Femtoliter Volumes Research Articles

Nanofilm Processors Controlled by Electrolyte Flows of Femtoliter Volume

Nanofilm processors are a new kind of smart system based on the lateral self-oxidation of nanoscale aluminum films. The time dependency of these devices is controlled by electrolyte flows of femtoliter volume which can be modulated by different mechanisms. In this paper, we provide a deeper investigation of the electrolyte transport in the nanofilm processor and the different possibilities to control the aluminum oxidation velocity. A method for the in situ investigation of the acidic characteristic of the channel electrolyte is demonstrated. The obtained results form a set of instruments for constructing more complex electrolyte circuits and should allow the creation of nanofilm processors of arbitrary time dependence. Because the nanofilm processor combines different functional blocks and can operate in a self-sustained manner, without requiring batteries, this smart system may serve as a basis for many potential applications.

Flexible Approach to Sensors Arrays Nanopatterning for Real-Time Water Contaminants Monitoring Platform

This paper reports on the development of a flexible nanopatterning approach using the NanoeNablerTM to manufacture miniaturised sensor arrays platform for real-time water quality assessment. Traditionally biosensors are fabricated by lithography, screen printing, inkjet printing, spin-or deep-coating methods to immobilize the sensing element onto substrate pre-patterned with electrodes. NanoeNablerTM patterning method is benchmarked against other currently adapted approaches for cost-effective sensors arrays manufacture. Sensors measuring ~1 µm diameter or more can be patterned for further employment in molecularly imprinted polymer structures. Notably, the dimensions of the sensor depend on the fluid being patterned and on the interaction forces between the substrate and the patterning tool. Thus, careful selecting of patterning parameters is vital for repeatable and controlled manufacture of sensors to guarantee superior sensitivity. The reported nanopatterning method is capable of accurately placing attoliter to femtoliter volumes of liquids, including proteins and DNAs, onto any substrate, thus making it an ideal technology for biomedical sensors. A custom-made 1 cm2 silicon wafer with 48 interdigited electrodes sensor heads was used as a platform for the multi-sensor array with potential use in a wide range of real-time monitoring applications.

J212013 ジャイアントリポソームを用いたフェムトリットル溶液操作システム([J212-01]マイクロ・ナノ材料創成とそのデバイス応用(1))

We report the utilization of giant unilamellar vesicles (GUVs) as a platform of handling chemical and biochemical reagents in femtoliter volumes. GUVs with diameter 5 to 10 μm containing chemical reagents together with inert polymers were subjected to electric pulses for fusion (electrofusion). After mixing of reagents, the fused GUV spontaneously deformed to a budding shape, separating the mixed solution into multiple sub-volumes^<(1)>. Here we utilized the microfluidic channel and the optical tweezers to select GUVs in interest, make them in contact, and fuse them together for mixing and dispensing chemical reagents. We also show that, by lowering the ambient temperature close to the phase transition temperature T_m of the lipid used, daughter GUVs completely detached (fission). This process completes all features for liquid handing used in biochemistry at femtoliter volumes, which have been unattained with the previous technologies.

Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems**DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949–2011). He was a valued colleague and friend at the University of Salford and made significant contributions to the development and applications of fluorescence lifetime imaging.

Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps.

Precipitation of ACC in liposomes—a model for biomineralization in confined volumes

Biomineralizing organisms frequently precipitate minerals in small phospholipid bilayer-delineated compartments. We have established an in vitro model system to investigate the effect of confinement in attoliter to femtoliter volumes on the precipitation of calcium carbonate. In particular, we analyze the growth and stabilization of liposome-encapsulated amorphous calcium carbonate (ACC) nanoparticles using a combination of in situ techniques, cryo-transmission electron microscopy (Cryo-TEM), and small angle X-ray scattering (SAXS). Herein, we discuss ACC nanoparticle growth rate as a function of liposome size, carbon dioxide flux across the liposome membrane, pH, and osmotic pressure. Based on these experiments, we argue that the stabilization of ACC nanoparticles in liposomes is a consequence of a low nucleation rate (high activation barrier) of crystalline polymorphs of calcium carbonate.

Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High-Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Embryos and Single Cells

AbstractChemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and clinical diagnosis of viral diseases and cancers.

Electrolyte-Doped Ice as a Platform for Atto- to Femtoliter Reactor Enabling Zeptomol Detection

Rapid freezing of an aqueous electrolyte in liquid nitrogen provides an effective way to fabricate uniform-sized liquid pores with the radius ranging from 0.15 to 3 μm (<1% rsd), corresponding to atto- to femtoliter volumes. The size of liquid pores depends on the temperature, and the concentration and type of a salt incorporated into an original aqueous solution. When the concentration of a salt is kept lower than 20 mM, liquid pores are discretely distributed in an ice matrix. Unlike usual small spaces accommodating liquid water, the pore size is tunable and continuously variable by changing the above experimental parameters. The liquid pore has been utilized as microreactors, in which the fluorescent complexation between Mg(2+) and 8-hydroxyquinoline-5-sulfonic acid (HQS) is studied. Under the optimum condition, fluorescence from Mg(2+) ions in the zeptomol level confined in a liquid pore is detected.

