WO2003040679A2 - Liaison reversible d'un fluorophore sur une surface pour detecter des phenomenes d'association ligat-ligand par extinction de fluorescence - Google Patents

Liaison reversible d'un fluorophore sur une surface pour detecter des phenomenes d'association ligat-ligand par extinction de fluorescence Download PDF

Info

Publication number
WO2003040679A2
WO2003040679A2 PCT/DE2002/004147 DE0204147W WO03040679A2 WO 2003040679 A2 WO2003040679 A2 WO 2003040679A2 DE 0204147 W DE0204147 W DE 0204147W WO 03040679 A2 WO03040679 A2 WO 03040679A2
Authority
WO
WIPO (PCT)
Prior art keywords
modified
fluorophore
ligates
ligate
ligand
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/DE2002/004147
Other languages
German (de)
English (en)
Other versions
WO2003040679A3 (fr
Inventor
Gerhard Hartwich
Thomas KRATZMÜLLER
Herbert Wieder
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Friz Biochem Gesellschaft fuer Bioanalytik mbH
Original Assignee
Friz Biochem Gesellschaft fuer Bioanalytik mbH
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 Friz Biochem Gesellschaft fuer Bioanalytik mbH filed Critical Friz Biochem Gesellschaft fuer Bioanalytik mbH
Priority to AU2002363452A priority Critical patent/AU2002363452A1/en
Publication of WO2003040679A2 publication Critical patent/WO2003040679A2/fr
Publication of WO2003040679A3 publication Critical patent/WO2003040679A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a method for the detection of ligate-ligand association events by fluorescence quenching.
  • Immunoassays and increasingly sequence analysis of DNA and RNA are used in disease diagnosis, in toxicological test procedures, in genetic research and development, as well as in the agricultural and pharmaceutical sectors.
  • serial methods with autoradiographic or optical detection parallel detection methods using array technology, so-called DNA or protein chips, are increasingly being used. With these parallel methods, too, the detection is based on optical, radiographic, mass spectrometric or electrochemical methods.
  • probe oligonucleotides For gene analysis on a chip, a library of known DNA sequences ("probe oligonucleotides”) is fixed in an ordered grid on one surface, so that the probe oligonucleotides.
  • Target oligonucleotides present in the test solution, the sequences of which are complementary to certain probe oligonucleotides on the chip, can be identified by
  • Radio label e.g. 32 P
  • a fluorescent dye e.g. fluorescein, Cy3 TM or
  • Fluorescence scanners are increasingly using radio labels.
  • the fluorescence scanners currently available on the market enable the detection of
  • Fluorophore in the subattomole range Fluorophore in the subattomole range.
  • labeled targets to detect hybridization events has some drawbacks.
  • the marking must take place before the actual measurement, which means an additional synthesis step and thus additional work. It is also difficult to ensure homogeneous marking of the sample material.
  • stringent washing conditions are necessary in order to remove non-specific or non-specifically bound material following hybridization.
  • targets antibodies or antigen or DNA fragment
  • the targets do not have to be modified with a detection label and after the hybridization no complicated washing steps are necessary are.
  • the probe molecules are labeled with appropriate fluorescent dyes.
  • the so-called molecular beacons work according to this principle. These single-stranded oligonucleotides have a hairpin structure (stem-and-loop) and carry a fluorophore (e.g. Fluorescein, TexasRed®) at one end of the sequence and a suitable fluorescence quencher (e.g. DABCYL) at the other end of the sequence. Due to the special geometric arrangement, the fluorescent group and the unit that leads to the quenching of the fluorescence are located in close proximity to one another. Therefore the probes show only an extremely low fluorescence.
  • a fluorophore e.g. Fluorescein, TexasRed®
  • DABCYL fluorescence quencher
  • gold nanoparticles are also used as efficient quenchers (cf. Nature Biotech. Vol. 19, 2001, page 365).
  • the quenching of fluorescence by metals is based primarily on a radiationless energy transfer from the dye molecule to the metal.
  • a greater sensitivity is observed when using gold nanoparticles than with organic quenchers.
  • dyes are efficiently quenched into the near infrared range.
  • a disadvantage of this method is that gold nanoparticles at temperatures are no longer stable above 50 ° C. Another disadvantage is that this method is limited to the investigation of solutions and therefore only a few sequences can be examined at the same time, so the degree of parallelization of this approach is low.
  • the object of the present invention is therefore to create a method for the detection of ligate-ligand association events by fluorescence quenching which does not have the disadvantages of the prior art.
  • PNA Peptide nucleic acid synthetic DNA or RNA in which the Sugar-phosphate unit is replaced by an amino acid.
  • synthetic DNA or RNA in which the Sugar-phosphate unit is replaced by an amino acid.
  • Nucleic acid contains two or more covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine).
  • the term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, e.g. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (e.g. phosphoramide, thio-phosphate or dithio-phosphate backbone).
  • An essential feature of a nucleic acid in the sense of the present invention is the ability for sequence-specific binding of naturally occurring cDNA or RNA.
  • Nucleic acid oligomer Nucleic acid of unspecified base length (e.g. nucleic acid octamer: A nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to one another). ns oligomer Nucleic acid oligomer
  • Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
  • Oligonucleotide equivalent to oligomer or nucleic acid oligomer for example a DNA, PNA or RNA fragment of a base length not specified in more detail.
  • Mismatch To form the Watson-Crick structure of double-stranded oligonucleotides, the two single strands hybridize in such a way that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (for RNA, T is by uracil ) replaced. Any other base pairing does not form hydrogen bonds, distorts the structure and is referred to as a "mismatch". ss Single Strand ds double Strand
  • Antibody complex binding partner of an antigen is Antibody complex binding partner of an antigen.
  • Antigen complex binding partner of an antibody is Antigen complex binding partner of an antibody.
  • Receptor complex binding partner of a hormone Association constant for the association of ligate and ligand.
