WO1996021154A1

WO1996021154A1 - Electrogenerated chemiluminescence through enhanced particle luminescence

Info

Publication number: WO1996021154A1
Application number: PCT/US1996/000493
Authority: WO
Inventors: David Talley
Original assignee: Igen, Inc.
Priority date: 1995-01-06
Filing date: 1996-01-11
Publication date: 1996-07-11
Also published as: AU4756296A

Abstract

An emission enhancing particulate support is disclosed where the optical properties of the support and the emissive characteristics of a metal luminescent tag are matched to provide enhanced luminescence in electrogenerated chemiluminescent assays.

Description

Title of the Invention

ELECTROGENERATED CHEMILUMINESCENCE THROUGH ENHANCED PARTICLE LUMINESCENCE Field Of Invention

The present invention relates to luminescent metal tagged supports and their use in assay, sequencing and separation

applications. In particular, the present invention relates to a solid phase support that provides reproducible surface presentation with enhanced light emission characteristics as a result of optical matching with a luminescent tag and used in immunoassays and DNA probe assays via electrogenerated chemiluminescent techniques.

Background Of Invention

Diagnostic techniques such as immunoassays, DNA probe assays and, fluorescent and separation assays frequently involve a solid phase support or substrate. The supports, often in particulate form, provide sites for

antibodies, antigens and probes, that may or may not be tagged. The supports maybe be inorganic, such as siliceous based glasses, or they may be polymeric in composition, such as polystyrene.

The solid phase support may contain metallic particles, such as iron oxide (Fe₂O₃), that are coated with a material that provides appropriate sites for adhering the tagged component that aids in determining the analyte of interest. For example, polymeric or

siliceous coatings will be applied. Instead of a coating, a particulate polymeric or siliceous material will incorporate Fe₂O₃ to provide the magnetic properties. The magnetic content allows the support to be more easily

concentrated and /or separated. Porous bodies of magnetic glass or crystals containing

magnetic crystals are also known. The glass materials serve as a site for immobilizing an antibody, antigen, protein or probe etc. The magnetic properties allow the particles to be used in assays or reactors and be easily

separated therein. See for example U.S. Patent Nos. 3,042,543 to Schuele, U.S. Patent No.

4,233,169 to Beall et al., 4,631,211 to Houghten, 4,812,512 to Buendia et al., 5,037,882 to Steel, 5,141,813 to Nelson and 5,128,204 to Charmot.

In PCT Application WO 93/10162 to Wong, magnetic porous inorganic supports are prepared for use in chromatography,

immunoassays, synthesis, separation and

purification techniques. The porous material of Wong has a particle size on the order of 1 to about 200 μ with a specific pore size. A variety of moiety components such as enzymes, antibodies, antigens, peptides, nucleotides, saccharides or cells are bonded to the support surface.

it is also known that enzyme, antibody, antigen, peptide, nucleotides,

saccharide or cell complements of the analyte of interest can be tagged with conventional tagging agents such as radioactive, colorimetric and/or luminescing agents. Of particular interest are labels which can be made to luminesce through photochemical, chemical, or electro-chemical reaction schemes. In particular, electro-chemiluminescent methods of determining the presence of labelled materials are of interest for the insensitive, nonhazardous and

inexpensive properties of the technique. Present electrochemiluminescent methods use organic compounds and metal

chelates. For example, Leland et al. in J. of Electrochemical Society, Vol. 137, No. 10, October 1990 report an ECL reaction between amines, such as, tripropylamine and Ru(bpy)₃ ²⁺ (bpy 2,2'bipyridine). It is proposed that upon electrochemical oxidation of both a luminophore and coreactant amine a strong emission is observed. Use of Os(bpy)₃ ²⁺ is also taught by Leland et al. Electrochemiluminescent

ruthenium- and osmium-containing labels have been used for detecting and quantifying analytes of interest in liquid media (U.S. Patent Nos. 5,310,687; 5,238,808; and 5,221,605). In addition, electrogenerated chemiluminescent (ECL) measurements to detect solution phase DNA intercalated with ruthenium-containing labels have been described (Carter, M.T. et al. (1990) Bioconiugate Chem 2:257-263). However,

detection of solution phase analytes is

diffusion controlled and has several drawbacks relative to detection of analytes on solid surfaces. Some of the advantages for solid phase techniques as opposed to solution

techniques are: (1) greater sensitivity

(detection of monolayer quantities); and (2) easier support separation. These luminescent systems are of increasing importance in

diagnostics.

