DNA covalent immobilization onto screen-printed electrode networks for direct label-free hybridization detection of p53 sequences

A new electrochemical biochip for the detection of DNA sequences was developed. The entire biochip-i.e., working, reference, and counter electrodes-was constructed based on the screen-printing technique and exhibits eight working electrodes that could be individually addressed and grafted through a simple electrochemical procedure. Screen-printed electrode networks were functionalized electrochemically with 1-ethyl-3-(3dimethylaminopropyl)carbodidiimide according to a simple procedure. Single-stranded DNA with a C6-NH(2) linker at the 5'-end was then covalently bound to the surface to act as probe for the direct, nonlabeled, detection of complementary strands in a conductive liquid medium. In the present system, the study was focused on a particular codon (273) localized in the exon 8 of the p53 gene (20 mer, TTGAGGTGCATGTTTGTGCC). The integrity of the immobilized probes and its ability to capture target sequences was monitored through chemiluminescent detection following the hybridization of a peroxidase-labeled target. The grafting of the probe at the electrode surface was shown to generate significant shifts of the Nyquist curves measured in the 10-kHz to 80-Hz range. These variations of the faradaic impedance were found to be related to changes of the double layer capacitance of the electrochemical system's equivalent circuit. Similarly, hybridization of complementary strands was monitored through the measurements of these shifts, which enabled the detection of target sequences from 1 to 200 nM. Discrimination between complementary, noncomplementary, and single-nucleotide mismatch targets was easily accomplished.     

Silicon nanowire arrays for label-free detection of DNA

Arrays of highly ordered n-type silicon nanowires (SiNW) are fabricated using complementary metal-oxide semiconductor (CMOS) compatible technology, and their applications in biosensors are investigated. Peptide nucleic acid (PNA) capture probe-functionalized SiNW arrays show a concentration-dependent resistance change upon hybridization to complementary target DNA that is linear over a large dynamic range with a detection limit of 10 fM. As with other SiNW biosensing devices, the sensing mechanism can be understood in terms of the change in charge density at the SiNW surface after hybridization, the so-called "field effect". The SiNW array biosensor discriminates satisfactorily against mismatched target DNA. It is also able to monitor directly the DNA hybridization event in situ and in real time. The SiNW array biosensor described here is ultrasensitive, non-radioactive, and more importantly, label-free, and is of particular importance to the development of gene expression profiling tools and point-of-care applications.     

Open bridge-structured gold nanoparticle array for label-free DNA detection

We focused on changes in the electrical property of the open bridge-structured gold nanoparticles array consisting of 46-nm parent and 12-nm son gold nanoparticles by hybridization and applied it for a simple electrical DNA detection. Since a target DNA of a 24-mer oligonucleotide was added to the probe DNA modified 12-nm Au nanoparticles, which was arranged on the gap between the 46-nm Au particles, the response was read by an electrical readout system. Even in a simple measuring method, we obtained a rapid response to the cDNA with a high S/N ratio of 30 over a wide concentration range and a detection limit of 5.0 fmol. Moreover, the array discriminated 1-base mismatches, regardless of their location in the DNA sequence, which enabled us to detect single-nucleotide polymorphism, which is one of the important diagnoses, without any polymerase chain reaction amplification, sophisticated instrumentation, or fluorescent labeling through an easy-to-handle electrical readout system.     

Semiconductor split-ring resonators for thermally tunable terahertz metamaterials

As the variation of temperature alters the intrinsic carrier density in a semiconductor, numerical simulations indicate that the consequent variation of the relative permittivity in the terahertz regime provides a way to realize thermally tunable split-ring resonators. Electromagnetic metasurfaces and metamaterials that are thermally tunable in the terahertz regime can thus be implemented.     

Optimizing label-free DNA electrical detection on graphene platform

The anodized epitaxial graphene (EG) electrode demonstrates a high level of performance for electrochemical impedance as well as differential pulse voltammetry detection of immobilized DNA and free DNA, respectively, at solid-liquid interfaces. On the anodized EG surface, because of the presence of oxygen functionalities as well as π conjugated domains, the anchoring of the DNA probe can be achieved by either covalent grafting or noncovalent π-π stacking readily. The effect of different binding modes on the sensitivity of the impedimetric sensing was investigated. Equivalent circuit modeling shows that the sensitivity of EG to DNA hybridization is controlled by changes in the resistance of the molecular layer as well as the space charge layer. The linear dynamic detection range of EG for DNA oligonucleotides is in the range of 5.0 × 10(-14) to 1 × 10(-6) M. In addition, with the use of differential pulse voltammetry, single stranded DNA, fully complimentary DNA, as well as single nucleotide polymorphisms can be differentiated on anodized EG by monitoring the oxidation signals of individual nucleotide bases.     

