Single-quantum-dot-based DNA nanosensor

Rapid and highly sensitive detection of DNA is critical in diagnosing genetic diseases. Conventional approaches often rely on cumbersome, semi-quantitative amplification of target DNA to improve detection sensitivity. In addition, most DNA detection systems (microarrays, for example), regardless of their need for target amplification, require separation of unhybridized DNA strands from hybridized stands immobilized on a solid substrate, and are thereby complicated by solution-surface binding kinetics. Here, we report an ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) capable of detecting low concentrations of DNA in a separation-free format. This system uses quantum dots (QDs) linked to DNA probes to capture DNA targets. The target strand binds to a dye-labelled reporter strand thus forming a FRET donor-acceptor ensemble. The QD also functions as a concentrator that amplifies the target signal by confining several targets in a nanoscale domain. Unbound nanosensors produce near-zero background fluorescence, but on binding to even a small amount of target DNA (approximately 50 copies or less) they generate a very distinct FRET signal. A nanosensor-based oligonucleotide ligation assay has been demonstrated to successfully detect a point mutation typical of some ovarian tumours in clinical samples.     

Fiber-optic hydrogen peroxide nanosensor

A fiber-optic sensor sensitive to hydrogen peroxide has been designed based on the electrostatic layer-by-layer self-assembly method. Meldola's blue and a catalyst hemin have been deposited in a polymeric structure formed by PAH+ and PAA/sup -/. The concentrations that can be detected range at least between 10/sup -7/ and 10/sup -1/ M, and recovery of the sensor after introduction in a reductive agent has been proved successfully. Some rules for estimation of the refractive index of the material deposited and the thickness of bilayers are also given.     

8-aminoquinoline functionalized silica nanoparticles: a fluorescent nanosensor for detection of divalent zinc in aqueous and in yeast cell suspension

Zinc is one of the most important transition metal of physiological importance, existing primarily as a divalent cation. A number of sensors have been developed for Zn(II) detection. Here, we present a novel fluorescent nanosensor for Zn(II) detection using a derivative of 8-aminoquinoline (N-(quinolin-8-yl)-2-(3 (triethoxysilyl)propylamino)acetamide (QTEPA) grafted on silica nanoparticles (SiNPs). These functionalized SiNPs were used to demonstrate specific detection of Zn(II) in tris-HCl buffer (pH 7.22), in yeast cell (Saccharomyces cerevisiae) suspension, and in tap water. The silane QTEPA, SiNPs and final product were characterized using solution and solid state nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption spectroscopy, transmission electron microscopy, elemental analysis, thermogravimetric techniques, and fluorescence spectroscopy. The nanosensor shows almost 2.8-fold fluorescence emission enhancement and about 55 nm red-shift upon excitation with 330 ± 5 nm wavelength in presence of 1 μM Zn(II) ions in tris-HCl (pH 7.22). The presence of other metal ions has no observable effect on the sensitivity and selectivity of nanosensor. This sensor selectively detects Zn(II) ions with submicromolar detection to a limit of 0.1 μM. The sensor shows good applicability in the determination of Zn(II) in tris-HCl buffer and yeast cell environment. Further, it shows enhancement in fluorescence intensity in tap water samples.     

Synthesis and characterization of ZnO nanowires for nanosensor applications

In this paper we report the synthesis of ZnO nanowires via chemical vapor deposition (CVD) at 650 °C. It will be shown that these nanowires are suitable for sensing applications. ZnO nanowires were grown with diameters ranging from 50 to 200 nm depending on the substrate position in a CVD synthesis reactor and the growth regimes. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), photoluminescence (PL), and Raman spectroscopy (RS) have been used to characterize the ZnO nanowires. To investigate the suitability of the CVD synthesized ZnO nanowires for gas sensing applications, a single ZnO nanowire device (50 nm in diameter) was fabricated using a focused ion beam (FIB). The response to H₂ of a gas nanosensor based on an individual ZnO nanowire is also reported.     

