sensors Article Matrix Effect in the Isolation of Breast Cancer-Derived Nanovesicles by Immunomagnetic Separation and Electrochemical Immunosensing—A Comparative Study Silio Lima Moura 1 , Mercè Martì 2 and María Isabel Pividori 1, * 1 Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; siliosilicio@hotmail.com 2 Laboratori d’Immunologia Cel·lular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; merce.marti@uab.cat * Correspondence: Isabel.Pividori@uab.cat; Tel.: +34-93-581-1976  Received: 17 November 2019; Accepted: 6 February 2020; Published: 11 February 2020  Abstract: Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods. Keywords: exosomes; breast cancer biomarker; liquid biopsy; immunomagnetic separation; ultracentrifugation; electrochemical immunosensor 1. Introduction Breast cancer is a highly lethal malignancy and the most commonly diagnosed cancer among women (WHO), with an estimated over 2 million new cases in 2018 [1]. Although treatments have increased the survival rate, it is still considered a major cause of morbidity and mortality in women. Treatment efficacy is highly related to early diagnosis. There is thus a growing demand for biomarkers that can help detect diseases at an early stage, as well as for follow-up of patients and therapeutic strategies. The exosomes [2] have received increasingly attention in the last years as a biomarker Sensors 2020, 20, 965; doi:10.3390/s20040965 www.mdpi.com/journal/sensors Sensors 2020, 20, 965 2 of 21 for early cancer detection and monitoring [3]. Exosomes are extracellular nanovesicles of 30–150 nm released by all types of cells during fusion of the multivesicular endosomes (MVEs) with the plasmatic membrane. They carry a cargo of active molecules to proximal and distal cells of the body as a mechanism of physiological communication for maintaining the natural homeostasis or even for a pathological response [4]. All types of cells use exosomes for this purpose. Importantly, one of the most remarkable features is that they are present in all the biological fluids, such as blood [5], saliva [6] and urine [7], among others. Their easy accessibility is one of the most compelling reasons for developing exosomes as clinical biomarkers. Another striking characteristic is their molecular cargo, which can be useful for diagnosis and prognosis of several diseases and conditions. During the biogenesis, some components of the cell of origin remain in the exosomes. Therefore, they carry specific surface markers that indicate their cell signatures, including surface proteins such as proteins related to transport and fusion (e.g., flotillin, caveolin-1), tetraspanins (e.g., CD9, CD63, CD81), heat shock proteins (e.g., Hsp90) and lipid-related proteins, as well as micro RNA (miRNA), messenger RNA (mRNA) and DNA [8]. However, realizing the potential of these vesicles as biomarker requires technical improvement, since the exosomes are exceptionally challenging to characterization with current technologies. The most common methods for targeting exosomes to date typically involve purification [9] followed by the specific characterization of their cargo [10]. The characterization firstly relies on morphological analysis 10 . The nanometric size of exosomes puts them out of the sensitivity range of most cell-oriented sorting or analysis platforms, as is the case of the classical flow cytometers. Exosomes can only be visualized with an electron microscope. Nanoparticle tracking analysis (NTA) is usually used to count the exosomes, followed by downstream processes for specific detection, including LC-MS/MS, Western Blot, and RT-PCR. Furthermore, due to their low concentration, conventional procedures for exosome characterization and detection usually require relatively large sample volumes and involve a preliminary purification and preconcentration step by ultracentrifugation, precipitation, size-exclusion chromatography or ultrafiltration, in order to prevent interferences from cells and free biomolecules in the sample. Differential centrifugation was the first technique described [2] and later optimized [10] by increasing g values (ultracentrifugation), being currently considered the gold standard for exosomes isolation. The main goal is to eliminate interferences of dead cells, cell debris and soluble protein by a physical separation. The major drawback is the lack of specificity, since it separates the whole population of exosomes, regardless their cell origin. Moreover, it can produce mechanical damage. The whole procedure is thus time consuming, requiring skilled personnel as well as laboratory facilities and benchtop instrumentation. Therefore, there is a growing need for novel methods to accurately characterize and specifically isolate exosomes in complex biological fluids. Since the early reports on magnetic separation technology [11], magnetic particles (MPs) have been used as a powerful and versatile preconcentration tool in a variety of analytical and biotechnology applications [12] and in emerging technologies including microfluidic devices and biosensors [13–16]. Magnetic particles (core such as iron, nickel, neodymium or magnetite) can be easily functionalized with biomolecules (for instance antibodies) to specifically bind the exosomes and concentrate them from the complex matrix under magnetic actuation, avoiding the interference of the biofluid matrix. Exosomes are thus preconcentrated while the interfering matrix is removed at the same time, increasing the sensitivity of the detection. Recent advances demonstrated the feasibility of the integration of MPs into bioassays for the detection and quantification of exosomes. We have recently demonstrated the feasibility of particle-based magnetic enrichment for exosomes isolation avoiding the ultracentrifugation steps in undiluted human serum using a magneto-actuated enzyme-linked immunosorbent assay (magneto-ELISA), which simplifies the conventional ELISA [17]. Furthermore, magnetic particles can be actuated on the surface of electrochemical transducers [13,16]. This kind of approach simplifies the analytical procedure in order to be used in resource-scarce settings by unskilled personnel, for the rapid detection of a condition, such as breast Sensors 2020, 20, 965 3 of 21 cancer, the most commonly diagnosed cancer among women [1]. Most of the currently available technologies for breast cancer diagnosis are based on imaging techniques [18]. The liquid biopsies based on circulating tumor cells (CTCs) can potentially simplify the equipment requirements and operational costs [19,20]. However, the clinical use of liquid biopsies is limited by their rarity in the peripheral blood [21] (1 CTC in 1 × 105–6 blood cells), hindering their detection even with benchtop equipment and requiring preconcentration from a large volume of sample [22]. However, a single breast cancer cell can release up to 60–65 exosomes per hour [23]. In this paper, we compare the analytical performance of immunomagnetic separation for the isolation of nanovesicles from breast cancer cell lines spiked in serum, and the further detection by a magneto-actuated electrochemical immunosensor. To achieve that, the culture supernatant from three breast cancer cell lines, including MCF7 (ATCC® HTB-22™), MDA-MB-231 (ATCC® CRM-HTB-26™) and SKBr3 (ATCC® HTB-30™) were purified by ultracentrifugation and spiked in phosphate buffer and depleted human serum at a wide concentration range, in order to carefully evaluate the effect of the human serum matrix and the presence of free receptors in the immunomagnetic separation (IMS). The results suggest that the use of CD81 modified magnetic particles can be useful for the specific isolation of exosomes, and further labeling using general as well as cancer-related receptors for the electrochemical readout. 2. Materials and Methods 2.1. Instrumentation Nanoparticle tracking analysis (NTA) was performed using the NanoSight LM10-HS system with a tuned 405 nm laser (NanoSight Ltd., Salisbury, UK). The cryogenic transmission electron microscopy (TEM) images were collected by a Jeol JEM 2011 (JEOL USA Inc, Peabody, MA, USA) transmission electron microscope at an accelerating voltage of 200 kV. The confocal images were collected on the microscope Leica, TCS SP5 (Leica Microsystems, Wetzlar, Germany). Flow cytometry was performed using BD FACSCANTO II (BD Biosciences, San Jose, CA, USA) equipment. The media fluorescence intensity (MFI) and beads count data of every sample-reading file were obtained by FlowJo analysis software. Optical measurements were performed on a TECAN Sunrise (TECAN AG, Männedorf, Switzerland) microplate reader with Magellan v4.0 software. Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, Denmark). All electrochemical experiments were performed using an AUTOLAB PGSTAT10 (Metrohm, Herisau, Switzerland) potentiostat/galvanostat electrochemical analyzer. The data were in all cases fitted using a nonlinear regression (four parameters logistic equation). 2.2. Chemicals and Biochemicals Magnetic particles (Dynabeads® M450 Tosylactivated, nº 14013, 4.5 µm diameter) were purchased from Thermo Fisher (Waltham, MA, USA). The antibodies are summarized in Table 1. The antibodies were selected towards different biomarkers that were previously reported to be expressed in the surface of exosomes, as shown in Table 2. The 10 mmol L−1 phosphate-buffered saline (PBS) buffer solution (pH 7.5) and boric acid buffer solution (pH 8.5) were prepared with ultrapure water and all other reagents were in analytical reagent grade (supplied from Sigma Aldrich, St. Louis, MO, USA). The Pierce™ TMB Substrate Kit (Ref. 34021) was purchased from Thermo Fisher. The Hoechst dye (No. 62249, Thermo Fisher) and Cy® 5 fluorophore dye (anti-mouse, No. ab97037, Abcam, Cambridge, UK) used in confocal microscopy were purchased from distributors in Barcelona, Spain. Ultrapure water (Millipore® System, resistivity 18.2 MΩ cm) was used throughout the experiments. Sensors 2020, 20, 965 4 of 21 Table 1. Summary of the antibodies used in this work. Antibody. Target Clonality Conjugate Host Reference Commercial Source Use/s antiCD24 CD24 monoclonal no mouse ab76514 Abcam 1, 2, 3 antiCD54 CD54 monoclonal no mouse ab2213 Abcam 1, 2, 3 antiCD326 CD326 monoclonal no mouse ab7504 Abcam 1, 2, 3 antiCD340 CD340 monoclonal no mouse Ab30 Abcam 1, 2, 3 antiCD9 CD9 monoclonal no mouse 10626D Thermo Fisher 1, 2, 3 antiCD63 CD63 monoclonal no mouse 10628D Thermo Fisher 1, 2, 3 antiCD81 CD81 monoclonal no mouse 10630D Thermo Fisher 1, 2, 3 antiCD44 CD81 monoclonal no mouse BMS113 eBioscience 1, 2, 3 antiCD81 CD81 polyclonal no rabbit HPA007234 Sigma-Aldrich 1, 2, 3 antimouse-HRP mouse IgG H&L polyclonal HRP rabbit ab6728 Abcam 4 antimouse-Cy5 mouse IgG H&L polyclonal Cy5® rabbit ab97037 Abcam 5 antiCD63-HRP CD63 monoclonal HRP mouse NBP2-42225H-100 BioNova 6 Uses: 1 Immunomagnetic separation when attached to tosyl-activated magnetic particles. 2 Indirect labeling in electrochemical immunosensing, as a primary antibody. 3 Indirect labeling in confocal microscopy and flow cytometry, as a primary antibody. 4 Indirect labeling in magneto-actuated enzyme-linked immunosorbent assay (magneto-ELISA) and electrochemical immunosensing, as a secondary antibody. 5 Indirect labeling in confocal microscopy and flow cytometry, as a secondary antibody. 6 Direct labeling in magneto-ELISA and electrochemical immunosensing, as a secondary antibody. Table 2. Description of the biomarkers selected in this work. Most Prominent Carcinomas Biomarker Length/Mass * Description * Function * Reported in 2018 ** Breast [24]/Prostate [25]/ CD9 141–228 aa/15.9–25.4 kDa Lung [26]/Colorectum [27]/ Stomach [28]/Liver [29] Cell-surface proteins member of the Involved in cell adhesion, migration Breast [30]/Prostate [31]/ transmembrane 4 superfamily, also known as the CD63 133–238 aa/14.3–25.6 kDa and motility and also platelet activation Lung [32]/Colorectum [33]/ tetraspanin family. Tetraspanins are extensively and aggregation. Stomach [34]/Liver [35] and variably glycosylated. Breast [36]/Prostate [37]/ CD81 51–274 aa/5.6–29.8 kDa Lung [38]/Colorectum [39]/ Stomach [40]/Liver [41] Sensors 2020, 20, 965 5 of 21 Table 2. Cont. Most Prominent Carcinomas Biomarker Length/Mass * Description * Function * Reported in 2018 ** Cell-surface small protein that is heavily and Involved in the regulation of cell Breast [42]/Prostate [43]/ distinctly glycosylated. It is a sialoglycoprotein CD24 37–129 aa/4.1–13 kDa binding capacity, proliferation, Lung [44]/Colorectum [45]/ expressed in B-cell development and B-cell maturation and tumor metastasis. Stomach [46]/Liver [47] neoplasia and a large variety of malignant tumors. Cell-surface transmembrane glycoprotein, receptor Involved in in cell activation, for hyaluronic acid (HA) and other ligands, such Breast [48]/Prostate [49]/ proliferation, differentiation, CD44 78–742 aa/8.8–81.5 kDa as osteopontin, collagens and matrix Lung [50]/Colorectum [51]/ recirculation and homing, metalloproteinases (MMPs). Frequently called Stomach [52]/Liver [53] hematopoiesis and tumor metastasis. HCAM (homing cell adhesion molecule) Cell-surface transmembrane glycoprotein of the Involved in leukocyte endothelial CD54 (ICAM-1, immunoglobulin superfamily. Typically expressed Breast [54]/Prostate [55] transmigration, cell signaling, adhesion, Intercellular Adhesion 180–532 aa/19.4–57.8 kDa on endothelial cells and also found in epithelial /Lung [56]/Colorectum [57]/ polarity, cell-cell interaction, tissue Molecule 1) cells and cells of the immune system. Frequently Stomach [58]/Liver [59] stability and tumor metastasis. called ICAM-1 (intercellular adhesion molecule 1). Cell-surface transmembrane glycoprotein that CD326 (Epithelial cell mediates cell adhesion in epithelia. Plays a role in Involved in cell signaling, migration, Breast [60]/Prostate [61]/ adhesion molecule 199–342 aa/21–37.9 kDa tumorigenesis and metastasis of carcinomas. It is proliferation, differentiation and Lung [62]/Colorectum [63]/ (EpCAM) also frequently called EpCAM (epithelial cell tumor metastasis. Stomach [64]/Liver [65] adhesion molecule). Involved in the binding with the Cell-surface transmembrane glycoprotein that is CD340 (HER2/erbB2, ligand-bound epidermal growth factor a receptor for members of the epidermal growth Breast [66]/Prostate [67]/ tyrosine kinase cell (EGF) receptor family members, 102–1255 aa/11.5–137.9 kDa factor (EGF) family. Plays a role in tumorigenesis Lung [68]/Stomach [69]/ surface receptor HER2, enhancing kinase-mediated activation and metastasis of carcinomas. Frequently called Liver [70] oncogene ERBB2) of downstream signaling pathways and HER2 (human epidermal growth factor receptor 2). tumor metastasis. * Data were taken from The Human Protein Atlas; https://www.proteinatlas.org/. ** Most prominent carcinomas in 2018 were taken from the Global Cancer Observatory: https://gco.iarc.fr/. The breast cancer cell lines were the following: MCF7 (ATCC® HTB‐22™), MDA‐MB‐231 (ATCC® CRM‐HTB‐26™) and SKBr3 (ATCC® HTB‐30™). Expansion of cell population was carried out from 1,000,000 cells in a T‐175 flask containing 32 mL of Dulbecco’s Modified Eagle Medium (DMEM) (Ref. 31966047, Thermo Fisher), supplemented with 10% exosome‐depleted fetal bovine Sensors 2020, 20, 965 6 of 21 serum (FBS) (Ref. 12007C, Sigma‐Aldrich, St. Louis, MO, USA), 100 U mL−1 penicillin‐streptomycin (Ref. 15140122, Thermo Fisher). The temperature was maintained at 37 °C in a humidified, concentrated 2.3. Cell Culture CO2 (5%) atmosphere. Once cells reached approximately 95% confluence on the T‐175 flask, the culture medium was removed and immediately centrifuged (300 g for 10 min, 2000 g for 10 The breast cancer cell lines were the following: MCF7 (ATCC® HTB-22™), MDA-MB-231 (ATCC® min and 10000 g for 30 min) and stored at −80 °C until exosome isolation was achieved. CRM-HTB-26™) and SKBr3 (ATCC® HTB-30™). Expansion of cell population was carried out from 1,000,000 cells in a T-175 flask containing 32 mL of Dulbecco’s Modified Eagle Medium (DMEM) 2.4 Exosome Isolation and Purification (Ref. 31966047, Thermo Fisher), supplemented with 10% exosome-depleted fetal bovine serum (FBS) (Ref.Exosomes were purifiedSt. 