A pore-Cavity-Pore Device to Trap and Investigate Single Nano-Scale Objects in Femto-Liter Compartments: Confined Diffusion and Narrow Escape

Spatial confinement from the nano- to the micro-scale is ubiquitous in nature. Striving to emulate biological compartmentalization and to understand the fundamental physical behavior of molecules in confined domains, micro- and nano-structuring techniques have been used extensively to create artificial devices comprising liquid-filled compartments and channels. In addition, the development of robust solid-state structures, which allow for the observation and manipulation of single nano-scale objects is key for the realization of future lab-on-chip devices with improved functionality. Here we introduce an electrically addressable nano-fluidic silicon device that consists of two stacked nanopores forming the in/outlets to a pyramidal cavity of micrometer dimensions, i.e. femto-liter volume. The electrical properties of the PCP structure are investigated by impedance spectroscopy. Furthermore, we present a FEM simulation of the electric field inside the device. We then demonstrate how individual fluorescent nano-particles and DNA can be actively (by electrical means) and passively (entropically driven) loaded into, trapped inside, and unloaded from the ‘pore-cavity-pore’ (PCP) device. A fundamentally important problem in biology is the escape of nano-objects from a micro-domain through a small opening (narrow escape problem). Using the PCP device it is possible for the first time to obtain data on the narrow escape time under well-defined geometrical and experimental conditions. Single particle tracking and residence time data are presented and quantitatively compared to random walk simulations and analytical theories on confined diffusion and the narrow escape problem. Furthermore, we extend the escape studies towards polymeric analytes like DNA.

Femtoliter and Attoliter Electrochemical Cells on Chips

Chip electrochemistry is one of the top ambitions of today's electrochemistry. Here, a study for manufacturing electrochemical microcells on chips in a cost-effective, facile, and mass-producible way is presented. The ultrasmall, planar electrochemical cells, ranging from 140 femtoliter to 14 attoliter, can work independently as electroanalytical devices with embedded functional microelectrodes. Electrochemical responses of the miniaturized cells have been characterized by cyclic voltammetry. Ideal steady-state voltammograms were recorded with femtoliter volume cells, indicating the domination of a radical diffusion regime and a greatly improved signal/background ratio. Quasi-thin-layer behavior was observed for attoliter volume cells, which exhibited a special capability of offering accurately confined domains for redox processes. Positive feedback effect of the cells indicated that interactions between the close-by working and reference/counter microelectrodes can be well developed and potentially utilized for trace level electroanalysis. This study vividly offers i) a new protocol of electrochemical chip for applications, ii) a new tool for trace electroanalysis, and iii) a more approachable insight for single molecule electrochemistry in the near future.

Use of near infrared femtosecond lasers as sub-micron radiation microbeam for cell DNA damage and repair studies

Laser induced radiation microbeam technology for radiobiology research is undergoing rapid growth because of the increased availability and ease of use of femtosecond laser sources. The main processes involved are multiphoton absorption and/or plasma formation. The high peak powers these lasers generate make them ideal tools for depositing sub-micrometer size radiant energy within a region of a living cell nucleus to activate ionising and/or photochemically driven processes. The technique allows questions relating to the effects of low doses of radiation, the propagation and treatment of deoxyribonucleic acid (DNA) damage and repair in individual live cells as well as non-targeted cell to cell effects to be addressed. This mini-review focuses on the use of near infrared (NIR) ca. 800nm radiation to induce damage that is radically different from the early and subsequent ultraviolet microbeam techniques. Ultrafast pulsed NIR instrumentation has many benefits including the ability to eliminate issues of unspecific UV absorption by the many materials prevalent within cells. The multiphoton interaction volume also permits energy deposition beyond the diffraction limit. Work has established that the fundamental process of the damage induced by the ultrashort laser pulses is different to those induced from continuous wave light sources. Pioneering work has demonstrated that NIR laser microbeam radiation can mimic ionising radiation via multiphoton absorption within the 3D femtolitre volume of the highly focused Gaussian beam. This light-matter interaction phenomenon provides a novel optical microbeam probe for mimicking both complex ionising and UV radiation-type cell damage including double strand breaks (DSBs) and base damage. A further advantage of the pulsed laser technique is that it provides further scope for time-resolved experiments. Recently the NIR laser microbeam technique has been used to investigate the recruitment of repair proteins to the sub-micrometre size area of damage in viable cells using both immuno-fluorescent staining of gamma-H2AX (a marker for DSBs) and real-time imaging of GFP-labelled repair proteins including ATM, p53 binding protein 1 (53BP1), RAD51 and Ku 70/80 to elucidate the interaction of the two DNA DSB repair pathways, homologous recombination and the non-homologous end joining pathway.

