A catalogue of the deMello Group publications
We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electro- phoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes fol- lowed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reprodu- cibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chro- matography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This meth- od is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout plat- forms for studies of the ribosomal proteome.
Calcium carbonate (CaCO3) is one of the most abundant minerals and of high importance in many areas of science including global CO2 exchange, industrial water treatment energy storage, and the formation of shells and skeletons. Industrially, calcium carbonate is also used in the production of cement, glasses, paints, plastics, rubbers, ceramics, and steel, as well as being a key material in oil refining and iron ore purification. CaCO3 displays a complex polymorphic behaviour which, despite numerous experiments, remains poorly characterised. In this paper, we report the use of a segmented-flow microfluidic reactor for the controlled precipitation of calcium carbonate and compare the resulting crystal properties with those obtained using both continuous flow microfluidic reactors and conventional bulk methods. Through combination of equal volumes of equimolar aqueous solutions of calcium chloride and sodium carbonate on the picoliter scale, it was possible to achieve excellent definition of both crystal size and size distribution. Furthermore, highly reproducible control over crystal polymorph could be realised, such that pure calcite, pure vaterite, or a mixture of calcite and vaterite could be precipitated depending on the reaction conditions and droplet-volumes employed. In contrast, the crystals precipitated in the continuous flow and bulk systems comprised of a mixture of calcite and vaterite and exhibited a broad distribution of sizes for all reaction conditions investigated.
We report an in-depth study of the long-term reproducibility and reliability of droplet dispensing in digital microfluidic devices (DMF). This involved dispensing droplets from a reservoir, measuring the volume of both the droplet and the reservoir droplet and then returning the daughter droplet to the original reservoir. The repetition of this process over the course of several hundred iterations offers, for the first time, a long-term view of droplet dispensing in DMF devices. Results indicate that the ratio between the spacer thickness and the electrode size influences the reliability of droplet dispensing. In addition, when the separation between the plates is large, the volume of the reservoir greatly affects the reproducibility in the volume of the dispensed droplets, creating “reliability regimes.” We conclude that droplet dispensing exhibits superior reliability as inter-plate device spacing is decreased, and the daughter droplet volume is most consistent when the reservoir volume matches that of the reservoir electrode.
Droplet-based microfluidic systems have emerged as a powerful platform for performing high-throughput biological experimentation. In addition, fluorescence polarization has been shown to be effective in reporting a diversity of bimolecular events such as protein–protein, DNA–protein, DNA–DNA, receptor–ligand, enzyme–substrate, and protein–drug interactions. Herein, we report the use of fluorescence polarization for high-throughput protein–protein interaction analysis in a droplet-based microfluidic system. To demonstrate the efficacy of the approach, we investigate the interaction between angiogenin (ANG) and antiangiogenin antibody (anti-ANG Ab) and demonstrate the efficient extraction of dissociation constants (KD = 10.4 ± 3.3 nM) within short time periods.
This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.
Current methods for screening libraries of compounds for biological activity are rather cumbersome, slow and imprecise. A method that breaks up a continuous flow of a compound's solution into droplets offers radical improvements.
Go with the (segmented) flow: A gas–liquid microfluidic reactor system has been developed to study Pd-catalyzed carbonylation reactions over a range of flow regimes and reaction conditions (see picture). The segmented gas–liquid flow regime, in comparison to annular flow, enables reactions to be studied over longer reaction times and without the buildup of unwanted Pd particles.
We describe the controlled synthesis of dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) using a stable passively-driven capillary-based droplet reactor. High quality highly crystalline particles were obtained with a narrow size distribution of mean diameter 3.6 nm and standard deviation 0.8 nm. The particles were evaluated for use in MRI, and found to exhibit a large saturation magnetisation of 58 emu/g and a high T2 relaxivity of 66 mM−1s−1 at 4.7 T, signifying good MRI contrast enhancement properties.
We present a novel method for the identification of live and dead T-cells, dynamically flowing within highly conductive buffers. This technique discriminates between live and dead (heat treated) cells on the basis of dielectric properties variations. The key advantage of this technique lies in its operational simplicity, since cells do not have to be resuspended in isotonic low conductivity media. Herein, we demonstrate that at 40 MHz, we are able to statistically distinguish between live and dead cell populations.
Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological/ chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chipbased microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cellbased assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.
The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1 In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented. Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.
The vast majority of droplet-based microfluidic devices are made from polydimethylsiloxane (PDMS). Unfortunately PDMS is not suitable for high frequency droplet generation at high operating pressure due to its low shear modulus. In this paper, we report the fabrication and testing of microfluidic devices using thermoset polyester (TPE). The optical characteristics of the fabricated devices were assessed and substrate resistance to pressure also investigated. TPE devices bonded using an O2 plasma treated PET substrate at 76 C were shown to function efficiently at pressures up to 18 MPa. TPE material retains many of the attractive features of PDMS such as ease of fabrication but significantly, has superior mechanical properties. The improved resistance of TPE to high pressures enabled investigation of high frequency droplet generation as a function of a wide range of flow-rates with three different oils as continuous phase.
Electron beam-induced shrinkage provides a convenient way of resizing solid-state nanopores in Si3N4 membranes. Here, a scanning electron microscope (SEM) has been used to resize a range of different focussed ion beam-milled nanopores in Al-coated Si3N4 membranes. Energy-dispersive X-ray spectra and SEM images acquired during resizing highlight that a time-variant carbon deposition process is the dominant mechanism of pore shrinkage, although granular structures on the membrane surface in the vicinity of the pores suggest that competing processes may occur. Shrinkage is observed on the Al side of the pore as well as on the Si3N4 side, while the shrinkage rate is observed to be dependent on a variety of factors.
We present a passive microfluidic strategy for sorting adult
C. elegans nematodes on the basis of age and size. The separation
mechanism takes advantage of phenotypic differences
between ‘adult’ and ‘juvenile’ organisms and their behaviour in
microfluidic architectures. In brief, the microfluidic device allows
worms to sort themselves in a passive manner.
» A microfluidic worm sorter.
Pipetting and dilution are universal processes used in chemical and biological laboratories to assay and experiment. In
microfluidics such operations are equally in demand, but difficult to implement. Recently, droplet-based microfluidics has
emerged as an exciting new platform for high-throughput experimentation. However, it is challenging to vary the
concentration of droplets rapidly and controllably. To this end, we developed a dilution module for high-throughput
screening using droplet-based microfluidics. Briefly, a nanolitre-sized sample droplet of defined concentration is trapped
within a microfluidic chamber. Through a process of droplet merging, mixing and re-splitting, this droplet is combined with
a series of smaller buffer droplets to generate a sequence of output droplets that define a digital concentration gradient.
Importantly, the formed droplets can be merged with other reagent droplets to enable rapid chemical and biological
screens. As a proof of concept, we used the dilutor to perform a high-throughput homogeneous DNA-binding assay using
only nanolitres of sample.
» A microfluidic droplet dilutor
» Nature Chemistry 2011 Cover
We have previously demonstrated that FT-IR spectroscopic imaging can be used as a powerful, label-free detection method for studying laminar flows. However, to date, the speed of image acquisition has been too slow for the efficient detection of moving droplets within segmented flow systems. In this paper, we demonstrate the extraction of fast FT-IR images with acquisition times of 50 ms. This approach allows efficient interrogation of segmented flow systems where aqueous dropletsmove at a speed of 2.5 mm/s. Consecutive FT-IR images separated by 120 ms intervals allow the generation of chemical movies at eight frames per second. The technique has been applied to the study of microfluidic systems containing moving droplets of water in oil and droplets of protein solution in oil. The presented work demonstrates the feasibility of the use of FT-IR imaging to study dynamic systems with subsecond temporal resolution.
The development of ultrafast Raman-based detection is one of the most interesting challenges underpinning the application of droplet-based microfluidics. Herein, we describe the use of surface-enhanced resonance Raman spectroscopy (SERRS) with submillisecond time resolution as a powerful detection tool in microdroplet reactors. Individual droplets containing silver nanoparticle aggregates functionalized with Raman reporters are interrogated and characterized by full spectra acquisitions with high spatial resolution in real time. Whereas previous works coupling SERRS with droplet-based microfluidics acquire a single spectrum over single or multiple droplets, we build upon these results by increasing our temporal resolution by 2 orders of magnitude. This allows us to interrogate multiple points within one individual droplet. The SERRS signals emitted from the aggregates are utilized to access the influence of flow rate on droplet size and throughput. Accordingly, our approach allows for high-throughput analysis that facilitates the study of other biological assays or molecular interactions.
The United Kingdom has a long history of innovation and development, much of which has its roots in the Industrial Revolution of the 18th and 19th centuries. Starting with the mechanisation of the textile manufacturing industry, major advances, particularly in the mechanical and chemical engineering fields, soon followed. One of the early pioneers of mechanical systems was James Watt who made significant improvements to steam engines, turning them into reliable, powerful, energy efficient machines that would drive the manufacturing centres of the country. His partner John Roebuck was one of the early pioneers (along with figures such as William Henry Perkin) to develop new methods for the mass production of chemicals, laying the foundations for the chemical industry as we know it today. Indeed, by the end of the 19th century Britain had large industrial plants manufacturing a large array of chemicals, and was exporting products and technology all over the world.
We demonstrate a compact, low cost and practical fluorescence detection system for lab-on-a-chip applications. The system comprises a commercially available InGaN light emitting diode (501 nm) as light source, an organic or silicon photodiode detector, absorptive dye coated colour filters and linear and reflective polarisers. An injection moulded polystyrene microfluidic chip is used as the platform for fluorescence immunoassays for cardiac markers myoglobin and CK-MB. The optical limit of detection (LOD) is measured using a TransFluoSphere suspension at 5.6e4 beads/µl which can be equated to ~3 nM fluorescein equivalent concentration. The LOD for the human plasma immunoassays is measured as 1.5 ng/ml for both myoglobin and CK-MB.
The behaviour of droplets entering a microfluidic chamber designed to house microelectrode detectors for real time analysis of clinical microdialysate is described. We have designed an analysis chamber to collect the droplets produced by multiphase flows of oil and artificial cerebral spinal fluid. The coalescence chamber creates a constant aqueous environment ideal for the placement of microelectrodes avoiding the contamination of the microelectrode surface by oil. A stream of alternating light and dark coloured droplets were filmed as they passed through the chamber using a high speed camera. Image analysis of these videos shows the colour change evolution at each point along the chamber length. The flow in the chamber was simulated using the general solution for Poiseuille flow in a rectangular chamber. It is shown that on the centre line the velocity profile is very close to parabolic, and an expression is presented for the ratio between this centre line velocity and the mean flow velocity as a function of channel aspect ratio. If this aspect ratio of width/height is 2, the ratio of flow velocities closely matches that of Poiseuille flow in a circular tube, with implications for connections between microfluidic channels and connection tubing. The droplets are well mixed as the surface tension at the interface with the oil dominates the viscous forces. However once the droplet coalesces with the solution held in the chamber, the no-slip condition at the walls allows Poiseuille flow to take over. The meniscus at the back of the droplet continues to mix the droplet and acts as a piston until the meniscus stops moving. We have found that the no-slip conditions at the walls of the chamber, create a banding effect which records the history of previous drops. The optimal position for sensors is to be placed at the plane of droplet coalescence ideally at the centre of the channel, where there is an abrupt concentration change leading to a response time <<16 ms, the compressed frame rate of the video. Further away from this point the response time and sensitivity decrease due to convective dispersion.
