Nanoliter Range Research Articles

Inkjet printing-based photo-induced electron transfer reaction on parchment paper using riboflavin as a photosensitizer

In this study, we report the photo-induced electron transfer (PET) on parchment paper using riboflavin as a photo inducer and ultraviolet lamp (362 nm) as the light source. To this end, a conventional inkjet printer equipped with 4 cartridges was used. Parchment paper was found to be a favorable substrate due to its insignificant self-absorption while assisting efficient sample interaction. Upon UV-irradiation, riboflavin generated superoxide anion radical (O2-·) and it was available to interact with superoxide dismutase present on the same spot. A decrease in NBT formazan in the reaction spot indicates increased O2-· scavenging activity of molecule. It was estimated that the well-plate based-colorimetric method used 12.5 μM (1.25 × 10-9 mole) of riboflavin and 0.25 mM (2.50 × 10-8 mole) of NBT to react with different superoxide dismutase or drug concentrations, while the printing technique consumed 3.19 × 10-13 mole of NBT and 2.98 × 10-13 mole of riboflavin to react with gradient superoxide dismutase or drug concentration. In contrast to the conventional well plate method, inkjet printing-based molecular assay provides automatic delivery in the nanoliter range with precise time, which ensures four-to five-order lower reagent consumption. The inkjet printing-based quantitative measurement specifies the amount of a particular drug/enzyme printed on a surface. Therefore, applicability of inkjet printing technique conjoined radical scavenging assay will be more competent to determine the radical scavenging potential of natural plant products. In addition, this inkjet printing approach offers easy, fast, cost-effective, and less time-consuming method to determine PET reaction on paper.

A Generic Polycarbonate Based Microfluidic Tool to Study Crystal Nucleation in Microdroplets

Crystal nucleation is important to control the product properties in industrial crystallization processes. To investigate crystallization phenomena, methods which rely on microscopic volumes have gained relevance over the last decade. Microfluidic devices are suitable for carrying out crystallization experiments based on a large set of individual droplets in the nanoliter range. In this work, we propose a simple method to manufacture such devices from polycarbonate as an alternative to conventional chips made of poly (dimethylsiloxane). The microfluidic device consists of two main functional parts: A T-junction for droplet generation and a section for storage and observation of up to 400 individual droplets. Using these manufactured devices, it is easy to produce and store highly monodisperse droplets of substances that require either a hydrophilic or hydrophobic surface of the microchannel. Since crystal nucleation is a stochastic process which depends on the sample volume, a reproducible droplet volume is of great importance for crystallization experiments. The versatile applicability of the manufactured devices is demonstrated for substances which are used in different crystallization applications, for example, solution crystallization (aqueous potassium nitrate solution) and melt crystallization (ethylene glycol distearate). Finally, we demonstrate that the manufactured microfluidic devices in our experimental setup can be used to conduct crystal nucleation measurements. Based on these measurements we discuss our results with respect to state-of-the-art nucleation models.

Design and Validation of a 150 MHz HFFQCM Sensor for Bio-Sensing Applications.

Acoustic wave resonators have become suitable devices for a broad range of sensing applications due to their sensitivity, low cost, and integration capability, which are all factors that meet the requirements for the resonators to be used as sensing elements for portable point of care (PoC) platforms. In this work, the design, characterization, and validation of a 150 MHz high fundamental frequency quartz crystal microbalance (HFF-QCM) sensor for bio-sensing applications are introduced. Finite element method (FEM) simulations of the proposed design are in good agreement with the electrical characterization of the manufactured resonators. The sensor is also validated for bio-sensing applications. For this purpose, a specific sensor cell was designed and manufactured that addresses the critical requirements associated with this type of sensor and application. Due to the small sensing area and the sensor’s fragility, these requirements include a low-volume flow chamber in the nanoliter range, and a system approach that provides the appropriate pressure control for assuring liquid confinement while maintaining the integrity of the sensor with a good base line stability and easy sensor replacement. The sensor characteristics make it suitable for consideration as the elemental part of a sensor matrix in a multichannel platform for point of care applications.

