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WO2008030071A1 - Méthode de détection d'hybridation de l'adn par diffusion - Google Patents

Méthode de détection d'hybridation de l'adn par diffusion Download PDF

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Publication number
WO2008030071A1
WO2008030071A1 PCT/KR2007/004356 KR2007004356W WO2008030071A1 WO 2008030071 A1 WO2008030071 A1 WO 2008030071A1 KR 2007004356 W KR2007004356 W KR 2007004356W WO 2008030071 A1 WO2008030071 A1 WO 2008030071A1
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WO
WIPO (PCT)
Prior art keywords
dna
nanoparticle
hybridization
detection
probe
Prior art date
Application number
PCT/KR2007/004356
Other languages
English (en)
Inventor
Du Soo Chung
Dai Sik Kim
Jong Won Kim
Jing Yu Piao
Original Assignee
Seoul National University Industry Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seoul National University Industry Foundation filed Critical Seoul National University Industry Foundation
Publication of WO2008030071A1 publication Critical patent/WO2008030071A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors

Definitions

  • the invention is about detection method of DNA hybridization and synthesis method of probe nanoparticle DNA used in above detection method, in detail is that detection method of DNA hybridization on DNA chip based on detection of light scattering of probe nanoparticle DNA.
  • DNA chip has been most widely developed and some used commercially.
  • DNA chip compared with traditional southernblotting and northernblotting, can detect a large amount of genes in one time and can be applied to detection of gene mutation, diagnosis of genetic gene and pharmagenetics.
  • trend in this field is focused on developing method of low cost, rapidity, accuracy, simplicity and convenience.
  • DNA chip immobilize capture DNA with specific sequence using various immobilization method and find out which kind of capture DNA hybridize with target DNA or RNA.
  • the hybridization between target DNA and capture DNA on chip is detected traditionally by radioisotope labels and expensive fluorescence dye scanned by microarray scanner.
  • radioisotope label method is very sensitive and applicable to some fields, it is substituted by fluorescence dye probe for its environmental problem.
  • fluorescence dye probe method is widely used for its advantage of high sensitivity, non toxicity, non destructivity and low cost, while in this method fluorescence dye molecules must be linked to biomolecules such as DNA and for signal readout the expensive laser or lamp scanner must be needed, so detection system using DNA chip is not widely used in commercial.
  • mass spectroscopy method does not need biomolecule probe modification and provides structure information, it has the disadvantage of expensive and complicated instrument needed. Therefore, it becomes important to find a very simple method to detect DNA hybridization easily on DNA chip.
  • Object of the invention is to provide detection method of DNA hybridization on DNA chip through detection of light scattering of nanoparticle probes hybridized on DNA chip.
  • the invention is to present new detection method of DNA hybridization on DNA chip, it can not only easily detect DNA hybridization on DNA chip using low cost lamp scanner without modification of biomolecules with fluorescent dye molecules as well as expensive optical instrument but also contribute to widely application of DNA chip. [Description of Drawings]
  • Figure 1 is to show general hybridization process used in detection of DNA hybridization between capture DNA and target DNA sample, target DNA and probe DNA on DNA chip (A) and modified hybridization process called sandwich hybridization (B) in which nanoparticle-DNA probe is used for detection of DNA hybridization according to light scattering of nanoparticle-DNA probes on DNA chip in the invention.
  • Figure 2 is synthesis of dye doped nanoparticle, which are modified further for preparation of probe nanoparticle DNA used in the invention, using water-in-oil microemulsion method (extra picture is TEM image of nanoparticle with diameter of about 50 run)
  • Figure 3 is the process of surface immobilization of dye-doped silica nanoparticles with probe DNA to make TMR-NP-DNA probe used for detection of DNA hybridization in the invention.
  • Figure 4 is the process of immobilization of oligonucleotide probe on glass slide through covalent bonding between sulfur and DNA to give capture DNA used for detection of DNA hybridization in the invention.
  • Figure 5 show hybridization of probe nanoparticle DNA with capture DNA directly which is different from Figure 1 (B) in which target DNA hybridized first with capture DNA, then probe nanoparticle DNA hybridize with other region of target DNA used for detection of DNA hybridization through light scattering of nanoparticle-DNA probes in the invention.
  • Figure 6 show format of HPV DNA chip used in the invention and laser scanning image of the result of sandwich hybridization with HPV16 target DNA using TMR-NP-DNA probe in the invention.
  • Figure 7 is the laser scanner image and digital camera image of the result of sandwich hybridization with HPV16 target DNA using TMR-NP-DNA probe in the invention.
  • Figure 8 is DNA hybridization result shown as graph of S/N ratio (signal/noise) in scattering signal of both TMR-NP-DNA probes (one nanoparticle
  • 3 4 contains about 10-10 dye molecules) hybridized on DNA chip and same concentration of TMR-DNA when hybridize on DNA chip (in case of TMR-NP-DNA probes, the ratio of (S/N)scattering to (S/N)fluorescence is between 0.7 and
  • Figure 9 is the laser scanner image and digital camera image of the result of DNA hybridization using several kinds of probes. [Best Mode]
