KR102332082B1 - Surface-enhanced Raman scattering (SERS) with magnetic microparticle-based assay for bio materials detection - Google Patents

Abstract

The present invention relates to a method for detecting multiple biomaterials by using Raman signals including magnetic microparticles (MMP) modified with a target capture probe, and more specifically, to comprise a step of hybridizing a biomaterial-specific target sequence and a signal probe to detect the modified MMP; a step of detecting each fluorescence signal from a solution including the hybridized MMP and the signal probe emitted from the MMP solution; and a step of detecting the Raman signals by adding metal nanoparticles to the emitted signal probe. The present invention specifically detects two or more types of biomaterials with high sensitivity. The present invention obtains the fluorescence signals and the Raman signals with the high sensitivity; facilitates multiple detection by simplifying a step of separating and cleaning the MMP and using various probes; reduces non-specific combination by separating the hybridizing step and the signal detecting step; and increases biomaterial detecting efficiency.

Description

Biomaterial detection method using magnetic microparticles and surface-enhanced Raman signal {Surface-enhanced Raman scattering (SERS) with magnetic microparticle-based assay for bio materials detection}

The present invention relates to a detection method using magnetic microparticles modified with a target capture probe and a surface-enhanced Raman signal, and the like.

Magnetic microparticles (MMP) have been used for a variety of purposes as a key factor in cell separation, protein purification, and assay protocols for the detection of protein or nucleic acid biomarkers. MMPs are monodisperse micro-sized polystyrene particles containing superparamagnetic nanoparticles therein. They are freely dispersed in a liquid phase, and can be quickly separated from the liquid phase by applying an external magnetic field. In addition, since the MMP surface has various surface chemistry such as carboxyl or amine, it can be utilized in the fields of simple cell analysis, immunoassay, and nucleic acid detection by modifying the MMP to include an antibody or nucleic acid. .

In the analysis process, after binding a target to a protein or nucleic acid, signal transduction is essential, and various signals including optical and spectroscopy can be directly measured using MMPs or signal molecules released from MMPs. MMP is multivalency, and since the signal molecule is in an enriched state in MMP, the target can be detected with high sensitivity. Previously, the potential utilization of MMP for the detection of pathogen-derived DNA in point-of-care diagnostics (POCD) including a fluorescence signal sent directly from the MMP was studied, and MMP was used as a scanometric method. It has been reported on an MMP-based detection system that can detect target DNA with a sensitivity level of 500 zeptomolar by detecting barcode DNA released from It has also been reported on the detection of barcoded DNA-mediated biomolecules with aM sensitivity by using mass spectrometry to detect DNA barcodes released from MMPs. The above studies include or exclude additional signal amplification methods, and can be viewed as assays that strongly depend on the specific properties of MMPs.

Despite various analyzes using MMP, Raman scattering as a signal readout has not yet been widely used. This is because the Raman scattering has a low signal intensity compared to the fluorescence signal. However, the Raman signal can be greatly increased by using surface-enhanced Raman dispersion, which is not only a promising method for signal transduction, but also a good strategy for signal amplification in MMP-based assays. However, a systematic comparison of the analytical capability between Raman dispersion and fluorescence signal has not yet been investigated in detail.

Therefore, in the present invention, nucleic acid targets of bacteria such as Escherichia coli (E. coli) , Enterococcus faecalis (E. faecalis) , and Staphylococcus aureus (S. aureus ), which are major pathogens known to cause sepsis, were selected. . In the case of sepsis, a simple, fast, and robust assay that does not rely on culture-based methods or conventional enzymatic amplification methods such as polymerase chain reaction (PCR). There is a strong demand for This is because bacterial culture is time-consuming and PCR is an enzymatic amplification method, which tends to produce false-positives. In this respect, MMP-based assays involving Raman scattering are promising as reliable non-enzymatic amplification methods and assay strategies.

The present invention devised a target capture probe DNA sequence and a signal probe DNA sequence modified with a fluorescent dye, both sequences being half-complementary to the target DNA sequence (Fig. 2). The fluorescence signal can be i) measured directly from a solution containing MMP, or ii) by a signal probe emitted from the MMP solution. The emitted signal probe may be quantified using surface-enhanced Raman scattering (SERS). Based on the above strategy, through rigorous comparison of analytical capabilities such as sensitivity and multidiagnostic capability between fluorescence and Raman scattering, the present inventors found that MMP-based methods including SERS detect bacterial target DNA in terms of sensitivity and multidiagnostic capability. It was confirmed that it is a promising new strategy to do this, and the present invention was completed.

KR 10-2156165

An object of the present invention is to provide a method for detecting multiple biomaterials including magnetic microparticles modified with a target capture probe.

Another object of the present invention is to provide an information providing method for diagnosing bacterial infectious diseases including magnetic microparticles modified with a target capture probe.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a method for detecting multiple biomaterials comprising the following steps.

(a) preparing magnetic microparticles (MMP) modified with a target capture probe;

(b) hybridizing with a biomaterial-specific target sequence and signal probe to detect the modified MMP with the target capture probe;

(c) detecting a fluorescence signal from a solution containing the hybridized MMP and a signal probe emitted from the MMP solution, respectively; and

(d) detecting a Raman (SERS) signal by adding metal nanoparticles to the emitted signal probe.

In one embodiment of the present invention, the target capture probe and the signal probe may be half-complementary to the target sequence, respectively.

In another embodiment of the present invention, the MMP of step (a) is modified with two or more different target capture probes, and can specifically bind to two or more target sequences.

As another embodiment of the present invention, the hybridization time in step (b) is 3 hours to 12 hours, and may be to treat the salt at a concentration of 0.15 M to 0.3 M, preferably the hybridization time is 3 hours, and the The concentration may be 0.3 M.

As another embodiment of the present invention, the signal probe emitted in step (c) is dispersed by applying a magnetic field to the hybridized MMP solution, and is emitted by heating to a temperature higher than _{the T m (melting temperature) of the target sequence.} can

In another embodiment of the present invention, the detection method may have a minimum detection limit of 30 fM, that is, 3 Х 10 ^-14 M.

As another embodiment of the present invention, the surface of the metal nanoparticles is cysteamine, APTES (3-Aminopropyl) triethoxysilane), APTMS (3-Aminopropyl) trimethoxysilane, AEAPTMS (N- [3- (Trimethoxysilyl) propyl) ]ethylenediamine, MPTMS(3- Mercaptopropyl)trimethoxysilane, MPTES((3-Mercaptopropyl)triethoxysilane), MPA(mercaptopropionic acid), MHA(6-mercaptohexanoic acid), BDT(1,4-benzenedithiol), MBA(4-mercaptobenzoic acid) ), and MBIA (2-mercaptobenzoimidazole-5-carboxylic acid) may be modified with any one or more selected from the group consisting of.

