Abstract
This work addresses a biosensor combining the immunomagnetic separation and the electrochemical biosensing based on the intrinsic ALP activity of the exosomes. This approach explores for the first time two different types of biomarkers on exosomes, in a unique biosensing device combining two different biorecognition reaction: immunological and enzymatic. Besides, the intrinsic activity of alkaline phosphatase (ALP) in exosomes as a potential biomarker of carcinogenesis as well as osseous metastatic invasion is also explored. To achieve that, as 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 individuals based on the immunomagnetic separation using specific epithelial biomarkers CD326 (EpCAM) combined with the intrinsic ALP activity electrochemical readout.
Highlights
Alkaline phosphatases (ALP, EC 3.1.3.1) are a family of ubiquitous enzymes present in most tissues
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 alkaline phosphatase (ALP) activity electrochemical readout
To the best of the author’s knowledge, this is the first electrochemical biosensor approach integrating the magnetic particles specific isolation to the detection of ALP activity, both in osteoblast-derived and breast cancer exosomes. This approach demonstrated to be useful for the discrimination of healthy and breast cancer patient, confirming the simultaneous expression of cancer biomarkers in exosomes, as is the case of epithelial CD326 (EpCAM), used for the specific isolation, and the ALP intrinsic activity, used for the sensitive electrochemical readout
Summary
Alkaline phosphatases (ALP, EC 3.1.3.1) are a family of ubiquitous enzymes present in most tissues. ALP are key enzymes involved in multiple biological and metabolic pathways, including the bone mineralization process, which increases the local concentration of phosphate ions (Anderson, 2003) by consuming pyrophosphate from the medium, known as inhibitor of the formation of hydroxyapatite crystals (Anderson et al, 2004) In this sense, the ALP activity is a well-established biomarker, routinely determined in clinical analysis, for the monitoring of a series of diseases. The method was firstly optimized using an in vitro model based on exosomes derived from human fetal osteoblastic (hFOB) cell line and compared with the gold standard colorimetric ALP assay in terms of the analytical performance In this first approach, the proposed biosensor combines the immunomagnetic separation of the exosomes based on the general tetraspanin (CD9, CD63, or CD81), followed by the electro chemical readout relying on the determination of ALP activity with pNPP substrate by using boron-doped microcrystalline diamond (BDD) electrodes. The exosomes were isolated using magnetic particles specific for CD326 (EpCAM) cancer-related biomarker, followed by the electrochemical biosensing of ALP content, which differentiates healthy donors and breast cancer patients based on specific epithelial biomarkers
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