Assay based on doped nanoparticles Number:7,410,810 from the United States Patent and Trademark Office (PTO) owispatent The invention relates to an assay based on resonance energy transfer (RET), comprising a first molecule grope A, which is marked with at least one energy donor, and at least one second molecule group B which is marked with at least one energy acceptor, the donor comprising a molecule or particle which can be energetically excited by an external radiation source and which is fluorescence enabled and the acceptor comprising a molecule or particle which can be excited by energy transfer via the donor with partial or complete quenching of the donor fluorescence, and the donor and/or acceptor comprise luminescing inorganic dope nanoparticles having an expansion of .ltoreq.50 nanometers, emitting electromagnetic radiation with stokes or anti-stokes scattering after energetic excitation.<H nanoparticles, doped, Assay, based, on, , 7410810.html, Patent, pto, 7410810

Title: Assay based on doped nanoparticles

Abstract: The invention relates to an assay based on resonance energy transfer (RET), comprising a first molecule grope A, which is marked with at least one energy donor, and at least one second molecule group B which is marked with at least one energy acceptor, the donor comprising a molecule or particle which can be energetically excited by an external radiation source and which is fluorescence enabled and the acceptor comprising a molecule or particle which can be excited by energy transfer via the donor with partial or complete quenching of the donor fluorescence, and the donor and/or acceptor comprise luminescing inorganic dope nanoparticles having an expansion of .ltoreq.50 nanometers, emitting electromagnetic radiation with stokes or anti-stokes scattering after energetic excitation.

Patent Number: 7,410,810 Issued on 08/12/2008 to Bohmann, et al.

Inventors: Bohmann; Kerstin (Koln, DE), Hoheisel; Werner (Koln, DE), Kohler; Burkhard (Leverkusen, DE), Dorn; Ingmar (Koln, DE)
Assignee: Bayer Technology Services GmbH (Leverkusen, DE)

Appl. No.: 10/494,390

Filed: November 4, 2002

PCT Filed: November 04, 2002

PCT No.: PCT/EP02/12256

371(c)(1),(2),(4) Date: April 30, 2004

PCT Pub. No.: WO03/040024

PCT Pub. Date: May 15, 2003

Foreign Application Priority Data


Nov 05, 2001 [DE]			101 53 829

Current U.S. Class: 436/524

Current International Class: G01N 33/551 (20060101)

Field of Search: 436/514,518,524-528,172,7.92 435/4,7.1,7.92

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Primary Examiner: Lam; Ann Y
Attorney, Agent or Firm: Norris McLaughlin & Marcus PA

Claims

The invention claimed is:

1. An assay kit for carrying out an assay method based on a resonance energy transfer (RET), said assay kit comprising: a) a first molecule group A which is labeled with at least one energy donor (donor), and b) at least a second molecule group B which is labeled with at least one energy acceptor (acceptor), wherein the donor comprises a molecule or particle which can be energetically excited by an external radiation source and is capable of fluorescing, the acceptor comprises a molecule or particle which can be excited by energy transfer by way of the donor, with partial or complete quenching of the donor fluorescence, and donor and/or acceptor comprise luminescent inorganic doped nanoparticles (lid nanoparticles) which have a breadth of >50 nanometers and which, after an energetic excitation, emit electromagnetic radiation with a Stokes shift or an anti-Stokes shift.

2. The assay kit as claimed in claim 1, wherein the acceptor is also capable of fluorescing.

3. The assay kit as claimed in claim 1, wherein the acceptor relaxes in a radiationless manner.

4. The assay kit as claimed in claim 1, wherein the RET is a fluorescence resonance energy transfer (FRET).

5. The assay kit as claimed in claim 1, wherein the RET is a Forster transfer or a transfer involving higher multiple orders.

6. The assay kit as claimed in claim 1, wherein the RET is a migration of charges or excitons.

7. The assay kit as claimed in claim 1, wherein the RET can be qualitatively and/or quantitatively recorded by a time-resolved or continuous measurement of a change in the luminescence properties.

8. The assay kit as claimed in claim 7, wherein the RET is qualitatively and/or quantitatively recorded by a time-resolved or continuous measurement of a change in an intensity of the fluorescent light, a change in the spectrum of the fluorescent light or a change in the decay time of the lid nanoparticles and/or of other donors and/or acceptors.

9. The assay kit as claimed in claim 7, wherein the RET is qualitatively and/or quantitatively recorded by a time-resolved or continuous measurement of a change in an intensity of the fluorescent light, a change in the spectrum of the fluorescent light or a change in the decay time of a chromophoric donor/acceptor.

10. The assay kit as claimed in claim 1, wherein either the donor or acceptor comprises lid nanoparticles which have a long fluorescence decay time and the other one of the donor or acceptor either also comprises lid nanoparticles whose fluorescence decay time is shorter than that of the other lid nanoparticles or comprises a molecular organic chromophore.

11. The assay kit as claimed in claim 10, wherein either the donor or acceptor comprises lid nanoparticles which exhibit a half life of more than 5 ns.

12. The assay kit as claimed in claim 11, wherein either the donor or acceptor comprises lid nanoparticles which exhibit a half life between 1 .mu.s and 50 ms.

13. The assay kit as claimed in claim 12, wherein either the donor or acceptor comprises lid nanoparticles exhibit a half life between 100 .mu.s and 10 ms.

14. The assay kit as claimed in claim 1, wherein the lid nanoparticles have a breadth in the range of from 1 nm to 50 nm.

15. The assay kit as claimed in claim 14, wherein the lid nanoparticles have a breadth in the range of from 1 nm to less than 20 nm.

16. The assay kit as claimed in claim 15, wherein the lid nanoparticles have a breadth in the range of from 2 nm to 10 nm.

17. The assay kit as claimed in claim 1, wherein the lid nanoparticles exhibit a needle-shaped morphology having a breadth in the range from 3 nm to 50 nm and a length in the range from 20 nm to 5 .mu.m.

18. The assay kit as claimed in one of claim 17, wherein the lid nanoparticles exhibit a needle-shaped morphology having a breadth in the range from 3 nm to 20 nm and/or a length in the range from 20 nm to 500 nm.