  • the invention is to provide a new detection method of DNA hybridization based on detection of light scattering of nanoparticle-DNA probes hybridized on DNA chip.
  • the invention of detection method of DNA hybridization on DNA chip for disease checking includes reaction step of hybridization of probe nanoparticle DNA with capture DNA to form hybridized probes; step of removing not hybridized probe nanoparticle DNA after above reaction; step of detection of light scattering degree of hybridized nanoparticle-DNA probes.
  • the invention of detection method of DNA hybridization on DNA chip for disease checking includes reaction step of hybridization of sample DNA with capture DNA to form hybridized complex; step of removing not hybridized sample DNA after reaction; reaction step of hybridizaton sample DNA on capture DNA chip with probe nanoparticle DNA; step of detection of light scattering degree of hybridized nanoparticle-DNA probes.
  • silica nanomaterials are much more desirable.
  • radioisotope and fluorescent dye as fluorescent component of nanoparticle, here, TMRCtetramethyl rhodamine are much more desirable.
  • FIG. 1 General detection method (A) of DNA hybridization on DNA chip and sandwich hybridization process (B) using probe nanoparticl DNA with light scattering of hybridized probe nanoparticle DNA as detection method in the invention. And figure 1 (B) shows detection method based on light scattering to detect hybridization between target DNA and capture DNA on DNA chip. That is, the detection method of DNA hybridization of the invention is actually similar with general detection method based on complementary DNA hybridization to hybridize target DNA with capture DNA, target DNA with nanoparticle-DNA probe. Detection method of general DNA hybridization process is as following.
  • Capture DNA which hybridize with complementary target DNA is immobilized on surface of silicon wafer, glass slide or plastic slide (capture DNA used in the invention is also called probe DNA in some papers, while in the invention we call it capture DNA which immobilized on chip surface and hybridize with target DNA), target DNA with specific sequence hybridize with capture DNA and the rest region of target DNA hybridize with probe nanoparticle DNA. Not hybridized target DNA in sample and buffer solution are removed, we can know what kind of target DNA are present in sample through detection of signaling probes. Although not shown in figure, sometimes for PCR amplification reaction, fluorescent dye molecules have been introduced from the beginning of the reaction to conjugate fluorescent dye molecule with target DNA.
  • our detection method of DNA hybridization is based on detection of light scattering and nanoparticles are used as signaling probes with no need of fluorescent dye molecules for signaling. Rather than, it is important to control nanoparticle size and concentration to get certain degree of light scattering, and the conjugation methodCfigure 1 (B)) of nanoparticle-DNA is important.
  • Light scattering phenomenon used in detection of DNA hybridization is dependent on nanoparticle size and in proportion to six square of nanoparticle radius, the volume and mass of nanoparticle increase 1,000 times when the radius of nanoparticle increases 10 times, while light scattering of nanoparticle increase one million times.
  • Detection of DNA hybridization on DNA chip based on light scattering in our invention is as following.
  • Sample is injected into chamber of DNA chip which contain capture DNA.
  • Target DNA hybridize with complementary capture DNA and not hybridized part of target DNA are left.
  • probe DNA conjugate with nanoparticle and we call it probe nanoparticle DNA.
  • DNA probe conjugated with nanoparticle hybridize with the remaining region of target DNA, such hybridization is called sandwich hybridization.
  • Nanoparticle is not just the meaning of particle with nanometer size. Particles of several micrometer can be seen by naked eye or by digital camera, smaller particles can also be observed by low cost laser scanner (flat bed scanner) based on light scattering. That is, even if the nanopart icles are small enough we can also detect light scattering phenomenon of nanopart icles using low cost instrument.
  • Figure 2 show scheme of the other kind of nanoparticle used in our invention-dye-doped silica nanoparticle synthesis in water-in-oil microemulsion. And TEM image of such nanoparticles with diameter of 50 nm.
  • Figure 3 is the scheme of conjugation of DNA probe with silica nanoparticle to form probe nanoparticle DNA used in our invention of detection method of DNA hybridization. The synthesis of probe nanoparticle DNA will be described more detail in following part.
  • silica nanoparticles are used and amine groups are introduced to the surface of silica nanoparticles.
  • amine groups are introduced to the surface of silica nanoparticles through silanization of 3- aminopropyltrimethoxysilane.
  • Silanization is widely used method of introducing amine, sulfide, hydrocarbon groups to the surface of silica nanoparticles. The process is as follow, in reaction on glass slide surface, the R is substituted with OH group.
  • the silanized amine group can link to specific protein through interaction with aldehyde group.
  • aldehyde group For example, through glutaraldehyde, avidin can be attached to the surface of silica nanoparticles. Such avidin attached silica nanoparticle can react with 3'-biotinalated DNA through specific avidin-biotin interaction to conjugate nanoparticle with DNA probe, thus probe nanoparticle DNA as mentioned above can be synthesized.
