Bioinspired Engineering of a Multivalent Aptamer‐Functionalized Nanointerface to Enhance the Capture and Release of Circulating Tumor Cells

Circulating tumor cell (CTC)‐enrichment by using aptamers has a number of advantages, but the issue of compromised binding affinities and stabilities in real samples hinders its wide applications. Inspired by the high efficiency of the prey mechanism of the octopus, we engineered a deterministic lateral displacement (DLD)‐patterned microfluidic chip modified with multivalent aptamer‐functionalized nanospheres (AP‐Octopus‐Chip) to enhance capture efficiency. The multivalent aptamer–antigen binding efficiency improves 100‐fold and the capture efficiency is enhanced more than 300 % compared with a monovalent aptamer‐modified chip. Moreover, the captured cancer cells can be released through a thiol exchange reaction with up to 80 % efficiency and 96 % viability, which is fully compatible with downstream mutation detection and CTC culture. Using the chip, we were able to find CTCs in all cancer samples analyzed.     

Reconfigurable Bioinspired Framework Nucleic Acid Nanoplatform Dynamically Manipulated in Living Cells for Subcellular Imaging

In nature, the formation of spider silk fibers begins with dimerizing the pH‐sensitive N‐terminal domains of silk proteins (spidroins) upon lowering pH, and provides a natural masterpiece for programmable assembly. Inspired by the similarity of pH‐dependent dimerization behaviors, introduced here is an i‐motif‐guided model to mimic the initial step of spidroin assembly at the subcellular level. A framework nucleic acid (FNA) nanoplatform is designed using two tetrahedral DNA nanostructures (TDNs) with different branched vertexes carrying a bimolecular i‐motif and a split ATP aptamer. Once TDNs enter acidic lysosomes within living cells, they assemble into a heterodimeric architecture, thereby enabling the formation of a larger‐size framework and meanwhile subcellular imaging in response to endogenous ATP, which can be dynamically manipulated by adjusting intracellular pH and ATP levels with external drug stimuli.     

Single‐Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells

DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single‐particle tracking. We found that the TDNs were rapidly internalized by a caveolin‐dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule‐dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA‐nanostructure‐based drug delivery nanocarriers for targeted therapy.     

Oriented Chiral DNA–Silica Film Guided by a Natural Mica Substrate

The formation of highly ordered chiral organic/inorganic films with high density and long‐range orientation is important in constructing chiral devices, such as broadband polarization devices, liquid‐crystal displays, or negative‐reflection materials. A feasible strategy is presented to fabricate three‐dimensional mesostructured chiral DNA–silica assemblies into large‐scale oriented arrangements. The highly ordered film was aligned by a mica crystal substrate with the bridging effect of suitable divalent metal ions, followed by the growth of the DNA–silica composite by bottom‐up assembly with a “quartet templating” method. This simple and effective route would perform well in the alignment and arrangement of highly charged biomolecules, such as polypeptides, proteins, viruses, and their inorganic assemblies, and furthermore could allow the fabrication of chiral optical materials with long‐range ordering.     

Microfluidic chip fabrication using hot embossing and thermal bonding of COP

The application of silicon mold inserts by micro‐hot embossing molding has been explored in microfluidic chip fabrication. For the mold insert, this study employed an SU‐8 photoresist to coat the silicon wafer. Ultraviolet light was then used to expose the pattern on the SU‐8 photoresist surface. This study replicates the microstructure of the silicon mold insert by micro‐hot embossing molding. Different processing parameters (embossing temperature, embossing pressure, embossing time, and de‐molding temperature) for the cycle‐olefin polymer (COP) film of microfluidic chips are evaluated. The results showed that the most important parameter for replication of molded microfluidic chip is embossing temperature. De‐molding temperature is the most important parameter for surface roughness of the molded microfluidic chip. The microchannel is bonded with a cover by thermal bonding processing to form the sealed microfluidic chip. The bonding temperature is the most important factor in the bonding strength of the sealed microfluidic chip. Copyright © 2009 John Wiley & Sons, Ltd.     

Microfluidics: Applications for analytical purposes in chemistry and biochemistry

In this review, we present recent advancements and novel developments in fluidic systems for applied analytical purposes in chemistry, biochemistry, and life science in general that employ and reflect the full benefits of microfluidics. A staggering rise in publications related to integrated, all‐in‐one microfluidic chips capable of separation, reaction, and detection have been observed, all of which realise the principal of micro total analysis systems or lab‐on‐a‐chip. These integrated chips actively adopt the scaling law concepts, utilising the highly developed fabrication techniques. Their aim is to multi‐functionalise and fully automate devices believed to assist the future advancements of point‐of‐care, clinical, and medical diagnostics.     

