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Microfluidic diffusional sizing

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Microfluidic diffusional sizing (MDS) is a method to measure the size of particles based on the degree to which they diffuse within a microfluidic laminar flow.[1] It allows size measurements to be taken from extremely small quantities of material (nano-grams) and is particularly useful when sizing molecules which may vary in size depending on their environment - e.g. protein molecules which may unfold or become denatured in unfavourable conditions.

Applications

MDS is primarily used in protein analyses, where size, concentration and interactions are important.

Protein size measurement

Measuring the size of a protein molecule is useful as an overall quality indicator, since misfolding, unfolding, oligomerization, aggregation or degradation can all affect size.

The literature specifically demonstrates the use of MDS in sizing protein-nanobody complexes, monitoring the formation of α-synuclein amyloid fibrils.[2] and in observing protein assembly into oligomers[3]

MDS can also be used to size membrane proteins, as the use of a protein specific labelling and detection system allows other species present in the solution (such as free lipid micelles or detergents) to be ignored.

Protein interactions

MDS has been used to characterise interactions between biomolecules under native conditions, and has been demonstrated to detect specific interactions within complex mixtures.[4] It has also been used in detecting and quantifying protein-ligand interactions[5] and protein-lipid interactions.[6]

Protein concentration

The concentration of purified protein solutions in the laboratory is useful in determining yield and measuring the success of a prep. MDS reports concentration as well as size for each test.

Since the detection is not based on inherent fluorescence of tryptophan or tyrosine residues, MDS has been used as an alternative to A280 UV-Vis quantification.[7]

Discover more about Applications related topics

Protein folding

Protein folding

Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproducible process, a polypeptide folds into its characteristic three-dimensional structure from a random coil. Each protein exists first as an unfolded polypeptide or random coil after being translated from a sequence of mRNA to a linear chain of amino acids. At this stage the polypeptide lacks any stable (long-lasting) three-dimensional structure. As the polypeptide chain is being synthesized by a ribosome, the linear chain begins to fold into its three-dimensional structure.

Oligomer

Oligomer

In chemistry and biochemistry, an oligomer is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers. The name is composed of Greek elements oligo-, "a few" and -mer, "parts". An adjective form is oligomeric.

Protein aggregation

Protein aggregation

Protein aggregation is a biological phenomenon in which intrinsically disordered proteins or mis-folded proteins aggregate either intra- or extracellularly. Mis-folded protein aggregates are often correlated with diseases. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including ALS, Alzheimer's, Parkinson's and prion disease.

Tryptophan

Tryptophan

Tryptophan is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic beta carbon substituent. It is essential in humans, meaning that the body cannot synthesize it and it must be obtained from the diet. Tryptophan is also a precursor to the neurotransmitter serotonin, the hormone melatonin, and vitamin B3. It is encoded by the codon UGG.

Tyrosine

Tyrosine

L-Tyrosine or tyrosine or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the Greek tyrós, meaning cheese, as it was first discovered in 1846 by German chemist Justus von Liebig in the protein casein from cheese. It is called tyrosyl when referred to as a functional group or side chain. While tyrosine is generally classified as a hydrophobic amino acid, it is more hydrophilic than phenylalanine. It is encoded by the codons UAC and UAU in messenger RNA.

Ultraviolet–visible spectroscopy

Ultraviolet–visible spectroscopy

UV spectroscopy or UV–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.

Advantages

If protein specific labelling is applied, MDS allows membrane proteins to be sized. This is particularly useful as it is an area where other biophysical techniques can struggle - for example dynamic light scattering (DLS) is of limited use, since free detergent molecules may also scatter light and affect the results.[8]

Furthermore, as the size reported is an average of all detectable species present there is no bias towards large species, as is found in DLS measurements.[9]

Another key advantage is that results can be obtained with very small quantities of material[10] which may be particularly important where samples are scarce or expensive.

With commercially available MDS instruments, testing is very simple and there is no need to input test parameters or sample conditions. This makes it a very repeatable method of testing as most of the functions such as flow rates, detector settings etc. are automated by the instrument rather than set by the operator.

In addition to size, MDS is able to calculate concentration so two parameters can be assessed in one test.

Finally the method does not require calibration, as it relies on a ratio-metric measurement to determine diffusion rate.[11]

Theory

In an MDS analysis, a stream of liquid containing the particles to be sized is introduced alongside an auxiliary stream in a laminar flow in a microfluidic channel. Because there is no convective mixing of the two streams, the only way particles can move to the auxiliary stream is by diffusion. The rate of this diffusion is dependent on the particle's size, as determined by the Stokes–Einstein equation, so small particles diffuse quicker than large particles.

After a period of diffusion the original and auxiliary streams are split and the degree of diffusion is fixed. The number of particles in each stream can then be detected (in the case of proteins this is achieved by addition of an amine reactive fluorogenic dye). The ratio between the two streams is used to determine the diffusion co-efficient, which is used to calculate the hydrodynamic radius. The sum of particles in both streams can also be used to measure the concentration of the analyte.[12]

Source: "Microfluidic diffusional sizing", Wikipedia, Wikimedia Foundation, (2022, November 25th), https://en.wikipedia.org/wiki/Microfluidic_diffusional_sizing.

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References
  1. ^ "What is Microfluidic Diffusional Sizing?". www.fluidic.com. Retrieved 2019-01-02.
  2. ^ Zhang, Yingbo (2016). "Protein Aggregate‐Ligand Binding Assays Based on Microfluidic Diffusional Separation". ChemBioChem. 17 (20): 1920–1924. doi:10.1002/cbic.201600384. PMID 27472818. S2CID 23410743.
  3. ^ Wright, Maya (2018). "Cooperative Assembly of Hsp70 Subdomain Clusters". Biochemistry. 57 (26): 3641–3649. doi:10.1021/acs.biochem.8b00151. PMC 6202011. PMID 29763298.
  4. ^ Arosio, Paolo (2016). "Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions". ACS Nano. 10 (1): 333–341. doi:10.1021/acsnano.5b04713. PMID 26678709.
  5. ^ Herling, Therese W. (2016). "A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions". Biophysical Journal. 110 (9): 1957–1966. Bibcode:2016BpJ...110.1957H. doi:10.1016/j.bpj.2016.03.038. PMC 4939750. PMID 27166804 – via NCBI.
  6. ^ Gang, Hongze (2018). "Microfluidic Diffusion Platform for Characterizing the Sizes of Lipid Vesicles and the Thermodynamics of Protein–Lipid Interactions". Analytical Chemistry. 90 (5): 3284–3290. doi:10.1021/acs.analchem.7b04820. PMID 29313342.
  7. ^ "Quantitation of low Tryptophan and Tyrosine peptides using the Fluidity One".
  8. ^ Jensen, Grant (2010). Methods in Enzymology - Volume 481 Cryo-EM, Part A. Elsevier. p. 28. ISBN 978-0-12-374906-2.
  9. ^ Stetefeld, Jorg (2016). "Dynamic light scattering: a practical guide and applications in biomedical sciences". Biophysical Reviews. 8 (4): 409–427. doi:10.1007/s12551-016-0218-6. PMC 5425802. PMID 28510011.
  10. ^ "Fluidity One". fluidic.com.
  11. ^ "How do I calibrate the Fluidity One?". fluidic.com.
  12. ^ "What is Microfluidic Diffusional Sizing?". fluidic.com.

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