Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

有关铜锌原电池的问题讨论

针对铜锌原电池中锌电极冒气泡的“异常”现象,通过分析不同类型原电池的工作原理,认为即使使用纯度极高的锌也会产生气泡,并给出其缘由。对在教学中如何处理这一棘手的难题,提出个人见解。同时,建议中学化学教材将“锌片上产生气泡”的现象明确地描述出来。     

Electrokinetically driven fluidic transport in integrated three-dimensional microfluidic devices incorporating gold-coated nanocapillary array membranes

Electrokinetically driven fluid transport was evaluated within three-dimensional hybrid nanofluidic-microfluidic devices incorporating Au-coated nanocapillary array membranes (NCAMs). Gold NCAMs, prepared by electroless gold deposition on polymeric track-etched membranes, were susceptible to gas bubble formation if the interfacial potential difference exceeded approximately 2 V along the length of the gold region. Gold membranes were etched to yield 250 mum wide coated regions that overlap the intersection of two orthogonal microfluidic channels in order to minimize gas evolution. The kinetics of electrolysis of water at the opposing ends of the gold region was modeled and found to be in satisfactory agreement with experimental measurements of the onset of gas bubble formation. Conditions to achieve electrokinetic injection across Au-coated NCAMs were identified, with significant reproducible injections being possible for NCAMs modified with this relatively thin gold stripe. Continuous gold films led to suppressed injections and to a variety of ion enrichment/depletion effects in the microfluidic source channel. The suppression of injections was understood through finite element modeling which revealed the presence of a significant electrophoretic velocity component in opposition to electroosmotic flow at the edge of the Au-dielectric regions.     

Label-free quantitation of peptide release from neurons in a microfluidic device with mass spectrometry imaging

Microfluidic technology allows the manipulation of mass-limited samples and when used with cultured cells, enables control of the extracellular microenvironment, making it well suited for studying neurons and their response to environmental perturbations. While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides for off-line coupling to microfluidic devices for characterizing small-volume extracellular releasates, performing quantitative studies with MALDI is challenging. Here we describe a label-free absolute quantitation approach for microfluidic devices. We optimize device fabrication to prevent analyte losses before measurement and then incorporate a substrate that collects the analytes as they flow through a collection channel. Following collection, the channel is interrogated using MS imaging. Rather than quantifying the sample present via MS peak height, the length of the channel containing appreciable analyte signal is used as a measure of analyte amount. A linear relationship between peptide amount and band length is suggested by modeling the adsorption process and this relationship is validated using two neuropeptides, acidic peptide (AP) and α-bag cell peptide [1-9] (αBCP). The variance of length measurement, defined as the ratio of standard error to mean value, is as low as 3% between devices. The limit of detection (LOD) of our system is 600 fmol for AP and 400 fmol for αBCP. Using appropriate calibrations, we determined that an individual Aplysia bag cell neuron secretes 0.15 ± 0.03 pmol of AP and 0.13 ± 0.06 pmol of αBCP after being stimulated with elevated KCl. This quantitation approach is robust, does not require labeling, and is well suited for miniaturized off-line characterization from microfluidic devices.     

Charge-injection device detection for improved performance in atomic-emission spectroscopy

Studies into the use of simultaneous multiwavelength detection over a broad wavelength region (220-520 nm) demonstrate the power and flexibility offered by a charge-injection device for detection in atomic-emission spectrometry. An echelle monochromator and a charge-injection device utilizing the general electric CID17B array detector are used in conjunction with a direct current plasma source to perform multi-line analysis for Mg, Sr, Fe, Dy, Ho and Yb, increasing the sensitivity and limits of detection. By monitoring the 341-nm OH band and background Ar emission lines, changes in the nebulization and excitation conditions are easily detected. The presence of an organic matrix component not present in the standards is detected by observing the C(I) emission at 247.8 nm. These diagnostic tools can be combined with the use of an internal standard to obtain a reliability not previously available in automated AES instrumentation.     

Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.     

Hyphenation of capillary separations with nuclear magnetic resonance spectroscopy

The hyphenation of small-volume separations to information-rich detection offers the promise of unmatched analytical information on the components of complex mixtures. Nuclear magnetic resonance (NMR) spectroscopy provides information about molecular structure, although sensitivity remains an issue for on-line NMR detection. This is especially true when hyphenating NMR to capillary separations as the observation time and analyte mass are decreased to the point where reduced information is obtained from the eluting analytes. Because of these limitations, advances in instrumental performance have a large impact on the overall performance of a separation–NMR system. Instrumental aspects and the capabilities of cLC–NMR, CEC–NMR and CE–NMR are reviewed, and applications that have used this technology highlighted. Recent trends towards small volume capillary scale separations are emphasized, as is the recent success of capillary-isotachophoresis (cITP)–NMR.     

Profiling pH gradients across nanocapillary array membranes connecting microfluidic channels

Nanocapillary array membranes (NCAMs), comprised of thin (d approximately 5-10 microm) nuclear track-etched polycarbonate sheets containing approximately 10(8) cm(-2) nearly parallel nanometer-diameter capillaries, may act to gate fluid transport between microfluidic channels to effect, for example, sample collection. There is interest in H+-transport across these NCAMs because there is significant practical interest in being able to process analyte-containing samples under different pH conditions in adjacent layers of an integrated microfluidic circuit and because protons, with their inherently high mobility, present a challenge in separating microfluidic environments with different properties. To evaluate the capability of NCAMs to support pH gradients, the proton transport properties of NCAMs were studied using laser scanning confocal fluorescence microscopy (LSCFM). Spatiotemporal maps of [H+] in microfluidic channels adjacent to the NCAMs yield information regarding diffusive and electrokinetic transport of protons. The NCAMs studied here are characterized by a positive zeta potential, zeta > 0, so at small nanocapillary diameters, the overlap of electrical double layers associated with opposite walls of the nanocapillary establish an energy barrier for either diffusion or electrokinetic transport of cations through the nanometer-diameter capillaries due to the positive charge on the nanocapillary surface. Proton transfer through an NCAM into microchannels is reduced for pore diameters, d < or = 50 nm and ionic strengths I < or = 50 mM, while for large pore diameters or solution ionic strengths, the incomplete overlap of electric double layer allows more facile ionic transfer across the membranes. These results establish the operating conditions for the development of multilevel integrated nanofluidic/microfluidic architectures which can support multidimensional chemical analysis of mass-limited samples requiring sequential operations to be implemented at different pH values.     

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleurobranchaea californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at –20 °C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites. Received: 23 August 2000 / Revised: 2 November 2000 / Accepted: 7 November 2000