High-Sensitivity Detection Methods

The general method to measure absorption spectra is based on the determination of the absorption coefficient a((A)) from the spectral intensity 

With a reference intensity 1 = Iq as produced, for example, by a 50% beam splitter in Fig.8,lb with a reflectivity R = 0.5, one can measure the absorption coefficient 

In the case of very small values of ax, this method cannot be very accurate since it measures a small difference of two large quantities and small fluctuations of the splitting ratio can already severely influence the measurement. Therefore several different techniques have been developed which allow increase of the sensitivity and accuracy of absorption measurements by several orders of magnitude. In the following we discuss some of these techniques. 

The first method is based on a frequency modulation of the monochromatic incident wave. It has not been designed specifically for laser spectroscopy but was taken from microwave spectroscopy where it is a standard method. The laser frequency is modulated at a modulation frequency f, which sweeps periodically from to When the laser is tuned through the absorp- 

Another very sensitive method directly monitors the absorbed energy rather than relying on a difference measurement (Ij -Ij). The energy IgaxA absorbed per second in a volume V = Ax can either be converted into fluorescence energy and monitored with a fluorescence detection system excitation spectroscopy) or it can be converted by collisions into thermal energy with a resultant temperature and pressure rise, which is monitored by a sensitive microphone photoacoustic spectroscopy). 

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Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments ia the types of columns and eluents for hplc appHcations, a significant amount of work has been done ia the kiads of detectioa methods for the vitamin. These detectioa methods iaclude direct detectioa by uv, fluoresceace after post-column reduction of the quiaone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

Applications for Highly-Sensitive Detection Method of Small Molecules by the Supramolecular Complexes between Antibodies... 

Here, we describe the design and preparation of antibody supramolecular complexes and their application to a highly sensitive detection method. The complex formation between antibodies (IgG) and multivalent antigens is investigated. When an antibody solution is mixed with divalent antigen, a linear or cyclic supramolecule forms [26-29]. With trivalent antigens, the antibody forms network structures. These supramolecular formations are utilized for the ampH-fication of detection signals on the biosensor techniques. 

Scheme 6. A highly sensitive detection method using an antibody specific for IgG (Fc) and gold nanoparticle...

Stains in the form of colloidal dispersions The need for highly sensitive detection methods for proteins after blotting (e.g., electroblotting, dot-blotting, slot blotting) applied to nitrocellulose or PYDF membranes drove scientists attention to develop new stains. It was discovered that the stains in the form of colloidal dispersions are suitable for such applications. The most commonly used colloidal stains are... 

Taniguchi, K. Nakamura, A. Tsurubuchi, K. O Hara, K. Sawai, T. Identification of Escherichia coli clinical isolates producing macrolide 2 -phos-photransferase by a highly sensitive detection method. FEMS Microbiol. Lett., 167, 191-195 (1998)... 

As an analytical tool, electrophoresis is simple, relatively rapid, and has unparalleled resolving power. It is used chiefly for analysis and purification of very large molecules such as proteins and nucleic acids. Highly sensitive detection methods have been developed to monitor and analyze electrophoretic separations. 

The use of building materials, furniture, carpets and household products produces an almost ubiquitous level of volatile organic compounds (VOCs) in indoor air. Several hundred different compounds have been identified in the indoor environment. Since most air pollutants occur in low concentrations of 1-1000 rg/m3, highly sensitive detection methods as well as efficient separation methods are needed to analyze air samples (Barro et al., 2009). 

However, p-jump techniques are not without fault (Takahashi and Alberty, 1969). Most chemical reactions are less sensitive to pressure than to temperature alterations. Thus, a highly sensitive detection method such as conductivity must be employed to measure relaxation times if p-jump is used. Conductometric methods are sensitive on an absolute basis, but it is also fundamental that the solutions under study have adequate buffering and proper ionic strengths. In relaxation techniques, small molar volume changes result, and consequently, even if a low level of an inert electrolyte is present, conductivity changes may be undetectable if pressure perturbations of 5-10 MPa are utilized (Takahashi and Alberty, 1969). 

The first two points represent a general motivation for miniaturization in separation science independent of the actual fabrication technology. The benefit of a reduction of the consumption of sample, reagents, and mobile phase in chemical and biochemical analysis is self-evident and does not need to be discussed further (reduced consumption of precious samples and reagents, reduced amounts of waste, environmental aspects). This advantage is, however, sharply contrasted by its severe implications on the detection side, as discussed elsewhere in this volume in detail. The detection of the separated zones of very small sample volumes critically depends on the availability of highly sensitive detection methods. It is not surprising that extremely sensitive laser-induced-fluorescence (LIF) has been the mostly used detection principle for chip-based separation systems so far. 

King G, Payne S, Walker F, et al. A highly sensitive detection method for immunohistochemistry using biotinylated tyramine. J. Pathol. 1998 183 237-241. 

High-quality reagents are available, with highly sensitive detection methods, but they must be employed properly in controlled fashion, and currently often are not. 

Peroxyoxalate chemiluminescence detection has been shown to be a highly sensitive detection method " for the determination of A-nitrosamines and secondary amines in combination with reversed-phase HPLC. Fentomole limits of detection can be obtained with conventional instrumentation. The principle of the reaction is illustrated in Figure 12.3. 

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... 

Table 1 summarizes selected parameters that are important for establishing a robust and reproducible technique for the separation and quantification of dsDNA products of PCR and RT-PCR using CE. Important advances have been made in a number of areas, including capillary coatings, sieving gel matrices, and high-sensitivity detection methods. Because of a... 

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