Self-assembled biosensor with universal reporter and dual-quenchers for detection of unlabelled nucleic acids.

A novel biosensor with universal reporter and dual quenchers was developed for rapid, sensitive, selective, and inexpensive detection of unlabelled nucleic acids. The biosensor is based on a single-strand DNA stem-loop motif with an extended universal reporter-binding region, a G-base rich stem region, and a universal address-binding region. The self-assembly of these stem-loop probes with fluorescence labeled universal reporter and a universal address region conjugated to gold nanoparticles forms the basis of a biosensor for DNA or microRNA targets in solution. The introduction of dual quenchers (G-base quenching and gold surface plasmon resonance-induced quenching) significantly reduces the fluorescence background to as low as 12% of its original fluorescence intensity and hence enhances the detection limit to 0.01 picomoles without signal ampilication.

Fabrication of MnFe2O4-CuInS2/ZnS Magnetofluorescent Nanocomposites and Their Characterization.

Magnetofluorescent nanocomposites (MFNCs) providing a single nanoscale platform with multimodal properties are gaining momentum in biological manipulation, biomedical imaging and therapy. In this work, we report the preparation of MFNCs integrating MnFe2O4 magnetic nanoparticles (MNPs), CuInS2/ZnS quantum dots (QDs) and poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) in a tetrahydrofuran (THF)/water solvent system. Through sonication and quick solvent displacement, multiple nanoparticles of each type are co-encapsulated within the hydrophobic core of PEG-PLGA micelles. The developed fabrication process is simple and fast. Moreover, due to the low toxicity of CuInS2/ZnS QDs, the fabrication process is environmentally benign. The fabricated MFNCs were further characterized regarding their fundamental physical, chemical and biological properties. Results reveal that the MFNCs possess high (Mn + Fe) recovery rates, and the optical properties and magnetic relaxivity of the MFNCs are sensitive to the MNP:QD mass ratios in the fabrication. Furthermore, the MFNCs present excellent stability in aqueous solutions, minimal cytotoxicity, and capability for bioconjugation. This study opens an avenue for the MFNCs to be employed in broad biological or biomedical applications.

Fluorescence signal transduction mechanism for immunoassay based on zinc ion release from ZnS nanocrystals.

In this work, a fluorescence signal transduction mechanism based on cation release from ZnS nanocrystals was developed for sandwich immunoassay. In this mechanism, ZnS nanocrystals as labels in immunoassay are dissolved by acid to release zinc ions. After pH adjustment of the dissolving solution using a basic solution, zinc-ion sensitive fluorescence indicator Fluozin-3 is added to bind with the released zinc ions for sensitive fluorescence measurement. Using mouse IgG as a model analyte, the immunoassay adopting this signal transduction mechanism demonstrates a low detection limit around 1 pM and a detection range with two orders of magnitude (1 pM to 0.5 nM).

Magnetic bead based assay for C-reactive protein using quantum-dot fluorescence labeling and immunoaffinity separation.

A magnetic bead based sensitive sandwich assay for C-reactive protein (CRP) has been developed. In this assay, magnetic microbeads conjugated with monoclonal anti-CRP are used to capture CRP in human serum, and then sequentially incubated with biotinylated monoclonal anti-CRP and streptavidin coated quantum dots (QDs) to form sandwich immunocomplexes. The immunocomplexes are further mixed with an immunoaffinity separation buffer to release QDs from the magnetic bead surface by dissociating antibody-antigen pairs. After magnetic bead separation, the fluorescence signal of QDs released in the immunoaffinity separation buffer is measured to quantify CRP. This proposed approach takes advantages of magnetic beads for fast magnetic separation, sandwich binding of two monoclonal antibodies for high specificity, and QD fluorescence labeling for high detection sensitivity. Moreover, it adopts immunoaffinity separation to avoid the interference of magnetic bead autofluorescence in signal transduction and thus enhances the detection resolution of the assay. The detection limit of CRP is 0.5 fM, corresponding to 10 pM CRP in 50 microL of sample volume, and the detection range is of around 3 orders of magnitude.

Compatibility of quantum dots with immunobuffers, and its effect on signal/background of quantum dot-based immunoassay.

