Cellular microarrays for use with capillary-driven microfluidics

We present a method for the facile arraying of cells on microstructured substrates which should be suitable for cellular assays in autonomous microfluidic capillary systems (CSs). The CSs, which were designed and microfabricated in Si, have various microfluidic functional elements including reaction chambers wherein cellular arrays are located. Two methods for arraying the cells were explored. In the first method, a hydrophobic alkanethiol was microcontact-printed on the bottom surface of a microfluidic reaction chamber. The subsequent adsorption of protein-repellent alkanethiols around the printed areas and the deposition from solution of fibronectin (FN) on the hydrophobic areas resulted in an adhesive pattern for the attachment of living human breast cancer cells. This method was limited by the formation of cellular clusters, which proved difficult to remove selectively. The second method employed a poly(dimethylsiloxane) elastomer having oval recessed microstructures. The selective coating of the inner walls of the ovals with FN and the blocking of the mesas around the ovals with bovine serum albumin (BSA) permitted single or multiple cells to be arrayed depending on the size of the ovals. The possibility of sealing CSs with cells arrayed on poly(dimethylsiloxane) may provide a versatile platform for high-throughput experimentation down to the single-cell level. Figure The deposition of one or a few living cells in fibronectin-coated poly(dimethylsiloxane) microstructures results in cellular arrays, which can be interfaced with capillary-driven microfluidics     

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.     

Relaxivity of Gd-Based MRI Contrast Agents in Crosslinked Hyaluronic Acid as a Model for Tissues

The efficiency of MRI contrast agents depends on the relaxation rate enhancement that they can induce at imaging fields. It is well known that, at these fields, large relaxation rates are obtained by binding of gadolinium(III) ions to large molecules. By the same token, the interaction of the gadolinium(III) complexes with macromolecules that are found in biological tissues can be responsible for an increase of the relaxation rate with respect to the value observed in liquids. We investigate here the relaxation enhancement of gadoteridol (Gd-HP-DO3A) in crosslinked hyaluronic acid, taken as model tissue, using fast field-cycling relaxometry. The analysis of the relaxation profiles as a function of the magnetic fields indicates that a sizable increase in the relaxation rates is due to a modest interaction of the contrast agent with the hydrogel and to the slower mobility of the water molecules outside the first-coordination sphere of the gadolinium(III) ion.     

Metal phosphonates applied to biotechnologies: a novel approach to oligonucleotide microarrays

A new process for preparing oligonucleotide arrays is described that uses surface grafting chemistry which is fundamentally different from the electrostatic adsorption and organic covalent binding methods normally employed. Solid supports are modified with a mixed organic/inorganic zirconium phosphonate monolayer film providing a stable, well-defined interface. Oligonucleotide probes terminated with phosphate are spotted directly on to the zirconated surface forming a covalent linkage. Specific binding of terminal phosphate groups with minimal binding of the internal phosphate diesters has been demonstrated. The mixed organic/inorganic thin films have also been extended for use arraying DNA duplex probes, and therefore represent a viable general approach to DNA-based bioarrays. Ideas for interfacing mixed organic/inorganic interfaces to other bioapplications are also discussed.     

Skeletal diversity via a branched pathway: efficient synthesis of 29 400 discrete,polycyclic compounds and their arraying into stock solutions

We report the synthesis and arraying of 29 400 structurally diverse and complex polycyclic carbocycles using diversity-oriented synthesis (DOS) and the "one bead-one stock solution" technology platform. Skeletal diversity, a difficult challenge in DOS, was achieved with a branching reaction pathway using one or two Diels-Alder reactions. This pathway yields small molecules having 10 different skeletons.     

Cell culture arrays using magnetic force-based cell patterning for dynamic single cell analysis

In order to understand the behavior of individual cells, single cell analyses have attracted attention since most cell-based assays provide data with values averaged across a large number of cells. Techniques for the manipulation and analysis of single cells are crucial for understanding the behavior of individual cells. In the present study, we have developed single cell culture arrays using magnetic force and a pin holder, which enables the allocation of the magnetically labeled cells on arrays, and have analyzed their dynamics. The pin holder was made from magnetic soft iron and contained more than 6000 pillars on its surface. The pin holder was placed on a magnet to concentrate the magnetic flux density above the pillars. NIH/3T3 fibroblasts that were labeled with magnetite cationic liposomes (MCLs) were seeded into a culture dish, and the dish was placed over the pin holder with the magnet. The magnetically labeled cells were guided on the surface where the pillars were positioned and allocated on the arrays with a high resolution. Single-cell patterning was achieved by adjusting the number of cells seeded, and the target cell was collected by a micromanipulator after removing the pin holder with the magnet. Furthermore, change in the morphology of magnetically patterned cells was analyzed by microscopic observation, and cell spreading on the array was observed with time duration. Magnetic force-based cell patterning on cell culture arrays would be a suitable technique for the analysis of cell behavior in studies of cell-cell variation and cell-cell interactions.     

Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL^?1, with a detection limit of 0.3 ng mL^?1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL^?1 of caspase-3 (for n?=?11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n?=?5).

Toward highly efficient CdS/CdSe quantum dots-sensitized solar cells incorporating ordered photoanodes on transparent conductive substrates

A series of ordered photoanodic architectures (including ordered TiO(2) nanotube arrays (TNT), ZnO nanorods, ZnO/TiO(2) core/shell nanostructures) for CdS/CdSe sensitized solar cells (QDSCs), were fabricated directly on transparent conductive oxide glasses by a facile sol-gel assisted template process. The morphologies, optical and electrical properties of TNTs and CdS/CdSe co-sensitized TNTs have been demonstrated. The effect of CdSe deposition time on the cell performance was clarified, and the growth mechanism of the CdSe quantum dots on the surface of the TNTs has been proposed as well. Furthermore, the evolution of open-circuit photovoltage (V(oc)) towards CdSe deposition time has been investigated by electrochemical impedance spectroscopy (EIS). A promising light-to-electricity conversion efficiency of up to 4.61% has been achieved with 3 μm long TNT arrays, which is the best record for sandwich-type ordered TNT-based QDSCs.     

Novel cytotoxic constituents of Orthosiphon diffusus

Chemical investigation on Orthosiphon diffusus resulted in the isolation of four relatively rare, novel polychiral furanopyrans, orthodiffenes A-D (1-4) which were characterized from detailed studies of their 1D and 2D NMR spectra. The X-ray crystallographic analysis of orthodiffene A (1) was accomplished. The in vitro cytotoxic activity of orthodiffenes A-C (1-3) was tested against Jurkat and HL-60 cells using camptothecin as a positive control. Orthodiffenes A (1) and B (2) showed comparable activity to camptothecin against HL-60 and Jurkat cells, respectively.     

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: a contribution to the optimization of gene and drug delivery

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.     

A microfluidic array with cellular valving for single cell co-culture

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (～99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (～70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.     

New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces

A new approach to oligonucleotide arrays is demonstrated that utilizes zirconium phosphonate-derivatized glass slides. The active slides are prepared by binding Zr(4+) to surfaces terminated with organophosphonate groups previously deposited using either Langmuir-Blodgett or self-assembled monolayer methods. Oligonucleotide probes modified with a terminal phosphate bind strongly to the active zirconium phosphonate monolayer, and arrays for detecting fluorescent targets have been prepared using commercial spotting and scanning instruments. Preferred binding to the surface of the terminal phosphate of the modified probes instead of the internal phosphate diester groups is demonstrated and shown to yield increased fluorescence intensity after hybridization with labeled targets. A significant decrease in background signal is achieved by treating the slides with bovine serum albumin after spotting and before hybridization. A further increase in fluorescence after hybridization is observed when using a poly-guanine spacer between the probe oligomer and the terminal phosphate. Combining these modifications, an intensity ratio of nearly 1000 is achieved when comparing 5'-phosphate-modified 33-mer probes with unmodified probes upon hybridization with fluorescent targets.     

Use of a gas-sensor array for detecting volatile organic compounds (VOC) in chemically induced cells

An application of gas sensors for rapid bioanalysis is presented. An array of temperature-modulated semiconductor sensors was used to characterize the headspace above a cell culture. Recombinant Saccharomyces cerevisiae yeast cells, able to respond to 17-estradiol by producing a reporter protein, were used as a model system. Yeast cells had the DNA sequence of the human estrogen receptor stably integrated into the genome, and contained expression plasmids carrying estrogen-responsive sequences and the reporter gene lac-Z, encoding the enzyme -galactosidase. The sensor-response profiles showed small but noticeable discrimination between cell samples induced with 17-estradiol and non-induced cell samples. The sensor array was capable of detecting changes in the volatile organic compound composition of the headspace above the cultured cells, which can be associated with metabolic changes induced by a chemical compound. This finding suggests the possibility of using cross-selective gas-sensor arrays for analysis of drugs or bioactive molecules through their interaction with cell systems, with the advantage of providing information on their bioavailability.     

Chemically responsive nanoporous pigments: colorimetric sensor arrays and the identification of aliphatic amines

A general method has been developed for the preparation of microspheres of nanoporous pigments, their formulation into chemically responsive pigment inks, and the printing of these inks as colorimetric sensor arrays. Using an ultrasonic-spray aerosol-gel synthesis from chemically responsive dyes and common silica precursors, 16 different nanoporous pigment microspheres have been prepared and characterized. New colorimetric sensor arrays have been created by printing inks of these chemically responsive pigments as primary sensor elements; these arrays have been successfully tested for the detection, identification, and quantitation of toxic aliphatic amines. Among 11 structurally similar amines, complete identification of each analyte without confusion was achieved using hierarchical cluster analysis (HCA). Furthermore, visual identification of ammonia gas was easily made at the IDLH (immediately dangerous to life or health), PEL (permissible exposure limits), and 0.1 PEL concentrations with high reproducibility.     

