Project Goal: A highly efficient continuous flow mixer, suitable for mass-production, has been sought for a long time by chemists and microbiologists wishing to study very rapid reactions. The project team set out to design, implement and evaluate continuous flow mixing devices capable of initiating and detecting reactions an order of magnitude faster than currently possible with stopped-flow instruments, such as those supplied by the our industrial Partner, TgK Scientific.

Relevance of the Research: A full characterisation of “reaction kinetics” underpins a myriad of activities in many important areas of the developed world, including the scaling up of manufacturing processes, product stability and decay in food-processing and the environmental impact of industrial waste.

In living organisms the complex network of reaction mechanisms is finely tuned by sequential and competitive reaction rates.  Proteins are essential in every process within cells - all proteins are “assembled” in ribosomes (small organelles found in every living cell) which they leave as long, straight amino-acid chains which then fold to appropriate 3D shapes in order to become active proteins. The vast majority of proteins do not fold directly into their proper conformation but do so in “steps” by firstly folding into intermediate conformations in a matter of microseconds and then changing into active protein. In fact and the earliest key events in protein folding often occur within a single microsecond.

Unfortunately, the process is not 100% reliable and some of the proteins may fold incorrectly.  Mis-folded proteins are inactive, but can aggregate forming ”clumps” which are dangerous to the cell - to date more than 30 diseases (including Alzheimer's, Parkinson’s and Creutzfeldt-Jakob disease) have been shown to be caused by protein mis-folding. Studying protein folding intermediates assists in the identification of the key steps responsible for protein mis-folding which can then be targeted by new drugs to slow down or even stop the disease progression.  An understanding of rates of molecular interactions is fundamental to many drug discovery programs. The most common means of initiating many biological and chemical reactions is by mixing two reactants in liquid solutions using a stopped-flow instrument, capable of allowing the investigation of reactions that occur in milliseconds to seconds. The drawback of the stopped flow method is that by the time the solutions are mixed, the flow stopped and the detection initiated, a valuable window of reaction time in the microsecond to the millisecond region is lost.

The Approach: The project team brought together micro-fabrication capabilities, computational fluid dynamic simulation and analysis, rapid CCD detection technologies and biological reactions knowledge.  The specific objectives were:

• To develop an efficient and robust continuous-flow mixing device (either by novel design or improvement of existing designs) suitable for mass production and which would enable the observation of microsecond duration protein folding reactions.
• To use the new device to carry out initial proving studies on the folding pathways of prion proteins.

Partner Involvement: TgK Scientific Ltd – a leading manufacturer of instruments for transient kinetics (www.hi-techsci.com), Bradford-on-Avon, UK

Research Outputs:

• Computational fluid dynamics (CFD) was used extensively in the prediction of mixing behaviour and the parametric optimisation for the mixer design.  The main challenge was achieving high accuracy in the predictions as the flow in the micro-channels can encounter flow regimes ranging from laminar through transition to fully turbulent.  It is particular difficult to predict the transition status.  The team’s approach successfully predicted the turbulent transition from laminar flow to turbulent flow in a rectangular micro-channel as shown in the Moody chart shown in Figure 1. The dots on the chart represent the numerical results.

  Three prototype mixing plates, comprising delivery tunnels and a reaction channel, were micro-fabricated using design parameters obtained from CFD.  The prototype plates led onto a final design  where reaction kinetics were measured in the sub-millisecond timescale, as illustrated in Figure 2 which shows the quenching reaction of a soluble tryptophan derivative N-acetyl tryptophan amine, (NATA), with N-bromosuccinimide (NBS).

• New understanding of flow properties in mixing devices was obtained through the CFD analysis developed in this project. The mean velocity and pressure field in the device can now be predicted and used as guidelines for design optimisation.
• CFD results confirmed that a combination of high velocity flow and reduced pressure could generate cavitation bubble clouds which might generate erosion in the mixing channel and/or promote mixing in certain situations.  This needs to be investigated in any follow on research. 

Publications: The project team has published three refereed journal papers to date and further publications are in preparation.

What Difference Will the Project Make?  The insight gained through CFD calculations not only aids the design of the new continuous flow mixers but it is also being used to fully characterise mixing of flows in stop flow devices. Ted: please expand[n1]  

Next Steps: The research outputs have provided a strong basis for the preparation of an application to EPSRC for follow-up funding.

Interested?  For further information and to discuss possible future collaboration please contact Dr Teresa Pinheiro, School of Life Sciences, University of Warwick, CV4 7AL.

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