WO2022216062A1 - Bandelette de dosage à écoulement latéral pour la détection d'un anticorps anti-covid-19 - Google Patents

Bandelette de dosage à écoulement latéral pour la détection d'un anticorps anti-covid-19 Download PDF

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WO2022216062A1
WO2022216062A1 PCT/KR2022/004987 KR2022004987W WO2022216062A1 WO 2022216062 A1 WO2022216062 A1 WO 2022216062A1 KR 2022004987 W KR2022004987 W KR 2022004987W WO 2022216062 A1 WO2022216062 A1 WO 2022216062A1
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corona
antibody
virus
detecting
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Korean (ko)
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심상준
이종욱
김상기
전명진
배건우
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고려대학교 산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561—Immunoelectrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • PCR technology has been used as a representative method for detecting and diagnosing the Corona 19 virus.
  • the amplified reaction product must be confirmed by electrophoresis, and finally, DNA sequencing must be performed.
  • specialized equipment such as a polymerase chain reactor (Thermocycler) and professional manpower to operate it are required, and high cost and high technology are required for sequencing.
  • it takes a lot of time to perform these series of processes and since it is not a detection method that can be identified with the naked eye, there is a problem that it is difficult to be utilized in a field where analysis equipment is not equipped.
  • the present invention is to solve the problems of the prior art described above, and one aspect of the present invention is a colorimetric method and a lateral flow immunoassay (LFA) through a metal nanoparticle probe that is double-labeled with an antigen and a fluorescent dye.
  • LFA lateral flow immunoassay
  • the side flow immunoassay strip for detecting Corona 19 virus antibody is a side surface in which a sample pad, a conjugation pad, a membrane, and an absorbent pad are sequentially connected
  • a first metal nanoparticle, a corona 19 virus (SARS-CoV-2) antibody in a sample bound to the surface of the first metal nanoparticle and loaded on the sample pad through an antigen-antibody reaction a first fluorophore comprising a detection antigen that specifically binds, and a first spacer having one end bound to the first metal nanoparticle and a first fluorescent marker bound to the other end of the first spacer, the junction a target probe disposed on the pad; and a reaction unit including a test line to which a target capture material specifically binding to the corona 19 virus antibody bound to the detection antigen is fixed, and disposed on the membrane.
  • SARS-CoV-2 corona 19 virus
  • the first fluorophore may further include a thiol group introduced into one end of the first spacer. have.
  • the single-stranded DNA may consist of any one nucleotide sequence of SEQ ID NOs: 1 to 4.
  • the target capture material may be anti-human immunoglobulin G (anti-human IgG).
  • control detection material is a streptavidin protein
  • control capture material is BSA-biotin (Bovine serum) albumin-biotin) polymer.
  • corona 19 virus infection can be easily diagnosed with the naked eye through a colorimetric signal using metal nanoparticles without additional equipment, and furthermore, the corona virus infection is more accurate by using the enhanced fluorescent signal to check whether the infection is doubled. 19 diagnostics can be performed. Through this, it can be applied to a technology for early diagnosis of COVID-19 that can be diagnosed faster than the PCR-based diagnosis required for the prior confirmation of Corona 19.
  • FIG. 1 is a diagram illustrating a side-flow immunoassay strip for detecting a corona 19 virus antibody and a detection mechanism thereof according to the present invention.
  • FIG. 2 is a view for explaining a dual-labeling process of the target probe shown in FIG. 1 .
  • FIG. 3 is a transmission electron microscope (TEM) image of gold nanoparticles synthesized according to Example 1.
  • FIG. 3 is a transmission electron microscope (TEM) image of gold nanoparticles synthesized according to Example 1.
  • 5 is a table showing the nucleotide sequence of the single-stranded DNA used in Example 3.
  • FIG. 6 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-ssDNA-SH groups are bound, prepared according to Example 3.
  • FIG. 6 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-ssDNA-SH groups are bound, prepared according to Example 3.
  • FIG. 8A to 8E are graphs of fluorescence signal intensity according to NaCl concentration (5 mM to 40 mM) of the gold nanoparticle-Cy3 complex prepared according to Example 3, and FIG. 8A is 5 mM In NaCl, Figure 8b shows 10 mM In NaCl, Figure 8c shows 20 mM In NaCl, Figure 8d is 30 mM In NaCl, Figure 8e is 40 mM Fluorescence signal intensity in NaCl.
  • FIG. 9 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-PEG-SH groups are bonded, prepared according to Example 4.
  • FIG. 9 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-PEG-SH groups are bonded, prepared according to Example 4.