  • Fluorophore chemical compound that is able to emit a longer-wave (red-shifted) fluorescent light when excited with light.
  • Fluorophores fluorescent dyes
  • UV ultraviolet
  • VIS visible
  • IR infrared
  • the absorption and emission maxima are usually shifted by 15 to 40 nm (Stokes shift).
  • Fluorescein resorcin phthalein fluorescent dye
  • Rhodamine 6G Basic Red 1 fluorescent dye
  • Texas Red® fluorescent dye from Molecular Probes, Inc.
  • Quench surface conductive (metal) surface that can quench fluorescence through an energy transfer (especially gold, silver, copper surfaces etc.)
  • EDTA ethylenediamine tetraacetate (sodium salt) ligand Term for molecules that are specifically bound by ligates;
  • ligands in the context of the present invention are substrates, cofactors or coenzymes of a protein (enzyme), antibodies (as ligand of an antigen), antigens (as ligand of an antibody), receptors (as ligand of a hormone), hormones (as ligand of a receptor) ) or nucleic acid oligomers (as ligand of the complementary nucleic acid oligomer).
  • Ligate Term for (macro) molecule with specific recognition and binding sites for the formation of a complex with a ligand (template).
  • linkers are commercially available as alkyl, alkenyl, alkynyl, hetero-alkyl, hetero-alkenyl or hetero-alkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent one in simple / known chemical reactions with the corresponding reaction partner chemical bond.
  • the reactive groups can also be photoactivatable, ie the reactive groups are only activated by light of certain or any wavelength.
  • Preferred linkers are those of chain length 1-60, in particular chain length 5-40, the chain length here being the shortest continuous connection between the structures to be connected, i.e. between the two molecules or between a surface atom, a surface molecule or a surface molecule group and another Molecule.
  • Spacer linker which is covalently bonded via the reactive groups to one or both of the structures to be connected (see linker).
  • Preferred spacers are those of chain length 1-60, in particular chain length 5-40, the chain length being the shortest continuous connection between the structures to be connected.
  • the terminal phosphate group of the oligonucleotide is esterified at the 3 'end with (HO- (CH 2 ) 2 -S) 2 to PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH, the SS bond being cleaved homolytically and each causes an Au-SR bond.
  • the probe oligonucleotide carries a covalently linked fluorophore (FP) such as Cy3 TM, Cy5 TM, Texas Red®, Rhodamine 6G, fluorescein etc.
  • FP covalently linked fluorophore
  • Au-S- (CH 2 ) 2 -ds-oligo-Au-S- (CH 2 ) 2 -ss-oligo-FP hybridizes with the oligonucleotide complementary to ss-oligo FP.
  • the present invention relates to a method for the detection of ligate-ligand association events by fluorescence quenching, which comprises providing a modified surface as a first step.
  • the modification of the surface consists in the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore, the Fluorophore forms a reversible bond with the modified surface.
  • the further steps of the method according to the invention are providing a sample with ligands, bringing the sample into contact with the modified surface, detection of the fluorescence of the fluorophore and comparison of the detected fluorescence intensity with reference values.
  • a comparison of the detected fluorescence intensities with reference values is required in the method according to the invention.
  • These reference values can already exist from previous measurements and therefore do not need to be detected in the most general case in the course of the method according to the invention.
  • the reference values should ideally be determined under exactly the same external conditions as the actual measured values of the fluorescence intensities, according to a preferred embodiment of the present invention, a first detection of the fluorescence of the fluorophore is carried out before the targets (sample) come into contact with the modified surface , The values obtained in this way are then used as reference values.
  • a normalization measurement is also carried out.
  • sites are applied to the modified surface to which a very specific degree of association can be assigned after adding the sample.
  • the signal obtained during the detection is then characteristic of this particular degree of association and can be used to normalize the signals from the test sites.
  • the present invention namely also encompasses methods in which a modified surface is used which has been modified by binding at least two types of modified ligates.
  • the different types of ligates are bound to the surface in spatially essentially separated areas.
  • “Substantially separated areas” are understood to mean areas of the surface that are largely modified by binding a certain type of ligate. Only in areas in which two such essentially separated areas adjoin one another can spatial mixing of different types of ligate occur.
  • the ligand is added to the sample before the sample is brought into contact with the modified surface, the ligand being a binding partner with a high association constant with a particular type of ligate, which is used in a particular Area (Site T 100 ) is bound to the surface.
  • the ligand is added to the sample in an amount that is greater than the amount of ligand that is necessary to fully associate the ligates of the T 100 sites.
  • the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the region T 10 o.
  • the value obtained for the area T 100 thus corresponds to the value when the association is complete (100%).
  • a modified surface which has been modified by attaching at least three types of ligates.
  • the different types of ligates are bound to the surface in spatially essentially separated areas.
  • at least one type of ligate is bound to the surface in a certain area (site T 0 ), from which it is known that there is no binding partner with a high association constant in the sample, that is to say the corresponding association partner or ligand does not appear in the sample ,
  • a ligand is added to the sample before the sample is brought into contact with the modified surface, the ligand being a binding partner with a high association constant with a certain type of ligate, which occurs in a certain region (site T 10 o) is bound to the surface.
  • the ligand is added to the sample in an amount that is greater than the amount of ligand that is necessary to fully associate the ligates of the T 10 o sites.
