Microspot Array Research Articles

Electroactive nanostructured antibacterial materials

Thin nanostructured metal (Au, Ag) films, magnetron-sputtered on semiconductor (n-type Si) substrate under 6 V voltage exposure for 15 min, exhibit high antibacterial effect against the food pathogens S. aureus and P. aeruginosa. Nanostructures were formed by femtosecond laser ablation, resulting in an array of microspots. The observed effect is caused by the emergence of submicron, laterally periodical static electric and magnetic fields, adjacent to the metal film, causing the abrupt voltage drops, which induce the hyperpolarization of the cell membrane and increase its permeability, resulting in the formation of pores (electroporation) in the membrane and the subsequent apoptosis of the bacterial cell. Additional factors, which enhance the antibacterial effect of the studied materials, are the volume convection in the liquid drop with bacterial culture, caused by the moderate heating of the substrate to 45 °C–50 °C during the electric current flow and electro-taxis of bacteria to the charged nanostructured metal film.

Bead-free digital immunoassays on polydopamine patterned perfluorinated surfaces

This paper reports sub-micron resolution patterning of perfluorinated surfaces and the application in bead-free digital immunoassays. We use the negative microcontact printing in conjugation with a composite stamp to fabricate polydopamine patterns with sub-micron resolution on the perfluorinated surfaces. To perform digital assays, polydopamine microspot arrays were fabricated. Using discontinuous dewetting, 106 femtoliter droplets are formed in situ on the microspots. The droplets are kept in air from evaporation for over 90 min. The polydopamine patterns on the perfluorinated surfaces are used not only as the template for microdroplet generation but also as the binding sites for various proteins without further modifications. The detection limit of the bead-free digital immunoassay for human interleukin-6 is 1.2 fM with a dynamic range from 1.2 fM to over 400 fM. The high resolution polydopamine patterned perfluorinated substrate offers a highly-sensitive and low-cost approach for digital immunoassays, showing great potential in high-throughput and digital bioassays.

Investigation of Electroless Plated Magnetic Thin Film for Application to Medical Magnetic Devices

Recently, pandemics due to new viruses and pathogens have occurred worldwide. New species of viruses and pathogens are less effective than conventional drugs, so more effective treatments are needed. A tailor-made drug discovery research has been actively investigated. Human immune cells have cells called B lymphocytes. If we can analyze the immune mechanism by stimulating B-lymphocytes with antigens, identifying and capturing cells that respond to B-lymphocytes, we can develop highly efficient and low side effect drugs that are effective only against the causative pathogens. Therefore, a method to capture B lymphocytes modified with magnetic beads on magnetic micro-spot arrays by magnetic force is proposed. This method has been shown to be one of the most promising candidates for obtaining accurate genomic data because it can capture cells more rapidly than conventional capture methods. However, this method requires a perpendicular residual magnetic flux density (Mr⊥) in the perpendicular magnetic field that does not allow the B lymphocytes to flow out, because, although it is possible to capture B lymphocytes when a magnet is installed, there is a problem that the B lymphocytes flow out during the subsequent rinsing process. In this study, the plating conditions were investigated to improve the perpendicular magnetic anisotropy of cobalt (Co) based magnetic thin films. Alkaline degreasing and acid-activated copper (Cu) plates were used as substrates for the experiments. CoNiP, CoNiMnP and CoNiMnReP thin films were prepared by electroless plating under the plating bath composition and plating conditions as shown in Table 1. Vibrating sample magnetometer (VSM), X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive X-ray analysis (EDS) were used for the evaluation. Fig. 1 shows the hysteresis curves of thin films plated with different bath compositions. Perpendicular residual magnetic flux density (Mr⊥) was 25 emu/cc for the CoNiP thin film and 219 emu/cc for the CoNiMnP thin film.The CoNiMnP thin film was found to improve Mr⊥ more than 8 times compared to the CoNiP thin film.It was found that the addition of MnSO4 5H2O improved the Mr⊥.The addition of MnSO4 5H2O may have enhanced Mr⊥ due to the effect of crystal magnetic anisotropy due to the vertical orientation of the c-axis of hcp Co.The perpendicular saturation magnetic flux density (Ms⊥) values were 818 emu/cc and 612 emu/cc for the CoNiMnP and CoNiMnReP thin films respectively.The results showed that the addition of NH4ReO4 reduced the Ms value with maintaining a high Mr⊥ values. Figure 1

A Multiplexed Microarray Platform for Targeted Proteomics

A Multiplexed Microarray Platform for Targeted Proteomics

An sp2 Patterned Boron Doped Diamond Electrode for the Simultaneous Detection of Dissolved Oxygen and pH.

