Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.     

Microfluidic device as a new platform for immunofluorescent detection of viruses

A bead-based microfluidic device was developed and demonstrated to achieve rapid and sensitive enzyme-linked immunosorbent assay (ELISA) with quantum dots as the labeling fluorophore for virus detection. In comparison to standard ELISA performed on the same virus, the minimal detectable concentration of the target virus was improved from 360 to 22 ng mL-1, the detection time was shortened from >3.25 h to <30 min, and the amount of antibody consumed was reduced by a factor of 14.3.     

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.     

Microfluidic device for capillary electrochromatography-mass spectrometry

A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4-8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement.     

Mixed DNA-functionalized nanoparticle probes for surface-enhanced Raman scattering-based multiplex DNA detection

Highly stable silver nanoparticle-oligonucleotide conjugates were prepared. Based on the mixed DNA-functionalized silver nanoparticles (AgNPs), multiplex DNA detections were demonstrated successfully by SERS.     

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.     

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300 amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.     

Microfluidic device for label-free measurement of platelet activation

In this work we demonstrate a new microfluidic method for the rapid assessment of platelet size and morphology in whole blood. The device continuously fractionates particles according to size by displacing them perpendicularly to the fluid flow direction in a micro-fabricated post array. Whole blood, labeled with the fluorescent, platelet specific, antibody PE-anti-CD41, was run through the device and the positions of fluorescent objects noted as they exited the array. From this, histograms of platelet size were created which show marked increases in size after exposure to thrombin or a temperature of 4 degrees C. We infer that the well known morphological changes that occur during activation are causing the observed increase in size.     

Microfluidic techniques for tumor cell detection

Cancer metastasis is the main cause of cancer‐related death. Early detection of tumor cell in peripheral blood is of great significant to early diagnosis and effective treatment of cancer. Over the past two decades, microfluidic technologies have been demonstrated to have great potential for isolating and detecting tumor cell from blood. The present paper reviews microfluidic techniques for tumor cell detection based on various physical principles. The specific methods are categorized into active and passive methods depending on whether extra force field is applied. Working principles of the two methods are explained in detail, including microfluidics combined with optical tweezer, electric field, magnetic field, acoustophoresis, and without extra fields for tumor cell detection. Typical experiments and the results are reviewed. Based on these, research characteristics of the two methods are analyzed.     

Microfluidic baker's transformation device for three-dimensional rapid mixing

We developed a new passive-type micromixer based on the baker's transformation and realized a fast mixing of a protein solution, which has lower diffusion constant. The baker's transformation is an ideal mixing method, but there is no report on the microfluidic baker's transformation (MBT), since it is required to fabricate the complicated three-dimensional (3D) structure to realize the MBT device. In this note, we successfully fabricate the MBT device by using precision diamond cutting of an oxygen-free copper substrate for the mould fabrication and PDMS replication. The MBT device with 10.4 mm mixing length enables us to achieve complete mixing of a FITC solution (D = 2.6 × 10(-10) m(2) s(-1)) within 51 ms and an IgG solution (D = 4.6 × 10(-11) m(2) s(-1)) within 306 ms. Its mixing speed is 70-fold higher for a FITC solution and 900-fold higher for an IgG solution than the mixing speed by the microchannel without MBT structures. The Péclet number to attain complete mixing in the MBT device is estimated to be 6.9 × 10(4).     

DNA detection with a polymeric nanochannel device

We present the development and the electrical characterization of a polymeric nanochannel device. Standard microfabrication coupled to Focused Ion Beam (FIB) nanofabrication is used to fabricate a silicon master, which can be then replicated in a polymeric material by soft lithography. Such an elastomeric nanochannel device is used to study DNA translocation events during electrophoresis experiments. Our results demonstrate that an easy and low cost fabrication technique allows creation of a low noise device for single molecule analysis.     

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol?L^?1 for all B19 genotype oligonucleotides and 650 pmol?L^?1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

Microfluidic device based on a micro-hydrocyclone for particle-liquid separation

This paper presents theoretical analysis, design, simulation, fabrication and test of a microfluidic device ('Micro-hydrocyclone') for separation of micron and submicron size solid particles from liquid in a particle liquid mixture. A theoretical analysis of the micro-hydrocyclone is performed to understand the physics and develop suitable design models. The structure of the proposed device is designed based on the Bradley model, as it offers lower cut-size thus making it suitable for microfluidics applications. The operational parameters are derived from the dimensional group model. The particle separation process inside the micro-hydrocyclone is simulated by solving fluid flows using Navier-Stokes equations and particle dynamics using a Lagrangian approach in a Eulerian fluid. The influence of inlet velocity and density on separation efficiency is investigated. The device is fabricated with SU-8 photoresist on a PMMA substrate using a combination of photolithography and micro-milling. Experiments are performed to demonstrate particle-liquid separation using polystyrene microbeads suspended in PBS as the feed sample. The influence of inlet velocity and particle size on particle separation efficiency is measured and compared with that obtained from simulations and a good match was found. The proposed device can be easily integrated with micro-environments thus it is suitable for lab-on-chip and microsystems development. The device may have applications in chemical analysis, materials research, point-of-care, blood sample preparation and other biomedical applications.     