In another embodiment of the present invention, the signal probe may be labeled with a fluorescent material, a color developing material, or a chemiluminescent material.

As another embodiment of the present invention, the fluorescent material is an ATTO-based dye, a cyanine-based dye, FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, fluorescein isothiocyanate (FITC), oregon green, Alexa Fluoro (alexa fluor), JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), It may be any one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), and thiadicarbocyanine.

In another embodiment of the present invention, the detection method may be performed in a well, a channel, a tube, or a combination thereof.

In another embodiment of the present invention, the biomaterial may be any one or more selected from the group consisting of proteins, cells, viruses, nucleic acids, organic molecules, and inorganic molecules.

In addition, the present invention provides an information providing method for diagnosing a bacterial infectious disease comprising the following steps.

(a) preparing magnetic microparticles (MMP) modified with a target capture probe;

(b) hybridizing with a biomaterial-specific target sequence and signal probe to detect the modified MMP with the target capture probe;

(c) detecting a fluorescence signal from a solution containing the hybridized MMP and a signal probe emitted from the MMP solution, respectively; and

(d) detecting the emitted Raman (SERS) signal by adding metal nanoparticles to the emitted signal probe.

In one embodiment of the present invention, the target capture probe and the signal probe may be half-complementary to the target sequence, respectively.

As another embodiment of the present invention, before step (a), it may include extracting genomic DNA from a patient sample suspected of having a bacterial infectious disease.

In another embodiment of the present invention, the patient sample may be any one or more selected from the group consisting of blood, serum, and plasma.

In another embodiment of the present invention, the MMP of step (a) is modified with two or more target capture probes of different types, and specifically binds to two or more target sequences, thereby producing a microorganism comprising the target sequence. It may be diagnosing two or more types of bacterial infectious diseases, each of which is a causative organism.

In another embodiment of the present invention, after step (d), determining the presence or absence of a bacterial infectious disease causative organism or the type of the causative organism using information of a fluorescence signal or a Raman signal by the biomaterial. can

In another embodiment of the present invention, the bacterial infectious disease is sepsis, sinusitis, pneumonia, food poisoning, dysentery, tuberculosis, P. aeruginosa infection, impetigo, purulent disease, acute dermatitis, bacteremia, endocarditis, enteritis, osteoarthritis, osteomyelitis, urinary tract infection, encephalitis , may be any one or more selected from the group consisting of meningitis, and nephritis.

In another embodiment of the present invention, the bacteria may be any one or more selected from the group consisting of gram-positive bacteria, gram-negative bacteria, and mixtures thereof.

The present invention relates to a method for multiple detection of a biomaterial using a Raman signal including magnetic microparticles (MMP) modified as a target capture probe, and more specifically, a biomaterial-specific target for detecting the modified MMP. After hybridization with a sequence and a signal probe, a fluorescence signal is detected from a solution containing the hybridized MMP and a signal probe emitted from the MMP solution, respectively, and then metal nanoparticles are added to the emitted signal probe to detect a Raman signal By including the step of, it is possible to specifically detect two or more types of biomaterials with high sensitivity. The present invention can obtain a fluorescence signal and a Raman signal with high sensitivity, and since the step of separating and washing MMP is simple, multiple detection using various probes is possible, and non-specific binding is reduced by separating the hybridization step and the signal detection step and increase the biomaterial detection efficiency.

1 shows an MMP-based assay for bacterial target DNA detection. The MMP modified with the target capture probe is hybridized with the biomaterial target sequence and the signal probe, and the MMP hybridized with the signal probe is separated in a magnetic field. Fluorescence intensity and emitted signal of the MMP can be measured through a fluorescence and SERS-based detection method.
Figure 2 shows the effect of the fluorescence signal according to the hybridization conditions. (A) shows the target binding process, (B) shows the fluorescence intensity and image of the MMP after hybridization of the target DNA according to the hybridization time (0, 1, 3, 6, 12 hours), (C) is The fluorescence intensity and image of MMP after hybridization of target DNA according to salinity (in case of no target, 0.15 M NaCl, 0.3 M NaCl) are shown. At this time, the scale bar is 5 μm.
3(A) shows an experimental technique for quantifying the amount of target capture probe in MMP using Cy5-modified DNA (5'-Cy5 dye-GTGCCAATGATCTTCGCTCG-3') complementary to the target capture probe, (B ) shows the calibration curve of the Cy5-DNA sequence, and (C) shows B/F and fluorescence images of COOH-functionalized MMP, Cy5-DNA hybridized MMP, and MMP after releasing Cy5-DNA.
4(A) shows a TEM image of Cys-AuNP, and (B) shows a UV-Vis spectrum of Cys-AuNP.
Figure 5 (A) is a schematic diagram of the extraction method of bacterial genomic DNA and the design of the target DNA, (B) is a growth curve of E. coli cultured in tryptic soy broth medium at ^{37 o C measured at 600 nm.} and (C) shows the UV-Vis spectrum of the extracted E. coli genomic DNA.
Figure 6(A) is a signal probe solution (5'-ATTO488 dye-GGATTTTCGACAGGATATTA-3' ( E.coli ), 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3' ( E. faecalis ), and 5'-ATTO647 dye-TCACAAACAGATAATGGCGT -3' ( S. aureus )) (10 ^-7 M) shows a fluorescence image, (B) is a B/F and fluorescence image of MMP alone, MMP excluding the target, and MMP hybridized with the target DNA of each bacteria is shown.
7(A) shows a signal probe solution (5'-ATTO488 dye-GGATTTTCGACAGGATATTA-3', 5'-cy5 dye-GGAT TTTCGACAGGATATTA-3', 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3', and 5'-ATTO647 dye -TCACAAACAGATAATGGCGT-3') (10 ^-6 M) shows the SERS spectrum, (B) is a mixed signal probe solution (5'-ATTO488 dye-GGATTTTCGACAGGATATTA-3', 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3' SERS spectrum of 5'-ATTO647 dye-TCACAAACAGATAATGGCGT-3', and 5'-cy5 dye-GGAT TTTCGACAGGATATTA-3', 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3', 5'-ATTO647 dye-TCACAAACAGATAATGGCGT-3') is shown.
8 is a comparison of the sensitivities of fluorescence-based detection and SERS-based detection. (A) shows the analysis technique of fluorescence and SERS, (B) shows the fluorescence intensity and image of the MMP hybridized with the signal probe, and the fluorescence intensity of the emitted signal probe, (C) shows the target DNA concentration It shows the SERS spectrum of the emitted signal probe, (D) shows the SERS intensity of the Raman shift (Raman shift) selected from the spectrum (C). At this time, the scale bar is 5 μm.
9 shows a multiplex diagnostic analysis of fluorescence-based and SERS-based detection for a single target DNA. (A) shows overall multiplex diagnostic assays including fluorescence-based and SERS-based detection of single targets (E.coli, E.faecalis, and S.aureus ), (B) single signal probes (ATTO 488; E Coli , ATTO 590; E. feacalis , and ATTO 647; S. aureus ) shows the fluorescence intensity and image of hybridized beads, (C) shows the fluorescence intensity of the emitted signal probe, (D) shows The SERS spectrum of the bacterial target DNA (10 ^{-9 M) is shown.} Reference spectrum of each signal probe is indicated by a dotted line. The reference spectrum was measured in 10 μL, ^{aggregated by mixing with 100 μL of signal probe (10 −7} M) and 100 μL of Cys-AuNP (OD 1.0), and centrifuged at 1800 rpm for 15 minutes. At this time, the scale bar is 5 μm.
Figure 10 shows E. faecalis target DNA at various target concentrations (10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, 10 ^-13 M, 10 ^-14 M, no target). It is the result of repeated measurement of the Raman spectrum obtained from three independent analyzes. (A) is 1 ^st attempt, (B) is 2 ^nd attempt, (C) shows a 3 ^rd attempt. (D) shows the intensity of the Raman shift (Raman shift (1333.29, 1520.53, and 1640.75 cm ^{-1 )) selected from the three independent analyzes.}
11 shows a multiplex diagnostic analysis for a plurality of target DNAs. (A) shows the overall multiplex diagnostic analysis of multiple target DNAs, (B) shows the fluorescence intensity and images of beads hybridized with multiple signal probes, (C) shows the fluorescence of multiple emitted signal probes Intensity is shown, and (D) shows SERS spectra of multiple target DNAs. At this time, the scale bar is 5 μm.
12 relates to the detection of E. coli genomic DNA. (A) shows the experimental procedure for detecting E. coli genomic DNA by fluorescence and SERS-based methods, (B) shows the fluorescence intensity of the signal probe and the bead hybridized with the emitted signal probe, (C) shows the SERS spectrum according to the concentration of E. coli genomic DNA (30 pM ~ 30 fM).

