Fast Deoxynivalenol Determination in Cereals Using a White Light Reflectance Spectroscopy Immunosensor

Deoxynivalenol (DON) is a mycotoxin produced by certain Fusarium species and found in a high percentage of wheat and maize grains cultured worldwide. Although not so toxic as other mycotoxins, it exhibits both chronic and acute toxicity, and therefore methods for its fast and accurate on-site determination are highly desirable. In the current work, we employ an optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the fast and sensitive immunochemical label-free determination of DON in wheat and maize samples. The assay is completed in 12 min and has a quantification limit of 2.5 ng/mL in buffer corresponding to 125 mu g/kg in whole grain which is lower than the maximum allowable concentrations set by the regulatory authorities for grains intended for human consumption. Several extraction protocols have been compared, and the highest recovery (>90%) was achieved employing distilled water. In addition, identical calibration curves were received in buffer and wheat/maize extraction matrix providing the ability to analyze the grain samples using calibrators in buffer. Recoveries of DON from spiked wheat and maize grain samples ranged from 92.0(+/- 4.0) to 105(+/- 4.0)%. The analytical performance of the WLRS immunosensor, combined with the short analysis time and instrument portability, supports its potential for on-site determinations

Microfabricated Tin–Film Electrodes for Protein and DNA Sensing Based on Stripping Voltammetric Detection of Cd(II) Released from Quantum Dots Labels

A novel disposable microfabricated tin–film electrochemical sensor was developed for the detection of proteins and DNA. The sensor was fabricated on a silicon wafer through photolithography to define the sensor geometry followed by tin sputtering. A sandwich-type immunoassay with biotinylated reporter antibody was employed for the determination of prostate-specific antigen (PSA) in human serum samples. For the detection of C533G mutation of the RET gene, biotinylated oligonucleotide probes were used. The biotinylated biomolecular probes were labeled with streptavidin (STV)-conjugated CdSe/ZnS quantum dots (QDs); quantification of the analytes was performed through acidic dissolution of the QDs and stripping voltammetric detection of the Cd­(II) released. The proposed QD-based electrochemical sensor overcomes the limitations of existing voltammetric sensors and provides a mercury-free sensing platform with scope for mass-production and further potential for application in clinical diagnostics

Photopatternable materials for guided cell adhesion and growth

Cells in vivo are grown in a highly organized environment defined by the interactions with other cells and the extracellular matrix proteins. Thus, in order to shed light in cellular functions such as adhesion, migration, proliferation, apoptosis and differentiation, the creation of cell culture surfaces that mimic the in vivo environment has emerged. To create such surfaces, many microfabrication techniques have been applied so far. In this review, we focus on application of light-assisted patterning techniques for construction of either 2D or 3D cell culture substrates. In the first section of the review, the principle of the main light-assisted patterning approaches is presented and their advantages and limitations are discussed. In the second section, the influence of micro/ nanopatterned surfaces fabricated with the described methods, on vital cell functions is presented, along with the significance of these findings in the field of tissue engineering and regenerative medicine

Photopatterned PLED arrays for biosensing applications

A solid-state optical biosensor integrated with a single layer flexible blue Polymer Light Emitting Diode (PLED) as the light source was demonstrated and used for the detection of biomolecules labeled with two different fluorescent dyes. An anti-rabbit IgG antibody labeled with the fluorescent dyes, that was adsorbed on the opposite side of a polyethylene tetraphthalate (PET) flexible substrate, was excited by light emitted from the PLED. Light is then absorbed by the dye-labeled antibody resulting in a red shift of the polymer emission spectrum. Emission originating from the dye is also observed. The emission spectral shift and its efficiency depend primarily on the concentration of the biomolecule solution, the overlap between the emission spectra of the polymer and the absorption spectra of the dye and the PLED emission characteristics. Biomolecules immobilized onto distinct areas of a plastic substrate might then selectively be detected with high sensitivity after reacting with fluorescently labeled counterpart molecules.</p

