Time-resolved analysis of biological reactions based on heterogeneous assays in liquid plugs of nanoliter volume

Anal Chem. 2013 Oct 15;85(20):9469-77. doi: 10.1021/ac401752j. Epub 2013 Oct 1.

Abstract

In this article, we present a concept which uses liquid plugs as reaction volumes for heterogeneous assay reactions to facilitate time-resolved analysis of biomolecular reactions. For this purpose, the reaction is first compartmentalized to a train of many identical plugs. Therefore, we established a simple fluidic setup build from off-the-shelf available tubing and connectors. It permits reliable formation of plugs and successive dosing of further assay reagents to these compartments (plug volume <5% CV). The time course of the reaction is obtained by routing the plugs successively through a detector. Thereby, the arrival time of a given plug at the detector represents the reaction time of the overall reaction at that moment. Thus, each analyzed plug represents a discrete state of the overall reaction. With this approach, we can achieve a temporal resolution as small as one second, which hardly can be met by conventional analytical methods for analysis of endogenous biological compounds. For analysis of the content of the plugs, we developed a method which allows for heterogeneous assays in two-phase flow. For this purpose, functionalized superparamagnetic beads are enclosed in the plugs for specific binding of the assay product. Purification from supernatant species is achieved by transferring the beads with bound analyte across the phase boundary between aqueous plugs and water-immiscible carrier fluid. We demonstrate this assay principle exemplarily for a sandwich immunoassay (cytokine IL-8). Time-resolved analysis is validated by monitoring a cell-free in vitro expression reaction (turboGFP) in plugs and conventionally in bulk solution. We show that our approach allows for analyzing the entire course of a reaction in a single run. It permits kinetic studies of biological processes with significantly reduced experimental effort and consumption of costly reagents.

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Cattle
  • Immunoassay
  • Interleukin-8 / analysis
  • Nanotechnology / methods*
  • Time Factors

Substances

  • Interleukin-8