The formation of conjugates with PEG–chitosan improves the biocatalytic efficiency and antitumor activity of L-asparaginase from <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The ChitoPEGylation method, which is a novel approach to regulating the catalytic properties of enzymes that is based on the formation of a covalent conjugate of an enzyme with branched copolymers of chitosan, has been developed. The efficiency of this method has been demonstrated using a new recombinant preparation of L-asparaginase from Erwinia carotovora (EwA) as a model. The molecular architecture and composition of EwA conjugates with PEG–chitosans have been optimized. It has been shown that the decisive factors that affect the activity of the EwA conjugates are the molecular weight of and PEGylation degree of chitosan. It has been found that the EwA conjugation with PEG–chitosan increases, its cytostatic activity against human chronic myeloid leukemia K562 cells, Burkitt’s lymphoma Raji cells, and acute lymphoblastic leukemia Jurkat cells. These data provide new approaches to the synthesis of L-asparaginase preparations with improved biocatalytic properties.     

A Solid‐State Electrochemiluminescence Sensor for Label‐Free Analysis of Leukemia Cells

A novel electrochemiluminescence (ECL) method for label‐free detection of cancer cells was proposed for the first time by capturing negatively charged Jurkat cells onto Ru(bpy′)${{{2+\hfill \atop 3\hfill}}}$ ‐immobilized indium tin oxide (ITO) electrode via electrostatic interaction. The ECL sensor exhibited excellent sensitivity, good stability and a linear response to Jurkat cells in the concentration range from 1×10³ to 2×10⁵ cells/mL, with a detection limit of 730 cells/mL. Furthermore, the method was successfully applied in the study of cell growth and cell apoptosis, which was supported by fluorescent images analysis. The proposed protocol is simple, rapid, inexpensive and universally targetable for tumors, offering a novel platform for the development of an ECL biosensor for cell detection.     

Transfer of protoporphyrin IX between cells

Human adenocarcinoma cells of the line WiDr and human leukemia T cells of the line Jurkat were incubated with 5-aminolevulinic acid and found to produce protoporphyrin IX (PpIX). They were able to transfer a fraction of the sensitizer to neighboring control cells. The transfer took place through direct membrane contact. Light exposures, inactivating about 20% of the sensitized cells, did not result in any acceleration of the transfer of PpIX. This is in contrast to what has been reported for PpIX in erythrocytes from patients with erythropoietic protoporphyria. In these cells light exposure transfers PpIX from the binding sites on hemoglobin to the plasma membrane and further to neighboring cells. The lack of light-induced transfer in the WiDr and Jurkat cells may be related to the binding sites of PpIX, supposedly membrane lipids and proteins embedded therein. Light exposure slightly increased the rate of loss of PpIX from WiDr cells.     

Morphology evolution of ZnO thin films from aqueous solutions and their application to solar cells

This paper reports a reproducible low-temperature solution-based process for the preparation of ZnO films of nanorod arrays and their application to dye-sensitized solar cells (DSSCs). A two-step approach was employed for the epitaxial growth of ZnO. We began with the preparation of a (002)-oriented ZnO seed layer by the electrochemical deposition method. After the treatment the substrate was soaked in an aqueous solution containing ZnCl2 and complex agents. A large-scale fabrication of ZnO nanorod arrays on transparent conductive oxides has been achieved after soaking at 95 degrees C for 1-48 h. The as-deposited ZnO film has a large surface area, therefore permitting a great amount of dye loading. The individually separated nanorod forms a linear nanoroad which should show more effective electron transportation than that in the film derived from ZnO powders. The DSSCs using these ZnO films as photoelectrodes show a conversion efficiency of about 0.6% at AM1.5.     

Optical microwell array for large scale studies of single mitochondria metabolic responses

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-μm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29 % and 35 % under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

A rational approach to minimal high-resolution cross-reactive arrays

We report a rational approach to the construction of cross-reactive arrays for steroids consisting of five to seven sensors incorporating modified oligonucleotides. The sensors for our arrays were selected to maximize their differential responses to the two steroids most different in an arbitrarily chosen parameter named "shape-length". The arrays incorporated three previously unreported types of sensors identified through a massive screening effort: (1) three-way junction sensors with neutralized charges within junction; (2) "self-aggregating sensors"; and (3) sensors incorporating fluorophore directly in a three-way junction as a spacer. The arrays were tested on seven steroids and an alkaloid (cocaine) over a range of concentrations, and achieved 92-96% accuracy in class assignments, despite the close structural similarities between steroids.