Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD_15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.     

Development of an SH-SAW sensor for the detection of DNA hybridization

We have developed SH (shear horizontal) surface acoustic wave (SAW) sensors for the detection of DNA (deoxyribonucleic acid) hybridization on the gold-coated delay line of transverse SAW devices. DNA hybridization experiments were performed with 15-mer oligonucleotides (probe and complementary target DNA). The sensor consists of twin SAW delay line oscillators (sensing channel and reference channel) operating at 100 MHz fabricated on 36° rotated Y-cut X-propagation LiTaO₃ piezoelectric single crystals. The relative change in the frequency of the two oscillators was monitored to detect hybridization between the target DNA and probe DNA immobilized on the delay line of the SAW sensor. The measurement results showed a good response of the sensor to the mass loading effects of the DNA hybridization with a sensitivity level up to 1.55 ng/ml/Hz.     

Multiplexed spectral coding for simultaneous detection of DNA hybridization reactions based on FRET

Fluorescence resonance energy transfer (FRET) is widely used in spectral codification of information at the molecular level, and can be used to generate several layers of information on a DNA chip. We used two oligonucleotides (probes) labeled with different donor (harvesting) molecules in hybridization experiments with complementary oligonucleotides labeled with four different acceptors (targets). By looking at the fluorescence response of the sample after “specific” excitation of each donor molecule (by “specific” we mean a wavelength where one of the donors is predominantly excited), we inspected the possibility to identify the complementary oligonucleotide hybridized to the probe, in mixtures containing two donor probe/acceptor target pairs. In most samples (13 out of the 16 possible), it is trivial to identify the complementary target that is hybridized to the excited donor probe in the mixtures. The major limitations of the chosen system arise when very different concentrations of donor probe/acceptor target pairs are present in the same sample.     

电化学DNA传感器中DNA的固定与杂交条件探讨

利用自组装单分子膜法,将人工合成的乙肝病毒单链DNA片断作为特异性探针固定在金电极表面,结合电活性指示剂Hoechst33258构成DNA电化学传感器。在乙肝特异性DNA探针的自组装固定过程中,探讨了自组装单分子膜的成膜条件,总结出在探针浓度为100mg/L,自组装固定时间为12h对DNA探针的固定较为有利。考察了单链DNA修饰电极的伏安特性和单链DNA修饰电极的电子传递性能。在对标准互补DNA溶液的杂交检测过程中,探讨了杂交时间、杂交温度、指示剂的作用时间等对DNA传感器检测的影响。当杂交时间为90min,杂交温度为25℃,杂交溶液中NaCl浓度为0.3mol/L时,指示剂的伏安信号较好。     

A rapid polymerisation process realizing 3D biocompatible structures in a microfluidic channel suitable for genetic analysis

Surface probe immobilisation is a complex and time consuming task undertaken prior to microfluidic integration, this requires surface functionalisation, biomolecule spotting, incubation and blocking steps. Traditional bonding techniques (anodic, thermal, etc.) or adhesives (UV cured) used to seal fluidic systems may denature biomolecules due to high temperature or vapour effects, thus bonding techniques such as thin film laminate or PDMS are used to seal systems, with substrate-fluidic alignment required prior to bonding. We propose a technique allowing probe DNA molecules to be immobilised in a sealed microfluidic system using (3D) hydrogel structures without any alignment steps. A prepolymer solution is introduced to the channels where photo-polymerisation is undertaken forming 3D structures covalently attached to the channel surface. We use a photo-initiated prepolymer material poly-ethylene-glycol (PEG) to form structures containing probe DNA. This process is fast compared to conventional biomolecule immobilisation techniques and is also biocompatible, this direct write approach removes overnight immobilisation/incubation of the probe DNA, it also facilitates immobilisation within a sealed fluidic system where conventionally DNA probe spots must be immobilised prior to channel sealing. We consider the transport of target DNA from bulk analyte to the 3D gel structure and evaluate hybridisation within the microfluidic system.     

