A ready-to-use, versatile, multiplex-able three-dimensional scaffold-based immunoassay chip for high throughput hepatotoxicity evaluation

Abstract

Hydrogel as three-dimensional (3D) substrate has been employed in miniaturized high throughput protein detection platforms to increase the number of effective antibodies and signal augmentation. However, the high water content of the hydrogel can dilute samples and create barrier to mass transfer, limiting hydrogel height to several microns in most platforms. Moreover, these platforms cannot achieve widespread use in common laboratories as they usually rely heavily on expensive robotic liquid handlers and custom-built components. Here we developed a ready-to-use, easy to store and handle, versatile and multiplex-able 3D scaffold-based immunoassay chip (3D immunoChip) possible for high throughput protein quantification using bench-top equipment in common laboratories. Sample dilution, mass transfer, signal scattering and storage problems can be avoided by using dry scaffolds that regain transparency upon rehydration. When combined with hydrophilic-hydrophobic patterned reagent loading slides, manual high throughput handling of samples can be achieved. As these micro-scaffolds are patterned without barriers in between, simultaneous and effortless washing of all the reaction zones is possible in a Petri dish. Such features aid the 3D immunoChip in saving up to 100 times reagent and about 6 times labour. The 3D immunoChip is able to detect albumin (ALB), as a model analyte, from 5 ng mL(-1) to 1000 ng mL(-1), making it comparable to the commercialized ELISA kit based on a 96-well plate (0.22-400 ng mL(-1)). This thus enables the 3D immunoChip to directly detect ALB secreted by HepaRG cells cultured in a 3D cell culture array chip for high throughput drug hepatotoxicity evaluation, which could potentially accelerate drug screening.

The Role of Tumor Microenvironment in Chemoresistance: 3D Extracellular Matrices as Accomplices

“…Ovarian cancer cells also show increased chemoresistance when grown as 3D spheroids compared to 2D culture [ 139 , 140 , 141 , 142 ]. It has now been established that 3D scaffolds generally better imitate the in vivo tumor microenvironment necessary for modelling cancer cell-ECM interactions and also for cancer cell-drug screening assays [ 136 , 143 , 144 , 145 , 146 , 147 ]. The interaction between cancer cells and their surrounding microenvironment plays a significant role in the acquisition of drug resistance in many cancers.…”

A Microfabricated Sandwiching Assay for Nanoliter and High‐Throughput Biomarker Screening

“…It has been shown that a microfluidic device with in‐channel micropatterning of antibodies enables highly sensitive and fast detection of biomolecules secreted from cells with samples of a few microliters. In addition, hydrogel microarrays have been employed as a reproducible and reliable high‐throughput immunoassay system . For instance, antibody‐containing lyophilized hydrogels were developed to evaluate hepatotoxicity responses to introduced drugs .…”

“…In addition, hydrogel microarrays have been employed as a reproducible and reliable high‐throughput immunoassay system . For instance, antibody‐containing lyophilized hydrogels were developed to evaluate hepatotoxicity responses to introduced drugs . By sandwiching cell culture chip microarrays and drug‐loaded immunochips, cell secreted biomarkers could be detected.…”

Development of cell metabolite analysis on microfluidic platform

“…Long-term culture was maintained as long as 30 days at single cell level, with the ability to retrieve any cell of interest for further examination. Collagen based 3D microtissue culture models for cell viability assessment [27] or immunoassay of cytotoxicity [28] under drug stimulation were also reported, which were compatible to microplate reader. This platform provides a high throughput and quantitative method for drug screening and better prediction of drug efficacy at the tissue level.…”