19. The assay kit as claimed in claim 1, wherein a host material of the lid nanoparticles comprises compounds of the type XY, where X is a cation consisting of one or more elements of main groups 1a, 2a, 3a or 4a, of the subgroups 2b, 3b, 4b, 5b, 6b or 7b, or of the lanthanides, of the Periodic Table, and Y is either a polyatomic anion consisting of one or more element(s) of the main groups 3a, 4a or 5a, or of the subgroups 3b, 4b, 5b, 6b, 7b and/or 8b, and also element(s) of the main groups 6a and/or 7, or else is a monoatomic anion from the main group 5a, 6a or 7a of the Periodic Table.

20. The assay kit as claimed claim 1, wherein a host material of the lid nanoparticles comprises at least one compound selected from the group consisting of sulfides, selenides, sulfoselenides, oxysulfides, borates, aluminates, gallates, silicates, germanates, phosphates, halophosphates, oxides, arsenates, vanadates, niobates, tantalates, sulfates, tungstates, molybdates, alkali metal halides and other halides, and nitrides.

21. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a doping material comprising one or more elements selected from the group consisting of elements of the main groups 1a or 2a, or Al, Cr, Tl, Mn, Ag, Cu, As, Nb, Nd, Ni, Ti, In, Sb, Ga, Si, Pb, Bi, Zn or Co, and/or elements of the lanthanides.

22. The assay kit as claimed in claim 21, wherein the doping material comprises a combination of two or more of said elements, in different relative concentrations to each other.

23. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a doping material in a concentration of the doping material in a host lattice of between 10.sup.-5 mol % and 50 mol %.

24. The assay kit as claimed in claim 23, wherein the concentration of the doping material in the host lattice is between 0.01 mol % and 30 mol %.

25. The assay kit as claimed in claim 24, wherein the concentration of the doping material in the host lattice is between 0.1 mol % and 20 mol %.

26. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a material selected from the group consisting of YVO.sub.4:Eu, YVO.sub.4:Sm, YVO.sub.4:Dy, LaPO.sub.4:Eu, LaPO.sub.4:Ce, LaPO.sub.4:Ce,Tb, LaPO.sub.4:Ce,Dy, LaPO.sub.4:Ce,Nd, ZnS:Th, ZnS:TbF.sub.3, ZnS:Eu ZnS:EuF.sub.3, Y.sub.2O.sub.3:Eu, Y.sub.2O.sub.2S:Eu, Y.sub.2SiO.sub.5:Eu, SiO.sub.2:Dy, SiO.sub.2:Al, Y.sub.2O.sub.3:Tb, CdS:Mn, ZnS:Tb, ZnS:Ag and ZnS:Cu.

27. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a material in which the host lattice has a cubic structure.

28. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a material selected from the group consisting of MgF.sub.2:Mn, ZnS:Mn, ZnS:Ag, ZnS:Cu, CaSiO.sub.3:Ln, CaS:Ln, CaO:Ln, ZnS:Ln, Y.sub.2O.sub.3:Ln, and MgF.sub.2:Ln (Ln=lanthanides).

29. The assay kit as claimed in claim 1, wherein the donor and/or the acceptor comprise lid nanoparticles which, after energetic excitation with electromagnetic radiation with wavelengths in the range of infrared light, of visible light, of UV, of X-ray light or of .gamma.-radiation, or particle radiation emit electromagnetic radiation with a Stokes shift or anti-Stokes shift.

30. The assay kit as claimed in claim 1, wherein the donor and/or the acceptor comprise lid nanoparticles which, after energetic excitation with electromagnetic radiation with wavelengths in the range of electron radiation, emit electromagnetic radiation with a Stokes shift or anti-Stokes shift.

31. The assay kit as claimed in claim 1, wherein the donor comprises lid nanoparticles and the acceptor comprises a conducting material.

32. The assay kit as claimed in claim 31, wherein the conducting material is a metal, a conducting oxide, or a conducting polymer.

33. The assay kit as claimed in claim 32, wherein the conducting material is a metal selected from the group consisting of gold, silver and platinum; the conducting oxide is indium tin oxide (ITO); and the conducting polymer is present in particulate form as nanoparticles or microparticles or as a planar, optionally structured, surface.

34. The assay kit as claimed in claim 1, which is capable of use in a homogeneous assay without any washing or separating steps.

35. The assay kit as claimed in claim 1, which is capable of use in a homogeneous immunoassay detecting at least one analyte in a sample.

36. The assay kit as claimed in claim 35, wherein the at least one analyte is selected from the group consisting of at least one monoclonal or polyclonal antibody, protein, peptide, oligonucleotide, nucleic acid, oligosaccharide, polysaccharide, hapten and low molecular weight synthetic or natural antigen; and/or the sample comprises at least one member selected from the group consisting of smears, sputum, organ punctate, biopsies, secretions, spinal fluid, bile, blood, lymph fluid, urine and feces.

37. The assay kit as claimed in claim 1, wherein a surface of the lid nanoparticle(s) is prepared such that affinity molecules can be coupled to it.

38. The assay kit as claimed in claim 37, wherein the surface of the lid nanoparticles is chemically modified and/or exhibits reactive groups and/or covalently or noncovalently bound connecting molecules, with the bound connecting molecules being able, for their part, to exhibit reactive groups.

39. The assay kit as claimed in claim 38, wherein the reactive groups are selected from the group consisting of amino groups, carboxylic acid groups, thiols, thioethers, disulfides, imidazoles, guanidines, hydroxyl groups, indoles, vicinal diols, aldehydes, alpha-haloacetyl groups, N-maleimides, mercurides, aryl halides, acid anhydrides, isocyanates, isothiocyanates, sulfonyl halides, imidoesters, diazoacetates, diazonium salts, 1,2-diketones, alpha-beta-unsaturated carbonyl compounds, phosphonic acids, phosphoric acid esters, sulfonic acids and azolides, and derivatives of said reactive groups.

40. The assay kit as claimed in claim 39, wherein the connecting molecules are selected from the group consisting of nucleic acid molecules, phosphonic acid derivatives, ethylene glycol, primary alcohols, amine derivatives, polymers or copolymers, polymerizable coupling agents, silica shells and catenate molecules having a polarity which is opposite to that of the surface of the lid nanoparticles.