In U.S. Pat. No. 4,372,745, chemiluminescent labels are used in

immunochemical applications where the labels are excited into a luminescent state by reaction of the label with H₂O₂ and an oxalate. However, though the system offers versatility, it lacks selectivity or specificity because typical biological fluids containing the analyte of interest also contain a large number of

potentially luminescent substances that can cause high background levels of luminescence.

Thus, a need exists for a solid phase system and method that (1) avoids the drawbacks of a diffusion controlled system; (2) is

selective; (3) provides a light transmission enhancing support; and (4) provides for

reproducible contact with an electrode. The present invention overcomes the limitations and drawbacks of the prior art.

Summary of Invention

The present invention enhances the emission characteristics of solid phase

particles that are used in immuno and DNA probe assays and other applications. The solid phase particles of glass or polystyrene of the present invention have a specified geometry to provide reproducible presentation of the particles to an electrode surface where they are concentrated. In addition, light transmitting characteristics of the particles are matched with the emission characteristics of an appropriate tag to enhance the transmission characteristics of the particle and therefore maximize the light transmission to the photodetector. The solid phase particles contain a covering or layer of a ruthenium- or osmium-containing chemiluminescent labeled ligand component on the surface thereof that will complex with an analyte of interest. The degree of coating is such that the particle surface is not completely saturated with the ligand-tag component. The light generated during electrogenerated chemiluminescent

reaction is detected to determine the analyte of interest.

Thus, an object of the present

invention is improve light emission

characteristics of solid phase-particulate supports for use in immuno and DNA probe assays.

An aspect of the present invention is to provide a complexible solid phase support formed from an optically suitable substrate with an exterior layer of a ruthenium or osmium containing label. The ruthenium or osmium label is reacted with a coreactant in the presence of an electrode to generate a detectable emission. The optically suitable substrate provides reproducible presentation of the substrate to an electrode surface to facilitate emission.

A further aspect of the present invention is to provide a solid phase support that enhances luminescence in ECL environments via surface geometry and particle transparency.

An object of the present invention is to detect an analyte of interest by providing a solid phase particle support containing a layer of bound luminescent metal label. The label complexes an analyte of interest and is

attracted to or collected on an electrode and thereafter reacted with a coreactant via

electron transfer from the electrode to emit a detectable emission.

These and other objects will become more apparent from the following detailed description and drawings. Brief Description Of The Drawings

Fig. 1 shows a tagged particle of the present invention in contact with an electrode. Fig. 2 shows a tagged particle that lacks surface geometry for reproducible presentation. Fig. 3 shows another embodiment of the present invention with a tagged particle providing reproducible presentation. Fig. 4 shows an analysis system for gravity separation.

Fig. 5 shows a flow cell for use with magnetically separable particles according to the present.

Fig. 6 shows the electrode

arrangement for ECL

analysis. Detailed Description Of Invention

The present invention provides enhanced particle luminescence by designing a particle with (1) geometry that provides reproducible presentation of the particle surface to an electrode; and (2) optical properties matched with the luminescent tag. By optical properties is meant that the particle must be transparent to facilitate light emitted light during the electrochemiluminescent

reaction and be matched to the emissive

characteristics of the tag. It was unexpected that matching the optical properties of the solid phase support with the emissive

characteristics of the luminescent tag would provide the results obtained.

The preferred solid phase particulate support of the present invention includes soda lime glass or polystyrene beads, with or without magnetic particles. The particle geometry must present a reproducible surface to an electrode. Fibrous or particulate material that maximizes surface contact is preferred, including but not limited to particles that are spherical,

ellipsoid, oval in shape can also be used.

Solid phase particles that are irregular in shape or are aggregates do not provide acceptable presentation and do not enhance the emission and luminescence of the reaction.

Also, uniformity in shape provides for improve concentration.

