Bead-based polymerase chain reaction on a microchip

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15?min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

MCU based real-time temperature control system for universal microfluidic PCR chip

The analysis of genetic materials in the post-human genome project era has become an ever-expanding branch of research and thus routinely employed in majority of biochemical laboratories. Most of the diagnostic research area emphasizes on polymerase chain reaction for detecting pathogenic organisms. However, the conventional polymerase chain reaction requires expensive and sophisticated thermal cycler and is not handy owing to its large dimensions. Therefore, we fabricated a continuous-flow polymerase chain reaction chip on a PDMS based microfluidic platform to ease the hardship of the conventional system. Temperature being the most crucial factor in polymerase chain reaction, was monitored and regulated by thermostatic action using an on-line computer system. Indium tin oxide coated glass platform was used for heating as it is transparent and has good thermal conductivity under the influence of DC bias. The heating circuit used an ATMega 128 MCU to control the temperature. As a result, a precise and quick heating environment was maintained on the microfluidic chip to amplify the target DNA. We successfully amplified Lambda phage and Escherichia coli DNA on our chip to prove the practicality of the device.     

Planar microreactor for biochemical application made from silicon and polymer films

Recently the progress of life science has been increasing rapidly, and the importance of the microfluidics for DNA analysis systems has been widely recognized, especially in medical fields. The polymerase chain reaction (PCR) is an essential technique for DNA assay of various diseases and it has been a strong requirement to shorten the total of PCR cycles more and more. We developed the microreactor with a single cell for PCR using fabrication technologies of MEMS. The reactor body and cover were sealed using high thickness PDMS prototyping film without using adhesive in order to achieve repeat grabbing motion for direct sample injection, resumption and cleansing the reaction cell. Good reproducibility of the heat cycling was obtained. The heating rate and cooling rate during PCR was 6.8 and 2.7°C, respectively, which well corresponds to the design parameters. The homogenous temperature distribution of variance less than 2.0°C was obtained. It is demonstrated that amplification of the DNA was successfully achieved by using the microreactor.     

A novel micro-fluidic biosensor for the rapid and sequence-specific detection of DNA with electrophoretic driving mode and laser-induced fluorescence detector

A novel DNA biosensor, which combines the merits of micro-fluidic chips, the electrophoretic driving mode, paramagnetic beads amplification, and laser-induced fluorescence detection was developed for the rapid and sequence-specific detection of DNA in this study. The proposed DNA biosensor has much higher discrimination ability for the detection of single-base mismatch and much stronger resistibility to the complex matrixes of real samples in comparison with previous biosensors. These features, as well as its ease of fabrication (the fabrication of the sensor takes only 10 min except the fabrication of micro-fluidic chip), operation convenience, stability, better re-usability (micro-fluidic chip can be reused without any extra treatment) and short analysis time (one determination only takes 15 min), make it a promising alternative to rapid detection of DNA in clinical diagnosis. With the help of the biosensor, we successfully determined DNA, which related to oral cancer, in a saliva sample without any pre-separation or dilution with a detection limit of 4.2 × 10^?11 M and a relative standard deviation (n = 5) <5 %. The success in the present biosensor served as a significant step toward the practical application of the biosensor in clinical diagnosis.     

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10² cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.     

An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers

This study reports a microfluidic system which automatically performs the systematic evolution of ligands by exponential enrichment (SELEX) process for rapid screening of aptamers which are specific to cancer stem-like cells. The system utilizes magnetic bead-based techniques to select DNA aptamers and has several advantages including a rapid, automated screening process, and less consumption of cells and reagents. By integrating a microfluidic control module, a magnetic bead-based aptamer extraction module, and a temperature control module, the entire Cell-SELEX process can be performed in a shorter period of time. Compared with the traditional Cell-SELEX process, this microfluidic system is more efficient and consumes fewer sample volumes. It only takes approximately 3 days for an entire Cell-SELEX process with 15 screening runs, which is relatively faster than that of a traditional Cell-SELEX process (1 week for 15 rounds). The binding affinity of this resulting specific aptamer was measured by a flow cytometric analysis to have a dissociation constant (K _d) of 15.32 nM. The capture rate for cancer stem-like cells using the specific aptamer-conjugated bead is better than that using Ber-EP4 antibody-conjugated bead. This microfluidic system may provide a powerful platform for the rapid screening of cell-specific aptamers.     

