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WO1996041177A9 - Procede de dosage simultane au moyen de composes chelates de lanthanides utilises comme luminophores pour marqueurs multiples - Google Patents

Procede de dosage simultane au moyen de composes chelates de lanthanides utilises comme luminophores pour marqueurs multiples

Info

Publication number
WO1996041177A9
WO1996041177A9 PCT/US1996/009870 US9609870W WO9641177A9 WO 1996041177 A9 WO1996041177 A9 WO 1996041177A9 US 9609870 W US9609870 W US 9609870W WO 9641177 A9 WO9641177 A9 WO 9641177A9
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WIPO (PCT)
Prior art keywords
lanthanide
ecl
coreactant
persulfate
ligand
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Ceased
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PCT/US1996/009870
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English (en)
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WO1996041177A1 (fr
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Priority to AU64766/96A priority Critical patent/AU6476696A/en
Publication of WO1996041177A1 publication Critical patent/WO1996041177A1/fr
Publication of WO1996041177A9 publication Critical patent/WO1996041177A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Definitions

  • the present invention relates to electrochemiluminescence (ECL) detection methods.
  • the invention has to do with a system using multiple ECL labels for simultaneous assays.
  • Emission intensity is also dependent upon the extent of non-radiative deactivation of the lanthanide excited state by solvent interactions. Therefore, the more luminescent lanthanide complexes are composed of heteroaromatic ligands which encapsulate the metal.
  • the photophysical properties of encapsulated lanthanides have recently been reviewed.
  • a cathodic electroluminescence technique using lanthanides has been described by Kankare (for example see Anal. Chim. Acta 266, 205, 1992, Anal. Chim. Acta 256, 17, 1992).
  • Cathodic electroluminescence is a different way of exciting than ECL techniques.
  • a common method of exciting lanthanide chelates is time resolved fluorescence; for example see I. Hemmilä, S. Dakubu, V.M. Mukkala, H. Siitari, T. Lovgren, Anal. Biochem. 137, 335-343 (1984).
  • An object of the invention was to develop an ECL detection method for simultaneous assays and we have developed a method which employs an excitation process for certain luminophores; particularly the lanthanides.
  • a new excitation method for the lanthanides was needed because previous methods do not provide satisfactory intensities for simultaneous assays.
  • the ECL emissions may be separated either by measuring at different emission wavelengths or by electrode potentials.
  • ECL can be obtained from various lanthanide complexes and with different ligands.
  • the level of the ECL signal for the Tb(2) 3+ complex is high enough to yield the lowest detection limit observed to date.
  • the ECL of lanthanides is achieved by "Antenna ECL".
  • This process consists of the generation of a ligand excited state via the reaction of a reduced ligand with an oxidizing radical, the “electrochemical excitation” (ECX) steps. Energy transfer from the ligand excited state to the emissive state (i.e., lanthanide excited state) yields the characteristic lanthanide emission.
  • ECX electrochemical excitation
  • Shown below are the reactions involved in the Antenna ECL process. In these reactions L represents the generic ligand, La represents a generic lanthanide metal, and an example of a coreactant which produces an oxidizing radical is presented, peroxydisulfate.
  • La represents lanthanide metal
  • L represents ligand
  • L represents a reduced ligand
  • L* represents a ligand excited state
  • La* represents a lanthanide excited state.
  • This feature allows the coupling of a species (such as the antenna or ligand) which is efficient at ECX and/or with the desired reduction potential (or peak potential) to another species (such as the lanthanide metal) with the desired excited state properties (i.e., highly emissive, appropriate wavelength of emission, or non-emissive).
  • a species such as the antenna or ligand
  • the desired reduction potential or peak potential
  • another species such as the lanthanide metal
  • the desired excited state properties i.e., highly emissive, appropriate wavelength of emission, or non-emissive
  • the first step of the Antenna ECL process involves a ligand reduction.
  • Tb(1) 3+ and Tb(2) 3+ Altering the ligand structure changes the reduction potential and correspondingly, the electrode potential for onset of ECL (excitation potential).
  • the emission spectra is unchanged because it is solely related to the type a metal lanthanide present in the chelate. This feature is illustrated by Tb(1) 3+ and Tb(2) 3+ .
  • ORIGEN® Analyzer available from IGEN, Inc., 16020 Industrial Drive, Gaithersburg, MD 20877 U.S.A.