With reference to figure 1, a solid phase support particle 3 formed from polystyrene containing Fe₂O₃, or soda lime glass, can be collected on electrode 1 by a magnet (not shown) or by gravity. Particles containing Fe₃O₄ are also contemplated. The polystyrene beads are

Dynabeads™ M-450, available from Dynal A.S., and are uncoated, uniform and magnetic with -OH resistance. They have a mean diameter of 4.5 μm (C.V. max 5%); s.g. = 1.5; magnetic

susceptibility - 10^-2 cgs units; and surface area of about 3-5 m²/g. Uniform glass microspheres, available from Duke Scientific Corporation are available in sizes of 1.5 to 40μ. The exterior surface of the bead contacts surface 2 of the electrode 1 at contact point 6. The particle 3 contains a plurality of ligand containing luminescent metal tags 4, although only one is shown for clarity. The size and surface

geometry of particle 3 permits electrons from electrode 2 to flow through the bead to provide the necessary electron transfer, i.e.,

activation for the reaction. In figure 2, an irregular shaped particle 7 has point contacts 8 that contact surface 2 of electrode 1. The point contacts do not necessarily provide the same degree of particle surface exposure as that of a spherical particle or bead. In addition, the irregularly shaped solid phase support particles precludes reproducible presentation of its surface to the electrode 1.

Figure 3 is an alternative embodiment where the bead 9 is elliptical in shape and provides reproducible presentation of the surface, though to a lesser degree than a spherical bead.

The size of the particulate support is also important because of the effective field distance of the electrode. It is believed that the electron transfer from the electrode to the support is most effective at a distance of up to about 100Å from the surface of the electrode.

The mean diameter of the particles suitable for the present invention between about 0.25 to about 10μ, preferably about 1 to 4.5μ. Particle size is one of the many factors that contribute to the luminescent enhancing support of the present invention. The metal luminescent tag of the present invention is preferably a ruthenium or osmium containing compound and may have

polydentate ligands or one or more monodentate ligands. A wide variety of these ligands are known to the art. Polydentate ligands of either ruthenium or osmium include aromatic and

aliphatic ligands. Suitable aromatic

polydentate ligands include aromatic

heterocyclic ligands. Preferred aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl. If the metal chelate has greater than one polydentate ligand, the polydentate ligands may be the same or different.

Suitable polydentate ligands may be unsubstituted, or substituted by any of a large number of substituents known to the art.

Suitable substituents include for example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing groups, phosphorous containing groups, and the carboxylate ester of N-hydroxysuccinimide. Suitable monodentate ligands include, for example, carbon monoxide, cyanides,

isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines. A more complete list of the ligands, e.g., monodentate and polydentate ligands, and methods of preparing that can be used in the present invention are set forth in U.S. Patent Nos. 5,310,687, 5,238,808 and

5,221,605, the subject matter of which are incorporated herein by reference. A preferred metal luminescent compound is 4-methyl-4-N-succinimdyloxycarbonylpropyl-2,2-bipyridine Ru(II) hexafluorophosphate.

According to the present invention, the metal label is excited by exposing the solid phase supported metal label chelate to

electrochemical energy. The potential at which the oxidation of the metal label will occur depends upon the exact structure of the metal label as well as factors such as the co-reactant utilized, the pH of the solution and the nature of the electrode used. Examples of suitable co-reactants which, when incubated with the solid phase supported metal label chelate in the presence of the electrochemical energy, will result in emission, include tripropylamine (TA), oxalate or other organic acid such as pyruvate, lactate, malonate, tartrate and citrate. This oxidation can also be performed chemically, with some strong oxidants such as PbO₂ or a Ce(IV) salt.

Those of ordinary skill in the art recognize how to determine the optimal potential and emission wave length of an

electrochemiluminescent system. The

electrochemiluminescent species may be measured by emitted electromagnetic radiation. For example, the rate of energy inputted into the system can provide a measure of the luminescent species. The measurements can be made either as continuous, rate-based measurements, or as cumulative methods which accumulate the signal over a long period of time. An example of rate-based measurements would be by using

photomultiplier tubes, photodiodes or

phototransistors to produce electric currents proportional in magnitude to the incident light intensity. Examples of cumulative methods are the integration of rate-based data, and the use of photographic film to provide cumulative data directly.