  • Avidin as well as streptavidin are widely used for their strong interaction with biotin.
  • Avidin has four reaction region due to its tetramer structure.
  • biotin is one kind of vitamin H small molecule and is widely used to link to one end of DNA. The whole process is to react avidin attached nanoparticle with 3'-biotinalated DNA and wash repeatedly to give probe nanoparticle DNA.
  • silica particles with different size can be ordered easily, amine-modified or carboxy-modified silica nanoparticles can also be synthesized or ordered easily.
  • FIG. 4 shows the silanization of nanoaparticle and immobilization process of capture DNA.
  • the above method is to introduce thiol group and immobilize oligonucleotide on glass slide to obtain capture DNA which hybridize with target DNA. It is possible to introduce amine, carboxy, sulfide and hydrocarbon group to the surface of nanoparticle through silanization to conjugate with DNA.
  • nanoparticle is conjugated with target DNA.
  • DNA chip on which capture DNA is immobilized, the nanoparticle-target DNA hybridize with capture DNA.
  • hybridization between target DNA and capture DNA can be detected.
  • Target DNA-nanoparticle can be synthesized as mentioned before, for example, through interaction between avidin and biotin after introduction of biotin to target DNA and introduction of avidin to nanoparticle.
  • FIG 1 (B) various method can be used dependent on individual obviously. Such example clearly show that using target DNA-nanoparticle can also detect hybridization between target DNA and capture DNA based on detection of light scattering of nanoparticles.
  • Hybridization between nanoparticIe-DNA and capture DNA is detected based on light scattering of nanoparticles hybridized on DNA chip in our invention.
  • the process is performed on commercial HPV(Human Papillomavirus) DNA chip.
  • HPV DNA probes immobilized.
  • DNA hybridization using different probes are also detected and the result image is taken by both digital camera and laser scanner(figure 9).
  • the result is that DNA hybridization using cy5 probes is not detectable by digital camera but only detectable by laser scanner.
  • DNA hyridization using plain nanoparticle probes is not detectable by laser scanner but only detectable by digital camera based on light scattering of nanoparticles. Detection of DNA hybridization is dependent on particle size, nanoparticle concentration and quality of material .
  • avidin is desirable to be used for conjugation of probe DNA
  • g1utaraldehyde is desirable to be used for conjugation with avidin after introduction of amine group through silanization of 3- aminopropyltriraethoxysilane on surface of silica nanoparticle.
  • the inventor have synthesized nanoparticle to further prepare probe nanoparticle DNA used in detection of DNA hybridization based on light scattering.
  • Silica nanoparticles or dye-doped silica nanoparticles are synthesized and used in this invention.
  • the inventor have synthesized dye-doped silica nanoparticles.
  • the synthesis procedure is shown in figure 2.
  • TMR(tetramethyl rhodamine) is used and dye-doped silica nanoparticles are prepared throug neutralization of silica monomer (TEOS), in detail, silica monomer (TEOS) is mixed with surfactant and stirred continuously. The result is that certain size of dye-doped nanoparticles are synthesized.
  • the inventor do not use the same synthesis method used in mode 1-1 to prepare plain silica nanoparticles, but ordered the plain silica particles(0.3 ⁇ m) from Bangs Laboratories, and call the particles plain silica nanoparticles.
  • Nanoparticle-DNA probe is synthesized using silica nanoparticles prepared in above Mode 1.
  • Amine group is introduced to the surface of silica nanoparticles which are synthesized in above Mode 1-1 and 1-2 (dye-doped siica nanoparticles and plain silica nanoparticles) through silanization of 3- aminopropyltrimethoxysilane.
  • avidin is attached to the surface of silica nanoparticles through glParaldehyde.
  • avidin immobilized silica nanoparticles react with 3'-biotinalated DNA to conjugate probe DNA with nanoparticles and give probe nanoparticle DNA. (figure 4) Because the procedure is clear and obvious to those skilled in the art, so omitted here.
  • Mode 2 synthesized probe nanoparticle DNA are used in detection of DNA hybridization based on light scattering on DNA chip. Following is about DNA chip of disease check and signal confirmation in detail.
  • the DNA chip used in the invention is from Biomedlab.Inc. Twenty-two types of Capture DNA (sequence 1: 5'H2N-TATGTGCTGCCATATCTACTTCAGAAACTACATA-3' ) are immobilized on surface of glass slide. HPV DNA is the major etiologica agent of cervical cancer and related to several malignancy tumor.
  • the result image taken by both digital camera and laser scanner is shown in figure 9 when using cy5 probes, TMR-doped silica nanoparticle probes and plain silica nanoparticle probes.
  • the result is that DNA hybridization using cy5 probes is not detectable by digital camera but only detectable by laser scanner.
  • DNA hyridization using plain nanoparticle probes is not detectable by laser scanner but only detectable by digital camera based on light scattering of nanoparticles.
  • TMR-doped silica nanoparticles DNA hybridization are detectable by both laser scanner and digital camera, thus it is no need to use high cost laser scanner.
  • the left bottom part is the region where DNA conjugated nanoparticles probes can be seen. It means that capture DNA hybridized with complementary target DNA with certain sequence, the event can also be seen by naked eye and digital camera, and low cost laser scanner can be used to detect the signal dependent on size of nanoparticles.

