Development and validation of a microfluidic immunoassay capable of multiplexing parallel samples in microliter volumes

Abstract

Immunoassays are widely utilized due to their ability to quantify a vast assortment of biomolecules relevant to biological research and clinical diagnostics. Recently, immunoassay capabilities have been improved by the development of multiplex assays that simultaneously measure multiple analytes in a single sample. However, these assays are hindered by high costs of reagents and relatively large sample requirements. For example, in vitro screening systems currently dedicate individual wells to each time point of interest and this limitation is amplified in screening studies when the investigation of many experimental conditions is necessary; resulting in large volumes for analysis, a correspondingly high cost and a limited temporal experimental design. Microfluidics based immunoassays have been developed in order to overcome these drawbacks. Together, previous studies have demonstrated on-chip assays with either a large dynamic range, high performance sensitivity, and/or the ability to process samples in parallel on a single chip. In this report, we develop a multiplex immunoassay possessing all of these parallel characteristics using commercially available reagents, which allows the analytes of interest to be easily changed. The device presented can measure 6 proteins in 32 samples simultaneously using only 4.2 µL of sample volume. High quality standard curves are generated for all 6 analytes included in the analysis, and spiked samples are quantified throughout the working range of the assay. In addition, we demonstrate a strong correlation (R2=0.8999) between in vitro supernatant measurements using our device and those obtained from a bench-top multiplex immunoassay. Finally, we describe cytokine secretion in an in vitro inflammatory hippocampus culture system, establishing proof-of-concept of the ability to use this platform as an in vitro screening tool. The low-volume, multiplexing abilities of the microdevice described in this report could be broadly applied to numerous situations where sample volumes and costs are limiting.

Lattice symmetries and the topologically protected transport of colloidal particles

“…We have shown that the driven transport of paramagnetic or diamagnetic colloidal particles above a two dimensional lattice is topologically protected by topological invariants of the modulation loops used to drive the transport [20,21]. Non-topological transport of particles in a dissipative environment is usually vulnerable because of a spreading of the driven motion with the distribution of properties of the classical particles [22][23][24][25][26][27][28] as well as due to the abundance of possible hydrodynamic instabilities [29,30] that limit the control over their motion. Topologically protected particle transport in contrast is robust against sufficiently small continuous modifications of the external modulation.…”

Topological protection of multiparticle dissipative transport

“…Topological protection might be used to transport a broad distribution of particles without dispersion, despite their different properties. High precision multiparticle transport is an important ingredient in, for example, multifunctional lab-on-a-chip devices 10 11 .…”

Emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development

“…As improvements in technology occur over time, early vaccine assays are often re-designed and bridged to assays with higher throughput, multiplexed detection, reduction of sample volumes, and automation to support testing of large numbers of specimens for late-stage clinical trials. [28][29][30][31][32][33][34][35] Often, a variety of tests are evaluated early in the program and, based on the usefulness of the data, a down-selection occurs so that the most relevant few remain to support large Phase 3 clinical trials and licensure of the vaccine. 2,35,36 Accepted "gold standards" of immunoassays for use at the onset of an EID vaccine program are rare.…”