The significance of this protocol is that it allows an accurate rheological characterization of non-Newtonian biological fluids, such as mucus, especially when only very small sample volumes are available. This technique facilitates the characterization of the apparent yield stress and viscoelasticity of complex structured biological fluids, such as gill raker mucus. This method enables the yield stress estimation of gill raker mucus, and facilitates the rheological characterization and protocol development of similar biological fluids, such as human, animal, and plant secretions.
Demonstrating the procedure with Kartik Bulusu will be Samantha Racan, a graduate student from our laboratory. To prepare approximately two milliliters of 100 milligrams per milliliter concentrated fish mucus solution, perform serial dilution of two milliliters of 400 milligrams per milliliter concentration by adding deionized water in one to two ratio twice. Place the vial of mucus solution onto a shaker until the solution is adequately homogenized and any mucus particulate agglomeration has been mitigated.
To calibrate the rheometer, launch the rheometer instrument control software and select the calibration and instrument tabs. Click calibration and calibrate to ensure that the calibration values are within 10%of each other and click accept. Turn the shaft on top of the rheometer to install the cone angle geometry.
The software will detect the 40 millimeter diameter, one degree, zero minute, 11 second cone angle geometry. To perform the rheometer geometry calibration, click calibration under inertia and click calibrate. Click accept to accept the calibration.
Click calibration under friction and click calibrate. Click accept to accept the calibration. The geometry inertia and friction calibration values should be recorded in the appropriate units.
To perform the zero gap initialization, click gap in options. In the pop-up window, set the axial force to one Newton and click OK, then click the zero gap icon. The initialization is complete when the axial force experienced by the geometry becomes greater than or equal to one Newton as it touches the Peltier plate.
To ensure that the rheometer gap is zeroed so that its reference position is accurate, use the up and down arrows to raise the geometry to any arbitrary height. The control screen on the rheometer instrument and the control panel of the rheometer instrument control software will both display the gap height. To perform the rheological experiment and the linear viscoelastic range of a mucus solution of interest, load approximately 300 microliters of the fish mucus solution into a one milliliter pipette tip and load the solution onto the Peltier plate.
Press the go to trim gap icon to lower the geometry onto the Peltier plate. Use the pipette to remove any excess mucus solution while applying a small angular velocity to the motor and click the stop and apply icons until the torque value reaches minimum. To perform oscillation amplitude experiments, in the procedures menu of the experiments tab, set the oscillation and amplitude and set the temperature to 22 degrees Celsius.
Set the soak time to 120 seconds and check wait for temperature. Set the frequency to one Hertz, the torque to 10 to 10, 000 micronewton meters, and the points per decade to 10. Next, click the insert duplicate step icon.
Change amplitude to frequency. Set the temperature to 22 degrees Celsius and set the soak time to zero seconds. Set the strain percent to one.
In the logarithmic sweep menu, set the frequency to 20 to 1 hertz and the points per decade to 10. To set up the flow sweep procedure, click the insert duplicate step icon and then change oscillation to flow and select sweep to set the temperature to 22 degrees Celsius and set the soak time to zero seconds. Set the shear rate to 1 to 10, 000 per second and the points per decade to 10 and check the steady state sensing box.
Press the go to geometry gap icon to lower the geometry to the preset suitable gap per specific geometry and click start in the instrument software and observe the motion of the cone geometry. A real-time plot that reports the loss in storage moduli will be generated by the rheometer in the main window after the temperature soak time is reached. Right-click to select the graph variables tab and set the x-axis of the plot to oscillation strain percentage to view the data of interest.
To view the angular frequency data, right-click frequency sweep and select send to new graph. To view the apparent viscosity versus shear stress data in real time, right-click flow sweep and select send to new graph, then save the file that contains both the experimental procedure and results in the native file format of the rheometer instrument control software when the experiment ends. For graphical analysis, export the mucus rheology data into a spreadsheet and run supplemental codes for the apparent viscosity for varying shear strain rates and loss modulus, storage modulus, and phase angle for varying oscillation stresses to generate graphs of the results.
As observed in dynamic oscillation amplitude data, the storage modulus of the 200 milligram per milliliter concentration decreased and crossed over the loss modulus within the stress range. The phase angle data for this concentration demonstrated a steady transition to viscoelastic liquid with a transitional region of yield stresses. Modulus data for the 400 milligrams per milliliter concentration demonstrated a yielding phenomenon with a crossover point between the storage and loss moduli that occurred at the apparent yield stress of approximately 0.27 pascals.
The phase angle data for the 400 milligrams per milliliter concentration demonstrated a sharp transition to viscoelastic liquid with a crossover point at approximately 0.27 pascals. As observed in the steady shear rate data, the 100 milligrams per milliliter concentration exhibited a predominantly constant viscosity outside the highlighted regions of instrument limitation, a negligible flat region, and a Newtonian fluid-like behavior. The 200 milligrams per milliliter concentration demonstrated non-Newtonian behavior with increasing shear rates and a propensity to yield with a pronounced flat region.
The 400 milligram per milliliter concentration is the closest to the gill raker mucus composition exhibiting a clear change in the state of the mucus from gel-like to a shear thinning fluid after yielding at approximately 0.27 pascals. As outlined in the protocol manuscript, it is important to determine the oscillation strain percentage value within the linear viscoelastic regime of the gill raker mucus before running the dynamic sweeps. Our protocol can be used to ascertain the apparent yield stress of sticky and gel-like biological fluids, and can be extended to tack and peel tests for a full characterization of the adhesivity of mucus-like materials.
This protocol paves the way for the hydrodynamic investigation of filter feeding with mucus-like materials, the creation of analytical models, and the advancement of cross-flow and membrane filtration technologies.