A hybrid sp2-sp3 electrochemical sensor comprising patterned regions of nondiamond-carbon (sp2) in a boron doped diamond (sp3) matrix is described for the simultaneous voltammetric detection of dissolved oxygen (DO) and pH in buffered aqueous solutions. Using a laser micropatterning process it is possible to write mechanically robust regions of sp2 carbon into a BDD electrode. These regions both promote the electrocatalytic reduction of oxygen and facilitate the proton coupled electron transfer of quinone groups, integrated into the surface of the sp2 carbon. In this way, in one voltammetric sweep (time of measurement ∼4 s) it is possible to determine both the DO concentration and solution pH. By varying the sp2 pattern the response can be optimized toward both analytes. Using a closely spaced sp2 microspot array, a linear response toward DO, across the range 0.0 to 8.0 mg L-1 (0.0 to 0.25 mM; sensitivity = -8.77 × 10-8 A L mg-1, R2 = 0.9991) and pH range 4-10 (sensitivity = 59.7 mV pH-1, R2 = 0.9983) is demonstrated. The electrode is also capable of measuring both DO concentration and pH in the more complex buffered environment of blood. Finally, we show how the peak position for ORR is independent of pH, and thus via measurement of the difference in ORR and pH peak position, internal referencing is possible. Such electrodes show great promise for use in applications ranging from biomedical sensing to water analysis.

An ultralow background substrate for protein microarray technology.

We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis.

Biosensing enhancement using passive mixing structures for microarray-based sensors

The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10–1000s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.

Magnetic Force-Based Microarray Chips on Soda Lime Glass Prepared by Plating Method for Medical Application

Abstract In medicine, microarray chips are desired in order to improve personal medicine. In this paper, we propose a fabrication method for microarray chips using electroplated soft magnetic films on glass substrates using a simple process. Electroless plated Ni–P films were formed on soda lime glass as conductive substrates and were subsequently annealed at 150 °C for 30 min to improve adhesion strength between the films and glass substrates. Microspot array photoresists were patterned on the Ni–P films by photolithography. Co–Ni–Fe alloy films were then electroplated and finely the photoresist removed. As determined by electron probe microanalysis, the Co–Ni–Fe films were composed of 15–74 atom % Co, 12–72 atom % Fe, and 1.6–24 atom % Ni under several plating bath compositions. Vibrating sample magnetometry showed saturation magnetic flux density of 1400–2200 emu cm−3, residual magnetic flux density of 1100–1600 emu cm−3 and coercive force of 90–100 Oe on the Co–Ni–Fe films. A microspot array prepared by electroplated Co–Ni–Fe soft magnetic films/electroless plated Ni–P films on soda lime glass with simple patterning successfully trapped pseudo B-lymphocytes conjugated with magnetic beads.

Scanned Pipette Techniques for the Highly Localized Electrochemical Fabrication and Characterization of Conducting Polymer Thin Films, Microspots, Microribbons, and Nanowires

AbstractThe limited toolbox for conducting polymer (CP) microscale fabrication and characterization hampers the development of applications such as sensors and actuators. To address this issue, a robust and integrated methodology is presented, capable of electrochemical fabrication and characterization of CPs in a highly localized manner, allowing for CP patterning and spatial mapping of voltammetric response. This is enabled by scanning probe microscopy (SPM) tipped with a single‐barreled micropipette to electrochemically polymerize CP microspot arrays, demonstrated for 3,4‐ethylenedioxythiophene and aniline monomers. Stationary electropolymerization produces individual microspots; lateral movement produces long microribbons; retraction produces extruded microstructures. Subsequently the same SPM setup is tipped with a double‐barreled micropipette to carry out localized cyclic voltammetry. The micropipettes are filled with saline solutions in contact with Ag/AgCl electrodes, forming a thin meniscus of solution at the micropipette tip, which enable an automated approach in air and subsequent contact with the surface. The flexibility of this novel technique is demonstrated by application to 2D poly(3,4‐ethylenedioxythiophene) (PEDOT) microspots, microribbons and nanowires, plus polyaniline (PANI) microstructures and self‐assembled thin films. Finally, setting up a dynamic electrochemical cell allowed for voltammetric–amperometric imaging, simultaneously mapping the morphology and current response of CPs. Future refinements towards the nanoscale through smaller‐tipped pipettes should open up new opportunities for voltammetric response mapping of individual CP nanostructures.

