Low-cost polymer microfluidic device for on-chip extraction of bacterial DNA

A polymer microfluidic device for on-chip extraction of bacterial DNA has been developed for molecular diagnostics. In order to manufacture a low-cost, disposable microchip, micropillar arrays of high surface-to-volume ratio (0.152 μm⁻¹) were constructed on polymethyl methacrylate (PMMA) by hot embossing with an electroformed Ni mold, and their surface was modified with SiO₂ and an organosilane compound in subsequent steps. To seal open microchannels, the organosilane layer on top plane of the micropillars was selectively removed through photocatalytic oxidation via TiO₂/UV treatment at room temperature. As a result, the underlying SiO₂ surface was exposed without deteriorating the organosilane layer coated on lateral surface of the micropillars that could serve as bacterial cell adhesion moiety. Afterwards, a plasma-treated PDMS substrate was bonded to the exposed SiO₂ surface, completing the device fabrication. To optimize manufacturing throughput and process integration, the whole fabrication process was performed at 6 inch wafer-level including polymer imprinting, organosilane coating, and bonding. Preparation of bacterial DNA was carried out with the fabricated PDMS/PMMA chip according to the following procedure: bacterial cell capture, washing, in situ lysis, and DNA elution. The polymer-based microchip presented here demonstrated similar performance to Glass/Si chip in terms of bacterial cell capture efficiency and polymerase chain reaction (PCR) compatibility.     

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.     

A completely automated sample preparation instrument and consumable device for isolation and purification of nucleic acids

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.     

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.     

Development of the automated gold-linked electrochemical immunoassay system for blood monitoring

This paper reports the development of fully automated miniaturized immunoassay system. The system consist of postage stamp sized microchip and compact (post card sized foot print) microchip driver. To realize easy sample loading into the microchip, surface modification of polydimethylsiloxane (PDMS) was developed, and life time of the modified surface up to 9 days is confirmed. The microchip just consumes a droplet of blood (2 μl) and the loading and metering of the sample is realized by capillary action, therefore the microchip is compatible with blood collection method by using lancet needle. Fully automated immunoassay protocol in the system is demonstrated within 15 min using whole blood sample. Finally, fully automated detection of antigen (insulin) was successfully demonstrated in the developed system.     

基于纳米磁珠技术的新型微全分析DNA芯片的研究

在微全分析系统的研究中,样品提取及DNA分析技术是非常重要的一个环节.也是目前国内外研究的热点之一.文中介绍了一种新型的基于单芯片的样品制备和扩增方法.采用多层微加工技术制作SU-8模具,通过注模成型,制作出有立体微柱结构的PDMS(聚二甲基硅氧烷)芯片,在芯片微池内填充超顺磁性磁珠,利用固相提取(solid phase extraction,SPE)法,将细胞裂解、DNA提取、PCR反应等功能集成在一个PDMS芯片上.整个流程快速有效,操作简便且易于芯片系统集成,提取产物可以不必洗脱,直接作为下一步PCR反应的模板,在同一芯片上进行扩增反应,实现了样品预处理、DNA提取和PCR扩增的集成.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

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.     

On-chip whole blood plasma separator based on microfiltration,sedimentation and wetting contrast

Miniaturized on-chip blood separators have a great value for point-of-care diagnosis. In our work, a combined design strategy—microfiltration, sedimentation in a retarded flow, and wetting contrast—was taken to overcome the known limitations of on-chip blood separators. Our microfluidic chip consists of a polydimethylsiloxane micropillar array and an etched glass with microchannel branches. The red blood cells are significantly slowed and gradually settled down due to micropillars and enlarged dimension of a chamber. An etched glass microchannel allows the extraction of blood plasma exclusively due to the capillary effect. The fabricated microfluidic device can separate blood plasma from a whole blood sample without any external driving force or dilution. The measured plasma separation efficiency was close to 100 % from human whole blood. Autonomous on-chip separation and collection of blood plasma was demonstrated.     