  • FIG. 10 is a graph of fluorescence signal intensity according to the PEG length of the gold nanoparticle-Cy3 complex prepared according to Example 4.
  • FIG. 10 is a graph of fluorescence signal intensity according to the PEG length of the gold nanoparticle-Cy3 complex prepared according to Example 4.
  • FIG. 11a to 11c are graphs showing changes in the UV spectrum of gold nanoparticles in each step of manufacturing a double-labeling probe according to Example 5.
  • FIG. 11a is a UV spectrum of gold nanoparticles to which Cy3-ssDNA-SH groups are bound
  • FIG. 11b is A UV spectrum of gold nanoparticles bound to Cy3-PEG-SH groups
  • FIG. 11c is a UV spectrum of double-labeled gold nanoparticles.
  • Example 12 is a signal intensity measurement result for each concentration for optimizing the concentration of the nucleocapsid corona antigen in Example 5.
  • Example 16 is a buffer stability test result for optimizing the pretreatment of the conjugation pad in Example 6.
  • Corona 19 is a result of the detection of the Corona 19 virus (SARS-CoV-2) antibody using the fluorescence method of the PEG double-labeling probe-based test strip according to Example 7.
  • Example 22 is a result of the detection of the Corona 19 virus (SARS-CoV-2) antibody using the colorimetric method of the PEG double-labeling probe-based test strip according to Example 8.
  • SARS-CoV-2 Corona 19 virus
  • Example 23 is an experimental result for optimizing the dilution ratio of a small amount of antibody and sample buffer in serum in Example 8.
  • FIG. 1 is a diagram for explaining a side flow immunoassay strip for detecting a corona 19 virus antibody according to the present invention and a detection mechanism thereof
  • FIG. 2 is a diagram illustrating a dual-labeling process of the target probe shown in FIG. It is a drawing.
  • the side flow immunoassay strip for detecting the Corona 19 virus antibody is a sample pad (sample pad, 10), a conjugation pad (conjugation pad, 20), a membrane (membrane, 30) ), and an absorbent pad (40) in the side flow immunoassay strip sequentially connected, the first metal nanoparticles (110) and the first metal nanoparticles (110) are bonded to the surface of the sample pad (10)
  • a detection antigen 120 that specifically binds to the corona 19 virus antibody 2 in the sample 1 loaded into the antigen-antibody reaction, and a first spacer, one end of which is coupled to a first metal nanoparticle 110 a target probe 100 including a first fluorophore 130 including a 131 and a first fluorescent marker 133 coupled to the other end of the first spacer 131 and disposed on the bonding pad 20; and a test line 210 to which a target capture material 211 that specifically binds to the corona 19 virus antibody 2 bound to the detection
  • the present invention relates to a side-flow immunoassay strip for detecting Corona 19 virus antibody.
  • RT-PCR technology which is used as a representative coronavirus diagnostic method, is difficult to apply to on-site diagnosis because analysis takes a long time and requires specialized equipment and personnel. Therefore, the Lateral Flow Assay (LFA), which can detect the virus in a short time, can be used for on-site diagnosis of the coronavirus, but there is a problem of low sensitivity and accuracy. As a method to solve this problem, the present invention This was not devised.
  • the side flow immunoassay strip for detecting Corona 19 virus antibody is a sample pad (sample pad, 10), a conjugation pad (20), a membrane (membrane, 30), and an absorbent pad (absorbent).
  • the pad 40 is a sensor strip sequentially connected, and includes a target probe 100 and a reaction unit 200 .
  • the sample pad 10 is a pad capable of diffusion flow by receiving the sample 1 loaded from the outside, and may be made of a porous material.
  • porous materials include, but are not limited to, fibrous papers, microporous membranes made of cellulosic materials, cellulose derivatives such as cellulose, cellulose acetate, fabrics such as nitrocellulose, glass fibers, cotton, nylon, etc. does not
  • the sample (1) is a biological material suspected of containing the corona 19 virus antibody (2) to be analyzed.
  • it may be human blood, saliva, mucus, and the like.
  • the sample 1 may be loaded into the sample pad 10 together with a predetermined buffer.
  • the buffer facilitates the flow of the sample 1 through the capillary.
  • the bonding pad 20 accommodates the sample 1 that diffuses and moves from the sample pad 10 , and the target probe 100 is disposed thereon. Like the sample pad 10 , the bonding pad 20 is made of a material capable of diffusing the sample 1 .
  • the membrane 30 provides a region through which the sample 1 that diffuses and moves from the bonding pad 20 passes.
  • the membrane 30 may include nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester, polypropylene, and the like.
