3 research outputs found
Label-Free Optical Biosensors Based on Aptamer-Functionalized Porous Silicon Scaffolds
A proof-of-concept
for a label-free and reagentless optical biosensing
platform based on nanostructured porous silicon (PSi) and aptamers
is presented in this work. Aptamers are oligonucleotides (single-stranded
DNA or RNA) that can bind their targets with high affinity and specificity,
making them excellent recognition elements for biosensor design. Here
we describe the fabrication and characterization of aptamer-conjugated
PSi biosensors, where a previously characterized his-tag binding aptamer
(6H7) is used as model system. Exposure of the aptamer-functionalized
PSi to the target proteins as well as to complex fluids (i.e., bacteria
lysates containing target proteins) results in robust and well-defined
changes in the PSi optical interference spectrum, ascribed to specific
aptamer-protein binding events occurring within the nanoscale pores,
monitored in real time. The biosensors show exceptional stability
and can be easily regenerated by a short rinsing step for multiple
biosensing analyses. This proof-of-concept study demonstrates the
possibility of designing highly stable and specific label-free optical
PSi biosensors, employing aptamers as capture probes, holding immense
potential for application in detection of a broad range of targets,
in a simple yet reliable manner
Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity
Ligands used on the surface of colloidal
nanoparticles (NPs) have
a significant impact on physiochemical properties of NPs and their
interaction in biological environments. In this study, we report a
one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized
CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated
methoxy polyethylene glycol (mPEG) molecules as a surface coordinating
ligand. The resulting mPEGāQdots were characterized by using
Ī¶ potential, FTIR, thermogravimetric (TG) analysis, and microscale
thermophoresis (MST) studies. We investigated the effect of mPEG molecules
and their grafting density on the Qdots photophysical properties,
colloidal stability, protein binding affinity, and in vitro cellular
toxicity. Moreover, cellular binding features of the resulting Qdots
were examined by using three-dimensional (3D) tumor-like spheroids,
and the results were discussed in detail. Promisingly, mPEG ligands
were found to increase colloidal stability of Qdots, reduce adsorption
of proteins to the Qdot surface, and mitigate Qdot-induced side effects
to a great extent. Flow cytometry and confocal microscopy studies
revealed that PEGylated Qdots exhibited distinctive cellular interactions
with respect to their mPEG grafting density. As a result, mPEG molecules
demonstrated a minimal effect on the ZnS shell deposition and the
Qdot fluorescence efficiency at a low mPEG density, whereas they showed
pronounced effect on Qdot colloidal stability, protein binding affinity,
cytotoxicity, and nonspecific binding at a higher mPEG grafting amount
Identification of the Target Binding Site of Ethanolamine-Binding Aptamers and Its Exploitation for Ethanolamine Detection
Aptamers are promising recognition
elements for sensitive and specific
detection of small molecules. We have previously selected ssDNA aptamers
for ethanolamine, one of the smallest aptamer targets so far. The
work presented here focuses on the determination of the binding region
within the aptamer structure and its exploitation for the development
of an aptamer-based assay for detection of ethanolamine. Sequence
analysis of the aptamers resulted in the identification of a G-rich
consensus sequence, which was able to fold in a typical two- or three-layered
G-quartet structure. Experiments with stepwise truncated variants
of the aptamers revealed that the consensus sequence is responsible
and sufficient for binding to the target. On the basis of the knowledge
of the aptamers binding site, we developed an aptamer-based microarray
assay relying on competition between ethanolamine and an oligonucleotide
complementary to the consensus sequence. Competitive binding of ethanolamine
and fluorescently labeled complementary oligonucleotides resulted
in fluorescence intensities dependent on ethanolamine concentration
with a limit of detection of 10 pM. This method enables detection
of small molecules without any labeling of analytes. The competitive
assay could potentially be transferred to other aptamers and thus
provides a promising system for aptamer-based detection of diverse
small molecules