Proximity or no proximity: That is the question—But the answer is more complex

THE significance of molecular interactions has been recognized since the dawn of molecular biology, and the real importance of the vast amount of information hidden in the human genome can only be understood if the interactome is also defined. Conventionally, molecular interactions are investigated by chemical crosslinking and coprecipitation. Although these approaches are widely used, they do not provide quantitative information, suffer from the lack of cell-by-cell resolution, and potential artifacts arising from high protein expression and membrane solubilization in the case of membrane proteins. Cytometry offers several alternative methods to assess protein–protein interactions. Fluorescence resonance energy transfer (FRET) has made its way to become the golden standard in quantitative analysis of protein interactions (1). In FRET, a fluorescent donor dye residing on a protein of interest transfers its excitation energy to a nearby acceptor molecule which labels another protein. The rate of this radiationless interaction decreases with the sixth power of the separation distance making FRET a sensitive tool for molecular interactions. In conventional FRET, the pairwise interaction between two spectroscopically distinct molecules is analyzed, and the principle has recently been applied for the simultaneous investigation of two pairwise interactions (2) and for the investigation of the three-way interaction among three molecules (3,4). Although FRET usually takes place between spectroscopically distinct molecules, the same process can also occur between molecules of the same type. This flavor of the interaction is called homo-FRET, and it has been used for the detection of molecular trimers (5) and even larger clusters (6,7). The combination of total internal reflection microscopy with FRET allowed the high-throughput screening of molecular interactions (8). A multitude of correlation techniques are available for the analysis of protein clustering. The mother of all such approaches is fluorescence correlation spectroscopy (FCS) in which the autocorrelation of fluorescence signals is measured in a femtoliter volume to determine the diffusion constant (9,10). FCS has been applied in confocal microscopy for the accurate and quantitative measurement of association states and concentrations in the intracellular space (11,12) and for the monitoring of dynamic properties of membrane proteins (13,14). Fluorescence cross-correlation spectroscopy (FCCS) has been used for the detection of the association of two fluorescently labeled proteins. Dynamic and transient interactions are more conveniently detected by FCCS than by FRET (15). However useful the aforementioned correlation techniques are, they require dedicated instrumentation. The realization that a temporal confocal image series taken by a conventional confocal microscope contains all the necessary information to calculate the diffusion constant and the association state of the fluorescently labeled substance has lead to the development of raster image correlation spectroscopy (RICS) (16) and number and brightness analysis (N&B) (17). Both of these techniques

Predictive model on micro droplet generation through mechanical cutting

We describe the step-by-step formation process of picoliter to femtoliter volume of microdroplets by using a mechanical valve. An experiment-based theoretical model has been proposed through a parametric study of droplet formation in a microchannel with variations of the channel height, the working pressure to drive the liquids in the microchannels, and the viscosity of the dispensed phase. Three steps in the process of droplet generation have been depicted. The present study provides a clear understanding of the droplet generation process based on physical cutting method by using a mechanical valve and could be applied to advanced design for highly flexible droplet-based microfluidic systems.

Zero-Mode Waveguides for Real-Time Observation of Single Nucleotide Incorporation

Single-molecule fluorescence studies on the incorporation of fluorescently labeled nucleotides by DNA polymerizing enzymes typically operate at nucleotide concentrations well below their Km values. While this is inevitable given the femtoliter observation volumes accessed via conventional fluorescence microscopy, the biological relevance of the insights gained into enzymatic kinetics may be compromised. Zero-mode waveguides (ZMWs), sub-wavelength holes in a thin metal film, provide an excellent solution to this problem by greatly reducing the observation volume [M. J. Levene, et al., 2003, Science 299, 682-686].We have successfully designed and fabricated ZMWs of about 100 nm in width. In addition we have developed a surface treatment protocol based on PEG functionalization to make the ZMWs biocompatible, and to facilitate the controlled tethering of biomolecules [A. Crut, et al., Nanotechnology, in press (2008)]. In these structures, we have observed the real-time observation of single nucleotide incorporation at biologically-relevant concentrations in ZMWs, The latest scientific results will be presented.