12007C, Sigma-Aldrich, according to Théry Louis, MO, USA),Error! U mL−1 100Bookmark not defined. and as schematically shown in penicillin-streptomycin (Ref. 15140122, Figure 1, panel A. The supernatant from MCF7, MDA‐MB‐231 ◦ Thermo Fisher). The temperature was maintained at 37 C in a humidified, and SKBr3 breast cancer cell lines concentrated CO2were(5%) subjected atmosphere. to differential centrifugation Once cells reached as follows: approximately 95%1300 g for 5 min confluence on the(removal of residual T-175 flask, cells),medium the culture 2000 g for was15removed min and 10,000 and g for 30 centrifuged immediately min (removal (300ofg cellular debris). for 10 min, 2000 gSubsequently, for 10 min anda 10000 Beckmang forCoulter 30 min) Optima TM L‐80XP ◦Ultracentrifuge was used at 100,000 g for 60 min with a 70Ti rotor to pellet and stored at −80 C until exosome isolation was achieved. exosomes. After that, the supernatant was carefully removed, and crude exosome‐containing pellets 2.4. Exosome were Isolation resuspended in and Purification 10 mmol L−1 phosphate‐buffered saline (PBS) solution and pooled. A second roundExosomes of the same ultracentrifugation were purified according setting was[10] to Théry carried and out, and the resulting as schematically shown exosome in Figurepellet was 1, panel resuspended A. The supernatantin 500 from μL (per 100 MDA-MB-231 MCF7, mL of supernatant) of 10 breast and SKBr3 mmol cancer L PBScell −1 buffer linessolution (0.22 μm were subjected to filtrated and sterile) and stored at −80 °C. All centrifugation steps were performed differential centrifugation as follows: 1300 g for 5 min (removal of residual cells), 2000 g for 15 min at a temperature of and4 °C. 10,000 g for 30 min (removal of cellular debris). Subsequently, a Beckman Coulter OptimaTM L-80XP Ultracentrifuge was used at 100,000 g for 60 min with a 70Ti rotor to pellet exosomes. After 2.5. Exosome‐Depleted Human Serum that, the supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 10The mmol L−1 phosphate-buffered exosome‐depleted salinewas human serum (PBS) solution used and pooled. for several A second matrix effect round studies. Theofdepletion the same of exosomes in human ultracentrifugation settingserum from healthy was carried out, and patients wasexosome the resulting performed aswas pellet described above,inand resuspended 500 µLis −1 schematically (per 100 mL ofshown in Figure supernatant) 1, panel of 10 mmol B.L This PBSexosome‐depleted human buffer solution (0.22 serum was µm filtrated and spiked sterile) at and a different −80 ◦ C. All centrifugation stored at concentration range with the exosomes steps obtained as were performed at aabove (Figure 1, temperature 4 ◦ C. A). of panel Figure 1. (A) Schematic procedure for the isolation of exosomes from cell culture supernatant of breast cancer cells. (B) Schematic procedure for the obtaining of depleted human serum. The undiluted serum Figure 1. as (as well (A)10%, Schematic procedure 25% and for the in 50% dilutions isolation of exosomes from phosphate-buffered cell(PBS)) saline culture is supernatant then spikedof breast with the cancer cells. (B) Schematic procedure for the obtaining of depleted human serum. purified exosomes, as well as PBS as a control, in order to perform the matrix effect study.The undiluted serum (as well as 10%, 25% and 50% dilutions in phosphate‐buffered saline (PBS)) is then spiked with 2.5. Exosome-Depleted the purified exosomes,Human Serum as well as PBS as a control, in order to perform the matrix effect study. The exosome-depleted human serum was used for several matrix effect studies. The depletion of exosomes in human serum from healthy patients was performed as described above, and is schematically shown in Figure 1, panel B. This exosome-depleted human serum was spiked at a different concentration range with the exosomes obtained as above (Figure 1, panel A). Sensors 2020, 20, 965 7 of 21 Sensors 2020, 20, x FOR PEER REVIEW 7 of 22 2.6. Covalent Immobilization on Magnetic Particles 2.6. Dynabeads® Dynabeads ® M450 tosylactivated superparamagnetic particles (MPs, 4.5 μm in diameter) have a M450 tosylactivated superparamagnetic particles (MPs, 4.5 µm in diameter) have acore coreofofiron ironoxide oxidesalt saltencapsulated encapsulatedby byaa polystyrene polystyrene polymer, polymer, which has a polyurethane polyurethane external external layer with the p‐toluenesulfonate group [71]. It is a good leaving group, which allows layer with the p-toluenesulfonate group [71]. It is a good leaving group, which allows an SN reaction an S N2 reaction to occur to occur inin the presence presence of a nucleophile [72]. A nucleophilic nucleophilic reaction by an antibody, protein, protein, peptide peptide or glycoprotein or glycoprotein removes removes and and replaces replacesthe thesulfonyl sulfonylester estergroups groupsfromfromthethepolyurethane polyurethanelayer. layer. Two different Two different approaches approaches were were used, used, asas depicted depicted in in Figure Figure 2. 2. The The first first one one involves involves the the direct direct covalent immobilization covalent immobilization of of exosomes exosomes onon magnetic magnetic particles particles (Figure (Figure 2,2, panel panel A). A). The second approach approach is based is based onon the the covalent covalent immobilization immobilization of of the the antibodies antibodies for for aa further further immunomagnetic immunomagnetic separation separation (IMS) of (IMS) of exosomes exosomes (Figure (Figure 2, 2, panel panelB). B). . Figure 2. (A) Direct covalent immobilization on Dynabeads® M450 tosylactivated. (B) Covalent Figure 2. (A) Direct covalent immobilization on Dynabeads® M450 tosylactivated. (B) Covalent immobilization of different antibodies (as depicted in Table 1) on Dynabeads®® M450 tosylactivated. immobilization of different antibodies (as depicted in Table 1) on Dynabeads M450 tosylactivated. Immobilization of exosomes. The immobilization of exosomes on Dynabeads® M450 tosylactivated Immobilization of superparamagnetic exosomes. particles The(Figure (MPs) immobilization 2, panel A)of was exosomes performedon Dynabeads as follows: M450 ® tosylactivated 3.5 × 10 10 exosomes superparamagnetic particles (MPs) (Figure 2, panel A) was performed as were added to 40 µL (1.6 × 107 MPs) tosylactivated Dynabeads® M450. The reaction kinetics follows: 3.5 × 10 10 exosomes were were added to 40 μL (1.6 × 10 7 −1MPs) tosylactivated Dynabeads ® M450. The reaction increased by adding a 0.1 mol L pH 8.5 borate buffer, in order to ensure the nucleophilic reaction kinetics were by increased by adding a 0.1 mol L −1 pH 8.5 borate buffer, in order to ensure the nucleophilic reaction the amine group. The incubation step was performed overnight with gentle shaking at 4 C. After that, ◦ aby0.5the molamine group. solution L−1 glycine The incubation was added steptowas performed ensure overnight the blocking of anywith gentle tosylactivated remaining shaking at 4 °C. After groups, that, by a 0.5 mol for incubation L glycine −1 2 h at 25solution ◦ C. After was added that, to ensure the blocking the exosome-modified of any particles magnetic remaining tosylactivated (exosomes-MP) groups, by incubation for 2 h at 25 °C. −1 After that, the exosome‐modified were resuspended in 160 µL of 10 mmol L PBS buffer solution in order to achieve 1 × 106 MPs magnetic particles per (exosomes‐MP) were resuspended in 160 μL of 10 mmol L −1 PBS buffer solution in order to achieve 1 6 10 µL. The calibration plots were performed by using 1 × 10 MPs per well plate and the number of × 106 MPs per 10 μL. The calibration plots were performed by using 1 × 106 MPs per well plate and exosomes ranged from 3.9 × 104 to 2 × 107 exosomes/µL−1 . the number of exosomes ranged from 3.9 × 104 to 2 × 107 exosomes/μL−1. Immobilization of antibodies. The different specific antibody (approximately 15 µg mL−1 antiCDX, Immobilization of antibodies. The different specific antibody (approximately 15 μg mL−1 antiCDX, optimized in each case as described in detail by our research group [17] to achieve full coverage) optimized in each case as described in detail by our research group Error! Bookmark not defined. to achieve full was added to 55 µL (2.2 × 107 MPs) Dynabeads® M450 tosylactivated (Figure 2, panel B). As above, coverage) was added to 55 μL (2.2 × 107 MPs) Dynabeads® M450 tosylactivated (Figure 2, panel B). 