太赫兹裂环谐振器的改进型TL-RLC模型

利用现代光刻技术工艺制备了一种圆形和两种分别在x和y方向伸展的椭圆形裂环谐振器,并在太赫兹时域系统进行测试,比较了尺寸结构参数和耦合系数对幅频特性的影响,提出包含LC谐振、偶极子谐振和第3个谐振的改进TL-RLC模型使模型与实验数据更为吻合。并对第3个谐振频率的表达式进行讨论。     

Indium microrod tags for electrochemical detection of DNA hybridization

The preparation and advantages of indium microrod tracers for solid-state electrochemical detection of DNA hybridization are described. The cylindrical metal particles were prepared by a template-directed electrochemical synthetic route involving plating of indium into the pores of a host membrane. The linear relationship between the charge passed during the preparation and the resulting particle size allows tailoring of the sensitivity of the electrical DNA assay. The resulting micrometer-long rods thus offer a greatly lower detection limit (250 zmol), as compared to common bioassays' spherical nanoparticle tags. Indium offers a very attractive electrochemical stripping behavior and is not normally present in biological samples or reagents. Solid-state derivative-chronopotentiometric measurements of the indium tracer have been realized through a "magnetic" collection of the DNA-linked particle assembly onto a thick-film electrode transducer. Factors affecting the performance, including the preparation of the microrods and pretreatment of the transducer surface, were evaluated and optimized. The resulting protocol offers great promise for other affinity bioassays, as well as for electrical coding and identification (through the plating of different metal markers and of multimetal redox-encoded tags).     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.     

Microphonic sensitivity of surface-acoustic-wave resonators

The shift in resonant frequency due to acceleration has been measured for surface-acoustic-wave resonators (SAWRs). SAWR devices were subjected to vibration from 5 Hz to 10 kHz with peak accelerations ranging from 10(-2) to 10(3) g. The vibration was applied both normal to and in the plane of propagating surface wave yielding microphonic sensitivities on the order of 1 ppb/g. To confirm the results of the direct measurement method, an oscillator loop was closed around the vibrated SAWR and the magnitudes of the FM sidebands were measured to indicate the resulting frequency shift. Several bulk crystal resonators were also measured and it was found that the microphonic sensitivities were generally comparable in magnitude to those of the SAWRs but spanned much wider ranges.     

Direct detection of DNA conformation in hybridization processes

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.     

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.     

Sensitive and label-free DNA methylation detection by ligation-mediated hyperbranched rolling circle amplification

Sensitive and specific detection of DNA methylation in CpG sites of genomic DNA is imperative for rapid epigenetic evaluation and early cancer diagnosis. Here, we employ for the first time the thermostable ligation for methylated DNA discrimination and hyperbranched rolling circle amplification (HRCA) for signal enhancement, without the need for restriction enzymes, PCR amplification, or fluorescence-labeled probes. After bisulfite treatment of methylated DNA, the methylation-specific linear padlock probe can be circularized only in the presence of methylated DNA and serves subsequently as a template for HRCA, whose products are easily detected using SYBR Green I and a standard fluorometer. While in the presence of unmethylated DNA, the linear padlock probe cannot be circularized because of the defectively matched substrate, and no HRCA occurs. This ligation-mediated HRCA-based method exhibits excellent specificity and high sensitivity with a detection limit of 0.8 fM and a detection range of 4 orders of magnitude, and it can even distinguish as low as 0.01% methylation level from the mixture, which is superior to most currently used methods for DNA methylation assay. This method can be further applied to analyze genomic DNA in human lung cancer cells.     

Acceleration sensitivity of surface wave resonators

In light of the substantial performance advantages of STW over SAW in various areas, theoretical and experimental studies of the acceleration sensitivities of STW and SAW resonators have been undertaken. The purpose of the studios has been to understand the fundamental nature of STW and SAW acceleration sensitivities, and to determine whether the performance advantages of STW seen in other areas extend to the case of acceleration sensitivity. The basic approach utilizes the perturbation theory developed by Tiersten to calculate the acceleration sensitivities of both STW and SAW resonators. The acceleration-induced bias is conveniently written in terms of acceleration-induced deformation gradients and factored elastic stiffness expressions. This representation clarifies important concepts regarding the frequency shift and the involved elastic constants, and provides the designer with insight into the basic nature of the problem. The dependencies of the normal acceleration sensitivities on substrate and mode shape parameters and the fundamental nature of plate flexure are discussed at length. The calculations compare favorably to recent experimental results     

Peptide-nucleic acid-modified ion-sensitive field-effect transistor-based biosensor for direct detection of DNA hybridization