The present inventors have completed the present invention by confirming the high sensitivity and multi-diagnostic capability of a detection method including magnetic microparticles and metal nanoparticles modified with a capture probe as a result of intensive research on a method for detecting multiple biomaterials.

As used herein, the term “biomaterials” refers to biomolecules representing a specific substrate, and may be interpreted as having the same meaning as target molecules or analytes. The biomolecule may be a protein, a cell, a virus, a nucleic acid, an organic molecule, or an inorganic molecule. In the case of the protein, any biomaterial such as an antigen, antibody, substrate protein, enzyme, coenzyme, etc. is possible. In the case of the nucleic acid, it may be DNA (gDNA and cDNA), RNA, PNA, LNA, or a combination thereof, and a nucleic acid molecule The nucleotide, which is the basic structural unit in , includes not only natural nucleotides, but also analogs in which sugar or base regions are modified. The biomaterial may preferably include bacteria, viruses, fungi, fungi, or a combination thereof.

As used herein, the term “target sequence” refers to a specific nucleic acid sequence specific to the biomaterial to be detected.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA consisting of several to hundreds of bases capable of specifically binding to the target sequence, and is labeled to determine the presence or absence of the target sequence. can be checked The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like.

As used herein, the term “target capture probe” refers to a chemical or biological agent having molecular properties capable of recognizing a specific region of the biomaterial and specifically binding thereto.

As used herein, the term "signal probe" refers to a probe comprising a sequence that is half-complementary to the target sequence. When the signal probe is bound to its target sequence, a detectable signal is detected. and, when the signal probe is not bound to its target sequence, it may generate no or little or a different signal. For example, the signal probe is a fluorescence-generating or fluorescent probe including a fluorescent substance and a quencher moiety, and when hybridized with a target sequence, a fluorescence change occurs.

The fluorescent material is an ATTO-based dye, a cyanine-based dye, FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, fluorescein isothiocyanate (FITC), oregon green, Alexa Fluoro (alexa fluor), JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), It may be any one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), and thiadicarbocyanine, but is not limited thereto.

As used herein, the term “hybridization” means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization may occur when the complementarity between two nucleic acid strands is perfect (perfect match) or may occur even in the presence of some mismatched bases. The degree of complementarity required for hybridization may vary depending on hybridization conditions.

As used herein, the term “metal nanoparticles” refers to nano-sized particles made of metal. The metal may be selected from the group consisting of gold, silver, copper, and mixtures thereof, but is not necessarily limited thereto, and any metal widely used in the art may be used without limitation. The diameter of the metal nanoparticles may be 0.1 nm or more and 1000 nm or less, and preferably gold nanoparticles (AuNP). The gold nanoparticles can be manufactured, and the size of the particles can be easily controlled, and have advantages of high biostability and low cytotoxicity.

As used herein, the term "Raman scattering" refers to a phenomenon in which a part of light deviates from a traveling direction and proceeds in another direction by changing the wavelength of light when light passes through a certain medium. As used herein, the term "Raman spectroscopy" is a useful spectroscopy method that obtains molecular level information by irradiating a short-wavelength laser to a sample to detect scattered light. It is widely used in the field of biochemistry/chemistry/materials as a method to obtain chemical structure information, and sample measurement is very simple compared to infrared spectroscopy. Raman spectroscopy provides information about the vibrational state of molecules. In most cases, absorbed radiation is re-radiated at the same wavelength, a process called Rayleigh scattering or elastic scattering. At this time, the energy difference between the re-radiated light rays appears as a shift in the wavelength between them, and the degree of this difference is measured as a unit of the wavenumber (the reciprocal of the wavelength) and expressed as a Raman shift. If the projected light has a short wavelength and uses a laser as its source, the scattered light (light) having a difference in frequency can be more easily distinguished from the Rayleigh scattered light (light). That is, the Raman shift corresponds to the vibration frequency of the molecule. Therefore, Raman spectroscopy is used to obtain information on the vibrational form and rotational state of molecules like infrared spectroscopy, but it is based on a different mechanism and selection rule than in infrared spectroscopy, and the measurement method is also different.

