Ultrafast surface enhanced resonance Raman scattering detection in droplet-based microfluidic systems

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.     

Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient

In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the β-galactosidase-catalyzed hydrolysis of resorufin-β-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.     

CMOS absorbance detection system for capillary electrophoresis

This paper presents a cost-effective portable photodetection system for capillary electrophoresis absorptiometry. By using a CMOS BDJ (buried double p–n junction) detector, a dual-wavelength method for absorbance measurement is implemented. This system includes associated electronics for low-noise pre-amplification and A/D conversion, followed by digital signal acquisition and processing. Two signal processing approaches are adopted to enhance the signal to noise ratio. One is variable time synchronous detection, which optimizes the sensitivity and measuring rate compared to a conventional synchronous detection technique. The other is a statistical approach based on principal component analysis, which allows optimal estimation of detected signal. This system has been designed and tested in capillary electrophoresis conditions. Its operation has been verified with performances comparable to those of a commercialized spectrophotometric system (HP-3D CE). With potential on-chip integration of associated electronics, it may be operated as an integrable detection module for microchip electrophoresis and other microanalysis systems.     

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.     

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.     

High-throughput microfluidic systems for disease detection

Accuracy and rapid response are critical to the detection of an acute infectious disease, not only because the detection results can affect the medical treatment, but also can prevent disease outbreaks. Since the current culture-based technology is time consuming and experience dependent, academia and industrial researchers are using microfluidics and nucleic acids as the fundamental ideas to build pioneering tools against infectious disease. While many point-of-care microfluidic systems have been realized to execute nucleic acid applications, high-throughput microfluidic systems are under development for various nucleic acid applications because of high efficiency and demand from the market. Building a high-throughput system is an interdisciplinary challenge because of the design concerns from science and the manufacturing concerns from engineering, but its realization will be a milestone. This article is aimed to review three essential steps of the nucleic acid-based detection realized in high-throughput formats, including polymerase chain reaction, capillary electrophoresis, and nucleic acid purification.     

Multiple open-channel electroosmotic pumping system for microfluidic sample handling

The development of a novel, fully integrated, miniaturized pumping system for generation of pressure-driven flow in microfluidic platforms is described. The micropump, based on electroosmotic pumping principles, has a multiple open-channel configuration consisting of hundreds of parallel, small-diameter microchannels. Specifically, pumps with microchannels of 1-6 microm in depth, 4-50 mm in length, and an overall area of a few square millimeters, were constructed. Flow rates of 10-400 nL/min were generated in electric-field-free regions in a stable, reproducible and controllable manner. In addition, eluent gradients were created by simultaneously using two pumps. Pressures up to 80 psi were produced with the present pump configurations. The pump can be easily interfaced with other operational elements of a micrototal analysis system (micro-TAS) device with multiplexing capabilities. A new microfluidic valving system was also briefly evaluated in conjunction with these pumps. The micropump was utilized to deliver peptide samples for electrospray ionization-mass spectrometric (ESI-MS) detection.     

Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation

We have developed a multipurpose microfluidic platform that allows for sensitive fluorescence detection on inexpensive disposable chips. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and covalent attachment of an unstructured flexible lid. This combines the virtues of elastomer technology with high-throughput compact disk injection molding. Using this technique, the time to produce 100 chips using a single master can be lowered from more than 1 week by standard PDMS technologies to only a couple hours. The optical properties of the fabricated chips were evaluated by studying individual fluorescence-labeled DNA molecules in a microchannel. Concatemeric DNA molecules were generated through rolling circle replication of circular DNA molecules, which were labeled by hybridization of fluorescence-tagged oligonucleotides. Rolling circle products (RCPs) were detected after as little as 5 min of DNA polymerization, and the RCPs in solution showed no tendency for aggregation. To illustrate the versatility of the platform, we demonstrate two additional applications: The flexible property of the lid was used to create a peristaltic pump generating a flow rate of 9 nL/s. Biocompatibility of the platform was illustrated by culturing Chinese hamster ovary cells for 7 days in the microfluidic channels.     

Optical detection strategies for centrifugal microfluidic platforms

Centrifugal microfluidic systems have become one of the principal platforms for implementing bioanalytical assays, most notably for biomedical point-of-care diagnostics. These so-called ‘lab-on-a-disc’ systems primarily utilise the rotationally controlled centrifugal field in combination with capillary forces to automate a range of laboratory unit operations (LUOs) for sample preparation, such as metering, aliquoting, mixing and extraction for biofluids as well as sorting, isolation and counting of bioparticles. These centrifugal microfluidic LUOs have been regularly surveyed in the literature. However, even though absolutely essential to provide true sample-to-answer functionality of lab-on-a-disc platforms, systematic examination of associated, often optical, read-out technologies has been so far neglected. This review focusses on the history and state-of-the-art of optical read-out strategies for centrifugal microfluidic platforms, arising (commercial) application potential and future opportunities.     

Addressable electric fields for size-fractioned sample extraction in microfluidic devices

Fraction collection following electrophoresis is of major importance for a variety of biological analyses. These assays typically need to identify specific fractions in the separated sample for further processing and require extraction of one or a group of fragments. In this paper, we have developed and characterized a technique to generate addressable electric fields for improved extraction during electrophoresis in microfluidic devices. The addressable electric field is achieved by applying a low bias voltage (1-2 V) to microelectrode pairs within the electrophoresis microchannel. Theoretical analysis shows the purity of the extracted sample can be improved as much as 30% over extraction without the shaped electric fields, and nearly 100% predicted yield can be achieved. We also describe the theoretical design of shaped electric fields by characterizing the optimal electrode geometry, field strength, channel configuration, and electrophoretic migration behavior needed for efficient band extraction.     

Real-time image acquisition for absorbance detection and quantification in thin-layer chromatography

This paper presents the first quantitative study of real-time acquisition of images of spots on thin-layer chromatographic plates during development. Procedures are described for imaging using a CCD camera and for image processing, incorporating corrections for fixed pattern effects and compensation for the moving solvent front, to measure the absorbance of the analyte. Imaging of Sudan II was carried out in transmission mode, and peak areas were found to be time-independent. Quantification of the relationship between peak area and sample loading was established over the range 1-50 ng. After averaging 55 images obtained during a single chromatographic run, which attenuates noise contributions from local nonuniformities in the sorbent layer, precision and detection limits were found to be comparable with values obtained in previous work using offline measurements.     

Formation of droplets of alternating composition in microfluidic channels and applications to indexing of concentrations in droplet-based assays

For screening the conditions for a reaction by using droplets (or plugs) as microreactors, the composition of the droplets must be indexed. Indexing here refers to measuring the concentration of a solute by addition of a marker, either internal or external. Indexing may be performed by forming droplet pairs, where in each pair the first droplet is used to conduct the reaction, and the second droplet is used to index the composition of the first droplet. This paper characterizes a method for creating droplet pairs by generating alternating droplets, of two sets of aqueous solutions in a flow of immiscible carrier fluid within PDMS and glass microfluidic channels. The paper also demonstrates that the technique can be used to index the composition of the droplets, and this application is illustrated by screening conditions of protein crystallization. The fluid properties required to form the steady flow of the alternating droplets in a microchannel were characterized as a function of the capillary number Ca and water fraction. Four regimes were observed. At the lowest values of Ca, the droplets of the two streams coalesced; at intermediate values of Ca the alternating droplets formed reliably. At even higher values of Ca, shear forces dominated and caused formation of droplets that were smaller than the cross-sectional dimension of the channel; at the highest values of Ca, coflowing laminar streams of the two immiscible fluids formed. In addition to screening of protein crystallization conditions, understanding of the fluid flow in this system may extend this indexing approach to other chemical and biological assays performed on a microfluidic chip.     

A high-throughput continuous sample introduction interface for microfluidic chip-based capillary electrophoresis systems

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with <4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.     

Multiplexed spectral signature detection for microfluidic color-coded bioparticle flow

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.     

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.     

Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.     

Demonstration of submersible high-throughput microfluidic immunosensors for underwater explosives detection

Significant security threats posed by highly energetic nitroaromatic compounds in aquatic environments and the demilitarization and pending cleanup of areas previously used for munitions manufacture and storage represent a challenge for less expensive, faster, and more sensitive systems capable of analyzing groundwater and seawater samples for trace levels of explosive materials. Presented here is an inexpensive high throughput microfluidic immunosensor (HTMI) platform intended for the rapid, highly selective quantitation of nitroaromatic compounds in the field. Immunoaffinity and fluorescence detection schemes were implemented in tandem on a novel microfluidic device containing 39 parallel microchannels that were 500 μm tall, 250 μm wide, and 2.54 cm long with covalently tethered antibodies that was engineered for high-throughput high-volume sample processing. The devices were produced via a combination of high precision micromilling and hot embossing. Mass transfer limitations were found in conventional microsystems and were minimized due to higher surface area to volume ratios that exceeded those possessed by conventional microdevices and capillaries. Until now, these assays were limited to maximum total volume flow rates of ~1 mL/min due in part to kinetics and high head pressures of single microchannels. In the design demonstrated here, highly parallelized microchannels afforded up to a 100-fold increase in total volume flow rate while maintaining favorable kinetic constraints for efficient antigen-antibody interaction. The assay employed total volume throughput of up to 6 mL/min while yielding signal-to-noise ratios of >15 in all cases. In addition to samples being processed up to 60 times faster than in conventional displacement-based immunoassays, the current system was capable of quantitating 0.01 ng/mL TNT samples without implementing offline preconcentration, thereby, demonstrating the ability to improve sensitivity by as much as 2 orders of magnitude while decreasing total analysis times up to 60-fold.     

Fabrication of microfluidic reactors and mixing studies for luciferase detection

We report the detection of luciferase by implementing a bioluminescent assay in microfluidic reactors. The reactors were fabricated in poly(methyl methacrylate) by hot embossing using a mold master with the reactor layouts made by high-precision micromilling. The overall fabrication process was simple to implement and had a quick turnaround time with low cost. Two reactors, one with smooth channels (called reactor I) and the other with staggered herringbone mixers (called reactor II), were studied for the bioluminescent assay. The assay was implemented by introducing a sample and an assay solution into the reactors and then mixing took place to achieve the enzymatic reactions. We found that the mixing efficiency in reactor II was 17.8 times higher than reactor I. Theoretical analysis of the experimental results indicated that the required channel length of mixing was linearly proportional to the flow rate. A calibration curve for luciferase was obtained for both reactors. We found that the detection sensitivity of reactor II was 3 times higher than reactor I. The limit of detection in reactor II was determined to be 0.14 microg/mL luciferase. The device was further exploited to determine the concentration of luciferase samples obtained from in vitro protein expression.     

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.     

CMOS chopper amplifier for chemical sensor

A highly sensitive CMOS chopper amplifier for an oxygen probe is described. It is integrated in a 2-μm double-poly p-well CMOS process. The chip uses only a single 5-V voltage source, and no external components are needed. It is realized by using the current mode chopper technique to overcome the low frequency noise and drift problems. The clock feedthrough generated by the chopper circuit is reduced by using the switched-capacitor-filter technique. The switched-capacitor technique is also used to implement the function of demodulation. The simulated current is converted into an output voltage with a slope of approximately 100 mV/nA. The current signal from 0.2 to 8 nA can be measured with a nonlinearity of 3.5%. Experimental results are given showing the performance of this amplifier