In this work, the compatibility of quantum dots (QDs) with immunobuffers was studied by investigating the fluorescence stability of QDs in immunobuffers (in this research immunobuffers were defined as buffers for immunoaffinity binding or separation). Experimentally, the fluorescence signals of QDs with different surface chemistries (amine-terminated, streptavidin-coated, or antibody-conjugated) in commonly used immunobuffers were monitored versus time. The effect of some buffer composition on the compatibility of QDs with these buffers was also explored. Based on experimental data, the QD compatibility with these buffers is summarized, and it is found that a trace amount of bovine serum albumin added to most of these buffers helps QDs to achieve compatibility with them. Moreover, with QD as fluorescence label and C-reactive protein as a model analyte, a magnetic bead-based assay was performed using compatible and incompatible QD-immunobuffer systems. It is shown that compatible QD-immunobuffer systems can be used to achieve a higher assay signal/background ratio.

Thermal Decomposition Based Synthesis of Ag-In-S/ZnS Quantum Dots and Their Chlorotoxin-Modified Micelles for Brain Tumor Cell Targeting.

Cadmium-free silver-indium-sulfide (Ag-In-S or AIS) chalcopyrite quantum dots (QDs) as well as their core-shell structures (AIS/ZnS QDs) are being paid significant attention in biomedical applications because of their low toxicity and excellent optical properties. Here we report a simple and safe synthetic system to prepare high quality AIS and AIS/ZnS QDs using thermal decomposition. The synthetic system simply involves heating a mixture of silver acetate, indium acetate, and oleic acid in dodecanethiol at 170 °C to produce AIS QDs with a 13% quantum yield (QY). After ZnS shell growth, the produced AIS/ZnS QDs achieve a 41% QY. To facilitate phase transfer and bioconjugation of AIS/ZnS QDs for cellular imaging, these QDs were loaded into the core of PLGA-PEG (5k:5k) based micelles to form AIS/ZnS QD-micelles. Cellular imaging studies showed that chlorotoxin-conjugated QD-micelles can be specifically internalized into U-87 brain tumor cells. This work discloses that the scalable synthesis of AIS/ZnS QDs and the facile surface/interface chemistry for phase transfer and bioconjugation of these QDs may open an avenue for the produced QD-micelles to be applied to the detection of endogenous targets expressed on brain tumor cells, or more broadly to cell- or tissue-based diagnosis and therapy.

Facilitated preparation of bioconjugatable zwitterionic quantum dots using dual-lipid encapsulation.

Zwitterionic quantum dots prepared through incorporated zwitterionic ligands on quantum dot surfaces, are being paid significant attention in biomedical applications because of their excellent colloidal stability across a wide pH and ionic strength range, antifouling surface, good biocompatibility, etc. In this work, we report a dual-lipid encapsulation approach to prepare bioconjugatable zwitterionic quantum dots using amidosulfobetaine-16 lipids, dipalmitoyl-sn-glycero-3-phosphoethanolamine lipids with functional head groups, and CuInS2/ZnS quantum dots in a tetrahydrofuran/methanol/water solvent system with sonication. Amidosulfobetaine-16 is a zwitterionic lipid and dipalmitoyl-sn-glycero-3-phosphoethanolamine, with its functional head, provides bioconjugation capability. Under sonication, tetrahydrofuran/methanol containing amidosulfobetaine-16, dipalmitoyl-sn-glycero-3-phosphoethanolamine, and hydrophobic quantum dots are dispersed in water to form droplets. Highly water-soluble tetrahydrofuran/methanol in droplets is further displaced by water, which induces the lipid self-assembling on hydrophobic surface of quantum dots and thus forms water soluble zwitterionic quantum dots. The prepared zwitterionic quantum dots maintain colloidal stability in aqueous solutions with high salinity and over a wide pH range. They are also able to be conjugated with biomolecules for bioassay with minimal nonspecific binding.

Numerical study of induced current perturbations in the vicinity of excitable cells exposed to extremely low frequency magnetic fields.