As an embodiment of the present invention, there is provided an information providing method for diagnosing bacterial infectious diseases, including measuring a surface-enhanced Raman scattering (SERS) signal. In the above step, the Raman signal may be measured by separating only the fluorescent material or the Raman active material bound to the end of the probe. In this case, the wavelength of the irradiated light may be in the range ^{of 400 cm -1} to 800 cm ^-1. For example, the wavelength of the light is 400 cm ^-1 to 800 cm ^-1 , 420 cm ^-1 to 800 cm ^-1 , 450 cm ^-1 to 800 cm ^-1 , 480 cm ^-1 to 790 cm ^-1 , or It may be 488 cm ^-1 to 785 cm ^{-1 .} In addition, the wavelength of the light may be an Ar ⁺ laser having a wavelength of 514 nm, a He-Ne laser having a wavelength of 633 nm, or a diode laser having a wavelength of 785 nm. That is, when a specific peak is observed from the Raman signal, it can be determined that the individual has a bacterial infectious disease, and the bacterial infectious disease is sepsis, sinusitis, pneumonia, food poisoning, dysentery, tuberculosis, Pseudomonas aeruginosa infection, impetigo, purulent disease, acute dermatitis, bacteremia, endocarditis, enteritis, osteoarthritis, osteomyelitis, urinary tract infection, encephalitis, meningitis, or nephritis. The target sequence can be detected through the above method, and quantitative detection is also possible.

In the present invention, "diagnosis" means determining the susceptibility of an individual to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or an individual suffering from a specific disease or disorder determining the prognosis of, or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy).

As used herein, the term “multiplexing detection or diagnosis” refers to the simultaneous detection of one or more types of biomaterials.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Example 1. Materials, Devices and DNA Information

All oligonucleotides were purchased from Integrated DNA Technologies (IDT Inc., Coralville, IA, USA). Dynabeads (M-270 carboxylic acid) were obtained from BBI Solutions invitrogen (Madison, Waltham, Massachusetts WI, USA). N- (3-dimethylaminopropyl) -N'- ethylcarbodiimide hydrochloride (EDC), 2- (N-morpholino) ethanesulfonic acid (MES) hydrate, Ethanolamine, sodium dodecyl sulfate (SDS), Gold (Ⅲ) chloride trihydrate (HAuCl 4 · 3H ₂ O), cysteamine, and sodium borohydride (NaBH ₄ ) were obtained from Sigma Aldrich (St. Louis, MO, USA). Sodium phosphate monobasic dihydrate (NaH ₂ PO ₄ ), sodium phosphate dibasic anhydrate (Na ₂ HPO ₄ ) and sodium chloride (NaCl) were obtained from DAEJUNG (Siheung, South Korea). Escherichia coli (KCCM 11234) was obtained from the Korean Culture Center of Microorganisms (KCCM, Daejeon, South Korea). The E. coli was grown in tryptic soy broth (Difco, Detroit, MI). The E. coli genomic DNA was extracted from a DNA purification kit and automated instrument (MX-8L Apintech, Seoul).

Extinction spectra were obtained with an Ultraviolet-visible (UV-Vis) spectrometer (SCINCO, South Korea). A transmission electron microscope (TEM) (H-7100, Hitachi, Tokyo, Japan) was used to characterize the nanoparticles. Bright field and fluorescence images were obtained under a microscope (Olympus, DP80, Tokyo, Japan) with a Х 20 air objective and Х 100 oil immersion lens objective (NA 1.3) and EzScan (NOST, South Korea) software was recorded using A Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Inc, Winooski, USA) was used to measure the fluorescence intensity. DNA extraction was performed using MX-8L nucleic acid extractor (Apintech, Seoul).

To design a bacterial-specific probe sequence, the present inventors selected target sequences (40 bp) of E. coli , E. faecalis , and S. aureus. Bacterial sequences were obtained from the NCBI database (https://blast.ncbi.nlm.nih.gov/), and gene alignment was performed manually using Mega 5 program. In silico tests for primer specificity were performed using the NCBI BLAST tool (https://blast.ncbi.nlm.nih.gov/). The target sequence is shown in Table 1 below.

E. coli
target sequence SEQ ID NO: 1 5'-TTTTGGCAACAGGGCTAGGTTAATATCCTGTCGAAAATCC-3' (T _m ; 65.5 ^o C) E. faecalis
target sequence SEQ ID NO: 2 5'-GTGCCAATGATCTTCGCTCGTAAGGCAAAAAGTTTAACCA-3' (T _m ; 65.5 ^o C) S. aureus
target sequence SEQ ID NO: 3 5'-CTTCAGAACCACTTCTATTTACGCCATTATCTGTTTGTGA-3' (T _m ; 63.5 ^o C)

The target capture probe sequence semi-complementary to the target sequence was modified with an amine group, an A10 spacer, and a short polyethylene group at the 3' end of the sequence. The target capture probes are shown in Table 2 below.

E. coli
capture probe SEQ ID NO: 4 5'-ACCTAGCCCTGTTGCCAAAA-PEG-A ₁₀ -NH ₂ -3' E. faecalis
capture probe SEQ ID NO: 5 5'-CGAGCGAAGATCATTGGCAC-PEG-A ₁₀ -NH ₂ -3' S. aureus
capture probe SEQ ID NO: 6 5'-AAATAGAAGTGGTTCTGAAG-PEG-A ₁₀ -NH ₂ -3'

For fluorescence-based detection, the signal probe sequences shown in Table 3 below were used. In addition, a Cy5-modified E. coli signal probe (5'-Cy5 dye-GGATTTTCGACAGGATATTA-3', SEQ ID NO: 10) was used for fluorescence-based detection of the extracted genomic DNA, and a Cy5-modified E. faecalis signal probe (5 '-Cy5 dye-GTGCCAATGATCTTCGCTCG-3', SEQ ID NO: 11) was used to quantify the number of capture probes to be modified in MMP.

E. coli
signal probe SEQ ID NO: 7 5'-ATTO488 dye-GGATTTTCGACAGGATATTA-3' E. faecalis
signal probe SEQ ID NO: 8 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3' S. aureus
signal probe SEQ ID NO: 9 5'-ATTO647 dye-TCACAAACAGATAATGGCGT-3'

For SERS-based detection, the ATTO488-modified E. coli signal probe was replaced with Cy5. This is because ATTO488 has a lower SERS intensity compared to ATTO590 and ATTO647, which is considered unsuitable for SERS-based detection. Therefore, the E. coli signal probe was 5'-cy5 dye-GGATTTTCGACAGGATATTA-3' (SEQ ID NO: 10), and the E. faecalis and S. aureus signal probes were used in the same way as the signal probes of the fluorescence-based assay.

Example 2. Construction of MMPs modified with target capture probes

MMPs modified with target capture probes were fabricated using the EDC coupling method. First, 100 μL of carboxylic acid-modified dynabeads (MMPs) (30 mg/mL) was washed by adding 100 μL of 25 mM MES buffer for 10 minutes. The MMP was dispersed by applying a magnetic field for 2 minutes, and the supernatant was removed. This step was repeated 2-3 times. An amine-modified DNA oligonucleotide ( E. faecalis capture probe, 10 ^-4 M, 60 μL) was added to the MMP solution (30 mg/ml, 100 μL). After incubation at room temperature for 30 minutes with a slow gradient rotation, an EDC (100 mM in MES buffer, 30 μL) solution was added to the MMP solution, and incubated overnight with a slow gradient rotation. Then, it was washed with 50 mM ethanolamine at room temperature with slow gradient rotation in PBS, pH 8.0 for 60 minutes. The washing step was repeated 4 times with PBS. Then, the MMPs modified with the target capture probe were redispersed in distilled water (10 mL), and the amount of the target capture probe in the MMP was measured with a fluorometer (FIG. 3).

Example 3. Synthesis of cysteamine modified with gold nanoparticles (AuNP)

To prepare AuNP-modified cysteamine (Cys-AuNPs), 200 mL of HAuCl ₄ 3H ₂ O solution (111.5 mg/200 mL of distilled water, 1.42 mM) was mixed with 2 mL of cysteamine solution (164 mg /10 mL of distilled water, 213 mM). At this time, the color of the solution changed from yellow to orange. After sufficient mixing, the solution was added to 50 μL NaBH ₄ solution (37.8 mg/10 mL of distilled water, 10 mM) and mixed overnight. At this time, the color of the solution changed from orange to red wine. Then, the solution was centrifuged at 1800 rpm for 15 minutes. UV-Vis extinction spectra were recorded at 530 nm. As a result of measuring the diameter of Cys-AuNPs using TEM, it was 30.6 nm ± 4.1 nm (FIG. 4).

Example 4. E. coli Genomic DNA Extraction

E. coli was grown in tryptic soy broth (Difco, Detroit, MI) and incubated at 37 °C. When E. coli reached the stationary phase of the growth curve, it was centrifuged at 6000 rpm for 10 minutes. The precipitate was redispersed in sterile saline and concentrated. The concentrated E. coli genomic DNA was extracted using the MX-8L cell-free DNA purification kit using bead beating. The DNA was strongly bound to the silica bead in the presence of a high concentration of guanidinium thiocyanate, and the residue remained on the column. Since other biomaterials (protein, RNA, etc.) were not bound, only genomic DNA was isolated. To remove beads from the extracted genomic DNA, centrifugation was performed at 12,000 rpm for 3 minutes. The molar concentration of E. coli genomic DNA was measured at absorbance at 260 nm and calculated by applying Beer-Lambert's law. The molar extinction coefficient (https://www.ncbi.nlm.nih.gov/genome/?term=E.coli) of E. coli ^{genomic DNA is 8.36 Х 10 10} /mol ^-1 cm ^-1 it was For example, when an E. coli genomic DNA solution is diluted 100 times and the absorbance is 0.84, the molar concentration of the genomic DNA is 1 nM. The molar concentration of E. coli genomic DNA calculated using the empirical rule, not the Beer-Lambert rule, was 1.32 nM. There was little difference in the molar concentrations calculated by applying the above two calculation methods. Therefore, the molar concentration of E. coli genomic DNA was calculated by applying the Beer-Lambert law (FIG. 5).

Example 5. Fluorescence measurement conditions

Fluorescence images of MMP hybridized with a signal probe were obtained using a microscope including GFP, TRITC, and Cy5 filters, respectively. The ATTO 488, ATTO 590, and ATTO 647 signals of the MMP are GFP filters (518-559 nm), TRITC filters (572-638 nm), and Cy5 filters (665-715) exposed for 0.5 sec, 0.25 sec, and 2 sec. nm) was detected. To measure the fluorescence intensity from the signal probe solution, the solution was excited at specific wavelengths (500 nm (ATTO 488), 610 nm (ATTO 590), and 650 nm (ATTO 647)), 530 nm, 640 nm, and at a wavelength of 680 nm (Fig. 6).

Example 6. SERS measurement conditions

SERS spectra were collected using an inverted Raman microscope (NOST, South Korea) with a × 60 objective (NA 0.7) (Olympus, Tokyo, Japan) and a 633 nm laser (6.7 mW, exposure time of 1 sec.) was obtained from the irradiated solution. The dispersed Raman signal is directed to a spectrometer (FEX-MD, NOST, South Korea) (1200 g nm ^-1 grating), and finally a spectroscopy charge-coupled device (CCD) camera (Andor (DV401A-DVF), Belfast, North Ireland) ) was detected with a confocal motorized pinhole (100 um) reaching the RAON Scan (NOST, South Korea) software was used to acquire Raman spectra.

For each SERS spectrum, the signal probe solution emitted from the MMP (100 μL) was mixed with 100 μL of cysteamine modified with AuNPs (OD 1.0) for 5 min with gentle shaking, and the solution was centrifuged at 1,800 rpm for 15 min. did. The centrifuged solution containing the precipitate was transferred to a well silicone isolator (2 mm diameter) of a cover glass for Raman analysis.

The main SERS peaks of Cy5, ATTO 488, ATTO 590, and ATTO 647 were obtained in pure or mixed state (ATTO 488, ATTO 590, ATTO 647 or Cy5, ATTO 590, ATTO 647). Raman spectra were compared in terms of sensitivity or multiple diagnostic capability. The main Raman scattering peaks of Cy5 were selected at 1187.08, 1359.22, 1465.1, and 1594.19 cm ⁻¹ . The main Raman scattering peaks of ATTO 590 were selected at ^{1333.29, 1520.53, and 1640.75 cm −1 .} The main Raman scattering peaks of ATTO 647 ^{were selected at 1423.6, and 1626.82 cm −1} ( FIG. 7 ).

Experimental Results 1. MMP-based analysis design for bacterial target DNA detection

Magnetic microbead (MMP)-based assays for nucleic acid detection are useful and simple methods in various fields. MMPs can be freely dispersed in a mixed solution due to their superparamagnetism, so they can be efficiently hybridized with target DNA, and can be rapidly dispersed by applying an external magnetic field. In the present invention, by utilizing the advantages of the above-described MMP, as shown in FIG. 1 , an MMP-based nucleic acid target detection method was devised.

First, the MMP was modified with a target capture probe semi-complementary to the target DNA sequence. MMPs can hybridize to a target sequence and a signal probe sequence that is semi-complementary to the target sequence (fluorescence-tagged). The fluorescence intensity of MMPs can be easily measured, and the fluorescence intensity is correlated with the amount of target DNA of the analyte. The fluorescence signal intensity can be measured with the emitted signal probe by raising the concentration of the MMP solution to 95 ^° C (>T _{m ).} In addition, SERS-based detection is also possible by measuring the SERS signal of fluorescent molecules in the emitted signal probe by mixing the fluorescent probe with AuNP-modified cysteamine (Cys-AuNPs). With the above method, the present invention strictly compared the analytical capabilities according to types such as fluorescence in beads, fluorescence in solution, and SERS intensity. In addition, the optimized assay platform will be utilized for a simple nucleic acid detection strategy.

