Analytical-scale microwave-assisted extraction

Microwave-assisted extraction (MAE) is a process of using microwave energy to heat solvents in contact with a sample in order to partition analytes from the sample matrix into the solvent. The ability to rapidly heat the sample solvent mixture is inherent to MAE and the main advantage of this technique. By using closed vessels the extraction can be performed at elevated temperatures accelerating the mass transfer of target compounds from the sample matrix. A typical extraction procedure takes 15-30 min and uses small solvent volumes in the range of 10-30 ml. These volumes are about 10 times smaller than volumes used by conventional extraction techniques. In addition, sample throughput is increased as several samples can be extracted simultaneously. In most cases recoveries of analytes and reproducibility are improved compared to conventional techniques, as shown in several applications. This review gives a brief theoretical background of microwave heating and the basic principles of using microwave energy for extraction. It also attempts to summarize all studies performed on closed-vessel MAE until now. The influences of parameters such as solvent choice, solvent volume, temperature, time and matrix characteristics (including water content) are discussed.     

Computer aided thermal analysis

Inverse numerical techniques have been applied in a range of different thermal studies in the past. These techniques require measurements of boundary conditions and temperatures at known position within the sample in order to determine thermal properties of the material of interest. Typically, they have been applied to highly specific applications and designs. In the current work the authors have designed a novel instrument in order to measure apparent specific heats of a range of different materials during continuous heating. Measurements of surface heat flux, surface and centre temperatures of the sample were obtained under controlled heating for temperatures of up to 1000°C. Measured data was used to quantify specific and latent heats by employing inverse numerical modelling technique. The instrument was calibrated with calorimetric calibration materials and results were compared with the literature values. The average experimental error was estimated to be approximately 0.9% for the reaction peak temperatures and 1.7% for the latent heats. Detailed experimental and calculation procedures as well as measured results of specific heat and enthalpy for a number of materials are presented here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spectrophotometric determination of osmium with ethylisobutrazine hydrochloride

The sheath flow cuvette is used for refractive index determinations of neat solutions within picoliter probe volumes. In this detector, a sample stream is injected as a narrow stream into the center of a flowing sheath stream under laminar flow conditions. The sample stream retains its identity as a thin cylinder through the center of a 250-m square flow chamber. The propagation properties of a focused helium-neon laser are perturbed by interaction with the sample stream. Detection limits of RI=3×10^–6 were obtained within a 20 micrometer radius sample stream, corresponding to about a 400 picoliter probe volume. For analyte with refractive index significantly different than the solvent, detection limits are possible which correspond to a few picograms of analyte within the probe volume.     

Conformational fluctuations and large fluorescence spectral diffusion in conjugated polymer single chains at low temperatures

The fluorescence of single chains of the conductive polymer poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylene vinylene] (MEH-PPV) was studied by means of single-molecule spectroscopy at 15 K. MEH-PPV was deposited onto a surface from a toluene solution and covered with a polymer cap layer of poly(vinyl alcohol) spin-coated from an aqueous solution for protection against air. Because MEH-PPV is insoluble in water, such sample preparation guarantees that MEH-PPV chains do not mix with the cap polymer. We found that this "host matrix free" environment results in substantially stronger fluorescence spectral diffusion than that observed for conjugated polymer single chains embedded into polymer matrices. The average spectral diffusion range was 500 cm(-1), and the maximum registered value reached 1100 cm(-1), which is approximately 6 times larger than the values reported before. We analyzed spectral diffusion by observation of temporal evolution of the fluorescence intensity, the position of the maximum, and the width of fluorescence spectra. We propose that the transition energy shifts are caused by the differences of the London dispersive forces in slightly different polymer chain conformations. Such conformational changes are possible even at low temperatures because the MEH-PPV single chains in our samples have more freedom for fluctuations than in the usual "in host" arrangement.     

Analytical detection techniques for droplet microfluidics—A review

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.     

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.     

Parallel mixing of photolithographically defined nanoliter volumes using elastomeric microvalve arrays

Portable microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed a monolithic polydimethylsiloxane (PDMS) microdevice that allows for storing and mixing subnanoliter volumes of aqueous solutions at various mixing ratios. Filling and mixing is controlled via two integrated PDMS microvalve arrays. The volumes of the microchambers are entirely defined by photolithography, hence volumes from picoliter to nanoliter can be fabricated with high precision. Because the microvalves do not require an energy input to stay closed, fluid can be stored in a highly portable fashion for several days. We have confirmed the mixing precision and predictability using fluorescence microscopy. We also demonstrate the application of the device for calibrating fluorescent calcium indicators. Due to the biocompatibility of PDMS, the device will have broad applications in miniaturized diagnostic assays as well as basic biological studies.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

Kinetics of thermo-induced micelle-to-vesicle transitions in a catanionic surfactant system investigated by stopped-flow temperature jump