We describe a simple technique for fabricating non-emissive colour filters based on the sensitisation of a highly porous nanostructured metal-oxide film with a monolayer of dye molecules. Ultrafast electron transfer at the oxide/dye interface induces efficient quenching of photogenerated excitons in the dye, reducing the photoluminescence quantum yield by many orders of magnitude. The resultant filters exhibit much less autofluorescence than conventional colour filters (where the chromophore is dispersed in a glass or polymer host), and are a viable low cost alternative to interference filters for microfluidic devices and other applications requiring non-emissive filtering.
We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.
Positron emission tomography (PET) is an imaging technique routinely used for screening, diagnosing and staging chronic conditions such as cancer and neurodegenerative diseases. In addition to clinical applications, PET is also widely used to gain a fundamental understanding of the underlying biology of these diseases and to discover new treatments. All PET scans require a positron emitting radioisotope to enter the body, usually in the form of an injected radiopharmaceutical.
Aqueous droplets were used as a tool to confine a molecular population and enable highly efficient detection at the single-molecule level. Picoliter-sized aqueous droplets were generated using classical multiphase microfluidics with an aqueous stream containing the analyte under investigation and an oil carrier phase. The droplets were then localized and isolated in specially designed trapping areas within the microfluidic channel to provide a static environment for detection of the encapsulated molecules. We show that by continuously flowing the carrier oil phase while holding the aqueous stationary, we can significantly improve on measuring repeat single-molecule events. Further, we find that the flowing oil stream induces a circulation within the trapped droplets which is proportional to the volumetric flow velocity. This circulation phenomenon allows a given molecule to be detected multiple times during an experiment and can therefore be used for performing time-dependent single-molecule analysis.
We report recent advances in the field of droplet-based microfluidics. Specifically, we highlight the unique features of such platforms for high-throughput experimentation; describe functional components that afford complex analytical processing and report on applications in synthesis, high-throughput screening, cell biology and synthetic and systems biology. Issues including the integration of high-information content detection methods, long term droplet stability and opportunities for large scale and intelligent biological experimentation are also discussed.
Naphyrone (naphthylpyrovalerone,O-2482) hasbeen recently advertised forpurchase on a number of websites. This compound has been viewed as a so-called ‘legal high’ and was classified as a controlled drug under the UK Misuse of Drugs Act 1971 in mid-July 2010. So far, naphyrone is commonly equated with 1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one (β-naphyrone) but analytical characterization of two naphyrone samples revealed the existence of a novel isomer consistent with 1- naphthalen-1-yl-2-pyrrolidin-1-yl-pentan-1-one (α-naphyrone). Analyses of both α- and β-naphyrone were carried out using gas chromatography ion trap (EI/CI)mass spectrometry and 1D/2D nuclear magnetic resonance spectroscopy. This provides the first report of α-naphyrone in the scientific literature and the ability to differentiate it from the β-isomer should be of interest to forensic and clinical communities.
Over the past few decades the concept of miniaturization has been earnestly applied to chemical and biological problems. For example, much interest has been focused on the development of lab-on-a-chip or microfluidic technology. Largely, this has been driven by a need to accomplish rapid analysis of the small sample volumes that are typical in genomics, drug discovery, highthroughput screening and medical diagnostics. However, at a basic level, the appeal of microfluidic technology has been motivated by the fact that physical processes can be more easily controlled, accelerated and exploited when instrumental dimensions are reduced to a micron or submicron scale. The idea of miniaturization, pushed to an even greater extreme, has led to the emergence of the field of nanotechnology. Approaching the nanometer scale, bulk properties start to give way to molecular and atomic interactions, often leading to novel phenomena. The past decade has seen enormous growth in our understanding of and ability to characterize objects with nanometer-range dimensions. Importantly, these recent advances in nanoscience now show great promise in a diverse array of endeavors such as drug delivery, electronics, optical detection and sensing. The current issue highlights some of the emerging themes in the fields of micro- and nanotechnology and provides a glimpse of how the concept of miniaturization is beginning to impact chemical and biological sciences.
The use of dielectrophoretic forces is crucially tied to the knowledge of Joule heating within a fluid, since the use of planar microelectrodes creates a temperature gradient within which the particle of interest is manipulated. Mapping temperature with sufficient spatial resolution within a dielectrophoretic trap is recognized to be of high importance. Herein, we demonstrate local temperature measurements in the vicinity of a trapped micrometer- size particle using confocal fluorescence spectroscopy. Such measurements are shown to provide a novel calibra- tion tool for screening temperature-mediated processes with high resolution.
We describe the use of two passive components to achieve controllable and alternating droplet generation in a microfluidic device. The approach overcomes the problems associated with irregularities in channel dimensions and fluid flow rates, and allows precise pairing of alternating droplets in a high-throughput manner. We study droplet generation and self-synchronization in a quantitative fashion by using high-speed image analysis.
Distinguishing specific cells is an essential technique in cell research and clinical diagnostics. We report
a novel method to passively isolate and extract cells in a microfluidic device. We utilise a droplet-based
microfluidic system to generate an aqueous two phase system in which aqueous droplets consist of two
phases in the form of a double emulsion. Specifically, we generate PEG droplets that completely
encapsulate DEX droplets within a microfluidic channel. Target cells can be introduced directly into the
droplets and driven to partition to the more favourable phase, whilst still being contained within the
aqueous droplet. Human T lymphoma cells, with diameters in the range of 10–15 μm, are chosen as
a model cell line to demonstrate the partitioning.
» Chemical Science 2010 Cover
For over a decade the chemical, biological and analytical sciences have exploited microfluidic technology for applications ranging from cell culture to DNA amplification and analysis. More recently, microfluidic technology has been applied to the rapid synthesis of radiolabelled compounds and radiopharmaceuticals used in positron emission tomography (PET). Interest in the application of microfluidics for high speed radiolabelling stems from the inherent advantages such miniaturised reaction environments have over conventional methods. This mini-review introduces, to the nonspecialist, the extensive area of microfluidics and some of the main challenges associated with radiolabelling procedures using short-lived positron emitting isotopes. It will include a discussion of the application of microfluidics for 18F radiolabelling reactions; an area that is relatively immature. The field of PET imaging is currently growing rapidly, with increasing demand for the production of existing PET tracers and the creation of new ones. It is widely expected that microfluidic technologies will make an important contribution to improving radiotracer synthesis for PET imaging.
The difficulty of preparing nanomaterials in a controlled, reproducible manner is a key obstacle to the proper exploitation of many nanoscale phenomena. An automated chemical reactor capable of producing (on demand and at the point of need) high-quality nanomaterials, with optimized physicochemical properties, would find numerous applications in nanoscale science and technology, especially in the areas of photonics, optoelectronics, bio-analysis, and targeted drug-delivery. In addition such a device would find immediate and important applications in toxicology, where it is essential to characterize the physiological effects of nanoparticles not only in terms of chemical composition but also in terms of size, shape, and surface functionalization. In this chapter, we describe recent advances in the development of microfluidic reactors for controlled nanoparticle synthesis and, more specifically, work in our group aimed at developing just such an automated reactor.
A versatile approach for the rapid prototyping of microfluidic devices suitable for use with FT-IR spectroscopic imaging is introduced. Device manufacture is based on the direct printing of paraffin onto the surface of an infrared transparent substrate, followed by encapsulation. Key features of this approach are low running costs, rapid production times, simplicity of design modifications and suitability for integration with FT-IR spectroscopic measurements. In the current experiments, the minimum width of channel walls was found to be ~120 µm and ~200 µm when a 25 µm and 12 µm spacer is used, respectively. Water and poly(ethylene glycol) are used as model fluids in a laminar flow regime, and are imaged in both transmission and attenuated total reflection (ATR) modes. It is established that adoption of transmission mode measurements yields superior sensitivity whilst the ATR mode is more suitable for quantitative analysis using strong spectral absorption bands. Results indicate that devices manufactured using this approach are suitable for use with in situ FT-IR spectroscopic imaging.
Microfluidic devices have many applications in chemistry and biology, but practical hitches associated with their use are often overlooked. One such device that optimizes catalysts tackles these issues head-on.
Microdroplets generated in microfluidic channels hold great promise for use as substrates in high-throughput chemical and biological analysis. These water-in-oil compartments can serve as isolated reaction vessels, and since they can be generated at rates in excess of 1 kHz, thousands of assays can be carried out quickly and reproducibly. Nevertheless, sampling the large amount of information generated from these platforms still remains a significant challenge. For example, considering the high droplet generation rates and velocities, reproducibility and micrometer resolution are challenging requirements that must be fulfilled. Herein we combine confocal fluorescence lifetime imaging microscopy with a statistical implementation that permits the analysis of mixing phenomena within microdroplets with a temporal resolution of 1 μs. Importantly, such exquisite resolution is only possible as a result of the large number of droplets sampled and their high structural reproducibility.
Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cells) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues.
We present a simple, automated method for high-throughput
formation of droplet interface bilayers (DIBs) in a microfluidic
device. We can form complex DIB networks that are able to fill
predefined three dimensional architectures. Moreover, we
demonstrate the flexibility of the system by using a variety of lipids
including 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC)
and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
» Chemical Communications 2010 Cover
We report recent progress in the development of surface-enhanced Raman scattering (SERS)-based
optofluidic platforms for the fast and sensitive detection of chemical and biological analytes. In the
current context, a SERS-based optofluidic platform is defined as an integrated analytical device
composed of a microfluidic element and a sensitive Raman spectrometer. Optofluidic devices for SERS
detection normally involve nanocolloid-based microfluidic systems or metal nanostructure-embedded
microfluidic systems. In the current review, recent advances in both approaches are surveyed and
assessed. Additionally, integrated real-time sensing systems that combine portable Raman
spectrometers with microfluidic devices are also reviewed. Such real-time sensing systems have
significant utility in environmental monitoring, forensic science and homeland defense applications.
» Analyst 2010 Cover
The evaluation and selection of the most appropriate catalyst for a chemical transformation is an important process in many areas of synthetic chemistry. Conventional catalyst screening involving batch reactor systems can be both time-consuming and expensive, resulting in a large number of individual chemical reactions. Continuous flow microfluidic reactors are increasingly viewed as a powerful alternative format for reacting and processing larger numbers of small-scale reactions in a rapid, more controlled and safer fashion. In this study we demonstrate the use of a planar glass microfluidic reactor for performing the three-component palladium-catalysed aminocarbonylation reaction of iodobenzene, benzylamine and carbon monoxide to form N-benzylbenzamide, and screen a series of palladium catalysts over a range of temperatures. N-Benzylbenzamide product yields for this reaction were found to be highly dependent on the nature of the catalyst and reaction temperature. The majority of catalysts gave good to high yields under typical flow conditions at high temperatures (150 oC), however the palladium(II) chloride-Xantphos complex [PdCl2 (Xantphos)] proved to be far superior as a catalyst at lower temperatures (75–120 oC). The utilised method was found to be an efficent and reliable way for screening a large number of palladium-catalysed carbonylation reactions and may prove useful in screening other gas/liquid phase reactions.
A droplet-based microfluidic system combined with high-sensitivity optical detection is used as a tool for high-throughput confinement and detection of single DNA molecules.