0.1 mol L−1 pH 8.5 borate buffer was added. The incubation step was performed overnight with gentle As above, 0.1 mol L−1 pH 8.5 borate buffer was added. The incubation step was performed overnight shaking at 37 ◦ C. After that, a blocking step with 0.5 mol L−1 glycine solution was performed for 2 h to with gentle shaking at 37 °C. After that, a blocking step with 0.5 mol L−1 glycine solution was ensure the blocking of the any remaining tosylactivated groups. After that, the antibody-modified MPs performed for 2 h to ensure the blocking of the any remaining tosylactivated groups. After that, the (herein, antiCDX-MPs, where antiCDX = antiCD9, antiCD24, antiCD44, antiCD54, antiCD63, antiCD81, antibody‐modified MPs (herein, antiCDX‐MPs, where antiCDX = antiCD9, antiCD24, antiCD44, antiCD326 or antiCD340) were resuspended in a 220 µL (10 µL per well in order to achieve 1 × 106 antiCD54, antiCD63, antiCD81, antiCD326 or antiCD340) were resuspended in a 220 μL (10 μL per particles per well) 10 mmol L−1 PBS buffer. It was not possible to immobilize CD326 satisfactorily well in order to achieve 1 × 106 particles per well) 10 mmol L−1 PBS buffer. It was not possible to on MPs. immobilize CD326 satisfactorily on MPs. Sensors 2020, 20, 965 8 of 21 2.7. Characterization of Exosomes by Nanoparticle Tracking Analysis Nanoparticle tracking analysis (NTA) was used as a gold standard method to count the exosomes. This information was used for the biosensing calibration plots [73,74]. NTA was also used to study the size distribution of exosomes purified from cell culture supernatant. The size distribution and concentration of exosomes were measured using the NanoSight LM10-HS system with a tuned 405 nm laser (NanoSight Ltd., Amesbury, UK). The purified exosomes were diluted in sterile-filtered PBS (50- to 100-fold). Nanosight NTA Software analyzed raw data videos by triplicate during 60 s with 50 frames per second and the temperature of the laser unit set at 24.8 ◦ C. 2.8. Characterization of Exosomes by Transmission Electron Microscopy A 10 µL aliquot of exosomes was directly laid on Formvar–carbon electron microscopy grids and frozen in ethanol. The Cryo-TEM images were collected on the on a Jeol JEM 2011 transmission electron microscope at an accelerating voltage of 200 kV. Exosomes were maintained at −182 ◦ C during the whole process. 2.9. Confocal Microscopy Study Confocal microscopy was used for the assessment of the molecular biomarkers expressed in the exosomes obtained by the three different cancer cell lines (MCF7, MDA-MB-231 and SKBr3). The presence of the following receptors was investigated: CD9, CD63, CD81, CD24, CD44, CD54, CD326 and CD340. As exosomes are between 30–200 nm in diameter, a size that makes them out of the sensitivity range of most cell-oriented sorting or analysis platforms, they were coupled to MPs to allow their characterization by confocal microscopy. In this study, the exosomes were attached on the surface of MPs by covalent immobilization (exosomes-MPs), as described in Figure 1, panel A. In order to achieve that, 3.5 × 1010 exosomes were covalently immobilized on 1.6 × 107 MPs overnight with gentle orbital shaking at 4 ◦ C. The indirect labeling was performed by incubation with 100 µL (5 µg mL−1 ) of the mouse antibodies towards the different cluster of differentiation antiCDX (CDX being either CD9, CD63, CD81, CD24, CD44, CD54, CD326 or CD340 biomarkers), for 30 min with gentle shaking at 25 ◦ C. Subsequently, three washing steps were performed. Afterwards, 100 µL (2 µg mL−1 ) of antimouse-Cy5 antibody (a far-red-fluorescent dye, excitation 647 nm, emission 665 nm [75]) was incubated for 30 min in darkness, with gentle shaking at 25 ◦ C, for further readout. In all instances, the percentage of labeled entities was normalized by the highest fluorescence value for a labeled receptor. The labeled exosomes-MP were resuspended in 200 µL of PBS with 0.1% bovine serum albumin (BSA) solution. After each incubation, washing steps with PBS with 0.1% BSA solution were performed. The confocal images were then collected. 2.10. Flow Cytometry Study The analysis of the molecular biomarkers CD9, CD24, CD44, CD54, CD63, CD81, CD326 and CD340 expressed in the exosomes derived from three different cancer cell lines (MCF7, MDA-MB-231 and SKBr3) was performed by flow cytometry. Accordingly, for the labeling of cell-derived exosomes, they were firstly immobilized on the surface of MPs by two different approaches, as described above (Figure 2). In Figure 2, panel A depicts the covalent immobilization of the 3.5 × 1010 exosomes on 1.6 × 107 MPs (exosomes-MPs). The exosomes-MPs were analyzed by indirect labeling by incubation with 100 µL (5 µg mL−1 ) of the antibodies antiCDX (mouse), (CDX being either CD9, CD24, CD44, CD54, CD63, CD81, CD326 or CD340 biomarkers), for 30 min with gentle shaking at 25 ◦ C. After that, three washing steps with PBS pH 7.5 0.5% BSA were performed. Afterwards, 100 µL (2 µg mL−1 ) of antimouse-Cy5 antibody (a far-red-fluorescent dye, excitation 647 nm, emission 665 nm) was incubated for 30 min at 25 ◦ C. The labeled exosomes-MPs were resuspended in PBS pH 7.5 0.5% BSA. In Figure 1, panel B, 1 × 106 MPs modified with rabbit antiCD81 (antiCD81-MPs) were incubated with 4 × 109 Sensors 2020, 20, 965 9 of 21 exosomes (1:4000 MP/exosomes ratio). Exosomes immunocaptured by antiCD81-MPs were detected using indirect labeling, as described above. 2.11. Matrix Effect Study by Immunomagnetic Separation and Optical Readout The evaluation of the matrix effect was firstly performed by immunomagnetic separation (IMS) of MCF7-derived exosomes spiked in 0% (10 mmol L−1 phosphate-buffered saline (PBS) (no serum)), 10%, 25%, 50% and 100% (undiluted) exosome-depleted human serum (obtained as schematically shown in Figure 1, panel B). The IMS was performed by antiCDX-MPs (CDX being either CD9, CD24, CD44, CD54, CD63, CD81, CD326 or CD340 biomarkers), followed by a direct labeling and optical readout. The procedure for the magneto-actuated immunoassay was previously described in detail by our research group [17]. To summarize, this approach involved a direct immunoassay format, and in all cases was performed in 96-well microtiter plates. The direct immunoassay format involved the following steps: (i) IMS of the exosomes with antiCDX-MPs. The antiCDX-MPs (CDX being any of CD9, CD24, CD44, CD54, CD63, CD81, CD326 or CD340 biomarkers) (containing 1 × 106 antiCDX-MPs per well) and the exosomes (4 × 109 exosomes per well spiked in 100 µL of any of the matrix: 0%, 10%, 25% or 50% diluted and undiluted human serum), were simultaneously incubated for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (ii) Direct labeling. The exosomes-coated MPs were incubated with the antiCD63-HRP antibody (100 µL, 1.24 µg mL−1 ) for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (iii) Optical readout. 100 µL of substrate solution (0.004% v/v H2 O2 and 0.01% w/v TMB in citrate buffer) was then added to each well and incubated for 30 min under dark conditions. The enzymatic reaction was stopped by adding 100 µL of H2 SO4 (2.0 mol L−1 ). Then, exosomes-MPs were separated using a magnet plate separator, and an exosomes-MPs pellet formed on the bottom tube, followed by supernatant separation. The absorbance measurement of the supernatants was thus performed with the microplate reader at 450 nm. After each incubation or washing step, a 96-well magnet plate separator was positioned under the microtiter plate until the pellet formation on the bottom corner, followed by supernatant separation. 2.12. Immunomagnetic Separation and Electrochemical Immunosensing of Exosomes in Human Serum Two different formats, classified accordingly to the labeling, were performed for the detection of exosomes derived in this instance from SKBr3 breast cancer cell lines, and spiked in both phosphate buffer and undiluted exosome-depleted human serum, and ranging from 0 to 2 × 109 exosomes, as determined by NTA. The whole procedure was performed in detail as follows: Direct format. The magneto-actuated electrochemical immunosensing involves the following steps: (i) IMS of the exosomes with antiCD81-MPs (rabbit) (containing 1 × 106 antiCD81-MPs per well) and the exosomes (100 µL, ranging from 0 to 2 × 109 exosomes per well), were simultaneously incubated for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (ii) Direct labeling. The modified-MPs were incubated with the antiCD63-HRP antibody (100 µL, 1.24 µg mL−1 ) for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (iii) Electrochemical readout, as described below. Indirect format. The magneto-actuated electrochemical immunosensing involves the following steps: (i) IMS of the exosomes with antiCD81-MPs (rabbit) (containing 1 × 106 antiCD81-MPs per well) and the exosomes (100 µL, ranging from 0 to 2 × 109 exosomes per well), were simultaneously incubated for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (ii) Incubation with the antiCDX