Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.     

Flow velocity detector in a microchip based on a photothermally induced grating.

A new photothermal technique was developed for measuring the flow velocity and making solute concentration measurements in a microchip by using the same optical and instrumental setup. Collinear pump and probe light were irradiated onto a microchip surface on which a grating pattern was fabricated. The pump light induced a temperature change with the grating pattern in a microchannel, and a refractive index change due to a subsequent temperature rise was monitored by a heterodyned diffraction signal of the probe light. The flow velocity and concentration were obtained by monitoring the motion and intensity change of the thermally induced grating, respectively. The dynamic range of the flow velocity measurement was 0.17 - 670 mm/s, which is sufficient for covering most chemical applications of a microchip. The detection limit of the concentration measurement was 2 x 10(-6) M for a rhodamine B solution.     

Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Subsecond chiral separations on a microchip

Fast chiral separation of DNS-amino acids could be realized using microchip electrophoresis with fluorescence detection. For this purpose, highly sulfated cyclodextrins (HS-gamma-CD) were used as chiral selectors enabling high selectivity. Even subsecond separation of DNS-tryptophan, DNS-norleucine, DNS-phenylalanine, DNS-methionine, and DNS-aspartic acid could be achieved. Baseline separation could be accomplished within 720 ms, which is the fastest separation of enantiomers reported to date. A more complex mixture consisting of three chiral DNS-amino acids could be separated within 3.3 s utilizing a separation length of only 7 mm and an electrical field strength of 2012 V/cm.     

Field-inversion electrophoresis on a microchip device

We have proposed a field-inversion electrophoresis on a microchip for a shorter effective length, and investigated the external frequency for the DNA analysis based on the field-inversion electrophoresis device. By using the optimized frequency, we demonstrated that the field-inversion electrophoresis has great potentials for the separation of DNA fragments with shorter effective length.     

Integration of multiple-ion-sensing on a capillary-assembled microchip

Multiple-ion-sensing functions are integrated on a capillary-assembled microchip (CAs-CHIP). Since the CAs-CHIPs are fabricated by embedding various chemically functionalized square capillaries onto a lattice PDMS channel plate having same channel dimensions as outer dimensions of square capillaries, integration of parallel multiple-ion-sensing is easily realized. Here, three ion-sensing capillaries are prepared and used for integrating these functions onto a single microchip. Ion-sensing square capillaries (sodium, potassium, calcium) are prepared by attaching ion-selective optode membranes to inner wall of capillaries, and are characterized in terms of response time, response range, and ion selectivity. Finally, fully characterized ion-sensing capillaries are embedded into PDMS channel plate in parallel to fabricate a multiple-ion-sensing chip. The CAs-CHIP-based strategy is promising for integrating multiple chemical sensing functions onto a single microchip.     

Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.     

Nanoparticle-based substrates for surface-enhanced Raman scattering detection of bacterial spores

The development of ultrasensitive and rapid methods for the detection of bacterial spores is important for medical diagnostics of infectious diseases. While Surface-Enhanced Raman Spectroscopic (SERS) techniques have been increasingly demonstrated for achieving this goal, a key challenge is the development of sensitive and stable SERS substrates or probes. This Minireview highlights recent progress in exploring metal nanoparticle-based substrates, especially gold nanoparticle-based substrates, for the detection of biomarkers released from bacterial spores. One recent example involves assemblies of gold nanoparticles on a gold substrate for the highly sensitive detection of dipicolinic acid (DPA), a biomarker for bacterial spores such as Bacillus anthracis. This type of substrate exploits a strong SERS effect produced by the particle-particle and particle-substrate plasmonic coupling. It is capable of accurate speciation of the biomarker but also selective detection under various reactive or non-reactive conditions. In the case of detecting Bacillus subtilis spores, the limit of detection is quite comparable (0.1 ppb for DPA, and 1.5 × 10(9) spores per L (or 2.5 × 10(-14) M)) with those obtained using silver nanoparticle-based substrates. Implications of the recent findings for improving the gold nanoparticle-based SERS substrates with ultrahigh sensitivity for the detection of bacterial spores are also discussed.     

Laser induced breakdown spectroscopy on meteorites

The classification of meteorites when geological analysis is unfeasible is generally made by the spectral line emission ratio of some characteristic elements. Indeed when a meteorite impacts Earth's atmosphere, hot plasma is generated, as a consequence of the braking effect of air, with the consequent ablation of the falling body. Usually, by the plasma emission spectrum, the meteorite composition is determined, assuming the Boltzmann equilibrium. The plasma generated during Laser Induced Breakdown Spectroscopy (LIBS) experiment shows similar characteristics and allows one to verify the mentioned method with higher accuracy. On the other hand the study of Laser Induced Breakdown Spectroscopy on meteorite can be useful for both improving meteorite classification methods and developing on-flight techniques for asteroid investigation.

