Off-chip passivated-electrode, insulator-based dielectrophoresis (OπDEP)

In this study, we report the first off-chip passivated-electrode, insulator-based dielectrophoresis microchip (OπDEP). This technique combines the sensitivity of electrode-based dielectrophoresis (eDEP) with the high-throughput and inexpensive device characteristics of insulator-based dielectrophoresis (iDEP). The device is composed of a permanent, reusable set of electrodes and a disposable, polymer microfluidic chip with microposts embedded in the microchannel. The device operates by capacitively coupling the electric fields into the microchannel; thus, no physical connections are made between the electrodes and the microfluidic device. During operation, the polydimethylsiloxan (PDMS) microfluidic chip fits onto the electrode substrate as a disposable cartridge. OπDEP uses insulting structures within the channel as well as parallel electrodes to create DEP forces by the same working principle that iDEP devices use. The resulting devices create DEP forces which are larger by two orders of magnitude for the same applied voltage when compared to off-chip eDEP designs from literature, which rely on parallel electrodes alone to produce the DEP forces. The larger DEP forces allow the OπDEP device to operate at high flow rates exceeding 1 mL/h. In order to demonstrate this technology, Escherichia coli (E. coli), a known waterborne pathogen, was trapped from water samples. Trapping efficiencies of 100 % were obtained at flow rates as high as 400 μL/h and 60 % at flow rates as high as 1200 μL/h. Additionally, bacteria were selectively concentrated from a suspension of polystyrene beads.

Novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives: Synthesis, crystal structure, fluorescence properties and cytotoxicity evaluation against human breast cancer cells

A series of novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II) have been designed and synthesized. The target compounds have been identified by elemental analysis and spectral (¹H NMR, IR, and MS) data and the absolute configuration of compound (II ₁ ) was confirmed by single crystal X-ray diffraction. The cytotoxicity of the target compounds have been evaluated in vitro against two human breast cancer cell lines MCF-7 and MDA-MB-231 by MTT assay. Most compounds exhibited good inhibition, and compounds II ₂₁ (IC₅₀ = 4.7 μM for MCF-7 and IC₅₀ = 9.3 μM for MDA-MB-231), II ₃₃ (IC₅₀ = 2.4 μM for MCF-7 and IC₅₀ = 4.2 μM for MDA-MB-231) and II ₄₀ (IC₅₀ = 3.3 μM for MCF-7 and IC₅₀ =8.6 μM for MDA-MB-231) displayed better inhibitory activity than 5-fluorouracil (IC₅₀ = 4.8 μM for MCF-7 and IC₅₀ = 9.6 μM for MDA-MB-231, respectively). Flow cytometric analysis and DNA fragmentation suggest that II ₃₃ is cytotoxic and able to induce the apoptosis of MCF-7 cells. The fluorescence properties of compounds II ₁ , II ₆ , II ₁₁ , II ₁₆ , II ₂₃ , II ₂₈ , and II ₃₅ were also studied and compound II ₂₈ afforded the highest photoluminescence quantum yield (38%).     

Synthesis, X-ray structural analysis, and cytotoxic activity of some new androstane d-homo lactone derivatives

A series of d-homo lactones 4?C10 from dehydroepiandrosterone 1 via 16-hydroximino derivatives 2 and 3 were synthesized. The d-homo lactone 4 was transformed by the Oppenauer oxidation to obtain compound 5. The (Z)-2-hydroxymethylene-4-en-3-one compound 6, was obtained through reaction of 4-en-3-one compound 5 with ethyl formate and sodium hydride. The epoxides 8 and 9 were prepared from compound 7 by oxidation with m-chloroperbenzoic acid. Compound 10 was obtained by treating epoxides 8 and 9 with chromium(VI)-oxide. The structure of compounds 6 and 10 were confirmed by X-ray structural analysis. These derivatives were screened for antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER?, MDA-MB-231, prostate cancer AR?, PC-3), and one human non-tumor cell line, MRC-5. Compounds 4, 7, 8, and 10 exhibited significant antitumor activity against MDA-MB-231 cells, while compound 5 showed strong cytotoxicity against MDA-MB-231. No compounds displayed toxicity against MRC-5 cells.     

