Indirect detection probe

Other detection methods can be applied to CEC. Indirect spectrophotometric detection methods are commonly used for ultraviolet (UV)-transparent ionic analytes (such as inorganic ions) in CE, but indirect detection in CEC tends to suffer from unstable baselines, caused by adsorption of the indirect detection probe onto the CEC ion-exchange stationary phases (SP). Fluorimetric detection is a very sensitive and robust method, but is limited to a relatively small... 

Inverse geometry Term used to describe the construction of a probe that has the 1H receiver coils as close to the sample as possible and the X nucleus coils outside these 1H coils. Such probes tend to give excellent sensitivity for 1H spectra at the expense of X nucleus sensitivity in 1-D techniques. They offer a lot of compensation in terms of sensitivity of indirectly detected experiments. 

Fig. 17.9. Indirect detection of analytes by the replacement of the probe (co-ion or background electrolyte) ions that are responsible for a constant high-background analytical signal.

AP- and/or horseradish peroxidase (HRP)-conjugated antibodies for the haptens used for labeling probes (a direct detection method) or unconjugated antibodies for the haptens and AP- and/or HRP-labeled anti-species antibodies (an indirect detection method) (Vector Laboratories, Inc., Bnrlingame, CA, USA) (see Note 8). 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

Fluorescence detection is widely employed in electromigration techniques for samples that naturally fluoresce or are chemically modified to produce molecules containing a fluorescent tag. Indirect detection incorporating a fluorescent probe into the electrolyte solution is also employed. One of the most common fluorophores used for this purpose is fluorescein, which is a water soluble, stable, and relatively cheap compound. 

E. S. Yeung and W. G. Kuhr, Indirect Detection Methods for Capillary Separations, Anal. Chem. 1991, 63, 275A J. Ren and X. Huang, Indirect Chemiluminescence Detection for Capillary Electrophoresis of Cations Using Co(III) as a Probe Ion, Anal. Chem. 2001, 731, 2663 M. Macka, C. Johns,... 

Electronic Absorption and Luminescence (Volume 12) Absorption and Luminescence Probes Fluorescence Imaging Microscopy Fluorescence Lifetime Measurements, Applications of Indirect Detection Methods in Capillary Electrophoresis Surface Measurements using Absorption/Luminescence... 

The application of indirect methods in biomedical analysis has been reviewed by Schill and Arvidsson.23 Reversed-phase HPLC is the main field of application for indirect detection, and both charged and uncharged species can be visualized, although sensitivity is better for ionic solutes. With indirect UV detection for reversed-phase HPLC, the eluent contains a chromophoric group (probe), and peaks are obtained for injected solutes as well as for the mobile phase additives (system peaks). For solutes that... 

Typical probes for the analysis of ionic solutes include 3-hydroxy-L-tyrosine (DOPA)24 and naphthalene-2-sulfonate,26 whereas those for use with uncharged solutes include nicotinamide,27 theophylline,28 and anthracene 29 Indirect detection is nonspecific and less suitable for the analysis of complex or impure samples, because unpurified biological samples, such as urine, contain a large number of hydrophilic solutes that will give problems such as extra system peaks. However, analyses of pharmaceutical products and quantification of impurities in substances are typical of applications.23... 

In conclusion, CO is an excellent probe molecule for the properties of coordinatively unsaturated surface Zn2+ sites, both isolated sites or those grouped into 2D patches. It is also emphasized that the prismatic faces also expose coordinatively unsaturated oxygen ions their presence, however, is indirectly detected only via their influence on the electrostatic field at the Zn2+ center. 

The most common probe head is a switchable probe head, which can be used to observe H and all NMR-active nuclei from the low-frequency limit up to the frequency of 31P. The proton coil can be tuned for the observation of 19F. The switch-able probe head is designed for either direct or inverse observation. The direct observation probe head is most sensitive for 1-D experiments on 13C and 31P. The inverse probe head in turn is most sensitive for the direct observation of H and indirect detection, for example of 31P, in 2-D experiments, taking advantage of polarization-transfer phenomena. 

When compounds are not optically active and are not easily derivatized, indirect detection is sometimes the best alternative. In indirect detection, an absorbing or fluorescing probe is added to the buffer. Displacement of the probe by the analyte produces a decrease in signal (10,11). Because of its universality and simplicity, indirect UV absorbance detection has been the predominant detection scheme for phos-phonic acid analyses by CE (12 17). 

Direct/Indirect Detection Systems. Direct detection of molecular probes requires physical attachment of the detection system component to the probe, such that binding of the probe to its target molecule results in immediate attachment of the detection system to the target-probe complex. In contrast, indirect detection requires that further separate steps be performed to identify target-probe complexes after they are formed. Direct and indirect target-probe-... 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

Is the probe direct, or inverse The former is good for direct observation with or without INEPT enhancement. The latter will give poor signal-to-noise in direct experiments since the sample does not fill the coil space, but is much preferred for indirect detection via, for example, a heteronnclear mnltiple qnantum coherence (HMQC) or heteronnclear single qnantum coherence (HSQC) experiment. 

L. L. M. Glavina and F. F. Cantwell, Origin of indirect detection in the liquid chromatography of a neutral sample with an ionic probe using an ODS bonded phase and aqueous mobile phase. Anal. Chem. 65 (1993), 268-276. 

The typical commercial PPG system consists of a probe with an added field gradient coil, a current amplifier, an interface to the acquisition computer, and software control. Probe manufacturers have produced a wide variety of probes containing a field gradient coil, including only, broadband indirect detection, and SmM and 8noM triple-resonance ( H, and probes. The amplifier must... 

Heteronuclear NMR experiments, which can be performed with the standard equipment of practically all modern spectrometers, require in general three separate radiofrequency (RF) channels for both spectrometer and probe head. The first two channels deliver the H (for decoupling) and "X frequencies to the sample, and the third channel is commonly tuned to D and operates the field frequency lock. In most standard probe heads, these three frequencies are delivered via two concentric coils. The inner coil with the higher Q factor is generally used for detection, the outer one only for the application of pulses and decouphng. TWo general designs are in use in normal or forward probe heads, which are optimized for direct detection of X nuclei, the inner coil is a tuneable X coil and the outer coil is normally double tuned to and the lock frequency, while in inverse probe heads which are optimized for indirect detection of "X resonances via H, this order is reversed. 

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