Open AccessJournal Article10.1039/C8LC00757H

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism.

- 01 Jan 2019

- Vol. 19, Iss: 1, pp 50-58

TL;DR: Free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism is used to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils to enable complex mixtures such as protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.

Abstract: Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.

AI Agents for this Paper

Find similar papers on Google Scholar, PubMed and Arxiv
Write a critical review of this paper
Analyze citations of this paper to find unaddressed research gaps

Most frequently asked questions

1. What are the contributions mentioned in the paper "Resolving protein mixtures using microfluidic diffu- sional sizing combined with synchrotron radiation cir- cular dichroism" ?

In this paper, the authors use the laminar flow properties inherent to operation in microfluidic devices to separate a complex mixture into well-resolved size-dependent fractions, using an H-filter configuration.

2. What have the authors stated for future works in "Resolving protein mixtures using microfluidic diffu- sional sizing combined with synchrotron radiation cir- cular dichroism" ?

Current biophysical methods and devices are usually not well-suited to study, in molecular details, heterogeneous mixtures in their native environment while retaining the temporal information.. In summary, the possibility to precisely separate a heterogeneous mixture into well-resolved, simpler fractions opens up a range of opportunities for the study of complex biological phenomena.. In particular, such developments open up interesting perspective to study protein misfolding and aggregation diseases such as Alzheimer ’ s and Parkinson ’ s diseases and may provide time-resolved information about the protein aggregation pathway.. In this manuscript, the authors show that the combination of microfluidic free-flow separation and label-free bulk measurement techniques such as synchrotron radiation circular dichroism can overcome such issues.

Fig. 4 Concentration profile of a solution of insulin monomers (2 mg/ml) across a 2000 µm wide microfluidic channel with flow rates of 30 and 300 µ l/h for the monomers and buffer solution respectively. The high energy photon beam was positioned using the motorised stage in 5 locations across the channel and the SRCD signal amplitude, taken at 208 nm, was measured continuously during one minute and averaged. The measured profile shows a good agreement with the expected (simulated) profile.

Fig. 3 Validation of the diffusion based separation using simple solutions of insulin monomers. (a) Simplified sketch of the device used with indication of the low (black circle) and high (grey circle) diffusion chambers. (b) Diffusion profile and fractions collected at the end of the diffusion channel. (c) SRCD spectra of insulin monomer in each chamber.

Fig. 6 Resolution of mixtures of insulin monomer and fibrils using analytical diffusion-based microfluidics separation and SRCD. It is not possible to resolve the mixture from its SRCD spectrum (a, left). However, by isolating the monomers (a, right), it is possible to reconstruct the fibrils spectrum after subtracting the spectrum of the resolved fraction from the that of the total mixture. (b). The spectrum shows good agreement with the measured spectrum in the same conditions (b) as confirmed by the analysis of their secondary structures (c).

Fig. 1 Schematic of the measurement principle. The combination of microfluidics-based free-flow separation with bulk measurement technique enables the study, in molecular details, of heterogeneous mixtures in their native environment while retaining the temporal information. In the manuscript we combine diffusion-based separation with the ability to measure SRCD using the highly collimated microbeam of B23 beamline to study proteins mixtures. (a) The complex mixture, here made up of monomers and fibrils, is flown alongside a buffer solution. The monomers, with a smaller hydrodynamic radius, diffuse faster than the fibres, enabling the separation of the mixture into well-defined fractions that can be studied separately. (b) Sketch of the diffusion-based separation microfluidic device and microbeam light to probe each fraction. (c) Diffusion profile of the monomers and fibrils normalised concentrations at the end of the microfludics device diffusion length. The fractions collected depend on the hydrodynamic resistance of the separation channels (normalised channel width).

Fig. 2 Schematic of a typical diffusion devices for the 2 architectures presented. In each case the measurement chambers are confined between quartz windows. The masters are represented on the left and the cross-section of the resulting device is show on the right (a) Architecture 1 consists in two-layer devices. The height of the measurement chamber (equivalent to the path length, l) can be tuned by adapting the height of each PDMS layer. (b) Architecture 2 is based on one-master, one-layer devices. The path length, l, is defined by the height of the channels.

Table 1 Comparison between expected (calculated) and measured fraction of insulin monomer and fibres in each chamber. A reference value of 208 nm was chosen for the monomers as it corresponds to the π → π* excitonic transition of α-helices; 222 nm is representative of the n → π* of β -sheet rich compounds such as insulin fibres.