41. The assay kit as claimed in claim 40, wherein the polymerizable coupling agents are selected from the group consisting of diacetylenes, styrenebutadienes, vinyl acetate, acrylates, acrylamides, vinyls, styrenes, silicone oxides, boron oxides, phosphorus oxides, borates, pyrroles, polypyrroles and phosphates, and also polymers of at least some of said polymerizable coupling agents.

42. The assay kit as claimed in claim 37, wherein the affinity molecules are selected from the group consisting of proteins, peptides, oligonucleotides or other nucleic acid molecules or nucleic acid-like molecules, oligosaccharides or polysaccharides, haptens, and low molecular weight synthetic natural antigens or epitopes.

43. The assay kit as claimed in claim 42, wherein the nucleic acid-like molecules are PNAs or morpholinos; and/or the haptens are biotin or digoxin.

44. The assay kit as claimed in claim 37, wherein the affinity molecules are able to interact with target molecules.

45. The assay kit as claimed in claim 44, wherein the target molecule is an enzyme, an antibody, a nucleic acid-binding molecule, a nucleic acid, a polynucleotide or a morpholino.

46. The assay kit as claimed in claim 45, wherein the enzyme is endonuclease, protease, kinase or phosphatase or an amino acid- or nucleic acid-modifying or cleaving enzyme.

47. The assay kit as claimed in claim 46, wherein an interaction of the affinity molecule with the target molecule results in a change in a spatial separation of molecule groups A and B.

48. The assay kit as claimed in claim 1, wherein the molecule groups A and B are constituents of one and the same molecule.

49. The assay kit as claimed in claim 1, wherein the molecule groups A and B are able to couple to the same affinity molecule.

50. The assay kit as claimed in claim 1, which is used for quantifying nucleic acids.

51. The assay kit as claimed in claim 1, wherein the molecule groups A and B are constituents of different molecules.

52. The assay kit as claimed in claim 51, wherein the molecule groups A and B are in each case coupled to their own affinity molecules.

53. The assay kit as claimed in claim 52, wherein the affinity molecules which are assigned to molecule groups A and B are able to interact specifically with the same target molecule.

54. The assay kit as claimed in claim 53, wherein an interaction of the affinity molecules which are assigned to molecule groups A and B with the common target molecule or with each other result in a change in the spatial separation of molecule groups A and B.

55. The assay kit as claimed in claim 52, wherein the affinity molecules which are assigned to molecule groups A and B are able to interact specifically with each other.

56. The assay kit as claimed in claim 1, wherein the lid nanoparticles comprise a material selected from the group consisting of LiI:Eu; NaI:Tl; CsI:Tl; CsI:Na; Lif:Mg; LiF:Mg,Ti; LiF:Mg,Na; KMgF.sub.3:Mn; Al.sub.2O.sub.3:Eu; BaFCl:Eu; BaFCl:Sm; BaFBr:Eu; BaFCl.sub.0.5Br.sub.0.5:Sm; BaY.sub.2F.sub.8:A (A=Pr, Tm, Er or Ce); BaSi.sub.2O.sub.5:Pb; BaMg.sub.2Al.sub.16O.sub.27:Eu; BaMgAl.sub.14O.sub.23:Eu; BaMgAl.sub.10O.sub.17:Eu; BaMgAl.sub.2O.sub.3:Eu; Ba.sub.2P.sub.2O.sub.7:Ti; (Ba,Zn or Mg).sub.3Si.sub.2O.sub.7:Pb; Ce(Mg or Ba)Al.sub.11O.sub.19; Ce.sub.0.65 Tb.sub.0.35MgAl.sub.11O.sub.19:(Ce or Tb); MgAl.sub.11O.sub.19:(Ce or Tb); MgF.sub.2:Mn; MgS:Eu; MgS:Ce; MgS:Sm; MgS:(Sm or Ce); (Mg or Ca)S:Eu; MgSiO.sub.3:Mn; 3.5MgO.0.5MgF.sub.2.GeO.sub.2:Mn; MgWO.sub.4: Sm; MgWO.sub.4:Pb, 6MgO.As.sub.2O.sub.5:Mn; (Zn or Mg)F.sub.2:Mn; (Zn.sub.4Be)SO.sub.4:Mn; Zn.sub.2SiO.sub.4:Mn; Zn.sub.2SiO.sub.4:Mn,As; ZnO:Zn; ZnO:Zn, Si,Ga; Zn.sub.3(PO.sub.4).sub.2Mn; ZnS:A (A=Ag, Al or Cu); (Zn or Cd)S:A (A=Cu, Al, Ag or Ni); CdBO.sub.4:Mn; CaF.sub.2:Mn; CaF.sub.2:Dy; CaS:A (A=lanthanides or Bi); (Ca or Sr)S:Bi; CaWO.sub.4:Pb; CaWO.sub.4:Sm; CaSO.sub.4:A (A=Mn or lanthanides); 3Ca.sub.3(PO.sub.4).sub.2.Ca(F or Cl).sub.2:Sb,M.sub.n; CaSiO.sub.3:(Mn or Pb); Ca.sub.2Al.sub.2Si.sub.2O.sub.7:Ce; (Ca or Mg)SiO.sub.3:Ce; (Ca or Mg)SiO.sub.3:Ti; 2SrO.6(B.sub.2O.sub.3).SrF.sub.2:Eu; 3Sr.sub.3(PO.sub.4).sub.2.CaCl.sub.2:Eu; A.sub.3(PO.sub.4).sub.2.ACl.sub.2:Eu (A=Sr, Ca or Ba); (Sr or Mg).sub.2P.sub.2O.sub.7:Eu; (Sr or Mg).sub.3(PO.sub.4).sub.2:Sn; SrS:Ce; SrS:Sm,Ce; SrS:Sm; SrS:Eu; SrS:Eu,Sm; SrS:(Cu or Ag); Sr.sub.2P.sub.2O.sub.7:Sn; Sr.sub.2P.sub.2O.sub.7:Eu; Sr.sub.4Al.sub.14O.sub.25:Eu; SrGa.sub.2S.sub.4A (A=lanthanides or Pb); SrGa.sub.2S.sub.4:Pb; Sr.sub.3Gd.sub.2Si.sub.6O.sub.18:Pb,Mn; YF.sub.3:Yb,Er; YF.sub.3:Ln (Ln=lanthanides); YLiF.sub.4:Ln (Ln=lanthanides); Y.sub.3Al.sub.5O12:Ln (Lu=lanthanides); YAl.sub.3(BO.sub.4).sub.3:(Nd or Yb); (Y or Ga)BO.sub.3:Eu; (Y or Gd)BO.sub.3:Eu; Y.sub.2Al.sub.3Ga.sub.2O.sub.12:Tb; Y.sub.2SiO.sub.5:Ln (Ln=lanthanides); Y.sub.2O.sub.3:Ln (Ln=lanthanides); Y.sub.2O.sub.2S:Ln (Lu=lanthanides); YVO.sub.4:A (A=lanthanides or In); Y(P,V)O.sub.4:Eu; YTaO.sub.4:Nb; YAlO.sub.3:A (A=Pr, Tm, Er or Ce); YOCl:(Yb or Er); LnPO.sub.4: (LnCe or Tb=lanthanides or mixtures of lanthanides); LuVO.sub.4:Eu; GdVO.sub.4:Eu; Gd.sub.2O.sub.2S:Tb; GdMgB.sub.5O.sub.10:(Ce or Tb); LaOBr:Tb; La.sub.2O.sub.2S:Tb; LaF.sub.3(Nd or Ce); BaYb.sub.2F.sub.8:Eu; NaYF.sub.4:(Yb or Er); NaGdF.sub.4:(Yb or Er); NaLaF.sub.4:(Yb or Er); LaF.sub.3:Yb, Er or Tm); BaYF.sub.5:(Yb or Er); Ga.sub.2O.sub.3:Dy; GaN:A (A=Pr, Eu, Er or Tm); Bi.sub.4Ge.sub.3O.sub.12; LiNbO.sub.3:(Nd or Yb); LiNbO.sub.3:Er; LiCaAlF.sub.6:Ce; LiSrAlF.sub.6:Ce; LiLuF.sub.4:A (A=Pr, Tm, Er or Ce); Li.sub.2B.sub.4O.sub.7:Mn, and SiO.sub.x:(Er or Al) (O.ltoreq.x.ltoreq.2).

