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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.
MDS is primarily used in protein analyses, where size, concentration and interactions are important.
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.
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]
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]
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]
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]
A biological membrane, biomembrane or cell membrane is a selectively permeable membrane that separates the interior of a cell from the external environment or creates intracellular compartments by serving as a boundary between one part of the cell and another. Biological membranes, in the form of eukaryotic cell membranes, consist of a phospholipid bilayer with embedded, integral and peripheral proteins used in communication and transportation of chemicals and ions. The bulk of lipids in a cell membrane provides a fluid matrix for proteins to rotate and laterally diffuse for physiological functioning. Proteins are adapted to high membrane fluidity environment of the lipid bilayer with the presence of an annular lipid shell, consisting of lipid molecules bound tightly to the surface of integral membrane proteins. The cell membranes are different from the isolating tissues formed by layers of cells, such as mucous membranes, basement membranes, and serous membranes.
In cell biology, the cytoplasm describes all material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The main components of the cytoplasm are the cytosol, the organelles, and various cytoplasmic inclusions. The cytoplasm is about 80% water and is usually colorless.
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Microfluidics refers to a system that manipulates a small amount of fluids using small channels with sizes ten to hundreds micrometres. It is a multidisciplinary field that involves molecular analysis, molecular biology, and microelectronics. It has practical applications in the design of systems that process low volumes of fluids to achieve multiplexing, automation, and high-throughput screening. Microfluidics emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro-propulsion, and micro-thermal technologies.
The molecular mass (m) is the mass of a given molecule. The unit dalton (Da) is often used. Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The derived quantity relative molecular mass is the unitless ratio of the mass of a molecule to the atomic mass constant (which is equal to one dalton).
Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
The fluid mosaic model explains various characteristics regarding the structure of functional cell membranes. According to this biological model, there is a lipid bilayer in which protein molecules are embedded. The phospholipid bilayer gives fluidity and elasticity to the membrane. Small amounts of carbohydrates are also found in the cell membrane. The biological model, which was devised by Seymour Jonathan Singer and Garth L. Nicolson in 1972, describes the cell membrane as a two-dimensional liquid that restricts the lateral diffusion of membrane components. Such domains are defined by the existence of regions within the membrane with special lipid and protein cocoon that promote the formation of lipid rafts or protein and glycoprotein complexes. Another way to define membrane domains is the association of the lipid membrane with the cytoskeleton filaments and the extracellular matrix through membrane proteins. The current model describes important features relevant to many cellular processes, including: cell-cell signaling, apoptosis, cell division, membrane budding, and cell fusion. The fluid mosaic model is the most acceptable model of the plasma membrane. In this definition of the cell membrane, its main function is to act as a barrier between the contents inside the cell and the extracellular environment.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed using the intensity or photon autocorrelation function. In the time domain analysis, the autocorrelation function (ACF) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. It has been shown that the intensity ACF is the Fourier transform of the power spectrum, and therefore the DLS measurements can be equally well performed in the spectral domain. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions.
In biology, membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane or a synthetic lipid membrane. Lipid packing can influence the fluidity of the membrane. Viscosity of the membrane can affect the rotation and diffusion of proteins and other bio-molecules within the membrane, there-by affecting the functions of these things.
Particle size analysis, particle size measurement, or simply particle sizing, is the collective name of the technical procedures, or laboratory techniques which determines the size range, and/or the average, or mean size of the particles in a powder or liquid sample.
Single-particle tracking (SPT) is the observation of the motion of individual particles within a medium. The coordinates time series, which can be either in two dimensions (x, y) or in three dimensions (x, y, z), is referred to as a trajectory. The trajectory is typically analyzed using statistical methods to extract information about the underlying dynamics of the particle. These dynamics can reveal information about the type of transport being observed (e.g., thermal or active), the medium where the particle is moving, and interactions with other particles. In the case of random motion, trajectory analysis can be used to measure the diffusion coefficient.
A model lipid bilayer is any bilayer assembled in vitro, as opposed to the bilayer of natural cell membranes or covering various sub-cellular structures like the nucleus. They are used to study the fundamental properties of biological membranes in a simplified and well-controlled environment, and increasingly in bottom-up synthetic biology for the construction of artificial cells. A model bilayer can be made with either synthetic or natural lipids. The simplest model systems contain only a single pure synthetic lipid. More physiologically relevant model bilayers can be made with mixtures of several synthetic or natural lipids.
Microscale thermophoresis (MST) is a technology for the biophysical analysis of interactions between biomolecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. The observed change in fluorescence is based on two distinct effects. On the one hand it is based on a temperature related intensity change (TRIC) of the fluorescent probe, which can be affected by binding events. On the other hand, it is based on thermophoresis, the directed movement of particles in a microscopic temperature gradient. Any change of the chemical microenvironment of the fluorescent probe, as well as changes in the hydration shell of biomolecules result in a relative change of the fluorescence detected when a temperature gradient is applied and can be used to determine binding affinities. MST allows measurement of interactions directly in solution without the need of immobilization to a surface.
Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. Alongside Surface Plasmon Resonance, BLI is one of few widely available label-free biosensing technologies, a detection style that yields more information in less time than traditional processes. The technology relies on the phase shift-wavelength correlation created between interference patterns off of two unique surfaces on the tip of a biosensor. BLI has significant applications in quantifying binding strength, measuring protein interactions, and identifying properties of reaction kinetics, such as rate constants and reaction rates.
There are many methods to investigate protein–protein interactions which are the physical contacts of high specificity established between two or more protein molecules involving electrostatic forces and hydrophobic effects. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are occurring in reality.
The cell membrane is a biological membrane that separates and protects the interior of a cell from the outside environment. The cell membrane consists of a lipid bilayer, made up of two layers of phospholipids with cholesterols interspersed between them, maintaining appropriate membrane fluidity at various temperatures. The membrane also contains membrane proteins, including integral proteins that span the membrane and serve as membrane transporters, and peripheral proteins that loosely attach to the outer (peripheral) side of the cell membrane, acting as enzymes to facilitate interaction with the cell's environment. Glycolipids embedded in the outer lipid layer serve a similar purpose. The cell membrane controls the movement of substances in and out of a cell, being selectively permeable to ions and organic molecules. In addition, cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity, and cell signalling and serve as the attachment surface for several extracellular structures, including the cell wall and the carbohydrate layer called the glycocalyx, as well as the intracellular network of protein fibers called the cytoskeleton. In the field of synthetic biology, cell membranes can be artificially reassembled.
A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.