Experimental result 2. Quantification of target capture probe DNA (capture DNA) amount in MMP

First, MMP was modified into an amine-modified target capture probe through a coupling reaction of a carboxyl group (COOH)-coated magnetic microparticle (MMP) using EDC coupling ( FIG. 3 ). The detailed manufacturing process was described above in Example 2.

To confirm the successful binding and the amount of target capture probe in MMP, a complementary DNA sequence including Cy5 dye was hybridized with the target capture probe in MMP in 0.3 M PBS for 3 hours, and excess unbound Cy5 dye DNA sequences including The hybridized Cy5 sequence was then released from the MMP by heating the solution to 95 ^°C. The fluorescence intensity of the released Cy5 DNA sequence was measured, and the amount of the Cy5 DNA sequence was calculated (FIG. 3A).

By using the Cy5 DNA sequence (1000, 500, 100, 50, and 10 pM) and the obtained calibration curve, the concentration of the Cy5 DNA sequence was determined, and the amount and number of target capture probes per MMP were calculated. Therefore, the number of target capture probe DNAs per MMP ^{was calculated to be 1.2Х10 4} , which is a suitable value for the binding ability of MMPs.

Experimental results 3. Optimization of analysis conditions

In order to efficiently detect a target nucleic acid, analysis conditions such as hybridization time and salinity are very important. The present invention optimizes the assay conditions before comparing the MMP-based assay capabilities involving both optical signals. As shown in Figure 2A, a dilution of MMP (100 μL, 0.03 mg/ml), a signal probe (50 μL, 10 ^-6 M, 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3', SEQ ID NO: 8), and target DNA After mixing all (15 μL of 10 ^-8 M ~ 10 ^-9 M final concentration, 5'-GTGCCAATGATCTTCGCTCGTAAGGCAAAAAGTTTAACCA-3'; E. faecalis target DNA, SEQ ID NO: 2), phosphate buffer (100 mM, pH 7.4) and 2 M NaCl was added to prepare 10 mM PB and 0.3 M NaCl to final concentrations. Then, the solution was ^{heated at 95 o} C for 2 minutes, and mixed at room temperature with different hybridization times (0, 1, 3, 6, and 12 hours). Finally, MMP was separated by removing the supernatant, washed 5 times in 0.15 M PBS buffer, and 10 μL of 0.15 M PBS buffer was added. The fluorescence intensity of ATTO590 dye was obtained from MMP (λ _ex = 610 nm, λ _em = 640 nm).

Fluorescence intensity increased linearly with increasing hybridization time. The fluorescence intensity reached saturation at the 12-hour incubation time starting from the 3-hour hybridization time (FIG. 2B). Since the intensity difference between 3 h and 12 h was about 20%, the hybridization time of 3 h was relatively reasonable considering the overall analysis time.

The salt (NaCl) concentration is also an essential condition for efficiently hybridizing the target DNA. All the conditions described above were the same, and only the concentration of the salt was changed from 10 mM PB to 0 M, 0.15 M, and 0.3 M NaCl. As shown in Fig. 2C, the hybridized fluorescence intensity in 0.15 M NaCl was 22.4% lower than that obtained in 0.3 M NaCl. As expected, higher salt conditions resulted in higher hybridization yields. Based on the above results, in the present invention, a hybridization time of 3 hours and a NaCl concentration of 0.3 M was adopted as an MMP-based assay.

Experimental Results 4. Comparison of Analysis Capabilities Between Fluorescence and SERS

The present invention compared the ability of MMP-based assays to include two different optical signals. First, while the concentration of the signal probe (10 ^-6 M) is fixed, the range of the target concentration (10 ^-9 , 10 ^-12 , 10 ^-15 , 10 ^-18 M) is changed and the sensitivity of the MMP-based assay is (Optimized analysis conditions were detailed in Experimental Result 3).

E. faecalis
target sequence SEQ ID NO: 2 5'-GTGCCAATGATCTTCGCTCGTAAGGCAAAAAGTTTAACCA-3' (T _m ; 65.5 ^o C) E. faecalis
capture probe SEQ ID NO: 5 5'-CGAGCGAAGATCATTGGCAC-PEG-A ₁₀ -NH ₂ -3' E. faecalis
signal probe SEQ ID NO: 8 5'-ATTO590 dye-TGGTTAAACTTTTTGCCTTA-3'

For fluorescence measurement, the fluorescence intensity of the MMP solution hybridized with the target and the signal probe was measured, and then the fluorescence intensity of the signal probe emitted from the MMP was measured (FIG. 8A). As shown in FIG. 8B , the fluorescence intensity of the MMP solution decreased significantly as the concentration of target DNA decreased. For ^{target concentrations of 10 −11} M, the fluorescence intensity did not differ from the control (no target), indicating the detection limit (0.1 nM) of the fluorescence-based method. The fluorescence images of MMP were consistent with the results obtained in the MMP solution. The fluorescence intensity of the emitted signal probe solution also showed the same trend, but was lower than that of MMP.

For SERS-based measurements, the emitted signal probes were mixed with Cys-AuNPs to form aggregates. Raman spectra were measured from the aggregate solution by irradiating a 633 nm laser. As shown in Fig. 8C, the SERS spectrum of the signal probe (ATTO 590) was clearly confirmed even at a target DNA concentration ^{of 10 -14 M.} The signal intensities of the three main Raman peaks (1333.29, 1520.53 and 1640.75 cm ^{−1 ) were obtained from various target concentrations (10 −9} M to 10 ⁻¹⁴ M), demonstrating the wide dynamic range of SERS-based detection (Fig. 8D). ). These results indicate the excellent sensitivity of SERS-based detection, which is an important capability for all assays in the field of bioanalysis. In addition, reproducibility is also a very important issue in any analysis. We have found that solution-based measurement of SERS greatly improves signal reproducibility. To measure the signal reproducibility, three independent measurements for the same target and various concentrations were performed, and as a result, the same SERS spectra were obtained from the emitted signal probe and the Cys-AuNP mixture (Fig. 6).

Experimental result 5. Multiplexing capability for single target DNA

Along with sensitivity, multidiagnostic capability is a very important capability for the development of bioanalytical fields. In this regard, since the target capture probe DNA sequence has a high concentration per MMP, the use of MMP is highly desirable. At this time, ^{it should be noted that the 1.2 Х 10 4} probe DNA sequence can be modified in a single MMP. To demonstrate the multidiagnostic ability of MMPs for bacterial target DNA, three different target capture probes must be modified in MMPs, each of which binds to their designed target sequences (E. coli , E. faecalis , and S. aureus). should be able to (Fig. 9A). Before simultaneously detecting various target sequences, the present invention first investigated a method for detecting a single target sequence using an MMP modified with three different target capture probes. For detection of E. coli , E. faecalis , and S. aureus , ATTO 488, A single probe including ATTO 590 and ATTO 647 dyes was used, and fluorescence images were obtained using GFP, TRITC, and CY5 filters under a microscope ( FIG. 7 ). The selected filter sets were suitable for the fluorescent dyes designed within a single probe.

