3-D Printing of EPR Flow Cells

Abstract

Electron Paramagnetic Resonance (EPR) is a spectroscopy technique that detects unpaired electrons. The highly reactive nature of organic radicals makes it difficult to detect. The necessity to mix reactions solutions quickly and efficiently has motivated us to design a flow cell using 3-D technologies. By designing our flow cells, we have more flexibility in the designs. Attempts to print a functioning flow cells have so far been unsuccessful, but a flow cell designed with a micropipette has allowed experiments on the flow rate effect on EPR signal detection.

Introduction

Free radical contain chemical species are of great interest due to their polymerization and biochemistry processes. These radicals are highly reactive with short lifetimes. The highly reactive nature of organic radicals makes it difficult to detect. To detect this spin trapping technique is used to react a normally unstable radical adduct with another more stable radical adduct. The stable radical adduct comes from the nitrone containing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)1. It is a necessity to mix reactions solutions quickly and efficiently to ensure the spin trapping of the radicals. Current market EPR flow cells limited and ordering custom glassware will be an expansive and time-consuming process. By designing our flow cells, we have more flexibility in the designs by incorporating microfluidic mechanics. Microfluidic flow behavior is found often in biological, chemical, or physical analysis. The short lifetime of radical will benefits of employing inertial driven mixing techniques. The radical forming reaction can occur right before or in the EPR cavity. This experiment focused on changing the flow rate of the two reagents. The reagents when flowing through the mixer experience laminar flow since there is nothing in the mixer to disturb the two liquids. When two flow next to each while experience laminar flow a sheer gradient force keeps the two liquids from homogenizing. By increasing the flow rate the sheer gradient force will be overcome and homogenizing will occur2. Enzymology studies using rapid-flow techniques had shown have improved observation of obligatory reaction intermediates with milligram quantities of enzymes3. By designing an EPR flow cell that takes advantage of microfluidics we hope to see higher-intensity EPR signals.

Materials and Methods

The SLA printer. The 3-D printed cell was printed using the Formlabs Form 2 3-D printer. Form 2 uses a stereolithography printing technique. Formlabs Clear (FLGCL04) resin was used. The resin is cured via a photolytic radical polymerization reaction using a focused UV laser. The clear resin can cause serious eye irritation, may cause an allergic skin reaction, toxic to aquatic life, and suspected of damaging fertility or an unborn child.4 The 3-D printed the Cell The mixing cells were designed using the Computer Assisted Design (CAD) program Autodesk Fusion 360. The mixing cell (Figure 1) is cylindrical with a 0.18 in. radius and a length of 7.00 in. The cut-ins are rectangular with a 2.50 in. length. 0.351 in. width, and a 0.140 in. depth. The cut-ins are centered on the midpoint of the mixing cell. The cells have to inlets and one outlet. The inlet channels are also cylindrical and have a radius of 0.0635 in. The center point of the inlet channels is 0.09 in. form the center point of the cell. The inlets channels center points are in parallel with each other. The inlet channels have and length of 2.15 in. and a bevel inward at 118° to form a T-junction with a 0.0393 in. radius. The mixing chamber was variable to determine a volume that will tune with the EPR. The corners have a 0.034 in. fillet. The outlet has a radius of 0.0635 in. Using 5-minute epoxy, silicone tubing was affixed to the inlets. The micropipette mixer A Drummond Scientific 50µL capillary micropipette was selected to the as the mixing chamber. Silicon tubing was connected to form the outlet. The inlets were formed using a silicon tubing T-junction. A glass tube was fitted over the micropipette to allow for stabilization in the EPR resonator (Figure 2). The inlets are connected to the same syringe pump as the 3-D printed cell. The ascorbic acid solution was made with a concentration of 1 mM. A 9.2 pH buffer was also prepared. Ascorbic acid generates a radical when pH is high. The ascorbic acid solution was held in one syringe and the pH buffer was also in another. EPR spectra were taken at varying flow rates. EPR spectra were recorded with a Bruker EMX spectrometer. The 3-D printed mixing cells were loaded into the EPR cavity with water to determine whether the cells were tunable. The micropipette mixer was loaded with a 1:1 mixer of the pH buffer and ascorbic acid. The EPR spectra were recorded under the following conditions: center field, 3498.00 G; sweep width, 20.00 G; modulation frequency, 1.00 G; time constant, 40.96; 20.48 conversion time. Each measurement was carried out two times.

Results and Discussion

Effectiveness of 3-D printed flow Cells We were unable to print a tunable flow cell. We were unable to find an acceptable volume for the mixing chamber to able to tune on the cell. Larger mixing chambers there is too much analyte volume to get an acceptable EPR signal. When trying to reduce the chamber volume, the resin inside of the channel does not clear. The SLA resin is viscous, the smaller mixing chamber volume, and the radii of the prevent clearing of the inner channels. The clear nature of the plastic mixer allows for the resin to cure inside of the inner channels making the resin to cure in the chambers. Often the pressure required to clear the viscous resin form the channel. A tunable blank cell was achieved but a loaded cell mixing chamber volume was not found. Further prototypes are required to find a printable mixing chamber volume. The Micropipette Mixer The prefabricated nature of the micropipette allowed for the small tunable volume required for an EPR spectrum. The results of the ascorbate spectra are given in figure 4. Figure 4 shows that as the flow rate decreased the EPR signal increase. The “flow stop” measurement show intensity of the signal when the reaction goes to completion. The trend is because the slower flowrate allowed for more radical adduct to form, intensifying the EPR signal. With the T-junction style of the micropipette mixer that result is to be suspected. Since there are no curves introduced in this style of mixer faster flow rates are required to break down the sheer gradient force keeping the two liquids separated. Experimenting with faster flow rates may better mixing in the mixing chamber to give an intensified EPR signal.

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References

1. Janzen, E. G. (1971). Spin trapping. Accounts of Chemical Research, 4(1), 31–40. https://doi.org/10.1021/ar50037a005
2. Nonlinear Microfluidics Daniel Stoecklein and Dino Di Carlo Analytical Chemistry 2019 91 (1), 296-341 DOI: 10.1021/asc.analchem.8b05042
3. Gutfreund, H. (1999). Rapid-flow techniques and their contribution to enzymology. Trends in Biochemical Sciences, 24(11), 457. https://doi.org/10.1016/S0968-0004(99)01468-1
4. Formlabs. (11.22.2019) Clear Resin [Material Safety Data Sheet]. Retrieved from https://formlabs-media.formlabs.com/datasheets/1801037-SDS-ENUS-0.pdf