An integrated superconductive magnetic nanosensor for high-sensitivity nanoscale applications

An integrated magnetic nanosensor based on a niobium dc SQUID (superconducting quantum interference device) for nanoscale applications is presented. The sensor, having a washer shape with a hole of 200?nm and two Josephson-Dayem nanobridges of 80?nm × 100?nm, consists of a Nb(30?nm)/Al(30?nm) bilayer patterned by electron beam lithography (EBL) and shaped by lift-off and reactive ion etch (RIE) processes. The presence of the niobium coils, integrated on-chip and tightly coupled to the SQUID, allows us to easily excite the sensor in order to get the voltage-flux characteristics and to flux bias the SQUID at its optimal point. The measurements were performed at liquid helium temperature. A voltage swing of 75?μV and a maximum voltage-flux transfer coefficient (responsivity) as high as 1?mV/Φ(0) were directly measured from the voltage-flux characteristic. The noise measurements were performed in open loop mode, biasing the SQUID with a dc magnetic flux at its maximum responsivity point and using direct-coupled low-noise readout electronics. A white magnetic flux noise spectral density as low as 2.5?μΦ(0)?Hz(-1/2) was achieved, corresponding to a magnetization or spin sensitivity in units of the Bohr magneton of 100?spin?Hz(-1/2). Possible applications of this nanosensor can be envisaged in magnetic detection of nanoparticles and small clusters of atoms and molecules, in the measurement of nanoobject magnetization, and in quantum computing.     

Folate conjugated fluorescent silica nanoparticles for labeling neoplastic cells

We describe a novel technique of using fluorescent silica nanoparticles (FSNPs) to detect over-expressed folate receptors, as typical for certain malignancies (metastatic adenocarcinoma, pituitary adenoma and others). Using St?ber's method with some modification, 135 nm size FSNPs were synthesized by a hydrolysis and co-condensation reaction of tetraethylorthosilicate (TEOS), fluorescein labeled (3-aminopropyl)triethoxysilane (APTS) and a water-dispersible silane reagent, (3-trihydroxysilyl)propyl methylphosphonate (THPMP) in the presence of ammonium hydroxide catalyst. Folic acid (folate) was covalently attached to the amine modified FSNPs by a carbodiimide coupling reaction. The characterization of folate-FSNPs was performed using a variety of spectroscopic (UV-VIS and fluorescence), microscopic (transmission electron microscopy, TEM) and light scattering techniques. Folate conjugated FSNPs were then targeted to human squamous cancer cells (SCC-9). Laser scanning confocal images successfully demonstrated the labeling of SCC-9 cells and the efficacy of FSNP based detection system.     

Electrochemical nanosensor for real-time direct imaging of nitric oxide in living brain

As gaseous nitric oxide (NO), a critical and multifaceted biomarker, diffuses easily once released, identifying the precise sources of NO release is a challenge. This study developed a new technique for real-time in vivo direct NO imaging by coupling an amperometric NO nanosensor with scanning electrochemical microscopy. This technique provides three-dimensional information of the NO releasing sites in an intact living mouse brain with high sensitivity and spatial resolution. Immunohistochemical analysis was carried out to confirm the anatomical reliability of the acquired electrochemical NO image. The real-time NO imaging results were well matched with the corresponding immunohistochemical analysis of neuronal NO synthase immunoreactive (nNOS-IR) cells, i.e., NO releasing sites in a living brain. The imaged NO local concentrations were confirmed to be closely related to the location in depth, the size of the nNOS-IR cell, and the intensity of nNOS immunoreactivity. This paper demonstrates the first direct electrochemical NO imaging of a living brain.     

Computational modeling of a carbon nanotube-based DNA nanosensor

During the last decade the design of biosensors, based on quantum transport in one-dimensional nanostructures, has developed as an active area of research. Here we investigate the sensing capabilities of a DNA nanosensor, designed as a semiconductor single walled carbon nanotube (SWCNT) connected to two gold electrodes and functionalized with a DNA strand acting as a bio-receptor probe. In particular, we have considered both covalent and non-covalent bonding between the DNA probe and the SWCNT. The optimized atomic structure of the sensor is computed both before and after the receptor attaches itself to the target, which consists of another DNA strand. The sensor's electrical conductance and transmission coefficients are calculated at the equilibrium geometries via the non-equilibrium Green's function scheme combined with the density functional theory in the linear response limit. We demonstrate a sensing efficiency of 70% for the covalently bonded bio-receptor probe, which drops to about 19% for the non-covalently bonded one. These results suggest that a SWCNT may be a promising candidate for a bio-molecular FET sensor.     

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.     