  • the present invention is not necessarily limited thereto, and there is no particular limitation as long as it is a material through which the sample 1 can pass.
  • the absorbent pad 40 is a pad connected to the membrane 30 to receive the sample 1 passing through the membrane 30 .
  • the absorbent pad 40 may promote capillary action and diffusion flow of the fluid through the membrane 30 .
  • the sample pad 10 , the bonding pad 20 , the membrane 30 , and the absorption pad 40 are sequentially connected.
  • the sample pad 10 , the bonding pad 20 , the membrane 30 , and the absorption pad 40 may be in contact with each other or overlap each other, or the other may be inserted into any one of them.
  • the sample pad 10 , the bonding pad 20 , the membrane 30 , and the absorbent pad 40 may be arranged on the same support.
  • the support is made of a material that can support the sample pad 10 , the bonding pad 20 , the membrane 30 , and the absorbent pad 40 , so that the diffusion and moving sample 1 do not leak through the support. It is preferable that it is a liquid impermeable material.
  • it may be a high molecular material such as polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate and polymelamine, glass, and the like.
  • the target probe 100 is a probe that detects the Corona 19 virus antibody 2 in the sample 1 .
  • the target probe 100 includes a first metal nanoparticle 110 , a detection antigen 120 , and a first fluorophore 130 .
  • the first metal nanoparticle 110 is a metal nanoparticle in which the detection antigen 120 and the first fluorophore 130 are double-labeled.
  • Metals of these nanoparticles include gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), ruthenium (Ru), titanium (Ti), and zirconium (Zr).
  • the metal nanoparticles are not necessarily limited thereto, and may preferably be gold nanoparticles.
  • the detection antigen 120 is an antigen that specifically binds to the Corona 19 virus antibody (2) through an antigen-antibody reaction.
  • the antigen may include any one or more selected from the group consisting of a nucleocapsid protein of Corona 19 virus (SARS-CoV-2), and a spike protein. These antigens may be bound to the surface of the first metal nanoparticles 110 through electrostatic bonding. Accordingly, a plurality of at least one of a nucleocapsid protein and a spike protein may be bound to the surface of the first metal nanoparticle 110 .
  • the first fluorophore 130 includes a first spacer 131 and a first fluorescent marker 133 . If the distance between the metal nanostructure and the fluorescent marker is properly maintained, the metal enhanced fluorescence (MEF) occurs in which the surface plasmon energy is transferred as fluorescence energy through resonance and the fluorescence is amplified, and a dramatic increase in the fluorescence signal occurs. Through this, the detection limit of the biosensor can be significantly lowered and the sensitivity can be increased. This MEF phenomenon is very sensitively affected by the distance between the metal nanostructure and the fluorescent marker.
  • MEF metal enhanced fluorescence
  • the first spacer 131 maintains a constant distance between the first metal nanoparticles 110 and the first fluorescent marker 133 in order to induce the MEF phenomenon.
  • one end of the first spacer 131 is coupled to the first metal nanoparticles 110
  • the other end of the first spacer 131 is coupled to the first fluorescent marker 133 , and the first metal nanoparticles 110 .
  • the first fluorescent marker 133 to always maintain a uniform distance.
  • the first fluorescent marker 133 is a fluorescent material, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tamla ( TAMRA), dichlorotriazinylamine fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride (dansyl chloride), quantum dots (quantum dots), phycoerythrin (phycoerythrin), fluorescein containing FAM (fluorecein amidite), etc. , Alexa fluor, and Cy3, Cy5, Cy7, and cyanine including indocyanine green, and one or more of them may be used in combination.
  • FITC fluorescein isothiocyanate
  • TAMRA tamla
  • dichlorotriazinylamine fluorescein diichlorotriazinylamine fluorescein
  • dansyl chloride dansyl chloride
  • quantum dots quantum dots
  • the first fluorescent marker 133 is not necessarily limited to the above material, and preferably Cy3 (Cyanine 3) may be used.
  • Cy3 Cy3
  • an adhesion mediator may be introduced to one end of the first spacer 131 , for example, a thiol group (thiol group). group) can be
  • the first spacer 131 may include any one or more selected from the group consisting of single-stranded DNA and polyethylene glycol (PEG). Since the distance between bases in DNA is always precisely maintained in DNA, the length can be predictably controlled by controlling the number of bases. In addition, a specific nucleotide sequence may affect the fluorescence intensity generated from the surrounding fluorescent markers and the hardness of the DNA strand. Accordingly, in the present invention, the base sequence of single-stranded DNA is designed so that MEF can be effectively generated, and this is adopted as the first spacer 131 .
