Nanoliter Dispenser Research Articles

Actinide and lanthanide thin-layer developments using a drop-on-demand printing system

Actinide and lanthanide thin layers with specific requirements regarding thickness, homogeneity, chemical purity, mechanical stability, and backing properties are applied in a multitude of physics and chemistry experiments. A novel target preparation method, the so-called “Drop-on-Demand” (DoD) technique, based on a commercial nanoliter (nL) dispenser is applied since a few years in the Nuclear Chemistry unit at Johannes Gutenberg University Mainz. The wetting behaviour of the nL droplets on the substrate’s surface is a key parameter determining the spatial distribution of the deposited material after evaporation. By switching from aqueous to organic solvents as well as by substrate surface modifications, the wetting behaviour can be influenced. Recent investigations on this influence and applications of the DoD method are presented. The produced actinide deposits were characterized by optical and scanning electron microscopy, by α spectroscopy as well as by radiographic imaging.

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Capacitive Sensing for Monitoring of Microfluidic Protocols Using Nanoliter Dispensing and Acoustic Mixing.

The development of protocols for bio/chemical reaction requires alternate dispensing and mixing steps. While most microfluidic systems use the opening of additional parts of the channel to allow the ingress of fixed volumes of fluid, this requires knowledge of the protocol before the design of the chip. Our approach of using a microfluidic valve to regulate the flow into an initially empty cavity allows for on-chip protocol development and refinement. Mixing is provided by way of surface acoustic wave excitation; this high-frequency vibration causes steady-state streaming flows. We show that capacitive sensing can be used to measure fluid levels, even if multiple fluid types are used, such that nanoliter dispensing accuracy is achieved. Also, the capacitive readout can be used to establish mixing quality and to monitor temperature fluctuations. These capabilities allow for protocols to be conducted without optical assessment and thus will allow for multiplexing, such that reactions could be conducted, simultaneously, in multiple chambers across a chip.

Rapid Analysis of Single Droplets of Lanthanide–Ligand Solutions by Electrospray Ionization Mass Spectrometry Using an Induction-Based Fluidics Source

Electrospray ionization mass spectra of lanthanide coordination complexes were measured by launching nanoliter-sized droplets directly into the aperture of an electrospray ionization mass spectrometer. Droplets ranged in size from 102 nL to 17 nL, while metal concentrations were 293 μM. The sample solution was delivered to a source capillary by a nanoliter dispenser at a rate of 21 nL/s, and droplets were ejected from the capillary by pulsing a potential onto the capillary. The end of the capillary was situated in front of the mass spectrometer and aimed directly at the aperture. The period and power of the electrical pulse was controlled by a digital energy source. The intensity of the extracted ion time profiles from the experiment showed reproducible production of lanthanide nitrato-anion complexes (Ce, Tb, and Lu). The integrated ion intensities of the complexes were reproducible, having relative standard deviations on the order 10% for anions, and 10-30% for cations. The integrated ion intensities were proportional to the droplet size, and the response was linear from about 100 to 650 pmol. However, the intercept is not zero, indicating a nonlinear response at lower analyte quantities or droplet sizes. Cation complexes were generated in separate experiments that corresponded to lanthanide nitrate ion pairs coordinated with the separations ligand octyl,phenyl,(N,N-diisobutylcarbamoyl)methylphosphine oxide (CMPO). Experiments showed a preference for formation of CMPO complexes with Ln(3+) having larger ionic radii. The relative standard deviation values of the cation abundance measurements were somewhat higher for the more highly coordinated complexes, which are also less stable. The mass spectral quality was high enough to measure the ratios of the minor isotopic ions to a high degree of accuracy. The approach suggests that the methodology has utility for analysis of solutions where the sample quantity is limited, or where the sampling efficiency of a normal ESI source is limiting on account of hazards derived from the sample solution.

Noncontact Determination of Velocity and Volume of Nanoliter Droplets on the Fly

We present a sensor for measuring volume and velocity of dispensed nanoliter droplets in a noncontact manner on the fly. The sensor-setup has a total thickness of 3.2 mm and can easily be mounted underneath any given nanoliter dispenser for continuous online monitoring of its dispensing performance. The principle is based on the interaction of dispensed single droplets of sample liquid passing the electric field of an open plate capacitor. The effect depends on droplet parameters like volume, velocity, and dielectric constant and is discussed in the paper. The presented data analysis enables a velocity independent volume determination of water droplets in the range from 26 to 82 nl with an accuracy of 3 nl. The sensor signal is sensitive to the alignment of the flight path of the nanoliter droplets within the 1.2-mm wide open capacitor and can lead to systematic volume errors of up to <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex Notation="TeX">$\Delta {\hbox {V}}\sim 12~{\hbox {nl}}$</tex></formula> . The impact of different dielectric constants can only be differentiated for very high variations; thus, the sensor needs to be calibrated to the different types of liquids.

