Multi-analyte assays

However, if a class-selective assay is desirable (for multi-analyte assays), the handle should be located at or near a position that differentiates members of the class and exposes features common to the class. Using the pyrethroid example, an ideal immunogen should retain the phenoxybenzyl moiety and link the protein from the distal acid end (Figure 9). Using such an immunogen hapten, a class-specific immunoassay was developed that was highly cross-reactive with the type I pyrethroids permethrin, phenothrin, resmethrin and bioresmethrin. ... 

Along this line Matsue et al. [37,45,78,79] have developed a number of biochips. Among them are multi-analyte assays for human placental lactogen (HPL) and human chorionic gonadotropin (HCG) [45] and leukocidin, a toxic protein produced by methicillin-resistant Staphylococcus aureus [79]. Figure 37.8 shows an example of a dual immunoassay with SECM detection. The analyte is defined by the position on the chip and the amount of analyte is quantified via the collection current at the UME. The current originates from the reduction of ferrocinium methanol (Fc+) at the UME. Fc+ is produced locally at the chip surface by the enzyme HRP under consumption of H202. 

Tschmelak, J., G. Proll, and G. Gauglitz. 2004. Verification of performance with the automated direct optical TIRF immunosensor (River Analyser) in single and multi-analyte assays with real water samples. Biosens. Bioelectron. 20 743-752. 

The amount of available sample needs to be considered both from the perspective of how this impacts the method and LLOQ and also from the standpoint of having enough sample for re-analysis in the event of run failure or other sample re-assay directives. Even in cases where an adequate amount of sample is available for re-assay, repeated run failures could lead to depletion of the sample. This is particularly problematic with multi-analyte assays where the probability of run failure due to one or more of the analytes in the assay increases as a function of the number of analytes in the method (Dadgar 1995). 

Sub-stocks are also used for multi-analyte assays where a mixture of analytes in a single solution is prepared for subsequent preparation of QCs, calibration solutions or spiking solutions used for the preparation of matrix matched calibrators. In this case, individual stock solutions are combined to make one or more sub-stock solutions containing all of the analytes in one solution. This practice will reduce the number of steps that would be required versus making an individual spiking solution for each analyte. However, for methods that required a different LLOQ for each analyte, the sub-stocks and spiking solutions need to be prepared at the corresponding appropriate concentration for each analyte. 

Mauriz, E., A. Calle, J.J. Manclus, et al. 2006. Single and multi-analyte surface plasmon resonance assays for simultaneous detection of cholinesterase inhibiting pesticides. Sens. Actuat. B Chem. 118 399M07. 

At this stage, any co-elution of the key analytes is corrected by fine-tuning the separation parameters, mostly by adjusting selectivity (a) (see Figure 2.17). This process may be quite challenging and iterative for multi-component assays of complex samples. Afterwards, efforts are focused on improving other method performance measurements such as sensitivity, peak shape, robust-... 

The length, material (mainly nitro-cellulose) and pore-size (50 nm to 12 pm, depending on the applied nanoparticles) of the detection and incubation pad define the incubation time [69], The detection and enrichment of the conjugates is achieved on the antibody-bearing lines. Analyte detection is performed on the test line and proof of assay validity on the control line. The readout is t5q)ically done by naked eye for absence (1 colored line) or presence (2 colored lines) of a minimum analyte amount. A readout with a reader enables quantitative analyte detection [70, 74]. For multi-analyte detection [69] or semi-quantitative setups [75] several test lines are applied. 

Numerical identifiability also becomes a problem with a poorly or inadequately designed experiment. For example, a drug may exhibit multi-exponential kinetics but due to analytical assay limitations or a sampling schedule that stops sampling too early, one or more later phases may not be identifiable. Alternatively, if sampling is started too late, a rapid distribution phase may be missed after bolus administration. In these cases, the model is identifiable but the data are such that all the model components cannot be estimated. Attempting to fit the more complex model to data that do not support such a model may result in optimization problems that either do not truly optimize or result in parameter estimates that are unstable and highly variable. 

MSPD is the next alternative for sample preparation of fruits, vegetables, herbs, cereals, and other plants. It is convenient for liquid and semi-liquid samples. This technique is a multi-residual assay which consists of matrix homogenization with a solid silica phase placed into a short column, as in SPE. Analytes and matrix interferences are retained on the mixed solid-phase material with completely new separation characteristics. Specific elution allows one to obtain analytes after the elimination of matrix compounds via washing steps. This... 

E. Fu, et al., A two-dimensional paper network format that enables simple multi-step assays for use in low-resource settings in the context of malaria antigen detection. Analytical Chemistry 84 (10) (2012) 4574-4579. 

Finally, in optical-based sensors and assays, there is the development of label-free bioanalytic detection on prepared membrane surfaces, ultra sensitive detection of microbial agents, the development of fiber-optic based enzyme sensors, multi-analyte breast cancer immunoassays, and photodynamic therapy of osteosarcoma in veterinary patients. 

Strip tests are simple, rapid and cost efficient. A disadvantage is the limited sensitivity of the assay and lack of multidimensional multi-analyte capabilities. Recently, micro -fluidic cluster chromatography devices proved to be promising candidates to overcome these limitations. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

In the last step of an assay, i.e. during the electrochemical detection of the electroactive species of interest, a new window appears in order to let the experimenter follow the detection on-line. As presented in Fig. 36.8, the software has been designed to perform chrono-amperometric measurements of the desired analyte. On the left part of the window the raw measurement data that provide the evolution of the current for each microchannel that is measured during a time period of generally 2 s appears. With the multi-potentiostat used, the eight channels are... 

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