Direct microcontact printing of oligonucleotides for biochip applications

Background  

A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips.     

Giant magnetoresistive biochip for DNA detection and HPV genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.     

A DNA biochip for on-the-spot multiplexed pathogen identification

Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole−probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.     

Adsorption of avidin on microfabricated surfaces for protein biochip applications

The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radiofrequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that a simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.     

SU-8-carbon composite as conductive photoresist for biochip applications

A composite photoresist has been developed for the direct photopatterning of electrodes useful as biochip substrates. The material is composed of SU-8 polymer added with graphite carbon filler which enables patterning of conductive thin films (22μm) on both glass substrate and transparency flexible film with a standard UV photolithography protocol. The resolution obtained using the conductive composite compared well with the bare resist, with lateral resolutions of 5 and 10μm for bare and conductive resists, respectively. The obtained electrodes, after an electrochemical pre-treatment, exhibited very good electrochemical behaviors, opening the path to various electrochemical detections and grafting possibilities. In order to demonstrate the potentialities of the developed material in the biosensors and biochips field, DNA probes were electrografted, using diazonium chemistry, directly at the composite photoresist surface. Target oligonucleotide interactions were detected using chemiluminescent labeling and a satisfactory detection limit of 0.25nM target sequence was demonstrated with a detection ranging over three orders of magnitude.     

A microfluidic-based electrochemical biochip for label-free diffusion-restricted DNA hybridization analysis

DNA hybridization detection in microfluidic devices can reduce sample volumes, processing times, and can be integrated with other measurements. However, as device footprints decrease and their complexity increase, the signal-to-noise ratio in these systems also decreases and the sensitivity is thereby compromised. Device miniaturization produces distinct properties and phenomena with greater influence at the micro-scale than at the macro-scale. Here, a diffusion-restriction model was applied to a miniaturized biochip nanovolume reactor to accurately characterize DNA hybridization events that contribute to shifts in both charge transfer resistance and diffusional resistance. These effects are shown to play a significant role in electrochemical impedance spectroscopy (EIS) analyses at these length scales. Our highly functional microfluidic biosensor enables the detection of ssDNA targets selectively, with a calculated detection limit of 3.8 nM, and cross-reactivity of 13% following 20 min incubation with the target. This new biosensing approach can be further modeled and tested elucidating diffusion behavior in miniaturized devices and improving the performance of biosensors.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

几种粉煤灰对磷素吸附与解吸特性的研究

通过吸附解吸和培养试验, 研究了几种粉煤灰对磷素吸附与解吸特性.结果表明,粉煤灰的全磷含量和有效磷含量分别为0.545～4.540 g·kg^-1和19.55～163.0 mg·kg^-1,显著高于土壤,粉煤灰对磷吸附量随着加入溶液磷浓度的增加而增加,但其吸附率随着加入溶液磷浓度的增加而减少;粉煤灰的吸磷率比土壤高,但其解吸率低.这主要是由于粉煤灰比土壤存在更多的磷吸附位点且结合能大,不易解吸.Langmuir方程、Freundlich方程和Temkin方程都能很好地拟合粉煤灰对磷吸附,其中Langmuir方程的MBC、Freundlich方程的a和Temkin方程的k2都可以表征粉煤灰对磷吸附能力, MBC、a和k2值越大,则吸磷能力越强.不同来源的粉煤灰的MBC、a和k2值不同,其大小顺序为：湘潭电厂(5 167.7,4 056.2,831.5)>岳阳纸厂(1 650.7,2 803.4,711.9)>华能电厂(303.0,1 677.6,368.7)>株洲电厂(76.2,464.2, 211.0) > 洞庭氮肥厂(34.7,413.48,213.8).粉煤灰对磷吸附固定作用随粉煤灰含水量的增加有增大的趋势.粉煤灰对磷吸附主要是专性吸附和化学沉淀反应,所以在施用粉煤灰改良土壤或利用粉煤灰制造复混肥时,须考虑粉煤灰对磷的固定作用及粉煤灰含水量的影响.     

磷酸盐在土壤中的竞争吸附与解吸机制

本文概述了近年来国内外有关磷酸盐的竞争吸附与解吸的研究成果。土壤中许多阴离子都能与磷竞争吸附点位,使得磷的吸附下降。有机质可促进或抑制磷的吸附,pH是影响竞争吸附的主要因子。磷被吸附后大多固持在表面而难于解吸,往往呈现明显的滞后现象。通常只有拟物理吸附的磷能被解吸,化学吸附的磷因与表面金属离子作用形成双齿配位而极难被淋洗下来。解吸受多种因素的影响,其中解吸剂的类型是主要因子之一。     

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.     

Laser desorption mass spectrometry for microbial DNA analysis.