  • the peak potential observed for Tb(1) 3+ was ca. -3 V while that for Tb(2) 3+ was approximately (sometimes abbreviated herein as "ca.") -5 V; however, both complexes have the
  • ECL from a Dy 3+ and Sm 3+ complex was first observed. Additionally, we observed for the first time a lanthanide ECL with a non-oscillating potential.
  • the lanthanide luminophores of the invention are used in a similar manner as with Ru(bpy) 3 2+ .
  • Linker arms for attachment to various biological molecules such as antibodies are used and sandwich assays for the analytes can be carried out in the usual manner.
  • Figure 1 illustrates the ligand structures 1 and 2.
  • Figure 2 is a cyclic voltammogram of Dy(2) 3+ in an acetonitrile solution 0.1 molar ("M”) in tetrabutylammonium perchlorate (“TBAP”) and millimolar (“mM”) in La 3 + recorded on platinum electrodes in volts ("V”) vs. 3M Ag/AgCl.
  • M acetonitrile solution 0.1 molar
  • TBAP tetrabutylammonium perchlorate
  • mM millimolar
  • Figure 3 is a cyclic voltammogram of Eu(2) 3+ using the same type of solution and electrodes as Figure 2.
  • Figure 4 is a graph of corrected ECL vs. potential (millivolts, abbreviated "mV”) for aqueous La(1) 3+ using Ramp ECL (wherein the potential was continuously increased at a rate of 4800 mV/s) with 1000 nanomolar ("nM”) Tb(1) 3+ and 10,000 nM Eu(1) 3+ .
  • mV millivolts
  • Figure 5 is a graph of corrected ECL counts vs. time (centiseconds, abbreviated
  • Figure 6 is a calibration curve for aqueous La(1) 3+ .
  • Figure 7 is a graph of corrected ECL counts vs. potential (mV) for aqueous La(2) 3+ using Ramp ECL.
  • Figure 8 is a graph of corrected ECL counts vs. time (cs) for aqueous La(2) 3+ using ECL Step (-5V).
  • Figure 9 is a calibration curve for aqueous La(2) 3+ .
  • the four lanthanide complexes Tb(2) 3+ , Dy(2) 3+ , Sm(2) 3+ , and Eu(1) 3+ ; can be quantified by reductive ECL in 0.1 M phosphate buffer (pH 6) which is 0.1% in surfactant and 50 micromolar (" M") in potassium persul fate.
  • a modified ORIGEN Analyzer was used for these analyses. The Analyzer was equipped with gold electrodes and a filter wheel running at ca. 10 hertz ("Hz") which had four narrow-band interference filters (613 nanometers ("nm”) for Eu 3+ , 545 nm for Tb 3+ , 644 nm for Sm 3+ , and 573 nm for Dy 3+ ).
  • the filter wheel was placed between the electrode and the photomultiplier tube ("PMT") (PMT model R1104 available from Hamamatsu, 360 Foothill Road, P.O. Box 6910, Bridgewater, NJ 08807 U.S.A.).
  • PMT photomultiplier tube
  • a potential step of -5 V for 10 seconds (“s") was applied to generate the ECL from these complexes. Since the ECL from these complexes does not totally decay over 10 s, this long of a pulse can be utilized for integration of the signals at each wavelength. Fluorescence discrimination of these lanthanides has been described by Y.-Y. Xu and I.A. Hemmila, Analytica Chimica Acta, 1992, 256, 9.
  • lanthanide complexes Based upon the highly luminescent properties of encapsulated lanthanides (La) and the narrowness of the emission bands (ca. 50 nm), six lanthanide complexes have been prepared with Eu 3+ , Tb 3+ , Dy 3+ , Sm 3+ , and two bipryidine based ligands, 1 and 2 (see Figure 1).
  • the complexes prepared were, Eu(1) 3+ , Eu(2) 3+ , Tb(1) 3+ , Tb(2) 3+ , Sm(2) 3+ , and Dy(2) 3+ .
  • the ECL from these complexes was evaluated using a persulfate system.
  • a 100 ⁇ L aliquot of the 2.00 mM solution was diluted to 100 mL with water to make a 2.00 ⁇ M solution; a 10 mL aliquot of the 2.00 ⁇ M solution was diluted to 100 mL with water to make a 0.200 ⁇ M solution.
  • 12.5 mg of [Sm(2)]Cl 3 was dissolved to make a 2.00 mM solution.
  • a 100 ⁇ L aliquot of the 2.00 mM solution was diluted to 100 mL with water to make a 2.00 ⁇ M solution; 10mL of the 2.00 ⁇ M solution was diluted to 100 mL with water to make a 0.200 ⁇ M solution.
  • UV-visible spectra were recorded with a HP 8452A Diode
  • Array spectrophotometer (available from Hewlett Packard Company, 2101 Gaither Road, Rockville, MD 20850 U.S.A.). A 100 ⁇ L aliquot of each mM aqueous solutions was diluted with 5.61 mL of water to make the following aqueous solutions: 37.8 ⁇ M Tb(2) 3+ and 35.0 ⁇ M of Eu(2) 3+ , Sm(2) 3+ , and Dy(2) 3+ . UV-visible spectra (180-820 nm) were measured for each of the ca. 35 ⁇ M aqueous La(2) solutions.
  • Potentiostat/Galvanostat (available from EG&G Princeton Applied Research, P.O. Box 2565, Princeton, NJ 08543 U.S.A.) controlled by a EG&G PARC 175 Universal
  • [La(2)](PF 6 ) 3 complexes were measured from 2.5 V to -2.3 V.
  • the complex concentrations were 2.3 mM (Tb 3+ ), 2.1 mM (Dy 3+ ), 1.9 mM (Sm 3+ ), and 2.6 mM (Eu 3+ ).