  • the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the area T 100 and with the value obtained for the area T 0 .
  • the value obtained for the area To corresponds to the value in the absence of association (0%).
  • At least one further type of ligand is added to the sample before the sample is brought into contact with the modified surface, it being known that this ligand is not contained in the original sample.
  • This further type of ligand has an association constant> 0 to a type of ligate that is bound to the surface in a certain region (site T n ).
  • the ligand is added to the sample in such an amount that after contacting the sample with the modified surface, n% of the ligates of the site T n are in an associated form.
  • the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the area T 100 , with the value obtained for the area T 0 and with those for the areas T n values obtained.
  • the value obtained for a particular test site T n thus corresponds to the value in the presence of n% ligate-ligand associates based on the total number of ligates of the type.
  • the amount of ligand which has to be brought into contact with the modified surface in order to bring about an n% association at the site T n can be determined by the person skilled in the art by simple routine tests. For this purpose, for example after detection of the values for T 0 and T 0 o, a calibrated measurement is carried out, in which the signal intensity is determined by (different) detection labels with which the ligate and the ligand are equipped. The ratio of the intensities of the ligand label signal to the ligate label signal corresponds to n%.
  • the present invention also includes a kit for carrying out a method for the detection of ligate-ligand association events by fluorescence quenching.
  • the kit comprises a modified surface, the modification being the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore and the fluorophore having a reversible bond with the modified surface.
  • the kit comprises a modified surface, the modification consisting in the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore and the fluorophore being modified by attachment of at least one chemical group, which forms a reversible bond with the modified surface.
  • the reference values are already included in the kit, so that the fluorescence of the fluorophore only has to be detected once by the end user. The values obtained in this detection then only need to be compared with the existing reference values.
  • the kit comprises a modified surface which has at least one area T 0 and at least one area T-100.
  • the modified surface additionally comprises at least one region T n .
  • modified fluorophores can also be used in the process according to the invention. According to a preferred embodiment, fluorophores are therefore used which are modified by attachment of at least one chemical group, the chemical group forming a reversible bond with the modified surface.
  • a chemical group that forms a reversible bond with the modified surface can not only be attached directly to the fluorophore, but also to the ligates.
  • ligates are used which are additionally modified by attachment of at least one chemical group, the chemical group forming a reversible bond with the modified surface.
  • Particularly favorable conditions for detecting the difference in the fluorescence intensity of ligand / ligate associates and ligates exist when the fluorophore is as close as possible to the modified surface.
  • the modified surface is additionally modified by attachment of chemical groups and at the same time the ligates are modified with chemical groups, because then the chemical groups bound to the surface with the chemical groups attached to the ligates have a reversible bond can enter into.
  • surface denotes any carrier material which is suitable for carrying fluorophore-derivatized ligates directly or after appropriate chemical modification, which are covalently immobilized on the surface and whose fluorescence is close to the surface (in approx. 1 to 50 ⁇ Distance to the surface) is significantly reduced by fluorescence quenching (radiation-free energy transfer between the fluorophore as emitter and the surface as absorber) (> 10% of the expected fluorescence intensity of the fluorophore in the absence of the surface under otherwise identical conditions). Gold and silver are particularly suitable as quench surface material.
  • the term surface is independent of the spatial dimensions of the surface and also includes nanoparticles (particles or clusters of a few individual to several hundred thousand surface atoms or molecules).
  • the surface can also be attached to a solid support such as e.g. Glass, metal or plastic are bound.
  • ligate molecules in particular biopolymers such as nucleic acid oligomers or antigens or antibodies
  • the immobilization can be carried out, for example, covalently via hydroxyl, epoxy, amino or carboxy groups of the support material with thiol, hydroxyl, amino or carboxyl groups which are naturally present on the ligate or are attached to the ligate by derivatization.
  • the ligate can be used directly or via a linker / spacer are connected to the surface atoms or molecules of a surface.
  • the ligate can be anchored by the methods customary in immunoassays, for example by using biotinylated ligates for non-covalent immobilization on avidin or streptavidin-modified surfaces.
  • nucleic acid oligomers are used as ligates, the chemical modification of the probe nucleic acid oligomers with a surface anchor group can already be introduced in the course of automated solid phase synthesis or in separate reaction steps.
  • the nucleic acid oligomer is also linked directly or via a linker / spacer to the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways known from the prior art (cf. for example Hartwich, G .: ELECTROCHEMICAL DETECTION OF
  • nucleic acid oligomers When connecting the nucleic acid oligomers, care must be taken that they are either bound to the surface completely without additional co-adsorbate or, if a co-adsorbate appears necessary, that it forms a layer as thin as possible above the surface. Either a direct attachment of the nucleic acid oligomer to the surface must be carried out or it must be coated together with short-chain co-adsorbates such as e.g. short chain thiols. Co-adsorbates of chain length 1 to 30, particularly preferably chain length 1 to 20, in particular chain length 1 to 10 are preferred.
  • a particular disadvantage in this connection is the binding of the nucleic acid oligomers in the form of a surface-biotin-avidin-biotin-oligomer compound.