All of these luminescence-based methods entail repeated luminescence by the ruthenium-containing compound. The repetitive nature of the detectable event distinguishes these labels from radioactive isotopes or bound chemiluminescent molecules such as luminol. The latter labels produce a detectable event only once per molecule (or atom) of label, thereby limiting their detectability.

Figures 4 and 5 exemplify systems that are suitable for electrogenerated

chemiluminescence. In figure 4, gravity

separation is relied upon. In the figure, an open vessel 21 contains an electrode 24 with a surface 25 upon which a metal tag containing luminescent support 23 will be collected or concentrated will rest. The bottom of the vessel may be sloped (not shown) to facilitate collection of the tagged supports 23 in solution 22 on the electrode surface 25. If supports 23 contain Fe₂O₃, a magnet 26 may be used.

In figure 5, a solution containing a metal luminescent tagged support 37, or a sandwiched support 39 formed from the reaction of, for example, an antibody-antigen pair, is fed through tube 32 of flow cell 31. The supports 37 and 39 are attracted to electrode 35 and maintained in position by magnet 34. Energy from electrode 35 across the surface 36 to the main support provides the energy for the reaction. If the device of figure 5 is used for gravity separation, the electrode 35 is located at bottom wall 38 (shown in broken lines).

The following examples illustrate various aspects of the invention but are in no way intended to limit the scope of the

invention.

Example A

45 μl of a DMSO solution of 4-methyl- 4-N-succinimdyloxy-carbonylpropyl-2,2-bipyridine Ru(II) hexafluorophosphate (0.5 mg in 62.5 μl) was added to a stirred solution of murine IgG in aqueous Physiologic Buffered Saline (182 μl of 1 mg/ml IgG to 318 μl PBS). The solution was incubated on a rotator at room temperature for approximately 30 minutes. The coupling reaction was stopped by adding 25 μl of a 2M glycine solution. The metal labelled antibody was separated from unreacted antibody and

derivitized ruthenium using a Sephadex G-25 column. The labelled antibody product was analyzed spectrophotometrically. Example B

To determine a degree of coating that does not saturate the beads surface area, Dynal particles (4.5 μm) were coated with different amounts of labelled antibody from Example A. Four 30 mg bead aliquots were washed with PBS and subsequently mixed in four separate tubes containing 530, 880, 952, and 988 μl of PBS containing 0.005% thimerosal. To their

respective bead mixtures, 470, 120, 48, and 12 μl of 500 μg/ml labelled antibody (Example A) was then added, and each mixture allowed to rotate over 1 hour. The beads were then

separated from their solutions using a magnet. The supernatant was removed and saved for electrochemiluminescence analysis. 1 ml of PBS containing 3% BSA was then added to the beads which were allowed to rotate for 1 hour to block any remaining unoccupied areas on the particles. This washing and separation process was repeated once more, and afterwards the beads were stored in PBS with 3% BSA.

Example C

Analysis of the

electrochemiluminesence generated from the labelled beads (Example B) was done using a ORIGEN Analyzer and reagents (Igen,

Incorporated) or could be performed in the systems described in figure 4 and 5 above. The analyzer first pumps the bead sample (150 μg/500μl) to a flow cell containing a gold working electrode, counter electrode, and a Ag/AgCl reference electrode shown in Figure 6. The beads are then allowed to settle (by

gravity) to the electrode surface where

electrochemical excitation is performed. The settling time is varied depending on the density of the beads and the distance it must travel to reach the electrode - 370 μ being the longest distance. Once the desired settling time is done, the excitation takes place with the subsequent generation of light. A

photomultiplier tube converts the light energy to electrical energy that is processed by a luminometer. Once the light has been collected and analyzed, the existing sample is washed away and the flow cell is cleaned and conditioned in preparation for the next sample of beads to be measured. Example 1

Dynal particles (4.5 μm) (Table I, Example 1) were coated with labelled antibody prepared in Example A. 30 mg. were washed with PBS and subsequently added to 988 μl of PBS containing 0.005% thimerosal. 12 μl of 500 μg/ml labelled antibody (Example B) was then added to this particle mixture and allowed to rotate over 1 hour. The beads were then

separated from the solution using a magnet. The supernatant was removed and saved for

electrochemiluminescence analysis. 1 ml of PBS containing 3% BSA was then added to the beads which were allowed to rotate for 1 hour to block any remaining unoccupied areas on the particles. The beads were separated again. This washing and separation process was repeated once more, and afterwards the beads were stored in PBS with 3% BSA.

