Aptamer-based detection of epithelial tumor marker mucin 1 with quantum dot-based fluorescence readout.

Mucin 1 (MUC1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this paper, we report an aptamer-based, quantitative detection protocol for MUC1 using a 3-component DNA hybridization system with quantum dot (QD)-labeling: in the absence of MUC1 peptides, strong fluorescence is observed upon mixing the three specially designed DNA strands (quencher, QD-labeled reporter, and the MUC1 aptamer stem); in the presence of MUC1 peptides, a successive decrease in fluorescence intensity is detected since the MUC1 peptide binds to the aptamer strand in such a way to allow the quencher and fluorescence reporter to be brought into close proximity (which leads to the occurrence of fluorescence resonance energy transfer, FRET, between the quencher and QD). The detection limit for MUC1 with this novel approach is in the nanomolar (nM) level, and a linear response can be established for the approximate range found in blood serum. This study also provided further insight into the aptamer/analyte binding site/mode for MUC1, which augments the possibility of improving this detection methodology for the early diagnosis of different types of epithelial cancers of large populations.

Charge conduction properties of a parallel-stranded DNA G-quadruplex: implications for chromosomal oxidative damage.

The charge-flow properties and concomitant guanine damage patterns of a number of intermolecular and wholly parallel-stranded DNA G-quadruplexes were investigated. The DNA constructs were structurally well-defined and consisted of the G-quadruplex sandwiched and stacked between two Watson-Crick base-paired duplexes. Such duplex-quadruplex-duplex constructs were designed to minimize torsional stress as well as steric crowding at the duplex-quadruplex junctions. When anthraquinone was used to induce charge flow within the constructs, it was found that the quadruplex served both as a sink and as a moderately good conductor of electron holes, relative to DNA duplexes. Most strikingly, the quadruplex suffered very little charge-flow generated oxidative damage relative to guanines in the duplex regions and, indeed, to guanines in antiparallel quadruplexes reported in prior studies. It is likely that these differences result from a combination of steric and electronic factors. A biological conclusion that may be drawn from these data is that if, as anticipated, G-quadruplex structures form in vivo at the telomeres and other loci in eukaryotic chromosomes, their ability to serve as protective sinks against chromosomal oxidative damage may depend on their specific character and topology. From a separate perspective, our results on the conduction properties of duplex-quadruplex-duplex DNA composites suggest the utility of G-quadruplexes as junction modules in the construction of DNA-based biosensors and nanocircuitry.

Design and testing of aptamer-based electrochemical biosensors for proteins and small molecules.

The fabrication of aptamer-based electrochemical biosensors as an emerging technology has made the detection of small and macromolecular analytes easier, faster, and more suited for the ongoing transition from fundamental analytical science to the early detection of protein biomarkers. Aptamers are synthetic oligonucleotides that have undergone iterative rounds of in vitro selection for binding with high affinity to specific analytes of choice; a sensitive yet simple method to utilize aptamers as recognition entities for the development of biosensors is to transduce the signal electrochemically. In this review article, we attempt to summarize the state-of-the-art research progresses that have been published in recent years; in particular, we focus on the electrochemical biosensors that incorporate aptamers for sensing small organic molecules and proteins. Based on differences in the design of the DNA/RNA-modified electrodes, we classify aptamer-based electrochemical sensors into three categories, for which the analyte detection relies on: (a) configurational change, i.e., the analyte binding induces either an assembly or dissociation of the sensor construct; (b) conformational change, i.e., the analyte binding induces an alteration in the conformation (folding) of the surface immobilized aptamer strands; and (c) conductivity change, i.e., the analyte binding "switches on" the conductivity of the surface-bound aptamer-DNA constructs. In each section, we will discuss the performance of these novel biosensors with representative examples reported in recent literature.

On the nature of DNA self-assembled monolayers on Au: measuring surface heterogeneity with electrochemical in situ fluorescence microscopy.