Citations

Cryo-EM structures of tau filaments from Alzheimer's disease

Anthony W. P. Fitzpatrick, +9 more

- 13 Jul 2017

TL;DR: It is demonstrated that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

...read moreread less

652

•Journal Article•10.1038/S41378-019-0080-3

Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

Oliver Vanderpoorten, +6 more

- 09 Sep 2019

- Microsystems & Nanoengineering

TL;DR: A hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices, enabling rapid nanof LU device fabrication and enhancement of existing microfluidic device architectures with nanofLUidic features.

...read moreread less

•Journal Article•10.1007/S12551-020-00679-4

Microfluidic approaches for the analysis of protein-protein interactions in solution.

William E. Arter, +3 more

- 08 Apr 2020

- Biophysical Reviews

TL;DR: The microfluidic tools described here provide the capability to yield insights on protein–protein interactions under physiological conditions, and are highlighted by highlighting future directions for the field, such as the integration of microfluids into high-throughput workflows for the investigation of protein interaction networks.

...read moreread less

•Journal Article•10.1002/JCTB.6920

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

M. Neves Sao Pedro, +3 more

- 30 Sep 2021

- Journal of Chemical Technology & Biotech...

TL;DR: In this paper, a review of existing methods to detect protein aggregation, such as dynamic light scattering, Raman spectroscopy and circular dichro-ism, is presented.

...read moreread less

•Journal Article•10.3390/MI11020135

Low-Cost Microfabrication Tool Box.

Jerome Charmet, +6 more

- 25 Jan 2020

- Micromachines

TL;DR: A range of low-cost approaches and equipment, including photolithography, micromilling, 3D printing, xurography and screen-printing used for the microfabrication of structural and functional materials are described.

...read moreread less

References

•Journal Article•10.1038/NPROT.2006.202

Using circular dichroism spectra to estimate protein secondary structure

Norma J. Greenfield

- 01 Jan 2006

- Nature Protocols

TL;DR: This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins.

...read moreread less

3.7K

Journal Article•10.1038/416507A

Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

Monica Bucciantini, +10 more

- 04 Apr 2002

- Nature

TL;DR: This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

...read moreread less

2.6K

Journal Article•10.1038/NRM3810

The amyloid state and its association with protein misfolding diseases

Tuomas P. J. Knowles, +2 more

- 01 Jun 2014

- Nature Reviews Molecular Cell Biology

TL;DR: The ability to form the amyloid state is more general than previously imagined, and its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.

...read moreread less

2.1K

•Journal Article•10.1038/NATURE23002

Cryo-EM structures of tau filaments from Alzheimer’s disease

Anthony W. P. Fitzpatrick, +9 more

- 05 Jul 2017

- Nature

TL;DR: Scheres et al. as mentioned in this paper presented a 3.4-3.5-resolution image of the brain of an individual with Alzheimer's disease and showed that the protein cores are made of two identical protofilaments comprising residues 306-378 of tau protein.

...read moreread less

1.7K

Journal Article•10.1126/SCIENCE.1105850

Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils

Aneta T. Petkova, +5 more

- 14 Jan 2005

- Science

TL;DR: Using electron microscopy and solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue β-amyloid peptide of Alzheimer's disease (Aβ1–40), it is shown that different fibril morphologies have different underlying molecular structures, that the predominant structure can be controlled by subtle variations infibril growth conditions, and that both morphology and molecular structure are self-propagating when fibrs grow from preformed seeds.

...read moreread less

1.7K

...

Expand

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism.

Chat with Paper

AI Agents for this Paper

Most frequently asked questions

1. What are the contributions mentioned in the paper "Resolving protein mixtures using microfluidic diffu- sional sizing combined with synchrotron radiation cir- cular dichroism" ?

2. What have the authors stated for future works in "Resolving protein mixtures using microfluidic diffu- sional sizing combined with synchrotron radiation cir- cular dichroism" ?

Figures

Citations

Cryo-EM structures of tau filaments from Alzheimer's disease

Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

Microfluidic approaches for the analysis of protein-protein interactions in solution.

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

Low-Cost Microfabrication Tool Box.

References

Using circular dichroism spectra to estimate protein secondary structure

Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

The amyloid state and its association with protein misfolding diseases

Cryo-EM structures of tau filaments from Alzheimer’s disease

Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils

Related Papers (5)

In Situ Measurements of Polypeptide Samples by Dynamic Light Scattering: Membrane Proteins, a Case Study.

Laser Light Scattering as an Indispensable Tool for Probing Protein Aggregation

Protein Sizing and Conformation Analysis with an Electro-Switchable DNA Chip

Rapid injection linear dichroism for studying the kinetics of biological processes.

Polymeric microfluidic continuous flow mixer combined with hyperspectral FT-IR imaging for studying rapid biomolecular events.