The kinetics of thermo-induced micelle-to-vesicle transitions in a catanionic surfactant system consisting of sodium dodecyl sulfate (SDS) and dodecyltriethylammonium bromide (DEAB) were investigated by the stopped-flow temperature jump technique, which can achieve T-jumps within ～2-3 ms. SDS/DEAB aqueous mixtures ([SDS]/[DEAB] = 2/1, 10 mM) undergo microstructural transitions from cylindrical micelles to vesicles when heated above 33 °C. Upon T-jumps from 20 °C to final temperatures in the range of 25-31 °C, relaxation processes associated with negative amplitudes can be ascribed to the dilution-induced structural rearrangement of cylindrical micelles and to the dissolution of non-equilibrium mixed aggregates. In the final temperature range of 33-43 °C the obtained dynamic traces can be fitted by single exponential functions, revealing one relaxation time (τ) in the range of 82-440 s, which decreases with increasing temperature. This may be ascribed to the transformation of floppy bilayer structures into precursor vesicles followed by further growth into final equilibrium vesicles via the exchange and insertion/expulsion of surfactant monomers. In the final temperature range of 45-55 °C, vesicles are predominant. Here T-jump relaxations revealed a distinctly different kinetic behavior. All dynamic traces can only be fitted with double exponential functions, yielding two relaxation times (τ(1) and τ(2)), exhibiting a considerable decrease with increasing final temperatures. The fast process (τ(1)～ 5.2-28.5 s) should be assigned to the formation of non-equilibrium precursor vesicles, and the slow process (τ(2)～ 188-694 s) should be ascribed to their further growth into final equilibrium vesicles via the fusion/fission of precursor vesicles. In contrast, the reverse vesicle-to-micelle transition process induced by a negative T-jump from elevated temperatures to 20 °C occurs quite fast and almost completes within the stopped-flow dead time (～2-3 ms).     

Reverse Intersystem Crossing of Single Deuterated Perylene Molecules in a Dibenzothiophene Matrix

Intersystem crossing to the long-lived metastable triplet state is often a strong limitation on fluorescence brightness of single molecules, particularly for perylene in various matrices. In this paper, we report on a strong excitation-induced reverse intersystem crossing (rISC), a process where single perylene molecules in a dibenzothiophene matrix recover faster from the triplet state, turning into bright emitters at saturated excitation powers. With a detailed study of single-molecule fluorescence autocorrelations, we quantify the effect of rISC. The intrinsic lifetimes found for the two effective triplet states (8.5±0.4 ms and 64±12 ms) become significantly shorter, into the sub-millisecond range, as the excitation power increases and fluorescence brightness is ultimately enhanced at least fourfold. Our results are relevant for the understanding of triplet state manipulation of single-molecule quantum emitters and for markedly improving their brightness.     

Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly(N-isopropyl acrylamide)

In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 degrees C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide-dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.     

A New Theoretical Approach to Steady State of Temperature Modulated Heat Flux DSC

The steady state of temperature modulated heat flux DSC, in which the sample temperature is controlled at a fixed frequency, a fixed amplitude and a constant underlying heating rate, is theoretically investigated for complex heat capacity of the sample, taking accounts of heat capacities of heat paths, heat loss to the environment and mutual heat exchange between the sample and the reference material. Rigorous and general solutions for the temperature difference oscillation are obtained in relation to the sample temperature as a reference oscillation. The results are quite different from those obtained in functions of the heat source temperature as a reference oscillation. From these solutions, application of the technique to heat capacity measurements is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Large-bore coated columns for sampling and concentration of organic volatiles in air, headspace and water analysis

Large-bore coated (LBC) columns were used as sampling and concentrating traps in analyses for traces of organic volatiles in air and water. This simple technique utilizes long metal columns thinly coated with SE-30 for direct trapping of the organics. The sample is simply passed through the LBC column; the trapped organics are then thermally desorbed onto a conventional porous polymer pre-column or onto a second LBC column. If desired, this can be shorter or narrower bore than the initial LBC sampling column. The sample is finally desorbed onto the gas chromatographic column for analysis. Multiple transfers between LBC columns are possible, with increased concentration at each transfer, resulting in a "concentration pump" effect. The technique offers the advantages of great simplicity, efficiency and ease of sample transfer. Samples are obtained with low back-pressure and minimal interfering artifacts. The system shows almost complete imperturbability to moisture. Indifference to moisture and the low back-pressure enable direct sampling of very large volumes of air and even breath. Direct sampling of aqueous systems was also possible. The latter area was not fully investigated but offers potential for water pollution analysis and in direct examination of biological fluids and aqueous flavor extracts where heat sensitivity is a problem. With LBC columns the sampling and concentration sequence exposes the substances sought to no more drastic conditions than those they will be subjected to in the process of gas chromatographic analysis.     

Enthalpic relaxation of convective desiccated trehalose-water glasses

Minimizing molecular mobility for desiccation preservation of biologics close to ambient temperature using trehalose glasses require quantitative characterization of their enthalpic relaxation at various end water contents. Differential scanning calorimetry (DSC) was used to characterize three different water contents: 0%, 1.5% and 10% over a wide range of aging temperatures. Results showed the characteristic time (τ) varies both with the water content and the aging temperature. τ increased with lowered aging temperature but showed a non-monotonous relationship as a function of water content. Fragility of trehalose glasses was analyzed using thermophysical parameters obtained from relaxation studies. The study showed trehalose to be a fragile glass former at all water contents, with 0% water samples showing a relatively stronger glass. A compromise between molecular mobility and glass fragility led to an optimal water content close to 1.5% and an aging temperature close to room temperature. This would ensure a τ value of 9000 h, which corresponds to a storage period of a year.     

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.     

Effect of the enthalpy,sample size and heating rate on the cure reaction of rubber in dsc

As a continuation of the recent studies devoted to the determination of profiles of temperature and state of cure developed through the sample when heated in a calorimeter working in scanning mode, the effect of the enthalpy of reaction, of rather low value, on these profiles was investigated. The effect of the cure enthalpy on the heat flux emitted through the sample-calorimeter interface was especially studied, for various heating rates and various sample sizes. Moreover, the effect of a change of this cure enthalpy on the heat flux and profiles of temperatures and state of cure was determined and reported. These results were obtained not only by experiments in case of the size of the sample available for our calorimeter, but also by calculation with the help of a numerical method with finite differences. This model took into account heat transferred by conduction and the heat generated by the cure reaction.     

Improvement of Fluorescence Lifetime from Er-Doped Sol-Gel Silica Glass by Dehydration in CCl4

Monolithic Er-doped silica xerogels with erbium content of 5000 ppm were prepared by sol-gel technique. Samples were densified by thermal treatment in O₂/CCl₄ environment. The 1.55 m photoluminescence lifetimes after annealing at different temperatures were measured, together with the relative content of hydroxyl groups inside the samples. It was found that hydroxyl groups can be removed by CCl₄ effectively. Consequently the photoluminescence lifetime of the samples increased significantly after the removal of hydroxyl groups. For sample treated in O₂/CCl₄ at 900°C, fluorescence lifetime as long as 6ms was obtained and is pretty stable when the sample is kept in ambient air.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

Sealed and temperature-controlled sample cell for inverted and confocal microscopes and fluorescence correlation spectroscopy

Precise temperature control of sample environments plays a key role while exploring biological systems or temperature-sensitive materials. We have developed a sample cell for inverted microscopes, which allows a temperature accuracy of ±0.05 K in a temperature range of 5 to 65 °C, with an absolute precession of ±0.1 K. Our sample cell is developed for requirements of single-molecule experiments, which comprises easy-to-clean and well-sealed devices to prevent solvent evaporation. The applied control algorithm permits a tunable independent setting of heat and cooling behavior and allows the application on microscopes without any objective heating. For measuring precise and absolute diffusion coefficients with two-focus fluorescence correlation spectroscopy, the exact control of the sample temperature is essential. We performed diffusion measurements of TetraSpeck 100-nm fluorescent latex particles and of temperature-sensitive microgels in aqueous solutions to demonstrate the excellent temperature stability and reproducibility of the device.     

Local heating of discrete droplets using magnetic porous silicon-based photonic crystals

This paper describes a method for local heating of discrete microliter-scale liquid droplets. The droplets are covered with magnetic porous Si microparticles, and heating is achieved by application of an external alternating electromagnetic field. The magnetic porous Si microparticles consist of two layers. The top layer contains a photonic code and it is hydrophobic, with surface-grafted dodecyl moieties. The bottom layer consists of a hydrophilic silicon oxide host layer that is infused with Fe3O4 nanoparticles. The amphiphilic microparticles spontaneously align at the interface of a water droplet immersed in mineral oil, allowing manipulation of the droplets by application of a magnetic field. Application of an oscillating magnetic field (338 kHz, 18 A rms current in a coil surrounding the experiment) generates heat in the superparamagnetic particles that can raise the temperature of the enclosed water droplet to >80 degrees C within 5 min. A simple microfluidics application is demonstrated: combining complementary DNA strands contained in separate droplets and then thermally inducing dehybridization of the conjugate. The complementary oligonucleotides were conjugated with the cyanine dye fluorophores Cy3 and Cy5 to quantify the melting/rebinding reaction by fluorescence resonance energy transfer (FRET). The magnetic porous Si microparticles were prepared as photonic crystals, containing spectral codes that allowed the identification of the droplets by reflectivity spectroscopy. The technique demonstrates the feasibility of tagging, manipulating, and heating small volumes of liquids without the use of conventional microfluidic channel and heating systems.