Biosensors and Bioelectronics 198 (2022) 113826 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios The activity of alkaline phosphatase in breast cancer exosomes simplifies the biosensing design Silio Lima Moura a, Arnau Pallarès-Rusiñol a, b, Luciano Sappia a, Mercè Martí b, María Isabel Pividori a, b, * a Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain b Biosensing and Bioanalysis Group, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain A R T I C L E I N F O A B S T R A C T Keywords: This work addresses a biosensor combining the immunomagnetic separation and the electrochemical biosensing Exosomes based on the intrinsic ALP activity of the exosomes. This approach explores for the first time two different types Alkaline phosphatase of biomarkers on exosomes, in a unique biosensing device combining two different biorecognition reaction: Breast cancer immunological and enzymatic. Besides, the intrinsic activity of alkaline phosphatase (ALP) in exosomes as a Liquid biopsy potential biomarker of carcinogenesis as well as osseous metastatic invasion is also explored. To achieve that, as Immunomagnetic separation Electrochemical biosensor an in vitro model, exosomes from human fetal osteoblasts are used. It is demonstrated that the electrochemical biosensor improves the analytical performance of the gold standard colorimetric assay for the detection of ALP activity in exosomes, providing a limit of detection of 4.39 mU L-1, equivalent to 105 exosomes μL-1. Furthermore, this approach is used to detect and quantify exosomes derived from serum samples of breast cancer patients. The electrochemical biosensor shows reliable results for the differentiation of healthy donors and breast cancer in­ dividuals based on the immunomagnetic separation using specific epithelial biomarkers CD326 (EpCAM) com­ bined with the intrinsic ALP activity electrochemical readout. 1. Introduction diagnosis of liver disorders, the assessment of its enzymatic activity is a key parameter because particular patterns of liver enzymes activity can Alkaline phosphatases (ALP, EC 3.1.3.1) are a family of ubiquitous be linked to common diseases (Giannini et al., 2005). It is also found that enzymes present in most tissues. These membrane-bound metal­ high levels of ALP in human saliva can be indicative of periodontal loenzymes catalyze the hydrolysis of phosphate monoesters in an alka­ disease (Luke et al., 2015; Melguizo-Rodríguez et al., 2020). line medium into inorganic phosphate and its corresponding alcohol Besides the abovementioned, several studies have demonstrated that (Sharma et al., 2014; Tang et al., 2019). Each active site of the enzymes the overexpression of ALP can be correlated with metastatic processes in contains two Zn2+ and one Mg2+ metallic-ions binding sites, necessary cancer disease, such as osteosarcoma (Kim et al., 2017), prostate (Rao for the folding of the enzyme and the binding of phosphate groups et al., 2017), breast (Tsai et al., 2000) and colorectal carcinomas (Saif (Dean, 2002; Sowadski et al., 1981). ALP are key enzymes involved in et al., 2005). multiple biological and metabolic pathways, including the bone The standard method for ALP activity determination in clinical fluids mineralization process, which increases the local concentration of is based on a spectrophotometric assay, in which the sample is mixed phosphate ions (Anderson, 2003) by consuming pyrophosphate from the with an alkaline buffer and a substrate (para-nitrophenyl phosphate), medium, known as inhibitor of the formation of hydroxyapatite crystals and measuring the product at 405 nm (Keiding et al., 1974). This sub­ (Anderson et al., 2004). strate is also widely used in electrochemical biosensors (Akyilmaz and In this sense, the ALP activity is a well-established biomarker, Turemis, 2010). Recently, our group reported an electrochemical routinely determined in clinical analysis, for the monitoring of a series of biosensor for the determination of ALP in clinical samples (Sappia et al., diseases. For example, ALP plays a role in bile production and in the 2019). We also demonstrated ALP activity on osteoblast-derived * Corresponding author. Grup de Sensors & Biosensors, Unitat de Química Analítica, Universitat Autònoma de Barcelona, Edificio Cn. Campus UAB, 08193, Bellaterra, Barcelona, Spain. E-mail address: isabel.pividori@uab.cat (M.I. Pividori). https://doi.org/10.1016/j.bios.2021.113826 Received 18 October 2021; Received in revised form 16 