Multiplexed Determinations

The possibility of determining different analytes in the same sample (multiplexing) is of paramount importance nowadays. When dealing with multianalyte determination, different strategies could be followed [172]  

Determination of the analytes as a whole, without differentiation among them. This is the simplest assay, since single label, one pot assay is carried out. If there are two analytes, A and B, a positive response may indicate that there is A, or B, or both, but without differentiation between them. This is very important in the case of determining the presence of pathogens in food, where the presence of only one of them imphes a health hazard. It does not matter if it is A or B the food has to be disposed or treated in a different manner. 

Monoclonal antibodies of bacteria Anodic stripping voitammetery 

CdS nanocrystal tagged anti E-coii antibody CuS nanocrystal tagged anti Saimoneiia antibody PbS nanocrystal tagged anti Campyiobacter antibody 

It is also possible to perform multiplexed analysis with a sequential detection, that is to say, separation in time of the measurements. In this case, the same surface (multispecific surface) is employed for the immunointeractions of analytes, for example, A and B with their respective antibodies. However, since the same label is used, measurement for A is first taken and then a second measurement corresponding to A and B is made. The difference between both is related to analyte B. With this strategy, since two different steps have to be performed for final interactions, analysis time is increased when compared with the spatial separation approach, especially when more than two analytes are determined. 

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FIGURE 3.5 Sketch of the indirect competition FLISA protocol used for the multiplex determination of drugs in pork tissue samples. DEX, dexamethasone MPA, medroxyprogesterone acetate GM, gentamicin CZP, clonazepam CEF, ceftiofur and dBSA, denatured bovine serum albumin. (Reprinted with permission from Peng, C. et al. 2009. 

Taking advantage of the relative simplicity, ease of miniaturization, possibility of in situ measurements, low cost, and high sensitivity of electroanalytical techniques, various electrochemical detection approaches have been coupled to FIA for the multiplexed determination of target analytes, with either direct or indirect detection. Commonly used electroanalytical techniques include potentiometry, conductometry, voltammetry, and amperometry, among others. Although amperometry has been the preferred option in most applications, potentiometry (Lee et al., 2001, 2002 Suwansa-Ard et al., 2005) and conductometry (Suwansa-Ard et al., 2005) have also been employed (Llorent-Martinez et al., 2011). 

In this section we will describe the multiplex determination of a number of substrates departing from a biosensor array formed by different enzyme biosensors incorporating different enzymes. In this definition class, there can be two extreme variants, when each associated enzyme biosensor is employed for its own substrate, just relying on the specificity of each biosensor, and when there is the crossresponse condition, and some extra ability to resolve the exact compounds involved is therefore obtained. 

These two transducer pairs are activated alternating. For this purpose an ultrasonic instrument is combined with a two channel multiplexer. Figure 8 presents a modified standard instrument USN52 which also implies a modified software. This system performs four measurements per second - alternating the velocity and the thickness are determined. The probe can be scanned over the surface and in every position both, the velocity and the wall thickness are indicated Using the serial interface of the instrument finally a two-dimensional map of velocity or thickness can be generated. 

Wong, K. S., Kenseth, J., Strasburg, R. Validation and long-term assessment of an approach for the high throughput determination of lipophilidty (log Pow) values using multiplexed, absorbance-based capillary electrophoresis. J. Pharm. Sci. 2004, 93, 916-931. 

Injection pump. An injection pump is used to force the waste into the injection zone, although in very porous formations, such as cavernous limestone, the hydrostatic pressure of the waste column in the well is sufficient. The type of pump is determined primarily by the well-head pressures required, the volume of liquid to be injected, and the corrosiveness of the waste. Single-stage centrifugal pumps are used in systems that require well-head pressures up to about 10.5 kg/cm2 (150 psi), and multiplex piston pumps are used to achieve higher injection pressures. 

If kinetic resolution is being studied, the ratio of pseudo-e nantiomers can be measured by MS, allowing for the determination of ee-values (and/or of selectivity factors E). The same applies to the reaction of pseudo prochiral compounds. This system has been used successfully in the directed evolution of enantioselective enzymes. However, it should work equally well in the case of asymmetric transition metal catalyzed reactions. In the original version about 1,000 ee-deter-minations were possible per day (Figure 6).94 The second-generation version based on an 8-channel multiplexed spray system enables about 10,000 samples to be handled per day, the sensitivity being 2% ee.96... 

DFT methods are valuable for determining the magnitude and phase of a complex mixture of frequency components simultaneously such as might be encountered in the multiplexed systems for collection of several frequencies. Once the discrete Fourier coefficients have been computed the uncorrected values of m and [Pg.91]

Yong and Rawliszyn [26] used a multiplex gas chromatograph with a hollow fibre membrane interface in a solventless method for the determination of traces of aliphatic chlorocompounds such as trichloroethane in raw sewage sludge. Down to 0.4pg L 1 of these compounds could be determined. 

Bayliss and co-workers [10] combined ultra-high flow rates, parallel LC columns, a multiplex electrospray source, and mass spectrometric detection for the rapid determination of pharmaceuticals in plasma using four narrow bore (50 mm x 1 mm, 30 pm Oasis HLB) or capillary (50 mm x 0.18 mm, 25 pm Oasis HLB) HPLC columns with large particle sizes (to avoid high system back-pressure) in parallel with a multiple probe injector and a MUX MS interface. Small sample aliquots were injected directly into the system without sample pre-treatment procedure, obtaining very low limits of quantification (from 1 to 5 ng/mL). 

Bayliss M. Little D. Mallett D. Plumb R. Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high-throughput. Rapid Communications in Mass Spectrometry, 2000, 14, 2039-2045. 

Another way is the intermediate method to bridge the two approaches above. This method determines biologically multiplexed activity profile data. It integrates biological complexity at multiple levels pathways, signal transductions, and environmental factors. 

Fig. 8.13 A multiplexing SAMDI-MS assay. In this case, a mixture of three substrates is immobilized on the SAM. After incubation with appropriate enzyme solutions, the enzymatic reaction is quenched by rinsing the surface. Subsequently, matrix is deposited on the surface and MALDI-MS is carried out. Consumption of all three substrates can thus be determined in parallel.

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