  • PEG polyethylene glycol
  • the single-stranded DNA that may serve as the first spacer 131 may consist of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4 in the 5' to 3' direction.
  • the first fluorescent marker 133 at the 5' end and the adhesion mediator at the 3' end may be introduced, respectively.
  • the first spacer 131 may also be implemented as PEG. Since PEG has a strong strength, it is possible to properly maintain a distance between the first metal nanoparticles 110 and the first fluorescent marker 133 .
  • the single-stranded DNA and PEG-based first spacer 131 may have a length of 3 nm to 33 nm, preferably 5 nm to 15 nm, more preferably 8 nm to 13 nm.
  • first fluorophore 130 labeled on the first metal nanoparticles 110
  • first fluorophores 130 are bound to the first metal nanoparticles 110
  • Each of the first fluorophores 130 includes the same single-stranded DNA or PEG-based first spacer 131, or some of them include the first spacer 131 based on single-stranded DNA and the other part
  • a PEG-based first spacer 131 may be included.
  • the reaction unit 200 is a region disposed on the membrane 30 and includes a test line 210 .
  • a target capture material 211 is fixed to the test line 210 .
  • the target capture material 211 is a material that specifically binds to the corona 19 virus antibody 2 bound to the detection antigen 120 of the target probe 100 .
  • the target capture material 211 may be anti-human immunoglobulin G (anti-human IgG).
  • the detection antigen Corona 19 virus antibody (2) is bound to 120
  • the target probe 100 moves along the membrane 30 together with the sample (1)
  • the corona 19 virus antibody (2) bound to the target probe 100 is captured by the target capture material 211 fixed to the test line 210 , and a detection signal is generated.
  • the detection signal is a colorimetric and fluorescence signal enhanced by MEF, and the signal may be analyzed with the naked eye, a colorimetric scanner, and a fluorescence scanner.
  • the side flow immunoassay strip for detecting the Corona 19 virus antibody according to the present invention may include a control probe 300 to check whether the reaction is normal.
  • the control probe 300 includes a second metal nanoparticle 310 , a control detection material 320 , and a second fluorophore 330 , and the second fluorophore 330 includes a second spacer 331 and a second fluorophore 330 .
  • 2 includes a fluorescent marker (333).
  • the second metal nanoparticles 310, the second spacers 331, and the second fluorescent marker 333 of the control probe 300 are, respectively, the first metal nanoparticles 110 of the above-described target probe 100, Since it corresponds to the first spacer 131 and the first fluorescent marker 133, a detailed description thereof will be omitted, and differences will be mainly described.
  • the control detection material 320 is a material that is bound to the surface of the second metal nanoparticle 310 and does not react with the corona 19 virus antibody 2 in the sample 1, for example, streptavidin protein.
  • the reaction unit 200 may further include a control line 220 , and a control capture material 221 that specifically binds to the control detection material 320 is fixed to the control line 220 . Accordingly, the control probe 300 flowing with the sample 1 is captured by the control capture material 221 , thereby generating a detection signal.
  • the control capture material 221 may be a BSA-biotin (Bovine serum albumin-biotin) polymer that specifically binds to a streptavidin protein.
  • BSA-biotin Bovine serum albumin-biotin
  • the present invention uses an optical-based biosensor as a platform capable of effectively diagnosing the Corona 19 virus, and more specifically, the Lateral Flow Assay (Lateral Flow Assay, A technology that can be applied to on-site diagnosis by accurately and quickly detecting the COVID-19 virus antibody (SARS-CoV-2 IgG), which is present at a very low concentration at the initial stage of infection, without false-positive or false-negative results by simultaneously performing colorimetric and fluorescence methods on LFA) is about
  • SARS-CoV-2 IgG COVID-19 virus antibody
  • corona 19 virus infection can be easily diagnosed with the naked eye through a colorimetric signal using metal nanoparticles without additional equipment, and furthermore, the corona virus infection is more accurate by using the enhanced fluorescent signal to check whether the infection is doubled. 19 diagnostics can be performed. Through this, it can be applied to a technology for early diagnosis of COVID-19 that can be diagnosed faster than the PCR-based diagnosis required for the prior confirmation of Corona 19.
  • 40 nm gold nanoparticles were prepared by reducing gold ions using trisodium citrate. After heating and boiling 10 ml of an aqueous solution of 1 mM chloroauric acid (HAuCl 4 ), when the solution started to boil, 1 ml of an aqueous solution of 2.27 ⁇ M sodium citrate was added and heated for another 5 minutes. Then, the solution was cooled to room temperature and filtered using a membrane filter (Cellulose acetate filter, Advantec). The size of the prepared nanoparticles was analyzed using UV-Vis spectroscopy and TEM.
