DNADerived Materials for Biosensors

To obtain improved biocompatibility and multifunctionality for surgical diagnosis, researchers are engineering molecules to build novel DNA-analogous materials based on DNA structures. For example, from advances in nanotechnology, a promising biomaterial has emerged recently, which is a bioinspired, self-assembled nanomaterial. Such materials have excellent biological properties and are easy to engineer for specific needs. As one of the many examples, rosette nanotubes (RNTs, Fig. 9) are novel biomimetic self-assembled DNA-derived structures, whose basic building blocks are guanine (G) and cytosine (C) DNA base pairs which can solidify into a viscous gel (Chun et al. 2004, 2005; Fenniri et al. 2001). The GAC heteroaromatic bicyclic base possesses the Waston-Crick donor-donor-acceptor properties of guanine and the acceptor-acceptor-donor of cytosine. GAC undergoes a hierarchical self-assembly process under physiological conditions to form a six-membered supermacrocycle by the formation of 18 hydrogen bonds.

Fig. 9 RNTs (with a lysine side chain) undergo spontaneous self-assembly under physiological conditions (adapted and redrawn from Chun et al. (2004, 2005))

Because of electrostatic forces, base stacking interactions, and hydrophobic effects, the rosettes form a stable stack with an inner channel 11 A in diameter. An amino acid side chain (lysine) was chosen to impose chirality and surface chemistry on the RNTs. Importantly, the lysine side chain (with an amine group and a carboxyl group) provides the possibility to functionalize a variety of peptides or proteins onto RNTs for enzymatic recognition. In addition, some small bioactive molecules could be trapped in the inner channel of RNTs by hydrogen bonds. This is another choice for the detection of small molecules using RNTs. In addition, RNTs have excellent cytocompatibility properties, suitable to be implanted into tissues for numerous sensor applications.

Therefore, RNTs not only have self-assembly and self-recognition properties (like DNA base pairs) but also have multiple functions, such as increasing viscosity when heated to low temperatures (40-60°C) and being able to connect with a high density of peptides or enzymes (Zhang et al. 2009). Moreover, RNTs are biocompatible with tissues because they can mimic the nanostructure of collagen and connective tissues to create a surface environment which improves protein (like collagen and fibronectin) adsorption as well as enhances cell adhesion and subsequent functions (Chun et al. 2004; Fenniri et al. 2001; Fine et al. 2009). RNTs are predicted to serve as a novel, multifunctional implant material for biosensors. In addition, in vivo toxicity studies have shown that RNTs lead to lower inflammatory responses than carbon nanotubes at the same dose (Journeay et al. 2008).

A previous study has further outlined a method to engineer RNTs with other materials (Chen et al. 2010a). Alginate gels, mixed with polyvinyl alcohol and polyethylene glycol, have been electrospun with RNTs. Transmission electron microscopy (TEM) imaging of this material clearly shows striations corresponding to RNTs on the electrospun fibers (arrows, Fig. 10a). In contrast, hydrogel composites without RNTs (Fig. 10b) were featureless. These observations suggested that the RNTs may behave as a novel nanoscaffolding on the surface of the hydrogel fibers with the ability of exposing bioactive functional groups (e.g., amino groups) to the surrounding tissues. Moreover, after electrospinning, the lysine side chains of the RNTs were labeled with a fluorescent probe to allow for the visualization of the RNT distribution in the hydrogel composite. Fluorescence microscopy imaging (Fig. 10c) showed that RNTs were present on the surface of the electrospun fibers, but not deep inside the fibers. In contrast, electrospinning fibers without RNTs (Fig. 10d) did not show any significant fluorescence. Importantly, these results confirmed the accessibility of the amino groups from the RNTs on the surface of the hydrogel fibers important for biosensor applications.

