Nanolitre droplet array for real time reverse transcription polymerase chain reaction

Recently, more and more effort has been put into the miniaturization of genetic tests such as quantitative PCR (qPCR), because it is no doubt a powerful tool for molecular diagnosis and quantitative biology. In this paper, we developed a low density nanolitre droplet array generated on a chemical modified silicon chip for gene quantification. Reliable and sensitive two step real time qRT-PCR assay for microRNA measurement was performed within 500 nL droplets. It has a dynamic range of six orders of magnitude, allowing for the quantification of microRNA input from 10(3) to 10(9) copies per reaction. We successfully applied the platform for quantitative measurement of mir-122 across five cultured cell lines. The minimum total RNA input was as low as 1 pg per assay, which showed great potential for gene quantification at single cell level. We envision the droplet based qPCR chip would be a universal and low-cost platform for gene quantification in ordinary biological laboratories.     

In situ electroporation of surface-bound siRNAs in microwell arrays

Gene silencing using RNA interference (RNAi) has become a prominent biological tool for gene annotation, pathway analysis, and target discovery in mammalian cells. High-throughput screens conducted using whole-genome siRNA libraries have uncovered rich sets of new genes involved in a variety of biological processes and cellular models of disease. However, high-throughput RNAi screening is not yet a mainstream tool in life science research because current screening platforms are expensive and onerous. Miniaturizing the RNAi screening platform to reduce cost and increase throughput will enable its widespread use and harness its potential for rapid genome annotation. With this aim, we have combined semi-conductor microfabrication and nanolitre dispensing techniques to develop miniaturized electroporation-ready microwell arrays loaded with siRNA molecules in which multiplexed gene knockdown can be achieved. Arrays of microwells are created using high-aspect ratio biocompatible photoresists on optically transparent and conductive Indium-Tin Oxide (ITO) substrates with integrated micro-electrodes to enable in situ electroporation. Non-contact inkjet microarraying allows precise dispensing of nanolitre volumes into the microwell structures. We have achieved parallel electroporation of multiple mammalian cells cultured in these microwell arrays and observed efficient knockdown of genes with surface-bound, printed siRNAs. Further integration of microfabrication and non-contact nanolitre dispensing techniques described here may enable single-substrate whole-genome siRNA screening in mammalian cells.     

Generation of dynamic chemical signals with pulse code modulators

The on-chip generation of dynamic chemical signals in a flow stream via pulse code modulation (PCM) is demonstrated. In this chip the output signal concentration is determined by dispersion and averaging of a serial stream of digitally encoded plugs of concentrated solute and pure solvent as the plugs flow through a long dispersive capillary. A two-bit PCM chemical signal generator was fabricated in two-level PDMS technology. The chip was capable of generating 31 distinct output levels with 10-plug cycles. Several example chemical waveforms (sawtooth and cosine) were generated at flow rates of 43.2 nL s(-1), and plug frequencies of up to 15 Hz, with maximum output signal bandwidth of up to about 1 Hz. The modulator chip was also used to synthesize physiological signals emulating intracellular beta-cell cytosolic Ca(2+) oscillations, extracellular beta-cell insulin release and rat-striatum dopamine release.     

Microfluidic platform for on-demand generation of spatially indexed combinatorial droplets

We propose a highly versatile and programmable nanolitre droplet-based platform that accepts an unlimited number of sample plugs from a multi-well plate, performs digitization of these sample plugs into smaller daughter droplets and subsequent synchronization-free, robust injection of multiple reagents into the sample daughter droplets on-demand. This platform combines excellent control of valve-based microfluidics with the high-throughput capability of droplet microfluidics. We demonstrate the functioning of a proof-of-concept device which generates combinatorial mixture droplets from a linear array of sample plugs and four different reagents, using food dyes to mimic samples and reagents. Generation of a one dimensional array of the combinatorial mixture droplets on the device leads to automatic spatial indexing of these droplets, precluding the need to include a barcode in each droplet to identify its contents. We expect this platform to further expand the range of applications of droplet microfluidics to include applications requiring a high degree of multiplexing as well as high throughput analysis of multiple samples.     

A printed nanolitre-scale bacterial sensor array

The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial "spots" in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 °C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.     

Fixed-volume metering microdispenser module

In this work, we present a novel fixed-volume metering microdispenser module using the sPROMs (structurally programmable microfluidic systems) technology. We have designed, simulated, fabricated and characterized an array of microdispensers with volumes ranging from 50 nL [nanoliter] to 150 nL. We have characterized several key components of the microdispenser, such as passive microvalves and the air-driven liquid column splitting process, using extensive simulations. The fabricated devices show extremely good accuracy (99.2%) and repeatability characteristics. We also present a simple technique for unloading the sub-microL [microliter] volumes from the microfluidic chip for measurement purposes. The dispensers realized in this work have immediate applications as a key ingredient of the lab-on-a-chip device.     

Micromachined filter-chamber array with passive valves for biochemical assays on beads

The filter-chamber array presented here enables a real-time parallel analysis of three different samples on beads in a volume of 3 nL, on a 1 cm2 chip. The filter-chamber array is a system containing three filter-chambers, three passive valves at the inlet channels and a common outlet. The design enables parallel sample handling and time-controlled analysis. The device is microfabricated in silicon and sealed with a Pyrex lid to enable real-time analysis. Single nucleotide polymorphism analysis by using pyrosequencing has successfully been performed in single filter-chamber devices. The passive valves consist of plasma-deposited octafluorocyclobutane and show a much higher resistance towards water and surface-active solutions than previous hydrophobic patches. The device is not sensitive to gas bubbles, clogging is rare and reversible, and the filter-chamber array is reusable. More complex (bio)chemical reactions on beads can be performed in the devices with passive valves than in the devices without valves.     

Nanoliter-scale non-invasive flow-rate quantification using micro-interferometric back-scatter and phase detection

The current trend toward miniaturization of fluid-handling systems, particularly those of micro-fluidic devices on the capillary-scale, will certainly lead to improvements in chemical and biochemical analyses. Unfortunately, when fluid volumes reach nano- and picoliter scale it is problematic to perform non-invasive fast and accurate volume flow or flow velocity measurements. Here a simple, non-invasive method is presented for detecting and measuring linear flow velocity within fluid-filled capillaries. A small fluid volume is repeatedly heated locally by means of an infrared laser diode and using the micro-interferometric back-scatter detector (MIBD) at a fixed distance downstream, a thermally induced change in refractive index is observed when the heated volume traverses the probe volume of the detector. Fluid velocity is calculated by monitoring the phase difference between the second harmonic of the heating function and the resulting MIBD output in the Fourier domain. In a probe volume of 40 nL flow rates between I and 10 microL min(-1) are quantifiable, with 3sigma detection limits determined to be 42.8 nL min(-1).     

Scanning electrochemical microscopy (SECM) of nanolitre droplets using an integrated working/reference electrode assembly

Scanning electrochemical microscopy (SECM) has been performed in the restricted space of nanolitre droplets with a robust and easy-to-handle coaxial electrode assembly centring a Pt microdisk in a circular Ag electrode. Straightforward and reproducible fabrication of the specially designed probe tips was achieved by using Tollens reaction to chemically deposit a uniform and well-adhering layer of silver on the body of a glass-insulated Pt microdisk electrode. The suitability of the novel dual-electrode SECM tip for measurement in small volumes was evaluated by imaging an array of four Pt band microelectrodes in 500 nL electrolyte.     

Active control of nanolitre droplet contents with convective concentration gradients across permeable walls

Nanolitre droplets in microfluidic devices can be used to perform thousands of independent chemical and biological experiments while minimizing reagents, cost and time. However, the absence of simple and versatile methods capable of controlling the contents of these nanolitre chemical systems limits their scientific potential. To address this, we have developed a method that is simple to fabricate and can continuously control nanolitre chemical systems by integrating a time-resolved convective flow signal across a permeable membrane wall. With this method, we can independently control the volume and concentration of nanolitre-sized drops without ever directly contacting the fluid. Transport occurring in these systems was also analyzed and thoroughly characterized. We achieved volumetric fluid introduction and removal rates ranging from 0.23 to 4.0 pL s(-1). Furthermore, we expanded this method to perform chemical processes. We precipitated silver chloride using a flow signal of sodium chloride and silver nitrate droplets. From there, we were able to separate sodium chloride reactants with a water flow signal, and dissolve silver chloride solids with an ammonia hydroxide flow signal. Finally, we demonstrate the potential to deliver large molecules and perform physical processes like crystallization and particle packing.     

Fabricating protein immunoassay arrays on nitrocellulose using dip-pen lithography techniques

Advancements in lithography methods for printing biomolecules on surfaces are proving to be potentially beneficial for disease screening and biological research. Dip-pen nanolithography (DPN) is a versatile micro and nanofabrication technique that has the ability to produce functional biomolecule arrays. The greatest advantage, with respect to the printing mechanism, is that DPN adheres to the sensitive mild conditions required for biomolecules such as proteins. We have developed an optimised, high-throughput printing technique for fabricating protein arrays using DPN. This study highlights the fabrication of a prostate specific antigen (PSA) immunoassay detectable by fluorescence. Spot sizes are typically no larger than 8 μm in diameter and limits of detection for PSA are comparable with a commercially available ELISA kit. Furthermore, atomic force microscopy (AFM) analysis of the array surface gives great insight into how the nitrocellulose substrate functions to retain protein integrity. This is the first report of protein arrays being printed on nitrocellulose using the DPN technique and the smallest feature size yet to be achieved on this type of surface. This method offers a significant advance in the ability to produce dense protein arrays on nitrocellulose which are suitable for disease screening using standard fluorescence detection.     

Nano-polypyrrole supercapacitor arrays prepared by layer-by-layer assembling method in anodic aluminum oxide templates

A novel method for preparing nano-supercapacitor arrays, in which each nano-supercapacitor consisted of electropolymerized Polypyrrole (PPy) electrode / porous TiO₂ separator / chemical polymerized PPy electrode, was developed in this paper. The nano-supercapacitors were fabricated in the nano array pores of anodic aluminum oxide template using the bottom-up, layer-by-layer synthetic method. The nano-supercapacitor diameter was 80 nm, and length 500 nm. Based on the charge/discharge behavior of nano-supercapacitor arrays, it was found that the PPy/TiO₂/PPy array supercapacitor devices performed typical electrochemical supercapacitor behavior. The method introduced here may find application in manufacturing nano-sized electrochemical power storage devices in the future for their use in the area of microelectronic devices and microelectromechanical systems.     

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 μL/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 μM to 10 mM using this method and Z′ values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method.     

Studying protein-protein interactions using peptide arrays

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.     

Quantifying chemical composition from two-dimensional data arrays

Many analytical instruments (e.g., videofluorimeter and liquid chromatographs with spectrometric detectors) can produce two-dimensional data arrays. Rank-annihilation factor analysis enables chemical composition to be quantified from such an array. Solution by this method requires a search for a minimum value of an eigenvector which cannot be calculated even iteratively. The problem is presented here as a generalized eigenvalue problem and a direct solution is found by using singular value decomposition.     

2D and 3D spatially addressed arrays for high-throughput automated synthesis of combinatorial libraries

One of the key elements in the drug discovery process is the use of automation to synthesize libraries of compounds for biological screening. The "split-and-mix" approaches in combinatorial chemistry have been recognized as extremely powerful techniques to access large numbers of compounds, while requiring only few reaction steps. However, the need for effective encoding/deconvolution strategies and demands for larger amounts of compounds have somewhat limited the use of these techniques in the pharmaceutical industry. In this paper, we describe a concept of directed sort and combine synthesis with spatially arranged arrays of macroscopic supports. Such a concept attempts to balance the number of reaction steps, the confidence in compound identity, and the quantity of synthesized compounds. Using three-dimensional arrays of frames each containing a two-dimensional array of macroscopic solid supports, we have conceptualized and developed a modular semiautomated system with a capacity of up to 100 000 compounds per batch. Modularity of this system enables flexibility either to produce large diverse combinatorial libraries or to synthesize more focused smaller libraries, both as single compounds in 12-15 micromol quantities. This method using sortable and spatially addressed arrays is exemplified by the synthesis of a 15 360 compound library.     

Automated nanoliter solution deposition for total reflection X-ray fluorescence analysis of semiconductor samples

In this study, a BioDot BioJet dispensing system was investigated as a nanoliter sample deposition method for total reflection X-ray fluorescence (TXRF) analysis. The BioDot system was programmed to dispense arrays of 20 nL droplets of sample solution on Si wafers. Each 20 nL droplet was approximately 100 μm in diameter. A 10 × 10 array (100 droplets) was deposited and dried in less than 2 min at room temperature and pressure, demonstrating the efficiency of the automated deposition method. Solutions of various concentrations of Ni and Ni in different matrices were made from stock trace element standards to investigate of the effect of the matrix on the TXRF signal. The concentrations were such that the levels of TXRF signal saturation could be examined. Arrays were deposited to demonstrate the capability of drying 100 μL of vapor phase decomposition-like residue in the area of a typical TXRF detector.     

Nanoliter-scale reactor arrays for biochemical sensing

A general approach is described for array-based biochemical sensing that uses contact-free dispersal of compounds into addressable microfabricated reactors. The arrays are composed of 1 to 100 nL volume open reactors that have been microfabricated on quartz substrates using lithography. The open architecture of these reactors allows them to be addressed in parallel or individually with an ink-jet arrayer that is capable of distributing 0.004 to 1 nL volumes of reagents. A seven-step biochemical assay has been conducted on a small array of reactors to demonstrate how they can be integrated with an ink-jet arrayer and optical detector. This nanoreactor assay format appears to overcome several limitations that chip-based microarray technology currently imposes on protein assays: the arrays can be created in a manner that does not expose the biochemical reagents to osmotic stress, independent reactions can be conducted in individual reactors, and the conditions in all of the reactors (e.g., concentration and pH) can be rapidly scanned. We believe that these nanoreactor arrays will be useful for biochemical sensing that involves delicate proteins and protein assemblies.