57. A method for detecting a target molecule, comprising the steps of: a) providing an assay kit as claimed in claim 1, wherein, in the assay kit, the molecule groups A and B are constituents of one and the same molecule and couple to the same affinity molecule, which is capable of interacting with a specific target molecule, and such an interaction brings about a change in the separation between the molecule groups A and B, b) adding a sample containing the target molecule to the assay kit, c) exciting the assay kit containing the sample with a source of electromagnetic or particulate radiation, and d) measuring the electromagnetic radiation emitted by the assay kit containing the sample, wherein the intensity or the spectrum of the emitted electromagnetic radiation, or the chronological course of the emission of the electromagnetic radiation, is a measure of the quantity of target molecule in the sample.

58. A method for detecting a target molecule, comprising the steps of: a) providing an assay kit as claimed in claim 1, wherein the molecule groups A and B are constituents of different molecules, and the affinity molecules which are assigned to molecule groups A and B are capable of specifically interacting with one and the same target molecule, or the affinity molecules which are assigned to molecule groups A and B are capable of specifically interacting with each other, and in both cases an interaction brings about a change in the separation between molecule groups A and B, b) adding a sample containing the target molecule to the assay kit, c) exciting the assay kit containing the sample with a source of electromagnetic or particulate radiation, and d) measuring the electromagnetic radiation emitted by the assay kit containing the sample, where the intensity or the spectrum of the emitted electromagnetic radiation, or the chronological course of the emission of the electromagnetic radiation, is a measure of the quantity of target molecule in the sample.

Description

This application is a 371 of PCT/EP02/12256, filed Nov. 4, 2002, and claims priority under 35 USC .sctn.119 on the basis of German Application No. 101 53 829.4, filed Nov. 5, 2001.

The present invention relates to a biotechnological assay which is based on a resonance energy transfer (RET) and which can be used to detect biological molecules such as enzymes, antibodies, nucleic acid-binding molecules, nucleic acids, polynucleotides or morpholino.

Immunoassays or nucleic acid detection methods are the basis of many applications in medical diagnosis, in the production control of biotechnologically produced products and in research. One method, which is used here, is that of resonance energy transfer (RET) between dyes.

The principle of resonance energy transfer (RET) is based on radiationless transfer of energy from a donor which is capable of fluorescing to an acceptor which is located in spatial proximity. This technique can be used to determine distances at the molecular level in a range between approx. 1 and 8 nm. The energy which has been transferred to the acceptor can, on the one hand, relax in a radiationless manner by means of internal conversion (RET) and then only leads to the donor fluorescence being quenched. On the other hand, the transferred energy can also be emitted by means of the acceptor fluorescing. This is referred to as fluorescence resonance energy transfer (FRET). These phenomena have been well understood for a long time now and, in the case of a dipole-dipole interaction between donor and acceptor, explained by Forster's theory (see, e.g., J. R. Lakowicz, "Principles of Fluorescence Spectroscopy", Kluwer Academic Press, New York, 1999, pages 368-445). The energy transfer reduces the intensity of the donor fluorescence and its decay time and correspondingly increases the fluorescence of the acceptor or else only excites or sensitizes it for the first time. The efficiency E of the energy transfer is very sensitive to the distance R between the donor and acceptor and declines proportionally to R.sub.0.sup.6/(R.sub.0.sup.6+R.sup.6). The mean range of the energy transfer, at which the efficiency is 50%, is defined by means of a material-specific constant, i.e. the Forster radius R.sub.0, and lies in the range of a few nanometers (less than 10 nm). When the excited state of the acceptor relaxes in a radiationless manner it is only donor fluorescence which suffers anything from a reduction through to complete quenching. In that which follows, the term (F)RET is used when the terms RET and FRET can be used alternately. (F)RET-capable donor/acceptor pairs are termed (F)RET pairs. In that which follows, the terms fluorescence and luminescence are used synonymously.

In biological systems, (FRET) is used to detect the spatial proximity to each other of appropriately labeled biomolecules or molecular groups. This method can be used as a method for detecting protein-protein interactions, e.g. as a method for detecting the antigen-antibody reaction in immune reactions, a receptor-ligand interaction, a nucleic acid hybridization or the binding of proteins to nucleic acids. The detection is itself effected by means of measuring the change in the intensity of, or the spectral change in, the donor fluorescence or acceptor fluorescence or by means of measuring a change in the decay time of the donor fluorescence. A large number of applications in this regard are described in the literature, such as the detection of specific antigens in immunofluorescence assays (U.S. Pat. Nos. 3,996,345; 4,160,016; 4,174,384; 4,199,559), the determination of the electrostatic potential in specific, localized regions on the surface of proteins (Yamamoto et al.; J. Mol. Biol. 241, 1994, pages 714-731), or the method involving high-throughput screening (Boisclair et al.; J. of Biomolecular Screening, 5, 2000, pages 319-328).