  • FIG. 3 is a transmission electron microscope (TEM) image of the gold nanoparticles synthesized according to Example 1, and it can be confirmed that the synthesized gold nanoparticles have a size of 40 nm through the TEM image.
  • Gold nanoparticles at 40 nm can exhibit excellent metal-enhanced fluorescence as well as high red colorimetric signal in dual-mode LFA.
  • signal visibility can be improved by increasing the diameter of gold nanoparticles, but the efficiency of corona protein binding by electrostatic force decreases.
  • the scattering efficiency increases as the size of gold nanoparticles increases according to the Mie theory and reaches a peak at 40 nm. That is, it suggests that the optimal size of the bimodal LFA gold nanoparticles is 40 nm.
  • FIG. 4 is a UV spectrum of gold nanoparticles synthesized according to Example 1.
  • FIG. The specific LSPR peak wavelength band of gold nanoparticles according to the size was measured using UV. The measured wavelength is 529 nm, and it can be confirmed that the size of the gold nanoparticles is 40 nm.
  • the stability of the gold nanoparticles was improved by replacing the surface of the gold nanoparticles made of citrate with Bis(p-Sulfonatophenyl)Phenylphosphine (BSPP). Gold nanoparticles coated with BSPP are effectively negatively charged, so that gold nanoparticles do not agglomerate through electrostatic repulsion.
  • BSPP Bis(p-Sulfonatophenyl)Phenylphosphine
  • a single strand of DNA in which Cy3 and a thiol group are bonded and PEG were combined on gold nanoparticles.
  • BSPP Bis(p-Sulfonatophenyl)Phenylphosphine
  • ssDNA single strand of DNA having various nucleotide sequences as shown in FIG. 5 was used.
  • 5 is a table showing the nucleotide sequence of the single-stranded DNA used in Example 3.
  • Cy3 since Cy3 is bonded to the 5' end of each DNA and a thiol group is bonded to the 3' end of each DNA, it can bind to the gold nanoparticles through Au-S bond.
  • a single-stranded DNA sequence was designed to maintain the distance between the gold nanoparticles and the fluorescent substance by taking advantage of the advantage of easy DNA synthesis.
  • Cy3 was selected as the phosphor with the best spectral overlap (the overlapping of the LSPR peak of the gold nanoparticle and the excitation/emission spectrum of the phosphor) with the 40nm gold nanoparticles.
  • a spacer capable of maintaining the distance between the gold nanoparticles and Cy3 single-stranded DNA was designed to be 5 nm, 10 nm, 15 nm, and 20 nm. Since thymine (T) has the lowest binding force to gold nanoparticles among nucleotides in DNA, the distance of each single-stranded DNA was uniformly controlled through thymine.
  • 'GGAAG' is applied to the five nucleotide sequences on the Cy3 side. ‘GGAAG’ increases the fluorescence signal of Cy3 through pi overlap, and maintains a constant straight line of DNA structure when thymine is applied.
  • DTT dithiothreitol
  • FIG. 6 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-ssDNA-SH groups are bound, prepared according to Example 3.
  • FIG. 1 To perform electrophoresis, 2 g of agarose and 100 ml of 0.5X TBE buffer were mixed in a 500 ml Erlenmeyer flask. Then, after heating the flask for 2 minutes and 30 seconds, 50 ml of the square gel tray was loaded and maintained at room temperature for about 30 minutes to form a gel. After injecting 300 ml of 0.5X TBE buffer into the electrophoresis port, the formed gel was combined.
  • FIG. 7 is a graph of fluorescence signal intensity according to the length of single-stranded DNA of the gold nanoparticle-Cy3 complex prepared according to Example 3.
  • FIG. The highest fluorescence signal was measured when the distance between the gold nanoparticles and the phosphor was adjusted with single-stranded DNA with a length of 10 nm.
  • the gold nanoparticles absorb the fluorescence energy of the phosphor and interfere with the emission of the phosphor. It is not transmitted effectively and the MEF effect is reduced, resulting in a decrease in the fluorescence signal.
  • FIG. 8A to 8E are graphs of fluorescence signal intensity according to NaCl concentration (5 mM to 40 mM) of the gold nanoparticle-Cy3 complex prepared according to Example 3, and FIG. 8A is 5 mM In NaCl, Figure 8b shows 10 mM In NaCl, Figure 8c shows 20 mM In NaCl, Figure 8d is 30 mM In NaCl, Figure 8e is 40 mM Fluorescence signal intensity in NaCl. The distance between the gold nanoparticles and the phosphor was maintained constant by adding NaCl to further stretch the single-stranded DNA.