November 2021; Accepted 19 November 2021 Available online 23 November 2021 0956-5663/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 exosomes from culture supernatants (Sanchez et al., 2020). KCl(sat.) reference electrode, a disc platinum counter electrode (geo­ The exosomes are nano-sized extracellular vesicles involved in metric area = 3.0 cm2) and a standard 500-μL one compartment three- intercellular communication mechanisms (Johnstone et al., 1987). The electrode cell was used in all experiments. exosome cargo is derived from the producing cells and includes tetra­ spanins (e.g. CD9, CD63, CD81 considered as ubiquitous exosome 2.2. Chemicals and biochemicals markers), DNA, RNA but also enzymes of cellular origin (Samanta et al., 2018). Exosomes are currently under study as biomarkers for cancer The magnetic particles (MPs) Dynabeads® M450 Tosylactivated diagnosis and monitoring (Halvaei et al., 2018; Raposo and Stoorvogel, (Ref. 14013) and the MPs modified with EpCAM antibody (Dynabeads™ 2013; Zhou et al., 2021). Most of the studies of exosome detection Epithelial Enrich, Ref. 16102) were purchased from Thermo Fisher. The involving biosensors use antibodies for the specific capturing and mouse monoclonal antibodies (antiCDX) were purchased from Thermo detection, for the discrimination of the cellular origin of the exosomes Fisher: CD9 (Ref. 10626D), CD63 (Ref. 10628D) and CD81 (Liu et al., 2021; Moura et al., 2020a, 2020b). Even though, the het­ (Ref. 10630D). A goat anti-mouse IgG H&L (Cy5®) (antimouse-Cy5) erogeneity of the exosomes can impair the use of a good antibody pair (Ref. ab97037) was purchased from Abcam. Calf intestine alkaline for capture and detection with high specificity, low background, and no phosphatase (ALP, ref. 10713023001), Dulbecco’s Modified Eagle’s/ cross-reactivity. Ham’s F-12 Nutrient (DMEM/F12, nº D9785) medium and fetal bovine Since there is a correlation among bone metastasis in breast cancer serum (FBS, ref. 12007C) were purchased from Sigma-Aldrich. All other and the increase of alkaline phosphatase in microcalcifications (Du reagents were in analytical reagent grade. et al., 2014; Kanakis et al., 2004; Zulauf et al., 2019), in this paper we address the use of exosome ALP activity instead of a labeled-antibody to 2.3. Cell culturing, exosome isolation and purification achieve the electrochemical readout. Several methods of ALP measure­ ment are commonly used in clinical practice, but none of them are Human fetal osteoblastic (hFOB) cell line (hFOB 1.19 ATCC® CRL- focused on the analysis of ALP in the serum-derived exosomes from 11372™) was grown as described in Supp. Data S2. Exosomes were epithelial cancer cells. To our knowledge, this is the first time that the purified from culture supernatant by differential ultracentrifugation as enzymatic activity of exosomes is proposed for the redout in a biosensor. previously reported by our research group (Moura et al., 2020a). Exo­ The method was firstly optimized using an in vitro model based on somes are resuspended in 10 mmol L− 1 TRIS buffer solution (pH 7.4) exosomes derived from human fetal osteoblastic (hFOB) cell line and (0.22 μm filtrated and sterile) and stored at –80 ◦ C. All exosomes puri­ compared with the gold standard colorimetric ALP assay in terms of the fication steps are described in Supp. Data S2. analytical performance. In this first approach, the proposed biosensor combines the immunomagnetic separation of the exosomes based on the 2.4. Characterization of exosomes by nanoparticle tracking analysis and general tetraspanin (CD9, CD63, or CD81), followed by the electro­ cryogenic transmission electron microscopy chemical readout relying on the determination of ALP activity with pNPP substrate by using boron-doped microcrystalline diamond (BDD) The size distribution and concentration of exosomes were measured electrodes. As a proof of concept in real samples, the biosensing by nanoparticle tracking analysis (NTA). The purified exosomes were approach was tested to discriminate breast cancer individuals from diluted in sterile-filtered PBS buffer solution (50- to 100-fold). Nano­ healthy donors by the analysis of serum samples (Moura et al., 2020a; sight NTA Software analyzed raw data videos by triplicate during 60 s Soysal et al., 2013). In this instance, the exosomes were isolated using with 25 frames/s and the temperature of the laser unit set at 24.8 ◦ C. For magnetic particles specific for CD326 (EpCAM) cancer-related the Cryo-TEM, the exosomes (2.0 × 109 exosomes) were directly laid on biomarker, followed by the electrochemical biosensing of ALP content, Formvar-Carbon EM grids and frozen in ethanol. Exosomes were which differentiates healthy donors and breast cancer patients based on maintained at –182 ◦ C during the whole process. specific epithelial biomarkers. 