Primer design for multiplex PCR using a genetic algorithm

Multiplex Polymerase chain reaction (PCR) is the term used when more than one pair of primers is used in a polymerase chain reaction. The goal of multiplex PCR is to amplify several segments of target DNA simultaneously and thereby to conserve template DNA, save time, and minimize expense. The success of the experiment is dependent on primer design. However, this can be a dreary task as there are many constrains such as melting temperatures, primer length, GC content and complementarity that need to be optimized to obtain a good PCR product. In our investigations, we found few primer design tools for multiplex PCR and there was no suitable tool for our partners who want to use a multiplex PCR genotypic assay. The tool draws on a genetic algorithm where stochastic approaches based on the concept of biological evolution, biological genetics and genetic operations on chromosomes are used to find an optimal solution for multiplex PCR. The presented experimental results indicate that the proposed algorithm is able to find a set of primer pairs that not only obey the design properties but also work in the same tube.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

On-chip polymerase chain reaction microdevice employing a magnetic droplet-manipulation system

An on-chip polymerase chain reaction (PCR) device employing a magnetic beads-droplet-handling system was developed. Actuation with a magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads by handling with a magnet. The device consists of a reaction chamber channel and two magnet-handling channels for the manipulation of micro-droplets containing magnetic beads. Micro-droplets were placed inside a reaction chamber filled with oil and manipulated with a magnet. When a droplet containing NaOH and magnetic beads was manipulated towards a droplet containing phenol red, a color change was observed after fusion. Sample preparation was performed by fusion of droplets containing a forward primer, reverse primer, template DNA and PCR mixture, using a droplet containing magnetic beads. PCR amplification or RT-PCR was also successfully performed, with efficiency comparable to manual methods that use this device by placing it on a thermal cycler for amplification. With a magnetic beads-manipulation step, purification of amplified DNA was also accomplished by using magnetic beads as the carrier. The amplified DNA was captured on streptavidin conjugated magnetic beads using a biotinylated primer, purified by washing and digested for separation of the target DNA.     

An automatic microfluidic sample transfer and introduction system

We report an easily setup, reliable and automatic microfluidic sample transfer and introduction system. Two different function liquid detection modules were developed to separately perform rapidly removing of a large approximate volume of air off chip and a low-speed high precision small volume of air purging process on chip incorporating liquid-on-chip handling module. As a proof of concept, we demonstrated that a small volume of radioactive sample as low as 5 μL could be successfully transferred and introduced from vials to the desired location in the microfluidic chip with minimal loss (2.1 ± 0.4 %, n = 3). The total time of the sample transfer and introduction was less than 1 min. The complete automation would facilitate the safe handling of the dangerous and toxic materials, such as radioactive compound.     

A microfluidic device for thermal particle detection

We demonstrate the use of heat to count microscopic particles. A thermal particle detector (TPD) was fabricated by combining a 500-nm-thick silicon nitride membrane containing a thin-film resistive temperature detector with a silicone elastomer microchannel. Particles with diameters of 90 and 200 μm created relative temperature changes of 0.11 and ?0.44 K, respectively, as they flowed by the sensor. A first-order lumped thermal model was developed to predict the temperature changes. Multiple particles were counted in series to demonstrate the utility of the TPD as a particle counter.     

一种多元聚合酶链反应的引物集选择算法

多元聚合酶链反应( multiplex PCR, MP-PCR )是一种运用多对引物同时扩增多条DNA序列或一条DNA序列上多个区域的生物学实验方法.引物集设计对于实验的成功至关重要.由于引物合成是实验成本的主要来源,且引物需要满足许多约束条件,因此设计满足多约束条件的最小引物集是保证实验成功、降低实验成本的有效手段.首先给出多约束最小引物集选择问题(minimum primer set selection problem with multiple constraints, MPSSPMC)的数学模型,通过引入新颖的遗传算子,提出一种求解该问题的单亲遗传算法MG-PGA.实验结果表明MG-PGA在满足多约束条件下能获得较小的引物集,为MP-PCR引物设计提供了一种有效的解决方法.     

A DNA methylation assay for detection of ovarian cancer cells using a HpaII/MspI digestion-based PCR assay in an integrated microfluidic system

Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10² cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.     

