High-throughput MALDI-MS based read-out of high-density droplet microarrays facilitating next generation on-chip drug discovery

Significance of the topic

High-density, low-volume on-chip platforms offer transformative potential for accelerating early-stage drug discovery. Traditional approaches rely on large reagent volumes and serial analyses, which limit throughput and increase costs. Integrating miniaturized synthesis with high-throughput, surface-compatible analytical read-outs addresses these constraints, enabling rapid screening of vast compound libraries while conserving materials.

Objectives and study overview

This study presents a proof-of-concept workflow combining AQUARRAY’s Droplet Microarray (DMA) technology with Bruker’s rapifleX MALDI PharmaPulse MALDI-TOF mass spectrometer. The main goals are:

• To demonstrate on-chip compound detection down to attomole levels in high-density nanodroplet arrays.
• To evaluate sensitivity gains achieved by reducing spot dimensions.
• To showcase an integrated workflow from solution dispensing to automated MALDI-MS read-out.

Methodology and instrumentation

The workflow consists of non-contact dispensing of compound solutions and matrix onto conductive, patterned DMA slides, followed by ambient drying and direct MALDI-TOF/TOF analysis. Key steps include:

• Preparation of two array formats: 80 spots of 2.83 mm diameter (500 nL each) and 1 152 spots of 900 µm diameter (50 nL each).
• Application of three lipidoid model compounds (m/z 644.522, 686.569, 672.553) over a concentration range corresponding to 0.05–1 000 fmol per spot.
• Dispensing of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution, followed by laser desorption/ionization with 10 000 Hz repetition rate and accumulation of 10 000 laser shots per spot.
• Automated peak detection and signal-to-noise evaluation to estimate limits of detection (LOD).

Used instrumentation

• I-DOT Mini AQ non-contact liquid dispenser (Aquarray GmbH) for precise nano- to microliter sample and matrix deposition.
• AQUARRAY Droplet Microarray conductive ITO-coated slides with omniphilic-omniphobic patterning.
• rapifleX MALDI PharmaPulse MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH) operating in positive reflector mode.

Main results and discussion

The on-chip MALDI-MS workflow achieved sensitive compound detection across both array formats. Key findings include:

• Limit of detection of ~10 fmol for spots of 2.83 mm diameter.
• Limit of detection improved to ~0.1 fmol for spots of 900 µm diameter, representing a 100-fold sensitivity enhancement.
• Higher array density (up to 1 152 spots per slide) directly correlates with improved throughput and reduced sample consumption.

Reducing spot dimensions concentrates analyte and matrix, boosting signal intensity and lowering noise. The DMA platform’s conductive, flat, and transparent properties ensure compatibility with direct MALDI analysis without additional sample transfer.

Benefits and practical applications

Integrating high-density microarrays with ultrafast MALDI-MS read-out offers:

• Substantial reductions in reagent use and per-compound analysis cost.
• Massive throughput gains, enabling parallel synthesis and screening of thousands of compounds.
• Versatility for multimodal assays, including UV-Vis, IR spectroscopy, and cell-based fluorescence microscopy.
• Elimination of sample transfer steps, minimizing cross-contamination and workflow complexity.

Future trends and potential applications

Advancements likely to further enhance on-chip drug discovery platforms include:

• Further miniaturization toward sub-nanoliter droplets to push sensitivity limits.
• Integration of automated data analysis pipelines using machine learning for rapid hit identification.
• Expansion to diverse chemical scaffolds and biological assays, including enzyme kinetics and phenotypic cell screening.
• Development of combined spectroscopic and mass spectrometric multimodal read-outs on a single slide.

Conclusion

This work demonstrates that combining Droplet Microarray technology with high-speed MALDI-TOF mass spectrometry enables attomole-level detection in high-density droplet arrays. The integrated platform streamlines compound synthesis, characterization, and screening into a unified on-chip workflow, offering significant gains in throughput, sensitivity, and cost-efficiency for next-generation drug discovery.

References

• Benz M, Asperger A, Hamester M, Welle A, Heissler S, Levkin PA (2020). A combined high-throughput and high-content platform for unified on-chip synthesis, characterization and biological screening. Nature Communications, 11, 5391.
• Benz M, Molla MR, Böser A, Rosenfeld A, Levkin PA (2019). Marrying chemistry with biology by combining on-chip solution-based combinatorial synthesis and cellular screening. Nature Communications, 10, 2879.