Quartz microfluidic chip for tumour cell identification by Raman spectroscopy in combination with optical traps

Three important technical innovations are reported here towards Raman-activated cell sorting. Firstly, a microfluidic chip made of quartz is introduced which integrates injection of single cells, trapping by laser fibres and sorting of cells. Secondly, a chip holder was designed to provide simple, accurate and stable adjustment of chips, microfluidic connections and the trapping laser fibres. The new setup enables to the collection of Raman spectra of single cells at 785 nm excitation with 10 s exposure time. Lastly, a new type of modelling the various background contributions is described, improving Raman-based cell identification by the classification algorithm linear discriminant analysis. Mean sensitivity and specificity determined by iterated 10-fold cross validation were 96 and 99 %, respectively, for the distinction of leucocytes extracted from blood, breast cancer cells BT-20 and MCF-7, and leukaemia cells OCI-AML3.      

A microfluidic system to study the cytotoxic effect of drugs: the combined effect of celecoxib and 5-fluorouracil on normal and cancer cells

We have investigated the response of normal and cancer cells to exposure a combination of celecoxib (Celbx) and 5-fluorouracil (5-FU) using a lab-on-a-chip microfluidic device. Specifically, we have tested the cytotoxic effect of Celbx on normal mouse embryo cells (Balb/c 3T3) and human lung carcinoma cells (A549). The single drugs or their combinations were adjusted to five different concentrations using a concentration gradient generator (CGG) in a single step. The results suggest that Celbx can enhanced the anticancer activity of 5-FU by stronger inhibition of cancer cell growth. We also show that the A549 cancer cells are more sensitive to Celbx than the Balb/c 3T3 normal cells. The results obtained with the microfluidic system were compared to those obtained with a macroscale in vitro cell culture method. In our opinion, the microfluidic system represents a unique approach for an evaluation of cellular response to multidrug exposure that also is more simple than respective microwell plate assays.

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L^?1. The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

A microfluidic device for open loop stripping of volatile organic compounds

The detection of volatile organic compounds is of great importance for assessing the quality of water. In this contribution, we describe a miniaturized stripping device that allows fast online detection of organic solvents in water. The core component is a glass microfluidic chip that facilitates the creation of an annular-flowing stream of water and nitrogen gas. Volatile compounds are transferred efficiently from the water into the gas phase along the microfluidic pathway at room temperature within less than 5 s. Before exiting the microchip, the liquid phase is separated from the enriched gas phase by incorporating side capillaries through which the hydrophilic water phase is withdrawn. The gas phase is conveniently collected at the outlet reservoir by tubing. Finally, a semiconductor gas sensor analyzes the concentration of (volatile) organic compounds in the nitrogen gas. The operation and use of the stripping device is demonstrated for the organic solvents THF, 1-propanol, toluene, ethylbenzene, benzaldehyde, and methanol. The mobile, inexpensive, and continuously operating system with liquid flow rates in the low range of microliters per minute can be connected to other detectors or implemented in chemical production line for process control.

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Andrographolide Exhibits Anticancer Activity against Breast Cancer Cells (MCF-7 and MDA-MB-231 Cells) through Suppressing Cell Proliferation and Inducing Cell Apoptosis via Inactivation of ER-α Receptor and PI3K/AKT/mTOR Signaling

Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.     

Insulator-based dielectrophoretic single particle and single cancer cell trapping

Trapping of individual cells at specific locations in a microfluidic lab-on-a-chip platform is essential for single cell studies, especially those requiring individual stimulation followed by downstream analysis. To this aim, we have designed microdevices based on direct current (DC) insulator-based dielectrophoresis (iDEP) acting as individual single cell traps. We present both the design of a negative iDEP trap and a positive iDEP trap using insulating posts integrated at microchannel intersections. We obtained electric field distributions via numerical simulations adapted to the intersection and trap geometry with which we predict single particle pathlines. With polystyrene particles of 10?μm diameter, we demonstrated an effective design for a single particle trap in the case of negative dielectrophoresis. The onset trapping voltage shows an inverse relation to the buffer conductivity, thus indicating the influence of electrokinetic effects on the trapping behavior. Additionally, we demonstrated the proof-of-principle of single MCF-7 breast cancer cell trapping in a positive iDEP trap. Our single particle trapping experiments were further in very good agreement with numerical simulations. To ensure that no significant damage occurred to the cells during the experiment, we further optimized medium conditions to ensure viability of the cells for at least 1?h, more than sufficient for microfluidic trapping experiments. Our results thus indicated the successful design of DC iDEP traps, which can easily be integrated into a variety of microchip operations for single cell analysis.     

