Wenbin Cao

Extraction, amplification and detection of DNA in microfluidic chip-based assays

AbstractThis review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references. FigureA graphical presentation of main PCR assays: DNA extraction from raw sample, target amplification by PCR and final product detection in conventional bench-top lab and miniaturized microfluidic chip.

Polydimethylsiloxane microfluidic chip with integrated microheater and thermal sensor

A microheater and a thermal sensor were fabricated inside elastomeric polydimethylsiloxane microchannels by injecting silver paint (or other conductive materials) into the channels. With a high-precision control scheme, microheaters can be used for rapid heating, with precise temperature control and uniform thermal distribution. Using such a microheater and feedback system, a polymerase chain reaction experiment was carried out whereas the DNA was successfully amplified in 25 cycles, with 1 min per cycle.

Design and integration of an all-in-one biomicrofluidic chip

We demonstrate a highly integrated microfluidic chip with the function of DNA amplification. The integrated chip combines giant electrorheological-fluid actuated micromixer and micropump with a microheater array, all formed using soft lithography. Internal functional components are based on polydimethylsiloxane (PDMS) and silvercarbon black-PDMS composites. The system has the advantages of small size with a high degree of integration, high polymerase chain reaction efficiency, digital control and simple fabrication at low cost. This integration approach shows promise for a broad range of applications in chemical synthesis and biological sensinganalysis, as different components can be combined to target desired functionalities, with flexible designs of different microchips easily realizable through soft lithography.

Continuous particle focusing in a waved microchannel using negative dc dielectrophoresis

We present a waved microchannel for continuous focusing of microparticles and cells using negative direct current (dc) dielectrophoresis. The waved channel is composed of consecutive s-shaped curved channels in series to generate an electric field gradient required for the dielectrophoretic effect. When particles move electrokinetically through the channel, the experienced negative dielectrophoretic forces alternate directions within two adjacent semicircular microchannels, leading to a focused continuous-flow stream along the channel centerline. Both the experimentally observed and numerically simulated results of the focusing performance are reported, which coincide acceptably in proportion to the specified dimensions (i.e. inlet and outlet of the waved channel). How the applied electric field, particle size and medium concentration affect the performance was studied by focusing polystyrene microparticles of varying sizes. As an application in the field of biology, the focusing of yeast cells in the waved mcirochannel was tested. This waved microchannel shows a great potential for microflow cytometry applications and is expected to be widely used before different processing steps in lab-on-a-chip devices with integrated functions.

Poly(L‑lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL‑g‑PMOXA‑c‑FA): A Bioactive Copolymer for Specific Targeting to Folate Receptor-Positive Cancer Cells

In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.

Mapping three-dimensional temperature in microfluidic chip.

Three-dimensional (3D) temperature mapping method with high spatial resolution and acquisition rate is of vital importance in evaluating thermal processes in micro-environment. We have synthesized a new temperature-sensitive functional material (Rhodamine B functionalized Polydimethylsiloxane). By performing optical sectioning of this material, we established an advanced method for visualizing the micro-scale 3D thermal distribution inside microfluidic chip with down to 10 ms temporal resolution and 2 ~ 6°C temperature resolution depending the capture parameters. This method is successfully applied to monitor the local temperature variation throughout micro-droplet heat transfer process and further reveal exothermic nanoliter droplet reactions to be unique and milder than bench-top experiment.

Facile and high spatial resolution ratio-metric luminescence thermal mapping in microfluidics by near infrared excited upconversion nanoparticles

A local area temperature monitor is important for precise control of chemical and biological processes in microfluidics. In this work, we developed a facile method to realize micron spatial resolution of temperature mapping in a microfluidic channel quickly and cost effectively. Based on the temperature dependent fluorescence emission of NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNPs) under near-infrared irradiation, ratio-metric imaging of UCNPs doped polydimethylsiloxane can map detailed temperature distribution in the channel. Unlike some reported strategies that utilize temperature sensitive organic dye (such as Rhodamine) to achieve thermal sensing, our method is highly chemically inert and physically stable without any performance degradation in long term operation. Moreover, this method can be easily scaled up or down, since the spatial and temperature resolution is determined by an optical imaging system. Our method supplied a simple and efficient solution for temperature mapping on a heteroge...

Simple and reusable picoinjector for liquid delivery via nanofluidics approach

Precise control of sample volume is one of the most important functions in lab-on-a-chip (LOC) systems, especially for chemical and biological reactions. The common approach used for liquid delivery involves the employment of capillaries and microstructures for generating a droplet which has a volume in the nanoliter or picoliter range. Here, we report a novel approach for constructing a picoinjector which is based on well-controlled electroosmotic (EO) flow to electrokinetically drive sample solutions. This picoinjector comprises an array of interconnected nanochannels for liquid delivery. Such technique for liquid delivery has the advantages of well-controlled sample volume and reusable nanofluidic chip, and it was reported for the first time. In the study of the pumping process for this picoinjector, the EO flow rate was determined by the intensity of the fluorescent probe. The influence of ion concentration in electrolyte solutions over the EO flow rate was also investigated and discussed. The application of this EO-driven picoinjector for chemical reactions was demonstrated by the reaction between Fluo-4 and calcium chloride with the reaction cycle controlled by the applied square waves of different duty cycles. The precision of our device can reach down to picoliter per second, which is much smaller than that of most existing technologies. This new approach, thus, opens further possibilities of adopting nanofluidics for well-controlled chemical reactions with particular applications in nanoparticle synthesis, bimolecular synthesis, drug delivery, and diagnostic testing.PACS85.85.+ j; 87.15.hj; 82.39.Wj

Digital microfluidic programmable stencil (dMPS) for protein and cell patterning

Patterning biomolecules and cells on substrates is usually a prerequisite for biological analysis and cell studies. A stencil is a versatile tool for sample patterning owing to its reusability and easy operation but it lacks addressable fluid control and programmable change of pattern. Here, we combined the advantages of a microfluidic chip and stencil to design a digitally controlled microfluidic programmable stencil. The key design is automatic passive matrix addressing based on combined application of two types of elastomeric valves. These two valves have distinct actuation thresholds, typically 13.4 psi for a round valve and much higher than 13.4 psi for a rectangle valve. Different types of protein and cell pattern on a 2D substrate could be obtained by controlling the fluid addressing code based on the passive matrix addressing method. An automatic microsampler was also applied to facilitate fast sample selection and introduction in the patterning process. A successful protein and cell pattern was obtained which could be used for downstream analysis and study.