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

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

Orthogonal Patterning of Multiple Biomolecules Using an Organic Fluorinated Resist and Imprint Lithography

The ability to spatially deposit multiple biomolecules onto a single surface with high-resolution while retaining biomolecule stability and integrity is critical to the development of micro- and nanoscale biodevices. While conventional lithographic patterning methods are attractive for this application, they typically require the use of UV exposure and/or harsh solvents and imaging materials, which may be damaging to fragile biomolecules. Here, we report the development of a new patterning process based on a fluorinated patterning material that is soluble in hydrofluoroether solvents, which we show to be benign to biomolecules, including proteins and DNA. We demonstrate the implementation of these materials into an orthogonal processing system for patterning multibiomolecule arrays by imprint lithography at room temperature. We further showcase this method’s capacity for fabricating patterns of receptor-specific ligands for fundamental cell studies

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach–Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 μg/mL for bovine k-casein, 1.0 μg/mL for peanut protein, 0.80 μg/mL for soy protein, and 0.10 μg/mL for gliadin. The assays were accurate (recoveries 88–118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations