Frequently Asked Questions

MDS exploits the unique properties of flow in microfluidic channels — specifically laminar flow, where streams can flow alongside one another with no convective mixing. On the chip, a sample containing a labeled protein of interest is injected alongside an auxiliary fluid that has no fluorescence. These two fluids meet at the top of the diffusion channel and then flow alongside each other down the diffusion channel. The flow is laminar, so the fluids do not mix, but molecules do diffuse as the fluids pass through the channel. At the end of the channel the two streams are separated again, physically encoding the amount of diffusion that took place and then flow into detection chambers where their fluorescence values are measured. The fluorescence values recorded report on the fraction of the protein of interest that diffused, and the average size of the protein of interest is then calculated from the observed amount of diffusion.

Watch the animation explaining MDS and the Fluidity One-M.

The hydrodynamic radius (R_h) determined by our Fluidity One-M instrument is the Stokes radius, or the size of a hard spherical particle that diffuses at the same rate as the protein(s) detected.

For proteins, this is largely determined by the molecular weight, but shape also plays a role, with compact, well-folded proteins diffusing faster than extended, poorly folded proteins, and thus having a smaller hydrodynamic radius. The size determined by gel permeation or size exclusion chromatography is also the hydrodynamic radius.

The correlation of hydrodynamic radius with protein molecular weight (M_w) is dependent on protein conformation. If proteins are globular, a strong correlation between R_h and M_w is observed. If proteins are unstructured or elongated the measured R_h will be larger than that of a globular protein of identical M_w. Consequently, comparison to a calibration curve of globular proteins will yield a good approximation of M_w for a globular protein of unknown M_w. This principle is used to calibrate SEC columns using samples of known molecular weights as the calibration standards.

Fluorescence polarization (FP) is a similar technique in being a solution-phase method to examine interactions, but one key difference is that Microfluidic Diffusional Sizing (MDS) can work with species that are closer together in terms of size.

FP works by exciting a fluorescently labeled molecule with polarized light and measuring the polarity of the emitted fluorescence. As the molecule tumbles in solution the polarity of the emitted light is scrambled, so the polarity of the emitted fluorescence reports on the tumbling rate of the protein of interest. The rate of tumbling is inverse to size, so if the labeled molecule is bound to another protein its tumbling rate will decrease. The main limitation of FP is that because it is based on detection of changes in rotational motion instead of a translational motion, there’s a requirement for a significant size change. Experimentally, this means the labeled species should be about 10-fold smaller than its binding partner and the is limited to interactions involving labeled species of 10 kDa or less. MDS can tolerate much smaller differences in size of the interacting components and can also work with far larger species than FP.

Finally, FP does not give absolute size information about the labeled species, so it can only provide the K_D and not the size of free or bound probe. MDS, on the other hand, reports these QC measures as standard.

The Fluidity One-M measures size (hydrodynamic radius, R_h) in solution of a fluorescently tagged species. This data is output, along with intensity data for each sample. If you are running a binding analysis then the instrument also carries out curve fitting on the data set to provide affinity (K_D) and the QC measurements of free and bound probe (R_h,free and R_h,complex). These data can all be conveniently viewed on-screen, or the data can be downloaded from the instrument using a USB flash drive.

The layout of the disposable chips is fixed due do the prohibitive cost of a new injection mold tool.

We do have chip prototyping capabilities however, and we’re always open to discussing potentially novel and innovative applications of our technology. Please contact us if you have a specific requirement and we will be happy to discuss.

Each circuit on a Fluidity One-M chip-plate can be used only once. This is because re-using circuits carries a high risk of carry-over and cross-contamination as well as likely fluidic failure due to obstructions in the very small channels of the circuit. It is worth noting, however, that a part-used chip-plate can be used to completion. To check which circuits are still available for use hold the chip-plate up to the NFC reader on the front of the Fluidity One-M instrument.

If the smaller peptide was labeled with a fluorescent label the size change between the small unbound peptide and the complex would be easily detected and determination of the binding affinity would be straightforward.

Yes. On the Fluidity One-M you have complete control of the fluid used as the auxiliary stream and so can adjust this to suit your experiment. The only constraint is that the viscosity of the auxiliary fluid must be similar to that of the sample fluid.

