls the size of the metal, and changes the shape of particles such as nanotubes, nanorods, nanowires, nanorings and nanocrescents. And metal NPs are arranged in dimers, trimers and aggregates, and are being developed in a number of directions by fusion of metals such as alloys. Among the various nanomaterials exhibiting SERS enhancement, Ag NP-introduced silica nanospheres have sufficient SERS signals that can be detected with only one particle due to the hotspot structure. These nanostructures, which can efficiently emit and maintain the SERS signal, enable high sensitivity to polycyclic aromatic hydrocarbons, flavonoids and other target molecules. In addition, the template-based nanostructures that are easy to synthesize can generate a uniform signal and be mass-produced. In addition, when the ligand and shell are applied to the structure, sensor stability and long-term stability are shown.

In the present invention, a stable SERS active nanomaterial capable of generating a stable and strong SERS signal was prepared, and the strength and frequency of the material depend on the concentration of the target material for quantitative analysis in chemical detection. The multilayer nanostructure is composed of a silica core coated with Ag NP, a Raman labeling compound, a silica shell and Ag NP. The internal Ag NP with RLC serves as an internal standard that generates a stable signal as a reference for quantitative analysis. External Ag NPs are used as sensor sites for analyte detection. The internal standard plays two roles: (1) providing the amount of multilayer particles in Raman intensity, and (2) providing the signal ratio of the _analyte and the internal standard (I _analyte /I _{internal standard} ). This approach makes it possible to estimate the target concentration from the particle's Raman signal without knowing the concentration of NP. First, a multilayer nanostructure was prepared and used for Raman-based detection of analytes. Using a multilayer Ag-immobilized silica nanostructure with an internal standard, it is possible to immediately quantify the amount of target material based on the number of particles measured based on a three-dimensional calibration curve.

The multilayer core-shell particle of the present invention is composed of a core, an inner layer and an outer layer. The core is made of a silica material, and the inner layer includes a first Ag particle introduced to the surface of the silica core particle, a Raman label compound introduced to the surface of the first Ag particle, and a silica shell surrounding them, and the outer layer includes a surface of the inner layer. The second Ag particles introduced into are included.

The silica core particles provided in the present invention may have a size of 10 nm to 10 mm, preferably 20 nm to 10 um, more preferably 50 nm to 500 nm, and most preferably 150 nm to 250 nm.

The first and second Ag particles present in the inner layer and the outer layer of the multilayer core-shell particle of the present invention, respectively, may be particles composed of Ag, or may be particles in the form of a shell coated with Ag, and independently size 1 to 200 nm, preferably 2 to 100 nm, more preferably 5 to 50 nm, and most preferably 8 to 15 nm.

The Raman-labeled compound introduced on the surface of the first Ag particle is a material containing a benzene ring in its chemical structure, and can be used without limitation, as long as it is a material having affinity with a metal, and a material that does not overlap with the absorbance band of the target analyte or A material exhibiting Raman bands is selected and used as an internal standard. Preferably 2-Amino-4-chlorobenzenethiol (2-ACBT), 2-Amino-4-(trifluoromethyl) benzenethiol (2-ATFT), 4-Aminothiophenol (4-ATP), 2-Bromobenzenethiol (2-BBT), 4-Bromobenzenethiol (4-BBT), Benzylmercaptane (BMT), Benzenethiol (BT), Benzyl disulfide (BZDSF), 3-Cyanobenzoic acid (3-CBA), 4-Cyanobenzaldehyde (4-CBAL), 2-Chlorobenzenethiol (2- CBT), 4-Chlorobenzenethiol (4-CBT), 3,4-Dichlorobenzenethiol (3,4-DCT), 3,5-Dichlorobenzenethiol (3,5-DCT), 3,4-Dimethoxybenzenethiol (3,4-DMOBT) , 3,5-Dimethoxylbenzenethiol (3,5-DMOBT), 3,4-Dimethylbenzenethiol (3,4-DMT), 3,5-Dimethylbenzenethiol (3,5-DMT), 2-Fluorobenzenethiol (2-FBT), 4 -Fluorobenzenethiol (4-FBT), 4-Isopropylbenzenethiol (4-IBT), 3-Mercaptobenzoic acid (3-MBA), 2-Mercapto-5-methyl Benzimidazole (2-MBI), 2-Mercapto-1-methylimidazole (2 -MMI), 2-mercapto-6-methylpyridine (2-MMP), 4-Methoxybenzenethiol (4-MOBT), 2-Mercaptopyrimidine (2-MPY), 4-Mercaptotoluene (4-MT), 4-Nitrophenyl disulfide (4 -NPDSF), 2-Naphthalenethiol (2-NT), 5-phenyl-1H-1, 2,4-triazole-3-thiol (5-PHTT), Phenyl isothiocyanate (PITC), 4-(pyridin-4-yl)pyridine (4-PPD), 1-Phenyltetrazole-5-thiol (1-PTET), 5-Phenyl-1,2,3-triazole-3-thiol (5-PTRT) and one selected from the group consisting of mixtures thereof (see Table 1 below), and more preferably 4-bromobenzenethiol ( 4-bromobenzenethiol, 4-BBT), 4-chlorobenzenethiol (4-CBT), or 4-fluorobenzenethiol (4-fluorobenzenethiol, 4-FBT).