Selective Deposition of Electrospun Alginate-Based Nanofibers onto Cell-Repelling Hydrogel Surfaces for Cell-Based Microarrays

This paper proposes selective deposition of electrospun alginate-based nanofibers through a charged shadow mask to form an array of nonwoven microspots on cell-repelling hydrogel surfaces; subsequently, the target cells are seeded onto these nanofiber-based extracellular matrix (ECM) scaffolds to create cell-based microarrays. The needle tip contains a drop of a blended solution of alginate and polyethylene oxide (PEO); an Al-coated glass shadow mask with micron-sized holes was used for selective deposition to form the patterned microspots of nonwoven mats. The shadow mask was applied with a suitable voltage to repel the nanofibers from its surface, while simultaneously forcing them into the micron-sized holes and onto the cell-repelling hydrogel surface. The electric-field distribution of the electrostatic focusing through the charged shadow masks was numerically simulated and characterized. The experimental results show that when the shadow mask is applied with a higher voltage, a larger dense central spot will be produced within the hole; in addition, the diameter is significantly reduced but the degree of diameter uniformity among the electrospun nanofibers improves. An array of 3 × 3 nonwoven microspots was successfully demonstrated to selectively deposit alginate-based nanofibers for cell patterning. BHK-21 fibroblast cells were shown to selectively adhere onto the nonwoven microspot surfaces due to the existence of the cell-repelling hydrogel around the microspots. The presented technique is simple and does not require complex surface modifications to selectively seed the target cells onto these micron-sized nanofiber-based ECM scaffolds for potential applications in cell biology and tissue engineering. Keywords: Electrospinning, electrostatic focusing, nanofiber, cell patterning, nonwoven microspots, shadow mask, cell-repelling

Microfluidic DNA microarray analysis: A review

Microarray DNA hybridization techniques have been used widely from basic to applied molecular biology research. Generally, in a DNA microarray, different probe DNA molecules are immobilized on a solid support in groups and form an array of microspots. Then, hybridization to the microarray can be performed by applying sample DNA solutions in either the bulk or the microfluidic manner. Because the immobilized probe DNA binds and retains its complementary target DNA, detection is achieved through the read-out of the tagged markers on the sample target molecules. The recent microfluidic hybridization method shows the advantages of less sample usage and reduced incubation time. Here, sample solutions are confined in microfabricated channels and flow through the probe microarray area. The high surface-to-volume ratio in microchannels of nanolitre volume greatly enhanced the sensitivity as obtained with the bulk solution method. To generate nanolitre flows, different techniques have been developed, and this including electrokinetic control, vacuum suction and syringe pumping. The latter two are pressure-driven methods which are more flexible without the need of considering the physicochemical properties of solutions. Recently, centrifugal force is employed to drive liquid movement in microchannels. This method utilizes the body force from the liquid itself and there are no additional solution interface contacts such as from electrodes or syringes and tubing. Centrifugal force driven flow also features the ease of parallel hybridizations. In this review, we will summarize the recent advances in microfluidic microarray hybridization and compare the applications of various flow methods.

Differentiation of liquid analytes in gel films by permeability-modulated double-layer chemo-chips

A double-layer chemo-chip for the characterization of liquid analytes by rapid fluorimetric imaging is described. The chemo-chip consists of an array of polymeric micro-spots prepared on a glass slide. Each spot is composed of a thin indicator layer made of PVA doped with an immobilized fluorescence dye and a top layer polymer spot with different permeation properties. The analytes can be differentiated by variations in the optical response rate of the indicator dye after its application. Consequently, different cross-linker concentrations were applied using the Nano-Plotter((TM)) which formed top layers of varying permeability. The chemo-chips were tested with the aqueous solutions of two model liquids (aqueous solutions of malonic acid and phenanthroline hydrochloride). It was found that the transition time of response had changed considerably (up to a factor of about 10) depending on different local cross linking degrees. This has resulted in time-dependent fluorescent patterns of the fluorescence images of the micro-array. The response was fast and the transition times were in the range between a few seconds to 30 s.

Parallel determination of phenotypic cytotoxicity with a micropattern of mutant cell lines