  • the fluorophore is always covered by a very thick layer of biotin Avidin-biotin is shielded from the surface, which is associated with corresponding disadvantages in the detection of fluorescence.
  • ligands Molecules that specifically interact with the ligate (probe) immobilized on a surface to form a complex are referred to as ligands.
  • ligands in the context of the present invention are substrates, cofactors or coenzymes as complex binding partners of a protein (enzyme), antibodies (as complex binding partners of an antigen), antigens (as complex binding partners of an antibody), receptors (as complex binding partners of a hormone), hormones (as complex binding partners) a receptor) or nucleic acid oligomers (as a complex binding partner of the complementary nucleic acid oligomer).
  • fluorescent dyes such as e.g. Texas Red®, rhodamine dyes, cyanine dyes such as Cy3 TM, Cy5 TM, fluorescein etc (see Fluka, Amersham and Molecular Probes catalog) are used.
  • Fluorescence quenching is the deactivation of an electronically excited species via a radiationless process. Deactivation can take place by means of impacts or by radiation-free energy transfer to metals. The energy released is dissipated as thermal energy. Gold is an example of a metal that has the ability to quench fluorescence. The quenching has a strong dependence on the distance of the fluorophore from the surface functioning as a fluorescence quencher (inversely proportional to a higher (third to sixth) power of the distance). The effect of fluorescence quenching can therefore only be measured at distances of less than 100 to 200 A. In the range greater than approx. 200 A, further changes in distance no longer lead to a measurable increase in the fluorescence intensity. Brief description of the drawings
  • Fig. 2 is a schematic representation of the detection of nucleic acid-oligomer hybridization events by specific interactions between groups on the fluorophore and the modified surface.
  • FIG. 1 shows a schematic representation of the detection of nucleic acid-oligomer hybridization events known from the prior art by means of “molecular beacons”.
  • the single-stranded oligonucleotides have a hairpin structure 101 (hairpin, stem-and-loop), carry a fluorophore at one end of the sequence
  • fluorescence quencher 103 e.g. DABCYL
  • the fluorescent group and the unit that leads to the quenching of the fluorescence are located in close proximity to one another. Therefore the probes show only an extremely low fluorescence.
  • the hybridization takes place in this area. This leads to a change in the conformation and the separation of fluorophore and quencher, which can be observed as a strong increase in fluorescence 106.
  • FIG. 2 shows a schematic representation of the detection of nucleic acid-oligomer hybridization events by specific interactions between groups 206 on the fluorophore 102 and the modified surface. Due to specific interactions between groups 206 on the fluorophore 102 and corresponding groups 207 on the surface 203, the single-stranded probe nucleic acid oligomer 201 is in a compressed form. The hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 205 between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher. This leads to a strong increase in the fluorescence intensity (see bar chart in FIG. 2). ,
  • nucleic acid-oligomer probes with different sequences are used tied to a support using the immobilization techniques described above.
  • the hybridization event of any target nucleic acid oligomer or (fragmented) target DNA is to be detected, for example in order to detect mutations in the target and to be demonstrated in a sequence-specific manner.
  • the surface atoms or molecules of a defined area are linked on a surface with DNA / RNA / PNA nucleic acid oligomers of known but any sequence, as described above.
  • the DNA chip can also be derivatized with a single probe oligonucleotide.
  • Nucleic acid oligomers eg DNA, RNA or PNA fragments
  • a base length of 3 to 70 preferably a length of 5 to 60, particularly preferably a length of 10 to 50, particularly preferably a length of 12 to 40, are used as probe nucleic acid oligomers used.
  • the target oligonucleotides can also comprise a larger number of bases, ie they can be longer than the probe oligonucleotides.
  • the expression “nucleic acid oligomer complementary to the probe oligonucleotide” is understood to mean a nucleic acid oligomer which has a base sequence which is complementary to the probe oligonucleotide in a partial region. The remaining portion (s) of the nucleic acid oligomer then protrude at the end (s) of the probe oligonucleotide beyond its base chain.
  • a reference measurement e.g. with a fluorescence scanner, the fluorescence intensity of the fluorophore-labeled probe oligonucleotides is determined in the single-stranded state.
  • the (as concentrated as possible) test solution with target oligonucleotide (s) is added to the surface with immobilized probe oligonucleotides.
  • Hybridization occurs only in the case in which the solution contains target nucleic acid oligomer strands which are complementary to the probe-nucleic acid oligomers bound to the surface or at least in many areas complementary.
  • the fluorescence intensity in the hybridized, double-stranded state is determined in a second fluorescence measurement.
  • a sequence-specific hybridization event can be performed by fluorescence-based methods such as e.g. Fluorescence microscopy or measurements with fluorescence scanners can be detected.
  • the difference between the reference measurement and the second measurement for each test site is proportional to the number of complementary (or in many areas complementary) target oligonucleotides originally present in the test solution for the respective test site.
  • the reference measurement can be omitted if the size of the reference signal is known beforehand (e.g. from previous measurements etc.).
  • a specific geometric arrangement of the probe nucleic acid oligomer relative to the modified surface can be achieved through specific interactions between groups on the dye that are naturally already present on the dye or that are introduced by chemical modification, and the (optionally modified) surface.
  • a geometrical arrangement can be realized by attaching suitable molecular groups to the dye (e.g. complexing groups such as bipyridyl groups or diamino groups), which can enter into a specific interaction that is stable under suitable conditions (e.g. complex formation) with corresponding groups. where the fluorophore is close to the surface in the non-hybridized state.
