Abstract
Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.
Highlights
While achieving a limit of detection (LOD) comparable to conventional ELISA, these integrated microfluidic immunoassay chips still require immobilizing capture antibodies on a solid phase and multiple washing, labeling, and signal amplification steps, retaining limitations associated with conventional ELISA in terms of assay complexity and time[14,15,16,17,18,19,20,21,22]
As a no-wash, homogeneous bead-based sandwich immunoassay, AlphaLISA eliminates washing and blocking steps required for heterogeneous sandwich immunoassays such as ELISA that often result in analyte dilution and human errors
Each circulation loop was divided into three compartments that were filled with (1) sample solutions containing target analytes, (2) anti-analyte AlphaLISA acceptor beads (1.2 mg mL−1) and biotinylated antibodies (125 nM), and (3) streptavidin-coated AlphaLISA donor beads (5 mg mL−1) (Fig. 1c)
Summary
While achieving a limit of detection (LOD) comparable to conventional ELISA, these integrated microfluidic immunoassay chips still require immobilizing capture antibodies on a solid phase and multiple washing, labeling, and signal amplification steps, retaining limitations associated with conventional ELISA in terms of assay complexity and time[14,15,16,17,18,19,20,21,22]. We developed a highly integrated microfluidic immunoassay chip incorporating a newly developed immunoassay technology, termed AlphaLISA (from PerkinElmer)[23,24,25,26], to achieve rapid, automated, parallel immunoassays with microliter quantities of samples. Using conventional microtiter plate assay platforms, the AlphaLISA technology requires only a small sample volume (down to 10 μ L) while retaining the capability of achieving an excellent LOD down to sub-pg mL−1 and a large dynamic range of five orders of magnitude, comparable to immunosensing performance of state of the art ELISA methods. To demonstrate its potential clinical application, the microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo
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