Automated analysis of DNA hybridization images for high-throughput genomics

The design and implementation of a computer vision system called DNAScan for the automated analysis of DNA hybridization images is presented. The hybridization of a DNA clone with a radioactively tagged probe manifests itself as a spot on the hybridization membrane. The imaging of the hybridization membranes and the automated analysis of the resulting images are imperative for high-throughput genomics experiments. A recursive segmentation procedure is designed and implemented to extract spotlike features in the hybridization images in the presence of a highly inhomogeneous background. Positive hybridization signals (hits) are extracted from the spotlike features using grouping and decomposition algorithms based on computational geometry. A mathematical model for the positive hybridization patterns and a Bayesian pattern classifier based on shape-based moments are proposed and implemented to distinguish between the clone-probe hybridization signals. Experimental results on real hybridization membrane images are presented.Received: 25 June 2002, Accepted: 11 November 2003, Published online: 17 February 2004 Correspondence to: Suchendra M. Bhandarkar     

An ant colony optimization algorithm for DNA sequencing by hybridization

The reconstruction of DNA sequences from DNA fragments is one of the most challenging problems in computational biology. In recent years the specific problem of DNA sequencing by hybridization has attracted quite a lot of interest in the optimization community. Several metaheuristics such as tabu search and evolutionary algorithms have been applied to this problem. However, the performance of existing metaheuristics is often inferior to the performance of recently proposed constructive heuristics. On the basis of these new heuristics we develop an ant colony optimization algorithm for DNA sequencing by hybridization. An important feature of this algorithm is the implementation in a so-called multi-level framework. The computational results show that our algorithm is currently a state-of-the-art method for the tackled problem.     

Application of Mismatch Detection Methods in DNA Computing

In many implementations of DNA computing, reliable detection of hybridization is of prime importance. We have applied several well-established DNA mutation scanning methods to this problem. Since they have been developed for speed and accuracy, these technologies are very promising for DNA computing. We have benchmarked a heteroduplex migration assay and enzymatic detection of mismatches on a 4 variable instance of 3SAT, using a previously described blocking algorithm. The first method is promising, but yielded ambiguous results. On the other hand, we were able to distinguish all perfect from imperfect duplexes by means of a CEL I mismatch endonuclease assay.     

Abstraction layers for scalable microfluidic biocomputing

Microfluidic devices are emerging as an attractive technology for automatically orchestrating the reactions needed in a biological computer. Thousands of microfluidic primitives have already been integrated on a single chip, and recent trends indicate that the hardware complexity is increasing at rates comparable to Moore’s Law. As in the case of silicon, it will be critical to develop abstraction layers—such as programming languages and Instruction Set Architectures (ISAs)—that decouple software development from changes in the underlying device technology. Towards this end, this paper presents BioStream, a portable language for describing biology protocols, and the Fluidic ISA, a stable interface for microfluidic chip designers. A novel algorithm translates microfluidic mixing operations from the BioStream layer to the Fluidic ISA. To demonstrate the benefits of these abstraction layers, we build two microfluidic chips that can both execute BioStream code despite significant differences at the device level. We consider this to be an important step towards building scalable biological computers.

An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

Experimental characterization of electrical current leakage in poly(dimethylsiloxane) microfluidic devices

Poly(dimethylsiloxane) (PDMS) is usually considered as a dielectric material and the PDMS microchannel wall can be treated as an electrically insulated boundary in an applied electric field. However, in certain layouts of microfluidic networks, electrical leakage through the PDMS microfluidic channel walls may not be negligible, which must be carefully considered in the microfluidic circuit design. In this paper, we report on the experimental characterization of the electrical leakage current through PDMS microfluidic channel walls of different configurations. Our numerical and experimental studies indicate that for tens of microns thick PDMS channel walls, electrical leakage through the PDMS wall could significantly alter the electrical field in the main channel. We further show that we can use the electrical leakage through the PDMS microfluidic channel wall to control the electrolyte flow inside the microfluidic channel and manipulate the particle motion inside the microfluidic channel. More specifically, we can trap individual particles at different locations inside the microfluidic channel by balancing the electroosmotic flow and the electrophoretic migration of the particle.     

Molecular detection based on the electrical conductance of gold nanoparticle arrays

We report a molecular sensing method based on changes in the electrical conductance of lithographically defined gold nanoparticle (NP) arrays immersed in an analyte solution. As the closely spaced NPs are enlarged due to the analyte-mediated deposition of gold ions onto their surfaces, the conductance increases steeply near the critical time (t_c) at which conducting pathways begin to form in the NP arrays. t_c decreases with increasing analyte concentration in the solution. The temperature dependence of the conductance of the modified NP arrays confirms the good electrical contacts established between the newly formed nanoclusters on the NP surfaces. Our results demonstrate that the electrical conductance through metal NP arrays can be employed as a sensitive and reliable analytical signal for NP-based sensors, which do not require any post-processing for the formation of electrical contacts between the NPs.     

