Sensitivity luminescence detection

The very first spectroscopic instruments, from Newton s prism and pinhole to Frauenhofer s simple spectroscope, were constructed to observe luminescence. Even though the great sensitivity of luminescence detection seemed to promise that luminescence would become an important tool for chemical analysis, the fact is that absorption spectroscopy was the first spectroscopic technique to be widely used. At first glance, this may seem surprising since absorption spectroscopy is inherently less sensitive and had to await the development of more complex instrumentation, especially, electronically amplified detection. 

H. Harma, T. Soukka, S.Lonnberg, J. Paukkunen, P. Tarkkinen, and T. Lovgren, Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence. Luminescence 15, 351-355 (2000). 

The variety of assays reported here displays the great versatility of luminescent detection systems. As already described, in all luminescent systems the main advantages are the high sensitivity and specificity, which reduce to the minimum the sample treatment, and the ease of use of the reagents and the luminometer. Immobilized systems greatly reduce the cost per assay on the other hand, their preparation requires expertise, especially in the surface activation step on nylon tubes. 

The real limitation of detection in spectrofluorimetry is not the sensitivity of the detector, but rather the stray light which result from imperfections of the monochromators and emissions by impurities in the solvents. The limiting quantum yields of luminescence detection are of about 10-4 in optimal conditions. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Ruthenium complexes have been applied successfully to the luminescent detection of proteins on blotting membranes like nitrocellulose [160]. The bipyridyl and phenanthroline complexes modified with aminoreactive NHS-ester or isothiocyanate groups are commercially available [161]. An even higher sensitivity and lower detection limit can be obtained by encapsulating... 

Luminescence detection has the advantage of very low background compared with fluorescent technologies, so assay sensitivity can be extremely high. 

Photoinduced absorption (PA) measurements provide useful information about relaxation processes of photocarriers and tail and gap states in a-Si H [Tauc, (1982) see also Chapter 9 by Tauc in Volume 21B]. Photoinduced absorption also depends on the recombination processes of those trapped electrons and holes that are created under optical excitation. Thus it is expected that PA will be spin-dependent, as is luminescence. Hirabayashi and Morigaki (1983a,b) have observed, for the first time, spin-dependent PA at 2°K in a-Si H, monitoring the PA intensity while irradiating the sample with microwaves at 9.6 GHz. This is called photoinduced absorption-detected ESR (PADESR), which provides complementary information to the conventional ODMR (luminescence-detected ESR). However, since the PADESR technique can be applied to nonluminescent materials, it may provide a more general means for highly sensitive detection of ESR than conventional ODMR. 

For EL measurements, the detection limit of weak EL is mainly determined by the detection sensitivity of an experimental apparatus used because no illumination is needed in this case. One can use various high-sensitivity detection methods such as a lock-in amplifier [16, 17], boxcar integration [17], and photon counting [17]. Care should, however, be taken when the time-averaging method is used because the luminescence intensity often changes with time even at a constant electrode potential, probably because the surface chemical structure of the semiconductor electrode... 

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