The creation of gold surfaces modified by single- or double-stranded DNA self-assembled monolayers (SAMs) is shown to produce heterogeneous surface packing densities through the use of electrochemical studies coupled with fluorescence imaging. The modified surfaces created by direct adsorption of thiolate DNA [followed by passivation with mecaptohexanol (MCH)] resulted in regions covered by a monolayer of DNA SAM and other regions that were coated by large particles of DNA. The difference in fluorescence intensity measured from these regions was dramatic. More importantly, a regional variance in fluorescence intensity in response to electrochemical potential was observed: the large aggregates showing a significantly different modulation of fluorescence intensity than the monolayer-coated regions. Electrochemical desorption and detection of the fluorescently tagged DNA provided clear evidence of a complete surface modification. These studies have implications for biosensor/biochip development using DNA SAMs. A modification in the method used to produce the DNA SAMs resulted in a significantly different surface with much fewer aggregates and more significant electromodulation of the fluorescence intensity, though at much lower DNA surface density (ca. 1% of maximum theoretical coverage). This method for forming the modified surfaces has clear advantages over the currently accepted practice and emphasizes the importance of studying the nonaveraged nature of the sensor surface using in situ imaging tools like electrofluorescence microscopy.

Aptamer-based biosensors for label-free voltammetric detection of lysozyme.

This paper reports a simple electrochemical approach for the detection of the ubiquitous protein lysozyme using aptamer-modified electrodes. Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly, for which the surface density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH3)6]3+) bound to the surface via electrostatic interaction with the DNA phosphate backbone. Upon incubation of the electrode with a solution containing lysozyme, the CV response of surface-bound [Ru(NH3)6]3+ changed substantially, and the relative decrease in the integrated charge of the reduction peak can be tabulated as a quantitative measure of the protein concentration. It is significant that the on-chip protein/aptamer binding constant and the optimized surface density to achieve the best detection limit can be evaluated. This biosensor is label-free and offers an alternative, sensitive, and versatile method for protein detection, which is beneficial to the ever-growing interests of fabricating portable bioanalytical devices with simple electrical readout protocols.

"Non-evaporating" microdroplets on self-assembled monolayer surfaces under ambient conditions.

A unique "non-evaporation" phenomenon, i.e., the unusually slow evaporation process of sessile microdroplets on self-assembled monolayer (SAM) surfaces, is reported. It has been observed that only droplets containing a certain proportion of a volatile and a less-volatile component undergo non-evaporation, which is characterized by hours-long existence of the droplets maintaining constant contact angle, contact area, and volume. We propose that for alcohol-water binary mixtures on SAM surfaces, the highly orientated and closely packed hydrophobic 1-decanethiol molecules induce a concentration gradient of alcohol in water, with a higher concentration of alcohol near the SAM surface. Initial evaporation of the alcohol (more volatile) increased the contact angle until the establishment of a new composition, which contains a strong hydrogen-bonding network among the water molecules in the presence of the alcohol alkyl chains. There is a lessened tendency for the alcohol to evaporate in the presence of a concentration gradient due to such interactions, which results in the observed "non-evaporating" phenomenon. This type of unusual evaporating profile was not observed on conventional substrates, such as polycarbonate sheets and microscope glass slides modified with alkyltrichlorosilanes.

Evaporation of microdroplets of ethanol-water mixtures on gold surfaces modified with self-assembled monolayers.

The wetting property and evaporation behavior of ethanol-water mixtures of various concentrations on gold surfaces modified with 1-decanethiolate self-assembled monolayers (SAMs) were studied by digital contact angle analysis. It has been shown that the initial contact angle decreases monotonically with increased concentration of ethanol in the mixture. Evaporation studies revealed a general trend with a preliminary increase in contact angle accompanied with a decrease in contact area, then a constant contact angle accompanied with a slower, linear decrease in contact area. At the very beginning of the evaporation process, the contact angles showed a rapid decrease for the microdroplets of a binary mixture with equal volume fractions (i.e., 50% ethanol). Three distinct stages of the evaporation profile for the ethanol-water mixtures were observed, which differ from the inclusive "pinning" and "shrinking" behavior observed for the pure liquid case. Ultimately, the study makes possible the use of an evaporation profile to monitor the change in concentration of a binary system and allows a better understanding of the interactions between liquid microdroplets with solid substrates.