2.5. Confocal microscopy and flow cytometry study 2. Experimental The analysis of the molecular biomarkers expressed in hFOB cell line 2.1. Instrumentation was carried out by flow cytometry. The cell-membrane and the exosome expression of CD9, CD63 and CD81 tetraspanins were compared. The Nanoparticle tracking analysis (NTA) was performed using the indirect labeling of 2 × 105 cells was performed by incubation of 100 μL NanoSight LM10-HS system with a tuned 405 nm laser (NanoSight Ltd, (5 μg mL− 1) of the antibodies antiCDX (mouse), (being CDX either CD9, UK). Cryogenic transmission electron microscopy (Cryo-TEM) images CD63 and CD81), for 30 min with gentle shaking at 25 ◦ C. After that, were collected by a Jeol JEM 2011 (JEOL USA Inc, USA) using an three washing steps with TRIS buffer solution containing 0.5% BSA so­ accelerating voltage of 200 kV. Flow cytometry was performed using BD lution were performed. Afterward, 100 μL (2 μg mL− 1) of the antimouse- FACSCANTO II (BD Biosciences, USA) equipment. The Media Fluores­ Cy5 antibody (a far-red-fluorescent dye, excitation 647 nm, emission cence Intensity and beads count data were obtained by FlowJo analysis 665 nm) was incubated for 30 min in darkness with gentle shaking at 25 software of every sample-reading file. Confocal images were collected ◦ C. The labeled cells were resuspended in 200 μL of TRIS buffer solution on the microscope Leica, TCS SP5 (Leica Microsystems, Germany). Op­ containing 0.5% BSA solution. tical measurements were performed on a TECAN Infinite m200 PRO The same procedure of labeling was performed in the case of the (TECAN AG, Switzerland) microplate reader with Magellan v7.0 soft­ osteoblast-derived exosomes, but in this approach, and due to their size ware. All electrochemical experiments were performed using an and resolution of the technique, the exosomes were firstly immobilized AUTOLAB PGSTAT10 potentiostat/galvanostat electrochemical on the surface of magnetic particles (MPs). To achieve that, 3.5 × 1010 analyzer (Metrohm AG, Switzerland). A boron-doped microcrystalline exosomes were covalently immobilized on 1.6 × 107 MPs, as detailed diamond (BDD) electrode (boron/carbon ratio of 20.000 ppm) as described in Supp. Data S5 (Fig. S4, panel A), followed by the indirect working electrode (geometric area = 0.5 cm2) was kindly supplied by labeling as described above, with antiCDX (mouse), (being CDX either Prof. Dr. Neidenei Ferreira from Group of Electrochemistry and Carbon CD9, CD63 or CD81 biomarkers). Materials, National Institute of Space Research (INPE), São José dos The same samples of cells and exosomes analyzed by flow cytometry Campos, São Paulo, Brazil. The construction of the BDD electrode is were subjected to confocal microscopy imaging for the study of the described in Supp. Data S1, as well as the characterization by Scanning binding pattern of antibodies. In the case of cells, the cellular DNA was Electron Microscopy, RAMAN and Cyclic Voltammetry. Ag/AgCl/ stained previously (before labeling with antibodies) with Hoechst dye (a 2 S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 blue-fluorescent dye, emission wavelength 490 nm). detailed described in Supp. Data S2 and S3. The two groups of samples were then evaluated by NTA and the protein content (as described in 2.6. ALP activity study in exosomes Supp. Data S4) and compared based on the same content of exosome protein (3.35 μg of protein per assay). The electrochemical biosensing 2.6.1. Spectrophotometric determination of the ALP activity in exosomes for ALP activity in exosomes from serum of breast cancer patient The gold standard colorimetric determination of the ALP activity was involved the following steps: i) IMS of the exosomes with antiCD326- performed in 96-well microtiter plates (Fig. 1, panel B). The colorimetric MPs (also known as EpCAM, a cancer-related biomarker) in order to assay for ALP activity in osteoblast-derived exosomes was detected and isolate only cancer-related exosomes, followed by ii) reaction of the ALP quantified by monitoring the enzymatic activity (rate of micromoles enzyme on the exosomes