Fabrication of DNA purification microchip integrated with mesoporous matrix based on MEMS technology

This paper presents a novel microfabricated DNA purification microfluidic chip with enhanced performance based on the principle of micro solid phase extraction. The microchip comprises a layer of mesoporous material as solid phase matrix which is fabricated on the internal wall of the channel of microfluidic chip by electrochemical etching Si in an electrolyte. The conditions of electrochemical etching and porosity of the mesoporous matrix have been investigated. The properties of mesoporous matrix have been characterized by scanning electron microscopy and by BET (Brunauer, Emmet, and Teller) nitrogen adsorption analysis. The pore size of the mesoporous matrix is in the range of 10–30 nm, and the surface area is about 300 m²/g. Compared with the microfluidic chips with micropillar array matrix or non-porous matrix, the microchip with mesoporous matrix is able to extract enough polymerase chain reaction-amplifiable DNA from cultures of rat mesenchymal stem cells in 20 min. This highly efficient, effortless, and flexible technology can be used as a lab-on-a-chip component for initial biologic sample preparation.     

Diamagnetic capture mode magnetophoretic microseparator for blood cells

This paper presents the characterization of a continuous diamagnetic capture (DMC) mode magnetophoretic microseparator for separating red and white blood cells from diluted whole blood based on their native magnetic properties. The DMC microseparator separated the blood cells using a high-gradient magnetic separation (HGMS) method without the use of additives such as magnetic beads. The microseparator was fabricated using microfabrication technology, enabling the integration of microscale magnetic flux concentrators in an aqueous microenvironment. Experimental results show that the DMC microseparator can continuously separate out 89.7% of red blood cells (RBCs) from diluted whole blood within 5 min using an external magnetic flux of 0.2 T from a permanent magnet. Monitoring white blood cells (WBCs) probed with a fluorescence dye show that 72.7% of WBCs were separated out within 10 min in the DMC microseparator using a 0.2 T external applied magnetic flux. Consequently, the DMC microseparator may facilitate the separation of WBCs from whole blood in applications such as a genetic sample preparation and blood borne disease detection. [1574].     

基于微球和微柱阵列芯片的乳滴数字PCR定量方法

BEAMing是一种基于磁珠表面核酸扩增的乳滴数字聚合酶链反应(PCR)技术,具有很高的灵敏度,然而后续检测目标磁珠比较困难.通过修饰链霉亲和素的聚苯乙烯微球捕获BEAMing实验中生物素修饰的目标磁珠并利用微柱阵列芯片拦截微球,可以达到统计磁珠的目的.微柱阵列芯片采用基于尺寸差异的拦截原理.该芯片组装简单成本低,降低了BEAMing技术的磁珠统计难度.利用该方法对不同浓度的特定DNA序列做了检测,验证了该方法的实用性.     

Sample Acquisition and Control On-Chip Microfluidic Sample Preparation

Microfluidic flow conditions allow the design of highly effective, yet simple devices for on-chip sample preparation and cleanup. Over the past few years, Micronics has developed a number of novel microfluidic structures that are compatible with complex samples such as whole blood or contaminated environmental fluids. The H-Filter is a technology based on the parallel laminar flow of two or more miscible streams in contact with each other. Such streams do not mix, but chemicals contained in these streams can diffuse from one stream into the other, with smaller molecules diffusing faster than larger ones. This principle can be used, for example, to remove salt from a solution containing DNA, or to extract smaller molecules from whole blood. The T-Sensor is based on the same laminar flow diffusion principle, but combines sample preparation with self-calibration and detection. These devices can be used not only in stand-alone research and point-of-use testing applications, but they can also be integrated, as sample preparation modules, into existing laboratory systems.     

Thread-based microfluidic system for detection of rapid blood urea nitrogen in whole blood

This study develops a thread-based microfluidic device with variable volume injection capability and 3-dimensional (3D) detection electrodes for capillary electrophoresis electrochemical (CE–EC) detection of blood urea nitrogen (BUN) in whole blood. A poly methyl methacrylate (PMMA) substrate with concave 3D electrodes produced by the hot embossing method is used to enhance the sensing performance of the CE–EC system. Results show that the chip with 3D sensing electrodes exhibits a measured current response nine times higher and signal-to-noise ratio five times higher when compared to the peak responses obtained using a chip with conventional 2D sensing electrodes. In addition, the developed thread-based microfluidic system is capable of injecting variable sample volumes into the separation thread simply by wrapping the injection thread different numbers of times around the separation thread. The peak S/N ratio can be further enhanced with this simple approach. Results also indicate that the CE–EC system exhibits good linear dynamic range for detecting a urea sample in concentrations from 0.1 to 10.0 mM (R ² = 0.9848), which is suitable for adoption in detecting the BUN concentration in human blood (1.78–7.12 mM). Separation and detection of the ammonia ions converted from BUN in whole blood is successfully demonstrated in the present study, with the developed thread-based microfluidic system providing a low-cost, high-performance method for detecting BUN in human blood.     

