Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes.

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes µL-1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

Peptide-based biosensing approaches for targeting breast cancer-derived exosomes.

Exosomes are nanovesicles present in all the biological fluids, making them attractive as non-invasive biomarkers for diseases like cancer, among many others. However, exosomes are complex to separate and detect, requiring comprehensive molecular characterization for their routine use in diagnostics. This study explores the use of peptides as cost-effective and stable alternatives to antibodies for exosome binding. To achieve that, phage display technology was employed to select peptides with high specificity for target molecules in exosomes. Specifically, a selected peptide was evaluated for its ability to selectively bind breast cancer-derived exosomes. Proteomic analysis identified 38 protein candidates targeted by the peptide on exosome membranes. The binding of the peptide to breast cancer-derived exosomes was successfully demonstrated by flow cytometry and magneto-actuated immunoassays. Furthermore, an electrochemical biosensor was also tested for breast cancer-derived exosome detection and quantification. The peptide demonstrated effective binding to exosomes from aggressive cancer cell lines, offering promising results in terms of specificity and recovery. This research shows potential for developing rapid, accessible diagnostic tools for breast cancer, especially in low-resource healthcare settings.

Magnetic Separation of Cell-Secreted Vesicles with Tailored Magnetic Particles and Downstream Applications.

The analysis of the receptors on the surface of the cell-secreted vesicles provides valuable information of the cell signature and may also offer diagnosis and/or prognosis of a wide range of diseases, including cancer.Due to their low concentration, conventional procedures for extracellular vesicle (EV) detection usually require relatively large sample volumes, involving preliminary purification or preconcentration steps from complex specimens. Here, we describe the separation and preconcentration in magnetic particles of extracellular vesicles obtained from cell culture supernatants from MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines, human fetal osteoblastic cell line (hFOB), and human neuroblastoma SH-SY5Y cell line, as well as exosomes from human serum. The first approach involves the covalent immobilization for the exosomes directly on micro (4.5 µm)-sized magnetic particles. The second approach is based on tailored magnetic particles modified with antibodies for further immunomagnetic separation of the exosomes. In these instances, micro (4.5 µm)-sized magnetic particles are modified with different commercial antibodies against selected receptors, including the general tetraspanins CD9, CD63, and CD81 and the specific receptors (CD24, CD44, CD54, CD326, CD340, and CD171). The magnetic separation can be easily coupled with downstream characterization and quantification methods, including molecular biology techniques such as immunoassays, confocal microscopy, or flow cytometry.

Advances in exosome analysis.

There is growing demand for novel biomarkers that detect early stage disease as well as monitor clinical management and therapeutic strategies. Exosome analysis could provide the next advance in attaining that goal. Exosomes are membrane encapsulated biologic nanometric-sized particles of endocytic origin which are released by all cell types. Unfortunately, exosomes are exceptionally challenging to characterize with current technologies. Exosomes are between 30 and 200nm in diameter, a size that makes them out of the sensitivity range to most cell-oriented sorting or analysis platforms, i.e., traditional flow cytometers. The most common methods for targeting exosomes to date typically involve purification followed by the characterization and the specific determination of their cargo. The whole procedure is time consuming, requiring thus skilled personnel as well as laboratory facilities and benchtop instrumentation. The most relevant methodology for exosome isolation, characterization and quantification is addressed in this chapter, including the most up-to-date approaches to explore the potential usefulness of exosomes as biomarkers in liquid biopsies and in advanced nanomedicine.

The activity of alkaline phosphatase in breast cancer exosomes simplifies the biosensing design.

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.

Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay.

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg µL-1/1.5 × 104 CFU mL-1). The LFA achieved visual detection of as low as 2.2 × 10-2 pg µL-1 using Av-CNPs and 8.4 × 10-2 pg µL-1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.