Hua-Zhong Yu

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.

Surface-enhanced raman scattering (SERS) from azobenzene self-assembled sandwiches

A silver island film was thermally deposited on the top of an azobenzene self-assembled monolayer on gold, yielding a SERS-active system possessing a “sandwiched” structure of Ag|R1−Azo−R2S−|Au. For the first time, the positions of the Azo groups were controlled by using azobenzenealkanethiols with different terminal groups (R1) or interchain spacers (R2), to clarify the relationship between the SERS effect and the structural nature of the system. As will be shown, increases in distances of Azo groups from the gold substrate and from the silver film both cause the enhancement factor to decay exponentially, indicating that the enhancement correlates to both the gold substrate underneath and the silver islands above.

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.

Immobilized DNA Switches as Electronic Sensors for Picomolar Detection of Plasma Proteins

The sensing principle of a new class of DNA conformational switches (deoxyribosensors) is based on the incorporation of an aptamer as the receptor, whose altered conformation upon analyte binding switches on the conductivity of an adjacent helical conduction path, leading to an increase in the measured electrical signal through the sensor. We report herein the rational design and biochemical testing of candidate deoxyribosensors for the detection and quantitation of a plasma protein, thrombin, followed by surface immobilization of the optimized sensor and its electrochemical testing in both a near-physiological buffer solution and in diluted blood serum. The very high detection sensitivity (in the picomolar range) and specificity, as well as the adaptability of deoxyribosensors for the detection of diverse molecular analytes both small and macromolecular, make this novel sensing methodology an extremely promising one. Such synthetic and robust DNA-based electronic sensors should find broad application in the rapid, miniaturized, and automated on-chip detection of many biomedically relevant substances (such as metabolites, toxins, and disease and tumor markers) as well as of environmental contaminants.

A Robust Electronic Switch Made of Immobilized Duplex/Quadruplex DNA†

In recent years, DNA and RNA have been used extensively as materials for the construction and self-assembly of a variety of nanoscale devices. DNA-based mechanical switches have been described recently. Extension–contraction transitions of DNA nanoconstructs (e.g., G-quadruplexes) upon ligand binding have also been reported. A G-quadruplex (G4DNA) is a quadruple-helical DNA secondary structure, in which guanines are paired by Hoogsteen hydrogen bonds to form guanine base quartets. G4-DNA is stabilized by the coordination of specific metal ions, such as K ions or Sr ions, which bind between successive G-quartets. G4-DNAbased nanoconstructs have been used as sensors to detect the blood protein thrombin, K ions, and target oligonucleotides by binding of these analytes to DNA aptamers (nucleic acid receptors that are themselves sometimes G4DNA). The ligand binding properties of G4-DNA, as well as its ability to conduct electrical charges, have been characterized extensively by using gel electrophoresis and spectroscopic methods. G4-DNAs typically form by the folding of G-rich singlestranded DNA (ssDNA), but such ssDNA to G4-DNA transitions are not easily reversed. In contrast, the duplex DNA (dsDNA) to G4-DNA transition can be kinetically more favored, brought about by the addition and removal of K ions (utilizing a chelator such as [18]crown-6). We recently reported a biochemical study of the charge-conducting properties of a contractile DNA nanoswitch, which is able to switch repeatedly between a structurally extended electronic “off” state (an extended duplex) and a contracted electronic “on” state (a G-quadruplex). The conductivity of the K-induced contracted duplex in solution was much higher than that of the extended conformational state. This biochemical study of conductivity, however, provided no direct indication of whether such a device could be adapted for practical use (i.e., whether electronic switching would still be observed if the device were to be localized on an electronic chip). Herein we report the results of our first chip-based strategy, which is able to directly and efficiently measure both the reversibility and the repeatability of the electronic switching behavior of the dsDNA/G4-DNA constructs. Specifically, we present an electrochemical (square-wave voltammetry) investigation of the DNA nanoswitch immobilized on a gold electrode. As shown in Figure 1, the contractile DNA duplex incorporates two short, separated motifs of G/G mismatches that are flanked by Watson–Crick base-paired DNA. The

Digitized molecular diagnostics: reading disk-based bioassays with standard computer drives.

We report herein a digital signal readout protocol for screening disk-based bioassays with standard optical drives of ordinary desktop/notebook computers. Three different types of biochemical recognition reactions (biotin-streptavidin binding, DNA hybridization, and protein-protein interaction) were performed directly on a compact disk in a line array format with the help of microfluidic channel plates. Being well-correlated with the optical darkness of the binding sites (after signal enhancement by gold nanoparticle-promoted autometallography), the reading error levels of prerecorded audio files can serve as a quantitative measure of biochemical interaction. This novel readout protocol is about 1 order of magnitude more sensitive than fluorescence labeling/scanning and has the capability of examining multiplex microassays on the same disk. Because no modification to either hardware or software is needed, it promises a platform technology for rapid, low-cost, and high-throughput point-of-care biomedical diagnostics.

A Mechano-Electronic DNA Switch

We report a new kind of DNA nanomachine that, fueled by Hg(2+) binding and sequestration, couples mechanical motion to the multiply reversible switching of through-DNA charge transport. This mechano-electronic DNA switch consists of a three-way helical junction, one arm of which is a T-T mismatch containing Hg(2+)-binding domain. We demonstrate, using chemical footprinting and by monitoring charge-flow-dependent guanine oxidation, that the formation of T-Hg(2+)-T base pairs in the Hg(2+)-binding domain sharply increases electron-hole transport between the other two Watson-Crick-paired stems, across the three-way junction. FRET measurements are then used to demonstrate that Hg(2+) binding/dissociation, and the concomitant increase/decrease of hole transport efficiency, are strongly linked to specific mechanical movements of the two conductive helical stems. The increase in hole transport efficiency upon Hg(2+) binding is tightly coupled to the movement of the conductive stems from a bent arrangement toward a more linear one, in which coaxial stacking is facilitated. This switch offers a paradigm wherein the performance of purely mechanical work by a nanodevice can be conveniently monitored by electronic measurement.

Metal cation-induced deformation of DNA self-assembled monolayers on silicon: vibrational sum frequency generation spectroscopy.

Nucleic acids possess charged phosphate groups in their backbones, which require counterions to reduce the repulsive Coulombic interactions between the strands. Herein we report how different mono- and divalent metal cations influence the molecular orientations of DNA molecules on silicon surfaces upon immobilization and hybridization. Our sum frequency generation (SFG) spectroscopy studies demonstrated that the degree of conformational variation of DNA self-assembled monolayers on silicon depends on the type of metal cations present. The molecular orientation change of immobilized single-stranded oligonucleotides correlates with DNA-cation affinity (Mg(2+) > Ca(2+) > K(+) approximately Na(+)): metal cations with the strongest affinity disrupt the structure of the underlying linker monolayer the most. Upon hybridization the trend is reversed, which is attributed to the greater ability of divalent cations to mask the negative charges on the DNA backbone. These findings provide useful information for the construction of more sensitive DNA biosensors, particularly the optimization of on-chip hybridization performance.

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.

Mobile app-based quantitative scanometric analysis.

The feasibility of using smartphones and other mobile devices as the detection platform for quantitative scanometric assays is demonstrated. The different scanning modes (color, grayscale, black/white) and grayscale converting protocols (average, weighted average/luminosity, and software specific) have been compared in determining the optical darkness ratio (ODR) values, a conventional quantitation measure for scanometric assays. A mobile app was developed to image and analyze scanometric assays, as demonstrated by paper-printed tests and a biotin-streptavidin assay on a plastic substrate. Primarily for ODR analysis, the app has been shown to perform as well as a traditional desktop scanner, augmenting that smartphones (and other mobile devices) promise to be a practical platform for accurate, quantitative chemical analysis and medical diagnostics.