A fucosyltransferase inhibition assay using image-analysis and digital microfluidics

Abstract

Sialyl-LewisX and LewisX are cell-surface glycans that influence cell-cell adhesion behaviors. These glycans are assembled by α(1,3)-fucosyltransferase enzymes. Their increased expression plays a role in inflammatory disease, viral and microbial infections, and cancer. Efficient screens for specific glycan modifications such as those catalyzed by fucosyltransferases are tended toward costly materials and large instrumentation. We demonstrate for the first time a fucosylation inhibition assay on a digital microfluidic system with the integration of image-based techniques. Specifically, we report a novel lab-on-a-chip approach to perform a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic disaccharide, 4-methylumbelliferyl β-N-acetyllactosaminide. As a proof-of-concept, guanosine 5 0 -diphosphate has been used to inhibit Helicobacter pylori α(1,3)-fucosyltransferase. An electrode shape (termed "skewed wave") is designed to minimize electrode density and improve droplet movement compared to conventional square-based electrodes. The device is used to generate a 10 000-fold serial dilution of the inhibitor and to perform fucosylation reactions in aqueous droplets surrounded by an oil shell. Using an image-based method of calculating dilutions, referred to as "pixel count," inhibition curves along with IC 50 values are obtained on-device. We propose the combination of integrating image analysis and digital microfluidics is suitable for automating a wide range of enzymatic assays.

Digital Microfluidics Chips for the Execution and Real-Time Monitoring of Multiple Ribozymatic Cleavage Reactions

“…4,54 However, regions with pointy edges and high electric fields (>108 V/cm) are known to cause a dielectric breakdown, 55 while ″skewed-wave″ edged electrodes have been proven to resolve this problem. 26 Droplet motion on the chip was achieved by actuating the electrodes for 0.7 s at a voltage of 178 V (15 kHz sine wave). To dispense droplets, a larger droplet was initially stretched by actuating four electrodes from the reservoir.…”

“…Adding surfactants to such solutions have proven to be effective even without any filler oil. 26 Hence, 0.05% Tetronic 304 was added to the aqueous cleavage buffer. Although the surfactant resulted in the improved movement of the cleavage buffer droplets (similar to our previous study 26 ), it was observed in this study that the droplets had left traces along their paths, which made chip reuse quite difficult.…”

“…26 Hence, 0.05% Tetronic 304 was added to the aqueous cleavage buffer. Although the surfactant resulted in the improved movement of the cleavage buffer droplets (similar to our previous study 26 ), it was observed in this study that the droplets had left traces along their paths, which made chip reuse quite difficult. Therefore, the reactions were done at larger volumes (2.5 μL) by directly dispensing cleavage buffer droplets on the detection points (to enable reusability of the chip) and then merging them with ribozyme-containing droplets (Figure 1).…”

“…25 Moreover, the portability and flexibility of these platforms when coupled with detection mechanisms such as fluorescence have made it easier to interface the devices with machines such as plate readers to automate entire experiments. 26 Ribozymes are enzymes composed of ribonucleic acid (RNA) molecules capable of catalyzing specific biochemical reactions within cells and in vitro. These molecules excise themselves 27 or a substrate 28 monitor the cleavage kinetics of different ribozymes.…”

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

“…Digital microfluidics (DMF) can alleviate the challenges associated with droplet‐in‐channel systems since DMF are able to address each droplet individually. Having this control is especially useful in multi‐step procedures, such as transformation and enzymatic assays, [ 42,43 ] drug and inhibitor screening, [ 44 ] and gene‐editing [ 27 ] Recently, we have combined both of these platforms together, in which we call a “hybrid” microfluidics, placing co‐planar electrodes (i.e., ground and activated electrodes on the same plate) under a network of microfluidic channels to enable individual control of the droplets in the channels. [ 45 ] Given the increased control over droplets and their contents, [ 46–51 ] there is an opportunity to use this hybrid‐based technology as a method to control the isolation of mammalian isoclones.…”