Detection of Single Enzymatic Events in Rare or Single Cells Using Microfluidics

Abstract

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling circle-enhanced enzyme activity single molecule detection assay with a custom designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in vast number of single cells thereby offering a valuable tool for basic research as well as theranostics.

Extraction of active enzymes from “hard-to-break-cells”: Evaluation by a RCA-based assay

“…Inspired by our previous finding that chemical reaction appears to be relatively more efficient in small microincubators than in large volume (such as in eppendorf tube) [9,10], we tested the enzyme extraction efficiency in microdroplets based extraction. As evident in Figure 3a, more signals, indicating higher enzymatic activity, was preserved from the microdroplets-based extraction, compared to the bead-beating lysis and bulk mixing.…”

One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis

“…26 Droplets of water in oil not only allow for the accumulation of products of single-cell enzymatic reactions in the same compartment as the genome but also provide an opportunity for the amplified detection of single enzymatic events from single cells. 96 In a recently reported assay, rolling circle amplification serves as an amplification method and empowers the detection of single DNA cleavage-ligation events. 96 Moreover, droplet microfluidics allows for fast screening of large libraries (10 8 in 10 h), 97 and studies focusing on the binding affinity of proteins have shown that the size of the library screened is correlated with the improvement achieved in the affinity of the selected proteins.…”

“…96 In a recently reported assay, rolling circle amplification serves as an amplification method and empowers the detection of single DNA cleavage-ligation events. 96 Moreover, droplet microfluidics allows for fast screening of large libraries (10 8 in 10 h), 97 and studies focusing on the binding affinity of proteins have shown that the size of the library screened is correlated with the improvement achieved in the affinity of the selected proteins. 98 Similarly, these types of correlations are also expected in studies on enzymes.…”

Refined Method for Droplet Microfluidics-Enabled Detection of Plasmodium falciparum Encoded Topoisomerase I in Blood from Malaria Patients

“…Two syringe pumps (Harvard Apparatus, Holliston, MA, USA) were used to control the flow rates of oil/surfactant and reagents independently. The droplet volume and generation frequency were controlled by the flow rate ratio, determined by the competition between continuous phase and disperse phase [2,6,7]. Subsequently, blood and lysis buffer (10 mM Tris pH 7.5, 5 mM EDTA supplemented with protease inhibitors) were subjected to droplet microfluidics essentially as described previously [2].…”