Parallel detection of chemical reactions in a microfluidic platform using hyperpolarized nuclear magnetic resonance

Abstract

The sensitivity of NMR may be enhanced by more than four orders of magnitude via dissolution dynamic nuclear polarization (dDNP), potentially allowing real-time, in situ analysis of chemical reactions. However, there has been no widespread use of the technique for this application and the major limitation has been the low experimental throughput caused by the time-consuming polarization build-up process at cryogenic temperatures and fast decay of the hyper-intense signal post dissolution. To overcome this limitation, we have developed a microfluidic device compatible with dDNP-MR spectroscopic imaging methods for detection of reactants and products in chemical reactions in which up to 8 reactions can be measured simultaneously using a single dDNP sample. Multiple MR spectroscopic data sets can be generated under the same exact conditions of hyperpolarized solute polarization, concentration, pH, and temperature. A proof-of-concept for the technology is demonstrated by identifying the reactants in the decarboxylation of pyruvate via hydrogen peroxide (e.g. 2-hydroperoxy-2-hydroxypropanoate, peroxymonocarbonate and CO₂). dDNP-MR allows tracing of fast chemical reactions that would be barely detectable at thermal equilibrium by MR. We envisage that dDNP-MR spectroscopic imaging combined with microfluidics will provide a new high-throughput method for dDNP enhanced MR analysis of multiple components in chemical reactions and for non-destructive in situ metabolic analysis of hyperpolarized substrates in biological samples for laboratory and preclinical research.

Fig. 1. Microfluidic multiwell plate for high-throughput DNP-MRSI experiments. (A) Assembling layers of the multiwell plate: i) 4 mm thick PDMS layer for distribution of the hyperpolarized substrate, ii) 5 and 1 mm thick PDMS layers with the detection chambers, and iii) 75 mm × 38 mm glass slide on the bottom of the PDMS chambers as a support. (B) Network of microfluidic channels that injects the polarized sample into one of the chambers. This design is replicated along all the chambers. (C) Photo of the microfluidic device. Microfluidic channels that distribute the polarized sample are filled with blue dye for easy identification. (D) Water volume inside each chamber after injecting 1.0, 1.4, 1.8, and 2.2 mL water into the device (each circle represents a chamber of the microfluidic plate).

Fig. 2. High-throughput DNP-MRSI assay. (A) Schematic illustration of the experimental setup consisted of i) polarizer equipment, ii) microfluidic multiwell plate, and iii) magnetic resonance imaging scanner. (B) T₂-weighted localizer images with axial, sagittal and coronal perspectives. (C) Stacked dynamic spectra of [1-¹³C]pyruvic acid of the well highlighted in blue along the experiment. Note that time 0 represents when the MRS data acquisition was initiated. The transfer time between dissolution and injection was 12 s on average, and MRS data acquisition was initiated 25 s after the sample came out from the polarizer. The longitudinal experiment was designed with a CSI sequence as 8 × 8 voxels matrix, with a FOV of 40 × 40 cm² and a slice thickness of 12 mm, 15° flip angle, echo time = 1.49 ms. T_acq = 51.2 ms and RT = 66.907 ms. Each point of the data set was acquired every 4 seconds.

Fig. 3. (A) Schematic image of the microfluidic device specifying the sample placed in each well. (B) Localizer with a coronal T₂-weighted image using a spin echo T₂ TurboRARE sequence with the corresponding voxels matrix overlapping. (C) Spectroscopic acquisition using a CSI pulse sequence to the microfluidic device at thermal equilibrium. (D) First spectroscopic acquisition point using a CSI pulse sequence to the microfluidic device after the hyperpolarized [1-¹³C]pyruvate injection. (E) Scheme of the oxidation reaction between pyruvic acid and H₂O₂, highlighting all products and intermediates displayed in the spectrum. (F) Representative spectrum of the decarboxylation of [1-¹³C]pyruvate reacted with H₂O₂, showing all the products and intermediate states of the reaction.

Fig. 4. (A) Plot showing the temporal evolution of the peak signal intensity, normalized by the signal intensity and volume of [1-¹³C]pyruvate, in wells containing [¹³C]urea (orange circle), wells containing H₂O₂ and sodium hydroxide solution (red square) and wells containing water and sodium hydroxide solution (blue triangle). (B) Plot showing the temporal evolution of the normalized peak signal intensity of [1-¹³C]pyruvate hydrate (orange circle), [1-¹³C]2-hydroperoxy-2-hydroxypropanoate (blue square), [1-¹³C]peroxymonocarbonate (red triangle), and ¹³CO₂ (inverted green triangle) over time in wells containing H₂O₂ and sodium hydroxide solution. Normalized peak signal intensity of [1-¹³C]pyruvate (dashed line) in well containing only sodium hydroxide is included in dashed line as a reference. The signal intensity displayed in each of the four voxels corresponding to a well were summed up and each well corresponds to a replicate. (C) Temporal evolution of the change in concentration of the reactants and products with respect to their initial concentration in the wells containing H₂O₂ and sodium hydroxide solution, corrected by the hyperpolarized signal T₁ decay. The legend is the same as in (B).