mouse monoclonal antibodies (100 µL, 0.50 µg mL−1 ) (CDX being any of the CD24 or CD340 biomarkers), which were simultaneously incubated for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. (iii) Indirect labeling. The modified-MPs were incubated with antimouse-HRP antibody (100 µL, 0.08 ng mL−1 ) for 30 min with shaking at 25 ◦ C, followed by three washing steps with PBS containing 0.5% BSA. Sensors 2020, 20, 965 10 of 21 (iv) Electrochemical readout, performed as described below, after the magnetic actuation of the modified-MPs on the surface of the m-GEC electrodes. Electrochemical measurements. All electrochemical experiments were performed using an AUTOLAB PGSTAT10 potentiostat/galvanostat electrochemical analyzer. A magneto-actuated graphite-epoxy carbon (m-GEC) as working electrode (geometric area = 0.5 cm2 ), a Ag/AgCl/KCl(satd.) reference Sensors 2020, 20, x FOR PEER REVIEW 10 of 22 electrode, a platinum counter electrode (geometric area = 3.0 cm2 ) and a standard one-compartment three-electrode compartment cell were used three‐electrode cellin all used were experiments. The enzymatic in all experiments. electrochemical The enzymatic signal issignal electrochemical based isonbased a hydroquinone on a hydroquinone mediator. Amperometric mediator. Amperometric measurements measurements were carried were carried outatat −0.1 out −0.1 VV vs. vs. Ag/AgCl/KCl(satd.) Ag/AgCl/KCl . Allexperiments . All (satd.) experimentswerewere performed performed using using aasingle singlem-GEC m‐GECelectrode electrodein in a 100 mmol a 100 mmol L−1 ◦ C. A reproducible steady-current was Lphosphate-buffered −1 phosphate‐buffered saline (PBS) saline (PBS) solution solutionwith withpHpH7.0 7.0at at25 25 °C. reproducible steady‐current was obtainedafter obtained after60 60ss and and used used for for the the calibration calibration curve. curve. An Anoutline outlineofofthis thisapproach approachbasedbasedononthethe magneto-actuated composite electrode and electrochemical readout magneto‐actuated composite electrode and electrochemical readout is shown in Figure 3.is shown in Figure 3. Schematicrepresentation Figure3.3.Schematic Figure representationofofthe theconstruction constructionofofthe themagneto‐actuated magneto-actuatedelectrode electrodebased basedon on rigidcomposites rigid composites(m‐GEC) (m-GEC)asaswell wellas asthe theenzymatic enzymaticreaction reactionbased basedon onhydroquinone hydroquinonemediator. mediator. 2.13. Safety Considerations 2.13. Safety Considerations All procedures involved in the manipulation of human cells were handled using Biosafety Level All procedures involved in the manipulation of human cells were handled using Biosafety Level 2 Laboratory (BSL-2) and containment. All works were performed in a Biosafety cabinet, and all 2 Laboratory (BSL‐2) and containment. All works were performed in a Biosafety cabinet, and all materials were decontaminated by autoclaving or disinfected before discarding in accordance with the materials were decontaminated by autoclaving or disinfected before discarding in accordance with U.S. Department of Health and Human Services guidelines for level 2 laboratory Biosafety [76]. the U.S. Department of Health and Human Services guidelines for level 2 laboratory Biosafety [76]. 3. Results and Discussion 3. Results and Discussion 3.1. Characterization of Exosomes by Nanoparticle Tracking Analysis 3.1. Characterization of Exosomes by Nanoparticle Tracking Analysis An estimation of the size diameter distribution of purified exosomes derived from the MCF-7 An estimation breast cancer of was cell line the size diameter performed by distribution NTA (Figureof4).purified exosomes A size from derived 50 to 300 from the MCF‐7 nm (considering 95.4% breast cancer cell line was performed by NTA (Figure 4). A size from 50 to 300 nm (considering of a Gaussian distribution), but with dominance around 105 and 153 nm (60% and 34% of the counted 95.4% of a Gaussian particles, distribution), respectively) wasbut with dominance obtained around for the MCF-7 105 and However, exosomes. 153 nm (60%it isand 34% of the important counted to highlight particles, respectively) was obtained for the MCF‐7 exosomes. However, it is important that NTA analysis is not able to distinguish isolated particles from aggregates. Similar results wereto highlight that NTA by obtained analysis NTA foris not the able to distinguish exosomes isolated derived from the particles from other breast aggregates. cancer cell linesSimilar results were (MDA-MB-231 and obtained by NTA for the exosomes derived from the other SKBr3) (as depicted in Figure 4, panels B and C, respectively). breast cancer cell lines (MDA‐MB‐231 and SKBr3) (as depicted in Figure 4, panels B and C, respectively). Sensors 2020, Sensors 2020, 20, 20, 965 x FOR PEER REVIEW 11 of 11 of 21 22 Sensors 2020, 20, x FOR PEER REVIEW 11 of 22 Figure 4. Nanoparticle tracking analysis (NTA) on size distribution on purified exosomes derived from Figure Figure (A) MCF7, 4.4. (B) MDA‐MB‐231 Nanoparticle Nanoparticle tracking tracking and (C) analysis analysis SKBr3 (NTA) (NTA) onbreast cancer on size size cellonlines. distribution distribution onThe purified purified purified exosomes exosomes exosomes derived were derived from diluted from (A) MCF7, in MCF7, (A) sterile‐filtered (B) MDA-MB-231 10 and mmol (B) MDA‐MB‐231 −1 PBS buffer (pH 7.5). Nanosight NTA Software analyzed raw (C)Land SKBr3(C)breast SKBr3cancer breastcell cancer lines.cell Thelines. The purified purified exosomesexosomes were were diluted data videos indiluted by triplicate in sterile‐filtered sterile-filtered −1 during 10 mmol L10 mmol 60 s with L PBS PBS buffer−1 50 frames (pH buffer per second (pH 7.5). NTA 7.5). Nanosight and Nanosightthe Softwaretemperature NTA of Software analyzed the laser rawanalyzed unit data videosraw set bydataat 24.8 triplicate videos°C.during 60 s with by triplicate 50 frames during per second 60 s with 50 frames andper thesecond temperature and the oftemperature the laser unitofset 24.8 ◦unit at laser the C. set at 24.8 °C. 3.2. Characterization of 3.2. Characterization of Exosomes Exosomes by by Transmission Transmission Electron Electron Microscopy Microscopy 3.2. Cryogenic Characterization of Exosomes by Transmission Electron Microscopy Cryogenic transmission transmissionelectron electronmicroscopy microscopy(TEM) provides (TEM) a tool for provides a high-resolution structural tool for high‐resolution analysis for structural frozen biostructures, analysistransmission Cryogenic avoiding for frozen biostructures, depression and avoiding depression electron microscopy even damage (TEM) providesand even to the sample. damage a tool for to Cryo-TEM the sample. high‐resolution micrographs Cryo‐TEM on exosomes structural micrographs derived on exosomes analysis for frozen from breast cancer derived avoiding biostructures, cell from breast lines are shown cancer celland depression linesin even Figure 5. are damage shown intoFigure 5. the sample. Cryo‐TEM micrographs on exosomes derived from breast cancer cell lines are shown in Figure 5. Figure 5.5. Cryogenic Cryogenictransmission transmissionelectron electron microscopy microscopy (Cryo‐TEM) (Cryo-TEM) on purified on purified exosomes exosomes derived derived from from (A, B)(A, Figure B)Cryogenic MCF-7, 5. MCF‐7, (C)transmission MDA‐MB‐231 (C) MDA-MB-231 and (D)microscopy andelectron (D) SKBr3 SKBr3 breastbreast cancercancer cell on cell lines. (Cryo‐TEM) lines. purified exosomes derived from (A, B) MCF‐7, (C) MDA‐MB‐231 and (D) SKBr3 breast cancer cell lines. The image comparatively shows TEM images of exosomes derived from MCF-7 MCF‐7 (panels A and B), MDA-MB-231 MDA‐MB‐231 (panel C) and SKBr3 (panel The image comparatively shows TEM images D) breast breast cancer cell cancer cell of exosomes lines. The micrographs lines. from MCF‐7 (panels derived revealed A and the B), typical exosome MDA‐MB‐231 consisting (panel C) andofSKBr3 well-shape (panelexosome well‐shape D) breastvesicles with cancer cell closed lines. Thecircular lipid bilayers micrographs revealed comprising packed the typicalpacked exosome membrane membrane consisting proteins proteins witha a110 with of well‐shape 110nmnm exosome diameter diameter (Figure (Figure vesicles with 5, 5, panel panel closed A). A). As expected, As expected, circular lipid the bilayers the TEM TEM micrographs micrographs comprising also packedalso reveal reveal the membrane the presence presence proteins withof of some some a 110 aggregates nmaggregates of exosomes of exosomes diameter (Figure (Figure 5, panel(Figure 5, panel 5, panel B), A). As expected, B), the confirming that the NTA TEM micrographs also analysis NTA reveal cannot analysisthe cannot distinguish distinguish presence of some vesicles vesicles and and vesicle aggregates vesicle aggregates. aggregates. of exosomes Similar (Figure results 5, panel B), confirming that the NTA analysis cannot distinguish vesicles and vesicle aggregates. Similar results Sensors 2020, 20, 965 12 of 21 Sensors 2020, 20, x FOR PEER REVIEW 12 of 22 were obtained for exosomes derived from other breast cancer cell lines (MDA-MB-231 and SKBr3, were Figure obtained 5, panels for exosomes C and derived from other breast cancer cell lines (MDA‐MB‐231 and SKBr3, D, respectively). Figure 5, panels C and D, respectively). 