Immunoassays (IAs) are analytical methods based on the specific recognition between the antigen (Ag), which molecular weight can vary in a wide range, and the variable regions of antibody (Ab), a Y shaped macromolecule composed by four polypeptide chains. IAs are applicable without analytes enrichment, purification, or pre-treatment having great advantages over standard methods. Immunoassays relate to medicine, food quality control, forensic, veterinary, and environmental analysis. A detectable label conjugated on one or both of the immunoreagents (IRs) reveals the recognition between Ag and Ab. The evolution of IAs corresponded to the evolution of the labelling technology and the optical detection is the simplest way to evaluate the analytical signal. Luminescence Immunoassays. The term luminescence defines the emission of light in the visible–near visible range (l ¼ 300–800 nm) occurring when electrons in a molecule, excited because of absorbed energy, relaxes to the ground state. New luminescent materials enlarged the emission spectra to the near-infrared region (l ¼ 300–1500 nm). The luminescence phenomena are defined depending on the source creating the excited state. Labels applied in IAs show: fluorescence (FL), chemiluminescence (CL), bioluminescence (BL), and electrochemiluminescence (ECL). The fluorescent labels emit light after photons absorbance; the chemiluminescent and bioluminescent ones after a chemical or biochemical exothermic reaction, respectively; the electrochemiluminescence ones after an electro-oxidation or reduction. The decay pathways (see Jablonski diagrams, Fig. 1) distinguish the photon-induced emission in: (1) fluorescence (S1–S0 decay, after 10-9 –10-7 s); (2) phosphorescence (decay to S0 through a metastable triplet state, after 10-3 –102 s) and (3) delayed fluorescence (the decay to S0 includes two intersystem crossings, S1–T1–S1). Because of the nonradiative loss of energy, emitted light has a longer wavelength (Stokes shift). Labels in fluorescence immunoassays (FIAs) must have a Stokes shift >50 nm to reduce interferences. The excitation light can be continuous or pulsed, from arc lamps, lasers, or light-emitting compounds through an excitation energy transfer. In CL, the excited state is generated in an exothermic chemical reaction but the decay transition is the same of FL (S1–S0 decay). Most of CL reactions involve O2, H2O2, and similar powerful oxidants. CL has various advantages over FL: no external light source, absence of high light background, signal amplification by enzymatic catalysis, simple and cheap instrumentation. CL has extensive application in immunoassays because of its high sensitivity, fast reaction rate, and wide dynamic range. BL is the light produced in living organisms by oxidation of a substrate (luciferin) catalyzed by an enzyme (luciferase). The most popular BL reaction occurs in the Firefly’s lantern but this phenomenon concerns many other terrestrial and marine organisms. In ECL the light signal originates from excited states generated by an electrochemical reaction at the electrodes’ surface. The luminescence immunoassays (LIAs) are carried out according to the classical IAs formats; the techniques used in enzyme-based assays are modified for the luminescent detection. The various IA formats will not be described here, mentioning only the specific luminescent protocols. The luminophores can be used to label Ags or Abs, bound to solid phases, or free in solution. IAs have undergone important changes in recent years, following the introduction of new molecules with recognizing capacity (recombinant Abs, molecularly imprinted polymers, nucleic acid aptamers, nanozymes, nanobodies, which are not discussed in this article) and the development of different nanomaterials, per se luminescent or modulating the fluorophores’ emission. LIAs include all these innovations in agreement with the contemporary trends: to develop miniaturized, reusable, high-throughput, multiplexed IAs for Point-of-Care (POC) tests. Luminescence based immunobiosensors, owing to the huge amount of recently developed solutions, are not discussed here.
Girotti S., Ferri E.N. (2019). Immunoassays | Luminescence immunoassays. Amsterdam : Elsevier [10.1016/B978-0-12-409547-2.14479-8].
Immunoassays | Luminescence immunoassays
Girotti S.
Primo
Writing – Review & Editing
;Ferri E. N.Ultimo
Writing – Original Draft Preparation
2019
Abstract
Immunoassays (IAs) are analytical methods based on the specific recognition between the antigen (Ag), which molecular weight can vary in a wide range, and the variable regions of antibody (Ab), a Y shaped macromolecule composed by four polypeptide chains. IAs are applicable without analytes enrichment, purification, or pre-treatment having great advantages over standard methods. Immunoassays relate to medicine, food quality control, forensic, veterinary, and environmental analysis. A detectable label conjugated on one or both of the immunoreagents (IRs) reveals the recognition between Ag and Ab. The evolution of IAs corresponded to the evolution of the labelling technology and the optical detection is the simplest way to evaluate the analytical signal. Luminescence Immunoassays. The term luminescence defines the emission of light in the visible–near visible range (l ¼ 300–800 nm) occurring when electrons in a molecule, excited because of absorbed energy, relaxes to the ground state. New luminescent materials enlarged the emission spectra to the near-infrared region (l ¼ 300–1500 nm). The luminescence phenomena are defined depending on the source creating the excited state. Labels applied in IAs show: fluorescence (FL), chemiluminescence (CL), bioluminescence (BL), and electrochemiluminescence (ECL). The fluorescent labels emit light after photons absorbance; the chemiluminescent and bioluminescent ones after a chemical or biochemical exothermic reaction, respectively; the electrochemiluminescence ones after an electro-oxidation or reduction. The decay pathways (see Jablonski diagrams, Fig. 1) distinguish the photon-induced emission in: (1) fluorescence (S1–S0 decay, after 10-9 –10-7 s); (2) phosphorescence (decay to S0 through a metastable triplet state, after 10-3 –102 s) and (3) delayed fluorescence (the decay to S0 includes two intersystem crossings, S1–T1–S1). Because of the nonradiative loss of energy, emitted light has a longer wavelength (Stokes shift). Labels in fluorescence immunoassays (FIAs) must have a Stokes shift >50 nm to reduce interferences. The excitation light can be continuous or pulsed, from arc lamps, lasers, or light-emitting compounds through an excitation energy transfer. In CL, the excited state is generated in an exothermic chemical reaction but the decay transition is the same of FL (S1–S0 decay). Most of CL reactions involve O2, H2O2, and similar powerful oxidants. CL has various advantages over FL: no external light source, absence of high light background, signal amplification by enzymatic catalysis, simple and cheap instrumentation. CL has extensive application in immunoassays because of its high sensitivity, fast reaction rate, and wide dynamic range. BL is the light produced in living organisms by oxidation of a substrate (luciferin) catalyzed by an enzyme (luciferase). The most popular BL reaction occurs in the Firefly’s lantern but this phenomenon concerns many other terrestrial and marine organisms. In ECL the light signal originates from excited states generated by an electrochemical reaction at the electrodes’ surface. The luminescence immunoassays (LIAs) are carried out according to the classical IAs formats; the techniques used in enzyme-based assays are modified for the luminescent detection. The various IA formats will not be described here, mentioning only the specific luminescent protocols. The luminophores can be used to label Ags or Abs, bound to solid phases, or free in solution. IAs have undergone important changes in recent years, following the introduction of new molecules with recognizing capacity (recombinant Abs, molecularly imprinted polymers, nucleic acid aptamers, nanozymes, nanobodies, which are not discussed in this article) and the development of different nanomaterials, per se luminescent or modulating the fluorophores’ emission. LIAs include all these innovations in agreement with the contemporary trends: to develop miniaturized, reusable, high-throughput, multiplexed IAs for Point-of-Care (POC) tests. Luminescence based immunobiosensors, owing to the huge amount of recently developed solutions, are not discussed here.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