  • the single-stranded probe nucleic acid oligomer is therefore in a form which is characterized by a small distance between the fluorophore and the quenching metal surface (low fluorescence intensity).
  • the distance changes as a result of the hybridization with the complementary nucleic acid oligomer strand (target) between the fluorescent dye molecule and the metal surface acting as quencher in such a way that the distance increases, the quenching decreases and a higher intensity of the fluorescence can be observed after the hybridization (see FIG. 2).
  • n nucleotide (nt) long nucleic acid probe (DNA, RNA or PNA, for example a 20 nucleotide long oligo) is near one of its ends (3 'or 5' end) directly or via any spacer a reactive group for covalent anchoring to the surface, e.g. as a 3'-thiol-modified probe oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold.
  • Other covalent anchoring options arise e.g. from amine-modified ligate oligonucleotide, which is used for anchoring to surface-bonded carboxylic acid functions (e.g.
  • thiols such as mercaptopropionic acid and activation e.g. as an active ester.
  • a fluorophore is covalently attached (see Example 1), which is modified with a functional group (e.g. a fatty acid, a bipyridyl group or a diamino group).
  • a functional group e.g. a fatty acid, a bipyridyl group or a diamino group.
  • Probe nucleic acid oligomers attached to the - or, if appropriate, derivatized - surface
  • a bifunctional linker for example a suitable diamino thiol compound or a mercaptopropionic acid activated as an active ester and then reacting with a corresponding triamino compound
  • the surface modified in this way is brought into contact with the corresponding bifunctional linker in solution (for example a suitable diaminothiol compound or a mercaptopropionic acid activated as an active ester and subsequent reaction with a corresponding triamino compound in phosphate buffer / EtOH mixtures in the case of thiol-modified probe oligonucleotides) binds the bifunctional linker via its reactive group to the surface, which may be correspondingly derivatized (see section "binding of the ligate to the surface").
  • a suitable diaminothiol compound or a mercaptopropionic acid activated as an active ester and subsequent reaction with a corresponding triamino compound in phosphate buffer / EtOH mixtures in the case of thiol-modified probe oligonucleotides
  • the (residual) fluorescence of the fluorophore on the probe oligonucleotide is detected by a suitable method, e.g. by fluorescence measurement with a fluorescence scanner using specific interactions between functional groups on the dye and, if appropriate, correspondingly functionalized groups on the gold surface.
  • a suitable method e.g. by fluorescence measurement with a fluorescence scanner using specific interactions between functional groups on the dye and, if appropriate, correspondingly functionalized groups on the gold surface.
  • One embodiment is e.g. bipyridyl groups attached to the dye molecule or in its vicinity, which enter into relatively stable interactions with the gold surface under suitable conditions.
  • a further embodiment are diamino groups attached to or in the vicinity of the dye molecule, which enter into a relatively stable interaction via a complex bond generated by copper (II) or nickel (II) to corresponding diamino groups attached to the gold surface.
  • a further embodiment are hydrophobic alkyl chains attached to the dye molecule (for example by binding a fatty acid), which enter into relatively stable interactions via hydrophobic interactions with the likewise hydrophobic gold surface.
  • the dissolved target is then added, potential hybridization events are made possible under suitable conditions known to the person skilled in the art (arbitrary, freely selectable stringency conditions of the parameters potential temperature / salt / chaotropic salts etc. for the hybridization) and the measurement is repeated to detect the fluorophore.
  • the difference in the measurement signal is proportional to the number of hybridization events between probe nucleic acid oligomer on the surface and suitable target nucleic acid oligomer in the test solution.
  • the described method can be used for a target type (eg a specific target oligonucleotide type with a known sequence) on a surface or - if different probe types are used for each test site - for several target types (same ligand groups as eg several different target Oligonucleotide types or different antibody types, antigen types etc., but also mixtures thereof) can be used.
  • a target type eg a specific target oligonucleotide type with a known sequence
  • target types eg several different target Oligonucleotide types or different antibody types, antigen types etc., but also mixtures thereof
  • oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
  • the oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
  • Modifications to the 5 'position of the oligonucleotides are carried out with a coupling step that is extended to 5 min.
  • the amino modifier C2 dT (Glen Research 10-1037) is built into the sequences with the respective standard protocol. The coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.
  • the oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h.
  • the oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS.
  • the amine-modified oligonucleotides are coupled to the corresponding activated fluorophores (eg fluorescein isothiocyanate) in accordance with conditions known to the person skilled in the art.
  • the coupling can take place both before and after the oligonucleotides have been bound to the surface.
  • oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
  • the oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
  • Modifications to the 5 'position of the oligonucleotides are carried out with a coupling step which is extended to 5 min.
  • the fluorophores are incorporated in the synthesizer in the penultimate coupling step as phosphoramidites (fluorescein-phosphoramidites Glen Research 10-1963) in the sequences with the respective standard protocol.
  • the amino modifier C2 dT (Glen Research 10-1037) is incorporated in the sequences with the respective standard protocol in the last coupling step.
  • the coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.
  • a fatty acid or an ethylenediamine function for example as ethylenediamine-N, N'-diacetic acid
  • the bipyridyl group for example as 2,2'-bipyridine-4,4'-dicarboxylic acid
