Nanoliter Volumes Research Articles

Microstructured Optical Fiber-based Biosensors: Reversible and Nanoliter-Scale Measurement of Zinc Ions.

Sensing platforms that allow rapid and efficient detection of metal ions would have applications in disease diagnosis and study, as well as environmental sensing. Here, we report the first microstructured optical fiber-based biosensor for the reversible and nanoliter-scale measurement of metal ions. Specifically, a photoswitchable spiropyran Zn(2+) sensor is incorporated within the microenvironment of a liposome attached to microstructured optical fibers (exposed-core and suspended-core microstructured optical fibers). Both fiber-based platforms retains high selectivity of ion binding associated with a small molecule sensor, while also allowing nanoliter volume sampling and on/off switching. We have demonstrated that multiple measurements can be made on a single sample without the need to change the sensor. The ability of the new sensing platform to sense Zn(2+) in pleural lavage and nasopharynx of mice was compared to that of established ion sensing methodologies such as inductively coupled plasma mass spectrometry (ICP-MS) and a commercially available fluorophore (Fluozin-3), where the optical-fiber-based sensor provides a significant advantage in that it allows the use of nanoliter (nL) sampling when compared to ICP-MS (mL) and FluoZin-3 (μL). This work paves the way to a generic approach for developing surface-based ion sensors using a range of sensor molecules, which can be attached to a surface without the need for its chemical modification and presents an opportunity for the development of new and highly specific ion sensors for real time sensing applications.

High-Precision Dispensing of Nanoliter Biofluids on Glass Pedestal Arrays for Ultrasensitive Biomolecule Detection.

Precise dispensing of nanoliter droplets is necessary for the development of sensitive and accurate assays, especially when the availability of the source solution is limited. Conventional approaches are limited by imprecise positioning, large shear forces, surface tension effects, and high costs. To address the need for precise and economical dispensing of nanoliter volumes, we developed a new approach where the dispensed volume is dependent on the size and shape of defined surface features, thus freeing the dispensing process from pumps and fine-gauge needles requiring accurate positioning. The surface we fabricated, called a nanoliter droplet virtual well microplate (nVWP), achieves high-precision dispensing (better than ±0.5 nL or ±1.6% at 32 nL) of 20-40 nL droplets using a small source drop (3-10 μL) on isolated hydrophilic glass pedestals (500 μm on a side) bonded to arrays of polydimethylsiloxane conical posts. The sharp 90° edge of the glass pedestal pins the solid-liquid-vapor triple contact line (TCL), averting the wetting of the glass sidewalls while the fluid is prevented from receding from the edge. This edge creates a sufficiently large energy barrier such that microliter water droplets can be poised on the glass pedestals, exhibiting contact angles greater >150°. This approach relieves the stringent mechanical alignment tolerances required for conventional dispensing techniques, shifting the control of dispensed volume to the area circumscribed by the glass edge. The effects of glass surface chemistry and dispense velocity on droplet volume were studied using optical microscopy and high-speed video. Functionalization of the glass pedestal surface enabled the selective adsorption of specific peptides and proteins from synthetic and natural biomolecule mixtures, such as venom. We further demonstrate how the nVWP dispensing platform can be used for a variety of assays, including sensitive detection of proteins and peptides by fluorescence microscopy or MALDI-TOF.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.

Photochemical Microscale Electrophoresis Allows Fast Quantification of Biomolecule Binding.

Intricate spatiotemporal patterns emerge when chemical reactions couple to physical transport. We induce electrophoretic transport by a confined photochemical reaction and use it to infer the binding strength of a second, biomolecular binding reaction under physiological conditions. To this end, we use the photoactive compound 2-nitrobenzaldehyde, which releases a proton upon 375 nm irradiation. The charged photoproducts locally perturb electroneutrality due to differential diffusion, giving rise to an electric potential Φ in the 100 μV range on the micrometer scale. Electrophoresis of biomolecules in this field is counterbalanced by back-diffusion within seconds. The biomolecule concentration is measured by fluorescence and settles proportionally to exp(-μ/D Φ). Typically, binding alters either the diffusion coefficient D or the electrophoretic mobility μ. Hence, the local biomolecule fluorescence directly reflects the binding state. A fit to the law of mass action reveals the dissociation constant of the binding reaction. We apply this approach to quantify the binding of the aptamer TBA15 to its protein target human-α-thrombin and to probe the hybridization of DNA. Dissociation constants in the nanomolar regime were determined and match both results in literature and in control experiments using microscale thermophoresis. As our approach is all-optical, isothermal and requires only nanoliter volumes at nanomolar concentrations, it will allow for the fast screening of biomolecule binding in low volume multiwell formats.

Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer.

We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term μFISH. This μFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes — the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-016-0064-0) contains supplementary material, which is available to authorized users.

Stick-Jump (SJ) Evaporation of Strongly Pinned Nanoliter Volume Sessile Water Droplets on Quick Drying, Micropatterned Surfaces.

We present an experimental study of stick-jump (SJ) evaporation of strongly pinned nanoliter volume sessile water droplets drying on micropatterned surfaces. The evaporation is studied on surfaces composed of photolithographically micropatterned negative photoresist (SU-8). The micropatterning of the SU-8 enables circular, smooth, trough-like features to be formed which causes a very strong pinning of the three phase (liquid-vapor-solid) contact line of an evaporating droplet. This is ideal for studying SJ evaporation as it contains sequential constant contact radius (CCR) evaporation phases during droplet evaporation. The evaporation was studied in nonconfined conditions, and forced convection was not used. Micropatterned concentric circles were defined having an initial radius of 1000 μm decreasing by a spacing ranging from 500 to 50 μm. The droplet evaporates, successively pinning and depinning from circle to circle. For each pinning radius, the droplet contact angle and volume are observed to decrease quasi-linearly with time. The experimental average evaporation rates were found to decrease with decreasing pining radii. In contrast, the experimental average evaporation flux is found to increase with decreasing droplet radii. The data also demonstrate the influence of the initial contact angle on evaporation rate and flux. The data indicate that the total evaporation time of a droplet depends on the specific micropattern spacing and that the total evaporation time on micropatterned surfaces is always less than on flat, homogeneous surfaces. Although the surface patterning is observed to have little effect on the average droplet flux-indicating that the underlying evaporation physics is not significantly changed by the patterning-the total evaporation time is considerably modified by patterning, up to a factor or almost 2 compared to evaporation on a flat, homogeneous surface. The closely spaced concentric circle pinning maintains a large droplet radius and small contact angle from jump to jump; the result is a large evaporation rate leading to faster evaporation.

Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

Convection-Enhanced Biopatterning with Recirculation of Hydrodynamically Confined Nanoliter Volumes of Reagents.

We present a new methodology for efficient and high-quality patterning of biological reagents for surface-based biological assays. The method relies on hydrodynamically confined nanoliter volumes of reagents to interact with the substrate at the micrometer-length scale. We study the interplay between diffusion, advection, and surface chemistry and present the design of a noncontact scanning microfluidic device to efficiently present reagents on surfaces. By leveraging convective flows, recirculation, and mixing of a processing liquid, this device overcomes limitations of existing biopatterning approaches, such as passive diffusion of analytes, uncontrolled wetting, and drying artifacts. We demonstrate the deposition of analytes, showing a 2- to 5-fold increase in deposition rate together with a 10-fold reduction in analyte consumption while ensuring less than 6% variation in pattern homogeneity on a standard biological substrate. In addition, we demonstrate the recirculation of a processing liquid using a microfluidic probe (MFP) in the context of a surface assay for (i) probing 12 independent areas with a single microliter of processing liquid and (ii) processing a 2 mm2 surface to create 170 antibody spots of 50 × 100 μm2 area using 1.6 μL of liquid. We observe high pattern quality, conservative usage of reagents, micrometer precision of localization and convection-enhanced fast deposition. Such a device and method may facilitate quantitative biological assays and spur the development of the next generation of protein microarrays.

A New Pico-Litre Fluid Dispensing Technology for New Possibilities

Biosensors more often than not require the manipulation of liquids, either to produce a biosensor itself, or to facilitate the biosensor’s operation. As technological progress is made, the volumes of fluid involved have reduced and continue to do so. Reactions which may have previously required milli-litres now are accomplished using micro-litre, nano-litre and pico-litre volumes of reagents and biomaterials. There is an increasing desire for more reaction data points, cost savings in terms of material usage, a reduction in physical laboratory space requirements and miniaturization in general. To this end, the use of pico-litre volumes of fluids, where the biology and chemistry support it, is a logical step. Recently a new fluid handling instrument has been made available which is capable of dispensing pico-litre and low volume nano-litre volumes of fluid with high precision and accuracy. The PolyPico technology is not novel in being able to dispense pico-litre and nano-litre volumes of fluids, however it is novel in being able to do so by using disposable plastic cartridges.

