On-Chip Assays

Field-amplified sample stacking is a fairly widely applicable method of achieving increased sensitivity for capillary and on-chip assays in a scheme that is easily integrated with electrophoretic separation techniques [4,34-43]. FASS is typically used as a preconcentration step that occurs before the electrophoretic separation of analyte ions. 

A cell assay is defined as measurement and analysis of cellular response to chemical and/or physical stimulus. Cellular responses are diverse alterations of intracellular and extracellular biochemistry, cell morphology, motility, and growth properties. These responses characterize the cell phenotype and are typically monitored in a culture dish or a multiwell plate, while more recently microfluidic devices have been employed. A cell assay performed in a microfiuidic device is sometimes termed an on-chip assay. 

The development of protein chip assays to determine protein function using purified components is a rapidly advancing area. Automated systems for the assay of protein function on chips in parallel for thousands of proteins simultaneously will likely be available in the next few years. These miniaturized arrays will be useful for basic research as well as for diagnostics and drug development. For instance, the combination of protein chips with combinatorial chemistry will allow the simultaneous screening of vast collections of small molecules against vast collections of potential target proteins. 

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

Several successful attempts were done to transfer classical CEIA to a microchip-based format. This kind of miniaturization is a trend that can overcome the limitations of CE in high-throughput systems. On-chip CE offers both parallel analysis of samples and short separation times. Koutny et al. showed the use of an immunoassay on-chip (32). In this competitive approach fluorescein-labeled cortisol was used to detect unlabeled cortisol spiked to serum (Fig. 8). The system showed good reproducibility and robustness even in this problematic kind of sample matrix. Using serum cortisol standards calibration and quantification is possible in a working range of clinical interest. This example demonstrated that microchip electrophoretic systems are analytical devices suitable for immunological assays that can compete with common techniques. 

Fig. 8 On-chip immunoassay for the quantification of cortisol in serum samples. Fluorescein-labeled cortisol (Ag ) was used in a competitive assay for the detection of unlabeled analyte cortisol. Three replicate injections for each of three samples with cortisol levels as indicated in the figure. Fluorescein was used as an internal standard (I.S.). (Reprinted with permission from Ref. 32. Copyright 1996 American Chemical Society.)...

J. Wang, On-chip enzymatic assays, Electrophoresis, 23 (2002) 713-718. J. Wang and M.P. Chatrathi, Microfabricated electrophoresis chip for bioassay of renal markers, Anal. Chem., 75 (2003) 525-529. 

With known analyte concentrations, the processed data provide calibration points, and Immusoft comprises fitting procedures to deduce the corresponding calibration curve that can be stored in the program for further experiments. Different calibration curves can be stored depending for instance on the assay protocol, on the specific features of the chip used or on the medium in which the assay is performed. In most cases, the calibration is performed with six independent chips of eight channels and cumulated in order to get a stable batch calibration. Then the results can be referred to this internal batch calibration. For routine control, one calibration each week is recommended to be sure that the chemistry is still in the specifications (e.g. + 10% of inter-assay standard deviation). 

In other work, using a range of different protein function microarrays, each fabricated in the form of BCCP fusion proteins as described here, we have been able to monitor a wide range of protein activities, including protein-protein interactions and protein-small molecule interactions, as well as carrying out on-chip phosphorylation assays (6) (see Note 15). 

Fig. 6. Measurement of the relative amount of ligand bound to each protein in the array. (A) Schematic of on-chip binding assay in which a fluorescently labeled interaction partner binds to the functional, arrayed protein immobilized to the streptavidin-coated surface via the biotinylated BCCP tag. (B) p53 protein function microarray probed with Cy3-labeled GADD45 duplex oligo. Quantification of the signal intensity from each spot allows the effect of polymorphic and functional variation on the DNA binding function of p53 to be determined.

In the on-chip theophylline homogeneous immunoassay, the antibody-antigen complex can be separated from the antigen. Why can such a separation be successful, but not in the case of the cortisol assay [330,1006] (2 marks)... 

Wakamoto, Y.C., Umehara, S., Matsumura, K., Inoue, I.,Yasuda, K., Development of non-destructive, non-contact single-cell based differential cell assay using on-chip microcultivation and optical tweezers. Sensors Actuators B 2003, 96, 693-700. 

Mao, H., Holden, M.A., You, M., Cremer, P.S., Reusable platforms for high-throughput on-chip temperature gradient assays. Anal. Chem. 2002, 74(19), 5071-5075. 

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