Realistic three-dimensional cell morphologies were modelled to determine the current density induced in excitable cell culture preparations exposed to 60 Hz magnetic fields and to identify important factors that can influence the responses of cells to these fields. Cell morphologies representing single spherical adrenal chromaffin cells, single elongated smooth muscle cells and chromaffin cell aggregates in a Petri dish containing culture medium were modelled using the finite element method. The computations for a spherical cell revealed alterations in the magnitude and spatial distribution of the induced current density in the immediate vicinity of the cell. Maxima occurred at the equatorial sides and minima at the poles. Proximity of cells to each other as well as cell aggregate shape, size and orientation with respect to the induced current influenced the magnitude and spatial distribution of the induced current density. For an elongated cell, effects on the induced current density were highly dependent on cell orientation with respect to the direction of the induced current. These results provide novel insights into the perturbations in induced current that occur in excitable cell culture preparations and lay a foundation for understanding the mechanisms of interaction with extremely low frequency magnetic fields at the tissue level.

Catecholamine release from cultured bovine adrenal medullary chromaffin cells in the presence of 60-Hz magnetic fields.

Effects of powerline frequency (50/60 Hz) electric and magnetic fields on the central nervous system may involve altered neurotransmitter release. This possibility was addressed by determining whether 60-Hz linearly polarized sinusoidal magnetic fields (MFs) alter the release of catecholamines from cultured bovine adrenal chromaffin cells, a well-characterized model of neural-type cells. Dishes of cells were placed in the center of each of two four-coil Merritt exposure systems that were enclosed within mu-metal chambers in matched incubators for simultaneous sham and MF exposure. Following 15-min MF exposure of the cells to flux densities of 0.01, 0.1, 1.0 or 2 mT, norepinephrine and epinephrine release were quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. No significant differences in the release of either norepinephrine or epinephrine were detected between sham-exposed cells and cells exposed to MFs in either the absence or presence of Bay K-8644 (2 microM) or dimethylphenylpiperazinium (DMPP, 10 microM). Consistent with these null findings is the lack of effect of MF exposure on calcium influx. We conclude that catecholamine release from chromaffin cells is not sensitive to 60-Hz MFs at magnetic flux densities in the 0.01-2 mT range.

A polymer encapsulation approach to prepare zwitterion-like, biocompatible quantum dots with wide pH and ionic stability.

A surface modification approach adopting polymer encapsulation was developed to prepare zwitterion-like quantum dots (ZWL-QDs). The fundamental physical, chemical, and biological properties of the ZWL-QDs were characterized. It is found that the ZWL-QDs almost preserve the quantum yield (QY) of native hydrophobic QDs in organic solvents, and also are compact in size (7 ~ 10 nm hydrodynamic diameter) and stable over wide pHs or in high salinity solutions. Further cellular study shows that the ZWL-QDs with a concentration less than 100 nM have a minimal cytotoxicity and thus are biocompatible. Characterizing and understanding these essential properties of the ZWL-QDs are an important step before employing them for various applications.

Cadmium and Zinc Alloyed Cu-In-S Nanocrystals and Their Optical Properties.

Cadmium (Cd) and zinc (Zn) alloyed copper-indium-sulfide (Cu-In-S or CIS) nanocrystals (NCs) in several nanometers were prepared using thermal decomposition methods, and the effects of Cd and Zn on optical properties, including the tuning of NC photoluminescence (PL) wavelength and quantum yield (QY), were investigated. It was found that incorporation of Cd into CIS enhances the peak QY of NCs whereas zinc alloying diminishes the peak. In contrast with Zn alloying, Cd alloying does not result in a pronounced luminescence blue shift. The further PL decay study suggests that Cd alloying reduces surface or intrinsic defects whereas alloying with Zn increases the overall number of defects.

Video imaging system for automated shaping and analysis of complex locomotory behavior.

Although many observational technologies have been developed for the study of behavior, most of these technologies have suffered from the inability to engender highly reproducible behaviors that can be observed and modified. We have developed ACROBAT (Automated Control in Real-Time of Operant Behavior and Training), a video imaging system and associated computer algorithms that allow the fully automated shaping and analysis of complex locomotory behaviors. While this operant conditioning system is particularly useful for measuring the acquisition and maintenance of complex topographies, it also provides a more general and user friendly platform on which to develop novel paradigms for the study of learning and memory in animals. In this paper we describe the instrumentation and software developed, demonstrate the use of ACROBAT to shape a specific topography, and show how the system can be used to facilitate the study of arthritic pain in mice.

Dynamic and quasi-static lung mechanics system for gas-assisted and liquid-assisted ventilation.