The results of an ECL analysis are set for in Table I.

Examples 2-9

The procedure described in Example 1 above was repeated using the particulate magnetic solid phase supports described for Examples 2-9 of Table I.

Example 10

Glass beads (2 μm) were coated with labelled antibody prepared in Example A. 30 mg of the beads were washed with PBS and

subsequently added to 988 μl of PBS containing 0.005% thimerosal. 12 μl of 500 μg/ml labelled antibody (prepared in Example B) was then added to this bead mixture and allowed to rotate over 1 hour. The mixture was then centrifuged at 2500 rpm for 5 minutes. The supernatant was removed and saved for electrochemiluminescence analysis. 1 ml of PBS containing 3% BSA was then added to the beads. The mixture was centrifuged again to separate the beads. This washing and centrifugation process was repeated once more, and afterwards the beads were stored in PBS with 3% BSA. Examples 11-14

The procedure described in Example 10 above was repeated using the particulate magnetic solid phase supports described for Examples 2-9 of Table I.

The result shown in Table I show that particle geometry and transmission properties for polystyrene containing Fe₂O₃ and soda lime glass provided significantly better and

unexpected results when compared to other materials.

Although the invention has been described in conjunction with specific

embodiments, it is evident that many

alternatives and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, the

invention is intended to embrace all of the alternatives and variations that fall with the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting an analyte

comprising:

a) providing a particulate solid phase support with a ligand containing luminescent metal label, said support and luminescent metal label having matched transmissive and emissive properties to enhance the emission from said metal label upon reaction with a coreactant;

b) contacting the solid phase support of step a) with an analyte of interest to form a luminescent labelled solid phase-analyte complex;

c) collecting said complex on an

electrode; and

d) exposing said complex to

electrochemical energy in the presence of a coreactant and measuring the resulting luminescence.

2. The method according to claim 1, wherein said electrogenerated chemiluminescence arises from a reaction of said label and said

coreactant.

3. The method according to claim 1, wherein said coreactant is an amine.

4. The method according to claim 1, wherein said amine is tripropyl amine.

5. The method according to claim 1, wherein said ligand is an antibody, antigen or DNA probe.

6. The method according to claim 1, wherein said particulate solid phase support is selected from magnetic glass and magnetic polystyrene and is contoured to provide reproducible

presentation of said magnetic particulate support to an electrode surface.

7. The method according to claim 6, wherein said ligand is an antibody, antigen or DNA probe.

8. The method according to claim 8, wherein said metal luminescent label is an aromatic, heterocyclic nitrogen-compound containing ruthenium or osmium compound.

9. The method according to claim 4 , wherein said metal luminescent label is formed from 4-methyl-4-N-succinimdyloxy-carbonylpropyl-2,2-bypyridine Ru(II).

10. An electrogenerated chemiluminescent solid phase support comprising:

a particulate substrate providing a

reproducible surface presentation to an

electrode surface and having an exterior layer of a metal luminescent label, said substrate and metal label having matched transmissive and emissive properties to enhance the emission from said metal luminescent label upon reaction with a coreactant.

11. The support to claim 10, wherein said metal label is attached to a ligand is an antibody, antigen or DNA probe.

12. The support according to claim 11, wherein said particulate substrate is selected from magnetic glass and magnetic polystyrene and is contoured to provide reproducible presentation of said magnetic particulate substrate to an electrode surface.

13. The support according to claim 10, wherein said ligand is an antibody, antigen or DNA probe.

14. The support according to claim 10, wherein said luminescent label is succinimdyloxy

carbonylpropyl-2,2-bypyridine.