Furthermore, studies have highlighted the potential of RNTs to serve as a bio-mimetic nanoprobe for sensor applications. Figure 11a shows that HA crystals rapidly formed on RNTs when mixed with CaCl2 and Na2HPO4 solutions for 2 min and were regularly aligned on RNTs in a pattern similar to the HA/collagen pattern in bone. Figure 11b shows randomly agglomerated HA on grids without RNTs (Zhang et al. 2008). Not only for ceramics, but metals have also been found to nucleate RNTs. Notably, the Au nanoparticles formed were nearly monodispersed clusters of gold particles (1.4-1.5 nm) nestled in pockets on the RNT surface

Fig. 10 Electrospun hydrogel fibers viewed in TEM (a, b) or under a fluorescence microscope (c, d). (a, c) with RNTs and (b, d) without RNTs. Arrows point to the RNTs on the hydrogel fiber surfaces (adapted and redrawn from Chen et al. (2010a))
Fig. 11 Scanning electron microscope (SEM) images of (a) biomimetic HA with a 0.1 mg/mL RNT coating on a porous carbon TEM grid; and (b) HA coating on a TEM grid (adapted from Zhang et al. (2008))

(Chhabra et al. 2010). Thus, RNTs are very promising DNA-derived materials for biosensors and should be further explored in this context.

Besides RNTs, another DNA-derived molecule is a triplex-forming oligonucleotide (TFO). Normally, DNA is formed by two nucleotide strings. However, in another process, a triplex nucleic acid can be formed through the sequence-specific interaction between a single-stranded homopyrimidine and homopurine TFO (Chen et al. 2010b; Duca et al. 2008). In this strategy, a TFO binds to the major groove of the target duplex in a sequence-specific manner through hydrogen bonding between its bases and exposed groups on the duplex base pairs, generating base triplets (Fig. 12). TFO has been the focus of considerable interest because of possible applications in developing new molecular biology tools as well as diagnostic agents (Duca et al. 2008; Igoucheva et al. 2004; Buchini and Leumann 2003).

An important application of TFO as a biosensor material is the recognition of nucleic acids. A big advantage is the fact that pyrimidine triplex formation is pH-dependent. Thus, the recognition of TFO is quite sensitive on the acidity of the environment and such a process is tunable. Triplexes can be used to select a sequence of DNA containing oligopurine/oligopyrimidine sequences in a mixture of duplex DNA (Duca et al. 2008; Ji and Smith 1993). Previous studies have realized this idea

minor groove minor groove H

Fig. 12 Specific recognition of DNA sequences by synthetic TFO (adapted and redrawn from Duca et al. (2008))

minor groove minor groove H

Fig. 12 Specific recognition of DNA sequences by synthetic TFO (adapted and redrawn from Duca et al. (2008))

by attaching a TFO to magnetic probes. In the presence of a mixture of duplexes (such as plasmids), a sequence able to form a triple helix can be selectively recognized inside a bacterial lysate (Ito et al. 1992; Schluep and Cooney 1998).

In another study, TFOs were combined with gold nanoparticles to detect DNA by a color change. Briefly, the pro-synthesized TFOs were adsorbed onto gold nanoparticles to prevent the individual red gold nanoparticles from forming blue aggregations under high-ionic strength conditions due to the low stability of the triplex structure. However, introducing a triplex binder/inducer stabilizes triplex formation through Hoogsteen-type hydrogen bonds. Thus, the triplex would not be able to bind and stabilize individual red gold nanoparticles, resulting in purple-blue gold nanoparticle aggregations (Fig. 13 (Chen et al. 2010b)).

In summary, DNA-derived materials have emerged for biosensor applications. They usually present unique properties, such as excellent biocompatibility (like RNTs) and high sensitivity for biomolecule recognition (like TFO). Therefore, based on nanotechnology and bioengineering, DNA-derived materials are outstanding candidates for a new generation of biosensors more effective for diagnosis.

Fig. 13 Schematic illustration of the structure and color change of TFO/gold nanoparticles with the detection of target DNA (adapted from Chen et al. (2010b))
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Diabetes 2

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