  • a 50 ⁇ M persulfate buffer solution was prepared from 10.2 mg of potassium persulfate (available from Aldrich, 1001 West Saint Paul Ave., Milwaukee, WI 53233 U.S.A.) and 750 mL of a 0.1 M phosphate buffer (pH 6.1-6.0) solution that was 0.1% in Triton X100 (available from Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178 U.S.A.). Aliquots of the Eu(1) 3+ aqueous stock, 151 ⁇ L and 15.1 ⁇ L, were diluted with 10 mL of the persulfate buffer to make 10 ⁇ M and 1 ⁇ M Eu(1) 3+ persulfate buffer solutions.
  • a 100 ⁇ L aliquot of the 10 ⁇ M Eu(1) 3+ persulfate buffer solution was diluted with 10 mL of persulfate buffer to make a 100 nM Eu(1) 3+ persulfate buffer solution.
  • the instrument program to measure ECL was used with a ramp to -5 V and a rate of 4800 mV/s. 800 V was applied to the PMT.
  • the following solutions were analyzed:
  • persulfate buffer 100 nM Eu(1) 3+ , 1 ⁇ M Eu( 1) 3+ , 10 ⁇ M Eu(1) 3+ , 10 nM Tb(1) 3+ , 100 nM Tb(1) 3+ , 1 ⁇ M Tb(1) 3+ , and 10 ⁇ M Tb(1) 3+ .
  • step analyses were performed with a step to -5 V and a pulse width of 10 s on persulfate buffer, 100 nM Eu(1) 3+ , 1 ⁇ M Eu (1) 3+ , 10 ⁇ M Eu(1) 3+ , 10 nM Tb(1) 3+ , 100 nM Tb(1) 3+ , 1 ⁇ M Tb(1) 3+ , and 10 ⁇ M Tb(1) 3+ .
  • 800 V was applied to the PMT.
  • the ECL from the La(2) 3+ complexes was studied with a ramp to -5 V at a rate of 4800m V/s as well as with a step potential to -5 V for 1 s.
  • 800 V were applied to the PMT.
  • a 50 ⁇ M persulfate buffer solution was prepared as above by dissolving solid K 2 S 2 O 8 in a 0.1 M phosphate buffer (pH 6) which was 0.1% in Triton X100.
  • a second set of Sm(2) 3+ - persulfate buffer solutions were prepared by diluting with 10 mL of persulfate buffer a 500 ⁇ L aliquot of the 2.0 ⁇ M aqueous solution, 5 ⁇ L and 50 ⁇ L aliquots of the 2.0 mM aqueous solution, and 250 ⁇ L and 500 ⁇ L aliquots of the 10000 nM Sm(2) 3+ - persulfate buffer solution to make 100 nM, 250 nM, 500 nM, 1000 nM, and 10000 nM Sm(2) 3+ - persulfate buffer solutions. Both sets of Sm(2) 3+ - persulfate buffer solutions were analyzed in triplicate with three tubes of persulfate buffer by ECL with a potential ramp and potential step.
  • (Tb) is the reported quantum efficiency for Tb(2) 3+ , 0.37 ⁇ 0.13
  • area represents the area under the emission bands and is estimated by the weight of the paper within the emission bands
  • Scale is the instrument scaling factor.
  • Each weight used in the calculation was an average weight from four separate emission spectra.
  • the relative quantum efficiencies are reported in Table I. Also included are the deviations which are based upon the reported deviation in the Tb(2) 3+ quantum efficiency and the standard deviations in each average weight. The largest portion of the listed quantum efficiencies was due to the reported 30% deviation in the quantum efficiency for Tb(2) 3+ .
  • the quantum efficiency for the Sm(2) 3 + complex is expected to be higher than reported in Table I since the largest emission band for this complex was obscured by the second harmonic of the excitation wavelength.
  • the quantum efficiency measurements will be repeated with a band ⁇ pass filter between the source and the sample to eliminate the second harmonic of the excitation wavelength.
  • the final one electron reductive wave is being assigned as the second reduction of one bpy arm, the other second reductions are presumed to be just beyond the potential window.
  • the cyclic voltammogram of Eu(2) 3+ is found in Figure 3 and shows a one electron reduction at -0.28 V and a set of three-one electron reductions between -1.7 V and -2.1 V.
  • the first reduction is assigned as the Eu III/II couple based upon previously reported Eu III/II couple. Alpha, B.; Lehn, J.-M.; Methis, G., Angew. Chem. Int. Ed. Engl. 1987, 26, 266.
  • the set of three reductions are assigned as the first reductions of each bpy arm.
  • ECL vs. potential curves for the Tb(2) 3+ , Sm(2) 3+ , and Dy(2) 3+ complexes are shown in Figure 7. The curves are similar for these complexes.
  • the ECL commences at ca. -2.2 V and increases until the edge of the potential window (-5 V) is reached. The peak potentials for these complexes are -5 V and are presumably associated with the ligand reduction.
  • a potential step-ECL decay curve for each of these complexes is shown in Figure 8.
  • the curves for the Tb(2) 3+ and Sm(2) 3+ complexes show that the ECL is noisy but constant over the 1 s pulse width; however, the ECL of Dy(2) 3+ decays over the pulse width.
  • Equation (6) La(L*) 3+ La*(L) 3+
  • ECX electrochemical excitation
  • this excitation mechanism can be a disadvantage for the use of lanthanides in immunoassays.
  • fewer biologically relevant species and the media will emit via ECX. If ECX and Antenna ECL are used to produce the lanthanide emission one would expect to have an assay with fewer interferences.
  • the ECL efficiency is defined as the moles of photons per 2 moles of electrons (2 electrons are needed to produce one reduced labels and one reduced persulfate).
  • the quantum efficiency is defined as the moles of photons per mole of excited states.

