Magnetic Bio-Sensing of Plasma-Derived Extracellular Vesicles for Cancer Screening

Abstract

In the last months, due to the COVID-19 pandemic, we have become familiar with the expression “rapid tests” referred to fast and easy-to-use bioanalytical devices to detect viral proteins or antibodies generated by the patient’s immune system in response to the infection. The same type of rapid tests is extremely interesting to detect cancer biomarkers for disease screening and early diagnosis. Besides the fast response, the optimal technology should be low-cost, easy to use, and portable.The rapid tests rely on the lateral flow immunoassay (LFIA) technique [1]. The LFIA substrate is a strip of nitrocellulose membrane along which the liquid sample containing the molecule of interest can flow by capillary action. Before this flow, the analyte is specifically tagged with fluorescent enzymes or particles to make it visible. Two lines of recognition molecules are immobilized across the strip: (i) the test line, formed by a molecule that captures the tagged molecule of interest, and (ii) the control line, at which the unbound tags are trapped. The LFIA technique is used for point-of-care detection of various biomarkers; the best known is the pregnancy hormone in the popular home pregnancy tests that we can buy in the pharmacy and use ourselves with no prescription.There are some limitations for LFIA expansion, mainly their sensitivity and limit of quantification, which are not sufficient for some biomedical applications. Despite their limitations, WHO highly encourages the research into their performance and utility for disease surveillance [2]. There are two crucial ways in which magnetism can contribute effectively to improve the performance of rapid diagnostic tests: magnetic nanotags and magnetic sensing.The use of magnetic particles to tag the biomolecule of interest can have several advantages compared to non-magnetic tags (used for optical detection): magnetic immuno-concentration, magnetic detection, and long-lasting signal. Once the biomolecule is tagged by magnetic particles, the magnetic pre-concentration or isolation of the target biomolecule can be achieved by a magnetic gradient that attracts them and allow their separation from the original medium. This is enormously advantageous to increase the sensitivity of any technique that is applied after it. We can then detect the particles through a magnetic sensor without interferences from the paper strip or the biological fluid. Finally, the magnetic signal coming from the particles in the test line perdures in time and is insensitive to the paper strip aging or staining [3].We recently developed a bio-detection method based on LFIA using superparamagnetic labels combined with an inductive sensor for reading out their signal. We proved the viability of the technique for quantifying prostatic cancer biomarkers [3] and toxic biogenic amines [4] in the range of interest.In this work, we have developed a lateral flow immunoassay (LFIA) system to detect plasma-derived extracellular vesicles (EV). These small (micro- or nano-sized) membrane structures are released by cells to mediate intercellular communication. Given that they carry proteins and micro-RNA specific to their cell of origin, they are nowadays thoroughly studied as disease biomarkers.We used superparamagnetic iron oxide nanoparticles coated with a double layer of oleic acid [5] as reporter labels. They were conjugated to antibodies against proteins expressed in the EVs surface, specifically CD-63 and CD-147. The latter is in higher concentrations in patients with colorectal cancer [6]. We used different concentrations of EVs purified from human plasma to perform LFIA tests to calibrate the system. Fig. 1 shows some of the LFIA strips obtained for such calibration. Subsequently, the strips were analyzed by a commercial reflectance sensor and our magnetic inductive sensor. The results for the assays with CD63 are shown in Fig. 2. We can appreciate that the linear range extends to all the concentration range understudy for the magnetic sensor. The tendency to saturation that we observe with the optical reader is typical of the immunoassays and is caused by the available antibodies' occupation. Given that the reflectance signal comes only from the particles at the surface of the membrane, the saturation is achieved at lower concentrations than in the magnetic one, which comes from the whole volume.Besides this obvious advantage, the magnetic sensor provides better linearity (even taking a reduced linear range of concentration for the optical signal) and a lower limit of detection (three times smaller for CD63 and two times smaller for CD147). We must emphasize that the analyte has not been magnetically pre-concentrated for these measurements. Such a technique will be studied in an ongoing investigation to increase further the sensitivity of the magnetic LFIA aiming at a rapid, portable technology for liquid biopsy applied to colorectal cancer screening.AcknowledgementsThis work was supported by the Spanish Ministry of Economy and Competitiveness (grant MAT2017-84959-C2-1-R), the Principality of Asturias (IDI/2018/000185) and the Council of Gijón/IUTA (SV-20-GIJON-1-22). **