Effect of micro tensile sample’s cross section shape on the strength of weld line in micro injection molding process

As a suitable mass and cost efficiency fabrication method, micro injection molding is doing a very good performance in micro plastic parts production. The mold design is an important part affecting micro parts properties. In this study, a micro injection mold with multi cavities of micro tensile bar is used. These micro cavities are fabricated by a micro milling process in different cross section shapes (semicircle R = 0.5 mm, equilateral triangle D = 0.3 mm, and trapezoid D = 0.336 mm t = 0.2 mm bottom angle = 95°). With an Arburg^® 320C injection molding machine, micro tensile test sample are prepared in different processing parameters so that a correlation between the cross section shapes with micro weld line strength in different conditions could be investigated by tensile test. Final results show that when the cross section shape is different, their corresponding weld line strength is also different. Equilateral triangle cross section is leading to strongest weld line, and then followed by trapezoid, semi-circle is the last. By analysis of these tensile test results, the quantitative factor a is defined as the ratio of perimeter to area of cross section shape, and higher a value is corresponding stronger weld line. After weld line strength comparison in different processing conditions, the results show that higher injection pressure induced to lower weld line strength whatever the cross section shape is. By higher mold and melt temperature, equilateral triangle cross section gives improved weld line strength. But mold and melt temperature affect weld line strength negative for other cross section shapes.     

A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes

This study presents a new thermal cycler using infrared (IR) heating and water impingement cooling for polymerase chain reaction (PCR) amplification of 10 l samples in thin glass capillary tubes. The thermal cycling system can achieve a temperature ramping-up rate of 65 °C/s and a ramping-down rate of 80 °C/s. Two other cooling mechanisms, natural convection and forced air convection, can also be used in the present system to obtain a ramping-down rate of 2 °C/s and 6 °C/s, respectively. The amplification of the 439 fragment of hepatitis B virus (HBV) DNA was successful. The PCR amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining for visualization. A comparison of results of the amplification products at three different ramping-down rates was made and the rapid thermal cycling of the present system can run DNA required amplification in 29 min for 20 thermal cycles that is only 1/3 the time spent in the conventional PCR machine used in comparison.This work was supported by the National Taiwan University College of Medicine in preparing the HBV DNA samples. We thank our research assistance W. L. Chen and Dr. Chen for their technical advice. We gratefully acknowledge the financial support by the Nation Science Council, No. NSC-88–2212-E-002–025.     

An Intersection Model for Multitolerance Graphs: Efficient Algorithms and Hierarchy

Tolerance graphs model interval relations in such a way that intervals can tolerate a certain degree of overlap without being in conflict. This class of graphs has attracted many research efforts, mainly due to its interesting structure and its numerous applications, especially in DNA sequence analysis and resource allocation, among others. In one of the most natural generalizations of tolerance graphs, namely multitolerance graphs, two tolerances are allowed for each interval—one from the left and one from the right side of the interval. Then, in its interior part, every interval tolerates the intersection with others by an amount that is a convex combination of its two border-tolerances. In the comparison of DNA sequences between different organisms, the natural interpretation of this model lies on the fact that, in some applications, we may want to treat several parts of the genomic sequences differently. That is, we may want to be more tolerant at some parts of the sequences than at others. These two tolerances for every interval—together with their convex hull—define an infinite number of the so called tolerance-intervals, which make the multitolerance model inconvenient to cope with. In this article we introduce the first non-trivial intersection model for multitolerance graphs, given by objects in the 3-dimensional space called trapezoepipeds. Apart from being important on its own, this new intersection model proves to be a powerful tool for designing efficient algorithms. Given a multitolerance graph with n vertices and m edges along with a multitolerance representation, we present algorithms that compute a minimum coloring and a maximum clique in optimal O(nlogn) time, and a maximum weight independent set in O(m+nlogn) time. Moreover, our results imply an optimal O(nlogn) time algorithm for the maximum weight independent set problem on tolerance graphs, thus closing the complexity gap for this problem. Additionally, by exploiting more the new 3D-intersection model, we completely classify multitolerance graphs in the hierarchy of perfect graphs. The resulting hierarchy of classes of perfect graphs is complete, i.e. all inclusions are strict.     

具有单一热源的对流双温PCR微流控装置的研究

为了克服传统连续流动聚合酶链式反应(PCR)微系统经常需要多个加热源来获取PCR所需要的温度,且集成或者非集成的泵系统也需要用来驱动PCR溶液连续流动,从而实现PCR扩增的缺点,研究了一种具有单一热源的对流双温PCR微系统.通过复合肋片技术在沟槽铜片上形成对流双温PCR所需要的温度,由流体密度差形成的自然对流来驱动闭环...