A nanoliter-scale nucleic acid processor with parallel architecture

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.     

Using a microfluidic device for 1 microl DNA microarray hybridization in 500 s

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 microl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA-DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 mul target was used to hybridize with an array that can hold 5000 probes.     

Using a microfluidic device for 1 μl DNA microarray hybridization in 500 s

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 μl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO₂ laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA–DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 μl target was used to hybridize with an array that can hold 5000 probes.     

基于微流控芯片的核酸等温扩增技术研究进展

核酸等温扩增技术是一种在恒温体系内对核酸进行高效扩增的分子扩增技术,它能够在短时间内实现目的基因的指数增长.微流控芯片(microfluidic chip)技术是把研究样品制备、核酸富集、纯化和检测等多个操作步骤集成到一块“微型化”的芯片上,经自动化处理,得出实验结果,即“样品进,结果出”.将核酸等温扩增技术与微流控芯...     

High throughput gene expression measurement with real time PCR in a microfluidic dynamic array

We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays.     

A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.     

Design and Validation of DNA Libraries for Multiplexing Proximity Ligation Assays

Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.     

基于微流控芯片的细胞-细菌相互作用光电检测技术

细胞/细菌及其相互作用研究对于生命科学、药物研发、医学诊疗等领域的研究具有重要意义。微流控芯片分析技术因微环境可控、生物相容性好、检测并行性、微型化等特性，正发展成为细胞/细菌及其相互作用研究的高效手段。本文在简要介绍基于微流控芯片分析技术的细胞-细菌分析方法和技术基础之上，对微流控芯片上细胞-细菌相互作用模型的建立进行了讨论，重点针对细胞-细菌及其相互作用过程的芯片检测进行了综述，尤其对芯片集成的光电检测技术及其测试效果进行总结和比较。通过芯片集成微流体控制、多种光电传感监测模块，使微流控芯片分析技术成为细胞/细菌及其相互作用过程分析和检测的支撑平台和优势手段。最后，对微流控光电检测技术在细胞-细菌相互作用检测中面临的挑战及发展趋势进行了讨论和展望。     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

Multiplexed Biomolecular Detection Based on Single Nanoparticles Immobilized on Pneumatically Controlled Microfluidic Chip

Plasmonics - A microfluidic chip integrated with pneumatically controlled valves was developed for multiplexed biomolecular detection via localized surface plasmonic resonance (LSPR) of single gold...     

基于微流控技术的微生物细胞梯度稀释分离方法

随着微流控分析技术的快速发展,集成化的微流控芯片在满足实验高通量的同时,还在微生物细胞分离领域呈现出独特的优势。本研究基于微流控技术,制备了以聚二甲基硅氧烷(PDMS)、玻片为材料的细菌细胞梯度稀释分离芯片。该芯片的核心是通过一系列复杂的梯度网络来实现对细菌悬液的连续稀释,最终被分离的细菌细胞进入通道末端的存储孔内。结果显示,该方法能分离出的最少细菌细胞数低于10个。此芯片平台操作简单、耗时短、成本低,为微生物单细胞研究提供了新的途径。     

Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus

The genotyping of hepatitis B virus (HBV) has become recently a valuable tool not only for epidemiological reasons but also for the clinical practice. Conventional methods for HBV genotyping typically include amplification of the target DNA sequences with a two-round nested PCR followed by separation of the amplified fragments by gel electrophoresis. A microfluidic chip that couples isotachophoresis (ITP) preconcentration and zone electrophoresis (ZE) separation may provide great advantages for sensitive, rapid and cost-effective clinical analysis. In this study, an HBV genotyping method with only one amplification round was developed by the application of the ITP-ZE chip. All the analysis steps of the ITP-ZE separation including sample injection, stacking and separation were performed continuously, controlled by sequential high-voltage switching. A 2.1cm sample plug was preconcentrated between discontinuous buffers in ITP process, followed by ZE separation. Sensitivity enhancement was obtained through the increase of sample loading volume. The average LOD value of the ITP-ZE microfluidic chip was determined to be 0.0021pg/muL. In a large-scale HBV genotyping test, single round PCR products were analyzed by ITP-ZE microfluidic chip, and the results were compared with that of the conventional method. Among the 200 cases studied, the classification rate obtained with microfluidic chip was 93%, which was 6% higher than that obtained with the conventional method. Method with ITP-ZE chip analysis provides HBV genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional method. This method holds great potential for extrapolation to the abundance of similar molecular biology-based techniques in clinical diagnosis.     

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.     

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.     

