HPLC 2025 Preview: Silicon-Micro-nanofabrication in Analytical Chemistry — Possibilities Beyond Micro-pillar Array Columns

We are increasingly recognizing that our lifestyles—and the way we conduct science—must evolve. We face immense challenges related to climate change, environmental degradation, and sustainability—and modern science is not without its shortcomings. For example, life science-related activities generate about 5 million tons of plastic waste each year (1). Other challenges related to solvent waste, for example, will undoubtedly be under increasing scrutiny regarding compliance to regulations, and by extension, attractability to user groups and investors. To illustrate, analytical methods based on conventional sample preparations such as protein precipitation and liquid–liquid extraction have rather gray “green-ness” scores of ca. 0.4–0.6 (on a scale of 0 to 1), calculated with the AgreePrep scoring system (2). But sustainability isn’t just about chemicals; for example, over 100 million animals are sacrificed for research yearly (3). The use of animal models is under more regulatory scrutiny, and their relevance/utilization in modern drug development is increasingly challenged (4)

Photo: Frøydis Sved Skottvoll and Steven Ray Wilson

Although life science faces its own sustainability challenges, it remains indispensable. Addressing monumental challenges such as antibiotic resistance, the threat of new pandemics, and the healthcare needs of an aging population will require the expertise of biologists, informaticians, and, of course, analytical chemists, to study larger numbers of often complex and limited samples. Such samples may arise from alternatives to animal models, for example, various microphysiological systems (MPS) such as organ-on-chip (OoC) platforms for high-throughput modeling, to personalized/patient group-specific therapeutics (5)

The Need for Separation Science and Robustness
Analyses of vast numbers of biosamples from small MPS samples would greatly benefit from approaches that are robust, miniaturized, high speed, and automated. To be compliant with sustainability, a reduced reliance on one-time consumables and an increased focus on inline, hyphenated systems can be key. Keeping the challenges and opportunities of sustainability through multi-usage (the very nature of inline analysis) in mind, we are exploring the possibilities of using silicon micro-nanofabrication as a platform for robust bioanalysis, for inline sample preparation, separation, and detection. Can advanced materials and fluidics reduce the use of consumables with robust, multi-use chip technology?

Silicon Micro-nanofabrication: A Brief Introduction
Micro-nanofabrication using glass/silicon is a cornerstone technology that enables the creation of miniaturized and multifunctional systems. Originating from the semiconductor industry, the micro-nanofabrication approach involves a series of highly controlled sequences of thin-film deposition, photolithography, and wet- or dry etching. These processes allow for the layer-by-layer fabrication of complex microscale components—such as microchannels, micropumps, valves, sensors, and actuators. The key characteristic of applying micro-nanofabrication technology in analytical chemistry is the potential of monolithic integration of several modalities on the same chip substrate. Furthermore, silicon micro-nanofabrication is highly scalable, enabling wafer-level production of devices with consistent quality and robust performance, a critical factor for future commercial applications. In the context of MPS such as OoCs (6), silicon-based microfabrication supports the integration of various actuation and sensing modalities, providing the tools for more physiologically accurate simulation of human organs. For instance, optical, acoustic, electrical, and mechanical stimuli can be precisely applied using microfabricated elements, while embedded sensors enable real-time monitoring of cellular responses. This level of multimodality integration is challenging to achieve with traditional polymer-based fabrication techniques.

Opportunities for Sample Preparation
Of course, the separation scientist will be familiar with previous utilizations of silicon micro-nanofabrication in chromatography, that is, micro-pillar array columns (9,10).µPACs have been shown to offer outstanding chromatographic performance through their highly ordered and reproducible features (11,12). µPACs have arguably found most influence in small-sample proteomics. However, we are exploring the possibilities of developing similar formats for other analytes, particularly for limited samples. Moreover, such separation devices may be integrated with online sample preparation devices. With its inherent advantages in scalability, reproducibility, and precise microchanneling, silicon micro-nanofabrication may well prove to be a pivotal technology for analytical chemistry in modern life sciences.

References
(1) Urbina, M. A.; Watts, A. J. R.; Reardon, E. E. Labs Should Cut Plastic Waste Too. Nature2015, 528 (7583), 479–479. DOI: 10.1038/528479c
(2) Wojnowski, W.; Tobiszewski, M.; Pena-Pereira, F.; Psillakis, E. AGREEprep – Analytical Greenness Metric for Sample Preparation. TrAC Trends Anal. Chem.2022, 149, 116553. DOI: 10.1016/j.trac.2022.116553
(3)Taylor, K.; Alvarez, L. R. An Estimate of the Number of Animals Used for Scientific Purposes Worldwide in 2015. Altern. Lab. Anim.2019, 47 (5–6), 196–213. DOI: 10.1177/0261192919899853
(4) Poh, W. T.; Stanslas, J. The New Paradigm in Animal Testing – “3Rs Alternatives.” Regul. Toxicol. Pharmacol.2024, 153, 105705. DOI: 10.1016/j.yrtph.2024.105705
(5) Ko, J.; Song, J.; Choi, N.; Kim, H. N. Patient‐Derived Microphysiological Systems for Precision Medicine. 2024, 13 (7), 2303161. DOI: 10.1002/adhm.202303161
(6) Skottvoll, F. S.; Escobedo-Cousin, E.; Mielnik, M. M. The Role of Silicon Technology in Organ-On-Chip: Current Status and Future Perspective. Adv. Mater. Technol.n/a (n/a), 2401254. DOI: 10.1002/admt.202401254
(7) Skottvoll, F. S.; Aizenshtadt, A.; Hansen, F. A.; et al. Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Anal. Sens. 2022, 2 (2), e202100051. DOI: 10.1002/anse.202100051
(8) Kogler, S.; Skottvoll, F. S.; Hrušková, H.; et al. Electromembrane Extraction Provides Unprecedented Selectivity for Drugs in Cell Culture Media Used in Organoid and Organ-on-Chip Systems. Anal. Chem. 2025, 97 (9), 4923–4931. DOI: 10.1021/acs.analchem.4c04994
(9) De Malsche, W.; Eghbali, H.; Clicq, D.; et al. Pressure-Driven Reverse-Phase Liquid Chromatography Separations in Ordered Nonporous Pillar Array Columns.Anal. Chem. 2007, 79 (15), 5915–5926. DOI: 10.1021/ac070352p
(10) Müller, J. B.; Geyer, P. E.; Colaço, A. R.; et al. The Proteome Landscape of the Kingdoms of Life.Nature 2020, 582 (7813), 592–596. DOI: 10.1038/s41586-020-2402-x
(11) Sandra, K.; Malsche, W. D.; t’Kindt, R.; et al. Evaluation of Micro-Pillar Array Columns (µPAC) Combined with High Resolution Mass Spectrometry for Lipidomics. Recent Developments in HPLC and UHPLC, LCGC Supplement 2017, 30, 6–13.
(12) Aizenshtadt, A.; Midtøy, L.; Thiede, B.; Krauss, S.; Reberg-Larsen, H.; Wilson, S. Micro-Pillar Array Column Separations for Proteomics of Liver Organoids. 2023, 36, 16–19.

Steven Ray Wilson is a Professor at the Department of Chemistry, University of Oslo. During his career, Wilson has developed and applied analytical systems with a focus on miniaturization, automation, and hyphenation. Application areas have been focused on biomedical research and applications, e.g. cancer stem cell-ness and related signal pathways (Hedgehog, Wnt). In recent years, Wilson has focused on the study of organoids and organ-on-a-chip systems, which are emerging alternatives to animal models in e.g. drug discovery. He engages in multi-disciplinary research as a PI in the Center of Excellence Hybrid Technology Hub, undertaking drug analysis and various omics approaches to the study of laboratory-grown organ models. Key visions are to develop analytical tools that will aid the reduction of animal models and the reduction of plastic waste in life science.

Frøydis Sved Skottvoll is a researcher in the BioMEMS and Medical Sensors group at SINTEF Digital, within the department of Smart Sensors and Microsystems. During her doctoral studies (2022, University of Oslo, Department of Chemistry), she developed an innovative platform for the automated drug analysis of organoids. Currently, she focuses on integrating micro- and nanofabrication technology with analytical chemistry, making current setup more suitable for small samples, high throughput, and enabling miniaturization. Her contributions to the field have recently earned her recognition as one of Norway's 50 most influential women in t