Yes. There is the option of having a label on the protein or on the DNA, and the decision of which species to label comes down to size. Because MDS detects change in size it is always best to fluorescently label the smaller of the two interacting species. Because the technique doesn’t rely on immobilization and interactions are analyzed in solution it is able to work with a wide range of biomolecules including proteins, nucleic acids, sugars or lipids.

Yes it is. This is an application that generates a lot of interest. The advantage of MDS is that because it is a solution phase measurement, membrane proteins can be presented in a more natural context such as in liposomes, lipid rafts or nanodiscs rather than relying on detergents. Because there is no need to fix the protein on a surface there is no risk of introducing conformational or steric artefacts. The workflow for membrane proteins is very simple: take your membrane protein of interest in its preferred solubilization format and titrate it against a labeled binding partner and measure those samples on the Fluidity One-M. The instrument will determine the sizes of each sample and directly produce a binding curve with fitting parameters of affinity (K_D) and QC size measures of the free and bound species (R_h,free and R_h,complex respectively).

Yes, Microfluidic Diffusional Sizing is able to perform measurements in crude biofluids. There are a number of publications showcasing the use of MDS to measure binding affinities directly in biological fluids, from saliva to plasma and serum. Our serum affinity and concentration (SAffCon) assay is specially designed for experiments in whole biofluids and determines both the binding affinity and concentration of binder in the biofluid. The assay requires no predilution or pretreatment of the biofluid sample.

Intrinsically disordered proteins (IDPs) are a fascinating class of proteins in that they function in a folded form but they spend the majority of the time in an unfolded form. The transition they make from folded to unfolded is functionally relevant. They play a role in a lot of binding interactions so they are really important as hub proteins and so are of interest to drug development researchers. The problem with IDPs is that, in terms of energy, they live in the boundary region between folded and unfolded states, just on the unfolded side. If their environment is changed too much then they can partially or completely fold, so the interaction you measure might not be representative of the one that takes place in a biological system.

This is always the risk of surface immobilization. If you attach an IDP to a surface or a matrix element like a gel, then that protein may adopt a fold of some sort. Because Microfluidic Diffusional Sizing measurements take place completely in solution, the only species that an IDP would meet would be its binding partner or other IDPs. By working with proteins in their natural state the Fluidity One-M can achieve more biologically relevant results.

One of the strengths of Microfluidic Diffusional Sizing is how easy it is to develop assays or set up experiments. The smaller of the two interacting species needs to be labeled, for which there are several kits that can be used. Because the Fluidity One-M has dual channel optics you can use most types of labels including but not limited to: FITC, Cy5, Alexa Fluor 488 or 647 or equivalent, or even GFP. For labeling chemistry either NHS or maleimide can be used. If you use a chemical label it is necessary to remove the free dye otherwise it suppresses the measured size of your protein. Once you have your labeled protein the experiment needs only a titration of the binding partner to be mixed with a constant concentration of the labeled species. After equilibration samples are transferred to a Fluidity One-M chip-plate and inserted into the Fluidity One-M for automated measurement and analysis.

Yes, competition assays are easily performed using Microfluidic Diffusional Sizing. The benefit of this approach is that it can prove coincident epitopes for binders, and also it provides a mechanism to measure interactions without labeling or immobilizing either of the species of direct interest.

This is not a difficult experiment to tackle using Microfluidic Diffusional Sizing. As measurement takes place in solution it is necessary for the lipids to be solubilized, for example as vesicles, micelles, rafts or nanodiscs, and then analysis can proceed as for any other soluble biomolecule. In most cases the lipid particle will be larger than its binding partner and so the binding partner should be labeled. In the unusual situations with a small lipid particle and large binding partner the lipid should be labeled. To measure affinity simply titrate the unlabeled species against a constant concentration of the labeled species, incubate to achieve equilibration, and measure on the Fluidity One-M.

To be clear, there are no barriers in the diffusion channel. Because the flow in the microfluidic circuits of the chip-plate is laminar there is no chaotic or convective mixing of the sample and the auxiliary streams when they meet in the diffusion channel. The chip material is compatible with the detergents used in bioscience and the only risk is of foaming or entrapment of bubbles which can be mitigated by being careful to avoid introduction of air when loading samples onto the chip-plate.