Periodic Reporting for period 1 - ACO-LC (Applying ACOustic streaming to enhance mass transfer in Liquid Chromatography of large molecules)
Reporting period: 2023-09-01 to 2025-08-31
High performance liquid chromatography (HPLC) is the most widespread analytical separation technique. The unique characteristics, make HPLC an indispensable tool in biological and pharmaceutical research, clinical diagnostics, and food quality inspection. Point-of-care (POC) testing is also a growing domain leading to the transition of diagnostics from the hospital to the primary setting. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke, lower limb amputation, and leads to premature deaths. At present, three methods are recognized for monitoring blood sugar, and consequently, diabetes diagnosing. The level of glycated hemoglobin (HbA1c) has been shown to assess long-term glycemic control and is the best marker for the risk of developing chronic complications of diabetes. However, neither the currently available routine laboratory methods nor POC tests enable to provide high quality HbA1c analysis and accurate quantification of other hemoglobin variants within a single run. The present project introduces a novel methodology to improve the separation efficiency of large molecules such as Hb variants, which is based on vortex generation inside the separation column using acoustic waves. This vortex-LC method enhances separation efficiency through the reduction of sample dispersion, and consequently, the separation resolution improves allowing for the quantification of HbA1c more accurately.
The induction of a lateral flow (orthogonal to axial flow) inside of the separation channel generates vortices. Lateral mixing can be achieved passively through the dedicated geometric designs, or more efficiently, using an active approach that requires additional power input, such as electroosmotic flow and acoustic wave. The latter is commonly established by matching the channel width with the applied acoustic wavelength (λw) to satisfy the resonance condition (characterization dimension =n*λw/2). Consequently, the channel width restricts to ~40μm (not applicable for chromatographic purposes) due to limitations in the commercially available equipment. In this project, hence, a novel strategy was proposed and validated, where the depth of the separation channel was matched with the λw to induce the vortices and the channel width was freely tuned to the chromatographically relevant dimensions. To perform the methodology successfully, it was necessary to consider different fabrication and operation aspects. First, the impact of curvature of the channel bottom was modeled in a simulation study. The results indicated that a curvature radius ranging from 0 to 2.0µm allows to achieving appropriate lateral flow. In practice, SEM images of the separation channel demonstrate the curvature radius in the range of 0.6-2µm. Second, the optimal aspect ratio (AR) of the separation channel (depth/width) was determined by consideration of sample dispersion, sample volume loadability, and availability of actuators. The AR of 7.5 (75/10) represents a practically optimal value, allowing a channel with a depth of 75.0μm to be acoustically matched with a commercially available 10.0 MHz ceramic piezoelectric (PZT) actuator. Third, the separation channel was coupled to the PZT with minimum damping effect and maximized acoustic energy transmission. Frequencies of 10.0 and 9.7 MHz were found to induce effective lateral flow at different potential amplitudes (0.5–2.0Vpp). However, a lower sample dispersion is achieved using the PZT actuating at a frequency of 10.0MHz and a potential of 2.0Vpp. By considering the critical operating aspects and maintaining optimal conditions, the acoustically vortex-mediated channel enables to reduce the sample dispersion up to ten-fold (see Fig.1).
The enhanced chromatographic performance of the methodology was demonstrated by separation of bovine serum albumin (BSA) and dextran (10kDa) as two large molecules in reverse-phase LC mode. Comparison of the obtained results using the same separation channel in the absence and the present of the acoustic-based vortices revealed that high dispersion of BSA leads to a faint peak in the absence of lateral flow (conventional LC), while vortex LC provides a nearly baseline separated chromatogram in half of the analysis time (see Fig.2).
The enhanced chromatographic performance of the methodology was demonstrated by separation of bovine serum albumin (BSA) and dextran (10kDa) as two large molecules in reverse-phase LC mode. Comparison of the obtained results using the same separation channel in the absence and the present of the acoustic-based vortices revealed that high dispersion of BSA leads to a faint peak in the absence of lateral flow (conventional LC), while vortex LC provides a nearly baseline separated chromatogram in half of the analysis time (see Fig.2).
The developed method does not only allow for an improved separation resolution, which can be applied to overcome the overlapping issue of Hb variants and other large molecules, but separations can also be performed in a shorter analysis time. Additionally, improved separation efficiency allows for the use of a larger sized microfluidic channel requiring lower operation pressure. Therefore, portable pumps (delivering pressures up to ~5bar) can be potentially used for POC applications, without the need for bulky systems in expert labs.
To protect the project results for future valorization, a patent application titled "Depth Matched Acoustic Microfluidic Device” was filed on 13/07/25 with application number of EP25189212.1. Further research and application development are needed to move to a higher Technology Readiness Level (TRL). The project team will therefore seek funding to develop the innovation to higher TRL, with the ambition to file for an EIC Transition on “Vortex Chromatography” in 2026. The ultimate ambition is to establish a spin-off company that will further develop and commercialize the acoustic-vortex LC technology.
The novel enhanced LC methodology with unique properties, including fast run, high accuracy, and compact size has high potential to be advanced as a POC system not only for diabetes diagnosis (via HbA1c determination) but also the diagnosis of other diseases through analysis of relevant biomarkers (e.g. proteins). Both the generic separation method (vortex chromatography, with the column and potentially (part of or an entire) instrument) as dedicated POC applications will be considered for valorization. An initial market study has been made by the project team and business development manager Filip Legein, but more in-depth work is needed to map existing and potentially new applications and rank them according to attractiveness, which is planned in the coming period.
To protect the project results for future valorization, a patent application titled "Depth Matched Acoustic Microfluidic Device” was filed on 13/07/25 with application number of EP25189212.1. Further research and application development are needed to move to a higher Technology Readiness Level (TRL). The project team will therefore seek funding to develop the innovation to higher TRL, with the ambition to file for an EIC Transition on “Vortex Chromatography” in 2026. The ultimate ambition is to establish a spin-off company that will further develop and commercialize the acoustic-vortex LC technology.
The novel enhanced LC methodology with unique properties, including fast run, high accuracy, and compact size has high potential to be advanced as a POC system not only for diabetes diagnosis (via HbA1c determination) but also the diagnosis of other diseases through analysis of relevant biomarkers (e.g. proteins). Both the generic separation method (vortex chromatography, with the column and potentially (part of or an entire) instrument) as dedicated POC applications will be considered for valorization. An initial market study has been made by the project team and business development manager Filip Legein, but more in-depth work is needed to map existing and potentially new applications and rank them according to attractiveness, which is planned in the coming period.