5 research outputs found
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 CdÂ(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 CdÂ(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 CdÂ(II), the target DNA can be detected
at levels as low as 0.11 pmol L<sup>–1</sup> 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, polyÂ(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 (H<sub>3</sub>PW<sub>12</sub>O<sub>40</sub> or α-(NH<sub>4</sub>)<sub>6</sub>P<sub>2</sub>W<sub>18</sub>O<sub>62</sub>), 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