The capability to screen a range of proteins at the single-molecule level with enhanced selectivity in biological fluids has been in part a driving force in developing future diagnostic and therapeutic strategies. The combination of nanopore sensing and nucleic acid aptamer recognition comes close to this ideal due to the ease of multiplexing, without the need for expensive labelling methods or extensive sample pre-treatment. Here, we demonstrate a fully flexible, scalable and low-cost detection platform to sense multiple protein targets simultaneously by grafting specific sequences along the backbone of a double-stranded DNA carrier. Protein bound to the aptamer produces unique ionic current signatures which facilitates accurate target recognition. This powerful approach allows us to differentiate individual protein sizes via characteristic changes in the sub-peak current. Furthermore, we show that by using DNA carriers it is possible to perform single-molecule screening in human serum at ultra-low protein concentrations.
Biomaterial
substrates can be engineered to present topographical
signals to cells which, through interactions between the material
and active components of the cell membrane, regulate key cellular
processes and guide cell fate decisions. However, targeting mechanoresponsive
elements that reside within the intracellular domain is a concept
that has only recently emerged. Here, we show that mesoporous silicon
nanoneedle arrays interact simultaneously with the cell membrane,
cytoskeleton, and nucleus of primary human cells, generating distinct
responses at each of these cellular compartments. Specifically, nanoneedles
inhibit focal adhesion maturation at the membrane, reduce tension
in the cytoskeleton, and lead to remodeling of the nuclear envelope
at sites of impingement. The combined changes in actin cytoskeleton
assembly, expression and segregation of the nuclear lamina, and localization
of Yes-associated protein (YAP) correlate differently from what is
canonically observed upon stimulation at the cell membrane, revealing
that biophysical cues directed to the intracellular space can generate
heretofore unobserved mechanosensory responses. These findings highlight
the ability of nanoneedles to study and direct the phenotype of large
cell populations simultaneously, through biophysical interactions
with multiple mechanoresponsive components.
The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.
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