The silica shell formed on the inner layer may have a thickness of 1 to 50 nm, preferably 5 to 20 nm, and more preferably 8 to 15 nm.

The second Ag particles of the outer layer introduced on the surface of the inner layer of the multilayer core-shell particle of the present invention are separated from the first Ag particles contained in the inner layer by the silica shell formed in the inner layer.

The multilayer core-shell particles of the present invention may have a size of 10 nm to 10 mm, preferably 50 nm to 10 um, more preferably 100 nm to 500 nm, and most preferably 200 to 300 nm.

The multilayer core-shell particles of the present invention exhibit a negative zeta potential value in a colloidal state, and may be -20 to -200 mV, preferably -50 to -150 mV, more preferably -70 to -100 mV. . The multilayer core-shell particles of the present invention exhibit a negative zeta potential value in a colloidal state, thereby exhibiting excellent dispersion stability and agglomeration-free properties.

In addition, the present invention comprises the steps of (a) introducing a thiol group on the surface of the silica core particle and then introducing the first Ag particle to prepare SiO ₂ @Ag particles; (b) preparing SiO ₂ @Ag/RLC particles by introducing a Raman label compound (RLC) to the SiO ₂ @Ag particles; (c) preparing SiO ₂ @Ag/RLC@SiO ₂ particles by encapsulating the SiO ₂ @Ag/RLC particles with a silica shell; And (d) introducing a thiol group to the surface of the SiO ₂ @Ag/RLC@SiO ₂ particles and then introducing the second Ag particles to prepare SiO ₂ @Ag/RLC@SiO ₂ @Ag particles. It provides a method for producing core-shell particles.

Step (a) is a step of preparing SiO ₂ @Ag particles by introducing a thiol group on the surface of the silica core particles and then introducing the first Ag particles. The silica core can be prepared by mixing tetraethylorthosilicate (TEOS) in anhydrous EtOH with NH ₄ OH and reacting at room temperature. In order to introduce a thiol group to the surface of the prepared silica particles, 3-mercaptopropyl trimethoxysilane (MPTS) and NH ₄ OH are reacted with the silica particles in EtOH to introduce a thiol group. Next, after mixing polyvinylpyrrolidone (PVP) and AgNO ₃ in ethylene glycol, octylamine (OA) is added to introduce Ag particles to the surface of the thiolated silica NP.

Step (b) is a step of preparing SiO ₂ @Ag/RLC particles by introducing a Raman-labeled compound to the SiO ₂ @Ag particles prepared in step (a). A Raman-labeled compound (e.g., 4-bromobenzenethiol (4-BBT)) is used as an internal standard material, and RLC can be introduced on the SiO ₂ @Ag surface by mixing it with a Raman-labeled compound in EtOH. I can.

Step (c) is by encapsulating the SiO ₂ @Ag/RLC particles prepared in step (b) with a silica shell, the first Ag particles introduced to the surface of the silica core particles, the Raman-labeled compound introduced to the surface of the first Ag particles , And by preparing an inner layer comprising a silica shell surrounding them, SiO ₂ @Ag/RLC@SiO _{2 It} is a step of preparing particles. In order to encapsulate SiO ₂ @Ag/RLC with silica, a Na ₂ SiO ₃ solution can be added to a suspension of SiO ₂ @Ag/RLC and reacted.

Step (d) includes the second Ag particles introduced to the inner layer surface by introducing a thiol group to the surface of the SiO ₂ @Ag/RLC@SiO ₂ particles prepared in step (c) and then introducing the second Ag particles. This is a step of preparing SiO ₂ @Ag/RLC@SiO ₂ @Ag particles by preparing an outer layer. To prepare the second Ag NP, thiol groups are introduced again on the surface of the resulting SiO ₂ @Ag/RLC@SiO ₂ , and the Ag NP is immobilized on the surface of the silica shell. To this end, SiO ₂ @Ag/RLC@SiO ₂ was reacted with MPTS and NH ₄ OH, the suspension was dispersed in an EG solution containing AgNO ₃ and PVP, OA was quickly added, and the resulting suspension was stirred. By doing so, ML _RLC dots can be synthesized.

In addition, the present invention provides a composition for detecting a target analyte including the multilayer core-shell particles.

In addition, the present invention provides a SERS-based chemical sensor comprising the multilayer core-shell particles.

In addition, the present invention provides a method for detecting an internal standard-based target analyte using the multilayer core-shell particle.

In the method for detecting a target analyte based on an internal standard of the present invention, the signal is stable and reproducible by using a ratiometric analysis method using a ratio analysis of the target analyte and an internal standard (I _analyte / I _{internal standard} ). Since it is generated, it is possible to analyze more accurately and quantitatively, and shows the characteristic that the concentration of an analyte can be obtained with a single measurement regardless of the number of particles.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Manufacturing and evaluation

1.1. materials

Tetraethylorthosilicate (TEOS), 3-mercaptopropyl trimethoxysilane (MPTS), 3-aminopropyl triethoxysilane, ethylene glycol (EG), silver nitrate (AgNO ₃ , 99.99%), octylamine (OA), sodium silicate solution, 4-bromobenzenethiol (4-BBT), 4-chlorobenzenethiol, 4-CBT), 4-fluorobenzenethiol (4-FBT), tetramethylthiuram disulfide (thiram, 99.9%) and ethyl alcohol (EtOH, 99.5 and 95%) It was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Aqueous ammonium hydroxide (NH ₄ OH, 27%) was purchased from Daejeong (Siheung, Korea).

1.2. Multilayer NP (ML _RLC Dot)

ML _RLC dots were prepared according to previous literature (Hahm, E. et al., Sci. Rep. 2016, 6, 26082.; Kim, H.-M. et al., J. Ind. Eng. Chem. 2016 , 33, 2227.). Silica NP was synthesized by the Stober method. TEOS (1.6 mL) in anhydrous EtOH was mixed with 3 mL of NH ₄ OH and stirred vigorously at room temperature for 20 hours. To remove excess reagent, the solution was centrifuged and washed 5 times with EtOH. A thiol group was introduced on the surface of the silica NP by the following method. MPTS (200 μL) and NH ₄ OH (40 μL) were reacted with silica NP (200 mg) in EtOH and stirred vigorously for 6 hours. The solution was centrifuged, washed with EtOH, and dispersed in 4 mL of EtOH. Next, 5 mg of polyvinylpyrrolidone (PVP) and 26 mg of AgNO ₃ in 50 ml of EG were mixed, and then 5 mM of OA was added, and Ag NP was added to thiolated silica NP (30 mg). Introduced on the surface. After 1 hour, the resulting SiO ₂ @Ag NP was centrifuged, washed 5 times with EtOH, and dispersed in 3 mL of EtOH. As an internal standard, RLC (4-BBT) was introduced on the SiO ₂ @Ag surface by mixing with 1 mM RLC in EtOH (1 mL). To encapsulate SiO ₂ @Ag/RLC with silica, a solution of 14.4 μL of Na ₂ SiO ₃ solution (0.036 wt %) dissolved in 15 mL of distilled water was added to the suspension of SiO ₂ @Ag/RLC for 12 hours. Reacted. After treatment with 60 mL of EtOH for 3 hours, the solvent was changed from water to EtOH by centrifugation and washing with EtOH. To prepare a second layer of Ag NP, thiol groups were introduced again on the surface of the resulting SiO ₂ @Ag/RLC@SiO ₂ , and Ag NPs were immobilized on the surface of the silica shell. For this, 10 mg of SiO ₂ @Ag/RLC@SiO ₂ was reacted with 40 μL of MPTS and 8 μL of NH ₄ OH for 12 hours. After washing several times with EtOH, the suspension was dispersed in 1 mL of EG solution containing 5 mg of AgNO ₃ and 1 mg of PVP for 1 hour. Then, a 50 μL portion of OA was quickly added, and the resulting suspension was stirred at 25° C. for 1 hour. The synthesized ML _RLC dot was centrifuged, washed with EtOH, and then dispersed in 5 mL of EtOH.

1.3. ML using TEM, EDX, dynamic light scattering and UV-visible absorbance spectrometer _RLC Characterization of dots

Transmission electron microscopy (TEM) images were obtained with a TEM microscope (LIBRA 120, Carl Zeiss), and TEM-energy dispersive X-ray spectroscopy (EDX) analysis was performed by JEM-2100F (JEOL Ltd). This was done using equipment. NP size was analyzed by digitized measurement using ImageJ software (Maryland, USA), and the average size of NP was calculated after analyzing at least 100 NPs. The zeta potential of the ML _RLC dot in EtOH was measured after about 3 days using a zetameter system (ELS Z-1000, Otsuka Electronics Co., Ltd.), and the UV-visible ray absorption spectrum was determined by an Optizen POP spectrophotometer (Mecasys Co., Ltd.) ) To obtain.

1.4. Raman measurement

To evaluate the SERS spectrum of the ML _RLC dot, the sample in solution was measured with a capillary tube using a DXR Raman microscope (Thermo Fisher Scientific, USA) and the SERS signal was measured with a ×10 objective lens to determine the backscattering geometry. Was collected. A 532 nm diode pumped solid state laser was used as a photoexcitation source with a 10 mW laser output for a sample with a spot size of about 2 μm. All SERS spectra were integrated for 5 seconds and the SERS signal of the sample was collected at random locations.

2. Results and discussion

To quantitatively analyze the concentration of the target material, the SERS signal of the NP and the SERS signal of the target analyte recognized as a relative SERS signal were compared in a single NP. For this approach, SiO ₂ @Ag/RLC@SiO ₂ @Ag(RLC), called multilayer (ML _RLC ) dots, was fabricated, which can generate a stable and strong SERS signal. The manufacturing strategy including immobilization of the internal assay material is shown in Fig. 1(a). When the laser is irradiated to the ML _RLC point having the surface on which the target analyte is adsorbed, Raman signals are generated from the internal standard (A) and the analyte (B). As shown in Fig. 1(b), Raman signal intensities of A and B could be obtained due to the open and accessible SERS shell surface and the localized internal standard of ML _RLC dots. Based on the Raman signal intensity of A and the ratiometric analysis, the concentration of the target analyte can be easily calculated from the relative SERS signal intensity with a single use of NP.