Recently, we demonstrated that a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) can be used to determine the molecular weight of polymerase chain reaction (PCR) products of intact 16S rRNA regions and to profile their restriction digests. This is the first time that MALDI-TOF MS with ultraviolet (UV) photoionization has been used to analyze a PCR product of approximately 1600 nucleotides in length.     

Discontinuous formation and desorption of clusters during particles adsorption at surfaces

A theoretical model was derived to describe the discontinuous formation and desorption of clusters during particle adsorption at surfaces. Two steps were investigated: (1) time-dependent adsorption, where we found that the initial slope and the limiting magnitude of an adsorption isotherm depend on the clusters' distribution. A higher magnitude of both the adsorption and desorption rates appear to contract the time scale and hence increase the initial slope. Decreasing the geometrical parameter, q, which represents the shape of an adsorbed cluster, enhances the growth of large clusters on the surface. (2) A concentration dependence model shows that the number of adsorbed molecules increases with increases in the value of n (nucleation capacity). Furthermore, higher rates of adsorption provide steeper initial slopes (higher affinity of, molecules to surface). Decreasing q from 2 to 1, i.e. from a circular to a linear cluster formation, slightly decreases the magnitude of the isotherms.     

外源腐殖酸对三种土壤磷吸附与解吸特性的影响

通过对3种土壤（红壤、棕壤和褐土）施入不同腐殖酸的室内培养试验,探讨了外源腐殖酸对不同土壤磷素吸附和解吸的影响。结果表明：与对照土壤相比,不同腐殖酸降低了3种供试土壤磷素的吸附量,其降低顺序为：褐土＞红壤＞棕壤;外源腐殖酸提高了红壤和棕壤磷素的解吸量和解吸率,提高的幅度与腐殖酸的种类有关;而腐殖酸对褐土磷素的解吸量则无明显促进作用。表明外源腐殖酸对3种土壤磷素吸附-解吸作用最强的为红壤,其次为棕壤,最弱的为褐土;同时表明腐殖酸可提高红壤和棕壤磷素的利用率。     

Kinetics of protein adsorption and desorption on surfaces with grafted polymers

The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.     

Root surface area measurements based on adsorption and desorption of nitrite

A method, based on the negative adsorption of NO₂ ^-, has been developed to determine surface area of roots. Young roots of 3–4 year old plants ofAcacia catechu, Eucalyptus camaldulensis andLeucaena leucocephala were up-rooted and cut into 18 cylindrical pieces. Each root piece was immersed individually for 10 sec in 0.05M, 0.10M and 0.15M aqueous NaNO₂ solution and the excess solution on the root surface allowed to drain off. It was then transferred to conical flask containing distilled water and shaken for 15 min for desorption of nitrite. A known quantity of this aliquot was reacted with 1% acidic sulphanilamide and 0.02% NED HCl. A pink colour developed, and its optical density was read at 540 nm. A positive linear correlation was noted between colour density and root surface area. The respective correlation coefficientvalues for 0.05M, 0.10M and 0.15M NaNO₂ solutions were 0.973, 0.963 and 0.964 forAcacia catechu, 0.933, 0.903 and 0.898 forEucalyptus camaldulensis and 0.968, 0.976 and 0.972 forLeucaena leucocephala (significant atp<0.001). The method was successfully adopted to determine the root surface area of seedlings ofAlbizia lebbek, A. procera, Acacia auriculiformis, A. nilotica, Dalbergia latifolia andD. sissoo.     

Ancient DNA applications for wildlife conservation

Ancient DNA analyses of historical, archaeological and paleontological remains can contribute important information for the conservation of populations and species that cannot be obtained any other way. In addition to ancient DNA analyses involving a single or few individuals, population level studies are now possible. Biases inherent in estimating population parameters and history from modern genetic diversity are exaggerated when populations are small or have been heavily impacted by recent events, as is common for many endangered species. Going directly back in time to study past populations removes many of the assumptions that undermine conclusions based only on recent populations. Accurate characterization of historic population size, levels of gene flow and relationships with other populations are fundamental to developing appropriate conservation and management plans. The incorporation of ancient DNA into conservation genetics holds a lot of potential, if it is employed responsibly.     

MELAS和MERRF综合征相关mtDNA突变位点检测集成芯片的建立

摘要: 文中建立了一种新型的寡核苷酸芯片, 用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers, MERRF)线粒体DNA所有已知突变位点的集成检测。将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面, 以多重不对称PCR方法制备Cy5荧光标记靶基因。利用此芯片对5例MELAS患者、5例MERRF患者及20例健康对照进行筛查, 结果发现, MELAS患者均为MT-T1基因A3243G突变; 在MERRF患者组, MT-TK基因A8344G突变4例, T8356C突变1例; 健康对照组均未发现31种相关mtDNA突变。芯片检测与DNA测序结果完全一致。结果表明, 这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测, 具有较高的灵敏度和特异性。这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断。