微流控层流技术的研究

基于微型通道自身的层流特点而发展起来的多相层流技术,从最初的液-液微萃取开始,由于其结构加工简单、操作方便和分析功能强大,已逐渐发展成为一种加工分析方法,为微流控分析的研究应用打开了一个崭新的局面。本文概述了层流的基本原理,总结了近10年来在这方面的研究,包括层流界面间的分子扩散、转移现象和化学反应,以及层流刻蚀加工技术及其在制备纳米材料和在生命医学方面的应用。具体介绍了应用层流技术进行微芯片的加工制作,微型反应器的制备,离子、分子的分离分析,聚合物薄膜的形成和应用,微通道内有机合成反应的控制,溶液的浓度梯度控制以及在免疫检测中的应用,对细胞、生物大分子的操作控制,以及对生物试剂的预处理分析等。     

微流控层流技术的研究

基于微型通道自身的层流特点而发展起来的多相层流技术,从最初的液-液微萃取开始,由于其结构加工简单、操作方便和分析功能强大,已逐渐发展成为一种加工分析方法,为微流控分析的研究应用打开了一个崭新的局面。本文概述了层流的基本原理,总结了近10年来在这方面的研究,包括层流界面间的分子扩散、转移现象和化学反应,以及层流刻蚀加工技术及其在制备纳米材料和在生命医学方面的应用。具体介绍了应用层流技术进行微芯片的加工制作,微型反应器的制备,离子、分子的分离分析,聚合物薄膜的形成和应用,微通道内有机合成反应的控制,溶液的浓度梯度控制以及在免疫检测中的应用,对细胞、生物大分子的操作控制,以及对生物试剂的预处理分析等。     

CE chips fabricated by injection molding and polyethylene/thermoplastic elastomer film packaging methods

This study presents a new packaging method using a polyethylene/thermoplastic elastomer (PE/TPE) film to seal an injection-molded CE chip made of either poly(methyl methacrylate) (PMMA) or polycarbonate (PC) materials. The packaging is performed at atmospheric pressure and at room temperature, which is a fast, easy, and reliable bonding method to form a sealed CE chip for chemical analysis and biomedical applications. The fabrication of PMMA and PC microfluidic channels is accomplished by using an injection-molding process, which could be mass-produced for commercial applications. In addition to microfluidic CE channels, 3-D reservoirs for storing biosamples, and CE buffers are also formed during this injection-molding process. With this approach, a commercial CE chip can be of low cost and disposable. Finally, the functionality of the mass-produced CE chip is demonstrated through its successful separation of phiX174 DNA/HaeIII markers. Experimental data show that the S/N for the CE chips using the PE/TPE film has a value of 5.34, when utilizing DNA markers with a concentration of 2 ng/microL and a CE buffer of 2% hydroxypropyl-methylcellulose (HPMC) in Tris-borate-EDTA (TBE) with 1% YO-PRO-1 fluorescent dye. Thus, the detection limit of the developed chips is improved. Lastly, the developed CE chips are used for the separation and detection of PCR products. A mixture of an amplified antibiotic gene for Streptococcus pneumoniae and phiX174 DNA/HaeIII markers was successfully separated and detected by using the proposed CE chips. Experimental data show that these DNA samples were separated within 2 min. The study proposed a promising method for the development of mass-produced CE chips.     

水凝胶微流控芯片的快速加工及在细胞培养检测中的应用

采用具有紫外光聚合性能的聚乙二醇(PEG)基水凝胶材料, 通过紫外光聚合作用快速加工双层水凝胶微流控芯片, 并验证了其对肿瘤细胞代谢液进行检测的可行性. 与传统微流控芯片材料相比, 该水凝胶芯片材料具有更好的生物相容性及可操控性, 可直接加工成形, 在生物学领域特别是细胞培养过程控制方面具有良好的应用前景. 实验结果表明, 该水凝胶微流控芯片可在微尺度空间有效模拟细胞生长环境, 并实现对细胞连续捕获后的原位培养. 将该芯片与卟啉可视阵列传感器系统结合, 经代谢特征分析可有效区分不同种类肿瘤细胞, 实现芯片细胞培养平台上的细胞代谢指纹快速可视化传感检测.     

