We report a continuous-flow, microfluidic mixer utilizing mid-infrared hyperspectral imaging detection, with an experimentally determined, submillisecond mixing time. The simple and robust mixer design has the microfluidic channels cut through a polymer spacer that is sandwiched between two IR transparent windows. The mixer hydrodynamically focuses the sample stream with two side flow channels, squeezing it into a thin jet and initiating mixing through diffusion and advection. The detection system generates a mid-infrared hyperspectral absorbance image of the microfluidic sample stream. Calibration of the hyperspectral image yields the mid-IR absorbance spectrum of the sample versus time. A mixing time of 269 μs was measured for a pD jump from 3.2 to above 4.5 in a D2O sample solution of adenosine monophosphate (AMP), which acts as an infrared pD indicator. The mixer was further characterized by comparing experimental results with a simulation of the mixing of an H2O sample stream with a D2O sheath flow, showing good agreement between the two. The IR microfluidic mixer eliminates the need for fluorescence labeling of proteins with bulky, interfering dyes, because it uses the intrinsic IR absorbance of the molecules of interest, and the structural specificity of IR spectroscopy to follow specific chemical changes such as the protonation state of AMP.

We report on a microfluidic mixer fabrication platform that increases the versatility and flexibility of mixers for biomolecular applications. A sandwich-format design allows the application of multiple spectroscopic probes to the same mixer. A polymer spacer is ‘sandwiched’ between two transparent windows, creating a closed microfluidic system. The channels of the mixer are defined by regions in the polymer spacer that lack material and therefore the polymer need not be transparent in the spectral region of interest. Suitable window materials such as CaF2 make the device accessible to a wide range of optical probe wavelengths, from the deep UV to the mid-IR. In this study, we use a commercially available 3D printer to print the polymer spacers to apply three different channel designs into the passive, continuous-flow mixer, and integrated them with three different spectroscopic probes. All three spectroscopic probes are applicable to each mixer without further changes. The sandwich-format mixer coupled with cost-effective 3D printed fabrication techniques could increase the applicability and accessibility of microfluidic mixing to intricate kinetic schemes and monitoring chemical synthesis in cases where only one probe technique proves insufficient.

The secondary conformational change of cellular prion protein (PrPc) into scrapie prion protein (PrPsc) is critical important for prion disease including bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. This study performed molecular dynamics simulations of both human prion protein (WT-PrPc) and pathogenic mutant prion protein (T188R) and analyze its conserved water molecules in equilibrium state. Although the secondary structure is not dramatically different, increase of Ca-RMSF occurs not only around mutation point but also in a wide range. This increase of intensity may result from the instability caused by point mutation. Next, we calculated mean residence time for each protein atoms to find conserved water molecules. Residence time is defined that the time from when the water molecule get into the sphere whose center is a protein atom to the evacuation of water molecule from the sphere. We found four conserved water molecule sites in WT-PrPc. Conserved water molecules also keep the higher rotational correlation than water molecules in bulk. These results shows that conserved water molecules are not only wrapped but also fixed by hydrogen bond between conserved water molecule and amino acids. On the other hand, every residence time of T188R decrease compared by WT-PrPc and the rotational relaxation is also faster than WT-PrPc although secondary structure is almost not different. These results show that conserved water molecules cannot survive related to the wide range fluctuation caused by point mutation.