This work presents a novel tool, the Continuous Flow Microspotter (CFM) and its use in patterning cellular microarrays of multiple cell types into the bottom of a tissue culture well. The CFM uses a system of isolated microfluidic channels to make an array of localized microspots of adhesion dependent cells in the bottom of a conventional tissue culture well. With this device we have created micropatterns of multiple cell lines in a single tissue culture well and used this system to conduct simultaneous cytotoxicity tests and recover dose survival curves in a parallel study. This mechanism of parallel testing allows the researcher to employ the use of positive and negative controls, as well as compare the chemical response of phenotypes in a tightly controlled microenvironment. For the experiments presented in this paper we have fabricated a CFM with a set of ten microchannels (five inlet channels and five outlet channels) to pattern a row of five microspots consisting of four cellular microspots and one empty spot for background measurements. Micropatterns containing a set of four different Chinese hamster ovarian cell (CHO) mutant phenotypes were deposited into the bottom of commercially available tissue culture wells then interrogated with mitomycin C, a chemotherapeutic agent. This study shows statistically significant (P < 0.05) hypersensitivity of the UV20 CHO mutant to a DNA interstrand cross-linking agent (mitomycin C). Because the CFM is also capable of depositing proteins and other biomolecules to the individual microspots of the array we foresee capabilities of the 48 microspot CFM to multiplex 48 cell types with 48 chemical reagents all within the confines of a 60 mm(2) area.

Modifying network chemistry in thiol‐acrylate photopolymers through postpolymerization functionalization to control cell‐material interactions

Thiol-acrylate photopolymers often contain pendant, unreacted thiol groups even following complete reaction of the acrylate functional groups. The results presented herein demonstrate a high throughput method for quantifying pendant thiol group concentrations using FTIR spectra of thiol-acrylate microspot arrays. Using this technique, more than 25% of the original thiol groups were detected as pendant groups in microspots made from monomer solutions containing at least 40 mol % thiol functional groups. Subsequent modification reactions allowed postpolymerization tailoring of the network chemistry. The extent of modification was controlled by the concentration of the pendant thiols (ranging from 0.01 to 0.4M) and the duration of the modification reaction (0-10 min for photocoupling reactions, 0-24 h for Michael-type addition reactions). Further, when photocoupling was used to modify the networks, spatial and temporal control of the light exposure facilitated the formation of chemical patterns on the surface and throughout the material.

Microfluidic arrangement with an integrated micro-spot array for the characterization of pH and solvent polarity

Micro-spot arrays with different pH and polarity sensitive fluorescence dyes were prepared using a piezo dispensing head. A polyethylene glycol matrix was used to immobilize the dyes covalently. The polymer matrix was cross-linked to ensure stable dye bonding and for controlling the swelling and permeability of the thin film. Analytical experiments were carried out in the fluidic phase with specially developed micro-fluidic and CCD imaging optical set-up. As a result, different solvents and solutions could be distinguished for their pH and polarity using fluorescence digital imaging. After calibration, a quantitative determination of the solution composition was also possible. The chemo-chips were tested against different solvent species leading to confirmation of the applicability of the developed spot array for the reproducible recognition of different liquid types focusing on polarity and pH as parameters.

Multiplexing molecular diagnostics and immunoassays using emerging microarray technologies

Enzyme-linked immunosorbent assays (ELISA) are frequently used for quantitative measurement of the presence of protein, for single-analyte testing, in a sample. The application of ELISA in a microarray format has the potential to simultaneously measure the presence and/or concentrations of numerous proteins, in multiplex testing, all contained in a small drop of test fluid. Microspot microarray technology, in combination with protein biomarkers and nucleic acid diagnostics, appears to be the future high-performance analytical platform of choice. Validation of a large number of disease markers in both molecular and protein diagnostics has paved the way for the emergence of the multiplex assay. Initially, simple low-throughput multiplex assays were tested using the immunoassay format. These were followed by low-level multiplexing and high-throughput array-based immunoassays. More recently, two types of high-level multiplexing and high-throughput diagnostic methods using microspot arrays and bead arrays have been successfully developed to complement single-analyte assays. The value in rapid diagnostic evaluation for high-throughput multiplex, diagnostic test systems based on sound assay design must take into account data screening, normalization and statistical evaluation of possible concentration measurement, data errors and automated operation. Benefits of using multiplex array platforms include improved-quality patient care, as well as cost effectiveness and time saving. These multiplex methods also set the stage for future protein/nucleic acid codetection. Currently, the one analyte at a time test scheme is still dominant; nonetheless, the multiplex microspot microarray tests evaluated in a single multiassay analyzer are expected to become a significant part of clinical diagnostic testing within the next 5–10 years. This review is focused on microspot array and bead array methods for providing high throughput and a high degree of multiplexing in diagnostic testing.

Trapping and Collection of Lymphocytes Using Microspot Array Chip and Magnetic Beads

A microspot array chip, which has microspots of a magnetic thin film patterned on a glass substrate, was fabricated for trapping individual cells and for measuring their cellular response. The chip was easily fabricated by conventional semiconductor fabrication techniques on a mass production level as a disposable medical device. When a solution of lymphocyte-bound-magnetic beads was poured into the magnetized chip, each lymphocyte was trapped on each microspot of the magnetic thin film. The trapped cells were easily recovered from the chip using a micromanipulator. The micro-spot array chip can be utilized for arraying live cells and for measuring the response of each cell. The chip will be useful for preparing on array of different kinds of cells and for analyzing cellular response at the single cell level. The chip will be particularly useful for detecting antigen-specific B-lymphocytes and antigen-specific antibody complementary deoxyribonucleic acid (cDNA).