  • FIG. 9 is an electrophoretic treatment result of gold nanoparticles (gold nanoparticles-Cy3 complex) to which Cy3-PEG-SH groups are bonded, prepared according to Example 4.
  • FIG. 1 To perform electrophoresis, 2 g of agarose and 100 ml of 0.5X TBE buffer were mixed in a 500 ml Erlenmeyer flask. Then, after heating the flask for 2 minutes and 30 seconds, 50 ml of the square gel tray was loaded and maintained at room temperature for about 30 minutes to form a gel.
  • the formed gel was combined. 10 ⁇ l of Cy3-PEG-SH unconjugated gold nanoparticles and 2 ⁇ l of glycerol were mixed and loaded in the left three lanes, and 10 ⁇ l of Cy3-ssDNA-SH-conjugated gold nanoparticles in the right three lanes and 2 ⁇ l of glycerol were mixed and loaded. As a result of electrophoresis at 240V for 15 minutes, the nanoprobes injected into the three lanes on the left had a smaller mass and lighter weight than the nanoprobes injected on the right.
  • FIG. 10 is a graph of fluorescence signal intensity according to the PEG length of the gold nanoparticle-Cy3 complex prepared according to Example 4.
  • S RBD Spike Receptor Binding Domain
  • FIG. 11a to 11c are graphs showing changes in the UV spectrum of gold nanoparticles in each step of manufacturing a double-labeling probe according to Example 5.
  • FIG. 11a is a UV spectrum of gold nanoparticles to which Cy3-ssDNA-SH groups are bound
  • FIG. 11b is A UV spectrum of gold nanoparticles bound to Cy3-PEG-SH groups
  • FIG. 11c is a UV spectrum of double-labeled gold nanoparticles.
  • 11A to 11C are results showing changes in the UV spectrum of gold nanoparticles according to the manufacturing process of the double-labeling nanoprobe, and the size of the probe was changed when the surface of the gold nanoparticles was modified with Cy3-DNA and Cy3-PEG. increased, a red-shift of about 2.5 nm was generated compared to the gold nanoparticles, and when the antigen was contacted, a red-shift of 3 nm was generated compared to the ligand-conjugated probe. Through this, it can be seen that the phosphor and antigen were conjugated to the surface of the gold nanoparticles.
  • 12 is a signal intensity measurement result for each concentration for optimizing the concentration of the nucleocapsid corona antigen in Example 5.
  • 0 ⁇ l, 1 ⁇ l, 2 ⁇ l, 4 ⁇ l, 8 ⁇ l, 12 N protein (30 kDa, 200 ⁇ g/ml) in a gold nano solution (Borate buffer (pH 9), 0.2 nM 300 ⁇ l) conjugated with DNA and PEG ⁇ l and 16 ⁇ l were added and reacted for 20 minutes. Thereafter, centrifugation was performed three times at 6000 rpm for 10 minutes to remove the supernatant, and then a colorimetric test was performed using a test strip prepared according to Example 6 to be described later.
  • the colorimetric signal intensity ratio was the highest when 12 ⁇ l of N protein was added.
  • FIG. 13 is a signal intensity measurement result for each pH for optimizing the pH conditions for conjugating nucleocapsid corona antigen to gold nanoparticles in Example 5.
  • FIG. After changing the pH of the DNA and PEG-conjugated gold nano solution (300 ⁇ l of 0.2 nM) from 5 to 11, 12 ⁇ l of N protein (30 kDa, 200 ⁇ g/ml) was added and reacted for 20 minutes. Thereafter, centrifugation was performed 3 times at 6000 rpm for 10 minutes to remove the supernatant, followed by a colorimetric test. After measuring the colorimetric signal of the test line and the control line of each test strip, the colorimetric signal intensity ratio of the test line to the colorimetric signal of the control line under each condition was measured. As a result, the colorimetric signal intensity ratio was the highest at pH 9.
  • Example 14 is a signal intensity measurement result for each concentration for optimizing the concentration of the spike RBD (Spike Receptor Binding Domain) corona antigen in Example 5; 0 ⁇ l, 2 ⁇ l, 4 ⁇ l, 8 ⁇ l, 16 ⁇ l, 20 ⁇ l of S RBD protein (50 kDa, 200 ⁇ g/ml) in a gold nano solution (Borate buffer (pH 7.4), 0.2 nM 300 ⁇ l) conjugated with DNA and PEG ⁇ l and 24 ⁇ l were added and reacted for 20 minutes. Thereafter, centrifugation was performed 3 times at 6000 rpm for 10 minutes to remove the supernatant, followed by a colorimetric test.
