Modification of an automated liquid-handling system for reagent-jet, nanoliter-level dispensing

Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solenoids and a liquid pressurization system to modulate volume delivery down to the nanoliter level. Additional modifications include the use of sapphire-tipped dispensing nozzles and a high-resolution substage to assist in the construction of DNA microarrays. Techniques for characterizing the dispensed volume are presented.     

Integrins may serve as mechanical transducers for low-frequency electric fields

A hypothesis is presented that a transduction mechanism for low frequency electric fields of physiological strength ( approximately 1 V/cm) is the same as that for sinusoidal fluid shear stresses, the force exerted on an integrin. Simple calculations show that the forces exerted on a model integrin by transverse electric fields and fluid shears that produce cellular effects are comparable in magnitude, about 1 fN. The electric force is provided by the interaction of the surface charges on the integrin with the tangential component of the applied field. The mechanical shear force is the transverse fluid drag force exerted on the cylindrical surface of the integrin. Either force is coupled mechanically to the actin cortex within the cell. The mechanical network which exists within a cell and connects a cell to its surroundings would then be directly coupled to an applied electric field. The fundamental transduction mechanism for some electric field effects may then be ultimately mechanical in nature.     

The fluid property dependency on micro-fluidic characteristics in the deposition process for microfabrication

The droplet impingement into a cavity at micrometer-scale is one of important fluidic issues for microfabrications, e.g. the inkjet deposition process in the PLED display manufacturing. The related micro-fluidic behaviors in the deposition process should be carefully treated to ensure the desired quality of microfabrication. The droplets generally dispensing from an inkjet head, which contains an array of nozzles, have a volume in several picoliters, while each nozzle responds very quickly and jets the droplets into cavities on substrates with micrometer size. The nature of droplet impingement depends on the fluid properties, the initial state of droplet, the impact parameters and the surface characteristics. The commonly chosen non-dimensional numbers to describe this process are the Weber number, the Reynolds number, the Ohnesorge number, and the Bond number. This paper discusses the influences of fluid properties of a Newtonian fluid, such as surface tension and fluid viscosity, on micro-fluidic characteristics for a certain jetting speed in the deposition process via a numerical approach, which indicates the impingement process consists of four different phases. In the first phase, the droplet stretching outwards rapidly, where inertia force is dominated. In the second phase, the recoiling of droplet is observed, where surface tension becomes the most important force. In the third phase, the gravitational force pulls the droplet surface towards cavity walls. The fourth phase begins when the droplet surface touches cavity walls and ends when the droplet obtains a stable shape. If the fluid viscosity is relatively small, the droplet surface touches cavity walls in the second phase. A stable fluid layer would not form if the viscosity is relatively small.     

DNA deposition on carbon electrodes under controlled dc potentials

The native calf-thymus DNA molecule fully dispersed in solution was deposited onto highly oriented pyrolytic graphite, carbon fiber column and disk electrodes under controlled dc potentials. X-ray photoelectron spectroscopy, atomic force microscopy and electrochemical investigations indicated that network structures of DNA could be formed on various carbon electrode surfaces resulting in significant surface enlargement. The conformation, conductivity and stability of the deposited DNA layer largely depended on the concentration of the DNA deposition solution, the applied dc potential and the mode of electric field. The optimal condition for deposition of the DNA on carbon fiber disk electrode was determined as a deposition potential of 1.8 +/- 0.3 V versus 50 mM NaCl-Ag/AgCl and a deposition DNA solution of 0.1 mg ml(-1). Under this condition, the DNA was covalently bonded on the electrode surface forming a three-dimensional modified layer, generating a 500-fold enlarged effective electrode surface area and similarly enlarged current sensitivity for redox species, such as Co(phen)3(3+). A possible mechanism for the formation of DNA networks is proposed.     

Fundamental study on a gene transfection methodology for mammalian cells using water-in-oil droplet deformation in a DC electric field

We have developed a gene transfection method called water-in-oil droplet electroporation (EP) that uses a dielectric oil and a liquid droplet containing live cells and exogenous DNA. When a cell suspension droplet is placed between a pair of electrodes, an intense DC electric field can induce droplet deformation, resulting in an instantaneous short circuit caused by the droplet elongating and contacting the two electrodes simultaneously. Small transient pores are generated in the cell membrane during the short, allowing the introduction of exogenous DNA into the cells. The droplet EP was characterized by varying the following experimental parameters: applied voltage, number of short circuits, type of medium (electric conductivity), concentration of exogenous DNA, and size of the droplet. In addition, the formation of transient pores in the cell membrane during droplet EP and the transfection efficiency were evaluated.     

Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays

DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.     

Cytoskeletal forces produced by extremely low‐frequency electric fields acting on extracellular glycoproteins

The physical mechanism by which cells transduce an applied electric field is not well understood. This article establishes for the first time a direct, quantitative model that links the field to cytoskeletal forces. In a previous article, applied electric fields of physiological strength were shown to produce significant mechanical torques at the cellular level. In this article, the corresponding forces exerted on the cytoskeleton are computed and found to be comparable in magnitude to mechanical forces known to produce physiological effects. In addition to the electrical force, the viscous drag force exerted by the surrounding medium and the restoring force exerted by the neighboring structures are considered in the analysis. For an applied electric field of 10 V/m, the force transmitted to the CD44 receptor of a hyaluronan chain in cartilage is about 1 pN at 10 Hz and 7 pN at 1 Hz. For an applied electric field of 100 V/m, the force transmitted to the cytoskeleton at one focus of the glycocalyx is about 0.5 pN at 10 Hz and 1.3 pN at 1 Hz. Mechanical forces of similar magnitude have been observed to produce physiological effects. Hence, this electromechanical transduction process is a plausible mechanism for the production of physiological effects by such electric fields. Bioelectromagnetics 31:77–84, 2010. © 2009 Wiley‐Liss, Inc.     

Fabrication of high quality microarrays

Fabrication of DNA microarray demands that between ten (diagnostic microarrays) and many hundred thousands of probes (research or screening microarrays) are efficiently immobilised to a glass or plastic surface using a suitable chemistry. DNA microarray performance is measured by parameters like array geometry, spot density, spot characteristics (morphology, probe density and hybridised density), background, specificity and sensitivity. At least 13 factors affect these parameters and factors affecting fabrication of microarrays are used in this review to compare different fabrication methods (spotted microarrays and in situ synthesis of microarrays) and immobilisation chemistries.     

Evaluation of surface chemistries for antibody microarrays

Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been derived primarily from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but there are no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 17 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyde silane, poly-l-lysine, or aminosilane (with or without activation with a crosslinker) consistently produce superior results in the sandwich ELISA microarray analyses we performed.     

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin-streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24mug/ml, quantitatively equal to 0.25 and 12.5pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.     

Impact of surface chemistry and blocking strategies on DNA microarrays

The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.     

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.     

EHD1 regulates cholesterol homeostasis and lipid droplet storage

Endocytic transport is critical for the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its trafficking. The C-terminal EHD protein, EHD1, controls receptor recycling through the ERC and affects free cholesterol distribution in the cell. We utilized embryonic fibroblasts from EHD1 knockout mice (Ehd1(-/-)MEF) and SiRNA in normal MEF cells to assess the role of EHD1 in intracellular transport of cholesterol. Surprisingly, Ehd1(-/-)MEFs displayed reduced levels of esterified and free cholesterol, which returned to normal level upon re-introduction of wild-type, but not dysfunctional EHD1. Moreover, triglyceride and cholesterol storage organelles known as 'lipid droplets' were smaller in size in cells lacking EHD1, indicating that less esterified cholesterol and triglycerides were being stored. Decreased cellular cholesterol and reduced lipid droplet size in Ehd1(-/-)MEFs correlated with ineffectual cholesterol uptake via LDL receptor, suggesting involvement of EHD1 in LDL receptor internalization.     

A comparison of microscope slide substrates for use in transfected cell microarrays

Transfected cell microarrays, arrays of mammalian cells expressing defined genes, offer enormous potential for the development of high-throughput cell-based detection technologies to monitor the presence of biological agents or environmental toxicants. The signals generated from these arrays are intimately linked to the efficiency of DNA uptake by the cells located on the micrometer-sized spots. However, quantitative analysis of the transfection efficiency on cellular microarrays has been limited. Further, little regard has been given to the role of the substrate in influencing the transfection efficiency of mammalian cells on transfected microarrays. In this report, we have quantified the transfection efficiency of mammalian cells on different microscope slide substrates. Using commercially available microscope slides bearing substrates that mediate cellular attachment (polystyrene, 3-aminopropylsilane, and poly-L-lysine), we have demonstrated the role of substrate hydrophobicity in determining the resulting spot size and the local DNA concentration when plasmid DNA is dispensed in a printing buffer containing gelatin and sucrose using a noncontact microarray printer. The mean spot diameter varied inversely with the substrate water contact angle (r2 = 0.970). Further, the relative local plasmid DNA concentration was a function of the mean spot diameter. The deposition of Rhodamine Red-labeled plasmid DNA revealed that, across all substrates, the average fluorescence signal within the spots varied inversely with the mean spot diameter (r2 = 0.976). The transfection efficiency of HEK 293T/17 cells varied in accord with the mean spot diameter, demonstrating that the uptake of DNA was a function of the local DNA concentration on each substrate.     

