Attempts to increase film stability lead to first stable multilayer

Modifying Protein Concentration Methods

Need for Improving Protein Impurity

Protein impurity has become a major headache and hindrance on our progress. If we acetone precipitate to concentrate then whilst there is no dewetting phenomenon and concentration yields are high, films are dominated by the strong aggregation tendency of urea to crystallize. Meanwhile, alternatively if we use dialysis in 10mM Tris, 0.1% Triton-X 100 pH 8.0 buffer in dialysis membrane, there are issues with tris preventing efficient solvent evaporation and wetting which lead to degradation and dewetting of the films which lead to in many cases non-uniformity and loss of colouration over time.

Alternative protein concentration methods were tried in order to help purity:

• Chloroform/methanol/water precipitation
• Microcentrifuge dialysis against 10mM Tris, 0.1% Triton-X 100 pH 8.0 buffer followed by vacuum centrifuge
• Microcentrifuge dialysis against 1X PBS buffer and pH 8 buffer followed by vacuum centrifuge
• Microcentrifuge dialysis against unbuffered D.I water followed by acetone precipitation

Samples from each method were then first drop cast onto silicon wafer to test for presence of protein and subsequently spun onto silicon substrate after dilution with 10-40 μl of HFIP with volume differing based on my expectation of protein yield.

Results

The results from this experiment shows that:

• Chloroform/methanol/water precipitation was too effective, not only did it get rid of contaminants it also removed the protein! However this outcome was within our calculations. From hydropathy plots Crookes et al determined that Loligo Reflectin A1 is a globally hydrophilic protein therefore there may be a possibility of it associating two strongly with the aqueous phase and therefore not precipitating in the interphase. Nevertheless I would still have expected more protein than nearly nothing, this I speculate is because the protocol should be implemented with all reagents at ice-cold temperatures to facilitate precipitation. We intend to repeat this protocol with ice cold reagents.

• Microcentrifuge dialysis though yielded much greater concentration of proteins than using the entire membrane was not as effective and much of the urea salt was retained in the microcentrifuge. Upon taking a sample of the crystals from the Tris buffer dialysis, diluting in 40μl HFIP and spinning at 3000 rpm for 1 min with 2μl of the reflectin-HFIP solution on silicon substrate one of the most beautiful films I have produced was seen. The film was a brilliant shade of green of perfect uniformity and thick enough to exhibit iridescence from different viewing angles, an effect seen more prominently when coated onto a layer of PDMS. Unfortunately soon after we found that the films crystallised indicating much of the resulting crystals after vacuum centrifuge are urea crystals. The brilliant colouration and wetting is believed to be the high concentration of proteins which should be close to the eluted concentration.

• The result of the dialysis with 1X PBS buffer again was not impressive with crystallisation within 10 seconds of spin coating and almost instantly when drop-casting making it unfeasible to work with.

• The result of unbuffered water too was not brilliant with very little iridescence after drop-casting.

Attempting to Stabilise Films

Based on our many different attempts to spin uniform, stable films we have noticed the following points which indicate some potential in at least delaying the onset of crystallizing.

• Crystallization is accelerated by heat
• Crystallization is promoted by greater solvent evaporation
• Crystallization takes much longer when using the first 2μl of a centrifuged reflectin-HFIP sample but rapidly got worse in the next few aliquots which suggests centrifugation may be fractionating some urea.

Several measures were investigated based upon the above observations:

• Dilution with more HFIP which provides a greater volume of fluid improving the possibility of contamination from the pellets produced in centrifugation. This was a technique used in the dialysed proteins to reduce the amount of insoluble tris contaminants.
• Attempting to filter the solutions particularly for the dialyzed proteins however the small working volumes makes this unreasonable
• Manually stopping the spin coater to leave sufficient solvent to delay crystallisaton and dewetting.
• Coat PDMS layer immediately after spin to not leave enough time for cystallisation or dewetting due to local humidity.

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