Hydrophobic Chip Research Articles

Trapping stable bubbles in hydrophobic microchannel for continuous ultrasonic microparticle manipulation

The application of bubbles in lab-on-chip devices has attracted considerable attention and is exerting an increasingly significant role in recent years due to its influential functions for fluid and particle manipulation. Herein, we demonstrated a simple air bubble trapping method based on the surface tension of hydrophobic chip material and demonstrated particle manipulation utilizing oscillating bubbles. The microfluidic device has a T-shaped junction structure with triangular obstacles, which allows us to transfer air-pocket generated due to non-synchronized liquid filling in the microchannel to stable bubbles attached to channel wall at the desired location. The trapped bubbles can maintain stable state at a flow rate of less than 50 μL/min. By activating bubbles with the sound wave at a specific frequency, we can achieve particle focusing, trapping, extraction and enrichment in a contact-free, label-free and continuous manner. The proposed bubble trapping method may serve as a reusable, detachable and biocompatible tool for broader bubble-based applications in chemical, biology, and engineering fields, such as drug delivery, cell focusing, as well as isolation of tumor cell.

Microfluidic emulsification: Process and formulation variables effects in flow behavior pattern on a flow-focusing device

Formulation and process variables in surfactant/oil/water (SOW) systems can strongly modify the water/oil interfacial tension leading to different behaviors. Pre-equilibration time of the SDS/toluene/1-butanol/NaCl(aq) system was studied as a process variable and the flow regimes were described at different continuous and dispersed flow rates through a flow focusing geometry. Salinity and oil nature for the SDS/n-alkane/alcohol/NaCl(aq) equilibrated system were studied as formulation variables at fixed dispersed flow rates, changing the continuous flow rate and the formulation variable in order to get O/W or W/O emulsions in hydrophilic and hydrophobic chips, respectively. Pre-equilibration time modifies the flow regimes depending on the differences observed in the interfacial tension at t = 0 and t = 24 h of equilibration. Under very hydrophilic conditions, when γt=24 > γt=0, the entire droplet area is slightly higher for equilibrated systems. At WIII condition, when γt=0 >> γt=24, droplets are formed only in jetting regime for not equilibrated systems. For salinity scan, monodispersed oil droplets were obtained at low salinities, S = 0−0.25 % and in the oil scan, water droplets were obtained when using cyclohexane and cyclohexane-hexane mixtures. In both cases, flow regimes are reported into the formulation maps, using the Hydrophilic Lipophilic Deviation as formulation variable (HLD vs flow rate ratio). Droplet size was fitted into the same equation ( di≈αCac-0.264), and it is noteworthy that the interfacial tension in CaC is lower than in most of reported works using flow-focusing geometries and both emulsions (W/O and O/W) were obtained with the same surfactant.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Approaches to the detection of whole cells using reflectometric interference spectroscopy

AbstractMethods for rapid and reliable detection of clinically relevant bacteria are of highest interest. For such purposes, many analytical methods, e.g. ELISA, have been developed. Also optical biosensors like reflectometric interference spectroscopy (RIfS) are suitable analytical platforms. Here, results are described to capture and detect Legionella pneumophila serogroup 1 on differently functionalized surfaces of RIfS glass chips. As a major problem, availability of suitable antibodies was found. Immobilization of capturing antibodies on the chip surface followed by trapping of L. pneumophila gave only insufficient results. For this approach, the chip surface was partially blocked with bovine serum albumin (BSA) in different ratios between BSA and the Legionella specific antibody in order to obtain a site‐directed immobilization. Further on, antibodies were immobilized by hydrophobic interaction followed by BSA treatment and exposition to L. pneumophila. However, also this approach did not allow sufficient capturing of bacteria cells. It can be assumed that binding forces between the antibody and bacteria are too low. Alternatively, L. pneumophila bacteria were directed immobilized on a hydrophobic chip surface. A sufficient sensor signal for quantification was obtained in a range between 8.25 × 106 and 8.25 × 108 bacteria cells/mL. This approach might open up the possibility for safe identification of L. pneumophila after subsequent addition of specific antibodies.

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.

PDMS–glass serpentine microchannel chip for time domain PCR with bubble suppression in sample injection

This paper reports on the development of a low-cost microreactor (10 µl) biochip for DNA polymerase chain reaction (PCR). The microbiochip (20 mm × 28 mm) is a hybrid type that is composed of a polydimethylsiloxane (PDMS) layer with a serpentine microchannel (360 µm × 100 µm) chamber and glass substrate integrated with a microheater and thermal microsensor. Because of the hydrophobic chip surface, bubbles are usually created during sample loading in the PMDS-based microchip. These bubbles disrupt the stable biochemical reaction. An improved microreactor chamber was designed using microfluidic simulation. The reactor has a rounded-corner serpentine channel architecture, which enables stable injection into the hydrophobic surface using only a micropipette. The reactor temperature needed for the PCR reaction is controlled within ±0.5 °C by the LabVIEW software proportional-integrative-derivative (PID) controller. PCR analyses of the sex-determining Y chromosome (SRY) gene and mouse GAPDH gene were successfully performed in less than 54 min by the fabricated microreactor chip.