Enzyme microarrays assembled by acoustic dispensing technology

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 μM ATP (3 μCi/μL ³²P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 μM substrate. The microarray was incubated at 30 °C (97% R_h) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 μM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were ∼20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development.     

基因芯片技术在检测肠道致病菌方面的应用

基因芯片技术具有高通量、自动化、快速检测等特点,因此被广泛地应用于各种研究领域,如细菌分子流行病学、细菌基因鉴定、致病分子机理、基因突变及多态性分析、表达谱分析、DNA测序和药物筛选等。现介绍基因芯片检测肠道致病菌方面的国外研究进展,基因芯片应用于检测肠道致病菌的3个方面:结合多重PCR对致病菌的毒力因子或者特异性基因进行鉴定;直接检测细菌的DNA或者RNA;以致病细菌核糖体RNA作为检测的靶基因同时检测多种肠道致病菌。由于其检测的高效率,该技术要优于其他分子生物学检测方法。基因芯片技术在肠道致病菌检测中有着巨大的应用价值,具有广阔的应用前景。     

Granulometric analysis of spots in DNA microarray images

As the topological properties of each spot in DNA microarray images may vary from one another, we employed granulometries to understand the shape-size content contributed due to a significant intensity value within a spot. Analysis was performed on the microarray image that consisted of 240 spots by using concepts from mathematical morphology. In order to find out indices for each spot and to further classify them, we adopted morphological multiscale openings, which provided microarrays at multiple scales. Successive opened microarrays were subtracted to identify the protrusions that were smaller than the size of structuring element. Spot-wise details, in terms of probability of these observed protrusions,were computed by placing a regularly spaced grid on microarray such that each spot was centered in each grid. Based on the probability of size distribution functions of these protrusions isolated at each level, we estimated the mean size and texture index for each spot. With these characteristics, we classified the spots in a microarray image into bright and dull categories through pattern spectrum and shape-size complexity measures. These segregated spots can be compared with those of hybridization levels.     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Automatic analysis of DNA microarray images using mathematical morphology

MOTIVATION: DNA microarrays are an experimental technology which consists in arrays of thousands of discrete DNA sequences that are printed on glass microscope slides. Image analysis is an important aspect of microarray experiments. The aim of this step is to reduce an image of spots into a table with a measure of the intensity for each spot. Efficient, accurate and automatic analysis of DNA spot images is essential in order to use this technology in laboratory routines. RESULTS: We present an automatic non-supervised set of algorithms for a fast and accurate spot data extraction from DNA microarrays using morphological operators which are robust to both intensity variation and artefacts. The approach can be summarised as follows. Initially, a gridding algorithm yields the automatic segmentation of the microarray image into spot quadrants which are later individually analysed. Then the analysis of the spot quadrant images is achieved in five steps. First, a pre-quantification, the spot size distribution law is calculated. Second, the background noise extraction is performed using a morphological filtering by area. Third, an orthogonal grid provides the first approach to the spot locus. Fourth, the spot segmentation or spot boundaries definition is carried out using the watershed transformation. And fifth, the outline of detected spots allows the signal quantification or spot intensities extraction; in this respect, a noise model has been investigated. The performance of the algorithm has been compared with two packages: ScanAlyze and Genepix, showing its robustness and precision.     

Mathematical design of prokaryotic clone-based microarrays

Background  

Clone-based microarrays, on which each spot represents a random genomic fragment, are a good alternative to open reading frame-based microarrays, especially for microorganisms for which the complete genome sequence is not available. Since the generation of a genomic DNA library is a random process, it is beforehand uncertain which genes are represented. Nevertheless, the genome coverage of such an array, which depends on different variables like the insert size and the number of clones in the library, can be predicted by mathematical approaches. When applying the classical formulas that determine the probability that a certain sequence is represented in a DNA library at the nucleotide level, massive amounts of clones would be necessary to obtain a proper coverage of the genome.