DNA芯片制作及其应用

阐述了DNA芯片的制作原理、杂交信号的检测方法及其在生物医学中的应用。DNA芯片的制造工艺主要分为两大类。DNA芯片主要通过杂交信号进行检测。DAN芯片在生物医学领域已经取得了一定成功并显示出巨大的应用前景     

A microfluidic platform for formation of double-emulsion droplets

A new method for actively controlling the number of internal droplets of water-in-oil-in-water (W/O/W) double-emulsion droplets was demonstrated. A new microfluidic platform for double-emulsion applications has been developed, which integrates T-junction channels, moving-wall structures, and a flow-focusing structure. Inner water-in-oil (W/O) single-emulsion droplets were first formed at a major T-junction. Then the droplets were sub-divided into smaller uniform droplets by passing through a series of secondary T-junctions (branches). The moving-wall structures beside the secondary T-junctions were used to control the number of the sub-divided droplets by selectively blocking the branches. Finally, double-emulsion droplets were formed by using a flow-focusing structure downstream. Experimental data demonstrate that the inner and outer droplets have narrow size distributions with coefficient of variation (CV) of less than 3.5% and 5.7%, respectively. Double-emulsion droplets with 1, 2, 3, and up to 10 inner droplets have been successfully formed using this approach. The size of the inner droplets and outer droplets could be also fine-tuned with this device. The development of this new platform was promising for drug delivery applications involving double emulsions.     

A novel method for concentration evaluation of reading behaviors with electrical activity recorded on the scalp

In this paper, a concentration evaluation of reading behaviors with electrical signal detection on the head is presented. The electrode signal is extracted by brain–computer-interface (BCI) to monitor the user's degree of concentration, where the user is reminded by sound to concentrate, or teaching staffs are reminded to help users improve reading habits, in order to facilitate the user's ability to concentrate. The digital signal processing methods, such as the Kalman Filter, Fast Fourier Transform, the Hamming window, the average value of the total energy of a frame, correlation coefficient, and novel judgment algorithm are used to obtain the corresponding parameters of concentration evaluation. Users can correct their manner of reading with reminders. The repeated test results may be expected to lie with a probability of 95%. Such model training results in better learning effect.     

用于液相检测的双参数压电DNA传感器研究

采用自组装技术,利用亲和素-生物素系统将25-mer的生物素标记的DNA探针固定于石英谐振器金电极上,与双参数压电传感器相结合,研制成了用于液相检测的压电DNA传感器。该传感器稳定性较好,3h频移在5Hz以内;液体的体积在一定范围内对双参数传感器未见明显影响;用双参数方法制作的传感器特异性等性能较好,为实现检测的自动化和生物的动力学测定打下了基础。     

Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.     

A novel integrated microfluidic platform based on micro-magnetic sensor for magnetic bead manipulation and detection

We present a novel integrated microfluidic platform based on micro-magnetic sensor for manipulating and detecting magnetic beads (MB). A micro-spiral planar coil in MB manipulating system microfabricated by micro-electro-mechanical system technology is implemented to manipulate MB, and a giant magnetoimpedance (GMI) based micro-magnetic sensor is employed to detect the trapped MB. In our work, MB can be efficiently trapped by trapping force generated from micro-coil in microchannel. Next, trapped MB are detected by the changing ratio of impedance, as well as the variation of resistance and reactance in GMI sensor for trapped MB induce weak stray magnetic field under the magnetization by external magnetic field. The maximum difference of GMI ratio between with beads condition and without beads condition is 4.0% at the optimum driving frequency of 20 MHz under the external magnetic field of 15 Oe, and resistance ratio varies more significantly than reactance ratio. In comparison with traditional MB detecting methods by GMI sensor, the integrated microfluidic platform based on GMI sensor can not only manipulate and detect MB signal sensitively, but also enhance detection efficiency and decrease the experiment errors. Furthermore, this platform avoids contamination from the solutions in chemically reactive layers and reduces assay time in future biomarker detection. In our work, the microfluidic platform based on GMI sensor has potential applications in biomarker detection via MB manipulation and detection.     

异常检测测试平台的设计

提出了一种可以测试不同算法的异常检测测试平台.为适合大规模分布式网络,将网络分成不同网段,每个网段放置一个探测器IC,把不同IC提供的网络数据汇总至异常检测部件,在此进行异常分析,并根据分析结果对可能的入侵行为进行实时报警,其中的异常检测算法可以替换.最后,针对一种基于统计的异常检测算法进行了实验,并给出异常检测结果.