Microfluidic protein detection and quantification using droplet morphology

Abstract

Sensitive, inline detection of proteins is required for post-chromatographic analyses in proteomics, cell-based assays, and drug discovery workflows. Among the common inline methods, post-column derivatization requires chemical labels, while label-free methods are either expensive (mass spectrometry) or have limited sensitivity at small length scales (UV–Vis). This paper presents a label-free detection technique based on the concept that dissolved proteins can function as surfactants and decrease the dynamic interfacial tension (IFT) of an immiscible (water–oil) interface. Existing methods for measuring IFT, such as axisymmetric drop shape analysis (ADSA), operate in batch mode and are not suitable for continuous detection. Here we show that a microfluidic flow-focusing droplet generator operating at a frequency of > 100 Hz can track IFT changes continuously, with high temporal resolution and small detection volumes. Variations in protein concentration alter the size and shape of the drops/plugs formed, and these changes can be quantified in time using a high-speed camera and in-house image processing software. Moreover, the continuously refreshing interface alleviates issues related to surface aging. Two applications are demonstrated: (1) direct injection of a single protein into a microfluidic chip. (2) post-column detection of protein mixtures separated by high performance size exclusion chromatography (SEC HPLC). Of interest, the dynamic range of protein (bovine serum albumin, BSA) was 50 -104 μg/ml without using HPLC unit. The lowest limit of detection without HPLC unit was ~ 1 μg/ml of thyroglobulin protein in a 1 nl droplet, which equates to 1 fg of total protein. When used as a detector, the aforementioned detection method offered a sensitivity of six orders of magnitude higher than conventional UV–VIS detectors.