(718b) Lithography Free Mem-Delisa Platform for Dual Color and Ultrasensitive Digital Detection of Colocalized Proteins on Extracellular Vesicles

Breast cancer stands as a prominent malignancy affecting women in the United States, ranking second in cancer-related mortality. Despite advancements in diagnostic methods such as mammography, magnetic resonance imaging (MRI), and whole-breast ultrasonography, their effectiveness is hindered by high rates of false positive results. Additionally, invasive procedures like tissue biopsies face challenges such as prolonged turnaround times, operator bias, and potential misinterpretations due to tumor heterogeneity, rendering them unsuitable for monitoring tumor response to anticancer treatments. In contrast, liquid biopsy presents a promising alternative by analyzing a diverse range of protein biomarkers found in biofluids like blood, saliva, or urine. This approach offers insights into disease status with minimal invasiveness, relatively modest cost, and scalability for high-throughput analysis. These molecular biomarkers are often carried on the surface of highly heterogenous and diverse membrane bound nano-micron size particles such as lipoproteins and Extracellular Vesicles (EVs) which also play key role in intercellular communication, immune responses, tissue repair and cancer progression. Consequently, the multiplex detection of various co-localized molecules serving as markers of tissue origin, cancer, drug resistance, or metastasis on these nanocarriers can provide more precise information about the disease.

The current gold standard for protein detection, Enzyme-Linked Immunosorbent Assay (ELISA), suffers from suboptimal limit of detection (~nM-pM), thereby limiting the identification of clinically relevant protein biomarkers (~pM-fM) across various pathological conditions. To surmount this limitation, single-molecule array (Simoa) technology has emerged as a promising alternative. Simoa involves the formation of an immunocomplex wherein a protein molecule is sandwiched between antibody-coated magnetic beads and an enzyme-conjugated detection antibody. Subsequently, this immunocomplex is partitioned into numerous femtoliter (fL) reaction chambers, ensuring that each chamber contains either one or zero molecules. The resulting digital readout provides precise quantification of protein biomarkers, thereby circumventing biases inherent in analog detection methods. However, numerous technical and practical challenges have impeded further development of multiplex digital protein assay for nanocarrier detection.

First, digital protein assay is performed in specially fabricated microwells using expensive and complex lithography techniques, thus increasing complexity and overall expense. Second, the total number of microwells in the device determines the sensor's dynamic range, which is constrained by the master mold's tunability and limits the application of these biosensors for extensive multiplexing applications. Third, air bubbles are often trapped within microwells, potentially affecting quantification accuracy. Resolving this issue often involves bulky vacuum pumps and costly surface modifications. Lastly and most importantly, Simoa-based multiplex digital protein assays are typically restricted to 3-plex immunoassays due to the use of color-coded magnetic beads and a single enzyme amplification reaction. This limits their suitability for applications requiring analysis of multiple protein signatures, such as protein colocalization assays on extracellular vesicles, lipoproteins, viruses, and proteins with multiple epitopes. Addressing these challenges is crucial for broader adoption of digital protein assays in various biomedical applications.

In this study, we introduce a novel dual-color Membrane Digital ELISA (Mem-dELISA) platform that overcomes the limitations of existing methods in a lithography-free and cost-effective manner (< $0.1 per assay). Our approach utilizes track-etched polycarbonate (PCTE) membranes, which possess through-pores (~5 µm) capable of effectively removing air bubbles via wicking. These membranes are sealed on one side through adhesion on the PDMS substrate to create microwells. Immunomagnetic bead-analyte complexes and substrate solution are then introduced into the microwells from the opposite side, achieving over 80% loading efficiency, before sealing with oil. We identified two enzyme candidates: beta galactosidase and alkaline phosphatase and their substrate combinations that demonstrate minimum cross reactivity and spectral overlap. Our Mem-dELISA platform demonstrates exceptional performance, achieving a dynamic range spanning 5 logs and an ultrasensitive limit of detection of 10 aM for both Biotinylated β-galactosidase (B-βG) and Biotin Alkaline Phosphatase Conjugated (B-ALP) proteins. As a proof-of-concept study, we showcase Mem-dELISA’s utility by illustrating that a higher dosage of paclitaxel (chemotherapy drug) effectively suppresses EpCAM-positive extracellular vesicles (EVs) but not GPC-1 positive EVs from breast cancer cells. This decrease in chemo-resistance is not discernible through conventional Western blot analysis of cell lysate. The Mem-dELISA platform offers significant potential for researchers, enabling ultrasensitive and high-throughput protein colocalization studies for disease diagnostics, treatment monitoring, and biomarker discovery. Its simplicity, affordability, and effectiveness position it as a valuable tool for advancing research in diverse biomedical applications.