As shown in FIGS. 2(a)-2(d), TEM analysis was performed after each synthesis step to confirm the multilayer core-shell structure with immobilized RLC. 2(a) and 3, the size of the silica NP is 200.8 ± 6.7 nm, whereas the size of the Ag NP on the surface of the silica NP is 12.9 ± 4.9 nm as shown in Fig. 2(b). to be. TEM and scanning electron microscope (SEM) images of the obtained ML _4-BBT dots are shown in Figs. 2(d) and 4, respectively. In the synthesized ML _4-BBT dots, the second introduced Ag NP showed a more uneven structure than the primary Ag coating. The final size of ML _4-BBT was 245.8 ± 25.6 nm. The ML _4-BBT dots are well dispersed, which is supported by the zeta potential data. As shown in Fig. 5, the ML _4-BBT colloid exhibits a high negative zeta potential value (-85.09 mV), which is associated with good dispersion stability and no aggregation. The TEM image of the silica shell-coated Ag NP-assembled silica NP is shown in Fig. 2(c), and the silica shell thickness between the two Ag layers is about 10 nm (see Fig. 2(f)). In Figs. 2(g) and (h), the bromine element of the internal standard 4-BBT is clearly observed although the amount of the bromine element is less than the amount of silica and Ag (see Fig. 6).

In addition, the UV-visible light absorption spectrum of the prepared particles was recorded in each synthesis step (see Fig. 2(i)). In the UV-visible absorption spectrum ([i], black line) of SiO ₂ @Ag, a wide band of 320 to 800 nm was observed, indicating the presence of Ag NPs on silica NPs. The UV-visible absorption spectrum ([ii], red line) of SiO ₂ @Ag@SiO ₂ shows a sharper peak than the above spectrum ([i]). The smaller the amount of silica NP used, the greater the density of Ag NPs on the silica surface. By the UV-visible absorption spectrum of each SiO ₂ @Ag, the formation of a bumpy shell structure that causes resonance dipole and multiple modes as the amount of Ag introduced on the surface of SiO ₂ increases. Therefore, it can be seen that broadening of the absorption band occurs. When the second layer of Ag NP was introduced into the silica shell, the UV-visible absorption spectrum of the ML dot was broadened ([iii], blue line).

In order to investigate the change of the SERS spectrum in each synthesis step, the SERS spectrum of the NP obtained in each synthesis step was recorded. Figure 7 (a) ([i]-[vi]) shows the SERS spectrum of each synthesis step (respectively SiO ₂ @Ag template, after adding 4-BBT, after silica coating, and the final ML _4-BBT dot). After immobilizing the 4-BBT as internal standard, SiO ₂ @ the SERS spectrum of the Ag / 4-BBT compared to the spectrum of the SiO ₂ Ag it showed a new sharp peaks at 491 and 721 cm ^-1. The strong peak at 491 cm ^-1 of 4-BBT (see Fig. 7(b)) shows that most of the added BBT molecules remain on the silica shell, and the thickness of the outer silica shell layer (10 nm) is that of 4-BBT. It indicates that it has no significant effect on signal strength. In addition, ML _4-BBT dots were mixed with 4-chlorobenzenethiol (4-CBT) or 4-FBT through external stimulation by vortexing to compare the SERS signal intensity of the target analyte and internal standard. . As shown in Fig. 7(c), each signal and an internal standard signal were well obtained, which proves that 4-BBT can be effectively used as an internal standard for SERS analysis. In addition, various chemicals such as 4-FBT and 4-CBT can be used as internal standards, which means that ML _RLC dots can have a variety of unique Raman bands. This makes it possible to detect a variety of substances by selecting a substance or Raman band that does not overlap with the target band.

To evaluate the accuracy of the quantification, the concentration of the analyte was determined using ratio-measured SERS signal analysis and ML dots. According to previous reports, in terms of the accuracy of SERS measurement, the ratio analysis of the target analyte and the internal standard (I _analyte /I _{internal standard} ) is more accurate than analyzing only the target _analyte . This method has proven to be more accurate because the signal is stable and reproducible. In addition, the limit of the adsorption site for the target analyte on the NP surface can be linked to a nonlinear relationship between the amount of NP and the target analyte, leading to a narrow dynamic range assay. 8 shows the change (a) of the internal standard signal according to the amount of ML _4-BBT dots and the change (b) of the signal ratio of the analyte and the internal standard according to the concentration of the analyte. In particular, the internal standard signal strength is directly proportional to the amount of ML _4-BBT dots, and the standard plot of FIG. 9 shows the highest linearity (R ² = 0.9969) and a low standard deviation (~ 5.3%). The above results indicate that the internal standard signal of the ML dot is sufficiently stable and reproducible to increase the accuracy of the detection method of the present invention.

