Label-Free Protein Analysis Using Liquid Chromatography with Gravimetric Detection

Abstract

The detection and analysis of proteins in a labelfree manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 μg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.

Analysis of the Effect of Electrode Materials on the Sensitivity of Quartz Crystal Microbalance

“…It is noteworthy that researchers nowadays mainly focus on application-oriented studies of QCM, such as the vacuum film thickness monitor [ 7 ]; Noi et al combined total internal reflection fluorescence microscopy with a quartz crystal microbalance to directly monitor the catabolic response of anthocyanins to individual amyloid β (Aβ) fibers [ 2 ]; Yao et al combined QCM and moisture-sensitive materials to make a QCM humidity sensor [ 8 , 9 , 10 ]; and Kartanas et al combined liquid chromatography with a QCM to achieve label-free quantitative analysis of protein mixtures [ 11 ]. The Sauerbrey equation is the basis for the study of QCM sensors [ 12 ], where , , and represent the frequency shift, mass change, and mass sensitivity, respectively.…”

A Pure Shift-Based Nuclear Magnetic Resonance Method for In-Phase Three-Dimensional Diffusion-Ordered Spectroscopy

“…Diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY) provides an effective analytical tool for studying mixtures by acquiring diffusion signals of individual components and separating these signals into different spectra based on their diffusion coefficient differences, thus achieving component separation and identification of mixtures in a noninvasive manner. − By contrast, other analytical methods for mixture studies are generally accompanied by physical sample pretreatments, such as sample extraction and gas and liquid chromatography . The performance of component separation by DOSY relies on the fact that signals acquired from the identical component theoretically have the same diffusion behavior.…”

Single-atom niobium doped BCN nanotubes for highly sensitive electrochemical detection of nitrobenzene

“…Numerous methods have been developed for detecting NB in water systems, which employ techniques such as high-phase liquid chromatography, meteorological chromatography, and electrochemical analysis. 8,9 Although rapid and selective NB detection is achievable through chromatography, several disadvantages including low recognition ability, complex operation, and bulky equipment prevent its application as a portable technique for on-site detection. 10 In contrast, electrochemical methods allow for rapid, portable, convenient analysis as well as exhibit low limits of detection (LODs).…”