MMPs modified with three target capture probes ^{were hybridized with a single target (10 −9} M) in 0.3 M PBS for 3 hours. After performing the usual washing steps, the fluorescence intensity of the MMP solution was measured. As shown in Fig. 9B, strong fluorescence intensities were obtained only when the correct target sequence was present ( E. coli (1), E. faecalis (2), and S. aureus (3)). In the absence of a target (4), no fluorescence signal was observed. The fluorescence image of the MMP also matched the solution-based measurement result, ie the intensity of the single emitted probe. The result obtained from the emitted signal probe was consistent with the MMP-based fluorescence measurement result (FIG. 9C). Finally, the SERS spectrum obtained from the mixture of Cys-AuNPs and single probes was ATTO 488, It matched typical Raman spectra of ATTO 590, and ATTO 647 dyes. The dotted line in the spectrum in Fig. 9D represents the Raman spectrum of each dye as a reference. All of the above results indicate the specific binding capacity of MMPs to the designed targets, which is an important starting point in multiplex diagnostic assays. The choice of Raman active molecule is also important for the sensitivity of the assay. For SERS, ATTO 488, ATTO 590, and ATTO 647 dyes and gold nanoparticles were excited at 633 nm. The absorption spectrum of ATTO 488 dye (400 - 534 nm) did not cause resonance at 633 nm excited state. As shown in FIG. 10 , the intensity of the SERS spectrum of ATTO 488 was very weak compared to the intensity of ATTO 590 and ATTO 647 dyes. This is not a desirable condition for multiplex diagnostic analysis. To overcome the above limitation, ATTO 488 was compared to cy5 dye (λ _abs ) as the cy5 dye had a high Raman cross-section at 633 nm laser excitation and showed ^{distinct Raman peaks at 1187.08, 1359.22, and 1465.7 cm −1 .} = 650 nm) (Fig. 10).

Experimental result 6. Multiple diagnostic ability for multiple target DNA

Next, MMP-based analysis of multiple target DNAs was performed. To demonstrate multiplex diagnostic capability for fluorescent signals, MMPs modified with multiple target capture probes were prepared and hybridized with a mixture of two target sequences ( E. Coli (1) and E. faecalis (2), or E. Coli (1) and S. aureus (3), or E. faecalis (2) and S. aureus (3)) ( FIG. 11A ). After performing the analysis (1.0 nM target concentration, 0.3 M PBS, 3 hours incubation), the analysis result is shown in FIG. 11B. Although the MMP probe was a mixture of E. faecalis (2) or S. aureus (3), it selectively bound to the E. Coli (1) target. The MMP probe also selectively bound to the E. faecalis (2) target, albeit a mixture of E. Coli (1) or S. aureus (3). However, in this case an increased fluorescence intensity can be observed due to the presence of non-specific binding during the assay. For the S. aureus (3) target, the MMP probe selectively bound to the S. aureus (3) target even though it was a mixture of E. coli (1) or E. faecalis (2). The fluorescence intensity of the emitted probe solution showed the same result, but the significant non-specific binding signal observed in E. faecalis (2) target detection was significantly reduced. MMP-based analysis including fluorescence signals not only shows high-accuracy results, but also has excellent multiplex diagnostic capability for three different targets.

Multidiagnostic ability using SERS-based signals was also investigated using a mixture of two target sequences ( E. Coli (1) and E. faecalis (2), or E. Coli (1) and S. aureus (3), or E. faecalis (2) and S. aureus (3)). After the signal probe was released from the MMP, a SERS spectrum was obtained from a mixture of a single probe and Cys-AuNP. As described above, as the SERS reporter, cy5 dye was used for the E. Coli (1) target, the ATTO 590 dye was used for the E. faecalis (2) target, and the ATTO 647 dye was used for the S. aureus (3) target. 11D , E. Coli (1) and E. faecalis (2), E. Coli (1) and S. aureus (3), or E. faecalis (2) and S. aureus (3) The SERS spectrum obtained from The specific Raman spectral patterns of cy5, ATTO 590, and ATTO 647 dyes were not easily distinguished by spectral overlap. The above results indicate that in the selection of SERS reporters for multiplex diagnostic analysis, it is most important not to overlap with each other. Commercial fluorescent dyes are structurally similar and have complex chemical structures, so spectral overlap cannot be avoided. Therefore, simple chemical structures with high Raman cross-section, such as 4,4'-dipyridyl, 4-azobis (pyridine) or alkyne dye, would be desirable as Raman reporters, especially for multidiagnostic Raman-based assays. .

Experiment Result 7. E. coli. dielectric detection of DNA

In order to apply the MMP-based assay capability to actual bacterial targets, the sensitivity of the assay including both optical signals for the extracted E. coli genomic DNA was investigated. 12A shows the process of extracting genomic DNA. A detailed extraction method and a method for determining the concentration of the extracted E. coli were detailed in Example 4. The concentrations of E. coli were adjusted to 30 pM, 3.0 pM, 300 fM, and 30 fM, and then the sensitivities were compared according to the same assay protocol. In this case, a signal probe coupled with a cy5 dye was used to accurately compare both optical signals.

As shown in Fig. 6B, the fluorescence signal was clearly observed in the MMP, and the signal probe was emitted at a target concentration of 300 fM. The standard deviation of the results is the intensity deviation of the results of three independent assays. The sensitivity of the fluorescence signal (300 fM) was 3 Х 10 ³ times higher compared to the results described above (results obtained from ATTO 590 dye and E. faecalis target). At this time, it should be noted that the molar extraction coefficient of Cy5 is two times higher than that of ATTO 590. As shown in Fig. 12C, SERS-based analysis can obtain a clear Raman spectrum of cy5 at a target concentration of 30 fM, and in the absence of a target sample, no signal was observed. All of the above results indicate that MMP-based assays with both optical signals have excellent ability to detect nucleic acid targets even in real bacterial samples. It took 4 hours to perform the entire analysis process, which is a very important process for identifying bacteria to prevent the onset of sepsis. For single-target detection, SERS-based signals were superior to fluorescence-based signals, but design of a Raman reporter is important for multiplex diagnostic assays using SERS, which will serve as a future development direction for MMP-based assays for more practical diagnostics.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Surface-enhanced Raman scattering (SERS) with magnetic microparticle-based assay for bio materials detection <130> DP-2020-0581, DHP20-K104 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Escherichia coli <400> 1 ttttggcaac agggctaggt taatatcctg tcgaaaatcc 40 <210> 2 <211> 40 <212> DNA <213> Enterococcus faecalis <400> 2 gtgccaatga tcttcgctcg taaggcaaaa agtttaacca 40 <210> 3 <211> 40 <212> DNA <213> Staphylococcus aureus <400> 3 cttcagaacc acttctattt acgccattat ctgtttgtga 40 <210> 4 <211> 20 <212> DNA <213> Escherichia coli <400> 4 acctagccct gttgccaaaa 20 <210> 5 <211> 20 <212> DNA <213> Enterococcus faecalis <400> 5 cgagcgaaga tcattggcac 20 <210> 6 <211> 20 <212> DNA <213> Staphylococcus aureus <400> 6 aaatagaagt ggttctgaag 20 <210> 7 <211> 20 <212> DNA <213> Escherichia coli <400> 7 ggattttcga caggatatta 20 <210> 8 <211> 20 <212> DNA <213> Enterococcus faecalis <400> 8 tggttaaact ttttgcctta 20 <210> 9 <211> 20 <212> DNA <213> Staphylococcus aureus <400> 9 tcacaaacag ataatggcgt 20 <210> 10 <211> 20 <212> DNA <213> Escherichia coli <400> 10 ggattttcga caggatatta 20 <210> 11 <211> 20 <212> DNA <213> Enterococcus faecalis <400> 11 gtgccaatga tcttcgctcg 20