Quantifying RNA-peptide interaction by single-quantum dot-based nanosensor: an approach for drug screening

The sequence-specific RRE RNA-Rev binding is essential for HIV-1 replication and provides a useful in vitro system for real-time evaluating the inhibitory effect of drugs on the RRE-Rev interaction. The rapid and sensitive detection of RRE-Rev interaction in complex biological systems represents a fundamental challenge. Here we report the development of a single-quantum-dot (QD)-based nanosensor for sensitively quantifying Rev peptide-RRE interaction and characterizing the potential inhibitors by virtue of single-molecule detection and QD-based fluorescence resonance energy transfer (FRET). We demonstrate that the stoichiometry of Rev peptide binding to RRE can be accurately determined by using this single-QD-based nanosensor. Importantly, this single-QD-based nanosensor can sensitively quantify the inhibitory efficacy of proflavin on the Rev peptide-RRE binding, even in the presence of substantial levels of interference fluorescence from high-concentration proflavin, which usually prevents the discrimination of FRET signals in ensemble measurements. The application of this nanosensor in the screening of libraries of small-molecule drugs will facilitate the development of new drugs against various diseases, cancers, and HIV.     

A new yellow fluorescent dopant for high-efficiency organic light-emitting devices

We describe the design and synthesis of a sterically hindered yellow dopant, tetra(t-butyl)rubrene (TBRb) which, when doped in either 1,4-bis[N-(1-naphthyl)-N′-phenylamino]-biphenyl or aluminum tris(8-hydroxyquinoline) (Alq₃) as emitter, shows nearly 25% increase in luminance efficiency over that of the state-of-the-art rubrene (Rb) device without significantly effecting its corresponding color. At 5% doping in Alq₃ and 20 mA/cm² current drive condition, the electroluminescence efficiency of TBRb reaches 8.5 cd/A and 2.8 lm/W with a yellow color of Commission Internationale de l'Eclairage chromaticity coordinates (CIE_x,y) = [0.52, 0.48], which is among the best ever reported for yellow electrofluorescence in organic light-emitting devices.     

A fluorescent chemosensor for sodium based on photoinduced electron transfer

A new optical sensor suitable for practical measurement of sodium in serum and whole blood samples is described. The optical sensor is based on a novel PET (photoinduced electron transfer) fluoroionophore immobilized in a hydrophilic polymer layer. The design concept of the fluoroionophore follows the receptor-spacer-fluorophore approach to sensor design using intramolecular PET-based signal transduction. Key to the development of this sensor is the identification of a nitrogen-containing, sodium-binding ionophore, coupled with a fluorophore having the correct spectral and electron-accepting properties. The slope of the sensor is approximately 0.5%/mM in the typical clinically significant range of 120-160 mM. This sensor has been implemented into a disposable cartridge, used for a commercially available critical care analyzer (Roche OPTI CCA) with precision better than +/- 1 mM (1 SD). The sensor displays excellent stability against hydrolysis and oxidation, leading to slope changes <5% after 9 months wet storage at 30 degrees C. On the basis of this design concept, fluoroionophores for other cations such as potassium, calcium and magnesium can be prepared by substitution of the ionophore.     

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways.

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling is described. Upon binding of insulin to cell-surface insulin receptor, the receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthetic phosphopeptide pY939 derived from the tyrosine phosphorylation domain of IRS-1 and its target protein SH2N containing an N-terminal SH2 domain of PI 3-kinase. The SH2N protein and pY939 phosphopeptide were labeled with fluorescein (F-SH2N) and tetramethylrhodamine (T-pY939), respectively. Formation of a F-SH2N-T-pY939 complex (termed a fluorescence resonance energy-transfer (FRET) pair) was evaluated from a change in a fluorescence emission spectrum based on FRET between the two fluorophores. The FRET pair was formed to dissociate in competition with the unlabeled pY939 phosphopeptide, resulting in a decrease in a pY939 phosphopeptide-dependent FRET emission at 580 nm and causing an increase in emission at 520 nm. Tyrosine phosphorylation by the partially purified insulin receptor of substrate peptide Y939 was detected with this formed FRET pair, and resulting changes in fluorescence emission spectra were observed for insulin concentration from about 1.0 x 10(-9) to 1.0 x 10(-6) M. These results indicated that the FRET pair served as a competitive fluorescent indicator for tyrosine phosphorylation-based insulin signaling.     

A fluorescent carbazole derivative: high sensitivity for quadruplex DNA

We have synthesized a novel molecule, 3,6-bis(1-methyl-4-vinylpyridium)carbazole diiodide (BMVC), for recognizing specific quadruplex structures, particularly the quadruplex of human telomeric sequence d(T(2)AG(3))(4). The fluorescence intensity of the BMVC molecule increases from 1 to almost 2 orders of magnitude upon interacting with various DNAs. At a concentration of BMVC of 10 microM, fluorescence bands with different colors of BMVC in electrophoresis gels of various DNAs can be observed. The fluorescence of BMVC can be used to discriminate between duplex and quadruplex DNAs. At the low concentration of 0.1 microM BMVC in prestained gels, the fluorescence is observed in the presence of quadruplexes with anti-anti-anti-anti and anti-anti-syn-syn arrangements. However, no fluorescence band is detected upon interacting with duplexes and quadruplexes with anti-syn-anti-syn arrangement. Moreover, the sensitivity assays show that as little as 0.2 pmol of quadruplex of d(T(2)AG(3))(4) can be revealed by BMVC.     