Based on these data, a three-dimensional calibration curve was derived to easily quantify the analyte concentration. As shown in Fig. 10(a), the concentration of the analyte can be easily measured from the intensity of the internal standard signal and the ratio of the signal to the Raman signal of the analyte. From this it can be seen that the concentration of the analyte can be obtained in a single measurement regardless of the number of particles. The following procedure was performed to obtain a quantitative SERS signal. First, SERS signals were measured in various amounts of ML _4-BBT dots (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg). Next, by measuring the SERS signal of 4-FBT (10 ^-3 ~ 10 ^-6 M) for each ML _4-BBT (z-axis), ML _4-BBT and 4-FBT (I _4-FBT /I _{4 -BBT} , y-axis) was obtained, and a 3D calibration SERS graph was created based on the obtained data. Finally, the unknown 4-FBT concentration at a specific amount of ML _4-BBT dots was predicted. The x-axis represents the signal strength of the internal standard according to the amount of ML _4-BBT dots. From the experimental data, it was possible to determine the concentration of the ML _4-BBT used (x-axis) and the ratio of the Raman signal of the internal standard to the target analyte. As a result, the concentration of the target analyte corresponding to the z-axis can be determined. The unknown concentration of 4-FBT was applied to the ML _4-BBT dots and the estimated ratio values were obtained, and the results are shown in Fig. 10(b). The concentration of 4-FBT was measured with an accuracy of less than 10% of the deviation.

To further evaluate the applicability of the ML _4-BBT dots, various concentrations of thiram used as pesticides were mixed with the ML _4-BBT dots. Then, Raman measurement was performed using a capillary tube to detect the signals of ML _4-BBT and Tiram. The ML dot solution reacted with the analyte was well dispersed (see FIG. 11). In order to prove that the ML dot solution was well dispersed in the course of analysis, the ML dot solution was mixed with the analyte (4-FBT and Tiram), and a sample was taken after 5 hours. The UV-visible light spectrum and z-potential graph are shown in Fig. 11 (Fig. 11(b) and (c)). As shown in Fig. 11(a), the ML dots mixed with the analyte are still well dispersed. In addition, the UV-visible spectrum of the ML dot solution mixed with the analyte (4-FBT, multiple concentration) was very similar to the UV-visible spectrum of the ML dot. Moreover, the z-potentiality of the ML dot mixed with 4-FBT showed a high negative value (-73.75 mV), indicating that the ML dot maintained good variance during the analysis process.

The ML _4-BBT dots showed strong Raman signals at 491 and 1379 cm ^-1 for 4-BBT and Thiram, respectively. Figure 12 shows the correlation between the SERS intensity ratio (I _thiram /I _4-BBT ) at the 491 and 1379 cm ^-1 peaks and various thiram concentrations (5 × 10 ^-5 to 1 × 10 ^-7 M). A good linear relationship between I _thiram /I _4-BBT and thiram concentration enables quantification in solutions with thiram concentrations as low as 1×10 ^-7 M. For the actual use of the ML _4-BBT dots, Tiram was sprinkled on the apple peel, and ML _4-BBT dots were dropped as shown in Fig. 13(a). Then, the SERS spectrum of the apple peel was measured to detect the signal of Tiram and the internal standard (4-BBT). ML _4-BBT dots showed strong Raman signals at 491 and 1379 cm ^-1 for 4-BBT and Tiram, respectively, on apple peels (see FIG. 13(b)). Moreover, the detectable amount of Tiram per apple peel was 24 ng cm ^-2 . According to Tiram data from the National Institution of Safety and Health, the lethal concentration of LC ₅₀ is 500 μg/mL. Using ML dots, 10 μL of tiram at 10 ^-8 mol/mL was detected, which is a very low concentration compared to the lethal concentration. This indicates that practical application may be possible by creating a 3D calibration curve by deriving the same range of analyte concentrations at different concentrations of ML _4-BBT dot concentrations.