Claims (18)

(a) preparing magnetic microparticles (MMP) modified with a target capture probe;
(b) hybridizing with a biomaterial-specific target sequence and signal probe to detect the modified MMP with the target capture probe;
(c) detecting a fluorescence signal from a solution containing the hybridized MMP and a signal probe emitted from the MMP solution, respectively; and
(d) detecting a Raman (SERS) signal by adding metal nanoparticles to the emitted signal probe;
It relates to a biomaterial multiple detection method comprising:
The target capture probe and the signal probe are each half-complementary to the target sequence, characterized in that the biomaterial multiple detection method.
According to claim 1,
The MMP of step (a) is modified with two or more different target capture probes, and specifically binds to two or more target sequences, a method for multiple detection of biomaterials.
According to claim 1,
The hybridization time in step (b) is 3 hours to 12 hours, and a method for multiple detection of biomaterials, characterized in that a salt having a concentration of 0.15 M to 0.3 M is treated.
According to claim 1,
The signal probe emitted in step (c) is dispersed by applying a magnetic field to the hybridized MMP solution, characterized in that it is released by heating to a temperature higher than _{the T m (melting temperature) of the target sequence, biomaterial multiple detection} Way.
According to claim 1,
The detection method is characterized in that the minimum detection limit is 30 fM, biomaterial multiple detection method.
According to claim 1,
The surface of the metal nanoparticles is cysteamine, APTES (3-Aminopropyl) triethoxysilane), APTMS (3-Aminopropyl) trimethoxysilane, AEAPTMS (N- [3- (Trimethoxysilyl) propyl] ethylenediamine, MPTMS (3- Mercaptopropyl) )trimethoxysilane, MPTES((3-Mercaptopropyl)triethoxysilane), MPA(mercaptopropionic acid), MHA(6-mercaptohexanoic acid), BDT(1,4-benzenedithiol), MBA(4-mercaptobenzoic acid), and MBIA(2-mercaptobenzoimidazole) - 5-carboxylic acid), characterized in that modified with any one or more selected from the group consisting of, biomaterial multiple detection method.
According to claim 1,
The signal probe is labeled with a fluorescent material, a chromogenic material, or a chemiluminescent material, a method for detecting multiple biomaterials.
8. The method of claim 7,
The fluorescent material is an ATTO-based dye, a cyanine-based dye, FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, fluorescein isothiocyanate (FITC), oregon green, Alexa Fluoro (alexa fluor), JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N- (1-Naphthyl) ethylenediamine), and thiadicarbocyanine (thiadicarbocyanine) at least any one selected from the group consisting of, biomaterial multiple detection method.
According to claim 1,
Wherein the detection method is performed in a well, a channel, a tube, or a combination thereof, a multiple detection method of a biomaterial.
According to claim 1,
The biomaterial is any one or more selected from the group consisting of proteins, cells, viruses, nucleic acids, organic molecules, and inorganic molecules, biomaterial multiple detection method.
(a) preparing magnetic microparticles (MMP) modified with a target capture probe;
(b) hybridizing with a biomaterial-specific target sequence and signal probe to detect the modified MMP with the target capture probe;
(c) detecting a fluorescence signal from a solution containing the hybridized MMP and a signal probe emitted from the MMP solution, respectively; and
(d) detecting an emitted Raman (SERS) signal by adding metal nanoparticles to the emitted signal probe;
It relates to a method for providing information for diagnosing bacterial infectious diseases, comprising:
The method for providing information, characterized in that the target capture probe and the signal probe are each half-complementary to the target sequence.
12. The method of claim 11,
Prior to the step (a), the information providing method comprising the step of extracting genomic DNA from a patient sample suspected of a bacterial infectious disease.
13. The method of claim 12,
The patient sample is any one or more selected from the group consisting of blood, serum, and plasma, information providing method.
12. The method of claim 11,
The MMP of step (a) is modified with two or more different target capture probes, and binds specifically to two or more target sequences, and two or more kinds of bacteria each having a microorganism containing the target sequence as the causative organism. A method of providing information for diagnosing infectious diseases.
12. The method of claim 11,
After the step (d), the information providing method comprising the step of determining the presence of a bacterial infectious disease causative organism or the type of the causative organism using information of a fluorescence signal or a Raman signal by the biomaterial.
12. The method of claim 11,
The surface of the metal nanoparticles is cysteamine, APTES (3-Aminopropyl) triethoxysilane), APTMS (3-Aminopropyl) trimethoxysilane, AEAPTMS (N- [3- (Trimethoxysilyl) propyl] ethylenediamine, MPTMS (3- Mercaptopropyl) )trimethoxysilane, MPTES((3-Mercaptopropyl)triethoxysilane), MPA(mercaptopropionic acid), MHA(6-mercaptohexanoic acid), BDT(1,4-benzenedithiol), MBA(4-mercaptobenzoic acid), and MBIA(2-mercaptobenzoimidazole) - 5-carboxylic acid) characterized in that modified with any one or more selected from the group consisting of, information providing method.
12. The method of claim 11,
The bacterial infectious disease is sepsis, sinusitis, pneumonia, food poisoning, dysentery, tuberculosis, Pseudomonas aeruginosa infection, impetigo, suppurative disease, acute dermatitis, bacteremia, endocarditis, enteritis, osteoarthritis, osteomyelitis, urinary tract infection, encephalitis, meningitis, and the group consisting of nephritis Any one or more selected from, information providing method.
12. The method of claim 11,
Bacteria are any one or more selected from the group consisting of gram-positive bacteria, gram-negative bacteria, and mixtures thereof, the information providing method.

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