A monolithic microfluidic probe for ambient mass spectrometry imaging of biological tissues.

Ambient mass spectrometry imaging (MSI) is a powerful technique that allows for the simultaneous mapping of hundreds of molecules in biological samples under atmospheric conditions, requiring minimal sample preparation. We have developed nanospray desorption electrospray ionization (nano-DESI), a liquid extraction-based ambient ionization technique, which has proven to be sensitive and capable of achieving high spatial resolution. We have previously described an integrated microfluidic probe, which simplifies the nano-DESI setup, but is quite difficult to fabricate. Herein, we introduce a facile and scalable strategy for fabricating microfluidic devices for nano-DESI MSI applications. Our approach involves the use of selective laser-assisted etching (SLE) of fused silica to create a monolithic microfluidic probe (SLE-MFP). Unlike the traditional photolithography-based fabrication, SLE eliminates the need for the wafer bonding process and allows for automated, scalable fabrication of the probe. The chamfered design of the sampling port and ESI emitter significantly reduces the amount of polishing required to fine-tune the probe thereby streamlining and simplifying the fabrication process. We have also examined the performance of a V-shaped probe, in which only the sampling port is fabricated using SLE technology. The V-shaped design of the probe is easy to fabricate and provides an opportunity to independently optimize the size and shape of the electrospray emitter. We have evaluated the performance of SLE-MFP by imaging mouse tissue sections. Our results demonstrate that SLE technology enables the fabrication of robust monolithic microfluidic probes for MSI experiments. This development expands the capabilities of nano-DESI MSI and makes the technique more accessible to the broader scientific community.

Surface-Enhanced Raman Spectroscopic Probing in Digital Microfluidics through a Microspray Hole.

We report a novel approach for surface-enhanced Raman spectroscopy (SERS) detection in digital microfluidics (DMF). This is made possible by a microspray hole (µSH) that uses an electrostatic spray (ESTAS) for sample transfer from inside the chip to an external SERS substrate. To realize this, a new ESTAS-compatible stationary SERS substrate was developed and characterized for sensitive and reproducible SERS measurements. In a proof-of-concept study, we successfully applied the approach to detect various analyte molecules using the DMF chip and achieved micro-molar detection limits. Moreover, this technique was exemplarily employed to study an organic reaction occurring in the DMF device, providing vibrational spectroscopic data.

Analysis of double-emulsion droplets with ESI mass spectrometry for monitoring lipase-catalyzed ester hydrolysis at nanoliter scale.

Microfluidic double-emulsion droplets allow the realization and study of biphasic chemical processes such as chemical reactions or extractions on the nanoliter scale. Double emulsions of the rare type (o1/w/o2) are used here to realize a lipase-catalyzed reaction in the non-polar phase. The surrounding aqueous phase induces the transfer of the hydrophilic product from the core oil phase, allowing on-the-fly MS analysis in single double droplets. A microfluidic two-step emulsification process is developed to generate the (o1/w/o2) double-emulsion droplets. In this first example of microfluidic double-emulsion MS coupling, we show in proof-of-concept experiments that the chemical composition of the water layer can be read online using ESI-MS. Double-emulsion droplets were further employed as two-phase micro-reactors for the hydrolysis of the lipophilic ester p-nitrophenyl palmitate catalyzed by the Candida antarctica lipase B (CalB). Finally, the formation of the hydrophilic reaction product p-nitrophenol within the double-emulsion droplet micro-reactors is verified by subjecting the double-emulsion droplets to online ESI-MS analysis.

On-the-Fly Mass Spectrometry in Digital Microfluidics Enabled by a Microspray Hole: Toward Multidimensional Reaction Monitoring in Automated Synthesis Platforms.

We report an approach for the online coupling of digital microfluidics (DMF) with mass spectrometry (MS) using a chip-integrated microspray hole (µSH). The technique uses an adapted electrostatic spray ionization (ESTASI) method to spray a portion of a sample droplet through a microhole in the cover plate, allowing its chemical content to be analyzed by MS. This eliminates the need for chip disassembly or the introduction of capillary emitters for MS analysis, as required by state-of-the-art. For the first time, this allows the essential advantage of a DMF device─free droplet movement─to be retained during MS analysis. The broad applicability of the developed seamless coupling of DMF and mass spectrometry was successfully applied to the study of various on-chip organic syntheses as well as protein and peptide analysis. In the case of a Hantzsch synthesis, we were able to show that the method is very well suited for monitoring even rapid chemical reactions that are completed in a few seconds. In addition, the strength of the low resource consumption in such on-chip microsyntheses was demonstrated by the example of enzymatic brominations, for which only a minute amount of a special haloperoxidase is required in the droplet. The unique selling point of this approach is that the analyzed droplet remains completely movable after the MS measurement and is available for subsequent on-DMF chip processes. This is illustrated here for the example of MS analysis of the starting materials in the corresponding droplets before they are combined to investigate the reaction progress by DMF-MS further. This technology enables the ongoing and almost unlimited tracking of multistep chemical processes in a DMF chip and offers exciting prospects for transforming digital microfluidics into automated synthesis platforms.

Quantification of Biocatalytic Transformations by Single Microbial Cells Enabled by Tailored Integration of Droplet Microfluidics and Mass Spectrometry.

Improving the performance of chemical transformations catalysed by microbial biocatalysts requires a deep understanding of cellular processes. While the cellular heterogeneity of cellular characteristics, such as the concentration of high abundant cellular content, is well studied, little is known about the reactivity of individual cells and its impact on the chemical identity, quantity, and purity of excreted products. Biocatalytic transformations were monitored chemically specific and quantifiable at the single-cell level by integrating droplet microfluidics, cell imaging, and mass spectrometry. Product formation rates for individual Saccharomyces cerevisiae cells were obtained by i) incubating nanolitre-sized droplets for product accumulation in microfluidic devices, ii) an imaging setup to determine the number of cells in the droplets, and iii) electrospray ionisation mass spectrometry for reading the chemical contents of individual droplets. These findings now enable the study of whole-cell biocatalysis at single-cell resolution.

Integration of segmented microflow chemistry and online HPLC/MS analysis on a microfluidic chip system enabling enantioselective analyses at the nanoliter scale.

In this work, we introduce an approach to merge droplet microfluidics with an HPLC/MS functionality on a single chip to analyze the contents of individual droplets. This is achieved by a mechanical rotor-stator interface that precisely positions a microstructured PEEK rotor on a microfluidic chip in a pressure-tight manner. The developed full-body fused silica chip, manufactured by selective laser-induced etching, contained a segmented microflow compartment followed by a packed HPLC channel, which were interconnected by the microfluidic PEEK rotor on the fused silica lid with hair-thin through-holes. This enabled the targeted and leakage-free transfer of 10 nL fractions of droplets as small as 25 nL from the segmented microflow channel into the HPLC compartment that operated at pressures of up to 60 bar. In a proof of concept study, this approach was successfully applied to monitor reactions at the nanoliter scale and to distinguish the formed enantiomers.