  • the quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is with (HO- (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2 -OH; modification 2: at the 5 'end is a fluorophore and an ethylenediamine group (see Ex. 2) installed according to the respective standard protocol) in 5x10 "5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) with the addition of approx.
  • 5 molar buffer solution phosphate buffer, 0.5 molar in water, pH 7
  • the free bifunctional thiol present at the same time in the incubation solution is also co-adsorbed by forming an Au-S bond (incubation step).
  • this single strand can also be hybridized with its complementary strand.
  • a state is then achieved by adding soluble nickel salts (e.g. Ni (II) chloride) or other suitable metal ions (such as copper) through the formation of a complex using the complex formation properties of the diamino group on the fluorophore and the diamino group on the surface , in which the fluorophore is in a geometric arrangement close to the surface.
  • soluble nickel salts e.g. Ni (II) chloride
  • suitable metal ions such as copper
  • the quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is with (HO- (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2 -OH; modification 2: at the 5 'end is a modified fluorophore and an ethylenediamine group (see example 2) Incorporated according to the respective standard protocol) in 5x10 "5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5 - 24 h.
  • the disulfide spacer PO- (CH 2 ) 2 -SS - (CH 2 ) 2 -OH of the oligonucleotide cleaved homolytically, the spacer forming a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol.
  • this single strand can also be hybridized with its complementary strand.
  • the gold surface thus modified is then completed with an approximately 10 "5 to 10 " 1 molar diaminothiol compound (or other suitable thiols or disulfides of suitable chain length, such as mercaptopropionic acid activated as an active ester, to which a triamino group is then coupled) wetted and incubated for 0.5 - 24 h.
  • the free thiol compound covers the free gold surface remaining after the incubation step by forming an Au-S bond.
  • Ni (II) chloride soluble nickel salts
  • suitable metal ions such as copper
  • An alternative production of Au-S (CH 2 ) 2 -ss-oligo-FP consists in the derivatization of the gold surface with the probe oligonucleotide (incubation step) without subsequent loading.
  • the quench surface here: gold plate
  • the quench surface is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is with (HO- (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2 -OH; modification 2: at the 5 'end is a modified fluorophore and a bipyridyl group (see example 2) Incorporated according to the respective standard protocol) in 5x10 "5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5 - 24 h.
  • the disulfide spacer PO- (CH 2 ) 2 -SS - (CH 2 ) 2 -OH of the oligonucleotide cleaved homolytically, the spacer forming a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol.
  • this single strand can also be hybridized with its complementary strand.
  • the bipyridyl group attached to the fluorophore or in its vicinity interacts with the unmodified gold surface, whereby a geometrical arrangement is achieved in which the fluorophore is present close to the surface.
  • Another alternative preparation of Au-S (CH 2 ) 2 -ss-oligo-FP consists in the derivatization of the gold surface with the probe oligonucleotide (incubation step) without subsequent loading.
  • the quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is with (HO- (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2 -OH; modification 2: at the 5 'end is a modified fluorophore and an alkyl chain (e.g. as a fatty acid unit, cf.
  • an alkyl chain e.g. as a fatty acid unit, cf.
  • Example 2 built in according to the respective standard protocol) in 5x10 "5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5-24 h.
  • 5 molar buffer solution phosphate buffer, 0.5 molar in water, pH 7
  • the disulfide spacer PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide cleaved homolytically, whereby the spacer forms a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy- mercaptoethanols is coming.
  • this single strand can also be hybridized with its complementary strand.
  • the gold surface can also be correspondingly hydrophobically modified by coadsorption (see Example 3) or subsequent coating (see Example 4) with hydrophobic alkylthiols (e.g. propanethiol or other thiols or disulfides of a suitable chain length).
  • hydrophobic alkylthiols e.g. propanethiol or other thiols or disulfides of a suitable chain length.
  • the fatty acid unit attached to or in the vicinity of the fluorophore interacts with the hydrophobic gold surface, whereby a geometric arrangement is achieved in which the fluorophore is present close to the surface.
  • the probe surface is produced analogously to Examples 3-6.
  • a modified oligonucleotide of the sequence 5'-fluorescein-AGC GGA TAA CAC AGT CAC CT-3 '[C 3 -SSC 3 -OH] is immobilized on gold (50 ⁇ mol oligonucleotide in phosphate buffer (K 2 HPO 4 / KH 2 PO 4 500 mM, pH 7) and in the form Au-S (CH 2 ) 2 -ss-oligo-Fluorescein the fluorescence intensity of the surface was determined with a fluorescence scanner from Lavision Biotech for the measurement of fluorescence in the presence of liquid media 150 ⁇ l of the medium are placed on the gold surface and then covered with a cover glass, or alternatively Hybriwells or imaging chambers can be used.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé pour détecter des phénomènes d'association ligat-ligand par extinction de fluorescence, ce procédé consistant d'abord à préparer une surface modifiée. La modification de surface consiste à établir une liaison d'au moins une catégorie de ligats 201 modifiés, lesquels sont modifiés par liaison avec au moins un type de fluorophore 102, qui établit une liaison réversible avec la surface modifiée. Les autres opérations du procédé de la présente invention sont les suivantes : préparation d'un échantillon de ligands, mise en contact de cet échantillon avec la surface modifiée, détection de la fluorescence du fluorophore et comparaison de l'intensité de la fluorescence détectée avec des valeurs de référence.
PCT/DE2002/004147 2001-11-09 2002-11-08 Liaison reversible d'un fluorophore sur une surface pour detecter des phenomenes d'association ligat-ligand par extinction de fluorescence Ceased WO2003040679A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002363452A AU2002363452A1 (en) 2001-11-09 2002-11-08 Reversibly binding a fluorophore to a surface for the purpose of detecting ligate/ligand association events by fluorescence quenching