Design of planar microcoil-based NMR probe ensuring high SNR

A microNMR probe for ex vivo applications may consist of at least one microcoil, which can be used as the oscillating magnetic field (MF) generator as well as receiver coil, and a sample holder, with a volume in the range of nanoliters to micro-liters, placed near the microcoil. The Signal-to-Noise ratio (SNR) of such a probe is, however, dependent not only on its design but also on the measurement setup, and the measured sample. This paper introduces a performance factor P independent of both the proton spin density in the sample and the external DC magnetic field, and which can thus assess the performance of the probe alone. First, two of the components of the P factor (inhomogeneity factor K and filling factor η) are defined and an approach to calculate their values for different probe variants from electromagnetic simulations is devised. A criterion based on dominant component of the magnetic field is then formulated to help designers optimize the sample volume which also affects the performance of the probe, in order to obtain the best SNR for a given planar microcoil. Finally, the P factor values are compared between different planar microcoils with different number of turns and conductor aspect ratios, and planar microcoils are also compared with conventional solenoids. These comparisons highlight which microcoil geometry-sample volume combination will ensure a high SNR under any external setup.

Experimental Characterization and Simulation of a Piezo-Actuated Micro Dispensing Valve

The dispensing behavior of a piezo-actuated micro-valve that closes the gap between the nanoliter range (e.g., inkjet technology) and the microliter range (e.g., standard displacement technology) has been investigated by experimental and numerical means. Water and different Newtonian model fluids with defined fluid properties were utilized for experimental characterization. The dispensed amount per single dispensing event could be freely adjusted from a few nanoliters to several hundred microliters showing the large working range and flexibility of the micro-valve, while maintaining a high accuracy with a low relative standard deviation. A correlation between fluid properties, dispensing parameters, and the resulting steady-state mass flow was established, showing good consistency of the experimental data. Furthermore, a three-dimensional numerical model for the quantitative simulation of the micro-valve's dispensing behavior regarding fluid mass flow was developed and validated, showing a high degree of correspondence between the experiments and simulations. Investigations of the transient behavior after the opening of the micro-valve revealed a nonlinear relationship between the valve opening time and dispensed mass for short opening times. This behavior was dependent on the working pressure but independent of the type of fluid.

Long Fragment Read (LFR) Technology: Cost-Effective, High-Quality Genome-Wide Molecular Haplotyping.

In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10-9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process.

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

Precise small volume sample handling for capillary electrophoresis.

CE is one of the most important analytical techniques. Although the injected sample volume in CE is only in the nanoliter range, most commercial CE instruments need approximately 50 μL of the sample in the injection vial to perform the analysis. Hence, in order to fully profit from the low injection volumes, smaller vial volumes are required. Thus experiments were performed using silicone oil, which has higher density than water (1.09 g/mL) to replace sample dead volume in the vial. The results were compared to those performed without using the silicone oil in the sample vial. As an example five standard proteins namely beta-lactoglobulin, BSA, HSA, myoglobin, and ovalbumin, and one of the coagulation cascade involved proteins called vitonectin were investigated using CE. Mobility ratios and peak areas were compared. However, no significant changes were observed (RSDs% for mobility ratios and peak areas were better than 0.9 and 5.8%, respectively). Afterwards, an affinity CE method was used to investigate the interactions of two proteins, namely HSA and vitronectin, with three ligands namely enoxaparin sodium, unfractionated heparin, and pentosan polysulfate sodium. Mobility shift precision results showed that the employment of the filling has no noticeable effect on any of the protein-ligand interactions. Using a commercial PrinCE instrument and an autosampler the required sample volume is reduced down to 10 μL, and almost this complete volume can be subsequently injected during repeated experiments.

Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts

Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion.

Cell dispensing in low-volume range with the immediate drop-on-demand technology (I-DOT).

