Under‐5‐Minute Immunoblot Assays by Vortex Fluidic Device Acceleration

Abstract

Unlocking the potential of personalized medicine in point‐of‐care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (<5 min), robust (comparatively lower CVs), and inexpensive (decreasing reagent use and cost by >70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps—blocking, binding, and washing—leverage the unique thin‐film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.

Universal and High-Speed Zeptomolar Protein Serum Assay with Unprecedented Sensitivity

“…In this work, the immunorecognition process was performed in an Eppendorf tube to avoid interference with the electrode interface. Additionally, PEGylated immunomagnetic beads formed a hydrated layer on the surface to resist the adhesion of nonspecific proteins. , Typically, routine immunorecognition processes demand a lengthy incubation period (>2 h), resulting in slow detection. − Therefore, a vortex mixer was employed to assist this process and dramatically accelerated the kinetics of movement and binding between antibodies and antigens. By optimizing the incubation time between antigens and the immunoprobe, the sandwich-structure of immunocomplex (Fe 3 O 4 /Ab 1 /PEG@antigen@SiO 2 /Ab 2 /3-ABA) was formed within 10 min.…”

Printable Hydrogel Arrays for Portable and High-Throughput Shear-Mediated Assays

“…The general process of testing was as follows. The SAV-HRP conjugate was first diluted (1:1000) in 5× ChonBlock (Chondrex Inc.), as per a previous study . The conjugate was either entrapped in the PAAm/Alg-Ca 2+ hydrogel precursor when cured by UV or entrapped in the Alg-Ca 2+ as a separate hydrogel layer cured on top of the preformed PAAm/Alg-Ca 2+ hydrogel array.…”

“…After each assay was completed, the insert was removed from the tube and placed in a PULUZ photo light box with constant top-down light for imaging. The images were converted into the grayscale mode with light-dark inversion, as in our previous methodology …”

“…We have previously established the vortex fluidic device (VFD)-accelerated immunoblot assay using a nitrocellulose membrane with a 10-fold acceleration in assay efficiency. 4 While this improves the cost-efficacy, further improvement is required because (i) the immunoblot nitrocellulose remains fragile and expensive, (ii) the spots only use a small amount of membrane area, which can add cost and waste, and (iii) the standard VFDs are cumbersome and not ideal for quick or onsite detection. Our previous works have also indicated that VFD is capable of facilitating affinity binding 5 in driving reactions in a hydrogel environment, 6 to develop hydrogel arrays as a high-throughput pathway to improve assay efficiency.…”

Diverse Supports for Immobilization of Catalysts in Continuous Flow Reactors

“…Raston and coworkers have developed the field of VFDs and widely expanded their applications in diverse areas (Figure 11). [94][95][96][97][98][99][100][101][102] In this section, recent contributions involving immobilization of catalytic units on tube walls via supports are selected and discussed (Figure 11c).…”