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10155053.7 2001-11-09
DE2001155053 DE10155053B4 (de) 2001-11-09 2001-11-09 Reversible Bindung eines Fluorophors an eine Oberfläche zur Detektion von Ligat-Ligand-Assoziationsereignissen durch Fluoreszenz-Quenchen

Publications (2)

Publication Number Publication Date
WO2003040679A2 true WO2003040679A2 (fr) 2003-05-15
WO2003040679A3 WO2003040679A3 (fr) 2004-05-06

Family

ID=7705170

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2002/004147 Ceased WO2003040679A2 (fr) 2001-11-09 2002-11-08 Liaison reversible d'un fluorophore sur une surface pour detecter des phenomenes d'association ligat-ligand par extinction de fluorescence

Country Status (3)

Country Link
AU (1) AU2002363452A1 (fr)
DE (1) DE10155053B4 (fr)
WO (1) WO2003040679A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2418018A (en) * 2004-09-01 2006-03-15 Perkinelmer Singapore Pte Ltd A method of analysing a sample of biological material that includes modifying fluorescent labels in the sample or the environmental conditions of the sample
CN100396790C (zh) * 2004-09-17 2008-06-25 北京大学 溶液识别、表面寻址蛋白质芯片及其制备和检测方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016000865A1 (de) * 2016-01-28 2017-08-03 Friz Biochem Gesellschaft Für Bioanalytik Mbh Funktionalisierte metallische Nanopartikel