3.3. Confocal Microscopy Study 3.3. Confocal Microscopy Study Confocal microscopy was performed to comparatively study the expression patterns of different Confocal microscopy was performed to comparatively study the expression patterns of different biomarkers on breast cancer cells’ exosomes derived from MCF-7, MDA-MB-231 and SKBr3 cell lines. biomarkers on breast cancer cells’ exosomes derived from MCF‐7, MDA‐MB‐231 and SKBr3 cell lines. The quantitative results The quantitative areare results summarized summarizedin in Figure Figure 6.6. FigureFigure 6. Confocalmicroscopy 6. Confocal microscopy toto evaluate the relative evaluate expression the relative of CD9, CD63, expression CD81, of CD9, CD24,CD81, CD63, CD44, CD24, CD54, CD326 and CD340 membrane protein markers in the exosomes derived from MCF7, MDA‐ CD44, CD54, CD326 and CD340 membrane protein markers in the exosomes derived from MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines. Magnetic particles appear stained in green while the membrane protein receptors, in red, represents a positive expression on the membrane of the exosomes. In all cases, the primary antibody was 5 µg mL−1 and 2 µg mL−1 of antimouse-Cy5 antibody. Sensors 2020, 20, 965 13 of 21 The expression of each cell line-derived exosomes was studied after the covalent immobilization on MPs (Figure 2, exosomes-MPs and Figure 6, panel A for exosomes derived from MCF7, panel B for MDA-MB-231 and panel C for SKBr3 cells lines). The intense green color of the magnetic particles is due to autofluorescence around 580 nm [77]. As expected, general tetraspanins (CD9, CD63 and CD81) [78,79] were expressed in exosomes from the MCF7 breast cancer cell line (Figure 6, panel A). The biomarkers related to breast cancer are weekly or not expressed. Negligible non-specific adsorption fluorescence was observed (Figure 6, negative control), indicating a good blocking procedure of the remaining tosyl-activated group after covalent immobilization. On the other hand, a poorer labeling pattern was achieved for other biomarkers in MCF7 (Figure 4, panel A), as well those derived from MDA-MB-231 and SKBr3 (Figure 6, panels B and C). This issue can be attributed to steric hindrance of the receptor after immobilization of the exosomes on the MPs, in agreement with the results achieved by flow cytometry as it will be further discussed. It is worth mentioning that the confocal images are the most representative and they only provide a qualitative analysis. 3.4. Flow Cytometry Study As in the case of confocal microscopy, the main goal of this set of experiments was to assess the expression of different biomarkers (including general exosome biomarkers such as CD9, CD63 and CD81, and cancer-related biomarkers such as CD24, CD44, CD54, CD326 and CD340) on exosomes derived from the three different breast cancer cell lines (MCF7, MDA-MB-231 and SKBr3), in order to determine the good design for the electrochemical immunosensor. The results of flow citometry are shown in Figure 7, panels A and B. In the first approach, the exosomes directly immobilized on MPs were assessed towards the expression of different biomarkers selected in this study, followed by an incubation with an antimouse-Cy5 secondary antibody. Then, the labeled exosomes-MPs were acquired by the flow cytometer and further analyzed. As shown in Figure 7, panel A, CD9, CD63 and CD81 were expressed on exosomes produced by MCF7 and SKBr3, but poorly expressed in the MDA-MB-231 cell line, in agreement with confocal microscopy and several other studies [78,79]. Besides, cancer-related biomarkers showed, in general, a poorer labeling pattern. In the second approach, the two biomarkers must be simultaneously expressed in the exosomes. One of the markers is involved in the IMS, while the other one is involved in the labeling. Accordingly, the general tetraspanin CD81 was used in this instance for the IMS by the antiCD81-MPs in order to achieve a massive capture of the exosomes, followed by further labeling by a second biomarker. As shown in Figure 7, panel B, a better performance was achieved in general with this approach. Increased tetraspanin labeling on the exosomes from SKBr3 and MCF7 was respectively observed, although MDA-MB-231 also expressed low levels of these general biomarkers. Cancer-related exosomal biomarkers (for instance CD44 and CD340), which were negative when immobilized covalently on MPs, were detected by this approach. This experiment suggests that CD81 tetraspanin can be successfully used for the IMS of the exosomes. Moreover, and as expected, Figure 7 shows that there is a different pattern for the expression of exosomes from different cell cancer lines. 3.5. Matrix Effect Study by Immunomagnetic Separation and Optical Readout The main goal of this study is to avoid the use of ultracentrifugation for the isolation of exosomes from the free proteins of the sample, and to replace it by immunomagnetic separation directly performed in undiluted human serum, in order to simplify the analytical procedure. This is a challenging task, since the free receptors (mainly proteins including tetraspanin) present in the undiluted serum can block—or even prevent—the separation of the exosomes by immunomagnetic separation. Accordingly, a rational study of the matrix effect of the human serum was performed, by spiking a known amount of MCF7 cancer-related exosomes (obtained as schematically shown in Figure 1, panel A), in undiluted human serum depleted of exosomes (obtained as schematically shown in Figure 1, panel B). The characterization of the interference of free molecules and proteins in the serum was then carefully Sensors 2020, 20, 965 14 of 21 evaluated by magneto-actuated immunoassay (optical readout), since it can be easily multiplexed. A preliminary study of the matrix effect of human serum was performed on exosomes purified by ultracentrifugation from the MCF7 cell line (as depicted in Figure 1, panel A) and spiked in 0% (PBS), 10%, 25%, 50% and 100% (undiluted) human serum. The signal was normalized by the highest absorbance value for a biomarker within each exosome immobilization approach, as shown in Figure 8. Sensors 2020, 20, x FOR PEER REVIEW 14 of 22 Figure 7. Flow cytometry study for exosomes derived from MCF7, MDA‐MB‐231 and SKBr3 breast Figure 7. Flow cytometry study for exosomes derived from MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines attached on magnetic particles (MPs), being (Panel A) covalent immobilization of the cancer cell lines attached on magnetic particles (MPs), being (Panel A) covalent immobilization of the exosomes on MPs (exosomes‐MPs) and (Panel B) IMS of the exosomes on antiCD81‐MPs, followed by exosomes on MPswith indirect labeling (exosomes-MPs) mouse antiCDX (5 and (Panel μg mL B) IMS −1), (CDX beingof theCD9, either exosomes on antiCD81-MPs, CD24, CD44, CD54, CD63, followed by CD326 or CD340 biomarkers) and antimouse‐Cy5−1 indirect labeling with mouse antiCDX (5 µg mL ), (CDX being either CD9, CD24, CD44, CD54, CD63, CD81, (2 μg mL −1). The concentrations of MPs and CD81,exosomes CD326were or CD340 106 MPs and 4 ×and set at 1 × biomarkers) 109 exosomes per assay, (2 antimouse-Cy5 µg mL−1 ). The concentrations of MPs and respectively. 6 9 exosomes were set at 1 × 10 MPs and 4 × 10 exosomes per assay, respectively. 