The isolation and characterisation of single cells from a heterogeneous population are important processes in cell biology, immunology, stem cell research, and cancer research. In the development of novel cell-based therapies, there is a considerable need to target specific cell types to allow for further analysis and amplification ex vivo. We introduce, herein, the use of droplet-based microfluidics as a platform technology for the identification and quantification of distinct cell phenotypes. Using molecular labelling of specific cell populations by antibodies and fluorescent dyes, detection of single cells encapsulated within picolitre-sized aqueous droplets can be performed using high-sensitivity confocal fluorescence detection. Specifically, rare progenitor cells were immunodetected within a heterogeneous population of cells isolated from human periosteal tissue. Using this model human cell population, the accuracy and reproducibility of the droplet system were tested and the results were verified using conventional flow cytometry. It was found that the quantitation of phenotypic subpopulations measured using both techniques is directly comparable. Accordingly, this study demonstrates the biological capacity of droplet-based microfluidics for cellular analysis and provides a necessary first step towards the development of a novel cell sorting technology.
We report a method for removing unwanted contributions to fluorescence signals from dyes absorbed in polydimethylsiloxane (PDMS) using fluorescence lifetime imaging microscopy (FLIM). By analysing experimental fluorescence decays using a bi-exponential decay model, we are able to discriminate between emission originating from dye molecules in free solution and those absorbed within the PDMS substrate. Simple image processing allows the unwanted background signal to be removed and thus enables a more accurate assessment of temperature. The efficacy of the approach is demonstrated by measuring temperature changes within a droplet-based PCR device.
We demonstrate that nanolitre-sized droplets are an effective tool in coupling two-dimensional separations in both time and space. Using a microfluidic droplet connector, chemically separated components can be segmented into nanolitre droplets. After oil filtering and droplet merging, these droplets are loaded into a second dimension for comprehensive separations.
Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.
We successfully demonstrate the merging of aqueous droplets within a microfluidic channel mediated by a difference in interfacial tension. Interfacial tension is shown to have a significant influence on the hydrodynamic forces associated with a segmented flow in a rectangular microchannel and results in the possibility of merging multiple droplets in a simple fashion. This facility is important in allowing droplet-based microfluidic systems to be used as synthetic tools in complex reaction processing.
Elucidating the chemical composition of microfluidic flows is crucial in both understanding and optimising reactive processes within small-volume environments. Herein we report the implementation of a novel detection methodology based on Attenuated Total Reflection (ATR)–Fourier Transform Infra-Red (FTIR) spectroscopic imaging using an infrared focal plane array detector for microfluidic applications. The method is based on the combination of an inverted prism-shape ATR crystal with a poly(dimethylsiloxane)-based microfluidic mixing device. To demonstrate the efficacy of this approach, we report the direct measurement and imaging of the mixing of two liquids of different viscosities and the imaging and mixing of H2O and D2O with consecutive H/D isotope exchange. This chemically specific imaging approach allows direct analysis of fluid composition as a function of spatial position without the use of added labels or dyes, and can be used to study many processes in microfluidics ranging from reactions to separations.
In this paper we describe a universal mechanism for merging multiple aqueous microdroplets within a flowing stream consisting of an oil carrier phase. Our approach involves the use of both a pillar array acting as a passive merging element, as well as built-in electrodes acting as an active merging element. The pillar array enables slowing down and trapping of the droplets via the drainage of the oil phase. This brings adjacent droplets into close proximity. At this point, an electric field applied to the electrodes breaks up the thin oil film surrounding the droplets resulting in merging.
One of the primary goals of current proteomics research is to
understand the physiological and metabolic pathways of cells
and thereby establish molecular diagnostic tools that are able
to identify the proteins associated with disease states. More
specifically, protein–protein interactions are critical for many
biological functions. For example, signal mediation between
the exterior and interior of a cell is normally a result of protein–
protein interactions. Such signal transduction plays an elementary
role in many biological processes and in many diseases
(such as cancer). Accordingly, the ability to probe protein–
protein interactions in a high-throughput manner is recognised
to be important in developing effective diagnostic techniques,
cultivating disease therapies and discovering new small-molecule
drug candidates.
» ChemBioChem 2009 Cover
We introduce microfluidics technologies as a key foundational technology for synthetic biology experimentation. Recent advances in the field of microfluidics are reviewed and the potential of such a technological platform to support the rapid development of synthetic biology solutions is discussed.
The epoxidation of styrene using a modularly constructed polymer immobilized Mn(III)-salen catalyst has been demonstrated within a continuous-flow glass fabricated microfluidic reactor.
We demonstrate that solution processed thin-film organic photodiodes (OPDs) can be used as compact and sensitive integrated detectors for antioxidant capacity screening. The OPDs were fabricated with blends of regioregular poly(3-hexylthiophene) (P3HT) and (6,6)-phenyl C61 butyric-acid methyl-ester (PCBM). The devices had a broadband photoresponse from 350 nm to 650nm with a peak responsivity of 0.25 A/W at 550nm and a dark current density of 0.59μA/cm2 under 10mV bias for a device area of 1mm2. The signal rise and fall times of the detectors were 0.51μs and 0.66μs, respectively. The detectorswere applied to an on-chip peroxyoxalate chemiluminescence (PO-CL) assay for antioxidant capacity determination. Antioxidant standards were injected into a stream of PO-CL reagents, resulting in a CL emission decrease that correlated with the antioxidant capacity. For the encountered CL signals the OPDs provided a comparable response to photomultiplier tubes (PMTs) commonly used in analytical applications. Antioxidant capacity screening results showed excellent consistency between the two detection methods. The compact and portable detection system is suited not only to low-cost in-the-field antioxidant capacity screening, but could have wider applications for chemiluminescence based diagnostic tests at the point-of-care.
We report a new method for the trace analysis of mercury (II) ions in water. The approach involves the use of droplet-based microfluidics combined with surface-enhanced Raman scattering (SERS) detection. This novel combination provides both fast and sensitive detection of mercury (II) ions in water. Specifically, mercury (II) ion detection is performed by using the strong affinity between gold nanoparticles and mercury (II) ions. This interaction causes a change in the SERS signal of the reporter molecule rhodamine B that is a function of mercury (II) ion concentration. To allow both reproducible and quantitative analysis, aqueous samples are encapsulated within nanoliter-sized droplets. Manipulation of such droplets through winding microchannels affords rapid and efficient mixing of the contents. Additionally, memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, are removed since the aqueous droplets are completely isolated by a continuous oil phase. Quantitative analysis of mercury (II) ions was performed by calculating spectral peak area of rhodamine B at 1,647 cm−1. Using this approach, the calculated concentration limit of detection was estimated to be between 100 and 500 ppt. Compared with fluorescence-based methods for the trace analysis of mercury (II) ions, the detection sensitivities were enhanced by approximately one order of magnitude. The proposed analytical method offers a rapid and reproducible trace detection capability for mercury (II) ions in water.
To realise effective size separations of nucleic acid fragments using CE, gel-based matrices are commonly employed. The separation of label-free dsDNA ladders and plasmid fragments in an uncross-linked semi-dilute poly (ethylene) oxide solution using multi-pixel UV detection at 254 nm is reported. Improvements in the sensitivity of UV detection of dsDNA using signal averaging over multiple pixels is demonstrated. Separations performed using a diode array detector also allow the progress of the separation to be monitored as a function of time. Several polymers were examined including methyl cellulose, linear polyacrylamide, hydroxy (propyl) methylcellulose and polyethylene oxide. Operations parameters investigated included UV transparency, selfcoating capacity and separation efficiency. The results show complete resolution of all fragments under a range of conditions, including short separation lengths.
Heavily-doped ZnO:Al films have been deposited on high temperature stable glass substrates using radio-frequency (RF) magnetron sputtering. The effect of growth temperature on physical properties of the films has been investigated. The microstructure evolved a columnar structure into a granular one with the increase in growth temperature and then a typical honeycomb-type microstructure representing huge grain formation indicating high densification. All Al-doped ZnO films exhibited high optical transparency and the absorption edge shifted to the short wavelength (blue-shift) as the growth temperature increased. The dense microstructure with a high crystallographic quality and large grains evolved at 500 °C enabled us to obtain 2.28 × 10–3 Ω cm and high visible transmittance over 90% even if the ZnO film was doped with an Al content of approximately 5.5 at%.
The quest for miniaturization of chemical reactors is leading to a tangled web of reaction vessels, each formed at the junction of polymer nanofibres.
We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools—SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.
We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 × 106-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.
» Continuous flow PCR in droplets
One of the primary motivations behind the development of miniaturised analysis devices has been to create new tools for modern day genomic and genetic analysis.1 At present, much effort is directed towards designing faster and more efficient DNA analysis devices that could potentially identify the genes responsible for specific diseases.2-4 One increasingly popular approach is confinement and detection of single analyte molecules within nanofluidic structures. Such devices, have at least one dimension of the channel measuring less than a few hundred nanometres.5 One of the main advantages of nanofluidics is in the ability to confine single molecules within a well defined space in order to be efficiently detected. Importantly, probing molecules at the single molecule level is essential if one wants to measure fluctuations usually lost in ensemble averaged techniques.
High-throughput screening (HTS) is a method of scientific experimentation widely used in drug discovery and relevant to the fields of biology. The development of micro- and nanofluidic systems for use in the biological sciences has been driven by a range of fundamental attributes that accompany miniaturization and massively parallel experimentation. We review recent advances in both arraying strategies based on nano/ microfluidics and novel nano/microfluidic devices with high analytical throughput rates.
We describe the design, fabrication and use of a single-layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in an array format controlled entirely by liquid flow. Aqueous picoliter droplets are trapped en masse and optically monitored for extended periods of time. Such an array-based approach is used to characterize droplet shrinkage, aggregation of encapsulated E. coli cells and enzymatic reactions. We also demonstrate that trapped droplets may be recovered from the microfluidic array for further processing.
We present a novel planar electrode geometry in which particles (typically 10 μm in diameter) are focused near a defined surface before being trapped using negative dielectrophoresis. The focusing element can deflect particles having speeds up to hundreds of micrometers per second. This trapping configuration results in improved trapping yields and a decrease in overall reagent consumption. Particles are trapped dynamically while flowing in a microfluidic channel.
A novel method is presented for controllably merging aqueous microdroplets within segmented
flow microfluidic devices. Our approach involves exploiting the difference in hydrodynamic
resistance of the continuous phase and the surface tension of the discrete phase through the use of
passive structures contained within a microfluidic channel. Rows of pillars separated by distances
smaller than the representative droplet dimension are installed within the fluidic network and
define passive merging elements or chambers. Initial experiments demonstrate that such a merging
element can controllably adjust the distance between adjacent droplets. In a typical scenario, a
droplet will enter the chamber, slow down and stop. It will wait and then merge with the
succeeding droplets until the surface tension is overwhelmed by the hydraulic pressure. We show
that such a merging process is independent of the inter-droplet separation but rather dependent on
the droplet size. Moreover, the number of droplets that can be merged at any time is also
dependent on the mass flow rate and volume ratio between the droplets and the merging chamber.
Finally, we note that the merging of droplet interfaces occurs within both compressing and the
decompressing regimes.
» Pillar-induced droplet merging in a microfluidic channel
We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide timeresolved kinetic measurements.
Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0e6 - 4.5e7 [1/(M.s)]
Fluorescence lifetime imaging (FLIM) is used to quantitatively map the concentration of a small molecule in three dimensions in a microfluidic mixing device. The resulting experimental data are compared with computational fluid-dynamics (CFD) simulations. A line-scanning semiconfocal FLIM microscope allows the full mixing profile to be imaged in a single scan with submicrometer resolution over an arbitrary channel length from the point of confluence. Following experimental and CFD optimization, mixing times down to 1.3±0.4 ms were achieved with the single-layer microfluidic device.