with pNPP substrate; (iii) electrochemical hydrolyzed per minute, μmol min− 1) with pNPP substrate in DEA buffer readout as described above. The detailed protocol for the electro­ into p-nitrophenol (pNP). A colorimetric calibration curve for pNP was chemical biosensing of the ALP activity in exosomes isolated by specific carried out to obtain the rate of hydrolyzing in enzyme activity (U L− 1), epithelial biomarker from serum of breast cancer patients is provided in as described in Supp. Data S12. The assay involved the following steps: i) Supp. Data S8. IMS of the exosomes with antiCDX-MPs (Fig. 1, panel A), followed by ii) reaction with pNPP substrate; iii) optical readout (Fig. 1, panel B). The 2.7. Statistical analysis protocol for the modification of magnetic particles and the spectro­ photometric assay are described in detail in Supp. Data S5.2 and S6, The statistical analyses were performed using GraphPad Prism 9 (San respectively. Diego, USA). The data of the ALP activity were statistically compared between the gold standard colorimetric and the electrochemical 2.6.2. Electrochemical biosensor for ALP activity in exosomes endpoint assays using a paired-sample Student’s t-test. The value p > The electrochemical biosensing for ALP activity in osteoblast-derived 0.05 was considered significant. exosomes involved the following steps: i) IMS of the exosomes with antiCDX-MPs (Fig. 1, panel A), followed by ii) reaction with pNPP 2.8. Safety considerations substrate and (iii) the electrochemical readout (Fig. 1, panel C). The detailed protocol for the electrochemical biosensor is provided in Supp. All works were performed in a Biosafety cabinet, and all material was Data S7. A standard 500-μL one compartment three-electrode cell was decontaminated by autoclaving or disinfected before discarding used. The readout was performed by monitoring the rate of micromoles following U.S. Department of Health and Human Services guidelines for hydrolyzed per minute (μmol min− 1) of pNPP substrate in DEA buffer level 2 laboratory Biosafety. into p-nitrophenol (pNP), followed by electrochemical conversion into p-aminophenol (pAP). A square wave voltammetry (SWV) calibration 3. Results and discussion plot for pAP was carried out to express the rate of hydrolyzing in enzyme activity (U L− 1), as described in Supp. Data S12. 3.1. Characterization of exosomes by nanoparticle tracking analysis and cryogenic transmission electron microscopy 2.6.3. Electrochemical biosensing of the ALP activity in exosomes isolated by specific epithelial biomarker from serum of breast cancer patients The diameter size distribution of osteoblast-derived nanovesicles Blood samples from anonymized healthy female donors (n = 10, derived from hFOB cell line and human serum samples ranged from 50 mean age 35/SD 5 years) and breast cancer female donors (n = 10, stage up to 460 nm (considering 95.4% of a Gaussian distribution). The IV, mean age 50/SD 6 years) were obtained from the Hospital del Mar, highest peaks are enriched with exosomes and correspond to 120–150 Barcelona, Spain. The work was carried out following the principles of nm in diameter (Fig. 2). The NTA profiles observed are characteristic of voluntariness and confidentiality. The samples (n = 10) each were each sample source, being more heterogenous samples from human pooled in two batches (healthy and breast cancer donors) and purified as serum (Fig. 2, panels B and C). Nevertheless, the particle concentration Fig. 1. Different approaches for the detection of alkaline phosphatase (ALP) activity in osteoblast-derived exosomes by optical readout and electrochemical biosensor. Further experimental details are provided in Supp. Data S6 and S7. 3 S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 Fig. 2. Characterization by NTA and Cryo-TEM of purified exosomes-derived from hFOB cell line (panel A) and human serum samples from healthy individuals (panel B) and breast cancer patients (panel C). The NTA characterization analyzed raw data videos by triplicate during 60 s with 25 frames per second and the temperature of the laser unit set at 24.8 ◦ C. Cryo-TEM images were obtained at an acceleration voltage of 200 kV. determined by NTA were similar in all cases, approximately 1010 par­ due to autofluorescence on the MPs at 580 nm, approximately (Agrawal ticles mL-1. Further details of NTA characterization are provided in et al., 2007). The percentage of labeled exosomes represents the total Supp. Data