Zhang-Run Xu

An osmotic micro-pump integrated on a microfluidic chip for perfusion cell culture

A novel microfluidic chip integrating an osmosis-based micro-pump was developed and used for perfusion cell culture. The micro-pump includes two sealed chambers, i.e., the inner osmotic reagent chamber and the outer water chamber, sandwiching a semi-permeable membrane. The water in the outer chamber was forced to flow through the membrane into the inner chamber via osmosis, facilitating continuous flow of fluidic zone in the channel. An average flow rate of 0.33microLmin(-1) was obtained within 50h along with a precision of 4.3% RSD (n=51) by using a 100mgmL(-1) polyvinylpyrrolidone (PVP) solution as the osmotic driving reagent and a flow passage area of 0.98cm(2) of the semi-permeable membrane. The power-free micro-pump has been demonstrated to be pulse-free offering stable flow rates during long-term operation. The present microfluidic chip has been successfully applied for the perfusion culture of human colorectal carcinoma cell by continuously refreshing the culture medium with the osmotic micro-pump. In addition, in situ cell immunostaining was also performed on the microchip by driving all the reagent zones with the integrated micro-pump.

DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system

A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.

Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient’s age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.

Microfluidic fabrication of multifunctional particles and their analytical applications.

Multifunctional particles have attracted extensive interest in scientific community in recent years for their capability in combining different functions within a single device. The present review focuses on the preparation methods of multifunctional particles using microfluidic techniques, and the applications of multifunctional particles in analytical and bio-analytical chemistry. As confirmed by most research works, microfluidic fabrication platforms can provide multifunctional particles with precisely controlled structure, high homogeneity and good reproducibility. Meanwhttp://live.elsevierproofcentral.com/authorproofs/macm84f82089f9eab0d214807a46cda8088e/supplier hile, multifunctional particles are proved to have enormous promise when applied in bio/chemical analysis. This paper aims to offer a path for the readers to get acquainted with state-of-the-art progress in these advanced materials from the viewpoint of microfluidics.

A microfluidic concentration-gradient droplet array generator for the production of multi-color nanoparticles

A microfluidic concentration-gradient droplet array generator (CDrAG) with parallel multi-channels and multi-layers was developed with 64 outlet channels producing 33 droplet gradient concentrations. A droplet production rate of 5 × 10(4) min(-1) was obtained, and the RSD value of droplet diameters in 64 groups is 5.5% (n = 64). Using the concentration gradient droplet array as parallel microreactors, 33 Au/Ag ratio nanoparticles were synthesized. The absorption spectra of the Au/Ag nanoparticles shifted from the spectrum of pure gold to one of pure silver. This demonstrates the CDrAG platforms promising potential to produce specific nanoparticle barcodes for high-throughput screening in chemistry, biology and a broad range of life science applications.

Fast DNA hybridization on a microfluidic mixing device based on pneumatic driving

A novel fluid mixing strategy was developed which significantly enhanced the efficiency of DNA hybridization. A pneumatic micro-mixing device consisting of two pneumatic chambers and an underneath DNA microarray chamber was built up. The fluid in the array chamber was pneumatically pumped alternately by the two pneumatic chambers. The chaotic oscillatory flow caused by the pumping greatly intensified the fluidic mixing. A homogeneous distribution of the tracer dye solution in the microarray chamber was observed after 2s mixing with a pumping frequency of 24 Hz. Microarray DNA hybridization was substantially accelerated using this device, and the fluorescence intensity showed a plateau after oscillating 30s at room temperature. The corresponding signal level of the dynamic hybridization was 12.5-fold higher than that of the static hybridization performed at 42°C. A signal-to-noise ratio of 117 was achieved and the nonspecific adsorption of the targets to the sample array was minimized, which might be attributed to the strong shearing force generated during the pneumatic mixing process.

On-chip preparation of calcium alginate particles based on droplet templates formed by using a centrifugal microfluidic technique.

A novel chip-based approach for the fabrication of oblate spheriodal calcium alginate particles was developed by combining the droplet template method and the centrifugal microfluidic strategy. Circular chips with multiple radial channels were designed. Sodium alginate solutions in radial channels were flung into CaCl2 solutions in the form of droplets under centrifugal force, and the droplets transformed into particles through cross-linking reaction. The size and morphology of particles could be controlled by regulating the centrifugal force, the channel geometry and the distance between the channel outlet and the CaCl2 solution. The throughput of particle production was evidently enhanced by increasing the number of radial channels to 48 and 64. The coefficients of variation of particle sizes were in the range of 5.2-5.6%, which indicated the monodisperse particles could be prepared by using the present method. With the chip configuration readily modified, the same platform could be used to produce Janus particles. The Janus particles showed clear interfaces owing to the high flight speed and the rapid gelling process of the droplets. This method would be capable of generating particles with complicated morphology and multifunction from diverse polymeric materials.

A miniaturized spatial temperature gradient capillary electrophoresis system with radiative heating and automated sample introduction for DNA mutation detection.

A miniaturized spatial temperature gradient CE system with automated sample introduction for DNA mutation detection was established. Continuous electrokinetic sample injection was achieved by combining an automated slotted‐vial array sample introduction device to the spatial temperature gradient CE system. The temperature gradient was produced by a radiative heating system with a single graphite block heater, and the stability of the temperature gradient was investigated. The temperature variation of each measure point was 0.12–0.21% RSD (n=7) within 6 h. A 14‐cm Teflon AF‐coated silica capillary was used both as the separation channel and as the liquid‐core waveguide tube of fluorescence signal. Under a temperature gradient from 54.8 to 59.5°C, a low range control mutation standard (209 bp) was separated within 4 min with only 5.6 nL sample consumption. Automated continuous sample introducing and changing were realized with a carryover of 3.3%. Utility of the system was further demonstrated by detecting K‐ras gene mutations in paraffin tissue sections from two colorectal cancer patients.

Controlled production of size-tunable Janus droplets for submicron particle synthesis using an electrospray microfluidic chip

Size-tunable Janus droplets of two miscible liquids were produced and further employed for synthesizing Janus particles with submicron size. An electrospray microfluidic chip was fabricated to form the Janus droplets from two-phase laminar flow, and to quickly regulate the droplet size by an electric field. With the aid of a direct-current electric field, we could obtain Janus droplets with various diameters in the range from 135 μm down to 3 μm. This regulation method was adaptive to the droplets made up of both aqueous solutions and organic polymers. The mechanism for the dependency of drop size on the high voltage was discussed comprehensively. Finally, submicron Janus particles were synthesized taking the minimized Janus droplets as templates.

SERS–Fluorescence Dual-Mode pH-Sensing Method Based on Janus Microparticles

A surface-enhanced Raman scattering (SERS)-fluorescence dual-mode pH-sensing method based on Janus microgels was developed, which combined the advantages of high specificity offered by SERS and fast imaging afforded by fluorescence. Dual-mode probes, pH-dependent 4-mercaptobenzoic acid, and carbon dots were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via a centrifugal microfluidic chip. On the basis of the obvious volumetric change of hydrogels in different pHs, the Janus microparticles were successfully applied for sensitive and reliable pH measurement from 1.0 to 8.0, and the two hemispheres showed no obvious interference. The proposed method addressed the limitation that sole use of the SERS-based pH sensing usually failed in strong acidic media. The gastric juice pH and extracellular pH change were measured separately in vitro using the Janus microparticles, which confirmed the validity of microgels for pH sensing. The microparticles exhibited good stability, reversibility, biocompatibility, and ideal semipermeability for avoiding protein contamination, and they have the potential to be implantable sensors to continuously monitor pH in vivo.