Here, we report the development of a peptide-nucleic acid (PNA)-modified ion-sensitive field-effect transistor (IS-FET)-based biosensor that takes advantage of the change in the surface potential upon hybridization of a negatively charged DNA. PNA was immobilized on a silicon nitride gate insulator by an addition reaction between a maleimide group introduced on the gate surface, the succinimide group of N-(6-maleimidocaproyloxy) succinimide, and the thiol group of the terminal cysteine in PNA. The surface was characterized after each step of the reaction by X-ray photoelectron spectroscopy analysis, and the kinetic analysis of the hybridization events was assessed by surface plasmon resonance. In addition, we measured the -potential before and after PNA-DNA hybridization in the presence of counterions to investigate the change in surface charge density at the surface-solution interface within the order of the Debye length. On the basis of the zeta-potential, the surface charge density, DeltaQ, calculated using the Grahame equation was approximately 4.0 x 10(-3) C/m2 and the estimated number of hybridized molecules was at least 1.7 x 10(11)/cm2. The I-V characteristics revealed that the PNA-DNA duplexes induce a positive shift in the threshold voltage, VT, and a decrease in the saturated drain current, ID. These results demonstrate that direct detection of DNA hybridization should be possible using a PNA-modified IS-FET-based biosensor. PNA is particularly advantageous for this system because it enables highly specific and selective binding at low ionic strength.     

Metal nanoparticle-based electrochemical stripping potentiometric detection of DNA hybridization

A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to the bridging of the gold nanoparticles to the magnetic beads. Further signal amplification, and lowering of the detection limits to the nanomolar and picomolar domains, are achieved by precipitating gold or silver, respectively, onto the colloidal gold label. The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.     

Anti-DNA:RNA antibodies and silicon photonic microring resonators: increased sensitivity for multiplexed microRNA detection

In this paper, we present a method for the sensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes and a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22-mer oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 amol). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis as well as a range of other RNA based detection applications.     

Ultrasensitive in situ label-free DNA detection using a GaN nanowire-based extended-gate field-effect-transistor sensor

In this study, we have successfully demonstrated that a GaN nanowire (GaNNW) based extended-gate field-effect-transistor (EGFET) biosensor is capable of specific DNA sequence identification under label-free in situ conditions. Our approach shows excellent integration of the wide bandgap semiconducting nature of GaN, surface-sensitivity of the NW-structure, and high transducing performance of the EGFET-design. The simple sensor-architecture, by direct assembly of as-synthesized GaNNWs with a commercial FET device, can achieve an ultrahigh detection limit below attomolar level concentrations: about 3 orders of magnitude higher in resolution than that of other FET-based DNA-sensors. Comparative in situ studies on mismatches ("hotspot" mutations related to human p53 tumor-suppressor gene) and complementary targets reveal excellent selectivity and specificity of the sensor, even in the presence of noncomplementary DNA strands, suggesting the potential pragmatic application in complex clinical samples. In comparison with GaN thin film, NW-based EGFET exhibits excellent performance with about 2 orders higher sensitivity, over a wide detection range, 10(-19)-10(-6) M, reaching about a 6-orders lower detection limit. Investigations illustrate the unique and distinguished feature of nanomaterials. Detailed studies indicate a positive effect of energy band alignment at the biomaterials-semiconductor hybrid interface influencing the effective capacitance and carrier-mobility of the system.     

Reflective interferometric detection of label-free oligonucleotides

New chip-based methods for the detection of unmodified biomolecular targets have significant potential as enabling technology in fundamental biology and biomedical analysis. We report a method based on changes in reflectivity from specially fabricated substrates that is capable of detecting the binding of as little as an average of 0.2 nm (i.e., a fraction of a monolayer) of biomolecules. We demonstrate the method on detection of femtomole quantities of untagged oligonucleotides in an array format, showing that the amount of target bound can be determined quantitatively. The simplicity of the approach promises to make it broadly applicable for any biomolecule for which suitable molecular recognition chemistry is available.     

PEMC-based method of measuring DNA hybridization at femtomolar concentration directly in human serum and in the presence of copious noncomplementary strands

Piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors having high-mode resonance near 1 MHz are shown to exhibit mass change sensitivity of 1-300 ag/Hz. Gold-coated PEMC sensors immobilized with 15-mer single-stranded DNA (ssDNA) were exposed to 10-mer complementary strands at concentrations of 1 fM, 1 pM, and 1 microM, both separately and sequentially at 0.6 mL/min in a sample flow cell housing the sensor. Decrease in resonance frequency occurred as complementary strands hybridized to the immobilized probe DNA on the sensor surface. Hybridization in three background matrixes--buffer, buffer containing 10,000 times higher noncomplementary strands, and 50% human plasma--were successfully tested. Sensor hybridization responses to 1 fM, 1 pM, and 1 microM complementary strand were nearly the same in magnitude in all three matrixes, but the hybridization rates were different. In each case, the sensor detected the presence of 2 amol of complementary 10-mer strand. The extent of hybridization calculated from resonance frequency change did not decrease in serum. The findings suggest ssDNA can be detected at 2 amol without a sample preparation step and without the use of labeled reagents.