We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 μl of reagents and sample, and is applicable to a wide range of analytes.
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This work was supported by Micronics Inc., the Washington Technology Center, and DARPA MicroFlumes program contract no. N660001-97-C-8632. We would like to thank Dr. Mark Holl for the majority of the design and implementation of the flow cell and fluidics system, Cathy Cabrera for developing image analysis software, Dr. Alex Goldstein for characterization of chemicals useful for pretreating blood samples, and Dr. Shelli R. Dennis for assistance exploring data analysis software. We thank Dr. Margaret Kenny, Diane Zebert, and Dr. Cai Cai Wu for work leading to immunoassay development in the T-sensor. Paul Yager has a financial interest in Micronics, Inc.
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Hatch, A., Kamholz, A., Hawkins, K. et al. A rapid diffusion immunoassay in a T-sensor. Nat Biotechnol 19, 461–465 (2001). https://doi.org/10.1038/88135
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