Handling and dosing of cells comprise the most critical step in the microfabrication of cell-based assay systems for screening and toxicity testing. Therefore, the immediate drop-on-demand technology (I-DOT) was developed to provide a flexible noncontact liquid handling system enabling dispensing of cells and liquid without the risk of cross-contamination down to a precise volume in the nanoliter range. Liquid is dispensed from a source plate within nozzles at the bottom by a short compressed air pulse that is given through a quick release valve into the well, thus exceeding the capillary pressure in the nozzle. Droplets of a defined volume can be spotted directly onto microplates or other cell culture devices. We present a study on the performance and biological impact of this technology by applying the cell line MCF-7, human fibroblasts, and human mesenchymal stem cells (hMSCs). For all cell types tested, viability after dispensing is comparable to the control and exhibits similar proliferation rates in the absence of apoptotic cells, and the differentiation potential of hMSCs is not impaired. The immediate drop-on-demand technology enables accurate cell dosage and offers promising potential for single-cell applications.

Very fast capillary electrophoresis with electrochemical detection for high-throughput analysis using short, vertically aligned capillaries.

A method for conducting fast and efficient capillary electrophoresis (CE) based on short separation capillaries in vertical alignment was developed. The strategy enables for high-throughput analysis from small sample vials (low microliter to nanoliter range). The system consists of a lab-made miniaturized autosampling unit and an amperometric end-column detection (AD) cell. The device enables a throughput of up to 200 separations per hour. CE-AD separations of a dye model system in capillaries of only 4 to 7.5cm length with inner diameters (ID) of 10 or 15μm were carried out under conditions of very high electric field strengths (up to 3.0kV/cm) with high separation efficiency (half peak widths below 0.2s) in less than 3.5s migration time. A non-aqueous background electrolyte, consisting of 10mM ammonium acetate and 1M acetic acid in acetonitrile, was used. The practical suitability of the system was evaluated by applying it to the determination of dyes in overhead projector pens.

Simple and reusable picoinjector for liquid delivery via nanofluidics approach

Precise control of sample volume is one of the most important functions in lab-on-a-chip (LOC) systems, especially for chemical and biological reactions. The common approach used for liquid delivery involves the employment of capillaries and microstructures for generating a droplet which has a volume in the nanoliter or picoliter range. Here, we report a novel approach for constructing a picoinjector which is based on well-controlled electroosmotic (EO) flow to electrokinetically drive sample solutions. This picoinjector comprises an array of interconnected nanochannels for liquid delivery. Such technique for liquid delivery has the advantages of well-controlled sample volume and reusable nanofluidic chip, and it was reported for the first time. In the study of the pumping process for this picoinjector, the EO flow rate was determined by the intensity of the fluorescent probe. The influence of ion concentration in electrolyte solutions over the EO flow rate was also investigated and discussed. The application of this EO-driven picoinjector for chemical reactions was demonstrated by the reaction between Fluo-4 and calcium chloride with the reaction cycle controlled by the applied square waves of different duty cycles. The precision of our device can reach down to picoliter per second, which is much smaller than that of most existing technologies. This new approach, thus, opens further possibilities of adopting nanofluidics for well-controlled chemical reactions with particular applications in nanoparticle synthesis, bimolecular synthesis, drug delivery, and diagnostic testing.PACS85.85.+ j; 87.15.hj; 82.39.Wj

Development and Characterization of a Capillary Gap Sampler as New Microfluidic Device for Fast and Direct Analysis of Low Sample Amounts by ESI-MS

Direct hyphenation of miniaturized sampling devices to electrospray ionization-mass spectrometry (ESI-MS) is attractive because ESI-MS is compatible with microfluidics and allows comprehensive sample analysis, yielding information that is orthogonal to that available from optical methods. We present a "capillary gap sampler" as a platform for directly connecting microfluidics to μ-ESI-MS. The sampler was designed to be robust, light and compact, and to allow precise and fast liquid handling. Sample introduction in the range of a few nanoliters is performed via an open liquid bridge as a new microfluidic element. This allows minimum contact of the sample with system surfaces during the infusion process. The system shows good performance characteristics such as symmetrical peak shapes, low sample carryover (below 1%), and total injection cycle times of less than 15 s. This new device thus has the potential for rapid analysis of biomedical and pharmaceutical samples with limited sample amounts in a high-throughput mode.

ChemInform Abstract: Small‐Volume Nuclear Magnetic Resonance Spectroscopy

AbstractReview: 117 refs.

Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy

BackgroundWhen conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later.ResultsA change in oil volume of ±1.75% from an optimum volume of 285 μl had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R2=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil.ConclusionsThis study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.

Analyzing small samples with high efficiency: capillary batch injection–capillary electrophoresis–mass spectrometry

We present an experimental approach to conducting fast capillary electrophoresis-mass spectrometry (CE-MS) measurements of very small samples in the nanoliter range. This is achieved by injecting sample very efficiently into a CE-MS system. Injection efficiency represents the ratio of injected sample to the amount of sample needed for carrying out the injection process (v/v). In order to increase this injection efficiency from typical values of 10(-3) to 10(-7), the concept of capillary batch injection is used to build an automated, small-footprint injection device for CE-MS. This device is capable of running true multi-sample measurement series, using minimal sample volumes and delivering an injection efficiency of up to 100 %. It is compatible with both aqueous and non-aqueous background electrolytes. As an additional benefit, CE-MS separations of a catecholamine model system in capillaries of 15 cm length under conditions of high electric field strength could be accomplished in 20 s with high separation efficiency. This report details design and specifications of the injection device and shows optimal parameter choices for injections with both high injection efficiency and high separation efficiency. Furthermore, a procedure is presented to coat the tip of a fused silica capillary with a silicone elastomer which acts as a seal between two capillaries.

A Cheap, Easy Microfluidic Crystallization Device Ensuring Universal Solvent Compatibility

Microfluidic devices are increasingly used for the screening of crystallization conditions. Their advantage is the generation of droplets, every droplet being an independent crystallizer with volumes in the nanoliter range. This enables a large number of experiments to be carried out under identical conditions necessitating only small quantities of materials. However, classic microfluidic crystallization devices are made of poly(dimethylsiloxane), only compatible with an aqueous medium. In addition, they generally involve very complicated setups, often inaccessible to nonmicrofluidics specialists. In this paper, we overcome these drawbacks, presenting a cheap and universally applicable microfluidic crystallization tool. This thermostatted device makes it possible to study nucleation in both aqueous and organic solvents, rendering microfluidic devices applicable to organic molecules such as APIs, explosives, and metal oxide nanoparticles.

Cultivation of Chlorella vulgaris in microfluid segments and microtoxicological determination of their sensitivity against CuCl2 in the nanoliter range

AbstractThe cultivation of the monocellular green alga Chlorella vulgaris was implemented into microfluid segments to demonstrate the possibility of an automated screening of toxic effects of the common algaecide CuCl2. Therefore, the nutritional as well as light and carbon dioxide requirements of the algae had to be adapted to the microfluidic device. Generally, sequences of about 350 fluid segments with single volumes of about 500 nL were applied for the dose–response experiments. The growth of algae cultures inside microfluidic segments was non‐invasively measured by microflow through techniques using two different optical channels. A multi‐endpoint detection was realized by the photometric characterization of cell density by transmission measurements and the measurement of density of autofluorescent cells. The different methods revealed comparable half maximal effective concentrations (EC50) in the range between 34.6 and 39.9 μg/mL for the toxicity of CuCl2 to the green algae C. vulgaris. By reference experiments in microtiter plates lower EC50 were achieved presumably caused by increased alkalinity of the growth medium due to higher photosynthesis. The results show that the microsegmented flow technique is well suited for the automated determination of dose/response functions for microorganisms like C. vulgaris and for the application of multi‐endpoint procedures at the nanoliter scale.

Small-Volume Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most information-rich analytical techniques available. However, it is also inherently insensitive, and this drawback precludes the application of NMR spectroscopy to mass- and volume-limited samples. We review a particular approach to increase the sensitivity of NMR experiments, namely the use of miniaturized coils. When the size of the coil is reduced, the sample volume can be brought down to the nanoliter range. We compare the main coil geometries (solenoidal, planar, and microslot/stripline) and discuss their applications to the analysis of mass-limited samples. We also provide an overview of the hyphenation of microcoil NMR spectroscopy to separation techniques and of the integration with lab-on-a-chip devices and microreactors.

Crystallizing membrane proteins for structure–function studies using lipidic mesophases

The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3½ years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.