Microfluidic chip for high efficiency DNA extraction

A high efficiency DNA extraction microchip was designed to extract DNA from lysed cells using immobilized beads and the solution flowing back and forth. This chip was able to increase the extraction efficiency by 2-fold when there was no serum. When serum existed in the solution, the extraction efficiency of immobilized beads was 88-fold higher than that of free beads. The extraction efficiency of the microchip was tested under different conditions and numbers of E. coli cells. When the number of E. coli cells was between 10(6) and 10(8) in 25 microl of whole blood, the extraction efficiency using immobilized beads was only slightly higher than that using free beads (10(0) to 10(1) fold). When the number of E. coli cells was in the range 10(4) to 10(6) in 25 microl of whole blood, the extraction efficiency of immobilized beads was greater than that of the free beads (10(1) to 10(2) fold). When the number of E. coli cells was lower, in the range 10(3) to 10(4) in 25 microl of whole blood, the extraction efficiency of immobilized beads was much higher than that of the free beads (10(2) to 10(3) fold). This study indicated that DNA could be efficiently extracted even when the number of bacterial cells was smaller (10(5) to 10(3)). This microfluidic extraction chip could find potential applications in rare sample genomic study.     

Microfluidic distillation chip for methanol concentration detection

An integrated microfluidic distillation system is proposed for separating a mixed ethanol-methanol-water solution into its constituent components. The microfluidic chip is fabricated using a CO₂ laser system and comprises a serpentine channel, a boiling zone, a heating zone, and a cooled collection chamber filled with de-ionized (DI) water. In the proposed device, the ethanol-methanol-water solution is injected into the microfluidic chip and driven through the serpentine channel and into the collection chamber by means of a nitrogen carrier gas. Following the distillation process, the ethanol-methanol vapor flows into the collection chamber and condenses into the DI water. The resulting solution is removed from the collection tank and reacted with a mixed indicator. Finally, the methanol concentration is inversely derived from the absorbance measurements obtained using a spectrophotometer. The experimental results show the proposed microfluidic system achieves an average methanol distillation efficiency of 97%. The practicality of the proposed device is demonstrated by detecting the methanol concentrations of two commercial fruit wines. It is shown that the measured concentration values deviate by no more than 3% from those obtained using a conventional bench top system.     

Microfluidic device for immunoassays based on surface plasmon resonance imaging

We have designed and fabricated a polydimethylsiloxane (PDMS) microfluidic device containing an array of gold spots onto which antigens or antibodies of interest can be attached. We use surface plasmon resonance (SPR) imaging to monitor the antibody-antigen recognition and binding events. This combination offers two significant advantages: (1) the microfluidic device dramatically reduces reaction time and sample consumption; and (2) the SPR imaging yields real-time detection of the immunocomplex formation. Thus, an immunoreaction may be detected and quantitatively characterized in about 10 min. The sensitivity of this method is at the subnanomolar level. When gold nanoparticles are selectively coupled to the immunocomplex to cause signal amplification, the sensitivity reaches the ten to one hundred picomolar level but the time required increases to about 60 min.     

Microfluidic fabrication of SERS-active microspheres for molecular detection

In this paper, we demonstrated a microfluidic system for fabricating microspheres with hierarchical surface nanopatterns for molecular detection based on surface-enhanced Raman scattering (SERS). Briefly, a photocurable silica suspension was emulsified into monodisperse droplets using a microfluidic device composed of two coaxial glass capillaries. The silica particles in each droplet protruded through the interface and spontaneously formed a hexagonal array. After polymerization of the droplets, we selectively decorated the exposed areas of the silica particles with silver nanoparticles through electroless deposition. The resulting hierarchically-structured microspheres showed high sensitivity and fast binding kinetics in molecular detection based on SERS, owing to the dense array of hot spots on each microsphere and high mobility of the microspheres, respectively. Notably, the SERS signals from molecules adsorbed on the microspheres could be detected in both the dried and suspension states. In addition, we demonstrated that the SERS-active microspheres can be functionalized into structural colored or magnetoresponsive microspheres for advanced applications.     

Microfluidic discharge-based optical sources for detection of biochemicals

This paper reports a discharge-based optical source for fluorescence of biochemicals in microfluidic systems. Its efficacy is demonstrated using a stacked microchip that integrates a microfluidic wavelength-tunable optical source, a biochemical sample reservoir and optical filters. It is shown to excite fluorescence in l-tryptophan and DNA samples labeled by SYBR green dye. The discharge is struck in ambient air, between a metal anode and a cathode cavity that is filled with an aqueous solution, which is doped with a metal salt selected for its emission characteristics. The characteristic line spectra, which arise from energetic transitions of the metal ions that are sputtered into the glow region of the discharge, are optically filtered and guided to the biochemical sample that resides in a separate on-chip reservoir. For DNA fluorescence, a barium chloride solution is used to emit light at 454 and 493 nm. For tryptophan fluorescence, the cathode contains lead (ii) nitrate solution to provide a 280 nm emission. The resulting fluorescence from the DNA and tryptophan samples is compared to reference data. This technique can also be used to excite other fluorophores by using appropriately doped liquid cathodes having the desired emission characteristics.