S9. counting of positive exosomes-MPs for each biomarker. The quantitative Cryo-TEM micrographs on osteoblast-derived exosomes show well- analysis showed, as expected, a strong labeling pattern of shape exosome vesicles with closed circular lipid bilayers (Fig. 2, inset osteoblast-derived exosomes to all the general tetraspanins studied panel A) of around 150 nm in diameter. As expected, the Cryo-TEM (CD9, CD63, and CD81) (Fig. 3, panel C). micrographs also reveal the presence of some aggregates of exosomes, The results for flow cytometry analysis are shown in Fig. 4. It is confirming the results obtained by NTA. In fact, Cryo-TEM micrographs noteworthy that the flow cytometric analysis was performed with the from serum derived exosomes (Fig. 2, insets panels B and C) showed same batch sample used in the confocal microscopy. The negative con­ higher aggregation of the vesicles, especially in cancer patient sample, trol, in which the signal appears onto the left side in blue, confirms that according to the NTA profiles (more micrographs in Supp. Data S9). there is a negligible (<0.1%) nonspecific adsorption of the secondary Finally, it is important to highlight that NTA analysis provides infor­ antibody (antimouse Cy®5 fluorophores) on the hFOB cells (Fig. 4, mation as counted entities, not only as isolated particles but also as panel A, control). Flow cytometry also showed strong labeling to CD9, aggregates. Therefore, the NTA analysis cannot clearly distinguish ves­ CD63, and CD81 biomarkers in hFOB cell line. The results of flow icles and vesicle aggregates. cytometry shown in Fig. 4, panel B and C, for the osteoblast-derived exosomes covalently immobilized on MPs also confirmed the high level of expression of CD9, CD63, and CD81 tetraspanins (Fig. 4, panel B 3.2. Confocal microscopy and C). The percentage of labeled entities (either the cells or the exosomes-MPs) represents the total counting of positive entities for each The expression of general exosome biomarkers such as CD9, CD63 biomarker, as shown in Fig. 4, panel C. and CD81 not always correlates between cell and exosome membranes. As for hFOB cells, the CD81 tetraspanin was most prominently dis­ Therefore, the characterization of the expression of those molecules was played in their derived exosomes. In agreement with several studies, performed on the hFOB cell line, as well as in their osteoblast-derived CD9, CD63, and CD81 as the most frequently identified proteins in exosomes for their further use in the immunomagnetic separation of exosomes and are considered classical biomarkers for exosomes (Chow the exosomes. et al., 2014). Therefore, any of these tetraspanins can provide good Expression patterns to CD9, CD63 and CD81 tetraspanins in hFOB performance for the immunomagnetic separation of osteoblast-derived cell line and their exosomes were evaluated qualitatively by confocal exosomes. microscopy. In the case of hFOB cells, the cellular DNA was stained with Hoechst dye (blue color). CD9, CD63, and with highlight the CD81 tetraspanins membrane receptors are shown with strong labeling in the 3.3. Determination of ALP activity hFOB cell line (Fig. 3, panel A and C). The percentage of labeled cells represents the total counting of positive cells for each biomarker, as Different parameters were optimized, and the results shown in Supp. shown in Fig. 3, panel C. The expression of osteoblast-derived exosomes Data S10, including the composition of the buffer (Tris or DEA), pH, was done after covalent immobilization on MPs due to their size (Fig. S4, Mg2+ concentration and the stop solution for ALP activity determination panel A) and was then studied and compared to the parental cell line on osteoblast-derived exosomes. According to the results, the optimized (Fig. 3, panel B). It is worth mentioning that the intense green color is parameters for the spectrophotometric determination of the ALP activity Fig. 3. Confocal microscopy images for (A) hFOB cell line and (B) their exosomes cova­ lently immobilized on MPs (exosomes-MPs), followed by indirect labeling with mouse antiCDX (5 μg mL− 1), (being CDX either CD9, CD63, and CD81 biomarkers) and antimouse-Cy5 (2 μg mL− 1). The concentra­ tion of MPs and exosomes were set in 1 × 106 MPs and 4 × 109 exosomes per assay, respectively. DNA in blue color, magnetic particles in green color, exosome protein membrane in red color. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4 S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 Fig. 4. Flow cytometry study represented in histograms for (A) hFOB cell line and (B) their exosomes covalently immobilized on MPs (exosomes-MPs), followed by indirect labeling with mouse antiCDX (5 μg mL− 1), (being CDX either CD9, CD63, and CD81 biomarkers) and antimouse-Cy5 (2 μg mL− 1). The concentration of MPs and exosomes were set in 1 × 106 MPs and 4 × 109 exosomes per assay, respectively. Population control onto the stained-blue regions on the left side and stained-red regions on the right side for a positive relative expression of membrane protein markers. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) in osteoblast-derived exosomes immunocaptured by antiCDX-MPs were the absorbance at 405 nm, and the analytical performance compared 10 mmol L− 1 pNPP in 1 mol L− 1 DEA buffer (pH 9.8) containing 6.0 with the electrochemical biosensing monitoring the current peak at -150 mmol L− 1 MgCl2 and 100 mmol L− 1 KCl. In all cases, the enzymatic mV. Fig. 5, panel A (spectrophotometric determination) and panel B reaction was performed for 60 min at 37 ◦ C and stopped by adding 25% (electrochemical biosensing) show the ALP activity of osteoblast- (v/v) of 5.0 mol L− 1 NaOH, and finally, the absorbance was measured at derived exosomes isolated by immunomagnetic separation base on 405 nm. (Supp. Data S10). antiCDX-MPs (being CDX: CD9, CD63 and CD81). These results Beside the characterization of BDD electrode by SEM and Raman, the demonstrated an improved separation performance of the IMS based on SWV-parameters were also optimized (Supp. Data S11). The optimal antiCD81-MPs and in the detection of ALP activity in the exosome experimental SWV-parameters for quantification of pAP in 1 mol L− 1 membrane, which can be attributed either to high expression of the DEA buffer (pH 9.8) containing 6.0 mmol L− 1 MgCl2 and 100 mmol L− 1 receptor or higher affinity constant (Ka) of the antibody. KCl using microcrystalline BDD electrode were found to be Esw = 200 Both set of data were fitted using nonlinear regression (Four mV pulse amplitude, = 200 Hz frequency, Esp = 8 mV pulse potential, Parameter Logistic Equation, GraphPad Prism Software). The osteoblast- Eap = -1.70 V and t = 10 s for accumulation potential and time, derived exosomes separated by using antiCD81-MPs provided improved respectively. Under these conditions, an excellent reproducibility was analytical performance, giving a limit of detection (LOD) of 825 exo­ achieved for the electrochemical readout, as also shown in Supp. Data somes μL− 1 (r2 = 0.9907) and 105 exosomes μL− 1 (r2 = 0.9949) for S11. The substrate also showed a good stability on the BDD electrode spectrophotometric assay and electrochemical biosensor, respectively. (Supp. Data S11). The LOD for the electrochemical biosensor corresponds to the 4.39 mU L− 1 or 13.47 mU mg− 1 for ALP activity and specific activity, normalized by the protein content, respectively. These LOD represent an improve­ 3.4. Comparative study of the electrochemical biosensor and the ment over the detection limits obtained by other methods for total spectrophotometric determination for ALP activity in exosomes isolated on exosome counting (López-Cobo et al., 2018; Oliveira-Rodríguez et al., magnetic particles 2016). These results demonstrate a more sensitive electrochemical platform than the gold standard spectrophotometric assay (p < 0.05). The ALP activity from osteoblast-derived exosomes was detected and Nonetheless, the pNPP substrate solution on the BDD electrode surface quantified by the gold standard spectrophotometric assay by monitoring Fig. 5. Comparative study of (A) spectrophoto­ metric gold standard determination and (B) electro­ chemical biosensor for the detection of ALP activity in osteoblast-derived exosomes ranging 0 from 2.35 × 107 exosomes μL− 1 in 10 mmol L− 1 TRIS buffer. Exosomes were immunocaptured by antiCDX-MPs (being CDX any of CD9, CD63, or CD81), followed by reaction with 10 mmol L− 1 pNPP in 1 mol L− 1 DEA buffer, containing 6.0 mmol L− 1 MgCl2 and 100 mmol L− 1 KCl. In all cases, the enzymatic reaction was during 60 min at 37 ◦ C, the concentration of antiCDX-MPs was fixed in 1 × 106 MPs. An enzy­ matic colorimetric assay was monitored at 405 nm and the electrochemical signal monitored the current peak at -150 mV vs. Ag/AgCl/KCl(satd.). BDD elec­ trode was used as a working electrode. SWV condi­ tions: Esw = 200 mV, Eap = -1.70 V, Esp = 8 mV, f = 200 Hz, t = 10 s, in a potential window from -0.4 to 