(F)RET systems are also used to determine absolute distances between two molecules or within a biomolecule. Two labels, which measurably interact in dependence on their distance from each other, are introduced for this purpose. Known applications of this method are the analysis of protein structure or DNA structure (Heyduk et al.; SPIE Vol. 3256, 1998, pages 218-222), the determination of distances within polypeptides (Lakowicz et al.; Biophys. Chem. 36, 1990, pages 99-115), proteins (K. Cai et al.; J. Biol. Chem. 271 1996, pages 27311-27320), polynucleotides (Hochstrasser et al.; Biophys. Chem. 45, 1992, pages 133-141 and Ozaki et al.; Nucl. Acids Res. 20, 1992, pages 5205-5214) or other macromolecules, the investigation of membranes and membrane proteins and their structure (S. Wang et al.; Biochemistry 27, 1988, pages 2033-2039), and the detection (U.S. Pat. Nos. 4,996,143; 5,532,129; 5,565,332) and quantification of nucleic acids which have been amplified by PCR (polymerase chain reaction) (U.S. Pat. Nos. 5,538,848; 5,723,591), e.g. for in vitro diagnosis, genetic analysis, forensics, foodstuff tests, agricultural product tests or parenthood tests. DNA or RNA is detected or quantified directly, i.e. without any additional separation steps.

The 5'-nuclease assay (U.S. Pat. Nos. 5,538,848; 5,210,015; Holland et al.; Proc. Natl. Acad. Sci USA 88, 1991, pages 7276-7280; Lee et al.; Nucleic Acids Res. 21, 1993, pages 3761-3766) which is termed the TaqMan.RTM. assay (Applied Biosystems Division of Perkin-Elmer Corp., Foster City, USA) is a quantitative nucleic acid determination by means of real-time PCR which uses (F)RET systems. The method of molecular beacons (Tyagi and Kramer, Nature Biotechnology 14, 1996, pages 303-306; U.S. Pat. No. 5,312,728) is based on a similar mechanism.

Organic dye molecules such as fluorescein, cyanine or rhodamine, for example, are classical, commercially available materials for making efficient (F)RET pairs. A general disadvantage of these organic fluorescent dyes is that they frequently exhibit a stability toward incident light which is inadequate for many applications. Particularly in the presence of oxygen or free radicals, some of them can already be irreversibly damaged or destroyed after a few million light absorption/light emission cycles. Furthermore, the fluorescent organic dye molecules can also have a phototoxic effect on biological material in the vicinity. On the one hand, the broad emission bands of the organic fluorescent dyes, with their additional ramifications into the long-wave region of the spectrum, are unfavorable for simultaneously reading several dyes, i.e. what is termed multiplexing. On the other hand, their usually small Stokes shift, i.e. the difference between the excitation maximum and the emission maximum of a dye and the relatively narrow spectral excitation bands within which an excitation is possible, is disadvantageous. As a result, several light sources and/or elaborate filter systems frequently have to be used, thereby additionally restricting the simultaneous reading of several dyes.

It is also possible to use fluorescent proteins as a FRET pair. In this case, the FRET process involved is also termed a bioluminescence resonance energy transfer (BRET). The fluorescent proteins include the green fluorescent protein GFP (U.S. Pat. No. 5,491,084) and its variants which possess other absorption and/or emission maxima, such as the yellow (YFP) or cyan (CFP) fluorescent proteins (U.S. Pat. No. 5,625,048). In this connection, GFPs can either be used as the donor and acceptor or in combination with other fluorophores such as fluorescein or luciferase (review article: Pollok and Heim; Trends Cell Biol. 9, 1999, pages 57-60). A problem is the small selection of different GFP proteins which satisfy the requirements for a suitable FRET pair (sufficiently large difference in the excitation wavelengths, sufficient overlapping of the donor and acceptor emission and excitation wavelengths). Thus, it has to date only been possible to successfully apply two combinations of GFPs as a FRET pair (review article: Pollok and Heim; Trends Cell Biol. 9, 1999, pages 57-60). Even in combination with other dyes or bioluminescent proteins, the small number and markedly different intensities of the GFPs is a limiting factor.

As an alternative to organic dyes, metal chelates or metal complexes are also used for FRET (see, e.g., Selvin; IEEE J. of Selected Topics in Quantum Electronics 2, 1996, pages 1077-1087).

Lanthanide chelates can be employed either as (F)RET pairs (Clark et al., Anal. Biochem. 210, 1993, pages 1 ff.) or as only the donor which transfers the energy to organic fluorescent dyes (Thomas et al.; Proc. Natl. Acad. Sci. 75, 1978, pages 5746 ff; S. G. Jones et al.; Journal of Fluorescence 11, 2001, pages 13-21) or to quenchers (Tanaka et al.; J. Photochem. Photobiol. A 74, 1993, pages 15 ff; Marko et al.; Biochemistry 31, 1992, pages 703 ff.).

Systems or assays which are based on energy transfer and on fluorochromes and chelates having a long lifetime have been disclosed in a number of patents (WO 8 707 955, EP 242 527, EP 439 036, WO 9201225, U.S. Pat. Nos. 4,822,733, 5,279,943, 5,622,821, 5,656,433, 5,998,146, 6,239,271). They use time gated fluorimetry (TGF) and/or time-resolved fluorimetry (TRF) for detecting an analyte.

In this connection, TGF is understood as being a measurement mode in which the excitation is effected using a pulsed light source (laser, photoflash lamp) and, after a defined delay time which then follows, the light emission is measured within a given time window. The delay time is sufficiently long to detect the long-lived fluorescence of the lanthanide chelates with an adequately high intensity. However, the delay time virtually complete discriminates against the short-lived background fluorescence (usually <1 .mu.s) which is elicited by intrinsic autofluorescence of the biological material, impurities in the solvent or surrounding vessel materials. In contrast to the TGF mode, measurements carried out in the TRF mode measure the fluorescence as a function of time at a fixed wavelength. In this connection, the donor is also excited by a pulsed light source or else by a light source which has been modulated in some other way.