3.5. Matrix Effect Study by Immunomagnetic Separation and Optical Readout Theapproach, In this main goal of the this study IMS was is to avoid the use ofby performed ultracentrifugation using a specific forbiomarker the isolation of exosomes(antiCDX-MPs), antibody from the free proteins of the sample, and to replace it by immunomagnetic separation directly followed by an optical readout using a general exosome biomarker (antiCD63-HRP antibody). performed in undiluted human serum, in order to simplify the analytical procedure. This is a This challenging format thus requires task, since the freethereceptors coexistence of twoincluding (mainly proteins biomarkers in the tetraspanin) same present exosomes to be in the magneto-actuated undiluted serum can andblock—or labeled.even Byprevent—the comparingseparation the patternof theofexosomes signal for each of the receptors used to by immunomagnetic separation. separated Accordingly, the the exosomes, a rational better study of the matrix analytical effect of the was performance human serum wasby achieved performed, using the by ubiquitous and spiking a known amount of MCF7 cancer‐related exosomes (obtained as schematically shown in highly expressed CD81 for the immunomagnetic separation with antiCD81-MPs, since no background Figure 1, panel A), in undiluted human serum depleted of exosomes (obtained as schematically was observed shown in Figureand 1,the value panel was B). The always negligible characterization at all theof matrix of the interference compositions free molecules and proteinsstudied. On the contrary, in the aserum highwasbackground then carefullywas observed evaluated in the case ofimmunoassay by magneto‐actuated using antiCD9-MPs. Thissince (optical readout), high value can be it can be attributed toeasily multiplexed. a cross A preliminary study reaction/interference of the matrix of some effect ofof component human serum was the serum withperformed the antibodies used in on exosomes purified by ultracentrifugation from the MCF7 cell line (as depicted in Figure 1, panel this approach. The results also show the interference of the free CD63 protein in the serum, which A) and spiked in 0% (PBS), 10%, 25%, 50% and 100% (undiluted) human serum. The signal was produces increasing normalized by the backgrounds highest absorbance as the percentage value of human for a biomarker serum within each increases exosome in the matrix. This free immobilization CD63approach, receptor as can shownthus block, in Figure 8. and therefore prevent by competition, the binding of the exosomes to the magneticIn thisparticles. approach, the IMS was These performed results were by using a specific confirmed biomarker antibody by performing the same(antiCDX‐MPs), experiments in depleted followed by an optical readout using a general exosome biomarker (antiCD63‐HRP antibody). This human serum (no exosomes present), and the results are shown in Figure 9. format thus requires the coexistence of two biomarkers in the same exosomes to be magneto‐actuated Once and again,Byitcomparing labeled. is demonstratedthe pattern that some for of signal soluble each ofproteins in the the receptors usedserum produce to separated thean increment in the signal, exosomes, especially the betterremarkable in the casewas analytical performance of CD9 andby achieved CD63. using Again, the CD81 the ubiquitous and commercial highly antibody expressed CD81 for the immunomagnetic separation with antiCD81‐MPs, (rabbit) is promising for the IMS of exosomes since no background is observed. since no background was observed and the value was always negligible at all the matrix compositions studied. On the contrary, These results are also in agreement with previous studies, reporting a higher biomarker expression of CD9, CD63 and CD81 tetraspanins as well as a lower biomarker expression of CD24, CD44, CD54, CD326 and CD340 cancer-related biomarkers on breast cancer exosomes. No results were obtained for Sensors free 2020,receptor CD63 20, x FOR PEER REVIEW can thus 15 of 22 block, and therefore prevent by competition, the binding of the exosomes to the magnetic particles. These results were confirmed by performing the same experiments in a high background depleted human serum was (no observed in the exosomes case of using present), antiCD9‐MPs. and the This high results are shown value 9. in Figure can be attributed to a cross reaction/interference of some component of the serum with the antibodies used in this approach. The Sensors 2020, 20, 965results also show the interference of the free CD63 protein in the serum, which 15 of 21 produces increasing backgrounds as the percentage of human serum increases in the matrix. This free CD63 receptor can thus block, and therefore prevent by competition, the binding of the exosomes CD340 to (Epcam), since the magnetic uponThese particles. immobilization on the results were MP, no binding confirmed was observed, by performing perhaps the same due to a bad experiments in orientation during covalent immobilization. depleted human serum (no exosomes present), and the results are shown in Figure 9. Figure 8. Evaluation of the matrix effect in exosome‐depleted human serum for MCF7 exosomes detection using antiCDX‐MPs modified with antibodies against CD9, CD24, CD44, CD54, CD63, CD81, CD340 and CD81R (rabbit polyclonal) biomarkers for the IMS, followed by direct labeling with an antiCD63‐HRP antibody (1.24 μg mL−1). Detection of exosomes in (A) 0%; 10 mmol L−1 phosphate‐ buffered saline/PBS (standard for no matrix effect), (B) 10%, (C) 25%, (D) 50% and (E) 100% exosome‐ Figure 8.human depleted Evaluation serumof(undiluted the matrix effect serum). human in exosome-depleted human The concentrations serum of MPs and for MCF7 exosomes exosomes were set detection Figure at using 1 × 108.6 MPs antiCDX-MPs Evaluation and 4 ×of10the modified matrix 9 exosomes effect with antibodies in exosome‐depleted per assay, against respectively. CD9, serum Thehuman CD24,(as background CD44, for a MCF7 CD54, CD63, exosomes negative control CD81, detectionCD340 using and CD81R antiCDX‐MPs (rabbit polyclonal) modified with biomarkers antibodies for the against IMS, CD9, followed CD24, without exosomes) are also shown in all the experiments. The error bars show the standard deviation by CD44, direct CD54, labeling CD63, with −1 ). Detection of exosomes in (A) 0%; 10 mmol L−1 for n =an CD81, 3. antiCD63-HRP CD340 and CD81Rantibody (1.24 µg mL (rabbit polyclonal) biomarkers for the IMS, followed by direct labeling with phosphate-buffered saline/PBS (standard for no an antiCD63‐HRP antibody (1.24 μg mL ). Detection of exosomes −1 matrix effect), (B) 10%, (C)0%; in (A) 25%, 10 (D) mmol 50%L−1and (E) 100% phosphate‐ exosome-depleted buffered Once saline/PBS again, human (standard it is demonstratedserum for(undiluted no thatmatrix human someeffect), serum). (B) soluble The25%, 10%, (C) proteins concentrations in (D) the 50% and serum of MPs and (E) 100% produce exosomes anexosome‐ increment in were set at 6 MPs and 4 × 109 exosomes per assay, respectively. The background (as a negative 1 × 10serum depleted the signal, human especially (undiluted remarkable human in the case serum). of CD9 Theandconcentrations CD63. Again,ofthe MPs and exosomes CD81 commercial were set antibody control at 1 × 10without 6 MPs and exosomes) 4 × 10 are also 9 exosomes shown per in all assay, the experiments. respectively. (rabbit) is promising for the IMS of exosomes since no background is observed. The The error background bars (as show a the negativestandard control for n = 3. are also shown in all the experiments. The error bars show the standard deviation deviationexosomes) without for n = 3. Once again, it is demonstrated that some soluble proteins in the serum produce an increment in the signal, especially remarkable in the case of CD9 and CD63. Again, the CD81 commercial antibody (rabbit) is promising for the IMS of exosomes since no background is observed. Figure 9. Evaluation of the matrix effect in exosome-depleted human serum using antiCDX-MPs modified with antibodies against CD9, CD24, CD44, CD54, CD63, CD81, CD340 and CD81R (rabbit polyclonal) biomarkers for the IMS, followed by direct labeling with an antiCD63-HRP antibody (1.24 µg mL−1 ) in depleted human serum. In this instance, no exosomes were spiked in the samples. All other conditions are as in Figure 8. The error bars show the standard deviation for n = 3. 