Sensitive detection of microRNA with isothermal amplification and a single-quantum-dot-based nanosensor

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. Here we develop a rapid, highly sensitive, and specific miRNA assay based on the two-stage exponential amplification reaction (EXPAR) and a single-quantum-dot (QD)-based nanosensor. The two-stage EXPAR involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide. Importantly, different miRNAs can be converted to the same reporter oligonucleotides, which can hybridize with the same set of capture and reporter probes to form sandwich hybrids. These sandwich hybrids can be assembled on the surface of 605 nm emission QDs (605QDs) to form the 605QD/reporter oligonucleotide/Cy5 complexes, where the 605QD functions as both a fluorescence resonance energy transfer donor and a target concentrator. Upon excitation with a wavelength of 488 nm, distinct Cy5 signals can be observed in the presence of target miRNA. This assay is highly sensitive and specific with a detection limit of 0.1 aM and can even discriminate single-nucleotide differences between miRNA family members. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same set of capture and reporter probes. This highly sensitive and specific assay has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.     

A genetically encoded fluorescent indicator capable of discriminating estrogen agonists from antagonists in living cells

A genetically encoded fluorescent indicator was developed for the detection and characterization of estrogen agonists and antagonists. Two different color mutants of green fluorescent protein were joined by a tandem fusion domain composed of LXXLL (L = leucine, X = any amino acid) motif from the nuclear receptor-box II of steroid receptor coactivator 1, a flexible linker sequence, and the estrogen receptor alpha ligand binding domain (ERalpha LBD). Monitoring real-time ligand-induced conformational change in the ERalpha LBD to recruit the LXXLL motif allowed screening of natural and synthetic estrogens in single living cells using fluorescence resonance energy-transfer technique. The indicator was named SCCoR (single cell-coactivator recruitment). The high sensitivity of the present indicator made it possible to distinguish between estrogen strong and weak agonists in a dose-dependent fashion, immediately after adding ligand to live cells. Discrimination of agonists from antagonists was efficiently achieved using the present study. The approach described here can be applied to develop biosensors for other hormone receptors as well.     

Optical nanosensor platform operating in near-physiological pH range via polymer-brush-mediated plasmon coupling

The nanosensors' platform made of a stimuli-responsive polymer/noble metal nanoparticle composite thin film exploits the combination of the swelling-shrinking transition in a poly(N,N'-dimethylaminoethyl methacrylate) brush and the localized surface plasmon resonance in metal nanoparticles to enable the transduction of changes in the solution pH in the near-physiological range into a pronounced optical signal.     

Differentiation of prostate cancer cells using flexible fluorescent polymers

Using water-soluble, fluorescent, flexible polymers, we have devised a novel methodology for identification and differentiation of prostate cancer cells. Using a stepwise linear discriminant analysis, we demonstrate that the differential modulations of the polymer emission intensities in the presence of conditioned cell culture media can be used to distinguish between prostate cancer subtypes and between cancerous and noncancer cells. The differences in the compositions of the conditioned cell culture media are likely contributing to different fluorescence spectral patterns of the polymers. This in vitro approach may provide a novel platform for the development of an alternative prostate cancer diagnostic and subtyping technique.     

Charge-driven selective localization of fluorescent nanoparticles in live cells

Covalent grafting of amino groups onto the carboxylic acid functionalities, naturally covering the surface of fluorescent nanoparticles produced from silicon carbide (SiC NPs), allowed tuning of their surface charge from negative to highly positive. Incubating 3T3-L1 fibroblast cells with differently charged SiC NPs demonstrates the crucial role of the charge in cell fluorescent targeting. Negatively charged SiC NPs concentrate inside the cell nuclei. Close to neutrally charged SiC NPs are present in both cytoplasm and nuclei while positively charged SiC NPs are present only in the cytoplasm and are not able to move inside the nuclei. This effect opens the door for the use of SiC NPs for easy and fast visualization of long-lasting biological processes taking place in the cell cytosol or nucleus as well as providing a new long-term cell imaging tool. Moreover, here we have shown that the interaction between charged NPs and nuclear pore complex plays an essential role in their penetration into the nuclei.