Abstract

L'invention concerne un procédé de détection par électrochimioluminescence (ECL), permettant d'effectuer plusieurs dosages en même temps. Un ou plusieurs luminophores sont excités au moyen d'un mécanisme d'antenne qui utilise un coréactif oxydant ou réducteur. Les luminophores peuvent être des composés chélatés de lanthanides, et un système persulfaté est utilisé pour générer l'électrochimioluminescence. Les émissions électrochimioluminescentes peuvent être séparées soit au moyen de mesures effectuées à différentes longueurs d'ondes d'émission soit au moyen d'électrodes de différents potentiels.
PCT/US1996/009870 1995-06-07 1996-06-06 Procede de dosage simultane au moyen de composes chelates de lanthanides utilises comme luminophores pour marqueurs multiples Ceased WO1996041177A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64766/96A AU6476696A (en) 1995-06-07 1996-06-06 Simultaneous assay method using lanthanide chelates as the l uminophore for multiple labels

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US48571595A 1995-06-07 1995-06-07
US08/485,715 1995-06-07

Publications (2)

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WO1996041177A9 true WO1996041177A9 (fr) 1997-02-06

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DE69902265T2 (de) * 1998-06-01 2003-03-27 Roche Diagnostics Corp., Indianapolis Redox-reversible imidazol-osmiumkomplex-konjugate
CN1055287C (zh) * 1998-11-27 2000-08-09 华东理工大学 双官能团穴状稀土铕络合物
EP1141404A1 (fr) * 1998-12-24 2001-10-10 Aclara BioSciences, Inc. Surfaces solides adressables individuellement pour operations multiplexees
US6136268A (en) * 1999-08-17 2000-10-24 Orion Diagnostica Method for luminescence measurements
GB0004852D0 (en) 2000-02-29 2000-04-19 Unilever Plc Ligand and complex for catalytically bleaching a substrate
WO2005113563A1 (fr) * 2004-05-19 2005-12-01 Merck Patent Gmbh Complexes metalliques
DE102008006610A1 (de) * 2008-01-29 2009-07-30 Universität Leipzig Verfahren zum sensitiven Nachweis von Polyaminosäuren und anderen Makromolekülen
WO2011154590A1 (fr) * 2010-06-11 2011-12-15 Sakari Kumala Puces intégrées à électrode de carbone pour l'excitation électrique de chélates de lanthanides, et procédés analytiques mettant en œuvre lesdites puces
US9213043B2 (en) 2012-05-15 2015-12-15 Wellstat Diagnostics, Llc Clinical diagnostic system including instrument and cartridge
US9625465B2 (en) 2012-05-15 2017-04-18 Defined Diagnostics, Llc Clinical diagnostic systems
US9075042B2 (en) 2012-05-15 2015-07-07 Wellstat Diagnostics, Llc Diagnostic systems and cartridges
TW202227813A (zh) * 2020-08-21 2022-07-16 美商梅梭刻度技術公司 輔助電極以及其使用及製造方法

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US5310687A (en) * 1984-10-31 1994-05-10 Igen, Inc. Luminescent metal chelate labels and means for detection
US5308754A (en) * 1988-03-21 1994-05-03 Kankare Jouko J Electrogenerated luminescence in solution

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