Microfluidics-Based PCR for Fusion Transcript Detection.

The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

Generation of droplets to serpentine threads on a rotating compact-disk platform

We generate stable monodisperse droplets of nano-liter volumes and long serpentine liquid threads in a single, simple “Y”-shaped microchannel mounted on a rotationally actuated lab-on-a-compact-disk platform. Exploitation of Coriolis force offers versatile modus operandi of the present setup, without involving any design complications. Based on the fundamental understanding and subsequent analysis, we present scaling theories consistent with the experimental observations. We also outline specific applications of this technique, in the biological as well as in the physical domain, including digital polymerase chain reaction (PCR), controlled release of medical components, digital counting of colony forming units, hydrogel engineering, optical sensors and scaffolds for living tissues, to name a few.

A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules

Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39fM (831 molecules in 35.4nl assay volume) for target DNA and 16fM (338 molecules) for target RNA after 1h on-chip hybridization.

Miniaturized Transmission-Line Sensor for Broadband Dielectric Characterization of Biological Liquids and Cell Suspensions

The nondestructive characterization of biological liquids and cell suspensions using electromagnetic waves in the microwave frequency range calls for accurate and sensitive measurement devices. Especially when reducing the sample volume, the sensitivity becomes a critical design parameter for broadband sensors. In this paper, a miniaturized transmission-line sensor based on a coplanar waveguide is used to characterize the permittivity of nanoliter volumes of biologically relevant liquids and cell suspensions. The sensor’s sensitivity is increased by means of electrically small discontinuities within the sensing section. The biological samples are guided across the sensor in a microfluidic channel, which is fabricated using microsystems technology. The sensor is used between 850 MHz and 40 GHz to detect the broadband permittivity of liquid samples such as aqueous salt and protein solutions. The experimentally detected contrast between living and dead Chinese hamster ovary cells in suspension is significant despite the small sample volume.

Characterization of Protein Using Two-Port STW Resonators

A surface acoustic wave (SAW) is an acoustic, mechanical wave that propagates on the surface of a piezoelectric crystal. Coupling to any medium that interacts the surface strongly affects the velocity and amplitude of the wave. In a typical surface transversal wave (STW) resonator, an electrical signal is converted at interdigital transducers (IDT’s) into polarized transversal waves travelling parallel to the sensing surface, utilizing the piezoelectric properties of the substrate material. This approach is very sensitive to biological interactions on the sensor surface. Typically, the wave transmitted is confined to the surface of the substrate. Thus, the acoustic energy is concentrated within the guiding layer rather than in the bulk of the piezoelectric material. The sensitivity of the sensor for surface modifications is increased, by the choice of material, and the design of the guiding layer as well as by the structure of the sensor and the transducers. The output signals are transformed in terms of their frequency or phase with respect to the input signals, due to the interaction of the input signal with the fluid contacting the sensitive surface. Surface acoustic wave (SAW) devices hold a promise in providing ultra fast sensing platform. Research focus is on studying methods to characterize conductance, susceptance, viscosity, and other properties of protein samples, like albumin using Two-Port Surface Transverse Wave (STW) resonator devices quickly and inexpensively using nano-liter volume. When a mass is deposited on the electrode the resultant decrease in resonant frequency can detect mass changes at nanogram level. We propose to advance the sensitivity of the characterization by use of quartz Two-Port STW resonator since they can operate at much higher frequency than bulk wave oscillators.

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample.

53. Bio-inspired cryoprotectants and microfluidics for cryopreservation

Cryopreservation of living cells, tissue, and organs has the potential to create a great impact on modern regenerative, transfusion, transplantation, and reproductive medicine. For instance, we have reported the preservation of human red blood cell morphology, mechanics, and function following vitrification in nanoliter volumes using an innovative cryo-printing approach that integrates naturally occurring cryo-inks, including ectoine and trehalose solutions. Further, we have demonstrated the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived embryonic bodies (EBs), the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality post freezing and thawing. These results present a platform for the development of a new therapeutic system for personalized medicine.

10. Bio-inspired cryoprotectants and microfluidics for cryopreservation

Cryopreservation of living cells, tissue, and organs has the potential to create a great impact on modern regenerative, transfusion, transplantation, and reproductive medicine. For instance, we have reported the preservation of human red blood cell morphology, mechanics, and function following vitrification in nanoliter volumes using an innovative cryo-printing approach that integrates naturally occurring cryo-inks, including ectoine and trehalose solutions. Further, we have demonstrated the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived embryonic bodies (EBs), the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality post freezing and thawing. These results present a platform for the development of a new therapeutic system for personalized medicine.

Soft compartmentalization: Combining droplet‐based microfluidics with freely accessible cells

Droplet‐based microfluidics allows the generation and manipulation of many independent microreactors (pico‐ to nanoliter volumes) optionally hosting human cells. However, one limitation is that mammalian cells within droplets are not fully accessible from outside. The contents of the plugs cannot be completely exchanged so that washing steps for immunoassays are not possible. Additionally, imaging of cells is difficult because cells move within the volume of the plugs. Here, we present an approach in which droplets are used for the specific delivery of compounds across a 150 μm thick polydimethylsiloxane (PDMS) membrane to cells grown on the opposite side of this thin membrane, resulting in spatially separated, parallelizable assays. As a first application, we used this setup to compartmentalize tetracycline within droplets and incubating them in a long channel for 16 h, after which the plugs were flushed out of the channels. Tetracycline diffused into the PDMS membrane and induced the expression of green fluorescent protein in HeLa cells within a large, freely accessible population in a pattern corresponding to the original positions of tetracycline droplets. This should allow the combination of droplet‐based microfluidics with cell‐based assays requiring long incubation times, studies of cell–cell interactions, and chemotaxis.

Passive removal of immiscible spacers from segmented flows in a microfluidic probe

Microfluidic probes (MFPs) are a class of non-contact, scanning microfluidic devices that hydrodynamically confine nanoliter volumes of a processing liquid on a surface immersed in another liquid. So far only chemical processes using a single processing liquid have been implemented using MFPs. In this letter, we present the design and implementation of a probe head that allows segmented two-phase flows to be used, which will enable different chemical species to be sequentially delivered to a surface in defined volumes and concentrations. Central to this probe head is a spacer-removal module comprising blocking pillars in the injection channel, a bypass and an orifice leading to the aspiration channel. We present a capillarity-based analytical model that provides insight into the functionality of the module based on geometrical parameters. In addition, we study the difference between two- and three-channel modules and predict a 30% reduction in fluctuation of the footprint of the confined liquid for the three-channel module. We show that such a module with a 15 μm pillar spacing, a 30 μm orifice width, and an oblique angle of 30° can remove immiscible spacers (Fluorinert FC-40) from an aqueous flow at a rate of up to 15 spacers per second while maintaining hydrodynamic confinement of processing liquid.

Droplet-based dielectrophoresis device for on-chip nanomedicine fabrication and improved gene delivery efficiency

In this article, we present the design, fabrication, and experimental verification of a droplet-based microfluidic device for effective on-chip fabrication and separation of polymer-based nanoparticles using dielectrophoresis (DEP) effect. The separated polyplexes nanoparticles were used in cells for improved gene transfection efficiency. By adjusting the flow rate of PEI600-CyD-FA (H1) and DNA plasmids, polyplexes products can be mixed and self-assembled inside droplets within approximately a nanoliter volume. This procedure ensures synthesized particles to have a narrow size distribution. In addition, a new microchannel design was developed to automatically coalesce two moving aqueous droplets and to directly extract aqueous polyplex products from oil. Finally, the H1-DNA polyplexes of ~116 nm diameter were separated via negative DEP force under 8 Vpeak–peak and 20 MHz conditions by passing three times through a non-uniform electric field. The biological findings demonstrated that the DEP-treated polyplexes still possessed the ability to enter HUVEC cells and that the gene transfection efficiency was raised to 15 %, as opposed to the control group’s 4 % where the polyplexes had no DEP treatment. The quantitative comparison was done by counting the number of cells produced via positive EPFG expression. These hydrodynamic and electrodynamic techniques provide an integrated microfluidic platform for fabricating and screening nanoscale drugs.