Microfluidics in silicon/polymer technology as a cost-efficient alternative to silicon/glass

We investigate TMMF photopolymer as a cost-efficient alternative to glass for the leak-tight sealing of high-density silicon microchannels. TMMF enables low temperature sealing and access to structures underneath via lamination and standard UV-lithography instead of costly glass machining and anodic bonding. TMMF is highly transparent and has a low autofluorescence for wavelengths larger than 400 nm. As the photopolymer is too thin for implementing bulky world-to-chip interfaces, we propose adhesive bonding of cyclic olefin copolymer (COC) modules. All materials were tested according ISO 10993-5 and showed no cytotoxic effects on the proliferation of L929 cells. To quantify the cost efficiency of the proposed techniques, we used an established silicon/Pyrex nanoliter dispenser as a reference and replaced structured Pyrex wafers by TMMF laminates and COC modules. Thus, consumable costs, manpower and machine time related to sealing of the microchannels and implementing the world-to-chip interface could be significantly reduced. Leak tightness was proved by applying a pressure of 0.2 MPa for 5 h without delamination or crosstalk between neighboring microchannels located only 100 µm apart. In contrast to anodic bonding, the proposed techniques are tolerant to surface inhomogeneities. They enable manufacturing of silicon/polymer microfluidics at lower costs and without compromising the performance compared to corresponding silicon/glass devices.

DNA microarrays for hybridization detection by surface plasmon resonance spectroscopy

We report on the development of a new platform technology for the detection of genetic variations by means of surface plasmon resonance (SPR) spectroscopy. TOPAS chips with integrated optics were exploited in combination with microfluidics. Within minutes, the detection of hybridization kinetics was achieved simultaneously at all spots of the DNA microarray. A nanoliter dispenser is used to deposit thiol-modified single-stranded probe DNA on the gold surface of the chips. We investigated the influence of different parameters on hybridization using model polymerase chain reaction (PCR) products. These PCR products comprised a single-stranded tag sequence being complementary to an anti-tag sequence of probes immobilized on the gold surface. The signals increased with increasing length of PCR products (60, 100 or 300 base pairs) as well as with their concentration. We investigated hybridizations on DNA microarrays comprising 90 spots of probe DNA with three different sequences. Furthermore, we demonstrate that sequences with possible hairpin structures significantly lower the binding rate, and thus, the SPR signals during hybridization.

TopSpot® Vario: a novel microarrayer system for highly flexible and highly parallel picoliter dispensing

The standard TopSpot technology has been successfully used in the recent years for the highly parallel nanoliter dispensing of bio-chemical substances for microarray printing. It is based on a pneumatically actuated printhead which enables non-contact microarray fabrication at a pitch of typically 500 microm. This paper reports on a new and improved way of operating the printheads termed TopSpot Vario technology, using an incompressible material between the piezo actuator and the dispensing medium. The advantage of the incompressible medium is the direct relation between the displacement amplitude and the ejected liquid volume. Earlier reports stated that the filling of the printheads is a key issue. In this paper we report on the implementation and characterization of a new printhead design including microchannels for bubble free priming of blind channels. Microarray fabrication was successfully conducted with four different types of protein.

Nanoliter Dispensing Method by Degassed Poly(dimethylsiloxane) Microchannels and Its Application in Protein Crystallization

This paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannels were reversibly bound to arrays of microwells. The PDMS elastomer was predegassed and served as an internal vacuum pumping source. Various aqueous solutions were infused into arrays of microwells through the reversibly sealed PDMS microchannels. Microwells fabricated in PDMS, poly(methyl methacrylate) (PMMA), and glass were all compatible with this dispensing method. By removing the PDMS microchannels, arrays of droplets confined in the microwells were obtained. Multiplex reaction and screening at the nanoliter scale were carried out by binding two such arrays of microwells to form microchambers. We applied this method to screening the crystallization conditions of four known proteins. Long-term incubation of over 2 months was achieved by employing glass microwells. An unknown protein was then crystallized using the screening method in microwells. The crystals with sufficient size were harvested from the reversibly bound microwells. X-ray diffraction with a resolution of 3.1 Angstrom was obtained.

An automated nanoliter dispenser for staining individual biopsies in tissue microarrays

In the field of molecular analysis of cancer, there exists a need for a clinical device that can automate protocols for immunohistochemical and in situ hybridization diagnostic staining on tissue microarrays. The tissue microarray antibody spotter (TMAS) has been developed to provide fundamental improvements over current histological staining techniques by enabling precision application of reagents to individual biopsies within a tissue microarray. This allows for multiplexed reactions on a single slide and promises to significantly reduce costs associated with immunohistochemistry and in situ hybridization based assays. Additionally, because TMAS allows for testing of different biomarkers on each element of a tissue array, a complete cancer profile can be obtained from a single TMA slide. Ultimately this may lead to cost-effective, faster and more accurate diagnosis of the patient.

Debunking the Myth: Validation of Fluorescein for Testing the Precision of Nanoliter Dispensing

Acoustic energy can precisely and accurately eject a droplet of liquid from a reservoir, enabling delivery of picoliter and nanoliter volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003, 8(5), 29–34). Acoustic droplet ejection has been shown to be extremely precise (coefficients of variation &lt; 2%) over a wide range of dispensed volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003, 8(5), 29–34). However, measuring the performance of low-volume fluid transfers can be difficult because the data are often masked by variability in bulk dispensers and fluorescence readers used as part of the overall measurement process (Petersen, J.; Nguyen, J. Comparison of absorbance and fluorescence methods for determining liquid dispensing precision. Journal of the Association for Laboratory Automation 2005, 10(2), 82–87; Rhode, H.; Schulze, M.; Renard, S.; Zimmerman, P.; Moore, T.; Cumme, G.; Horn, A. An improved method for checking HTS/uHTS liquid handling systems. Journal of Biomolecular Screening 2004, 9, 726–733). The fluorophore used must also be stable so that thermal bleaching and photobleaching do not contribute additional variability to the measurements. This study assesses the suitability of fluorescein to measure the precision of fluid transfers of 2.5-nL DMSO droplets. The short-term and long-term stabilities of fluorescein are first qualified using a reference standard. Next, we determine the noise contribution of the filler and reader. Lastly, data are presented for the precision of 5- and 50-nL fluid transfers using this fluorescein measurement process. (JALA 2006;11:233–9)

Microbead‐based affinity chromatography chip using RNA aptamer modified with photocleavable linker

A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.

Mosquito®: An Accurate Nanoliter Dispensing Technology

Mosquito® from TTP LabTech Ltd. is an innovative nanoliter dispenser that combines the liquid transfer capability of a fixed head pipette with the elimination of cross-contamination, using disposable tips. For many applications required in genomics, proteomics and drug discovery, Mosquito can reduce assay cost by minimizing reagent and sample usage.

Impact of medium properties on droplet release in a highly parallel nanoliter dispenser

We report about the correlation between satellite free droplet release and liquid viscosity in a highly parallel, pressure driven nanoliter dispenser. In extensive studies we found that for liquids of different viscosities the duration of the pressure pulse is the predominant effect compared to pressure amplitude. This result is of essential importance when actuation parameters have to be adopted for different media like oligonucleotides, DNA or protein solutions as it is the case for the non contact high throughput fabrication of microarrays [J. Ducrée, H. Gruhler, N. Hey, M. Mueller, S. Békési, M. Freygang, H. Sandmaier, R. Zengerle, TopSpot—A new method for the fabrication of microarrays, Technical Digest, in: Proceedings of the Thirteenth IEEE Annual International Conference on Micro Electro Mechanical Systems, Mizyazaki, Japan, 23–27 January 2000, pp. 317–322]. For oligonucleotides printing we found coefficient of variations (CVs) of droplet diameter to be better than 1% within one single dispensing channel and 1.5% within all 24 channels for each used printing buffer. The CVs for protein printing were slightly higher but likewise for the oligonucleotides below 1%. Experiments with oligonucleotides as well as with different proteins showed ascertained carry-over and cross-talk free printing of DNA and protein microarrays. With it a prime critical point of microarray production is solved, leading to high quality whilst high throughput microarray fabrication.

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser.

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.

Automated mass spectrometry: a revolutionary technology for clinical diagnostics.

For various diagnostic analyses and the studies of functional genomics, the use of an accurate and cost-effective analytic platform to analyze large numbers of samples is essential. An automated platform called MassArray (Sequenom, Inc, San Diego, CA), designed for high-throughput diagnostic analyses, has recently been validated. The platform combines miniaturized, two-dimensional chip arrays with proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Nanoliter dispensing of samples in high-density formats of 384 or greater results in improved throughput and reduced costs. Automation prevails from the initial assay design through sample processing and data analysis, for the most part eliminating the labor component of assay development and implementation. The MassArray platform is being used in the following areas: (1) molecular diagnosis of genetic disease and infectious agents, (2) pharmacogenomics, (3) paternity and/or identity testing, and (4) agriculture (e.g., marker-assisted breeding). MALDI-TOF mass spectrometry can also be used for analyzing proteins; therefore, genotype/phenotype testing can be performed on a single platform.

Application of a microchamber array for DNA amplification using a novel dispensing method.

We recently developed a microchamber array chip for DNA amplification by adopting semiconductor microfabrication technology; a polymerase chain reaction (PCR) was performed in the microchamber array, and the amplified DNA was detected using a fluorescent dye. In order to manipulate a single cell or sample into each microchamber individually in this system, the chip was directly sealed with a cover glass slip which impeded the retrieval of the products from each chamber. The present study was therefore carried out to improve the system by developing methods for covering the microchambers and introducing the reaction solution. First, we fabricated a microchamber array chip, and the oil layer was coated on the whole chip instead of the cover glass slip. The solution for DNA amplification was introduced into each chamber through an oil layer using a nano-liter dispenser. Following this, the microarray chip was placed onto the thermal cycling system for DNA amplification, and the amplified DNA was subsequently detected by fluorescence microscopy. In this system, the products were easily retrieved using a micromanipulator for further analysis.