Water-in-oil microdroplets within fluidic channels have the potential to serve as isolated reaction compartments for monitoring real-time dynamics with high efficiency and repeatability. Droplets, usually generated from aqueous and oil solutions using standard microfluidic formats, can be produced at frequencies in excess of 1 kHz. Although mixing within such microdroplets is normally enhanced by chaotic advection, the mixing pattern from droplet to droplet is almost identical and reproducible in form. Herein, we demonstrate that fluorescence lifetime imaging can be used to reconstruct mixing patterns within a droplet with a time resolution of 5 μs.
The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for applications within the chemical and biological sciences. Interest in microfluidic systems has been stimulated by a range of fundamental features that accompany system miniaturization. Such features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit cost, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Moreover, microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the added advantage of allowing ultra-high throughput experimentation and being able to mimic conditions similar to that of a single cell (in terms of volume, pH, and salt concentration) thereby compartmentalizing biological and chemical reactions. This review provides an overview of methods for generating, controlling and manipulating droplets. Furthermore, we discuss key fields of use in which such systems may make a significant impact, with particular emphasis on novel applications in the biological and physical sciences.
We numerically assess the light propagation and distribution characteristics of electromagnetic fields on nanopores formed in dielectric and metal/dielectric membranes using a frequency-domain finite element method (3D full-wave electromagnetic field simulation). Results of such studies were used to identify aluminum as an ideal material for use in optically thick metal/dielectric membranes. The comparison between SiN and Al/SiN membranes (with and without a submicron sized aperture) was numerically and experimentally shown to verify the effect of optically thick metal layers on light propagation and fluorescence excitation. The cut-off behavior for Al/SiN membranes with varying pore diameters was investigated in terms of light propagation, distribution of electromagnetic fields, and light attenuation characteristics.
Wall adsorption is a common problem in microfluidic devices, particularly when proteins are used. Here we show how superhydrophobic surfaces can be used to reduce protein adsorption and to promote desorption. Hydrophobic surfaces, both smooth and having high surface roughness of varying length scales (to generate superhydrophobicity), were incubated in protein solution. The samples were then exposed to flow shear in a device designed to simulate a microfluidic environment. Results show that a similar amount of protein adsorbed onto smooth and nanometer-scale rough surfaces, although a greater amount was found to adsorb onto superhydrophobic surfaces with micrometer scale roughness. Exposure to flow shear removed a considerably larger proportion of adsorbed protein from the superhydrophobic surfaces than from the smooth ones, with almost all of the protein being removed from some nanoscale surfaces. This type of surface may therefore be useful in environments, such as microfluidics, where protein sticking is a problem and fluid flow is present. Possible mechanisms that explain the behaviour are discussed, including decreased contact between protein and surface and greater shear stress due to interfacial slip between the superhydrophobic surface and the liquid.
In this chapter we describe the use of organic semiconductor-based light sources and photodiodes for integrated optical detection in lab-on-a-chip devices. The application of this low-cost detection technology to portable diagnostic devices for the point-of-care is also outlined.
We report that highly compact, bright light sources and sensitive detectors made from solution processed thin-film polymer organic light emitting diodes (OLEDs) and organic photodiodes (OPDs) can be widely used for on-chip absorbance based detection of a wide range of analytes for in-the-field bio-analytical and point-of-care clinical diagnostic applications.
The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reac- tors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spec- troscopy as a high-efficiency detection method for droplet- based microfluidics. Issues such as surface contamina- tion, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow- based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet com- positions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentra- tions. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.
We describe a novel approach for optically detecting DNA translocation events through an array of solid-state nanopores that potentially allows for ultra high-throughput, parallel detection at the single-molecule level. The approach functions by electrokinetically driving DNA strands through sub micrometer-sized holes on an aluminum/silicon nitride membrane. During the translocation process, the molecules are confined to the walls of the nanofluidic channels, allowing 100% detection efficiency. Importantly, the opaque aluminum layer acts as an optical barrier between the illuminated region and the analyte reservoir. In these conditions, high-contrast imaging of single-molecule events can be performed. To demonstrate the efficiency of the approach, a 10 pM fluorescently labeled λ-DNA solution was used as a model system to detect simultaneous translocation events using electron multiplying CCD imaging. Single-pore translocation events are also successfully detected using single-point confocal spectroscopy.
We describe an autonomous ‘black-box’ system for the controlled synthesis of fluorescent nanoparticles. The system uses a microfluidic reactor to carry out the synthesis and an in-line spectrometer to monitor the emission spectra of the emergent particles. The acquired data is fed into a control algorithm which reduces each spectrum to a scalar ‘dissatisfaction coefficient’ and then intelligently updates the reaction conditions in an effort to minimise this coefficient and so drive the system towards a desired goal. In the tests reported here, CdSe nanoparticles were prepared by separately injecting solutions of CdO and Se into the two inlets of a heated y-shaped microfluidic reactor. A noise-tolerant global search algorithm was then used to efficiently identify—without any human intervention—the injection rates and temperature that yielded the optimum intensity for a chosen emission wavelength.
In 1828, when attempting to prepareammoniumcyanate from silver cyanide andammoniumchloride, Friedrich Wöhler accidentally synthesized urea. Contemporary wisdom at the time held that organic compounds could only be created from a “vital force,” which existed within living organisms. In a letter to Jöns Jakob Berzelius, Wöhler’s excitement was apparent; “I can no longer, so to speak, hold my chemical water and must tell you that I can make urea without needing a kidney, whether of man or dog; the ammonium salt of cyanic acid is urea.” Although Wöhler’s discovery was not the first synthesis of an organic compound it sparked huge interest in making organic compounds from nonliving substances and marked the beginning of organic chemistry as an academic and industrial discipline. Since that time, organic chemistry (the study of the structure, properties, composition, reactions, and synthesis of carbon-containing compounds) has flourished and is of vital importance to the pharmaceutical, chemical, cosmetic, petrochemical, and textile industries. For the vast majority of the time, since (and for centuries before) Wöhler’s synthesis of urea, the chemist’s toolkit has predominantly consisted of macroscopic components fabricated from glass. Examples of such components include round-bottomed flasks, test tubes, distillation columns, reflux condensers, Erlenmeyer flasks, drying tubes, separation funnels, recrystallization tubes, and burettes. Despite the enormous advances that have been made in experimental, mechanistic, and theoretical organic chemistry over the past 150 years it is noteworthy that the basic experimental techniques and associated laboratory equipment remain largely unaffected. Perhaps this is unsurprising, since standard laboratory glassware provides a fitting environment in which to perform the vast majority of synthetic transformations. Glass as a generic material is robust, possessing good chemical inertness. It also exhibits high thermal conductivity, low electrical conductivity, and good transparency within the visible region of the electromagnetic spectrum. This means that standard glassware can be used to process most chemical reagents under a wide range of temperatures and pressure. A more pragmatic reason for the adoption of macroscale glassware in organic synthesis relates to the fact that chemists as individuals feel comfortable performing reactions in environments, which they can easily manipulate, control, and observe. Nevertheless, although a chemist may prefer to perform a reaction within a round bottom flask, at the molecular scale it makes little difference if a reaction is processed within a volume of 100 mL or 100 pL. What is more important is that the “ideal” chemical reactor should provide an environment in which chemical state functions are precisely controllable, allowing the rapid synthesis of a desired product in high yield. To this end, over the past decade, the application of micromachining techniques cultivated within the semiconductor and microelectronics industries have allowed the creation of a new instrumental platform able to efficiently process and analyze molecular reactions on the micron to nanometer scale. This chapter aims to provide an introduction to the concept of reaction miniaturization. The theory of miniaturization is discussed and followed by a practical assessment of the use of microfluidic systems in synthetic chemistry.
We report polymer light emitting diodes fabricated on flexible poly(ethyleneterephthalate) substrates coated with a layer of poly(3,4-ethylene-dioxythiophene) : poly(styrenesulfonate) that was lithographically patterned to define the anode structure. A blend of poly(9,9-dioctylfluoreneco- benzothiadiazole) and poly(9,9-dioctylfluorene-co-N-(4-butylphenyl)diphenylamine) was then spin-coated on top as the emissive layer and the device was completed by vacuum deposition of a LiF/Al bilayer cathode. The resulting yellow light emitting diodes had typical peak power and current efficiencies of 13.7 lm/W and 8.8 cd/A respectively, which compare well with values for similar devices fabricated on ITO-coated rigid glass substrates. A maximum luminance in excess of 7300 cd/m^2 was achieved.
We present a method for improving detection efficiencies in single molecule confocal fluorescence spectroscopy with subfemtoliter probe volumes within microfluidic channels. Our approach is based on hydrodynamically focusing an analyte stream within a microfluidic channel down to a width on the same order of magnitude as that of the confocal probe volume. Experiments are performed in which fluorescently labeled polystyrene microspheres 930 nm diameter are motivated through a microchannel and passed through a focused laser beam at a variety of flow rates 0.1 −11 μL/min. Hydrodynamic focusing of the analyte stream is achieved by introduction of two sheath flow streams that flank the central analyte stream. Through variation of the relative flow rates in each input stream the analyte stream can be controlled with micron resolution. A maximum hydrodynamic focusing width of 3 μm was achieved within a 50 μm wide microfluidic channel; hence, a larger proportion of molecules traveling through the microfluidic device were detected. Furthermore, simple statistical methods are used to investigate the resulting fluorescence bursts and generate single-particle burst width and burst area distributions. From these data, the total detection efficiencies are shown to be an order of magnitude better than those achievable in conventional unfocused systems.
The development of new strategies for the synthesis of positron-emitting radiolabeled compounds is of great current interest owing to the increased use of positron-emission tomography (PET) in medical imaging.
The authors present a simple and direct analysis method for the discrimination between different cell populations in fluidic media. The methodology is based on analysis of single particle interphoton burst recurrence times and has potential use in high precision single cell sizing and counting applications. The approach requires registration of only a few hundred photons from single fluorescent particles to distinguish between different molecular populations. The technique is simple to implement and can be designed to extract information in real time within microfluidic environments.
We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.
Recent years have seen significant progress in the development of microfabricated systems for use in the chemical and biological sciences. Much of this development has been driven by a need to perform rapid measurements on small sample volumes in areas such as chemical synthesis, DNA analysis, drug discovery, pharmaceutical screening, proteomics, and medical diagnostics. It is well recognized that, when compared to macroscale instruments, microfluidic systems engender a number of distinct advantages with respect to speed, analytical throughput, reagent usage, process control, automation, and operational and configurational flexibility. Although all these advantages are directly facilitated by system downscaling (and the associated improvements in both mass and thermal transfer), the instantaneous-reaction volumes that characterize microfluidic systems typically range from a few picoliters to hundreds of nanoliters. This means that analyte detection and identification is a significant challenge and often defines the principal limitations of a microfluidic system. Despite this problem, a variety of detection methods have been successfully transferred and integrated with microfluidic systems.
We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes. The measured burst width characteristics are predominately governed by the fluorescence quantum yield and absorption cross section of the proteins used. It is these characteristics which were used to distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimes originating from single cells could also be determined. Average fluorescence lifetimes are determined using standard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst width distributions is expected to be extremely valuable for single-cell sorting experiments.
