Measurements of Label Free Protein Concentration and Conformational Changes Using a Microfluidic UV‐LED Method

Abstract

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

“…16 However, the addition of fluorescent labels to proteins can affect their ligand binding, solubility and stability, and it is also difficult to ensure the attachment of only a single label per protein molecule. 17,18 The measurement of intrinsic protein fluorescence has also been demonstrated previously in microchannels for the label-free detection of proteins and their conformational changes, by using lasers [19][20][21] or light-emitting diodes (LED's) 22,23 for the excitation of samples. However, these techniques have not yet been sufficiently accurate to obtain thermodynamic parameters such as the free-energy of protein unfolding, as is required for use in drug-discovery, formulation or protein engineering applications.…”

Parallel Detection of Intrinsic Fluorescence from Peptides and Proteins for Quantification during Mass Spectrometric Analysis

“…In previous studies, interrogation of biomolecules by UV-IF and LC-MS utilized LC flow-splitting, microliter flow rates, microfluidic devices, or separate analyses 23,26,27. However, LC-MS analyses are routinely performed in capillary columns in the nanoflow regime for enhanced ESI-MS sensitivity 13.…”

Electroosmosis Performance Analysis of Microchip Electrophoresis Fabricated by the Ultra-precision CNC Milling

“…), compares favorably with the EOF value of the LIGA based PMMA chip ( )[6].4. SURFACE XPS ANALYSIS OF PMMA…”