In this paper certified meteorites belonging to different typologies have been investigated by LIBS: Dofhar 461 (lunar meteorite), Chondrite L6 (stony meteorite), Dofhar 019 (Mars meteorite) and Sikhote Alin (irony meteorite).     

Hydroxyapatite nanoparticle based fluorometric turn-on determination of dipicolinic acid,a biomarker of bacterial spores

Hydroxyapatite nanoparticles (HAP-NPs) were rendered fluorescence by doping with Eu(III) ion. The resulting fluorescent NPs are shown to be viable probes for sensitive and selective determination of dipicolinic acid (DPA), a major constituent of bacterial spores as used in bioterrorism. It is found that the addition of DPA to solutions of such HAP-NPs result in an enhancement of fluorescence due to the coordination of DPA with the Eu(III) dopant. The assay allows DPA to be detected in the 0.1 to 40 μM concentration range and with a 77 nM detection limit. The assay was applied to the detection of spores of Bacillus subtilis. The attractive properties of the probe make it a promising candidate for used in rapid detection of pathogenic bacterial spores.

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Graphical abstract Fluorescent hydroxyapatite nanoparticles (HAP-NPs) are shown to be a viable probe for detection of dipicolinic acid, a major constituent of bacterial spores. The red asterisks represent the fluorescence intensity of the HAP-NPs.

     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

Pulsed microchip laser induced fluorescence for in situ tracer experiments

A compact system for remote and non intrusive in situ analysis of fluorescent tracers using a newly developed pulsed microchip laser coupled to fiber optics was used for in situ rhodamine determinations. By using a crystal doubling in front of the microchip Nd-YAG laser, it is possible to obtain 532 nm at 5 kHz with an energy of 0.6 μJ in a 0.5 ns pulse. Using fiber optics and a passive optode, it was possible to analyze remotely the fluorescence of rhodamine with a compact detection system (monochromator and photomultiplier). Limits of detection down to 10^–10–10^–11 mol/L can be reached depending on the rhodamine studied. Such a laser can be directly implanted in the optode avoiding laser losses when exciting in the U.V. Received: 30 July 1997 / Revised: 6 October 1997 / Accepted: 10 October 1997     

Integrated multilayer flow system on a microchip.

We utilized microchip technology and found that the multilayer flow of liquids can be formed in microchannels. Liquid/liquid interfaces were formed parallel to the side wall of the microchannels, because the surface tension and friction force are stronger than the force of gravity. A water/ethylacetate/water interface was formed in a 70-microm-wide and 30-microm-deep channel. The interface was observed to be quite stable and to be maintained for a distance of more than 18 cm. As an example of a multilayer flow application, we demonstrated the liquid/liquid extraction of Co-dimethylaminophenol complex in a microchannel. The solvent-extraction process of the complex into m-xylene in the multilayer flow was found to reach equilibrium in 4 s, while it took 60 s in a simple two-phase extraction.     

Pulsed microchip laser induced fluorescence for in situ tracer experiments

A compact system for remote and non intrusive in situ analysis of fluorescent tracers using a newly developed pulsed microchip laser coupled to fiber optics was used for in situ rhodamine determinations. By using a crystal doubling in front of the microchip Nd-YAG laser, it is possible to obtain 532 nm at 5 kHz with an energy of 0.6 μJ in a 0.5 ns pulse. Using fiber optics and a passive optode, it was possible to analyze remotely the fluorescence of rhodamine with a compact detection system (monochromator and photomultiplier). Limits of detection down to 10^–10–10^–11 mol/L can be reached depending on the rhodamine studied. Such a laser can be directly implanted in the optode avoiding laser losses when exciting in the U.V. Received: 30 July 1997 / Revised: 6 October 1997 / Accepted: 10 October 1997     

Infection control by antibody disruption of bacterial quorum sensing signaling

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.     

激光诱导荧光检测微流控芯片毛细管电泳分离多肽的研究

微流控分析系统是分析科学及分析仪器重要的发展前沿,是90年代初发展起来的微分析系统的主要组成部分及目前较为活跃的领域.将微流控分析系统应用于电泳分离,与传统的电泳分离手段相比较而言,具有微型化、可集成化、速度快、进样量小等特点.它的检测方法有多种,其中激光诱导荧光(Laser induced fluorescence,LIF)法因其灵敏度高,成为微流控芯片分析检测目前最广为采用的方法[1].     

Capillary electrophoresis on microchip

Capillary electrophoresis and related techniques on microchips have made great strides in recent years. This review concentrates on progress in capillary zone electrophoresis, but also covers other capillary techniques such as isoelectric focusing, isotachophoresis, free flow electrophoresis, and micellar electrokinetic chromatography. The material and technologies used to prepare microchips, microchip designs, channel geometries, sample manipulation and derivatization, detection, and applications of capillary electrophoresis to microchips are discussed. The progress in separation of nucleic acids and proteins is particularly emphasized.