We report a new lithographic procedure that enables the patterning of as-received semiconducting
polymers and small molecules at the near micron level without causing discernible degradation of
the patterned material. The method involves a minimum of processing steps, requires no
modification of the active layer, and is compatible with both rigid and flexible substrates. The
technique makes use of an intermediate resist layer between the substrate and the active layer, i.e.
underneath the active layer, and involves the simultaneous patterning of the resist and active layers
in a single expose/develop step. The technique has been successfully applied to the fabrication of
flexible ITO-free light-emitting diodes and photodiodes, yielding peak quantum efficiencies of
8.8 cd A-1 and 57% respectively comparable to similar devices fabricated on ITO-coated glass. It
is also readily extendible to the patterning on a single substrate of multiple devices incorporating
different component materials, e.g. the red, green and blue pixels of a colour display.
» Journal of Materials Chemistry 2007 Cover
A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis- (phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SAHP of 3.27× 10−3 μmol−1 L), followed by α-tocopherol (SAHP of 2.36× 10−3 μmol−1 L) and quercetin (SAHP of 0.31×10−3 μmol−1 L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements.
We report the use of solution-processed thin-film organic photodiodes for microscale
chemiluminescence. The active layer of the photodiodes comprised a 1 : 1 blend by weight of the
conjugated polymer poly(3-hexylthiophene) [P3HT] and [6,6]-phenyl-C61-butyric acid-methylester
[PCBM]—a soluble derivative of C60. The devices had an active area of 1 mm 6 1 mm, and a
broad-band response from 350 to 700 nm, with an external quantum efficiency of more than 50%
between 450 and 550 nm. The photodiodes have a simple layered structure that permits facile
integration with planar chip-based systems. To evaluate the suitability of the organic devices as
integrated detectors for microscale chemiluminescence, a peroxyoxalate based chemiluminescence
reaction (PO-CL) was monitored within a poly(dimethyl-siloxane) (PDMS) microfluidic device.
Quantitation of hydrogen peroxide indicated excellent linearity and yielded a detection limit of
10 mM, comparable with previously reported results using micromachined silicon microfluidic
chips with integrated silicon photodiodes. The combination of organic photodiodes with PDMS
microfluidic chips offers a means of creating compact, sensitive and potentially low-cost
microscale CL devices with wide-ranging applications in chemical and biological analysis and
clinical diagnostics.
» Lab on a Chip 2007 Cover
We investigate the influence of poly3,4-ethylenedioxythiophene-polystyrenesulfonate PEDOT:PSS on the optoelectronic properties of polymer light-emitting diodes containing poly9,9-dioctylfluorene PFO. Electromodulation and IV luminance measurements are reported for a series of devices with bare indium tin oxide ITO or PEDOT:PSS-coated ITO anodes and Ba or Al cathodes. The ITO/PFO/Al, ITO/PFO/Ba, and ITO/PEDOT:PSS/PFO/Al devices all exhibit conventional field-induced electromodulation behavior, in both forward and reverse bias, consistent with the Stark effect SE. The ITO/PEDOT:PSS/PFO/Ba devices by contrast exhibit conventional behavior only for applied biases below the flat-band voltage; at higher biases, the field-induced SE features vanish and are replaced by anomalous charge-induced electromodulation features. This anomalous behavior is observed only when PEDOT:PSS is used in conjunction with a strongly electroninjecting cathode such as Ba, and is attributed to the presence of trapped electrons at the PEDOT:PSS-emitter interface, which screen the electric field from the bulk of the device. The enhanced field at the interface increases the rate of field-assisted hole injection into the highest occupied molecular orbital HOMO of the PFO, resulting in lower drive voltages and increased electroluminescence efficiencies.
Recent years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Much development has been driven by a need to perform rapid measurements on small sample volumes. However, at a more primary level, interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micrometre scale. Such systems define new operational paradigms and provide predictions about how molecular synthesis might be revolutionized in the fields of high-throughput synthesis and chemical production.
Microfluidic devices show much promise for the controlled synthesis of materials on the nanoscale. In this work the first report of a synthetic method for the preparation of nanoparticles of gold stabilized by an adsorbed monolayer of a thiol (monolayer protected clusters) using a microfluidic device is described. Improvements in monodispersity are observed relative to bulk synthetic methods.
We report efficient polymer photodiodes fabricated on flexible polyethyleneterephthalate (PET) substrates. The PET substrates were coated with a layer of poly(3,4-ethylene-dioxythiophene) : polystyrenesulfonate (PEDOT : PSS) that was lithographically patterned to define the anode structure. A blend of poly(3-hexylthiophene) (P3HT) and 1-(3-methoxycarbonyl)-propyl-1-phenyl- (6,6)C61 (PCBM) was then spin-coated from a 1 : 1 mixture by weight of the two components in dichlorobenzene, and the device was completed by vacuum deposition of an aluminium electrode in vacuum. The resulting photodiodes had short-circuit quantum efficiencies of 45% and peak power efficiencies of 3%, which compare favourably with values for similar devices fabricated on rigid indium tin oxide (ITO) coated glass substrates.
We report the fabrication of high quality monolithically integrated optical long-pass filters, for
use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules
directly into the microfluidic chip substrate, thereby providing a fully integrated solution that
removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a
poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured
SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with
1200 μg mL-1 Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4),
>80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare
favourably with commercially available Schott-glass long-pass filters, indicating that high quality
optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The
filters were found to be robust in use, showing only slight degradation after extended illumination
and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of
low cost high quality integrated filters represents a key step towards the development of
high-sensitivity disposable microfluidic devices for point-of-care diagnostics.
» Lab Chip 2006 Cover
We investigate the influence of carrier injection on the electric field distribution in polyfluorene-based polymer light-emitting diodes containing poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS). The devices show strong charge-induced electromodulation spectra due to the accumulation of trapped electrons close to the PEDOT:PSS/polyfluorene interface. The trapped electrons cause the potential to drop preferentially at the interface, enhancing hole injection and substantially reducing the magnitude of the electric field in the bulk semiconductor. The detailed operating mechanisms of such “trap-rich” devices are poorly understood, and in this paper we perform a series of temperature-dependent current-voltage sweeps and electromodulation measurements to clarify the role of the injected charge. We find that the devices show strong field redistribution only at room temperature and that devices operating at lower temperatures (<100 K) resemble trap-free light-emitting diodes with a uniform electric field that extends through the bulk. We consider also the effects of pixel aging and show that field redistribution effects are reduced after extended device operation.
A simple and robust derivatisation system for glass and silica microdevices is described. The device surface is coated in a onestep treatment with a highly cross-linked polystyrene/divinylbenzene/allylsiloxane copolymer. The surface derivatisation is highly resistant to solvents, acids, bases and oxidising or reducing agents.
We describe a novel method for quantitatively mapping fluidic temperature with high spatial resolution within microchannels using fluorescence lifetime imaging in an optically sectioning microscope. Unlike intensity-based measurements, this approach is independent of experimental parameters, such as dye concentration and excitation/detection efficiency, thereby facilitating quantitative temperature mapping. Micrometer spatial resolution of 3D temperature distributions is readily achieved with an optical sectioning approach based on two-photon excitation. We demonstrate this technique for mapping of temperature variations across a microfluidic chip under different heating profiles and for mapping of the 3D temperature distribution across a single microchannel under applied flow conditions. This technique allows optimization of the chip design for miniaturized processes, such as on-chip PCR, for which precise temperature control is important.
We report that polymer light emitting diodes (pLEDs) and polymer photodetectors can be integrated on disposable polydimethylsiloxane [PDMS] microfluidic flowcells to form hybrid microchips for bioluminescence applications. PLEDs were successfully employed as excitation light sources for microchip based fluorescence detection of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease. To circumvent the use of optical filters, fluorescence was detected perpendicular to the biolabel flow direction using a CCD spectrophotometer. Prior to investigating the suitability of polymer photodiodes as integrated detectors for fluorescence detection, their sensitivity was tested with on-chip chemiluminescence. The polymer photodetector was integrated with a PDMS microfluidic flowcell to monitor peroxyoxalate based chemiluminescence (CL) reactions on the chip. This work demonstrates that our polymer photodetectors exhibit sensitivities comparable to inorganic photodiodes. Here we prove the concept that thin film solution-processed polymer light sources and photodetectors can be integrated with PDMS microfluidic channel structures to form a hybrid microchip enabling the development of disposable low-cost diagnostic devices for point-of-care analysis.
Carbonylative cross-coupling reactions of arylhalides to form secondary amides were rapidly carried out on a glass-fabricated microchip—the first time a microstructured device has been used to perform a gas–liquid carbonylation reaction.
We present the application of wide-field time-resolved fluorescence imaging methods for the study of solvent interactions and mixing in microfluidic devices. Time-resolved imaging of fluorescence polarization anisotropy allows us to image the local viscosity of fluorescein in three dimensions in order to directly monitor solvent mixing within a microfluidic channel. This provides a viscosity image acquisition time of the order of minutes, and has been applied to a steady-state laminar flow configuration. To image dynamic fluid mixing in real-time, we demonstrate high-speed fluorescence lifetime imaging at 12.3 Hz applied to DASPI, which directly exhibits a solvent viscosity-dependant fluorescence lifetime. These two methods facilitate a high degree of quantification of microfluidic flow in 3-D and/or at high speed, providing a tool for studying fluid dynamics and for developing enhanced microfluidic assays.
As a first step towards a fully disposable stand-alone diagnostic microchip for determination of urinary human serum albumin (HSA), we report the use of a thin-film organic light emitting diode (OLED) as an excitation source for microscale fluorescence detection. The OLED has a peak emission wavelength of 540 nm, is simple to fabricate on flexible or rigid substrates, and operates at drive voltages below 10 V. In a fluorescence assay, HSA is reacted with Albumin Blue 580, generating a strong emission at 620 nm when excited with the OLED. Filter-less discrimination between excitation light and generated fluorescence is achieved through an orthogonal detection geometry. When the assay is performed in 800 μm deep and 800 μm wide microchannels on a poly(dimethylsiloxane) (PDMS) microchip at flow rates of 20 μL min-1, HSA concentrations down to 10 mg L-1 can be detected with a linear range from 10 to 100 mg L-1. This sensitivity is sufficient for the determination of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease (clinical cut-off levels: 15–40 mg L-1)
We report electromodulation sEMd studies of polyfluorene-based light-emitting diodes containing polys3,4- ethylene-dioxythiophened-polysstyrene-sulfonated sPEDOT:PSSd, in which the barrier to hole injection is large (~0.7 eV). Measurements are reported on devices fabricated with aluminium and barium cathodes to provide respectively poor and efficient electron injection into the active layer. The Al devices exhibit low currents, indicating low rates of electron and hole injection, whereas the Ba devices exhibit high currents and high electroluminescence efficiencies, implying efficient injection of both electrons and holes despite the large hole injection barrier. The Al devices show conventional field-induced EM behavior consistent with the Stark effect sSEd. The Ba devices show conventional SE behavior for low applied biases but, above turn-on, the sfieldinducedd SE features vanish, indicating suppression of the internal field, and are replaced by charge-induced bleaching and absorption features. The behavior of the devices is attributed to the presence of electron traps close to the PEDOT:PSS/organic interface. The experimental findings are consistent with earlier findings by Murata et al., Van Woudenbergh et al., Poplavskyy et al., and Lane et al.
Microreactors incorporating thin film resistive heating elements for continuous flow organic synthesis are presented. Internal thermal conditions were monitored in real time using reflectance spectra of temperature sensitive thermochromic liquid crystals (TLC) in a collateral microfluidic network. To demonstrate the precise temperature control provided by this method, the thermal optimisation of the Reimer–Tiemann formylation of b-naphthol was performed under hydrodynamic pumping regimes.
We investigate the influence of annealing conditions on the physical properties of thin films of poly(3,4-ethylenedioxythiophene)/poly(styrene sulfonate) (PEDOT/PSS). In particular, we describe how annealing temperature, the ambient gas, and choice of dopant affect the conductivity, morphology, and work function of the films. Two specific dopants are considered, sorbitol and glycerol, and broad guidelines are developed for using PEDOT/PSS as a hole-injection electrode in polymeric light-emitting devices, solar cells, and photodetectors.
An extremely rapid tool for continuous flow synthetic process optimisation is described. A microfluidic reaction system operating in continuous flow is used in conjunction with confocal Raman microscopy to afford rapid molecule synthesis and product quantitation. Accordingly, the approach allows for rapid reaction optimisation within a continuous flow system. Specifically, the catalytic oxidation of isopropyl alcohol (IPA) to acetone using tetra-N-propylammonium perruthanate (TPAP)/N-methylmorpholine N-oxide (NMO) in a radial interdigitated micromixer is studied as a model reaction system. The composition of the reaction effluent can be determined with great facility and information relating to catalyst/co-oxidant ratios, catalyst turnovers and reaction endpoints extracted. Specifically, variation of catalyst and co-oxidant volumetric flow rates between 0 and 50 μL min-1 is used to vary reactant concentrations, define reaction residence times and control product conversions between 0 and 100%. The rapid nature of the system allows chemical information to be gathered and utilised on a sub-minute timescale.
We report the use of thin-film organic photodiodes as integrated optical detectors for microscale chemiluminescence. The copper phthalocyanine–fullerene (CuPc–C60) small molecule photodiodes have an external quantum efficiency of ~30% at 600–700 nm, an active area of 2mm×8mm and a total thickness of ~2 mm. Simple detector fabrication, based on layer-by-layer vacuum deposition, allows facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, CuPc–C60 photodiodes were used to monitor a peroxyoxalate based chemiluminescence reaction (PO-CL) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Optimum results were obtained for applied reagent flow rates of 25 μL/min, yielding a CL signal of 8.8 nA within 11 min. Reproducibility was excellent with typical relative standard deviations (R.S.D.) below 1.5%. Preliminary quantitation of hydrogen peroxide yielded a detection limit of ~1mM and linearity over at least three decades.With improved sensitivity and when combined with enzymatic assays the described integrated devices could find many applications in point-of-care diagnostics.
The application of micro total analysis systems has grown exponentially over the past few years, particularly diversifying in disciplines related to bioassays. The primary focus of this review is to detail recent new approaches to sample preparation, nucleic acid amplification and detection within microfluidic devices or at the microscale level. We also introduce some applications that have as yet to be explored in a miniaturised environment, but should benefit from improvements in analytical efficiency and functionality when transferred to planar-chip formats. The studies described in this review were published in commonly available journals as well as in the proceedings of three major conferences relevant to microfluidics (Micro Total Analysis Systems, Transducers and The Nanotechnology Conference and Trade Show). Although an emphasis has been placed on papers published since 2002, pertinent articles preceding this publication year have also been included.
We use electromodulation (EM) spectroscopy to probe the electric field distribution in polymer light-emitting diodes. The EM spectrum below the turn-on bias is dominated by electroabsorption of the emissive layer but vanishes completely above the turn-on bias. The EM spectrum above turn-on is due entirely to absorption and bleaching effects arising from injected charge. The total elimination of the electroabsorption signal indicates that the internal electric field is effectively screened by the injected charge, and this effect is attributed to accumulation of trapped electrons close to the anode.
In this paper the development of lightsources and detectors for integrated Microchip based optical detection is reported. A polyfluorene based pLED with peak emission at 488 nm was successfully employed as a lightsource for microchip electrophoresis. With a pLED drive voltage of 5.5 V, separations of fluorecein and 5-carboxyfluorescein could be detected at concentrations down to 1 mu M. In separate experiments, thin-film organic photodiodes were employed as integrated detectors for microscale chemiluminescence. The copper phthalocyanine-fullerene (CuPc-C-60) small molecule photodiodes have an external quantum efficiency of similar to 30% at 550 to 650 nm. The photodiodes were used to monitor a peroxyoxalate based chemiluminescence reaction (PO-CL) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Preliminary PO-CL based quantitation of hydrogen peroxide yielded a detection limit of 1 mM.
We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino-group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on-chip labeling applications. Other alternatives are available such as o-phthaldehyde (OPA), however, the inferior photophysical properties and the UV λmax present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on-chip mixing and reaction) of < 60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples.
Microfluidic devices for spatially localised heating of microchannel environments were designed, fabricated and tested. The devices are simple to implement, do not require complex manufacturing steps and enable intrachannel temperature control to within ± 0.2 degC. Ionic liquids held in co-running channels are Joule heated with an a.c. current. The nature of the devices means that the internal temperature can be directly assessed in a facile manner.
The online analysis of cadmium selenide nanoparticle formation in continuous-flow microfluidic reactors is described. The as-produced particles exhibit sharp excitonic absorption and emission peaks (~30–40 nm) with relatively high quantum efficiencies (~10%). The mean size and dispersity of the particles, determined using on-line fluorescence detection, may be controlled by varying the reaction temperature and/or the flow rate. The microfluidic approach provides considerable control over nucleation/growth processes and is a promising strategy for the direct production of near-monodisperse nanoparticles without recourse to further size selection.
We use electromodulation spectroscopy and modeling studies to probe the electric-field distribution in polyfluorene-based polymer light-emitting diodes containing poly(3,4-ethylenedioxythiophene) poly(styrene sulfonate). The bulk internal field is shown to be zero under ordinary operating conditions, with trapped electrons close to the anode fully screening the bulk semiconductor from the external field. The effect has far-reaching implications for the understanding and optimization of organic devices.
Downsizing of analytical components allows the integration of entire diagnostic processes within credit-card sized devices. However, the realisation of portable diagnostic devices for point-of-care testing has been hampered by the lack of suitable miniaturised optical detection systems. Low-cost solution processable organic semiconductors with tuneable optical properties may provide the answer. To this end, we demonstrate the application of polymer light emitting diodes (pLEDs) and organic photodiodes as integrated detection components. As a step towards the development of disposable, quantitative diagnostic tests, a prototype comprising an analytical microchip, integrated optical detection and support electronics is presented.
Nanoscale science (or nanotechnology) is the exploration and exploitation of the physical, chemical, and biological properties of systems in which phenomena length scales are comparable to the dimensions of the structure. Nanotechnology has been widely recognized as one of the key research topics of the 21st century, and one that will only realise its full potential by the development of new tools for manipulating matter at the atomic/molecular scale. Over the past decade the discovery of novel phenomena, properties and processes at the “nanoscale” has opened revolutionary opportunities for the creation of novel materials and devices with superior chemical, physical, optical, electronic and/or biological properties. Nanocrystalline semiconductors are of particular interest in this regard owing to their tuneable optical and electronic properties. They are seen as tailored precursors in creating functional materials for use in a variety of applications including biological sensing, optoelectronics, electroluminescent displays, fibre optic communications and lasers.
We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 μm x 1000 μm and a thickness of ~ 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 1026 M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.
A procedure for the continuous purification of volatile liquids within microfluidic systems is reported.
A novel approach to the monitoring and analysis of bubbles in gas/liquid binary systems is described. The method is based on the varying extent of refraction experienced by radiation as it passes through a gas or liquid. Specifically, carbonated liquids are hydrodynamically motivated through a microfluidic channel network (40 μm wide and 30 μm deep) and directed through the path of laser beam. As a gas bubble passes through this region, the reduction in local refractive index leads to an increased displacement of the laser beam which is monitored using a position-sensitive detector. Statistical analysis of multiple bubble events (in terms of autocorrelation curves, burst width distributions and interburst time curves) yields information relating to both the bubble size and the frequency of bubble formation.
A statistical method based on the maximum likelihood estimator is used in order to discriminate between single particle flow velocities using confocal spectroscopy. Particles or molecules travelling at specific flow rates through a confocal volume are expected to have a definable burst widths, heights, and areas. Hence, single particles travelling with different velocities should be distinguishable based on such parameters. With this approach we show that for particles travelling with a flow rate difference of 20x, there is virtually a 0% error rate in assigning a particle to a given flow velocity, based on the analysis of a single fluorescence burst. When a flow rate difference of 4x is used, 90% of the particles travelling through the confocal probe volume are still assigned a correct flow velocity. Use of this method technique allows a statistically meaningful flow velocity to be assigned from the analysis of a photon burst from a single particle.
We describe the use of continuous-flow, microfluidic reactors for the controllable synthesis of compound semiconductor nanoparticles. Specifically, CdS nanoparticles are synthesised by rapid mixing of Na2S and Cd(NO3)2.4H2O flow streams within a silicon/glass micromixer based on the principle of flow lamination. Room temperature syntheses at a variety of volumetric flow rates yield CdS nanoparticles of variable polydispersity. Analysis of absorption spectra of the CdS nanoparticles suggests that varying volumetric flow rates of reagent streams provides a simple and direct method of controlling population dispersity. Simple, single layer glass microstructures are also used to synthesis CdSe nanoparticles in continuous flow at temperatures between 180 and 240°C.
This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (200-930 nm diameter) passing through a focused laser beam at a variety of flow rates (100-1000 nL/min). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate single-particle burst width and burst area distributions. Analysis of such distributions demonstrates that the average burst width and burst area decrease as particle size increases. In addition, both burst width and burst area (for a given particle size) are observed to decrease as volumetric flow rate is increased. The dependence of such distributions on particle size is proposed as a potential route to sizing single particles and molecules in microfluidic systems.
Monolithic microfluidic reactors for the safe, expedient, and continuous synthesis of products involving unstable intermediates were fabricated and assessed. The formation of diazonium salts in anhydrous conditions and their subsequent in situ chlorination within microfluidic channels under hydrodynamic pumping regimes is presented. Significant enhancements in yield were observed due to enhanced heat and mass transfer in microfluidic systems. Analysis performed using off-line GC and GC-MS was compared with on-line, on-chip Raman spectroscopy for the direct determination of analytes.
It is fair to say that the development of microfluidic systems for use in the physical and biological sciences has progressed at a significant rate since the early 1990’s. The potential benefits afforded by system downsizing are documented and have provided persuasive reasons for the transferral of instrumentation from the macroscale to planar chip formats. For example, huge leaps in the application, flexibility and efficiency of separation techniques have been realised by miniaturising column dimensions and creating monolithic fluidic networks on planar substrates. Indeed, today almost all separation techniques based on electrophoretic or chromatographic discrimination have been successfully transferred to chip-based formats.
The polymerase chain reaction is widely used to amplify samples of DNA for genetic analysis, and fast, high throughput is the ideal. Microscale, chip-based devices are now proving themselves in this arena.
The polymerase chain reaction is widely used to amplify samples of DNA for genetic analysis, and fast, high throughput is the ideal. Microscale, chip-based devices are now proving themselves in this arena.
A novel method for performing in-column field-amplified sample stacking (FASS) in chip-based electrophoretic systems is presented. The methodology involves the use of a narrow sample channel (NSC) injector. NSC injectors allow sample plugs to be introduced directly into the separation channel, and subsequent stacking and separation can proceed without any need for leakage control. More importantly, stacking and separation occur in a single step negating the requirement for complex channel geometries and voltage switching to control sample plugs during the stacking procedure. The chip is composed of six paralleled systems. Using the NSC injector design, the number of reservoirs in the multiplexed chip is reduced to N + 2, where N is the number of paralleled systems. This design feature radically reduces the complexity in chip structures and associated chip operation. The approach is applied to the analysis of fluorescently labelled biogenic amines affording detection at concentrations down to 20 pM.
Analytical chemistry is an expansive field, encompassing a myriad of methods and techniques employed to provide discrimination of an analyte of interest from its surroundings. A generic analytical procedure can be broken down into three broad categories: the analytical principle on which the measurement is based, the analytical method (i.e. the concept of optimising the conditions for the analytical principle chosen), and finally the analytical procedure (that encompasses all considerations from analyte to analytical result).
This paper describes the use of reversed-phase, reversed-polarity head-column field-amplified sample stacking (HCFASS) for on-line sample concentration in conventional capillary electrophoresis. The effective stacking efficiency was determined as a function of sodium hydroxide concentration in the sample matrix. Results concur with theoretical predictions where stacking efficiency depends on the conductivity (electric field strength) and electrophoretic mobility in the sample matrix solution. Fluorescein isothiocyanate-derivatized aniline and 2,4-dimethylaniline were dissolved in sodium hydroxide (800 μM), separated in a phosphate running buffer (0.05 M, pH 9.0) and detected utilising laser-induced fluorescence. The use of reversed-phase, reversed-polarity HCFASS with laser-induced fluorescence detection yielded sensitivity improvements with respect to normal injection schemes in excess of three orders of magnitude, and a limit of detection as low as 10-13 M.
We report emissive devices exhibiting electroluminescence in the solution phase. The principle operating mechanism for these devices - direct electronic carrier injection from the electrodes into the carrier bands of the dissolved polymer - resembles that of a conventional solid-state organic lightemitting diode and is distinct from the solvent-mediated electrochemical devices recently reported by Chang et al.
The analysis of alcohol dehydrogenase (ADH) at low concentration using capillary electrophoresis is described. Several simple and effective ways to improve detection limits and sensitivity are investigated. These include large volume sample stacking, head column field amplified sample stacking, and sweeping. Results indicate that by using a combination of head-column field amplified sample stacking and sweeping, fluorescently labelled alcohol dehydrogenase can be pre-concentrated online by dissolving samples in water or other low conductivity matrices, and injecting into a high conductivity micellar buffer. The abrupt changes in conductivity cause narrowing of the analyte length and thus enhance the detection sensitivity. Combination of this approach with laser induced fluorescence detection yields a limit of detection of 5e-13 M. Both qualitative and quantitative aspects of this method are investigated.
A polydimethylsiloxane-glass capillary microchip is fabricated for the rapid analysis of a mixture of common biogenic amines using indirect fluorescence detection. Using a running buffer of phosphate and 2-propanol, and Rhodamine 110 as a background fluorophore, both co-ionic and counter-ionic systems are explored. Studies demonstrate the separation and analysis of cations using indirect fluorescence detection for the first time in a chip-based system. Resulting electrophoretic separations are achieved within a few tens of seconds with detection limits of approximately 6 M. The reduced sample handling and rapid separations afforded by the coupling of indirect fluorescence detection with chip-based capillary electrophoresis provide a highly efficient method for the analysis and detection of molecules not possessing a chromophore or fluorophore. Furthermore, limits of detection are on a par with reported chip-based protocols that incorporate precolumn derivatisation with fluorescence detection. The current device circumvents lengthy sample preparation stages and therefore provides an attractive alternative technique for the analysis biogenic amines.
A microfluidic procedure for the controlled production of cadmium sulfide nanoparticles is described
Bradley M. Stone and Andrew J. de Mello ponder the fundamental question of the ‘origin of life’ in the universe, and discuss whether miniaturised analytical instruments could be put to use within instrument payloads in upcoming space missions that aim to search for biogenic precursor molecules.
At its most fundamental level, the great interest in lab-on-a-chip technology stems from the inherent performance gains that arise when most analytical systems are downsized to the micron scale. It can be argued that the reduced physical size of many chip-based systems is attractive and important in niche areas, such as ‘point-of-care’ diagnostics and ‘in-the-field’ analysis, but even then performance gains determine ultimate applicability. Over the past decade, the most active field (as judged by publication output) of microsystem development has been in transferring conventional, macroscale separation techniques to planar chip formats. This in large part has been directly due to the unmistakable improvements in separation performance as system features are diminished.
Recent years have seen great progress in the development of microfabricated systems for use in the chemical and biological sciences. Much of this development has been driven by a need to perform analytical measurements on small sample volumes. Reducing the physical size of the measurement system naturally yields advantages such as improved efficiency with respect to sample size, response times, cost per analysis, experimental throughput and automation. However, at an even more primary level, interest in miniaturised analytical systems has been stimulated by the fact that scientists can more easily control physical processes (such as chemical reactions and molecular separations) when instrumental dimensions are reduced to the micron scale.
Without doubt, the conceptualization and development of the miniaturized total chemical analysis system (μTAS) over the past decade has revolutionized the way scientists can address a variety of complex chemical and biological problems. Since Andreas Manz and Michael Widmer presented their visionary concept in 1990 the evolution of the field has occurred at a truly exponential rate. Today, Lab-on-a-Chip technologies are routinely used in a wide variety of application areas including separation science, protein analysis, process control, environmental monitoring, chemical synthesis, DNA amplification, immunoassays, DNA sequencing, and cell manipulations. The rapid acceptance of miniaturized systems for analytical and measurement applications has been motivated by a number of reasons. At a fundamental scientific level, miniaturized analysis systems exhibit clear advantages when compared to their conventional (macroscale) counterparts. These include improved efficiency with respect to sample size, response times, analytical performance, process control and throughput. Furthermore, the fabrication methods necessary to create system features on the micron scale had been defined, developed and refined within the microelectronics industry for over almost half a century. This foundation of technological expertise in bulk and surface micromachining of silicon and silicon-compatible materials very simply meant that the creation of microfluidic chip devices (containing elements such as flow-manifolds, valves, reactors, electrodes, detectors and filters) was achieved in a relatively short timescale.
Singlet oxygen was effectively and safely generated in a nanoscale reactor and used for the synthesis of ascaridole. The technique allows for the generation of singlet oxygen without the inherent dangers of large quantities of oxygenated solvents. The methodology allows for facile scale-out of the process.
Monolithic nanoreactors for the safe and expedient continuous synthesis of products requiring unstable intermediates were fabricated and tested by the synthesis of azo dyes under hydrodynamic pumping regimes.
The advent of the polymerase chain reaction (PCR) has, without a shadow of a doubt, hugely accelerated the progress of studies on the genetic structure of a diversity of organisms. PCR is an enzyme catalyzed amplification technique that allows any nucleic acid sequence to be generated in vitro and in abundance. It was first reported in early 1986 at the 51st Cold Spring Harbour laboratory Symposium on Quantitative Biology by Kary Mullis, and since has become an indispensable tool in basic molecular biology, genome sequencing, clinical research and evolutionary studies.
In his now celebrated lecture on the 29th December 1959 Richard Feynman pondered the potential of miniaturization in the physical sciences. His vision, based on known technology, examined the limits set by physical principles and proposed a variety of new nano-tools including the concept of “atom by atom” fabrication. In the intervening decades, many of these predictions have become reality; microelectronic systems have shrunk to sizes close to the molecular level, scanning probe microscopes (e.g. STM and AFM) enable us to image and manipulate individual atoms, and the molecular machinery of living systems is now being more fully understood and harnessed.
A PEEK interface for use in microfluidic applications is designed, fabricated and tested. The interface allows for the facile, non-permanent coupling of standard capillary tubing to silicon/glass micromixer chips. Importantly, the interface provides for a secure connection between capillary lines and chip reservoirs without the need for any adhesive materials. Furthermore, when used in conjunction with silicon/glass micromixer chips fluidic transport is stable over a wide range of volumetric flow rates (1–1500 μL min-1), and the entire construct can be rapidly assembled and disassembled at any time during the course of experimentation.
A novel method to detect refractive index variations within microchannel environments using an evanescent wave probe is described. Experiments, with and without a fluorescent buffer, demonstrate that 10 mM sucrose plugs can be detected. This method is also applied to the detection of sucrose plugs flowing hydrodynamically through microchannels and for chip-based electrophoretic separations of saccharide mixtures.
This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (0.93 μm diameter) passing through a focused laser beam at a variety of flow rates (50 nL min-1 – 8 μL min-1). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate velocity data for the flowing particles. Fluid manipulation is achieved by hydrodynamically pumping fluid through microchannels (150 μm wide and 50 μm deep) structured in a polydimethylsiloxane (PDMS) substrate. The mean fluorescence burst frequency is shown to be directly proportional to flow speed. Furthermore, the Poisson recurrence time and width of recovered autocorrelation curves is demonstrated to be inversely proportional to flow speed. The component-based confocal fluorescence detection system is simple and can be applied to a diversity of planar chip systems. In addition, velocity measurement only involves interrogation of the fluidic system at a single point along the flow stream, as opposed to more normal multiple-point measurements.
This paper presents a novel approach for performing spectroscopic refractive index detection within microfluidic channel environments. Based on the principle of total internal reflection (TIR), changes in the refractive index of an analyte stream passing through a microfabricated channel are detected through interaction with an optical evanescent field formed at the channel wall. Refractive index variations within the microchannel environment modify the critical angle at the liquid–solid interface, thereby altering the characteristics of evanescent field formation in solution. These variations are evidenced through measurement of fluorescence intensities. Initially, the design and testing of the method are described. Subsequently, refractive index values for bulk sucrose solutions (0–35% w/v sucrose in water) are measured using the single point evanescent wave probe and compared with values obtained through conventional refractometry and the literature. Close agreement between all three approaches is demonstrated. The method is then applied to the detection of sucrose plugs (10–500 mM) hydrodynamically flowing through microfabricated channels on a planar glass chip. The evanescent wave probe is also used to selectively monitor specific analytes within a multicomponent system, by precise angular control in the vicinity of the critical angle. Although detection limits using the prototype system are non-ideal ( ~ 5 μM carbohydrate), they compare favourably with existing methods for on-chip refractive index detection.
A miniaturised-SYNthesis and Total Analysis System (μSYNTAS) integrating a silicon-machined chemical microprocessor and time-of-flight mass spectrometry (TOF-MS) is used for the generation of compound libraries based on sub-reactions of an Ugi multicomponent reaction (MCR). The microreactor—based on the concept of an AND logic operator—allowed the coupling of serially-switched solution-phase library generation with on-line compound analysis and identification. In addition, the μSYNTAS allowed real-time parallel-processing of MCR sub-reactions; in contrast to combinatorial techniques employing a solid support for reagent and product isolation, the μSYNTAS protocol required no additional preparation or work-up procedures.
The separation and detection of both print and film developing agents (CD-3 and CD-4) in photographic processing solutions using chip-based capillary electrophoresis is presented. For simultaneous detection of both analytes under identical experimental conditions a buffer pH of 11.9 is used to partially ionise the analytes. Detection is made possible by indirect fluorescence, where the ions of the analytes displace the anionic fluorescing buffer ion to create negative peaks. Under optimal conditions, both analytes can be analyzed within 30 s. The limits of detection for CD-3 and CD-4 are 0.17 mM and 0.39 mM, respectively. The applicability of the method for the analysis of seasoned photographic processing developer solutions is also examined.