3. Conclusion

In summary, in the present invention, a multi-layered SiO ₂ @Ag/RLC@SiO ₂ @Ag core-shell NP was prepared for quantitative SERS analysis. The ML _RLC dot with the internal standard immobilized generated the SERS signal of the target analyte when the target analyte was adsorbed onto the open shell structure. The immobilized RLC, protected from the external environment by the outer shell, provides a reference value for calibrating the SERS signal. Thus, ML _RLC dots can provide quantitative SERS signals of target analytes (4-FBT and Thiram) by ratio-measurement analysis through normalization of relative SERS intensities. The novel ratio-measurement analysis strategy of the present invention using ML _RLC dots containing an internal SERS standard can be usefully used for the quantitative detection of various analytes and the development of new types of nanoprobes.

4. Summary

Surface-enhanced Raman scattering (SERS) spectroscopy is attractive for a variety of detection assays. However, the quantitative method using SERS spectroscopy still has many areas to be developed. An important problem in developing a quantitative analysis method using SERS spectroscopy is to prepare a reliable SERS active material, such as a particle-based structure, from which SERS signals are collected without interference that changes the SERS signal strength and frequency. In the present invention, smooth multilayer core-shell nanoparticles without a connecting surface having a Raman-labeled compound contained as an internal standard (ML _RLC dot) were prepared for quantitative SERS analysis. The nanostructured nested Raman-labeled compound provides a reference value for calibrating the SERS signal. By using ML _RLC dots, different concentrations of the target analyte signal can be obtained while maintaining the Raman signal of the internal standard. ML _4-BBT dots containing 4-bromobenzenethiol (4-BBT) as an internal standard quantitative analysis of 4-fluorobenzenethiol (4-FBT) and Tiram, a model insecticide Has been successfully applied to In addition, the ratio-measurement analysis proved practical through standardization of the relative SERS intensity. Ratio-measurement strategies can be applied to various SERS substrates for quantitative detection of various targets.

Claims (10)

Silica core particles;
An inner layer including a first Ag particle introduced to the surface of the silica core particle, a Raman label compound (RLC) introduced to the surface of the first Ag particle, and a silica shell surrounding the first Ag particles; And
The outer layer including the second Ag particles introduced on the inner layer surface
Multilayer core-shell particles comprising a. The multilayer core-shell particle of claim 1, wherein the silica core particle has a size of 10 nm to 10 mm. The multilayer core-shell particle of claim 1, wherein the first Ag particles and the second Ag particles each independently have a size of 1 to 200 nm. The multilayer core-shell particle of claim 1, wherein the silica shell has a thickness of 1 to 50 nm. The multilayer core-shell particle according to claim 1, wherein the multilayer core-shell particle has a size of 10 nm to 10 mm. (a) preparing SiO ₂ @Ag particles by introducing a thiol group to the surface of the silica core particles and then introducing the first Ag particles;
(b) preparing SiO ₂ @Ag/RLC particles by introducing a Raman label compound (RLC) to the SiO ₂ @Ag particles;
(c) preparing SiO ₂ @Ag/RLC@SiO ₂ particles by encapsulating the SiO ₂ @Ag/RLC particles with a silica shell; And
(d) preparing SiO ₂ @Ag/RLC@SiO ₂ @Ag particles by introducing a thiol group on the surface of the SiO ₂ @Ag/RLC@SiO ₂ particles and then introducing the second Ag particles
A method for producing a multilayer core-shell particle comprising a. A composition for detecting a target analyte comprising the multilayer core-shell particles of any one of claims 1 to 5. The SERS-based chemical sensor comprising the multi-layered core-shell particles of any one of claims 1 to 5. A method for detecting an internal standard-based target analyte using the multilayer core-shell particle of any one of claims 1 to 5. 10. The method of claim 9, wherein a ratiometric analysis method is used.

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