Bead‐based Enzymatic Assay On‐a‐chip

Obesity is a complex metabolic disorder which leads to several other physiological complications. Microfluidics is an upcoming field which uses microphysiological environments also known as organs‐on‐a‐chip that can be controlled using less space, low volumes of medium, and cells. One of their biggest advantages is that organs‐on‐chips can be used to study the interrelation between the tissue types of interest but eliminating the cross‐talk between the various other tissues that cannot be controlled in in vivo studies. Use of microfluidics generates low volumes of sample analytes which can be challenging to analyze using the traditional biochemical assays requiring large of sample volumes. Enzyme based assays are important for clinical measurements and in in vitro systems to assess glucose and lipid metabolism. Measurement of cholesterol and other lipids is carried out by etherification and oxidation using enzymes to esterify and oxidize the lipids to form hydrogen peroxide which can be detected either colorimetrically or fluorimetrically. Detection of secretory lipids using conventional methods requires volumes ranging from 20–50ul, whereas microfluidic devices deal with volumes between 1–5ul, making it challenging to carry out conventional biochemical assays, hence, the need to develop an enzymatic assay that can detect the biomolecule of interest in low sample volumes. Clark et al (2009) used lipid detection enzymatic assay for glycerol in a microfluidic device in their studies on perfusion chamber cultured adipocytes. Schrell et al (2016) detail the use of microfluidic devices and assays for quantifying the cellular secretions. These approaches use enzyme based assays which form fluorescent solutions. The detection is achieved by photomultiplier tube and laser beam as illumination source similar to the microplate readers used for conventional biochemical analysis. The main drawback of this approach is complex data processing and analysis. We have designed a bead‐based enzymatic assay that takes place in a microfluidic device. We use the assay to measure the lipids secreted from both cells that are also cultured on a microfluidic device. Using beads for the enzymatic assay addresses the problem of the conventional assays requiring high volumes of samples and reagents and to detect the lipid secretion temporally. The detection is simple because it uses standard fluorescent microscopy to capture the individual fluorescent beads. The images were analyzed using ImageJ, which enabled us to quantify the lipids secreted by the cells. Our assay is simpler than current techniques and is very versatile for all enzyme based assays such as for glucose, triglyceride, and cholesterol. Figure 1 represents our enzymatic bead assay in microfluidic device. The commercially available Amplex UltraRed forms fluorescent dye Resorufin after reacting with hydrogen peroxide in the presence of HRP was chosen for our study as it is highly sensitive and can detect low concentrations (2μM) of the lipids in the sample. Our study showed that the assay retained its sensitivity after immobilizing the enzyme on the beads and measured the cellular secretion in near real‐time. The assay can serve as a model for transforming enzymatic‐assays used for glucose and other lipids in microfluidic format to measure cellular response to inflammatory cytokines, hormones, and drugs. Support or Funding Information This work was supported by NIH grant (DK095984). 1Enzymatic assay to detect cholesterol and other lipids on a bead in a microfluidic device.