Paper-Based Microfluidic Device with Integrated Sputtered Electrodes for Stripping Voltammetric Determination of DNA via Quantum Dot Labeling

This work reports a microfabricated electrochemical paper-based analytical device (ePAD) for the voltammetric determination of DNA. The device is patterned by wax-printing on paper and features a circular assay zone connected to an inlet zone and a sink via grooved microfluidic channels for accelerated flow rate. An electrochemical cell with integrated electrodes is formed on the reverse side of the paper by sputtering of thin metal films (Sn, Pt and Ag as the working, counter and reference electrode, respectively). Proof-of-principle of the ePAD for biosensing is demonstrated for a DNA assay involving attachment of capture DNA, hybridization with biotinylated target oligonucleotide and labeling with streptavidin-conjugated CdSe/ZnS quantum dots (QDs). After the acidic dissolution of the QDs, the released Cd­(II) is quantified by anodic stripping voltammetry (ASV) at the Sn-film working electrode. Thanks to the synergistic effects of QDs amplification, the inherent sensitivity of ASV and the excellent detection capabilities of the Sn-film working electrode for Cd­(II), the target DNA can be detected at levels as low as 0.11 pmol L^–1 using sample volumes as low as 1 μL. The developed microfluidic ePAD costs only 0.11$ and presents favorable fabrication and operational features that make it an excellent candidate biosensor for simple and ultrasensitive point-of-need testing

Protein-Resistant Cross-Linked Poly(vinyl alcohol) Micropatterns via Photolithography Using Removable Polyoxometalate Photocatalyst

In the last years, there has been an increasing interest in controlling the protein adsorption properties of surfaces because this control is crucial for the design of biomaterials. On the other hand, controlled immobilization of proteins is also important for their application as solid surfaces in immunodiagnostics and biosensors. Herein we report a new protein patterning method where regions of the substrate are covered by a hydrophilic film that minimizes protein adsorption. Particularly, poly­(vinyl alcohol) (PVA) cross-linked structures created by an especially developed photolithographic process are proved to prevent protein physisorption and they are used as a guide for selective protein adsorption on the uncovered areas of a protein adsorbing substrate such as polystyrene. The PVA cross-linking is induced by photo-oxidation using, as a catalyst, polyoxometalate (H₃PW₁₂O₄₀ or α-(NH₄)₆P₂W₁₈O₆₂), which is removed using a methyl alcohol/water mixed solvent as the developer. We demonstrate that the polystyrene and the cross-linked PVA exhibit dramatically different performances in terms of protein physisorption. In particular, the polystyrene areas presented up to 130 times higher protein binding capacity than the PVA ones, whereas the patterning resolution could easily reach dimensions of a few micrometers. The proposed approach can be applied on any substrate where PVA films can be coated for controlling protein adsorption onto surface areas custom defined by the user

Detection of ochratoxin A in beer samples with a label-free monolithically integrated optoelectronic biosensor

An optical biosensor for label-free detection of ochratoxin A (OTA) in beer samples is presented. The biosensor consists of an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources on the same Si chip (37mm2). The chip was transformed to biosensor by functionalizing the MZIs sensing arms with an OTA-ovalbumin conjugate. OTA determination was performed by pumping over the chip mixtures of calibrators or samples with anti-OTA antibody following a competitive immunoassay format. An external miniaturized spectrometer was employed to continuously record the transmission spectra of each interferometer. Spectral shifts obtained due to immunoreaction were transformed to phase shifts through Discrete Fourier Transform. The assay had a detection limit of 2.0ng/ml and a dynamic range 4.0-100ng/ml in beer samples, recoveries ranging from 90.6 to 116%, and intra- and inter-assay coefficients of variation of 9% and 14%, respectively. The results obtained with the sensor using OTA-spiked beer samples spiked were in good agreement with those obtained by an ELISA developed using the same antibody. The good analytical performance of the biosensor and the small size of the proposed chip provide for the development of a portable instrument for point-of-need determinations