  • S RBD Spike Receptor Binding Domain
  • the colorimetric signal intensity ratio of the test line to the colorimetric signal of the control line under each condition was measured.
  • the colorimetric signal intensity ratio was the highest when 20 ⁇ l of S RBD protein was added.
  • FIG. 15 is a result of measuring the signal intensity for each pH to optimize the pH conditions for conjugating the spike receptor binding domain (RBD) corona antigen to the gold nanoparticles in Example 5.
  • FIG. After changing the pH of the DNA and PEG-conjugated gold nano solution (0.2 nM 300 ⁇ l) from 5 to 11, 20 ⁇ l of S RBD protein (50 kDa, 200 ⁇ g/ml) was added and reacted for 20 minutes. Thereafter, centrifugation was performed 3 times at 6000 rpm for 10 minutes to remove the supernatant, followed by a colorimetric test.
  • the test strip used for detection of the SARS-CoV-2 antibody was constructed as shown in FIG. 1 .
  • a secondary antibody anti-human IgG, 2 mg/ml, 0.7 ⁇ l/cm
  • BSA- on a nitrocellulose membrane (2.5 cm ⁇ 4 mm) using a bio jet dispenser (XYZ platform, Biojet 3000kit, BioDot)
  • biotin 1.2 mg/ml, 0.5 ⁇ l/cm
  • 16 is a buffer stability test result for optimizing the pretreatment of the conjugation pad in Example 6.
  • the conjugate pad was treated with 10% (w/v) sucrose, 1% (w/v) BSA, 0.05% Tween-20 (v/v), respectively.
  • This was pre-treated in PBS buffer containing borate buffer, 10% (w/v) sucrose, 1% (w/v) BSA, and 0.05% Tween-20 (v/v) for 1 hour.
  • 10 ⁇ l of the double-labeled nanoprobe was loaded onto the bonding pad and dried for 1 hour.
  • the picture on the right is a double-labeled nanoprobe dried on a bonding pad reacted in PBS buffer.
  • the picture on the left shows a red color due to the stability of the nano-probe, while the picture on the right shows a gray-colored result because the concentration of NaCl in PBS rapidly increases when drying, and the stability of the nano-probe decreases accordingly. showed According to the result, in order to secure the stability of the double-labeled nanoprobe in the test strip, pretreatment was performed in borate buffer.
  • sample buffer PBS, 0.05% BSA, 0.1% Tween 20
  • sample buffer PBS, 0.05% BSA, 0.1% Tween 20
  • Example 17 is an experimental result for optimizing the amount of the sample buffer that causes the flow of the sample in Example 7.
  • the amount of Tween-20 added to the sample buffer to induce capillary action in dual mode LFA was optimized.
  • Tween- at 0%(v/v), 0.1%(v/v), 0.5%(v/v), 1%(v/v), 2%(v/v), 5%(v/v)
  • colorimetric signals were compared.
  • the highest result was obtained when 0.5% (v/v) Tween-20 was added to the PBS buffer.
  • 18 is a result of the detection of the Corona 19 virus (SARS-CoV-2) antibody using the fluorescence method of the DNA double-labeling probe-based test strip according to Example 7.
  • 18 shows the results of performing dual-mode LFA according to the Cy3-DNA-modified nanoprobe. The figure on the left shows the colorimetric signal, and the figure on the right shows the fluorescence signal.
  • 19 is a result of the detection of the Corona 19 virus (SARS-CoV-2) antibody using the fluorescence method of the PEG double-labeling probe-based test strip according to Example 7.
  • 19 shows the results of performing dual-mode LFA according to the Cy3-PEG-modified nanoprobe. The figure on the left shows the colorimetric signal, and the figure on the right shows the fluorescence signal.
  • Example 20 is a sensitivity measurement result of a test strip according to Example 7. Colorimetric and fluorescence signals were observed by applying various target concentrations from 100 pg/ml to 10 ⁇ g/ml to dual mode LFA under optimal conditions. In the case of the colorimetric test, it was quickly and easily observed that a red band appeared when the concentration of the target was 1 ⁇ g/ml or more. On the other hand, in the case of the fluorescence test, it was observed that green fluorescence was emitted when the concentration of the target was 1 ng/ml or more, suggesting that the target can be measured at a lower concentration. These results demonstrate the advantages of dual-mode LFA, which allows simple and rapid diagnosis, quantitative analysis, and effective prevention of false-negative results. It also sheds light on the possibility of building other infectious viruses and multi-detection platforms besides coronavirus.