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19811730A1 (de) * 1998-03-18 1999-09-23 November Ag Molekulare Medizin Verfahren und Vorrichtung zum Identifizieren einer Markierung
US20020086289A1 (en) * 1999-06-15 2002-07-04 Don Straus Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms
US6645733B1 (en) * 1999-06-25 2003-11-11 Ingeneus Corporation Fluorescent intensity method for assaying binding between proteins or peptides
AU5601800A (en) 1999-08-09 2001-03-05 Motorola, Inc. Adaptive delay lock loop tracking
JP2003517842A (ja) * 1999-12-21 2003-06-03 オーソ−クリニカル・ダイアグノスティックス・インコーポレイテッド 核酸の検出方法
US6265170B1 (en) * 2000-01-24 2001-07-24 Ingeneus Corporation Homogenous assay of duplex of triplex hybridization by means of multiple measurements under varied conditions
AU2001293232A1 (en) * 2000-08-29 2002-03-13 The Rockefeller University Methods employing fluorescence quenching by metal surfaces

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2418018A (en) * 2004-09-01 2006-03-15 Perkinelmer Singapore Pte Ltd A method of analysing a sample of biological material that includes modifying fluorescent labels in the sample or the environmental conditions of the sample
GB2418018B (en) * 2004-09-01 2007-09-12 Perkinelmer Singapore Pte Ltd A method of analysing a sample including fluorescent labels and apparatus therefor
CN100396790C (zh) * 2004-09-17 2008-06-25 北京大学 溶液识别、表面寻址蛋白质芯片及其制备和检测方法

Also Published As

Publication number Publication date
WO2003040679A3 (fr) 2004-05-06
AU2002363452A1 (en) 2003-05-19
DE10155053A1 (de) 2003-08-14
DE10155053B4 (de) 2005-11-24

Similar Documents

Publication Publication Date Title
DE69637065T2 (de) Die bestimmung von nukleinsäuren und nukleinsäure-einheiten
DE102007044664B4 (de) Verdrängungsassay zur Detektion von Nukleinsäureoligomer-Hybridisierungsereignissen
DE69736667T2 (de) Verfahren zum nachweis und amplifikation von nukleinsäuresequenzen unter verbrauch von modifizierten oligonukleotiden mit erhöhter zielschmelztemperatur (tm)
DE60223276T2 (de) Verfahren zur Blockierung von unspezifischen Hybridisierungen von Nukleinsäuresequenzen
DE69231853T2 (de) Hybridisierung von mit chromophoren und fluorophoren konjugierten polynukleotiden zur erzeugung eines donor-zu-donor energietransfersystems
DE69031665T2 (de) Nachweis von Nukleinsäuresequenzen unter Verwendung von Fluoreszenz-Polarisation
DE69737598T2 (de) Nanopartikel mit daran angehefteten oligonukleotiden sowie deren verwendungen
DE69231124T2 (de) Schnelle bestimmungsverfahren für amplifikationsprodukte
DE69032847T2 (de) Hydrophobe Nukleinsäure-Sonde
DE3888653T2 (de) Verfahren und Testsatz zum Nachweis einer Nukleinsäure-Sequenz.
EP1230398A1 (fr) Oligonucleotide marque par colorant pour le marquage d'une molecule d'acide nucleique
DE102011056606B3 (de) Verfahren zur elektrochemischen Detektion von Nukleinsäureoligomer-Hybridisierungsereignissen
EP1554396B1 (fr) Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
DE10142643A1 (de) Detektion von Wechselwirkungen auf Sonden-Arrays
DE102009044795B3 (de) Kompetitionsassay zur Detektion von Nukleinsäureoligomer-Hybridisierungsereignissen
DE60221964T2 (de) Verfahren und testkit zur quantitativen bestimmung von variationen der polynukleotidmengen in zell- oder gewebeproben
DE102006014879B4 (de) RNA-Markierungsverfahren
DE10155053B4 (de) Reversible Bindung eines Fluorophors an eine Oberfläche zur Detektion von Ligat-Ligand-Assoziationsereignissen durch Fluoreszenz-Quenchen
DE102014203266B4 (de) Zusammensetzung mit FRET-Paar in definierter Geometrie
DE19806962B4 (de) Markierung von Nukleinsäuren mit speziellen Probengemischen
DE102012203964B3 (de) Verfahren und Kit zur Detektion von Nukleinsäuren
WO2003040678A2 (fr) Extinction de fluorescence pour detecter des phenomenes d'association ligat-ligand dans des champs electriques
DE10155055A1 (de) Fluoreszenz-Quenchen zur Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen bei hohen Salz-Konzentrationen
WO2003018834A2 (fr) Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
DE60120118T2 (de) Test- und nachweisverfahren für einen mikroarray

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU BR CA CN CU HU IL JP MX NO PL SI US ZA

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2002802611

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2002802611

Country of ref document: EP

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP