The detection of p53 gene via fluorescence quenching of quantum dot in microfluidic chip

Recently, quantum dot (QD) has been used widely in the field of bio assay including cell imaging, biomarker, and fluorescence resonance energy transfer (FRET) sensor. The DNA assay without labeling process has several advantages including low cost, short time, and simplicity. Microbeads of agarose, glass, and polystyrene have been used as a solid support in microfluidic devices to trace molecules. The main advantages of microfluidics include high throughput, short analysis time, small sample volume, and high sensitivity. PDMS based microfluidic chips were prepared for the detection of p53 gene by using QD-DNA conjugate. The microfluidic chip has a weir in the channel to trap microbeads to which QD-DNA probes bind. Carboxylated CdSe/ZnS QDs (wavelength of emission: 605 nm) could bind to microbeads of polystyrene/divinyl benzene via EDC/NHS crosslinking reaction. The target gene and DNA intercalating dye (TOTO-3) were loaded into the micro-channel. Fluorescence quenching from QDs by intercalating dye was observed after hybridization of DNA at the weir in the channel of microfluidic chip. The fluorescence quenching from QDs by TOTO-3 was dependent on the concentration of target gene. This experiment shows the possibility of rapid detection of DNA via bead-QD complex on microfluidic chip.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

Direct visualization of dye and oligonucleotide diffusion in silica filaments with collinear mesopores

The diffusion dynamics of terrylene diimide (TDI) dye molecules and dye-labeled double-strand DNA were studied in micrometer long silica filaments containing collinear, oriented mesopores using single molecule fluorescence microscopy. TDI was used as a stable and hydrophobic probe molecule for single molecule structural analysis. We used template-free mesoporous silica filaments with 4 nm pore diameter and chemical functionalization with one or two types of trialkoxysilane groups to enhance the affinity between the host system and the guest molecules. Insights about the mesoporous structure as well as the translational and orientational diffusion dynamics of the guest molecules observed along micrometer long trajectories could be obtained. Additionally, the stability of DNA oligomers (15 base pairs, bp, about 5.3 nm long) within the mesopores was examined, showing no degradation of the oligonucleotide upon incorporation into the mesopores. Diffusion of both guest molecules could be controlled by exposure to vapors of water or chloroform; the latter both induced a reversible on-off control of the translational movement of the molecules.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

A microfluidic system for large DNA molecule arrays

Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.     

High-throughput flow cytometric DNA fragment sizing

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.     

A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions

This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.     

Microfluidic electrochemical aptameric assay integrated on-chip: a potentially convenient sensing platform for the amplified and multiplex analysis of small molecules

Aptamers are artificial oligonucleotides that have been widely employed to design biosensors (i.e., aptasensors). In this work, we report a microfluidic electrochemical aptamer-based sensor (MECAS) by constructing Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules. In combination with the microfluidic channels covering on the glass chip, different targets are transported to the Au electrodes integrated on different positions of the chip. These electrodes are premodified by different kinds of aptamers, respectively, to fabricate different sensing interfaces which can selectively capture the corresponding target. It is an address-dependent sensing platform; thus, with the use of only one electrochemical probe, multitargets can be recognized and detected according to the readout on a corresponding aptamer-modified electrode. In the sensing strategy, the electrochemical probe, [Ru(NH(3))(6)](3+) (RuHex), which can quantitatively bind to surface-confined DNA via electrostatic interaction, was used to produce chronocoulometric signal; Au nanoparticles (AuNPs) were used to improve the sensitivity of the sensor by amplifying the detection signals. Moreover, the sensing interface fabrication, sample incubation, and electrochemical detection were all performed in microfluidic channels. By using this detection chip, we achieved the multianalysis of two model small molecules, ATP, and cocaine, in mixed samples within 40 min. The detection limit of ATP was 3 × 10(-10) M, whereas the detection limit of cocaine was 7 × 10(-8) M. This Au-Ag dual metal electrochemical chip detector integrated MECAS was simple, sensitive, and selective. Also it is similar to a dosimeter which accumulates signal upon exposure. It held promising potential for designing electrochemical devices with high throughput, high automation, and high integration in multianalysis.     

Generation of Femtoliter Reactor Arrays within a Microfluidic Channel for Biochemical Analysis

We present a simple microfluidic method to generate high-density femotoliter-sized microreactor arrays within microfluidic channels. In general, we designed a main channel with many small chambers built into its walls. After sequentially infusing aqueous solution and organic solvent from a single tube into the device, aqueous droplets are confined in the chambers by the solvent flow. The generated reactors are small and stable enough for carrying out ultrasensitive biochemical assays at single molecule levels. As a demonstration, in this paper, we optically observed hydrolysis activity of β-galactosidase enzymatic molecules in the reactor arrays at single molecule levels. Further, this method has the following advantages: (1) the droplets are observable immediately after formation and (2) its simple procedure is sufficiently robust such that even handy infusion of the preloaded solutions is reproducible. We believe our method provides a platform attractive to a variety of single molecule studies and sensing applications such as clinical diagnostics.     

High-throughput DNA droplet assays using picoliter reactor volumes

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 reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, 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 compositions 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 concentrations. 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.     

Microfluidic DNA fragmentation for on-chip genomic analysis

We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ～ 5k?bp fragment lengths by applying low hydraulic pressures (≤1?bar) across micromachined constrictions positioned in larger microfluidic channels that create point-sink flow with large velocity gradients near the constriction entrance. Long constrictions (100?μm) produce shorter fragment lengths compared to shorter constrictions (10?μm), while increasing the hydrodynamic pressure requirement. Sample recirculation (10 ×) in short constrictions reduces the mean fragment length and fragment length variation, and improves yield compared to single-pass experiments without increasing the hydrodynamic pressure.     