Fabrication, modification, and application of poly(methyl methacrylate) microfluidic chips

Poly(methyl methacrylate) (PMMA) is particularly useful for microfluidic chips with the features of low price, excellent optic transparency, attractive mechanical and chemical properties, ease of fabrication and modification, biocompatibility, etc. During the past decade, significant progress in the PMMA microfluidic chips has occurred. This review, which contains 120 references, summarizes the recent advances and the key strategies in the fabrication, modification, and application of PMMA microfluidic chips. It is expected that PMMA microchips should find a wide range of applications and will lead to the creation of truly disposable microfluidic devices.     

Programmable Engineering of a Biosensing Interface with Tetrahedral DNA Nanostructures for Ultrasensitive DNA Detection

Self‐assembled DNA nanostructures with precise sizes allow a programmable “soft lithography” approach to engineer the interface of electrochemical DNA sensors. By using millimeter‐sized gold electrodes modified with several types of tetrahedral DNA nanostructures (TDNs) of different sizes, both the kinetics and thermodynamics of DNA hybridization were profoundly affected. Because each DNA probe is anchored on an individual TDN, its lateral spacing and interactions are finely tuned by the TDN size. By simply varying the size of the TDNs, the hybridization time was decreased and the hybridization efficiency was increased. More significantly, the detection limit for DNA detection was tuned over four orders of magnitude with differentially nanostructured electrodes, and achieved attomolar sensitivity with polymeric enzyme amplification.     

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody‐based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF‐7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF‐7‐containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.     

Review: Aptamers in microfluidic chips

This review, covering reports published from 2002 to August 2010, shows how aptamers have made significant contributions in the improvements of microfluidic chips for affinity extraction, separations and detections. Furthermore, microfluidic chip methods for studying aptamer-target interactions and performing aptamer selections have also been summarized. Accordingly, research vacancies and future development trends in these areas are discussed.     

Analytical and numerical study of Joule heating effects on electrokinetically pumped continuous flow PCR chips

Joule heating is an inevitable phenomenon for microfluidic chips involving electrokinetic pumping, and it becomes a more important issue when chips are made of polymeric materials because of their low thermal conductivities. Therefore, it is very important to develop methods for evaluating Joule heating effects in microfluidic chips in a relatively easy manner. To this end, two analytical models have been established and solved using the Green's function for evaluating Joule heating effects on the temperature distribution in a microfluidic-based PCR chip. The first simplified model focuses on the understanding of Joule heating effects by ignoring the influences of the boundary conditions. The second model aims to consider practical experimental conditions. The analytical solutions to the two models are particularly useful in providing guidance for microfluidic chip design and operation prior to expensive chip fabrication and characterization. To validate the analytical solutions, a 3-D numerical model has also been developed and the simultaneous solution to this model allows the temperature distribution in a microfluidic PCR chip to be obtained, which is used to compare with the analytical results. The developed numerical model has been applied for parametric studies of Joule heating effects on the temperature control of microfluidic chips.     

基于单螺旋微流控芯片的细菌气溶胶的高效富集

设计了一种单螺旋通道的聚二甲基硅氧烷(Poly(dimethylsiloxane),PDMS)微流控芯片,用于副溶血性弧菌气溶胶的快速有效富集。该芯片的特征在于其通道呈螺旋分布,且通道内部含有均匀分布的鱼骨形结构。结果表明,在不同富集时间段内,采用该芯片方法捕获的细菌总数均远高于传统落板法。对于传统落板法无法有效捕获的低浓度样本(10~4CFU/mL)的缺陷,该方法的优势在于:芯片内部的螺旋通道可增大对气溶胶中微生物的离心力;鱼骨形结构的设计增加了待测样品与芯片内壁间的接触几率。此外,以无鱼骨形的螺旋芯片作为对照,验证了鱼骨形结构对于高效富集的意义。此芯片设计巧妙、易于制备、高效便携、富集效果较好,在气溶胶污染严重的水产加工等场所具有较大的应用前景。     

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(?) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.     

Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining

In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).     

Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.     

Chip integrated strategies for acoustic separation and manipulation of cells and particles

Acoustic standing wave technology combined with microtechnology opens up new areas for the development of advanced particle and cell separating microfluidic systems. This tutorial review outlines the fundamental work performed on continuous flow acoustic standing wave separation of particles in macro scale systems. The transition to the microchip format is further surveyed, where both fabrication and design issues are discussed. The acoustic technology offers attractive features, such as reasonable throughput and ability to separate particles in a size domain of about tenths of micrometers to tens of micrometers. Examples of different particle separation modes enabled in microfluidic chips, utilizing standing wave technology, are described along a discussion of several potential applications in life science research and in the medical clinic. Chip integrated acoustic standing wave separation technology is still in its infancy and it can be anticipated that new laboratory standards very well may emerge from the current research.