0.1 V. Further details of the conversion of absorbance and current into ALP enzymatic activity are provided in Supp. Data S12. The error bars show the standard deviation for n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 5 S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 has an excellent hydrolytic stability with no formation of pNP/pAP, remaining colorless in absence of ALP enzyme (Fig. S14, Supp. data). Previous studies reported the monitoring of ALP activity in human serum using a glassy carbon electrode by the rate of hydrolysis of ascorbic acid 2- phosphate (AAP) (Sun and Jiao, 2005). ALP enzymatic hydrolysis product of AAP produced ascorbic acid (AA), which was monitored at 0.38 V (vs Ag/AgCl/KCl(sat.)) by using differential pulse voltammetric (DPV) and ALP assay exhibited a LOD of 0.3 U L− 1. Although a broad operating range (0.4–2000 U L− 1) was covered as mentioned by the authors, the use of carbon electrode needs of a me­ chanical polishing and the spontaneous oxidation of the enzymatic product ascorbic acid in presence of atmospheric air turn the assay into not appropriate for implementation as a portable system for ALP activity determination. Nonetheless, the ascorbic acid undergoes oxidation at 0.38 V (vs Ag/AgCl/KCl(sat.)), which is a high potential, not desirable and relatively complicated in the determination of an electroactive an­ alyte due to possible interfering molecules. This probably happens in the same way as in the dimerization of aniline (Sapurina et al., 2015) and dapsone (Moura et al., 2015). Other researchers have addressed the electrochemical determination of ALP activity using indium–tin oxide (ITO) electrode (Qin et al., 2017), graphite screen-printed electrode (Sappia et al., 2019), graphite-IrO2 composites (Wang et al., 2009), Fig. 6. Electrochemical biosensing for detection of the ALP activity from pu­ graphene oxide-modified gold electrode (Shen et al., 2016), copper rified exosomes derived from healthy donors (n = 10, pooled) and breast cancer (n = 10, pooled) patients. The exosomes were preconcentrated on the surface of sulfide-decorated graphene sheet (Peng et al., 2015). Although these the electrode by immunomagnetic separation based on antiCD326-MPs (1 × works and others reported in the literature have demonstrated advances 106 MPs per assay), followed by reaction with 10 mmol L− 1 pNPP in 1 mol L− 1 in the strategy towards ALP-based assays, some drawbacks are crucial in DEA buffer, containing 6.0 mmol L− 1 MgCl2 and 100 mmol L− 1 KCl. The the ALP determination as to the cost-effectiveness, non-reusable and electrochemical signal monitored the current peak at -150 mV vs. Ag/AgCl/ electrode surface fouling, high potential detection to phenolic-type KCl(satd.). BDD electrode was used as a working electrode. SWV conditions: Esw substrates, which current peaks overlap with the oxygen-evolution re­ = 200 mV, Eap = -1.70 V, Esp = 8 mV, f = 200 Hz, t = 10 s, in a potential gion (>1.0 V vs. Ag/AgCl/KCl(sat.)). window from -0.4 to 0.1 V. In all cases, the enzymatic reaction was during 120 min at 37 ◦ C. Further details of the conversion of current in ALP enzymatic 3.5. Electrochemical biosensing of the intrinsic activity of ALP in activity are provided in Supp. Data S12. exosomes isolated by specific epithelial biomarker from serum of breast cancer patients These advantages are attributed to the conductivity, stability, very low adsorption, and excellent catalytic properties of the boron-doped dia­ The analysis of purified exosomes from healthy donors and breast mond (BDD) electrode for ALP determination. Furthermore, the use of a cancer patients is shown in Fig. 6. The approach is based on the boron-doped microcrystalline diamond (BDD) electrode enables the immunomagnetic separation of the exosomes using the specific CD326 sample volume minimization, preventing the fouling of nitro-phenolic (also known as EpCAM) cancer-related biomarker, followed by the derivatives, an effect usually observed in conventional-based elec­ detection of exosome-derived ALP enzyme by reaction with pNPP trodes (Sappia et al., 2019). Furthermore, this approach simplified the substrate. conventional sandwich immunosensing format, which uses two anti­ Accordingly, and to compare the expression of the receptors on bodies for the detection, and in most of the instances, two incubation exosomes from the two populations, the electrochemical biosensing was and a further enzymatic reaction (Moura et al., 2020a, 