Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

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.

Electrochemistry-based approaches to low cost, high sensitivity, automated, multiplexed protein immunoassays for cancer diagnostics

“…Examples of such tools are fluidic-integrated microarrays 22,23 and microplate-based tools 24 , bead-based fluidic systems and fluidic nanoarrays 25 . These tools are low volume, moderate sensitivity that mainly represents biomarker levels in several clinical conditions including cancer.…”

“…The major advantage of this chip is that it allows the immunophenotyping of the cell samples to examine cytokine secretion behavior. 23 …”

Automated 3D-printed unibody immunoarray for chemiluminescence detection of cancer biomarker proteins

“…The overall assay cost for two biomarker proteins is ~$2.00 in assay time 30 min. While other techniques for protein detection may offer competitive or better sensitivity and detection limits 15,21–32,54,55 the CL immunoarray described here can provide fast quality results at very low cost, which is crucial to future diagnostic applications of multiprotein analyses in the clinic. Additional improvements such as microprocessor/micropump control, data acquisition and reporting could lead to a low cost point-of-care device suitable for widespread use in cancer diagnostics.…”

Centrifugal microfluidics for sorting immune cells from whole blood

“…CMCs were fabricated using soft lithography [11, 13–15]. CAD designs were printed out as transparency masks (CAD/Art Services, Inc., Bandon, OR).…”

“…5a for illustrations of different CMC configurations) [11, 13–15]. For no-glass, bottom-glass, and glass-sandwich CMCs with a 2 mm thick PDMS channel-layer, PDMS prepolymer with a 10:1 monomer to curing agent ratio was poured over the Si mold and degassed.…”