A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme β-galactosidase and rotary motor protein F(1)-ATPase enclosed in a droplet.     

Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays

In this communication, single molecules of beta-galactosidase were captured on a 1 mm femtoliter array using biotin-streptavidin binding. The femtoliter arrays, containing 24 000 individual reaction chambers, permit digital concentration readout as the percentage of reaction vessels that successfully capture a target molecule is correlated to the bulk target concentration. This capture and readout approach should prove useful for DNA and antibody assays that utilize an enzyme label to catalyze the generation of a fluorescent signal.     

Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies

We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.     

Fast mixing and reaction initiation control of single-enzyme kinetics in confined volumes

A device with femtoliter-scale chambers and controlled reaction initiation was developed for single-molecule enzymology. Initially separated substrate and enzyme streams were rapidly mixed in a microfluidic device and encapsulated in an array of individual microreactors, allowing for enzyme kinetics to be monitored with millisecond dead times and single-molecule sensitivity. Because the arrays of chambers were fabricated by micromolding in PDMS, the chambers were monodisperse in size, and the chamber volume could be systematically controlled. Microreactors could be purged and replenished with fresh reactants for consecutive rounds of observation. Repeated experiments with statistically identical initial conditions could be performed rapidly, with zero cross-talk between chambers in the array.     

Mass action at the single-molecule level

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.     

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.     

Analytical Chemistry on the Femtoliter Scale

The compartmentalization of reactions in femtoliter (fL) containers and integration of fL containers into arrays not only enhances and accelerates chemical and biochemical analysis but also leads to new scientific methods and insights. This review introduces various fL container and array formats and explores their applications for the detection and characterization of biologically relevant analytes. By loading analytes, sensing elements, or cells into fL arrays, one can perform thousands of analytical measurements in parallel. Confining single enzyme molecules in fL arrays enables one to analyze large numbers of individual enzyme molecules simultaneously in solution. New nanofabrication techniques and progressively more sensitive detection methods drive the field of fL analytical chemistry. This review focuses on the progress and challenges in the field of fL analytical chemistry with examples of both basic and applied research.     

Bio-solvents change regioselectivity in the synthesis of disaccharides using Biolacta β-galactosidase

Bio-solvents are good alternative solvents that avoid the use of classical organic solvents when performing enzymatic reactions. A noticeably change in regioselectivity was observed in the synthetic behaviour of Biolacta β-galactosidase using bio-solvents derived from dimethylamide and glycerol as co-solvents. Under these conditions, the enzyme changes its well known tendency to produce β-(1→4) to β-(1→6) disaccharides. An evaluation of the bio-solvent concentration and the effects of the non proteic additives in commercially available Biolacta β-galactosidase was undertaken in order to optimize the reaction conditions to improve the yield of the β-(1→6) product.     

SERS in Salt Wells

We report herein a simple, inexpensive fabrication methodology of salt microwells, and define the utility of the latter as nanoparticle containers for highly sensitive surface‐enhanced Raman scattering (SERS) studies. AFM characterization of Ag and Au loaded salt microwells reveal the ability to contain favorable nanostructures such as nanoparticle dimers, which can significantly enhance the Raman intensity of molecules. By performing diffraction‐limited confocal Raman microscopy on salt microwells, we show high sensitivity and fidelity in the detection of dyes, peptides, and proteins, as a proof of our concept. The SERS limit of detection (accumulation time of 1 s) for rhodamine B and TAT contained in salt mircowells is 10 pM and 1 nM , respectively. The Raman characterization measurements of salt microwells with three different laser lines (532 nm, 632.81 nm, 785 nm) reveal low background intensity and high signal‐to‐noise ratio upon nanoparticle loading, which makes them suitable for enhanced Raman detection. SERS mapping of these sub‐femtoliter containers show spatial confinement of the relevant analyte to a few microns, which make them potential candidates for microscale bioreactors.     

Probing redox photocatalysis of trapped electrons and holes on single Sb-doped titania nanorod surfaces

We used a fluorogenic reaction to study in conjunction the photocatalytic properties for both active sites (trapped photogenerated electrons and holes) on individual Sb-doped TiO(2) nanorods with single-molecule fluorescence microscopy. It was found that active sites around trapped holes show higher activity, stronger binding ability, and a different dissociation mechanism for the same substrate and product molecules in comparison with the active sites around trapped electrons. These differences could be elucidated by a model involving the charged microenvironments around the active sites.     

Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels

Improving the sensitivity of DNA biosensors is extremely important in clinical diagnostics, gene therapy, and a variety of other biomedical studies. In this regard, we have developed a highly sensitive single molecule DNA assay platform with a 1fM experimental detection limit using enzymatic amplification in an array of femtoliter-sized reaction wells. To validate the utility of this technology in our study, we employed a fiber optic array to create thousands of femtoliter-sized reaction wells, each specifically functionalized with oligonucleotide probes capable of capturing biotinylated target DNA. After hybridization, the fiber was incubated with streptavidin-labeled enzyme solution. The bound single enzyme molecules were confined to individual reaction vessels containing excess fluorogenic substrate and catalyzed the production of a sufficient number of fluorescent product molecules to generate a detectable signal. At low target DNA concentrations with relatively short incubation times, only a small percentage of the capture sites bind target DNA, enabling a binary readout of target concentration from the high-density fiber array. This simple binary readout-based scheme is easy to perform and exhibits a high signal-to-noise ratio in the presence of trace amounts of DNA target. Furthermore, it also should be possible to extend this technology to protein detection by modifying the reaction wells with specific capture antibodies. We expect this assay to be useful in a number of biomedical applications where accurate and highly sensitive target analysis is critical.     

A prototypic system of parallel electrophoresis in multiple capillaries coupled with microwell arrays

We present a microfluidic system that can be directly coupled with microwell array and perform parallel electrophoresis in multiple capillaries simultaneously. The system is based on an array of glass capillaries, fixed in a polydimethylsiloxane (PDMS) microfluidic scaffold, with one end open for interfacing with microwells. In this capillary array, every two adjacent capillaries act as a pair to be coupled with one microwell; samples in the microwells are introduced and separated by simply applying voltage between two electrodes that are placed at the other ends of capillaries; thus no complicated circuit design is required. We evaluate the performance of this system and perform multiple CE with direct sample introduction from microwell array. Also with this system, we demonstrate the analysis of cellular contents of cells lysed in a microwell array. Our results show that this prototypic system is a promising platform for high-throughput analysis of samples in microwell arrays.     

Spatial confinement of avidin domains in microwell arrays

Microwell arrays were chemically etched across the distal faces of coherent fiber-optic bundles. A typical 1.6 mm diameter array comprised approximately 3000 individual microwells that were approximately 1-14 microm deep and approximately 22 microm wide. A methodology involving organosilane functionalized microwell surfaces and site-selective photobiotin chemistry was developed to partially fill microwells with a thin avidin layer. Avidin microwell arrays were characterized using charge coupled device optical microscopy and scanning electron microscopy. The avidin microwell arrays had individual well volumes that were six orders of magnitude smaller and up to 30-fold more numerous than commercially available avidin-coated microtiter plates. Preliminary results indicated that individual avidin microwells were ideally suited to house single biological cells. Using standard epifluorescence microscope optics and a mercury-arc lamp, an individual 22 microm wide microwell could be optically addressed and selectively filled with avidin without the use of a photolithographic mask. The ability to control both the size and position of avidin domains on the microwell array surface demonstrates the utility of this methodology towards fabricating a single microwell array with multianalyte sensing capabilities.     

Single Molecule Assay of Escherichia coli β-Galactosidase Using Two Competing Substrates Simultaneously,DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside

Single enzyme molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.     

Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy

Raman spectroscopy was tested for the identification of biomolecules (glycine, L-alanine, β-alanine, L-serine, and γ-aminobutyric acid) trapped in fluid inclusions inside halite model crystals. The investigated biomolecules represent important targets for future astrobiological missions. We know from terrestrial conditions that organic molecules and microorganisms can be sealed within fluid inclusions and can survive intact even for hundreds of millions of years. Raman spectroscopy is currently being miniaturized for future extraterrestrial planetary exploration (ExoMars 2018). Raman spectroscopy has shown the ability to detect investigated aminoacids nondestructively without any sample preparation, in short measurement times, and in relatively low concentrations. The number of registered Raman bands of investigated aminoacids and their intensity clearly correlate with the given concentration of biomolecules within fluid inclusions.     

Micro-well arrays for 3D shape control and high resolution analysis of single cells

In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.     

Organized arrays of individual DNA molecules tethered to supported lipid bilayers

An unappreciated aspect of many single-molecule techniques is the need for an inert surface to which individual molecules can be anchored without compromising their biological integrity. Here, we present new methods for tethering large DNA molecules to the surface of a microfluidic sample chamber that has been rendered inert by the deposition of a supported lipid bilayer. These methods take advantage of the "bio-friendly" environment provided by zwitterionic lipids, but still allow the DNA molecules to be anchored at fixed positions on the surface. We also demonstrate a new method for constructing parallel arrays of individual DNA molecules assembled at defined positions on a bilayer-coated, fused silica surface. By using total internal reflection fluorescence microscopy to visualize the arrays, it is possible to simultaneously monitor hundreds of aligned DNA molecules within a single field-of-view. These molecular arrays will significantly increase the throughput capacity of single-molecule, fluorescence-based detection methods by allowing parallel processing of multiple individual reaction trajectories.     

Patterning reactive microdomains inside polydimethylsiloxane microchannels by trapping and melting functional polymer particles

This paper describes a facile technique to pattern reactive microdomains inside polydimethylsiloxane microchannels by utilizing polymer particles as the carrier of functional groups. The air/liquid interface formed in microchannels equipped with microwells exerts lateral force on the particles, trapping particles only inside the wells. We then fix the polymer matrix on the wells by melting the trapped particles to form reactive domains with flexible shapes and high resolution. We employed monodisperse poly(styrene-co-glycidyl methacrylate) microparticles having an epoxy group and patterned various types of microdomains with a resolution of several micrometers. Several tests confirmed the presence of the epoxy group and the flatness of the patterned domain. The presented scheme provides a new way of preparing highly functional microsystems by using simple operations and would be useful for various applications, including local patterning of graft polymers and the site-specific cultivation of cells in a confined space.     

Droplet-based microfluidic device for multiple-droplet clustering

We present a multiple-droplet clustering device that can perform sequential droplet trapping and storing. Shape-dependent droplet manipulation in forward and backward flows has been incorporated to achieve high trapping and storing efficiency in a 10 × 12 array of clustering structures (e.g., storing well, storing chamber, trapping well, and guiding track). In the forward flow, flattened droplets are trapped in each trapping well. In the backward flow, the trapped droplets are released from the trapping well and follow the guiding tracks to their corresponding storing wells. The guided droplets float up out of the confining channel to the super stratum of the storing chamber due to interfacial energy and buoyancy effects. This forward/backward flow-based trapping/storing process can be repeated several times to cluster droplets with different contents and samples in the storing chambers. We expect that the proposed platform will be a valuable tool to study complex droplet-based reactions in clustered droplets.     

A microchamber array for single cell isolation and analysis of intracellular biomolecules

We present a microfluidic device that enables the determination of intracellular biomolecules in multiple single cells. The cells are individually trapped and isolated in a microchamber array. Since the microchambers can be opened and closed reversibly, the cells can be exposed to different solutions sequentially, e.g. for incubation, washing steps, labelling and finally, for lysis. The tightly sealed microchambers enable the retention and analysis of cell lysate derived from single cells. The performance of the device is demonstrated by monitoring the levels of the cofactors NADPH and NADH both in healthy mammalian cells and in cells exposed to oxidative stress. The platform was also used to determine the toxic impact of the alkaloid camptothecin on the intracellular enzyme glucose-6-phosphate dehydrogenase levels. In general, the device is applicable for the analysis of cell auto-stimulation and the detection of intracellular metabolite concentration or expression levels of proteins.