However, a disadvantage of the lanthanide chelates or metal complexes is the fact that their chemical stability is low for a number of applications or that their fluorescence properties depend on the chemical environment of the particles. Frequently, additional separation steps, or an additional complex formation, is/are also required in order to be able to measure a fluorescence.

FRET effects have also been observed in the case of particulate label systems which are based on semiconductor nanocrystals, what are termed quantum dots: (Bawendi et al. and C. Kagan et al.; Phys. Rev. Lett. 76, 1996, pages 1517-1520). Quantum dots are also able to interact with organic fluorophores (O. Schmelz et al.; Langmuir 17, 2001, pages 2861-2865).

It is possible to exploit FRET effects between quantum dots themselves or else between quantum dots and other substances (e.g. dyes). WO 00/29617 discloses that it is possible to detect proteins and nucleic acids using quantum dots as labels. In particular, the patent also discloses their use as fluorophores in the case of the hairpin-like DNA structures known as "molecular beacons".

However, a disadvantage of the quantum dots is that they have to be produced with the highest possible degree of precision. Since the emission wavelength of the fluorescent light depends on the size of the quantum dots, it is necessary to achieve a very narrow particle size distribution in a sample. In order to ensure that the fluorescent light is of the narrow band width which is required for the multiplexing, the differences in size between quantum dots of one species can only be a few Angstroms, i.e. amount to only a few monolayers. This places high demands on the synthesis. In addition, due to radiationless electron/hole pair recombinations on their surface, quantum dots normally exhibit relatively weak quantum efficiencies. For this reason, it is necessary to produce core-shell structures (Xiaogang Pent et al.; J. Am. Chem. Soc. 119, 1997, pages 7019-7029), which require a more elaborate synthesis, in order to increase the quantum efficiencies.

Furthermore, in the case of quantum dots, the decay time of the fluorescence is very short and is in the lower nanosecond range. For this reason, it is not possible to make any measurements in the TGF mode and only possible to make measurements in the TRF mode using relatively elaborate technology and equipment. Another disadvantage of the quantum dot systems is their composition, with many of the systems containing toxic elements such as cadmium, selenium or arsenic.

Nano-scale phosphors which are of less than 50 nm in size, and which are designated luminescent inorganic doped nanoparticles (lid nanoparticles) below, have been described many times in scientific publications.

The published lid nanoparticles consist of oxides, sulfides, phosphates or vanadates which are doped with lanthanides or with Mn, Al, Ag or Cu. These lid nanoparticles fluoresce, due to their doping, in a narrow spectral range. The preparation of the following lid nanoparticles has been published, inter alia: LaPO.sub.4:Ce,Th; (K. Riwotzki et al.; Angewandte Chemie, Int. Ed. 40, 2001, pages 573-576); YVO.sub.4:Eu, YVO.sub.4:Sm, YVO.sub.4:Dy (K. Riwotzki, M. Haase; Journal of Physical Chemistry B; Vol. 102, 1998, pages 10129-10135); LaPO.sub.4:Eu, LaPO.sub.4:Ce, LaPO.sub.4:Ce,Tb; (H. Meyssamy, K. Riwotzki, A. Kornowski, S. Naused, M. Haase; Advanced Materials, Vol. 11, Issue 10, 1999, pages 840-844); (K. Riwotzki, H. Meyssamy, A. Kornowski, M. Haase; Journal of Physical Chemistry B Vol. 104, 2000, pages 2824-2828); ZnS:Tb, ZnS:TbF.sub.3, ZnS:Eu, ZnS:EuF.sub.3, (M. Ihara, T. Igarashi, T. Kusunoki, K. Ohno; Society for Information Display, Proceedings 1999, Session 49.3); Y.sub.2O.sub.3:Eu (Q. Li, L. Gao, D. S. Yan; Nanostructured Materials Vol. 8, 1999, pages 825 ff); Y.sub.2SiO.sub.5:Eu (M. Yin, W. Zhang, S. Xia, J. C. Krupa; Journal of Luminescence, Vol. 68, 1996, pages 335 ff.); SiO.sub.2:Dy, SiO.sub.2:Al, (Y. H. Li, C. M. Mo, L. D. Zhang, R. C. Liu, Y. S. Liu; Nanostructured Materials Vol. 11, Issue 3, 1999, pages 307-310); Y.sub.2O.sub.3:Tb (Y. L. Soo, S. W. Huang, Z. H. Ming, Y. H. Kao, G. C. Smith, E. Goldburt, R. Hodel, B. Kulkarni, J. V. D. Veliadis, R. H. Bhargava; Journal of Applied Physics Vol. 83, Issue 10, 1998, pages 5404-5409); CdS:Mn (R. N. Bhargava, D. Gallagher, X. Hong, A. Nurmikko; Physical Review Letters Vol. 72, 1994, pages 416-419); ZnS:Tb (R. H. Bhargava, D. Gallagher, T. Welker; Journal of Luminescence, Vol. 60, 1994, pages 275 ff.). Ullmann's Encyclopedia of Industrial Chemistry, WILEY-VCH, 6.sup.th edition, 2001 Electronic Release, The "Luminescent Materials: 1. Inorganic Phosphors" chapter, provides a review of the known luminescent inorganic doped materials which are of a few micrometers in size, and of their use as industrial phosphors.

In many cases, a (F)RET between a donor (sensitizer) and an acceptor (emitter) is also responsible for the light emission which is elicited in luminous phosphors as are used, for example, for fluorescent lamps (D. Dexter; J. Chem. Phys. 21, 1953, pages 836-850, T. Justel et al.; Angewandte Chemie, International Edition 37, 1998, pages: 3084-3103). Since, however, donor and acceptor are present in a shared crystal lattice in the case of a luminescent phosphor, the (F)RET system of the luminescent phosphors cannot be used for detecting a parameter change resulting from a biochemical process.

U.S. Pat. No. 5,043,265 discloses that it is possible to detect biological macromolecules which are coupled to nanoscale luminescent phosphor particles by measuring the fluorescence. The patent explains that the fluorescence of the particles will rapidly lose its intensity as the diameter becomes smaller and that the particles should therefore be larger than 20 nm and preferably even larger than 100 nm.