Over the past decade the miniaturization of analytical techniques and methods has become a highly visible and dominant trend in the physical and biological sciences. Development in this area has primarily been driven by a need for rapid, on-line measurements at low concentrations within fields such as DNA analysis, drug discovery, pharmaceutical screening, medical diagnostics, environmental analysis and chemical production. The advantages associated with shrinking analytical systems are well known and include improved efficiency with respect to sample size, application, response times, cost, analytical performance, integration, throughput and automation.
A miniaturised-SYNthesis and Total Analysis System (μSYNTAS) was used for the solution-phase synthesis and on-line analysis (TOF-MS) of Ugi multicomponent reaction (MCR) products. This approach provides an unusually high degree of control of the MCR and delivers detailed, novel information on reaction intermediates in real-time. Specifically, the Ugi 4 component condensation (4CC) involving the reaction of an amine, acid, aldehyde and isocyanide species was performed at room temperature in a controllable fashion. Furthermore, observation of the nitrilium intermediate, cyclohexyl(2-piperidin-1-ylethylidyne)ammonium chloride, is presented for the first time.
We have constructed a simple component-based confocal detection system, that is capable of fluorescence detection at the single-molecule level. The component-based format maximises flexibility and reduces start-up costs. A new model for the optical probe volume is proposed, that is based on the 1/e2 Gaussian intensity contour of a laser beam focused to the diffraction limit. Observation of the onset of single-molecule detection in our experimental system confirms that this model is more appropriate than a simple cylindrical approximation for a probe volume defined by a wide, tightly focused laser beam.
This review article discusses and documents the basic philosophies, concepts and current advances in the field of μ-TAS development, with special emphasis on applications in the arena of biosciences. After a brief overview of miniaturization theory and fabrication techniques, areas of microfluidic component development, detection protocols, biochemical assays, and integrated biological analyses are addressed.
A capillary electrophoresis microchip is developed for the rapid determination of 4-amino-3-methyl- N-ethyl-N-(b-methane sulfonamidoethyl)aniline (CD-3) in commercial colour photographic processing solutions and the applicability of the method is examined. The use of indirect fluorescence as an on-chip detection method is also demonstrated. Using a running buffer at pH 11.9 prepared from disodium hydrogenphosphate and fluorescein the quantitative determination of CD-3 is achieved, resulting in an analysis time of approximately 7 s. Under these conditions, a detection limit of about 5 mg L-1 is obtained, with good linearity between signal and concentration over a range of 5–20 mg L-1.
This paper presents a micromixer for the laminar flow regime based on the principle of flow lamination. The structure is made up from a glass/silicon/glass sandwich, has a total internal volume of ~ 600 nL and measures 5 mm x 10 mm. Flow rates between 1–200 µL/min have successfully been used. Fluorescence quenching experiments were carried out for quantification and showed 95% mixing within 15 ms.
We report photoluminescence studies of poly(3-dodecylthiophene) (P3DT) in solution. In a good solvent the polymer exhibits luminescence with a high quantum efficiency and a decay time of 500 ps. In a poor solvent the emission is red shifted with a 20-fold reduction in quantum efficiency and a decay profile that is non-monoexponential, but has an average lifetime that is very similar to the good solvent environment. The data indicate a large increase in the natural radiative lifetime from approximately 1 ns in a good solvent to 20 ns in a poor solvent, which implies an emitting state that is different in the two situations. In the poor solvent the spectrum is almost identical to that of the thin film, suggesting that the polymer aggregates in the solution and the emitting species is the same in both environments. The data are consistent with the formation of an excited state that is not localized on a single chain but is delocalized over more than one chain.
The concentration distributions of fluorescein and acridine orange, at a water/silica interface, are determined using the technique variable-angle time-resolved evanescent wave-induced fluorescence spectroscopy. It is demonstrated theoretically that the radiative fluorescence lifetime of a molecule is affected in only a minor way by the presence of a dielectric interface. Consequently, time-resolved evanescent wave-induced fluorescence spectroscopy is used to measure the variations in the fluorescence quantum yield caused by the pressence of the interface. Knowledge of these variations is then used to determine molecular concentration distributions. The concentration distribution of fluorescein is found to be uniform and homogeneous as a function of distance away from the hydrophilic silica surface. However, it is found that a water/silica interface has a definite affect on the concentration distribution of acridine orange. A “surface associated” acridine orange population distinguishable from “bulk” acridine orange is demonstrated by spectral and kinetic analysis of EWIF emission. This information is then used to create concentration profiles of both molecular populations.
The fluorescence properties of 2-aminoacridone (2-AMAC) and some polysaccharide derivatives have been studied in the steady state and with time-correlated single-photon counting. 2-AMAC in methanol has an absorption maximum at 425 nm and a fluorescence emission maximum at 530 nm. The absorption spectrum shows a sufficiently broad tail for satisfactory excitation by the light from an argon ion laser (488 nm). Lifetime decay analysis shows that the fluorescence lifetime of the aminoacridone moiety is not changed significantly by the addition of a sugar chain, or by the length or structure of that chain. 2-AMAC exhibits a fluorescence decay that may be fitted to a single exponential in both methanol (t ≈ 12 ns) and water (t ≈ 10 ns); in an equal mixture of the solvents the behaviour is best described by a sum of two exponential terms. We have also shown that the absorbance of a complex mixture of 2-AMAC tagged glycans is directly proportional to their fluorescence emission at 525 nm. These results have confirmed our previous studies that 2-AMAC is a useful and non-selective fluorescent tag in the analysis of polysaccharide chains.
A novel detection scheme for capillary electrophoresis on planar glass microchips is presented. The application of a holographic-based refractive index detector to the electrophoretic separation of carbohydrates is described. The microchip device consists of a cyclic (square) separation channel having a circumference of 80 mm, a width of 40 mm and a depth of 10 µm. The volume of the injection scheme is approximately 16 pl. Separation and refractive index detection of a mixture of sucrose, N-acetylglucosamine and raffinose, each at a concentration of 33 mm, was achieved within 17 s of injection. Preliminary results demonstrate the feasibility of using hologram-based refractive index detectors in microchip separation systems. Although the initial detection limits are poor in comparison with alternative techniques, the potential of a universal detector of this kind is clear.
Time-integrated and time-resolved fluorescence spectroscopies have been used to probe the photophysical properties of ethidium bromide (Eb) complexed to calf thymus DNA (ct-DNA). Fluorescence decay profiles are obtained using the technique of time-correlated single photon counting (TCSPC), and subsequently analysed using conventional sum-of-exponential (SOE) routines and also the maximum entropy method (MEM). Through use of these methods and simulated decay data, it is demonstrated that the kinetics of Eb in the presence of ds-DNA are best described by a generic model consisting of three exponential terms. At all DNA:Eb ratios and NaCl concentrations studied, free Eb is detected. Furthermore, Eb is found to interact with ds-DNA through two mechanisms, each distinguishable by its fluorescence decaytime. Eb is shown to interact with DNA through classic intercalation, and also through binding at secondary sites. The component decaytimes are shown to be a function of NaCl concentration but independent of DNA:Eb molar ratio.
A micromachined chemical amplifier was successfully used to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device is analogous to an electronic amplifier and relies on the movement of sample through thermostated tem- perature zones on a glass microchip. Input and output of material (DNA) is continuous, and amplification is independent of input concentration. A 20-cycle PCR amplification of a 176 – base pair fragment from the DNA gyrase gene of Neisseria gonorrhoeae was performed at various flow rates, resulting in total reaction times of 90 seconds to 18.7 minutes.
A small volume, electrochemical cell for the generation and detection of electrochemiluminescence from tris(2,2'-bipyridyl) ruthenium(II) has been fabricated. The flowcell is a poly(methyl methacrylate) (PMMA)–acetate–PMMA sandwich construct, containing two platinum, thin-film electrodes. Operation of the microchip establishes sub-microliter detection of tris(2,2'-bipyridyl) ruthenium(II) electrochemiluminescence in continuous flow. Initial experiments demonstrate a detection limit of 5e-13 M at an effective cell volume of 100 nl. This corresponds to the detection of only 30 000 molecules.
Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This con- struct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 °C/s heating, 2.5 °C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photo- lithographically fabricated channel filled with hydroxyeth- ylcellulose sieving matrix. Electrophoretic injection di- rectly from the PCR chamber through the cross injection channel was used as an “electrophoretic valve” to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a -globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was per- formed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target am- plification in these integrated PCR-CE devices is also feasible. Amplification of the -globin target as a function of cycle number was directly monitored for two different reactions starting with 4 × 107 and 4 × 105 copies of DNA template. This work establishes the feasibility of perform- ing high-speed DNA analyses in microfabricated inte- grated fluidic systems.
Time-integrated and time-resolved evanescent wave-induced fluorescence spectroscopies (EWIFS) have been used to probe the photophysical properties of Rhodamine 101 at two solution/solid interfaces. Interaction of Rhodamine 101 with a fused silica surface leads to a reduction in the molecular fluorescence quantum efficiency in both cases. The fluorescence kinetics of interfacial species are shown to be complex (non-exponential), a function of bulk solution concentration, and a function of distance normal to the interface. The application of the maximum entropy method to the analysis of EWIF decays is presented. Recovered lifetime distributions expose inherent complexity and heterogeneity that is hidden to conventional analysis techniques.
We report photoluminescence studies of poly( 3-dodecylthiophene) (P3DT) in solution. In a good solvent the polymer exhibits luminescence with a high quantum efficiency and a decay time of 500 ps. In a poor solvent the emission is red shifted with a 20-fold reduction in quantum efficiency and a decay profile that is non-monoexponential, but has an average lifetime that is very similar to the good solvent environment. The data indicate a large increase in the natural radiative lifetime from approximately 1 ns in a good solvent to 20 ns in a poor solveni, which implies an emitting state that is different in the two situations. In the poor solvent the spectrum is almost identical to that of the thin film, suggesting that the polymer aggregates in the solution and the emitting species is the same in both environments. The data are consistent with the formation of an excited state that is not localized on a single chain but is delocalized over more than one chain.
For tetrasulfonated aluminum phthalocyanine (AlPcS4), dimer formation is characterized in the absorption spectrum by a broadening of the Q-band and the appearance of a new band a t the red edge of the spectrum. The high concentrations required to produce dimers, however, often leads to anomalous observations in fluorescence spectroscopy. In the present study, we have examined the photophysical characteristics of two dye systems; AlPcS4 in a 66% ethanol/water mixture and disulfonated aluminum phthalocyanine in methanol. Using absorption spectroscopy, the formation of dimers is shown to be prevalent only in the case of AIPcS4. The fluorescence emission spectra in both cases, however, exhibit similar spectral changes with increasing dye concentration. The measured fluorescence decay profiles for both dyes also show similar trends: They are monoexponential, invariant with emission wavelength and have decay times that increase with dye concentration. These distortions are sometimes incorrectly attributed to dimer fluorescence. We find no evidence for the existence of dimer fluorescence and demonstrate that these data can be readily explained, by taking into consideration the effects of reabsorption of fluorescence.
The techniques of evanescent wave induced fluorescence spectroscopy (EWIFS) and time-correlated single-photon counting have been combined to investigate the photophysical properties of auramine-O at a solid/solution interface. Interaction of the dye with the surface causes an enhancement of the fluorescence quantum efficiency through the restriction of intramolecular rotations. The fluorescence kinetics of the adsorbed molecules are shown to be a function of surface and bulk solution concentration. The application of the maximum entropy method to the analysis of EWIFS decays is also introduced and discussed