Integratable non-clogging microconcentrator based on counter-flow principle for continuous enrichment of CaSki cells sample

This study addresses the need to reduce the risk of clogging when preparing samples for cell concentration, i.e., the CaSki Cell-lines (epidermoid cervical carcinoma cells). Aiming to develop a non-clogging microconcentrator, we proposed a new counter-flow concentration unit characterized by the directions of penetrating flows being at an obtuse angle to the main flow, due to employment of streamlined turbine blade-like micropillars. Based on the optimization results of the counter-flow unit profile, a fractal arrangement for the counter-flow concentration unit was developed. A counter-flow microconcentrator chip was then designed and fabricated, with both the processing layer and collecting layer arranged in terms of the honeycomb structure. Visualized experiments using CaSki cell samples on the microconcentrator chip demonstrated that no cell-clogging phenomena occurred during the test and that no cells were found in the final filtrate. The test results show an excellent concentration performance for the microconcentrator chip, while a concentrating ratio of >4 with the flow rate being below 1.0 ml/min. As only geometrical structure is employed in the passive device, the counter-flow microconcentrator can be easily integrated into advanced microfluidic systems. Owing to the merit of non-clogging and continuous processing ability, the counter-flow microconcentrator is not only suitable for the sample preparation within biomedical field, but also applicable in water-particle separation.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

On-chip HA/SSCP for the detection of hereditary haemochromatosis

Microfluidics provides a promising tool for meeting the growing demand for high-throughput and low-cost mutation detection technology. With conventional instrumentation, this need is often addressed by the combination of the single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) methods. This paper describes an effective microchip-based method to analyse the three most commonly tested gene mutations (C282Y, H63D, and S65C) associated with hereditary haemochromatosis by simultaneously performing microchip-based SSCP and HA, directly upon samples of polymerase chain reaction (PCR) product. We have increased the sensitivity of mutation detection considerably by adapting and combining SSCP with HA. We are able to perform the analysis within several minutes by avoiding off-chip sample preparation steps for SSCP and HA (apart from the PCR). The most important mutation in the screening of populations for this disease is the C282Y mutation and this mutation has not previously been detected with methods of HA/SSCP suitable for microchip implementation. This is, to the best of our knowledge, the first microchip-based test applying SSCP and HA for all three of the most common HFE mutations.     

Optimized design and fabrication of a microfluidic platform to study single cells and multicellular aggregates in 3D

A microfluidic platform for cell motility analysis in a three-dimensional environment is presented. The microfluidic device is designed to study migration of both single cells and cell spheroids, in particular under spatially and temporally controlled chemical stimuli. A layout based on a central microchannel confined by micropillars and two lateral reservoirs was selected as the most effective. The microfluidics have an internal height of 350 μm to accommodate cell spheroids of a considerable size. The chip is fabricated using well-established micromachining techniques, by obtaining the polydimethylsiloxane replica from a Si/SU-8 master. The chip is then bonded on a 170-μm-thick microscope glass slide to allow high spatial resolution live microscopy. In order to allow the cost-effective and highly repeatable production of chips with high aspect ratio (5:1) micropillars, specific design and fabrication processes were optimized. This design permits spatial confinement of the gel where cells are grown, the creation of a stable gel–liquid interface and the formation of a diffusive gradient of a chemoattractant (>48 h). The chip accomplishes both the tasks of a microfluidic bioreactor system and a cell analysis platform avoiding critical handling of the sample. The experimental fluidic tests confirm the easy handling of the chip and in particular the effectiveness of the micropillars to separate the Matrigel? from the culture media. Experimental tests of (i) the stability of the gradient, (ii) the biocompatibility and (iii) the suitability for microscopy are presented.