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.     

Resolution of multiple ssDNA structures in free solution electrophoresis

By using high concentrations of buffer, electroosmotic flow within uncoated channels of a microfluidic chip was minimized, allowing the free solution electrophoretic separation of DNA. More importantly, because of the ability to efficiently dissipate heat within these channels, field strengths as high as 600 V/cm could be applied with minimal Joule heating (<2 degrees C). As a result of the higher field strengths, separations within an 8-cm-long channel were achieved within a few minutes. However, when the electrophoretic separation of single-stranded DNA (ssDNA) less than 22 bases in length was performed, containing the fluorophore Texas Red as an end label, more than the expected single peak was observed at this high electric field. On the other hand, the free solution electrophoresis of a double-stranded DNA (dsDNA) consisting of a random sequence did exhibit the expected single peak. The appearance of these multiple peaks for ssDNA is shown to be dependent upon the base content and sequence of the ssDNA as well as on the chemical structure of the fluorophore used to tag the DNA for detection. Specifically, the peaks can be attributed to different secondary structures that result either from hydrophobic interactions between the DNA bases and an uncharged fluorescent dye or from G-quadruplexes within guanine-rich strands.     

Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 min was achieved. Further development of the system may allow application to high-throughput applications such as drug screening.     

Electrostretching DNA molecules using polymer-enhanced media within microfabricated devices

In this paper, we demonstrate immobilization and stretching of single lambda-phage DNA molecules within microfluidic systems using ac fields. We present a novel "thiol-on-gold"-based immobilization technique for fixing one specific end (3' end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (approximately 3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 microm) of fluorescently stained lambda-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 microm for stretching DNA molecules with an ac field (1 MHz, 3 x 10(5) V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device.     

Single-molecule DNA amplification and analysis in an integrated microfluidic device

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.     

Detection of Individual Molecules and Ions by Carbon Nanotube‐Based Differential Resistive Pulse Sensor

This paper presents a new method of sensing single molecules and cations by a carbon nanotube (CNT)‐based differential resistive pulse sensing (RPS) technique on a nanofluidic chip. A mathematical model for multichannel RPS systems is developed to evaluate the CNT‐based RPS signals. Individual cations, rhodamine B dye molecules, and ssDNAs are detected successfully with high resolution and high signal‐to‐noise ratio. Differentiating ssDNAs with 15 and 30 nucleotides are achieved. The experimental results also show that translocation of negatively charged ssDNAs through a CNT decreases the electrical resistance of the CNT channel, while translocation of positively charged cations and rhodamine B molecules increases the electrical resistance of the CNT. The CNT‐based nanofluidic device developed in this work provides a new avenue for single‐molecule/ion detection and offers a potential strategy for DNA sequencing.     

Sensitized luminescent terbium nanoparticles: preparation and time-resolved fluorescence assay for DNA

A highly luminescent terbium nanoparticle as the biolabel based on the sensitization of a dye molecule was prepared. The luminescent complexes included in the particles were composed of a quinolone-based dye molecule as the light-energy transfer donor and a polyaminocarboxylate-based chelator with excellent water-solubility and a high binding constant for lanthanides. The structure of two functional entities in the single molecule made the complex highly luminescent in aqueous solution. Silica nanoparticles containing terbium complexes were prepared by the reverse microemulsion method. Such a terbium nanoparticle is as bright as about 340 free terbium complexes, and it has a 1.5-ms fluorescence lifetime that enables it to be used in the time-resolved fluorescence assays. The conjugate of the nanoparticle with oligonucleotide was prepared and used to carry out a DNA sandwich hybridization assay based on magnetic microbeads as solid-phase carrier. The experimental results showed that the detection sensitivity with the nanoparticles is more than 100-fold as high as that with dye Fluorescein isothiocyanate (FITC) molecules.     

Art‐on‐a‐Chip: Preserving Microfluidic Chips for Visualization and Permanent Display

“After a certain high level of technical skill is achieved, science and art tend to coalesce in aesthetics, plasticity, and form. The greatest scientists are always artists as well.” said Albert Einstein. Currently, photographic images bridge the gap between microfluidic/lab‐on‐a‐chip devices and art. However, the microfluidic chip itself should be a form of art. Here, novel vibrant epoxy dyes are presented in combination with a simple process to fill and preserve microfluidic chips, to produce microfluidic art or art‐on‐a‐chip. In addition, this process can be used to produce epoxy dye patterned substrates that preserve the geometry of the microfluidic channels—height within 10% of the mold master. This simple approach for preserving microfluidic chips with vibrant, colorful, and long‐lasting epoxy dyes creates microfluidic chips that can easily be visualized and photographed repeatedly, for at least 11 years, and hence enabling researchers to showcase their microfluidic chips to potential graduate students, investors, and collaborators.     

Microfabricated polymer analysis chip for optical detection

A coupling between multimode polymer waveguides and microfluidic channels on a polymethylmethacrylate (PMMA) capillary electrophoresis (CE)-chip for optical analytical applications has been successfully realised. This technology allows the integration of polymer optical waveguides together with hermetically sealed fluidic channels. The microchannels and waveguides are made in PMMA by the approved hot-embossing technology. The technology developed for the fabrication of polymer waveguides on the microfluidic chip offers the possibility of great flexibility in the choice of core materials, design and alignment of the polymer waveguides. The integration of polymer waveguides on an analysis chip enables highly spatially resolved optical detection without the large and expensive conventionally used apparatus. The optical properties of the analytical system developed are verified by transmission and propagation loss measurements. The results of measurements prove the suitability of the presented device for optical applications between 440 and 800 nm. This was shown with absorbance measurements of the dye Sulfanilazochromotrop (SPADNS) within 50 microm fluidic channels.