  • sample buffer PBS, 0.05% BSA, 0.1% Tween 20
  • 21 is a result of detection of a corona 19 virus (SARS-CoV-2) antibody using the colorimetric method of the DNA double-labeling probe-based test strip according to Example 8.
  • SARS-CoV-2 corona 19 virus
  • LFA was performed according to the Cy3-DNA-modified nanoprobe.
  • a red line was formed in the test zone.
  • the colorimetric signal intensity ratio of the test line and the control line was obtained through ImageJ, and the colorimetric signal was generated by varying the ratio of the sample buffer to the serum containing the target from 1:0 to 1:10,000.
  • Example 22 is a result of the detection of the Corona 19 virus (SARS-CoV-2) antibody using the colorimetric method of the PEG double-labeling probe-based test strip according to Example 8.
  • SARS-CoV-2 Corona 19 virus
  • LFA was performed according to the Cy3-PEG-modified nanoprobe.
  • a red line was formed in the test zone.
  • the colorimetric signal intensity ratio of the test line and the control line was obtained through ImageJ, and the colorimetric signal was generated by varying the ratio of sample buffer and serum containing the target from 1:0 to 1:10,000.
  • Example 23 is an experimental result for optimizing the dilution ratio of a small amount of antibody and sample buffer in serum in Example 8.
  • the ratio of the colorimetric signal of the control line to the colorimetric signal intensity of the test line was obtained according to the ratio of the serum containing the antibody diluted with PBS, and the most ideal result was obtained when the serum was diluted 100 times.
  • There are various factors that block the flow of LFA in serum and this problem can be solved by diluting the serum with PBS.
  • the concentration of the target in serum also decreases, so detection in LFA may be difficult. Therefore, 1:100, in which the dilution ratio is lowered as much as possible and the signal intensity ratio is kept constant, was selected as the optimal dilution ratio.
  • Example 24 is a specificity verification result of a test strip according to Example 9; Specificity was observed by loading the target antibody, severe fever with thrombocytopenia syndrome (SFTSV) antibody, dengue virus antibody, avian influenza A (H7N9) antibody, and normal human antibody into dual mode LFA. As a result of the experiment, colorimetric and fluorescent signals were observed in the test containing the target, and no signal was measured due to failure of antigen-antibody interaction when other antibodies were loaded. This result indicates that there was no cross-reaction between SARS-CoV-2 and other viruses, indicating that the bimodal LFA has excellent specificity.
  • SFTSV severe fever with thrombocytopenia syndrome
  • H7N9 avian influenza A
  • 25 is a reproducibility verification result of a test strip according to Example 9;
  • the ratio of the colorimetric and fluorescent signal intensity of the test line to the colorimetric and fluorescent signal of the control line was measured with at least 10 identical samples.
  • Ten tests showed a constant signal intensity ratio, indicating that the developed dual-mode LFA satisfies good reproducibility.
  • the present invention is a corona 19 virus antibody present at a very low concentration in the initial stage of infection by simultaneously performing a colorimetric method and a fluorescence method on a Lateral Flow Assay (LFA) through a metal nanoparticle probe labeled with an antigen and a fluorescent dye.
  • LFA Lateral Flow Assay

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Abstract

La présente invention concerne une bandelette de dosage à écoulement latéral pour la détection d'un anticorps anti-COVID-19. La bandelette est une bandelette de capteur dans laquelle un tampon d'échantillon (10), un tampon de conjugaison (20), une membrane (30) et un tampon absorbant (40) sont connectés de manière séquentielle, et comprend une sonde cible (100) et une partie de réaction (200).
PCT/KR2022/004987 2021-04-06 2022-04-06 Bandelette de dosage à écoulement latéral pour la détection d'un anticorps anti-covid-19 WO2022216062A1 (fr)

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US20160346208A1 (en) * 2015-05-28 2016-12-01 University Of South Carolina Dual responsive brain targeted nanoparticles and their applications

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US20160346208A1 (en) * 2015-05-28 2016-12-01 University Of South Carolina Dual responsive brain targeted nanoparticles and their applications

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CHAO HUANG, TIAN WEN, FENG-JUAN SHI, XIAO-YAN ZENG, YONG-JUN JIAO: "Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay", ACS OMEGA, ACS PUBLICATIONS, US, vol. 5, no. 21, 2 June 2020 (2020-06-02), US , pages 12550 - 12556, XP055756472, ISSN: 2470-1343, DOI: 10.1021/acsomega.0c01554 *
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