Nanoscale Corona Discharge in Liquids, Enabling Nanosecond Optical Emission Spectroscopy

My corona! A nonthermal corona discharge inside a liquid medium around electrodes with ultrasharp tips or elongated nanoparticles allows simultaneous chemical analysis of multiple dissolved elements within nanoseconds. The proposed optical emission spectroscopy method can be applied for ultrafast time-resolved multielemental analysis of femtoliter volumes of liquid with a one-micrometer spatial resolution. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z802891_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Controlled electrospray pulsation for deposition of femtoliter fluid droplets onto surfaces

The ability to atomize a liquid sample into femtoliter droplets and deposit them precisely on a surface is a key problem in microfluidics and chemical analysis. Here we show that control of stable oscillations in an unforced electrospray is a high accuracy drop-on-demand method of depositing femtoliter droplets. Examples are presented of a liquid jet, formed for 35 μ s , in a discontinuous spray mode controlled using electrostatic fields of short duration; no liquid pump was employed. Each transient jet ejects femtoliter volumes of material, which was deposited on a nearby surface. The volumes ejected by pulsating sprays on a range of nozzle sizes are predicted from electrospray scaling laws. Using the modified nanoelectrospray method, we have printed 1.4 μ m wide features onto a surface in a drop-on-demand fashion with a placement accuracy of a few micrometers. We anticipate that our technique could produce biological microarrays and precisely deliver ultra-small samples for lab-on-a-chip analysis.

Effects of salinity on the clam Chamelea gallina. Part I: alterations in immune responses

In the present study, the effects of differing salinities on some important functional responses of haemocytes from the clam, Chamelea gallina, were investigated. The animals were kept for 7 days at 28‰ (hyposalinity), 34‰ (control) and 40‰ salinity (hypersalinity), and total haemocyte count (THC), haemocyte volume, phagocytosis, lysozyme-like activity (in both haemocyte lysate and cell-free haemolymph) were measured. The survival-in-air test was also performed. Clams kept at 28‰ showed significantly increased THC with respect to animals kept at 34 and 40‰. The analysis of haemocyte size frequency distribution highlighted that in clams kept at 28‰ the haemocyte fraction of about 5 μm in diameter and 50–100 femtolitre in volume increased markedly. Conversely, in animals kept at 40‰ an increase was observed in the haemocyte fraction having about 8–10 μm diameter and 400–500 femtolitre volume. Higher phagocytic activity was recorded in haemocytes from control clams, with respect to that of clams kept at 28 and 34‰. Lysozyme-like activity in haemocyte lysate was shown to increase significantly in animals kept at 28‰ with respect to that of clams kept at 40‰, whereas enzyme activity in cell-free haemolymph from clams kept at 34‰ was significantly higher with respect to that of clams maintained at 40‰. A relationship between phagocytosis and lysozyme secretion is suggested. The resistance to air exposure of clams kept at 28 and 40‰ was shown to decrease significantly; LT50 values fell from 7 days in clams kept at 34‰ to 4 and 5 days in those kept at 28 and 40‰, respectively. Results demonstrated that salinity values far from 34‰ affects the functional responses of haemocytes and reduce the resistance of clams to exposure to air.

Toward the detection of single virus particle in serum

There is a grand challenge for the detection of target molecules at single molecule sensitivity in a bulk body fluid for the early diagnosis of diseases. We report our progress on tackling this challenge via the combination of fluorescence cross-correlation spectroscopy (FCCS) and micro fabricated devices toward highly sensitive detection of the dengue virus. We demonstrate that by using a dengue-specific antibody, we can probe the individual dengue virus in a nanomolar bulk solution by following the specific association of dengue antibody using FCCS. Consequently, we designed and fabricated a microfluidic chamber array structure and were able to compartmentalize the bulk aqueous dengue sample into femtoliter volumes using such a device. More importantly we demonstrate that we can differentiate between the compartments containing the dengue virus and the virus-free compartments. Our experiment suggests that by expanding the throughput using microfluidic devices integrated with FCCS, both of which can be achieved practically, we should be able to detect single virus particle in human body fluids in the near future.

Optically trapped aqueous droplets for single molecule studies

We demonstrate a technique for creating, manipulating, and combining femtoliter volume chemical containers. The containers are surfactant-stabilized aqueous droplets in a low index-of-refraction fluorocarbon medium. The index-of-refraction mismatch between the container and fluorocarbon is such that individual droplets can be optically trapped by single focus laser beams, i.e., optical tweezers. Here, we trap and manipulate individual droplets, detect the fluorescence from single dye and red fluorescent protein molecules encapsulated in droplets, and observe fluorescence resonance energy transfer from a single dye pair on a deoxyribonucleic acid molecule encapsulated in a droplet.

Characterization of three-dimensional crystallographic distributions using polychromatic X-ray microdiffraction

Abstract Polychromatic microdiffraction uses small X-ray beams to characterize the local crystallographic structure of materials. When combined with a depth resolving technique called differential aperture microscopy, the phase and local orientation of femto-liter volumes (0.5 × 0.5 × 0.7 μm 3 ) can be resolved beneath the surface of a sample. In addition, the local elastic strain and dislocation tensors can also be determined and the local dislocation type can be modeled. Here we present recent technical developments, including example applications and emerging research directions.

Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >105.