Our aim was to develop a computerized system for real-time monitoring of lung mechanics measurements during both gas and liquid ventilation. System accuracy was demonstrated by calculating regression and percent error of the following parameters compared to standard device: airway pressure difference (Delta P(aw)), respiratory frequency (f(R) ), tidal volume (V(T)), minute ventilation (V'(E)), inspiratory and expiratory maximum flows (V'(ins,max), V'(exp,max)), dynamic lung compliance (C(L,dyn) ), resistance of the respiratory system calculated by method of Mead-Whittenberger (R(rs,MW)) and by equivalence to electrical circuits (R(rs,ele)), work of breathing (W(OB)), and overdistension. Outcome measures were evaluated as function of gas exchange, cardiovascular parameters, and lung mechanics including mean airway pressure (mP(aw)). Delata P(aw), V(T), V'(ins,max), V'(exp,max), and V'(E) measurements had correlation coefficients r = 1.00, and %error < 0.5%. f(R), C(L,dyn), R(rs,MW), R(rs,ele), and W(OB) showed r > or = 0.98 and %error < 5%. Overdistension had r = 0.87 and %error < 15%. Also, resistance was accurately calculated by a new algorithm. The system was tested in rats in which lung lavage was used to induce acute respiratory failure. After lavage, both gas- and liquid-ventilated groups had increased mP(aw) and W(OB), with decreased V(T), V'(E), C(L,dyn), R(rs,MW), and R(rs,ele) compared to controls. After 1-h ventilation, both injured group had decreased V(T), V'(E) , and C(L,dyn), with increased mP(aw), R(rs,MW), R(rs,ele), and W(OB) . In lung-injured animals, liquid ventilation restored gas exchange, and cardiovascular and lung functions. Our lung mechanics system was able to closely monitor pulmonary function, including during transitions between gas and liquid phases.

Stem-loop probe with universal reporter for sensing unlabeled nucleic acids.

In this article, we present the design principles and application of a motif composed of a stem-loop probe (SP) hybridized to a fluorescently labeled universal reporter (UR) for sensing unlabeled nucleic acids. At room temperature, SP-UR is in the hairpin-closed form in which the fluorophore of UR is in proximity to the G bases of the hairpin, where consequently the fluorescent emission is quenched significantly. On hybridization with target, SP-UR is trapped in the hairpin-opened configuration in which the fluorophore and the G quenchers are apart. This turns off quenching, increases emission intensity, and signals the presence of target. Compared with the common approach that employs an oligonucleotide probe with a covalently linked fluorophore, the use of a fluorescently labeled universal reporter strand hybridized to an unlabeled stem-loop probe provides a more efficient approach to the fabrication of nucleic acid sensors and microarrays potentially useful for real-time analysis.

Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields.

This study examined whether 60 Hz magnetic field (MF) exposure alters intracellular calcium levels ([Ca(2+)](i)) in isolated bovine adrenal chromaffin cells, a classic model of neural responses. [Ca(2+)](i) was monitored by fluorescence video imaging of cells loaded with the calcium indicator fluo-4 during exposures to magnetic flux densities of 0.01, 0.1, 1.0, 1.4, or 2.0 mT. MFs generated by Helmholtz coils constructed from bifilar wire allowed both 60 Hz field and sham exposures. Following a 5 min monitoring period to establish baseline patterns, cells were subjected for 10 min to a 60 Hz MF, sham field or no field. Reference calcium responses and assessment of cell excitability were obtained by the sequential addition of the nicotinic cholinergic receptor agonist dimethylphenylpiperazinium (DMPP) and a depolarizing concentration of KCl. Throughout an 8 day culture period, cells exhibited spontaneous fluctuations in [Ca(2+)](i). Comparisons of the number of cells exhibiting transients, the number and types of calcium transients, as well as the time during monitoring when transients occurred showed no significant differences between MF exposed cells and either sham exposed or nonexposed cells. With respect to the percentage of cells responding to DMPP, differences between 1 and 2 mT exposed cells and both nonexposed and sham exposed cells reached statistical significance during the first day in culture. No statistically significant differences were observed for responses to KCl. In summary, our data indicate that [Ca(2+)](i) in chromaffin cells is unaffected by the specific 60 Hz MF intensities used in this study. On the other hand, plasma membrane nicotinic receptors may be affected in a manner that is important for ligand-receptor interactions.