Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function of field-effect transistors

We present a label-free method for the detection of DNA hybridization, which is monitored by non-metallized silicon field-effect transistors (FET) in a microarray approach. The described method enables a fast and fully electronic readout of ex situ binding assays. The label-free detection utilizing the field-effect is based on the intrinsic charge of the DNA molecules and/or on changes of the solid–liquid interface impedance, when biomolecules bind to the sensor surface. With our sensor system, usually a time-resolved, dc readout is used. In general, this FET signal suffers from sensor drift, temperature drift, changes in electrolyte composition or pH value, influence of the reference electrode, etc. In this article, we present a differential ac readout concept for FET microarrays, which enables a stable operation of the sensor against many of these side-parameters, reliable readout and a possibility for a quick screening of large sensor arrays. We present the detection of point mutations in short DNA samples with this method in an ex situ binding assay.     

High-throughput SNP genotyping on universal bead arrays

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.     

Simultaneous positioning of cells into two-dimensional arrays using ultrasound

Contactless simultaneous positioning of micrometer-sized particles in suspension (e.g., copolymer beads, living cells, silicon microparts) can be performed using ultrasound. Current devices are capable of collecting particles into planes or lines by exciting a resonance in the fluid by means of a piezoelectric transducer located beneath the fluidic cavity and are designed such that a one-dimensional pressure field is created. The focus of this work is to collect cells in distinct point locations for potential drug screening array applications. A device to create two-dimensional arrays of cells within a micromachined chamber is described. The chamber is etched into a silicon wafer and sealed with glass; on the underside of the silicon layer a piezoelectric actuator is attached. A signal is applied to each of two orthogonally aligned strips electrodes defined on the surface of the piezoelectric plate. These two strip electrodes create independently addressable approximately one-dimensional pressure fields. It is shown that by applying the same signal to each electrode a diagonally aligned grid of cells can be produced. However, the independence of the two electrodes allows the application of two signals with slightly different frequencies to be applied which creates a grid of circular cell clumps highly suitable for the identified application. (c)     

Fabrication,Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques ^{1, 2}, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization ³.     

Localized transfection with magnetic beads coated with PCR products and other nucleic acids

The bead transfection method involves binding nucleic acids onto 3-microm-diameter paramagnetic beads, treating the beads with transfection reagent, and using them as scaffolds to direct transfection to individual cells or regions in a population. Typically, PCR products are used because they can be conveniently generated using biotinylated primers and can introduce site-directed mutations, without the need for cloning or plasmid purification. However, the method can be adapted to transfect plasmid DNA or RNA. The magnetic properties of the beads allows magnets to direct the loci of transfection in cell culture; magnetic arrays are built in cell culture chambers to allow multiple parallel transfections on the same microscope coverslip. The PCR reaction and transfection can be carried out in 1 d, and transfection results can be viewed in 24-48 h.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.     

Monoclonal antibody selection for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays

A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.     

Membrane biochips

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.     

NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue?), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. ? 2012 International Society for Advancement of Cytometry.     

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO₂ beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.     

Fabrication of silicon nanowire devices for ultrasensitive, label-free, real-time detection of biological and chemical species

Detection and quantification of biological and chemical species are central to many areas of healthcare and the life sciences, ranging from diagnosing disease to discovery and screening of new drug molecules. Semiconductor nanowires configured as electronic devices have emerged as a general platform for ultra-sensitive direct electrical detection of biological and chemical species. Here we describe a detailed protocol for realizing nanowire electronic sensors. First, the growth of uniform, single crystal silicon nanowires, and subsequent isolation of the nanowires as stable suspensions are outlined. Second, fabrication of addressable nanowire device arrays is described. Third, covalent modification of the nanowire device surfaces with receptors is described. Fourth, an example modification and measurements of the electrical response from devices are detailed. The silicon nanowire (SiNW) devices have demonstrated applications for label-free, ultrasensitive and highly-selective real-time detection of a wide range of biological and chemical species, including proteins, nucleic acids, small molecules and viruses.     

Immunoassays based on electrochemical detection using microelectrode arrays

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.     

A spatially addressable bead-based biosensor for simple and rapid DNA detection

A simple glass-polymer-based biosensor that allows arrays of beads to be immobilized, separated and identified without any prior encoding is developed. To do so, distinct bead types that are conjugated with different oligonucleotide probes are sequentially spotted onto a polymeric matrix (or gel pad) on the surface of the device. The spotted beads are firmly immobilized to the gel pad, acquiring spatial codes that allow them to be identified. Throughput is enhanced by spotting different bead types onto hundreds of different gel pads. The bead-based biosensor was applied for the DNA-based detection of 10 model bacterial species and two single-nucleotide polymorphisms, and based on passive hybridization the final signals are obtained with single-mismatch specificity within 10 min.     

Advances in functional protein microarray technology

Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, protein-nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening.     

Silicon nanopillar substrates for enhancing signal intensity in DNA microarrays

The use of ordered, high-aspect ratio nanopillar arrays on the surface of silicon-based chips to enhance signal intensity in DNA microarrays is reported. These nanopillars consisting either of a single silicon dioxide substrate or a dual silicon/silicon dioxide substrate are fabricated using deep-UV lithography followed by reactive ion etching. These pillar type arrays provide a three-dimensional high surface-density platform that increases the immobilization capacity of captured probes, enhances target accessibility and reduces background noise interference in DNA microarrays, leading to improved signal-to-noise ratios, sensitivity and specificity. Consequently, it was found that the use of such nanopillars enhanced the hybridization signals by up to seven times as compared to silicon dioxide thin film substrates. In addition, hybridization of synthetic targets to capture probes that contained a single-base variation showed that the perfect matched duplex signals on dual-substrate nanopillars can be up to 23 times higher than the mismatched duplex signals, allowing the targets to be unambiguously identified. These results suggest that the nanopillars, particularly the dual-substrate pillars, are able to enhance the hybridization signals and discrimination power in nucleic acids-based detection, providing an alternative platform for improving the performance of DNA microarrays.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.     

Fibrillogenesis of human β2‐microglobulin in three‐dimensional silicon microstructures

The authors describe the interaction of biological nanostructures formed by β₂‐microglobulin amyloid fibrils with three‐dimensional silicon microstructures consisting in periodic arrays of vertical silicon walls (≈3 μm‐thick) separated by 50 μm‐deep air gaps (≈5 μm‐wide). These structures are of great interest from a biological point of view since they well mimic the interstitial environment typical of amyloid deposition in vivo. Moreover, they behave as hybrid photonic crystals, potentially applicable as optical transducers for label‐free detection of the kinetics of amyloid fibrils formation. Fluorescence and atomic force microscopy (AFM) show that a uniform distribution of amyloid fibrils is achieved when fibrillogenesis occurs directly on silicon. The high resolution AFM images also demonstrate that amyloid fibrils grown on silicon are characterized by the same fine structure typically ensured by fibrillogenesis in solution. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)