Porous PDMS‐Based Microsystem (ExoSponge) for Rapid Cost‐Effective Tumor Extracellular Vesicle Isolation and Mass Spectrometry‐Based Metabolic Biomarker Screening

Abstract

communication. Their membrane contains and protects proteins, nucleic acids, and metabolites. Hence, they can serve as active cargo delivery vehicles and messengers of genetic information. [1] There has been an increased interest in the clinical use of EVs as biomarkers due to their significance in cellular signaling, disease progression, and therapeutics. The isolation and characterization of exosomes allow their use as reliable biomarkers for minimally invasive disease diagnosis, called liquid biopsy. One of the promising opportunities for disease diagnosis is the diagnosis of cancer since EV secretion is increased by many cancer types. Their presence in a variety of biosamples -that is, blood, urine, saliva -makes them an attractive avenue for exploration for liquid biopsy to provide a simple, in vitro analysis of a patient's tumor status. [2,3] Deregulated cellular metabolism has been established as a hallmark of cancer which supports the use of metabolite characterization as a sensitive, inexpensive, and origin-agnostic tool for minimally-invasive diagnosis of cancer. [4] However, the large amounts of biosample contaminants and the size of the desired endosome-derived small EVs (sEV), also called exosomes, at just 30-150 nm diameter makes isolation difficult and challenging [5] using conventional EV isolation methods.Polydimethylsiloxane (PDMS) is an inexpensive robust polymer that is commonly used as the fundamental fabrication material for soft-lithography-based microfluidic devices. Owing to its versatile material properties, there are some attempts to use PDMS as a porous 3D structure for sensing. However, reliable and easy fabrication has been challenging along with the inherent hydrophobic nature of PDMS hindering its use in biomedical sensing applications. Herein, a cleanroom-free inexpensive method to create 3D porous PDMS structures, "ExoSponge" and the effective surface modification to functionalize its 3D porous structure is reported. The ability of ExoSponge to recover cancer-associated extracellular vesicles (EVs) from complex biological samples of up to 10 mL in volume is demonstrated. When compared to ultracentrifugation, the ExoSponge shows a significant increase in cancer EV isolation of more than 210%. Targeted ultra-high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further employed to profile 70 metabolites in the EVs recovered from the lung cancer patient's plasma. The resulting profiles reveal the potential intraexosomal metabolite biomarker, phenylacetylglutamine (PAG), in non-small cell lung cancer. The high sensitivity, simple usage, and cost-effectiveness of the ExoSponge platform creates huge potential for rapid, economical and yet specific isolation of exosomes enabling future diagnostic applications of EVs in cancers.