Integration of microfluidics with a four-channel integrated optical Young interferometer immunosensor

This report describes an optical sensing hybrid system obtained by bonding a microfluidic system to an integrated optical (IO) four-channel Young interferometer (YI) chip. The microfluidic system implemented into a glass plate consists of four microchannels with cross-sectional dimensions of 200 microm x 15 microm. The microfluidic system is structured in such a way that after bonding to the IO chip, each microchannel addresses one sensing window in the four-channel YI sensor. Experimental tests show that the implementation of the microfluidics reduces the response time of the sensor from 100s, as achieved with a bulky cuvette, to 4s. Monitoring the anti-human serum albumine/human serum albumine (alpha-HSA/HSA) immunoreaction demonstrates the feasibility to use the microfluidic sensing system for immunosensing applications. In this case, a better discrimination between the bulk refractive index change and the layer formation can be made, resulting into higher accuracy and offering the prospect of being able to use the kinetics of the immunoreaction. The microfluidic sensing system shows an average phase resolution of 7 x 10(-5) x 2pi for different pairs of channels, which at the given interaction length of 4 mm corresponds to a refractive index resolution of 6 x 10(-8), being equivalent to a protein mass coverage resolution of 20 fg/mm2.     

A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples

Our POC (Point of Care) device is intended to be a diagnostic tool for routine use in the clinical sector. The validation of the whole procedure, including bacterial genomic DNA isolation and the Real Time detection of Salmonella spp., was conducted on 29 clinical stool samples that had been diagnosed with Salmonella spp. by a routine culture technique. The entire process was achieved in a single microfluidic chip within 35 min. In comparison to the culture reference method that is used in the clinical laboratories, this new device performed well in regards to the analytical parameters of sensitivity, specificity and accuracy. Therefore, the POC device reported in this study proved to be very appropriate for the fully integrated analysis system. To the best of our knowledge, this is the first work to report the sample preparation and followed by Real Time PCR (Polymerase Chain Reaction) on a single 2.5 μl chamber chip for the detection of Salmonella spp. bacteria in stool samples.     

Microfluidic components and bio-reactors for miniaturized bio-chip applications

In this paper miniaturized disposable micro/nanofluidic components applicable to bio chip, chemical analyzer and biomedical monitoring system, such as blood analysis, micro dosing system and cell experiment, etc are reported. This system includes various microfluidic components including a micropump, micromixer, DNA purification chip and single-cell assay chip. For low voltage and low power operation, a surface tension-driven micropump is presented, as well as a micromixer, which was implemented using MEMS technology, for efficient liquid mixing is also introduced. As bio-reactors, DNA purification and single-cell assay devices, for the extraction of pure DNA from liquid mixture or blood and for cellular engineering or high-throughput screening, respectively, are presented.     

High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode

A novel enzyme-linked DNA hybridization assay on an interdigitated array (IDA) microelectrode integrated into a microfluidic channel is demonstrated with sub-nM detection limit. To improve the detection limit as compared to conventional electrochemical biosensors, a recyclable redox product, 4-aminophenol (PAP) is used with an IDA microelectrode. The IDA has a modest and easily fabricated inter-digit spacing of 10 μm, yet we were able to demonstrate 97% recycling efficiency of PAP due to the integration in a microfluidic channel. With a 70 nL sample volume, the characterized detection limit for PAP of 1.0 × 10?1? M is achieved, with a linear dynamic range that extends from 1.0 × 10?? to 1.0 × 10?? M. This detection limit, which is the lowest reported detection limit for PAP, is due to the increased sensitivity provided by the sample confinement in the microfluidic channel, as well as the increased repeatability due to perfectly static flow in the microchannel and an additional anti-fouling step in the protocol. DNA sequence detection is achieved through a hybridization sandwich of an immobilized complementary probe, the target DNA sequence, and a second complementary probe labeled with β-galactosidase (β-GAL); the β-GAL converts its substrate, 4-aminophenyl-d-galactopyranoside (PAPG), into PAP. In this report we present the lowest reported observed detection limit (1.0 × 10?1? M) for an enzyme-linked DNA hybridization assay using an IDA microelectrode and a redox signaling paradigm. Thus, we have demonstrated highly sensitive detection of a targeted DNA sequence using a low-cost easily fabricated electrochemical biosensor integrated into a microfluidic channel.     

Peptide nucleic acid-modified carbon nanotube field-effect transistor for ultra-sensitive real-time detection of DNA hybridization

We have developed a nanosensor array composed of carbon nanotube field-effect transistors (CNTFETs) on SiO₂/Si substrates. Unlike previously reported CNTFETs where the recognition event occurred directly on the CNT, in this case, the reverse surface of the substrate was utilized for the biomolecular functionalization. A self-assembled monolayer (SAM) of peptide nucleic acid (PNA) probes associated with the tumor necrosis factor-α gene (TNF-α) was attached onto the gold electrode on the reverse side of the CNTFET device. A time-dependent conductance increase was monitored upon sequential introduction of wild-type (WT) DNA samples through a microfluidic channel of the poly(dimethylsiloxane) (PDMS) chip. High selectivity of PNA probes only toward the full-complementary WT DNA samples enabled rapid and simple discrimination against single-nucleotide polymorphism (SNP) or non-complementary (NC) DNA. Concentration-dependent measurements indicated a limit-of-detection (LOD) of 6.8 fM WT DNA. Our CNTFET-based biochip is a promising candidate for the development of an integrated, high-throughput, portable device for nucleic acid-based diagnostics.     

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.