Overlap Detection System

The ICP has proliferated as a method of converting chemical compounds into their elemental constituents which subsequently emit light of characteristic wavelengths. Accordingly, ICP has been used extensively as an emission source for optical detection systems in order to perform elemental analysis. Since each element can emit hundreds of optical lines, the use of ICP/AES for multiple element analysis, or for the detection of elements in unknown or concentrated matrices, can suffer from interferences due to spectral overlap. By contrast, ICP-MS provides inherently simpler spectral Information. An example of such a spectrum is demonstrated in Figure 2 showing a typical ICP-MS scan for a 10 ug ml"l solution of mixed transition metals. The demonstrated sensitivity here is 10 to 10 counts s l per ug m1"l and, coupled with the nearly universal ionization efficiency of the ICP ion source, provides typical detection limits in a narrow range between 0.1 to 10 ng.ml" for most elements. In fact over 90% of the elements in the periodic table are accessible for such analytical determinations. 

Fig. 13.4 Fluorescent bleed-through. A problem in fluorescent microscopy occurs when the filters cannot separate emission wavelengths, for example, when an emission from 488 fluorophore is detected in the 555 channel and in the 555 emission. The emission spectrum for the 488 fluorophore (black dashed line) is shown with a tail that extends into the 555 emission filter (gray solid line). If the intensity of the 555 fluorophore emission is low as seen by the maximum height at 40% and the 488 fluorophore has high-intensity emission, then the amount of overlap in emission can be significant (dark gray area). To compound the problem, if the detection system gain is increased in the red 555 emission, then the 488 fluorophore intensity is about 50% of the light as from the 555 fluorophore intensity.

Spectral interferences involve a change in the amount of light that reaches the detection system by concomitants in samples. They can be classified as spectral overlaps, scatter, and molecular absorption. 

An alternative homogenous fluorescence-based detection system is fluorescence resonance energy transfer (FRET). This phenomenon occurs when two fluorophores are in close proximity, and the donor fluorophore has an emission spectrum that overlaps the excitation spectrum of the acceptor fluorophore. When the donor fluorophore is excited, energy is transferred from donor to acceptor with the result that the intensity of emission from the donor is reduced (quenched). If both the analyte and antibody are coupled to fluorophores with overlapping spectra, then FRET will occur only when the complex forms. Thus, FRET has not been applied widely in conventional immunoassays. 

The measured emission spectra were not corrected for the response function of the detection system. The total detection system was sensitive to radiation between 200 nm and 800 nm (the maximum sensitivity is between 200 nm and 400 nm). Since the intensity of the recorded spectra changed somewhat from shot to shot, the 30-nm-spectra were recorded with a certain overlap and normalized to each other. 

---