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.

Osteoblastic exosomes. A non-destructive quantitative approach of alkaline phosphatase to assess osteoconductive nanomaterials.

Alkaline phosphatase (ALP) is an essential biomarker of osteoblastic activity. Currently, ALP activity has been used to study bone mineralization mechanisms and osteoactive biomaterials among others. The ALP quantification is usually performed by destructive methods either on growing cells or cells lysate in which the osteoconductive biomaterial is being assessed. This work addresses the evaluation of a non-destructive colorimetric approach for the determination of ALP activity on osteoblast-derived exosomes from culture supernatants. The efficiency of the method was evaluated on osteoconductive electrospun scaffolds of PCL compounded with ZnO as a reference biomaterial. The results demonstrated that the osteoblast cell line mineralization induced by osteoconductive scaffolds can be monitorized over time by the non-destructive measurement of ALP activity on osteoblast derived exosomes. Consequently, this non-destructive approach suggested to be a reliable alternative technique for the quantification of biomaterials osteoconductivity or even evaluation of osteoblastic response at stem cells.

PEDOT-polyamine composite films for bioelectrochemical platforms - flexible and easy to derivatize.

We report a straightforward route for the preparation of flexible, electrochemically stable and easily functionalizable poly(3,4-ethylenedioxythiophene) (PEDOT) composite films deposited on PET foils as biosensing platforms. For this purpose, poly(allylamine) hydrochloride (PAH) was blended with PEDOT to provide amine-bearing sites for further biofunctionalization as well as to improve the mechanical properties of the films. The conducting PEDOT-PAH composite films were characterized by cyclic voltammetry, UV-vis and Raman spectroscopies. An exhaustive stability study was carried out from the mechanical, morphological and electrochemical viewpoint. Subsequent sugar functionalization of the available amine groups from PAH allowed for the specific recognition of lectins and the subsequent self-assembly of glycoenzymes (glucose oxidase and horseradish peroxidase) concomitant with the prevention of non-specific protein fouling. The platforms presented good bioelectrochemical performance (glucose oxidation and hydrogen peroxide reduction) in the presence of redox mediators. The developed composite films constitute a promising option for the construction of all-polymer biosensing platforms with great potential owing to their flexibility, high transmittance, electrochemical stability and the possibility of glycosylation, which provides a simple route for specific biofunctionalization as well as an effective antifouling strategy.

MOF@PEDOT Composite Films for Impedimetric Pesticide Sensors.

Due to its deleterious effects on health, development of new methods for detection and removal of pesticide residues in primary and derived agricultural products is a research topic of great importance. Among them, imazalil (IMZ) is a widely used post-harvest fungicide with good performances in general, and is particularly applied to prevent green mold in citrus fruits. In this work, a composite film for the impedimetric sensing of IMZ built from metal-organic framework nanocrystallites homogeneously distributed on a conductive poly(3,4-ethylene dioxythiophene) (PEDOT) layer is presented. The as-synthetized thin films are produced via spin-coating over poly(ethylene terephtalate (PET) substrate following a straightforward, cost-effective, single-step procedure. By means of impedance spectroscopy, electric transport properties of the films are studied, and high sensitivity towards IMZ concentration in the range of 15 ppb to 1 ppm is demonstrated (featuring 1.6 and 4.2 ppb limit of detection, when using signal modulus and phase, respectively). The sensing platform hereby presented could be used for the construction of portable, miniaturized, and ultrasensitive devices, suitable for pesticide detection in food, wastewater effluents, or the assessment of drinking-water quality.

Biomimetic magnetic sensor for electrochemical determination of scombrotoxin in fish.

This work addresses a novel, rapid and cost-effective approach for the electrochemical sensing of scombrotoxin (histamine) in fish based on magnetic molecularly imprinted polymer (magnetic-MIP). The histamine magnetic-MIP was synthesized by the core-shell method using histamine as a template, and 2-vinyl pyridine as functional monomer. The magnetic-MIP was characterized by TEM, SEM, and confocal microscopy. Additionally, the binding capacity of magnetic-MIP towards histamine was investigated and compared with magnetic non-molecularly imprinted polymer (magnetic-NIP). This biomimetic material merged the advantages of MIPs and magnetic particles (MPs), including low cost of production, stability, high binding capacity and can be easily separated by the aid of a permanent magnet. The magnetic-MIP was integrated into magneto-actuated electrodes for the direct electrochemical detection of histamine preconcentrated from fish samples. The results revealed that this approach succeeded in the preconcentration and determination of histamine with a LOD as low as 1.6 × 10-6 mg L-1, much lower than the index for fish spoilage (50 mg kg-1) accordingly to the legislation. Furthermore, the analytical performance was validated for the determination of histamine in scombroid fish samples with recovery values ranging from 96.8 to 102.0 %, confirm so it can be applied easily for routine food examination.

Controlled degradability of PCL-ZnO nanofibrous scaffolds for bone tissue engineering and their antibacterial activity.

Up to date, tissue regeneration of large bone defects is a clinical challenge under exhaustive study. Nowadays, the most common clinical solutions concerning bone regeneration involve systems based on human or bovine tissues, which suffer from drawbacks like antigenicity, complex processing, low osteoinductivity, rapid resorption and minimal acceleration of tissue regeneration. This work thus addresses the development of nanofibrous synthetic scaffolds of polycaprolactone (PCL) - a long-term degradation polyester - compounded with hydroxyapatite (HA) and variable concentrations of ZnO as alternative solutions for accelerated bone tissue regeneration in applications requiring mid- and long-term resorption. In vitro cell response of human fetal osteoblasts as well as antibacterial activity against Staphylococcus aureus of PCL:HA:ZnO and PCL:ZnO scaffolds were here evaluated. Furthermore, the effect of ZnO nanostructures at different concentrations on in vitro degradation of PCL electrospun scaffolds was analyzed. The results proved that higher concentrations ZnO may induce early mineralization, as indicated by high alkaline phosphatase activity levels, cell proliferation assays and positive Alizarin-Red-S-stained calcium deposits. Moreover, all PCL:ZnO scaffolds particularly showed antibacterial activity against S. aureus which may be attributed to release of Zn2+ ions. Additionally, results here obtained showed a variable PCL degradation rate as a function of ZnO concentration. Therefore, this work suggests that our PCL:ZnO scaffolds may be promising and competitive short-, mid- and long-term resorption systems against current clinical solutions for bone tissue regeneration.