U.S. Pat. No. 5,674,698 discloses special types of luminous phosphors for use as biological labels. These luminous phosphors are "upconverting phosphors" which have the property of emitting light, whose wavelength is shorter than that of the absorbed light, by way of a two-photon process. Using these particles makes it possible to work almost free of background since such autofluorescence is to a very large extent suppressed. The particles are prepared by milling and then annealing. The particle size is between 10 nm and 3 .mu.m, preferably between 300 nm and 1 .mu.m. These particles are primarily used in immunoassays (see, for example, Niedbala et al.; Analytical Biochemistry 293, 2001, pages 22-30). One disadvantage of these particles is their broad size distribution resulting from the preparation process. Another is that, in the case of the smaller particles, there are frequently qualitative restraints which result from the preparation and which are reflected in the preferred particle size of 300 nm-1 .mu.m. In general, a higher excitation intensity than in the case of the one-photon process is required for exciting a two-photon process in order to achieve comparable emission intensity.

U.S. Pat. No. 6,159,686 discloses special phosphor/dye complexes for carrying out photophysical catalysis or photodynamic therapy. Upconverting phosphors are initially excited with innocuous infrared light. Energy in the visible light range is then transferred to the dye, which in turn, acting as a catalyst, transfers its energy to a target molecule. It is also possible to detect target analytes using such pairs of upconverting phosphors and suitable dyes. For this, a complex composed of target analyte, phosphor and dye is formed in the presence of the target analyte, with this complex then permitting, as a result of the spatial proximity, an energy transfer from the phosphor to the dye. This patent also discloses the use of these upconverting phosphors, together with a corresponding "matched label" in homogeneous, heterogeneous and competitive assays. In this connection, the phosphors can be used either as donor or as acceptor.

The object according to the invention consists in providing an assay for detecting a biological target molecule, which assay does not suffer from the disadvantages described in the prior art.

The object is achieved, according to the invention, by means of an assay which is based on resonance energy transfer (RET) or on fluorescence resonance energy transfer (FRET) and which contains a first molecule group A, which is labeled with at least one energy donor according to the invention, and at least one second molecule group B, which is in each case labeled with at least one energy acceptor.

Within the meaning of the invention, a donor is understood as being a molecule or particle which is energetically excited, continuously or in a time-modulated manner, by an external radiation source (electromagnetic radiation or particle radiation) and which is capable of fluorescence.

Within the meaning of the invention, an acceptor is understood as being a molecule or particle which is excited by energy transfer by way of the donor, which completely or partly quenches donor fluorescence and which can, but which does not have to, itself be capable of fluorescence. A donor which is not capable of fluorescence relaxes in a radiationless manner.

According to the invention, donor and/or acceptor comprise lid nanoparticles which have a breadth of .ltoreq.50 nanometers and which, after an energetic excitation, emit electromagnetic radiation with a Stokes or Antistokes shift.

The advantage of an assay which is based on lid nanoparticles having a breadth of 50 nanometers or less is that the particles exhibit less potential for steric problems or undesirable sedimentation in an assay than can be the case when using larger particles. In addition, the presence of the lid nanoparticle has less influence on the kinetics of a binding reaction (e.g. immune reaction or DNA hybridization) or of a biochemical process which is to be investigated.

In this, larger lid particles suffer from disadvantages when making measurements in the TRF mode. In the case of a (F)RET, it is only possible for energy to be transferred from, for example, a lid particle, which is acting as donor, to a molecule which is located in spatial proximity, and which is acting as acceptor, within a distance extending a few nanometers. This means that, in the case of relatively large particles, a significant part of the particle volume, and consequently of the doping ions in the particle, is not within range of the acceptor which is located in front of the particle surface and is therefore not involved in the (F)RET. As a result, the effect of the decay time change (TRF mode) caused by a (F)RET is less pronounced or possibly no longer measurable. For the same reasons, a complete, or at least significant, quenching of the donor fluorescence by the acceptor would no longer be possible.

A RET or FRET can be effected by means of a dipole/dipole interaction (Forster transfer), by means of an interaction with involvement of higher multipoles, or by means of the migration of charges or excitons. In the case of a Forster transfer, the spectral overlap between the donor emission and the acceptor absorption must be sufficiently large. The distance between the donor and acceptor can consequently be measured since the efficiency of the energy transfer depends on the distance.

Preference is given to at least one of the two partners (donor or acceptor) being a luminescent inorganic doped nanoparticle having a long fluorescence decay time (>5 ns). The other partner in each case either contains a molecular, organic chromophore or a luminescent inorganic doped nanoparticle which preferably exhibits a shorter fluorescence decay time. In this connection, the lid nanoparticle having a long fluorescence decay time has a halflife of more than 5 ns, preferably between 1 .mu.s and 50 ms, and particularly preferably between 100 .mu.s and 10 ms. In an assay of this design, the donor can be excited with a pulsed light source of suitable wavelength. When the donor/acceptor pair are in appropriate spatial proximity to each other (a few nanometers), a FRET can now take place, i.e. the acceptor, e.g. a molecular chromophore, is sensitized and can release its energy by means of light emission. Since the decay time e.g. of the donor fluorescence is very long, the decay time of the sensitized fluorescence of the acceptor is also very long, on account of the FRET, and consequently very much longer than as a result of the direct excitation of the acceptor by the pulsed light source. When the fluorescence is measured in the TGF mode, it is therefore possible, by masking out the short-lived acceptor fluorescence, to detect the sensitized acceptor fluorescence virtually in the absence of background and consequently with a high degree of sensitivity. By using a light source which is modulated with a suitable frequency, it is also possible to carry out phase-sensitive measurements. The donor can also exhibit short-lived fluorescence and the acceptor exhibit long-lived fluorescence, as can be observed, for example, with the system of doped LaPO.sub.4 nanoparticles. In this case, those nanoparticles which are doped with Cer ions act as donor and those which are doped with terbium ions act as acceptor.

In another embodiment, the donor consists of a lid nanoparticle and the acceptor consists of a conducting material. These materials can be metals, such as gold (Au), silver (Ag) or platinum (Pt), or conducting oxides, such as indium tin oxide (ITO), or conducting polymers. In this connection, they can be present in particulate form as nanoparticles or microparticles, or consist of a planar surface, which can also be structured.

