Application of Metal-organic frameworks synthesized by Microfluidics in Microextraction for Metabolomics: development of a non-invasive bioanalytical method for early diagnosis of fatty Liver diseases

Project Information

M4Liver

Grant agreement ID: 101059391

DOI

10.3030/101059391

Project closed

EC signature date 15 July 2022

Start date 1 September 2023

End date 31 August 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 187 624,32

Coordinated by

ACADEMISCH ZIEKENHUIS GRONINGEN
Netherlands

Periodic Reporting for period 1 - M4Liver (Application of Metal-organic frameworks synthesized by Microfluidics in Microextraction for Metabolomics: development of a non-invasive bioanalytical method for early diagnosis of fatty Liver diseases)

Reporting period: 2023-09-01 to 2025-08-31

Summary of the context and overall objectives of the project

Non-alcoholic fatty liver diseases (NAFLD) are the most common and undiagnosed liver disorders worldwide that can only be diagnosed via risky and complex liver biopsies. The determination of metabolites in biological fluids is one of the most promising alternatives to these invasive procedures. Despite several studies have identified branched amino acids in blood plasma as potential biomarkers, there are a lot of inconsistencies between the results. This is because of the complexity of the analysis given the high hydrophilicity of the amino acids and other metabolites and their low concentration, which hampers their extraction from the plasma and leads to time-consuming and laborious procedures. Thus, there is a need to develop a simple, selective and sensitive analytical method for a reliable diagnosis of NAFLD by detecting key biomarkers. Microextraction techniques are very simple and present high preconcentration, clean-up and sustainability features for sample preparation. There are a few commercially available extraction phases, but they lack selectivity, stability and cannot target highly hydrophilic compounds. In this context, metal-organic frameworks (MOFs) are materials that have attracted much attention because of their high surface area and synthetic tunability, which might lead to better extraction efficiency and tailored selectivity.
The M4Liver project aimed to develop a new analytical platform for the direct determination of hydrophilic biomarkers in plasma samples, which can be implemented for the diagnosis of liver diseases. Here, MOFs were systematically studied in terms of extraction capacity and selectivity towards branched amino acids. Then, the MOFs were incorporated in a microextraction device and evaluated for the analysis of plasma samples. Ultimately, the MOF-based microextraction device could be used as a simple and sensitive tool not only for the determination of amino acids, but also for obtaining the metabolomics profile of the samples, which could be expanded for the simple monitoring of other diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

MOFs of aluminum and zirconium were evaluated because of their stability in water and high surface areas. The screening studies together with the adsorption kinetics and isotherms for the extraction of amino acids revealed that stronger interactions were involved in the extraction with zirconium MOFs, thus better recoveries were achieved when using aluminum MOFs with high surface areas. The MIL-68(Al) was used to prepare the microextraction device that consisted of 1 mg of MOF particles immobilized on nitinol sheets with the aid of polyacrylonitrile. In the microextraction method, the device was directly stirred in the plasma to extract the biomarkers, followed by the immersion of the device in a low volume of water for the desorption, which was then subjected to analysis by liquid-chromatography and fluorescence detection. The method was optimized and validated for the analysis of plasma, observing little effects of proteins in the extraction performance thanks to the anti-fouling features of polyacrylonitrile. In a comparative study, the MOF device outperformed all the commercial sorbents, including HLB, which is commonly used for metabolomics studies. Quantification of branched amino acids in donor samples demonstrated the applicability of the method, which also presented high precision when using different devices. Finally, the microextraction method was applied in a preliminary metabolomics study, showing the possibility of expanding the use of MOFs not only for the determination of hydrophilic compounds but also hydrophobic, covering a wide range of metabolites in a simple and fast methodology in comparison with conventional protocols.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The M4Liver project demonstrated the potential of MOFs to be used as sorbents for the extraction of highly hydrophilic compounds from aqueous samples, which is a long-standing challenge in the sample preparation field. This microextraction device and methodology can be also applied for more complete analysis that require the determination of a wide variety of compounds, as it is the case of metabolomics. This can be expanded to other diseases besides liver diseases. According to the initial objectives of the project, further research is required to show the applicability of this platform to diagnose and monitor liver diseases. Thus, the methodology needs to be applied for the analysis of plasma from patients and evaluate the different metabolomics profile to discriminate the different stages of the diseases by this simple blood analysis.
Given the simplicity of the technique, the low requirements of materials and the absence of organic solvents or other toxic materials, this methodology can be easily implemented in routine laboratory analysis. For further commercialization of the device, the only challenge is the acquisition and/or synthesis of the MOFs, which still face sustainability and scalability issues and was beyond this research project.