2020b). The performed with the same amount (3.35 μg) of exosome protein content difference among healthy donors and breast cancer patients is in per assay, for healthy donors and breast cancer patients (n = 10 each). accordance with the highly expressed epithelial biomarker CD326 pre­ Fig. 6, shows that breast cancer patients overexpressed ALP enzyme in viously reported by our research group (Moura et al., 2020a, 2020b) and CD326-positive exosomes and can be well discriminated from exosome- ALP enzyme in breast cancer patients (Chen et al., 2017; Tsai et al., derived healthy donors (mean 4.5-fold, p < 0.05), without the need of a 2000; Zulauf et al., 2019; Sanchez et al., 2020). Although this is a pre­ secondary labelling antibody for detection of the vesicles. liminary study, the proposed biosensing approach, which combines the exosome immunocapture and the intrinsic enzymatic detection, pro­ 4. Conclusion vides a new device aimed at the simplification of the analytical pro­ cedure. In the case of nanovesicles with multiple biomarkers, the need of To the best of the author’s knowledge, this is the first electrochemical two or more antibodies for capturing and detection implies expensive biosensor approach integrating the magnetic particles specific isolation and time-consuming assays to find a good antibody pair while avoiding to the detection of ALP activity, both in osteoblast-derived and breast cross-reactivity. Moreover, some subpopulation can be excluded since cancer exosomes. This approach demonstrated to be useful for the the two biomarkers should be equally expressed in a single exosome. The discrimination of healthy and breast cancer patient, confirming the use of the intrinsic enzymatic activity of the vesicles is a novel strategy simultaneous expression of cancer biomarkers in exosomes, as is the case that can be applied in other devices not only for the electrochemical of epithelial CD326 (EpCAM), used for the specific isolation, and the readout, but also with visual detection in paper-based RDTs using the ALP intrinsic activity, used for the sensitive electrochemical readout. proper substrates for ALP activity. The ALP-based electrochemical biosensor for exosomes with a limit of detection of 105 exosomes μL− 1 (4.39 mU L− 1 or 13.47 mU mg− 1) rep­ CRediT authorship contribution statement resents an improvement in LODs regarding other exosome quantifica­ tion (Xia et al., 2017; Yadav et al., 2017), and is more sensitive than the Silio Lima Moura: Data curation, Investigation, Validation, Writing gold standard spectrophotometric assay. The low monitored potential of – original draft. Arnau Pallarès-Rusiñol: Visualization, Formal anal­ -150 mV vs. Ag/AgCl/KCl(sat.) avoids possible interfering molecules. ysis, Writing – original draft. Luciano Sappia: Investigation, Validation. 6 S.L. Moura et al. Biosensors and Bioelectronics 198 (2022) 113826 Mercè Martí: Conceptualization, Methodology, Supervision. María Keiding, R., Hörder, M., Denmark, W.G., Pitkänen, E., Tenhunen, R., Strömme, J.H., Theodorsen, L., Waldenström, J., Tryding, N., Westlund, L., 1974. Scand. J. Clin. Isabel Pividori: Conceptualization, Methodology, Supervision, Funding Lab. Invest. 33, 291–306. acquisition. Kim, S.H., Shin, K.H., Moon, S.H., Jang, J., Kim, H.S., Suh, J.S., Yang, W.I., 2017. Cancer Med. 6, 1311–1322. Declaration of competing interest Liu, C., Qie, Y., Qin, W., Zhao, K., Zhu, J., Zhao, L., Li, M., Guo, L.H., 2021. Sensor. Actuator. B Chem. 345, 130336. López-Cobo, S., Campos-Silva, C., Moyano, A., Oliveira-Rodríguez, M., Paschen, A., The authors declare that they have no known competing financial Yáñez-Mó, M., Blanco-López, M.C., Valés-Gómez, M., 2018. J. Nanobiotechnol. 16, interests or personal relationships that could have appeared to influence 47. Luke, R., Khan, S.N., Iqbal, P.S., Soman, R.R., Chakkarayan, J., Krishnan, V., 2015. J. Int. the work reported in this paper. Oral Health 7, 54–57. Melguizo-Rodríguez, L., Costela-Ruiz, V.J., Manzano-Moreno, F.J., Ruiz, C., Illescas- Acknowledgments Montes, R., 2020. Int. J. Mol. Sci. 21, 1–17. Moura, S.L., Martín, C.G., Martí, M., Pividori, M.I., 2020a. Talanta 211, 120657. Moura, S.L., Martín, C.G., Martí, M., Pividori, M.I., 2020b. Biosens. Bioelectron. 150, This work was funded by the Ministry of Economy and Competi­ 111882. tiveness, Madrid (Project BIO2016-75751-R), and Ministry of Science Moura, S.L., Santos Júnior, J.R. 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