A new generation of scanners for DNA chips

Today, most of the DNA chips are used with fluorescent markers. Associated with fluorescence confocal scanners, this technology achieves remarkable performances in terms of sensitivity and accuracy. The main technical issues related to these scanners have already been reviewed. However, these scanners are costly, especially when high density chips are used. In this case, a mechanical precision of 1 microm or less is required to achieve the measurement precision required. This cost level prevents the spread of this technology in the diagnostic market. We will present a new concept for scanners with equivalent or superior performances, with a cost cut of 5-10. This concept is inspired from the field of optical disk and reader. Basically, an optical format is added to the chip, before DNA deposition. This format contains tracks which are superimposed to the DNA features. These tracks define the path that an optical head of a CD player must follow in order to scan the surface of the DNA chip. Such a head is a very cheap component, and has a precision of less than 100 nm thanks to real-time focus and tracking. These functions are fulfilled by electromagnetic actuators mounted on the support of the frontal lens. We show here that it is possible to use such a head to build a fluorescence confocal scanner with equivalent or even better performances than conventional scanners.     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.     

聚合物微流控芯片消毒灭菌技术研究进展

聚合物微流控芯片成本低、易加工,目前在医药、生物检测和化学合成等领域得到了普遍应用。以热塑性聚合物聚甲基丙烯酸甲酯(polymethylmethacrylate,PMMA)和热固型聚合物聚二甲基硅氧烷(polydimethy lsiloxane,PDMS)为基材的高分子聚合物材料因具有较好的生物相容性和光学透明性,已逐渐成为聚合物微流控芯片加工的主导材料,被广泛应用于生物医药类微流控芯片的制备。鉴于该类芯片应用场景的特殊性,需在使用前进行消毒灭菌处理以避免微生物干扰。目前,针对PMMA和PDMS的消毒灭菌方法包括高压蒸汽灭菌、紫外线灭菌、电子束、60Co γ射线辐射灭菌、超临界二氧化碳灭菌、乙醇消毒、环氧乙烷灭菌、过氧化氢低温等离子体灭菌、绿原酸消毒、清洗剂消毒。本文从基本原理、消毒灭菌方法、应用场景等方面,回顾和总结了相关技术在PMMA和PDMS基体微流控芯片中的实现方法,并在芯片材质、适用范围等方面分析了所适用的消毒灭菌方法,为以聚合物为基材的生物医药类微流控芯片的消毒灭菌提供有益参考。     

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.     

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.     

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence

A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nano-assembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

几种新型生物芯片的研究进展

随着生物芯片技术的迅速发展,一些新型生物芯片,如生物电子芯片、凝胶元件微阵列芯片、药物控释芯片、毛细管电泳或层析芯片、PCR芯片及生物传感芯片等应运而生,这些芯片不同于常规的分子微阵列芯片,而是以各种结构微阵列为基础,用于分子杂交与扩增,以检测突变、分析多态性及测序,通过电泳及层析分离生物样品,控制药物释放以治疗疾病,作为生物传感器检测分子行为等,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域的新工具.     

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.     

Online optical detection of food contaminants in microdroplets

To accommodate the considerable increase of disease based on microbial food contaminants in the last decade, a modulated, fast optical fluorescence detection combined with microdevices is created. This method, which consists of five different steps, first selects contaminants, mainly bacteria, in the food matrix. This process is based on a biomagnetic separation technique developed by our collaborators at the Technical University of Dresden. By the steps of binding antibody functionalized magnetic beads and fluorescent capsules on the target cell, a magnetic bead‐target cell‐microcapsule complex (MTM) is generated. The well‐established pipe‐based bioreactors (pbb) platform enables the generation of droplets with a volume between 60 and 160 nL and the detection of the target cell with an integrated microscopic and spectroscopic detection system. The module used for generating droplets is based on the segmented flow principle and is chip‐ or probe‐based. In this context, the successful use of polydimethylsiloxane (PDMS) as a cost‐effective alternative to the well‐established glass‐chips is introduced. To quantify the detection based on a yes‐ or no‐decision, the most important step is to separate one MTM‐complex per droplet. This equalized the quantity of the fluorescent signals with the quantity of the contaminants in the cell sample. The feasibility of microscopic and spectroscopic detection with only one fluorescent capsule per droplet is shown. Also the first results of a special prototyping optical detection set‐up that is already in an advanced stage of development, will be presented. This easy‐to‐use device implemented a software‐controlled, automatic documentation for every fluorescent signal of a droplet to guarantee the quality control. Here are the advantages of an integration of microdevices in a rapid detection of food pathogens presented. Obviously, the modular set‐up of this detection platform enables a wide range of high‐throughput applications.     

Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Development of a high-throughput Candida albicans biofilm chip

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.     

Gene-based identification of bacterial colonies with an electric chip

A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.     

Overview of DNA chip technology

DNA chip technology utilizes microscopic arrays (microarrays) of molecules immobilized on solid surfaces for biochemical analysis. Microarrays can be used for expression analysis, polymorphism detection, DNA resequencing, and genotyping on a genomic scale. Advanced arraying technologies such as photolithograpy, micro-spotting and ink jetting, coupled with sophisticated fluorescence detection systems and bioinformatics, permit molecular data gathering at an unprecedented rate. Microarray-based characterization of plant genomes has the potential to revolutionize plant breeding and agricultural biotechnology. This review provides an overview of DNA chip technology, focusing on manufacturing approaches and biological applications.     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。     

A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids

Oligonucleotide microarrays or oDNA chips are effective decoding and analytical tools for genomic sequences and are useful for a broad range of applications. Therefore, it is desirable to have synthesis methods of DNA chips that are highly flexible in sequence design and provide high quality and general adoptability. We report herein, DNA microarray synthesis based on a flexible biochip method. Our method simply uses photogenerated acid (PGA) in solution to trigger deprotection of the 5′-OH group in conventional nucleotide phosphoramidite monomers (i.e. PGA-gated deprotection), with the rest of the reactions in the synthesis cycle the same as those used for routine synthesis of oligonucleotides. The complete DNA chip synthesis process is accomplished on a regular DNA synthesizer that is coupled with a UV-VIS projection display unit for performing digital photolithography. Using this method, oDNA chips containing probes of newly discovered genes can be quickly and easily synthesized at high yields in a conventional laboratory setting. Furthermore, the PGA-gated chemistry should be applicable to microarray syntheses of a variety of combinatorial molecules, such as peptides and organic molecules.     

Achieving differentiation of single-base mutations through hairpin oligonucleotide and electric potential control

A novel assay for surface DNA hybridization, which is free of sample and probe labeling, convenient and of low cost, sensitive and capable of differentiation of single-base mutations, is reported. Hairpin oligonucleotides are carefully designed as probes and are covalently attached to Si chips. Segments of the human p53 gene are chosen to demonstrate the major features of the novel technique. Impedance measurement is used to detect the hybridization. To further optimize the performance, electric potential is applied on the chip. The apparently different responses of the chip to the complementary strand and the single-base mutant are shown under electric potential control. The criteria on the design of the hairpin oligonucleotides are discussed.     

Possibility of using DNA chip technology for diagnosis of human papillomavirus

To explore the application of DNA chip technology for the detection and typing of Human Papillomavirus (HPV), the HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by a PixSys 5500 microarrayer as probes to prepare the HPV gene chips. HPV samples, after being labeled with fluorescent dye by restriction display PCR (RD-PCR) technology, were hybridized with the microarray, which was followed by scanning and analysis. The experimental condition for preparing the HPV gene chips was investigated, and the possibility of HPV genotyping using gene chips was discussed. The technique that was established in this study for preparing HPV gene chips is practical. The results of the present study demonstrated the versatility and inspiring prospect of using this technology to detect and genotype HPV.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

Anthracene based base-discriminating fluorescent oligonucleotide probes for SNPs typing: synthesis and photophysical properties

2- and 9-Anthracenecarboxamide labeled 2'-deoxyuridines were synthesized and their photophysical properties were examined. These oligonucleonucleotide probes are capable of detecting adenine base on a target DNA sequence. It was also found that 2-anthracene based oligonucleotide probe is more efficient than the corresponding 9-anthracene based oligonucleotide in the application for DNA chip based SNP detection, due to its longer emission wavelength and high fluorescence intensity.