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Abstract

Cette invention concerne une méthode de détection d'hybridation de l'ADN sur une puce à ADN, c'est-à-dire une détection fondée sur la diffusion de la lumière d'une sonde d'ADN à nanoparticules de l'invention. L'invention concerne une nouvelle méthode de détection d'hybridation de l'ADN sur puce à ADN, qui permet de détecter facilement une hybridation de l'ADN sur puce à ADN en utilisant un scanneur à lampe bon marché sans modification de biomolécules avec des molécules de colorant fluorescent ou un instrument optique onéreux, ce qui contribue à élargir le champ d'application de la puce à ADN.
PCT/KR2007/004356 2006-09-09 2007-09-08 Méthode de détection d'hybridation de l'adn par diffusion WO2008030071A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020060087116A KR100904825B1 (ko) 2006-09-09 2006-09-09 산란 현상을 이용한 ｄｎａ혼성화 측정방법
KR10-2006-0087116 2006-09-09

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WO2008030071A1 true WO2008030071A1 (fr) 2008-03-13

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3180464A4 (fr) * 2014-08-12 2018-03-14 The Regents of The University of Michigan Détection d'acides nucléiques
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030186240A1 (en) * 2002-03-14 2003-10-02 Xing Su Methods to increase nucleotide signals by raman scattering
US20040234970A1 (en) * 2001-01-27 2004-11-25 Yoo Jae Chern Nucleic hybridization assay method and device using a cleavage technique responsive to the complementary double strand or the single strand of nucleic acids or oligonucleotides
US20050053966A1 (en) * 1999-10-06 2005-03-10 Vladimir Poponin Apparatus and method for the analysis of nucleic acids hydbridization on high density NA chips
US20060166249A1 (en) * 2003-05-16 2006-07-27 University Of Rochester Methods for separating short single-stranded nucleic acid from long single-and double-stranded nucleic acid, and associated biomolecular assays

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050053966A1 (en) * 1999-10-06 2005-03-10 Vladimir Poponin Apparatus and method for the analysis of nucleic acids hydbridization on high density NA chips
US20040234970A1 (en) * 2001-01-27 2004-11-25 Yoo Jae Chern Nucleic hybridization assay method and device using a cleavage technique responsive to the complementary double strand or the single strand of nucleic acids or oligonucleotides
US20030186240A1 (en) * 2002-03-14 2003-10-02 Xing Su Methods to increase nucleotide signals by raman scattering
US20060166249A1 (en) * 2003-05-16 2006-07-27 University Of Rochester Methods for separating short single-stranded nucleic acid from long single-and double-stranded nucleic acid, and associated biomolecular assays

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3180464A4 (fr) * 2014-08-12 2018-03-14 The Regents of The University of Michigan Détection d'acides nucléiques
US10093967B2 (en) 2014-08-12 2018-10-09 The Regents Of The University Of Michigan Detection of nucleic acids
EP3705609A1 (fr) * 2014-08-12 2020-09-09 The Regents of The University of Michigan Détection d'acides nucléiques
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

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Publication number Publication date
KR100904825B1 (ko) 2009-06-25
KR20080023278A (ko) 2008-03-13

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