Paper-based three-dimensional microfluidic device for monitoring of heavy metals with a camera cell phone

A 3D paper-based microfluidic device has been developed for colorimetric determination of selected heavy metals in water samples by stacking layers of wax patterned paper and double-sided adhesive tape. It has the capability of wicking fluids and distributing microliter volumes of samples from single inlet into affrays of detection zones without external pumps, thus a range of metal assays can be simply and inexpensively performed. We demonstrate a prototype of four sample inlets for up to four heavy metal assays each, with detection limits as follows: Cu (II)?=?0.29 ppm, Ni(II)?=?0.33 ppm, Cd (II)?=?0.19 ppm, and Cr (VI)?=?0.35 ppm, which provided quantitative data that were in agreement with values gained from atomic absorption. It has the ability to identify these four metals in mixtures and is immune to interferences from either nontoxic metal ions such as Na(I) and K(I) or components found in reservoir or beach water. With the incorporation of a portable detector, a camera mobile phone, this 3D paper-based microfluidic device should be useful as a simple, rapid, and on-site screening approach of heavy metals in aquatic environments.

A micro-discharge photoionization detector for micro-gas chromatography

We report on a small (20?×?10 mm) micromachined device for the detection of gases in micro-gas chromatography (GC). It incorporates a micro-discharge across a 20-μm gap, and a remote electrode in the micro cavity that generates an electrical signal corresponding to the photo-ionization of gaseous analytes in a stream of carrier gas. Multi-component mixtures were detected and the results compared to those obtained with a flame ionization detector. The minimum detectable limit is 350 pg.μL^?1 of n-octane in air when applying a 1.4 mW discharge. The combination of wet etching of glass (as used for microfluidic channels) with a lift-off process for detector electrodes by a robust batch process results in a universal, non-destructive, and sensitive microdetector for micro-GC.

Expression of miRNAs Targeting <Emphasis Type="Italic">mTOR</Emphasis> and <Emphasis Type="Italic">S6K1</Emphasis> Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3′-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.     

High-throughput,multiparameter analysis of single cells

Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca²⁺ waves are propagation of Ca²⁺ transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca²⁺ waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca²⁺ signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca²⁺ waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.

Bioelectrochemical probing of intracellular redox processes in living yeast cells—application of redox polymer wiring in a microfluidic environment

Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. “Wiring” of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione–PVI-Os double mediator system provides an effective means to conduct automated microbial assays.

Micro-/nanofluidic device for tunable generation of a concentration gradient: application to Caenorhabditis elegans chemotaxis

In this study, we propose a novel micro-/nanofluidic device that can generate a chemical concentration gradient using a parallel nanochannel as gradient generator. This device is easy to fabricate, showing high reproducibility. Its main feature is the multiple-nanochannel-based gradient generator, which permits the diffusion of small molecules and tunably generates concentration gradients. The nanopattern for the nanochannels can be rapidly and easily fabricated by wrinkling a diamond-like carbon thin film which is deposited on a polydimethylsiloxane substrate; the generation of the concentration gradient can be adjusted by controlling the dimensions of the nanochannels. The developed gradient generator is embedded into a microfluidic device to study chemotaxis in the nematode Caenorhabditis elegans, which has a highly developed chemosensory system and can detect a wide variety of chemical molecules. This device shows good performance for rapid analysis of C. elegans chemotaxis under sodium chloride stimuli.

Colloidal silica beads modified with quantum dots and zinc (II) tetraphenylporphyrin for colorimetric sensing of ammonia

Colloidal crystal beads (CCBs) were fabricated by assembling monodisperse silica nanoparticles via a microfluidic device. The pore size of the CCBs was tuned by using different nanoparticles. The CCBs were then coated with cadmium telluride quantum dots and zinc(II) meso-tetraphenylporphyrin for the purpose of optical sensing. Ammonia causes the color of the sensor to change from green to red. The method has a dynamic range of 0–2500 ppm, good reversibility, and is not sensitive to humidity. The limit of detection is 7 ppm. The sensor has the advantage of a porous microcarrier structure and that pore sizes can be well controlled and thus can fulfill various demands in gas detection.

Sample loading and retrieval by centrifugation in a closed-loop PCR microchip

We report on a novel concept of sample loading for microfluidic devices using a benchtop centrifuge and a magnetically actuated circular closed-loop PCR microchip as a model system. The PCR mixture and the ferrofluid were loaded into a specially designed microchip. The microchip was then placed in an off-the-shelf 50-mL tube and centrifuged. The strong centrifugal force drives the PCR mixture and the ferrofluid into the microchannels of the microchip, and simultaneously expels any trapped microbubbles. PCR was successfully carried out on single and parallel closed-loop PCR microchips. The addition of a few off-chip handling steps allows great simplification of the device design. This new loading concept may be useful for designing robust and low-cost lab-on-a-chip devices because benchtop centrifuges are quite common in most laboratories.

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.