The lid nanoparticles which are used in the assay according to the invention are doped with foreign ions such that the can be excited by narrow-band or broad-band, pulsed, modulated or continuous electromagnetic radiation with wavelengths in the range of infrared light, of visible light, of UV, of X-ray light or of .gamma.-radiation or particle radiation, such as electron radiation, or by a particle beam, and the acceptor can be qualitatively and/or quantitatively detected by time-resolved or continuous measurement of material-specific fluorescent light or its change.

The biochemical reaction is detected by measuring a RET or FRET, i.e. by measuring the change in the luminescence properties (intensity, spectral or by a change in the decay time) of the lid nanoparticles and/or the other chromophores involved. In this way, it is possible to detect, in an assay, the changes in the spacing of the (F)RET partners involved.

The spatial proximity of an acceptor can be detected, in a (F)RET system, from the change in the donor decay time. Because of the presence of another decay channel, due to the transfer of energy to the acceptor, the decay time of the donor fluorescence is significantly shortened. This change can be measured both in the case of the donor fluorescence and in the case of the sensitized acceptor fluorescence (measurement in the TRF mode). The emission decay time, as an observed quantity for FRET, offers an alternative to measuring intensities. It makes a measurement which is independent of concentration effects, quantum efficiency of the chromophore, incomplete labeling and partial or complete quenching of the acceptor fluorescence. Virtually every photon which is detected is a contribution to the useful signal. In the case of a Forster transfer, and when using the mathematical relationships which are known for this purpose, it is also possible to infer the spatial distance between the donor and the acceptor from the decrease in the decay time of the donor fluorescence. An important advantage of using lid nanoparticles is their intrinsically long decay time, which frequently extends into the range of a few milliseconds and can therefore be conveniently recorded using simple experimental means.

The lid nanoparticles have a virtually spherical morphology, with breadths in the range from 1 nm to 50 nm, preferably in the range from 1 nm to less than 20 .mu.m, and particularly preferably in the range from 2 nm to 10 nm. Breadths are understood as meaning the maximum separation of two points lying on the surface of a lid nanoparticle. The lid nanoparticles can also have an ellipsoidal morphology or be faceted, with breadths which lie within the abovementioned limits.

In addition to this, the lid nanoparticles can also exhibit a pronounced needle-shaped morphology, with a breadth of from 3 nm to 50 nm, preferably of from 3 nm to less than 20 nm, and a length of from 20 nm to 5 .mu.m, preferably of from 20 nm to 500 nm. In this case, breadth is understood as meaning the maximum separation of two points which lie on the surface of a needle-shaped lid nanoparticle and, at the same time, in a plane which is perpendicular to the longitudinal axis of the needle-shaped lid nanoparticle. The particle size can be determined by the method of ultracentrifugation or of gel permeation chromatography or by means of electron microscopy.

Materials which are suitable, within the meaning of the invention, for the lid nanoparticles are inorganic nanocrystals whose crystal lattices (host material) are doped with foreign ions. These materials include, in particular, all the materials and material classes which are used as what are termed phosphors, e.g. in luminescent screens (e.g. for cathode-ray tubes) or as coating material in fluorescent lamps (for gas-discharge lamps), as are mentioned, for example, in Ullmann's Encyclopedia of Industrial Chemistry, WILEY-VCH, 6.sup.th edition, 2001 Electronic Release, the "Luminescent Materials: 1. Inorganic Phosphors" chapter, and also the lid nanoparticles which are known from the above-cited prior art. In these materials, the foreign ions serve as activators for the emission of fluorescent light following excitation by UV light, visible light or IR light, X-rays or gamma rays, or electron beams. In the case of some materials, several types of foreign ions are also incorporated into the host lattice in order, on the one hand, to produce activators for the emission and, on the other hand, to make the excitation of the particle system more efficient or in order to adjust the absorption wavelength by shifting it to the wavelength of a given excitation light source (what are termed sensitizers). The incorporation of several types of foreign ions can also be used to select a particular combination of fluorescence bands which are to be emitted by a particle.

The host material of the lid nanoparticles preferably consists of compounds of the type XY. In this connection, X is a cation derived from elements of the main groups 1a, 2a, 3a and 4a, of the subgroups 2b, 3b, 4b, 5b, 6b or 7b, or of the lanthanides, of the periodic system. In some cases, X can also be a combination or mixture of said elements. Y can be a polyatomic anion which contains one or more element(s) of the main groups 3a, 4a or 5a, or of the subgroups 3b, 4b, 5b, 6b, 7b and/or 8b, and also elements of the main groups 6a and/or 7a. However, Y can also be a monoatomic anion from the main group 5a, 6a or 7a of the periodic system. The host material of the lid nanoparticles can also consist of an element from main group 4a of the periodic system. Elements of the main groups 1a and 2a, or from the group containing Al, Cr, Tl, Mn, Ag, Cu, As, Nb, Nd, Ni, Ti, In, Sb, Ga, Si, Pb, Bi, Zn and Co, and/or elements from the lanthanides, can be used as doping. Combinations of two or more of these elements can also be used, in different relative concentrations to each other, as doping material. The concentration of the doping material in the host lattice is between 10.sup.-5 mol % and 50 mol %, preferably between 0.01 mol % and 30 mol %, particularly preferably between 0.1 mol % and 20 mol %. The doping material is selected such that the decay time of the fluorescence which it induces is long (>100 ns).

Sulfides, selenides, sulfoselenides, oxysulfides, borates, aluminates, gallates, silicates, germanates, phosphates, halophosphates, oxides, arsenates, vanadates, niobates, tantalates, sulfates, tungstates, molybdates, alkali metal halides and also other halides, or nitrides, are preferably used as host materials for the lid nanoparticles. Examples of these material classes are cited, together with the corresponding dopings, in the following list (materials of the type B:A, with B=host material and A=doping material):

LiI:Eu; NaI:Tl; CsI:Tl; CsI:Na; Lif:Mg; LiF:Mg,Ti; LiF:Mg,Na; KMgF.sub.3:Mn; Al.sub.2O.sub.3:Eu; BaFCl:Eu; BaFCl:Sm; BaFBr:Eu; BaFCl.sub.0.5Br.sub.0.5:Sm; BaY.sub.2F