Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics

Abstract

Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

“…Electrochemiluminescence (ECL), in particular, provides a sensitive and useful methodology for detection of target materials. ECL employs metal chelates, such as ruthenium(I1) trisbipyridal chelate (Ru(bpy)3 *+), in redox reactions which are conducted on the surface of an anode and are mediated by electron carriers such as tripropylamine (TPA) (5)(6)(7)(8). Both R~( b p y )~~' and TPA are oxidized on the anode resulting in TPA+ and Ru(bpy)3 3-t.…”

Detection Methods Using Chemiluminescence

“…Electrochemiluminescent labels for DNA hybridization assays have been utilized in a highly sensitive, simple, and versatile assay system. Oligonucleotide probes, synthesized with a free 5'-amino group, are readily labeled with Ru(bpy)2+-NHS ester (Blackburn et al, 1991;Kenten et al, 1991). Alternatively, oligonucleotide probes may be labeled during synthesis by incorporating labeled phosphoramidites DiCesare et al, 1993).…”

“…Electrochemiluminescent labels are relatively small molecules (--~ 1000 dalton) that are extremely stable and may be coupled to nucleic acids, haptens, or proteins without affecting immunoreactivity or hybridization characteristics. The dynamic range for detection of these labels has been reported to be over six orders of magnitude (Blackburn et al, 1991). These advantages, compared with other nonisotopic detection methods, provide potential wide utility in automated nonradioactive clinical diagnostic assays, including both DNA hybridization and immunoassay formats.…”

“…A disadvantage of electrochemiluminescence, however, is a need for specialized instrumentation that can induce generation of electrochemically-excited states coupled with sensitive light detection. Blackburn et al (1991) used electrochemiluminescence detection with a DNA probe assay to quantify polymerase chain reaction (PCR)-amplified HIV-1 gag sequences. Double-stranded biotinylated PCR product was captured on streptavidin-coated microparticles and treated with alkali.…”

“…Electrochemiluminescence detection has been used in both manual (Blackburn et al, 1991;Gudibande et al, 1992;Kenten et al, 1992) and automated (QPCR System 5000; Wages et al, 1993) post-PCR amplification DNA hybridization assays for the detection of HIV-1 and HPV .…”