시료주입시 기포발생이 억제된 반응조 형태의 중합효소연쇄반응용 PDMS/유리 바이오칩

This paper reports low-cost microreactor (10 ㎕) biochip for the DNA PCR (polymerase chain reaction). The microbiochip (20㎜ × 28㎜) is a hybrid type which is composed of PDMS (polydimethylsiloxane) layer with serpentine micochannel (360 ㎛ × 100 ㎛) chamber and glass substrate integrated with microheater and thermal microsensor. Undesirable bubble is usually created during sample loading to PMDS-based microchip because of hydrophobic chip surface. Created bubbles interrupt stable biochemical reaction. We designed improved microreactor chamber using microfluidic simulation. The designed reactor has a cornerrounded serpentine channel architecture, which enables stable injection into hydrophobic surface using micropipette only. Reactor temperature needed to PCR reaction is controlled within ±0.5℃ by PID controller of LabVIEW software. It is experimentally confirmed that SRY gene PCR by the fabricated microreactor chip is performed for less than 54 min.

Detection and analysis of the cyanobacterial peptide hepatotoxins microcystin and nodularin using SELDI-TOF mass spectrometry

Surface-enhanced laser desorption ionization mass spectrometry (SELDI-TOFMS) was used to develop a new and useful method for determination and identification of the cyanobacterial (blue-green algae) toxins: microcystin and nodularin. The technique, combining chromatography and MS, enables microcystin/nodularin capture, purification, analysis, and processing from complex biological mixtures directly onto a hydrophobic chip. Factors affecting ion intensities, including matrix concentration and laser intensity, were investigated to optimize sensitivity of the method. Microcystins and nodularin were analyzed for femtomolar sensitivity (about 2.5 pg microcystin-LR in 2 μl water). Samples of blood sera and liver tissue were spiked with microcystin-LR and analyzed. The detection limit was 1 ng in 2 μl blood sera solution. Reactions of microcystins by compounds containing mercaptan groups, such as dithiothreitol, aminoethanethiol and protein phosphatase 1, were examined on the chip by mass spectrometry. Formation of the microcystin–dithiothreitol conjugate was used to confirm the target compounds. The MS/MS data obtained showed the presence of the microcystin conjugate. The reaction position of the toxin with target compound was confirmed by a series of MS/MS fragment ions. The protein profile of microcystins reacting with protein phosphatase 1 was also obtained from the SELDI-TOF mass spectra.

The discovery of stannin in rat dorsal root ganglia using an integrated proteohistological approach

The use of proteomic analysis to discover proteins (previously identified or unknown) in a tissue sample is a valuable tool. However, there is a limit to the extent one can validate a discovery with any single technology. In an effort to obviate this inherent constraint and to add value and dimension to protein profiling, we have coupled the information obtained through proteomic techniques with the validation provided by in situ hybridization and immunohistochemistry techniques. This approach can be illustrated by our efforts in the discovery of stannin in rat dorsal root ganglia (DRG). In this study, we initially used the Ciphergen ProteinChip® to perform protein profiling on the DRG of rats in a carrageenan-induced paw inflammation study. In an effort to discover new potential targets in inflammatory pain models, we profiled many potential peaks unique to the ipsilateral DRG of interest. One protein, found to bind to a hydrophobic chip at a molecular mass of 9500 Dalton, was preliminarily identified as stannin. To confirm its identification, we performed in situ hybridization and immunohistochemistry on the source DRG tissue to investigate the presence of stannin mRNA and protein expression, respectively. In addition to confirming the presence of stannin in these DRGs, we observed the upregulation of stannin in the DRGs over the course of carrageenan-induced inflammation, suggesting a possible role of stannin in inflammatory hyperalgesia. Taken together, these results illustrate the synergistic benefits of coupling 0 proteomic and histochemical techniques in identifying and validating targets and biomarkers for drug discovery.

Screening of the Bacillus thuringiensis Cry1Ac δ-endotoxin on the artificial phospholipid monolayer incorporated with brush border membrane vesicles of Plutella xylostella by optical biosensor technology

The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant ( K 1) of 508 nM, followed by the second uni-molecular step with an equilibrium constant ( K 2) of 0.472. The overall affinity constant K d was determined to be 240 nM. The binding was markedly inhibited by N-acetyl- d-galactosamine ( K i=8 mM). The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosensor technology.

Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.

A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.