3.6. Immunomagnetic Separation and Electrochemical Immunosensing of Exosome in Human Serum The detection of exosomes in non-diluted human serum is critical for a routine clinical diagnosis, since it can suffer from interferences of free biomarkers. In order to avoid ultracentrifugation, Sensors 2020, 20, 965 16 of 21 the evaluation of the analytical performance of the electrochemical immunosensor coupled with IMS based on a CD81 receptor for the detection of SKBr3 exosomes spiked in depleted non-diluted human serum is shown in Figure 10. The exosomes concentration ranged from 2.00 × 107 to 2.00 × 1010 exosomes/mL−1 in order to cover the matrix effect in a wide concentration range. The exosomes concentration in healthy individuals determined by NTA in biological fluids varies notably in the literature, from 1.505 to 2.245 × 108 vesicles/mL−1 (plasma of healthy male individuals) [80]; 9.25 × 109 /2.4 × 1010 /1.8 × 1010 vesicles/mL−1 (depending on the mode of NTA in plasma of healthy individuals) [81]; 0.7 × 108 /5.3 × 108 vesicles/mL−1 (serum)/2.3 × 108 /9.9 × 108 vesicles/mL−1 Sensors 2020, (plasma) 20, x FOR PEER (depending on REVIEW the mode of NTA in plasma of healthy individuals) [82]; and 0.88 × 10 178ofto22 9 −1 1.34 × 10 exosomes/mL (serum or plasma of healthy individuals) [83]. Figure 10. Electrochemical immunosensor for the detection of exosomes derived from the SKBr3 breast Figurecancer cell line spiked in 10. Electrochemical exosome-depleted immunosensor for thehuman serum detection of and a phosphate exosomes derivedbuffer. fromIMS the of the SKBr3 exosomes on antiCD81-MPs, breast cancer cell line spikedfollowed by indirect labeling in exosome‐depleted humanwithserum (A)and antiCD24, (B) antiCD340 a phosphate and buffer. IMS of by the direct labeling exosomes with (C) antiCD63-HRP on antiCD81‐MPs, followed antibody. by indirectInlabeling all cases, the(A) with concentration of MPs antiCD24, (B) was fixed antiCD340 andin by × 106 MPs, 1 direct 0.50 µg mL −1 of primary antibody, 0.08 ng mL−1 of antimouse-HRP and 1.24 µg mL−1 of labeling with (C) antiCD63‐HRP antibody. In all cases, the concentration of MPs was fixed in 1 antiCD63-HRP × 106 MPs, 0.50 antibody. μg mL−1Enzymatic of primaryelectrochemical antibody, 0.08signals ng mLwere −1 of monitored at −0.1and antimouse‐HRP V vs.1.24 Ag/AgCl μg mL −1 of (sat.) . InantiCD63‐HRP all cases, the replicates antibody.were obtained with Enzymatic three differentsignals electrochemical samplesweremeasured with different monitored at −0.1m-GEC V vs. electrodes. The Ag/AgCl(sat.). Inerror bars show all cases, the standard the replicates weredeviation obtainedfor n = three with 3. different samples measured with different m‐GEC electrodes. The error bars show the standard deviation for n = 3. The results show the performance of the magneto-actuated electrochemical immunosensor for the detection 4. Conclusions of exosomes spiked on PBS (10 mmol L−1 ) and on human serum (exosome-depleted), and performed by IMS on antiCD81-MPs and the detection of cancer-related biomarkers (CD24 and CD340The for most Figureimportant 10, panelsfeature A and that should be considered to simplify the analytical procedure for B, respectively). the detection of exosomes at low concentration The limit of detection (LOD) for each plot were levels involves estimated bynovel fittingsolid‐phase the raw data separation methods using a nonlinear to avoid ultracentrifugation. regression (four parameters logisticIn this paper, we demonstrated equation), by processingthat theparticle‐based negative control magnetic samplesenrichment (n = 10) simplifies exosomes isolation and can be easily coupled with emerging and obtaining the mean value for each plot. The cutoff value was then determined in all cases technologies as is thewith case with electrochemical immunosensing. However, the interferences of free receptors a one-tailed t-test at a 95% confidence level. These values were interpolated in each plot to obtain present in the serum, the LODs. including the tetraspanins CD9 and CD63, can prevent or interfere with the immunomagnetic separation The LOD based on these for the receptors. detection approachInterestingly, the immunomagnetic using antiCD24 was found to beseparation 1.94 × 105ofandthe1.73 exosomes × 105 based on CD81 is not affected by the serum (even if it is undiluted), which exosomes per µL−1 for exosomes spiked in human serum and phosphate buffer, respectively. The LOD can be easily detected by an the for electrochemical detection approachimmunosensor using cancer using antiCD340 wasrelated foundbiomarkers, to be 1.02 ×such106 as andCD24 5.64and× 10CD340. 5 exosomes Moreover, −1 the exosome electrochemical immunosensor per µL for exosomes spiked in human serum and PBS, respectively. shows an outstanding LOD as low as 104 exosomes Finally,per theμL −1 directly in undiluted immunosensor human serum for CD63 recognition, whenofthe a LOD 1.24detection × 105 and is as based low on CD63, as 2.34 this × 10 4 LOD being compatible −1 with the levels of exosomes present in serum exosomes per µL was obtained with antiCD63-HRP (Figure 10, panel C) in non-diluted human and plasma and, importantly, without serum andany previous buffer. a phosphate isolation step using Although fromultracentrifugation. the results shown inThe electrochemical Figure immunosensor, 10, there is a matrix effect in coupled with immunomagnetic separation, offers an exciting alternative, the serum, this approach demonstrated a good performance of the electrochemical immunosensor especially in resource‐ scarce settings that can be handled by unskilled personnel at the community care level. Author Contributions: M.M. and M.I.P. conceived of the presented idea. S.L.M. verified the analytical methods. M.I.P. and M.M. encouraged S.L.M. to investigate and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript. S.L.M. carried out the experiment. M.I.P. wrote the manuscript with support from M.M. and S.L.M. All authors have Sensors 2020, 20, 965 17 of 21 towards cancer-related exosomes isolated by IMS in non-diluted human serum samples, without any further pretreatment (such as ultracentrifugation). 4. Conclusions The most important feature that should be considered to simplify the analytical procedure for the detection of exosomes at low concentration levels involves novel solid-phase separation methods to avoid ultracentrifugation. In this paper, we demonstrated that particle-based magnetic enrichment simplifies exosomes isolation and can be easily coupled with emerging technologies as is the case with electrochemical immunosensing. However, the interferences of free receptors present in the serum, including the tetraspanins CD9 and CD63, can prevent or interfere with the immunomagnetic separation based on these receptors. Interestingly, the immunomagnetic separation of the exosomes based on CD81 is not affected by the serum (even if it is undiluted), which can be easily detected by an electrochemical immunosensor using cancer related biomarkers, such as CD24 and CD340. Moreover, the exosome electrochemical immunosensor shows an outstanding LOD as low as 104 exosomes per µL−1 directly in undiluted human serum when the detection is based on CD63, this LOD being compatible with the levels of exosomes present in serum and plasma and, importantly, without any previous isolation step using ultracentrifugation. The electrochemical immunosensor, coupled with immunomagnetic separation, offers an exciting alternative, especially in resource-scarce settings that can be handled by unskilled personnel at the community care level. Author Contributions: M.M. and M.I.P. conceived of the presented idea. S.L.M. verified the analytical methods. M.I.P. and M.M. encouraged S.L.M. to investigate and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript. S.L.M. carried out the experiment. M.I.P. wrote the manuscript with support from M.M. and S.L.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Ministry of Economy and Competitiveness (MINECO), Madrid (Projects Sens4All BIO2016-75751-R) and the CNPq—Conselho Nacional de Desenvolvimento Científico e Tecnológico, of the Ministry of Science, Technology, Innovation and Communications of Brazil (CNPq/CsF–233595/2014-7) is also gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J. Clin. 2018, 68, 394–424. [CrossRef] [PubMed] 2. Johnstone, R.M.; Adam, M.; Hammond, J.R.; Orr, L.; Turbide, C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J. Boil. Chem. 1987, 262, 9412–9420. 3. 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