Characterisation of lithographically patterned organosilane monolayers by preferential adsorption of dye molecules

A method for the visualisation of micropatterned spots of molecular organosilane monolayers by adsorption of dyes is presented. Therefore, microspot arrays consisting of different types of organosilane monolayers are exposed to dye solutions. After this immersion, the chips are rinsed in a solvent, resulting in a locally differentiated fluorescence intensity. The contrast between silanized and non-silanized surface areas depends on the applied dyes and their solution concentrations. Charge and polarity seems to play an important role in the adsorption. In result, the pattern of molecular organosilane monolayers can easily be identified by intensity differences of fluorescence radiation from the different regions of chip surfaces.

Monitoring β-galactosidase activity by means of scanning electrochemical microscopy

The activity of β-galactosidase from Escherichia coli was characterized by scanning electrochemical microscopy (SECM). β-Galactosidase microspots were patterned by immobilizing biotin-labelled β-galactosidase on streptavidin-coated paramagnetic beads and depositing the beads as microscopic microspots on a hydrophobic surface. The enzyme activity was mapped with SECM in the generation-collection mode by monitoring the oxidation of p-aminophenol formed in the galactosidase-catalyzed hydrolysis at the surface of the beads. The electrochemical properties of the enzyme substrate and product were studied with a platinum ultramicroelectrode, and the imaging conditions were optimised. The suitability of the procedure for forming β-galactosidase microspot arrays and the capability of SECM to resolve enzyme activity over individual enzyme microspots were demonstrated.

Analyzing effects of extra-virgin olive oil polyphenols on breast cancer-associated fatty acid synthase protein expression using reverse-phase protein microarrays

Inhibitors of fatty acid synthase (FASN), a key enzyme involved in the anabolic conversion of dietary carbohydrates to fat in mammals, are receiving increasingly more attention as they may provide therapeutic moieties for the treatment of human malignancies. Natural compounds, such as the green tea polyphenol epigallocatechin-3-gallate, have been shown to induce anti-cancer effects by suppressing FASN, which may account for the epidemiologically observed inverse correlation between green-tea drinking and cancer risk in Oriental populations. Since extra-virgin olive oil (EVOO)-derived phenolics have been suggested to possess biological activities that may explain the health-promoting effects of the 'Mediterranean diet', we evaluated their effects on the expression of FASN protein in human breast epithelial cell lines. First, we developed a reverse phase protein microspot array (RPPA) capable of rapidly assessing the relative amount of FASN protein in whole lysates from cultured human cells. Then we tested the effects of phenolic fractions from EVOO and its main constituents including single phenols (i.e. tyrosol, hydroxytyrosol, vanillin), phenolic acids (i.e. caffeic acid, p-coumaric acid, vanillic acid, ferulic acid, elenolic acid), lignans (i.e. 1-[+]-pinoresinol, 1-[+]-acetoxy-pinoresinol), flavonoids (i.e. apigenin, luteolin), or secoiridoids (i.e. deacetoxyoleuropein aglycone, ligstroside aglycone, oleuropein glycoside, oleuropein aglycone) on FASN protein expression. EVOO polyphenols lignans, flavonoids and secoiridoids were found to drastically suppress FASN protein expression in HER2 gene-amplified SKBR3 breast cancer cells. Equivalent results were observed in MCF-7 cells engineered to overexpress the HER2 tyrosine kinase receptor, a well-characterized up-regulator of FASN expression in aggressive sub-types of cancer cells. EVOO-derived lignans, flavonoids and secoiridoids were significantly more effective than the mono-HER2 inhibitor trastuzumab ( approximately 50% reduction) and as effective as the dual HER1/HER2 tyrosine kinase inhibitor lapatinib (> or =95% reduction) at suppressing high-levels of FASN protein in HER2-overexpressing SKBR3 and MCF-7/HER2 cells. EVOO single phenols and phenolic acids failed to modulate FASN expression in SKBR3 and MCF-7/HER2 cells. These findings reveal for the first time that phenolic fractions, directly extracted from EVOO, may induce anti-cancer effects by suppressing the expression of the lipogenic enzyme FASN in HER2-overexpressing breast carcinoma cells, thus offering a previously unrecognized mechanism for EVOO-related cancer preventive effects.