Graphene Oxide Nanosheets Modified with Single‐Domain Antibodies for Rapid and Efficient Capture of Cells

Peripheral blood can provide valuable information on an individual’s immune status. Cell‐based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells. 1 Current methods to classify leukocytes, such as recovery on antibody‐coated beads or fluorescence‐activated cell sorting require long sample preparation times and relatively large sample volumes. 2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single‐domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (～30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.     

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N) 3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Sample pretreatment microfluidic chip for DNA extraction from rat peripheral blood

A sample pretreatment microfluidic chip was described based on the principle of solid phase extraction and micro electro mechanical system technology. Oxidized porous silicon with the large surface area as the solid phase matrix for absorption of DNA from a biological sample can greatly improve the DNA yield. The factors that could affect the DNA yield were analyzed and the preparation technology and the experiment procedure were improved. The DNA purification process from the rat peripheral blood can be achieved and the DNA yield is 24 ng/(μL whole blood), which can reach the level of the commercial DNA purification kits. Furthermore, the DNA extracted from the whole blood can be amplified by polymerase chain reaction, which can achieve a high efficiency of the amplification. Translated from Chemical Journal of Chinese Universities, 2006, 27(4) (in Chinese)     

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on‐demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary‐ and chip‐based methods offering low consumable costs (<0.1$) circumventing cost‐intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2–4 μL in volume, a semi‐contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non‐contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR‐grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi‐circular cross section of adaptable width (∼200–600 μm) and height (∼30–80 μm) as well as a typical length of 15–55 mm. Layout of such liquid structures is adaptable on‐demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label‐free DNA ladder (100–1000 bp) at 100 V/cm via in‐line staining and laser induced fluorescent end‐point detection using an automated prototype.     

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating‐Tumor‐Cell Isolation

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.     

用于提取外周血DNA微流控样品预处理芯片的研制

基于固相萃取原理和微电子机械系统(Micro-Electro-Mechanical System, MEMS)技术研制了一种多孔氧化硅微流控样品预处理芯片, 并利用具有大比表面积的多孔氧化硅作为提取DNA的固相载体, 从而大大提高了DNA的提取产率. 分析了影响DNA提取产率的因素, 改进了芯片制备工艺和DNA提取实验方案, 成功地提取了小鼠外周血DNA, 提取产率为24 ng/(μL全血), 达到商用试剂盒水平. 同时以该DNA作为PCR扩增模板, 扩增效果良好.     

Immuno-capture and in situ detection of Salmonella typhimurium on a novel microfluidic chip

The new method presented in this article achieved the goal of capturing Salmonella typhimurium via immunoreaction and rapid in situ detection of the CdSe/ZnS quantum dots (QDs) labeled S. typhimurium by self-assembly light-emitting diode-induced fluorescence detection (LIF) microsystem on a specially designed multichannel microfluidic chip. CdSe/ZnS QDs were used as fluorescent markers improving detection sensitivity. The microfluidic chip developed in this study was composed of 12 sample channels, 3 mixing zones, and 6 immune reaction zones, which also acted as fluorescence detection zones. QDs–IgG–primary antibody complexes were generated by mixing CdSe/ZnS QDs conjugated secondary antibody (QDs–IgG) and S. typhimurium antibody (primary antibody) in mixing zones. Then, the complexes went into immune reaction zones to label previously captured S. typhimurium in the sandwich mode. The capture rate of S. typhimurium in each detection zone was up to 70%. The enriched QDs-labeled S. typhimurium was detected using a self-assembly LIF microsystem. A good linear relationship was obtained in the range from 3.7 × 10 to 3.7 × 10⁵ cfu mL⁻¹ using the equation I = 0.1739 log (C) − 0.1889 with R² = 0.9907, and the detection limit was down to 37 cfu mL⁻¹. The proposed method of online immunolabeling with QDs for in situ fluorescence detection on the designed multichannel microfluidic chip had been successfully used to detect S. typhimurium in pork sample, and it has shown potential advantages in practice.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.     

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF‐7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF‐7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.     

Modular fluidic resistors to enable widely tunable flow rate and fluidic switching period in a microfluidic oscillator

Microfluidic systems with modular components are attractive alternatives to monolithically integrated microfluidic systems because of their flexibility. In this study, we apply the modular concept on a water‐head‐pressure‐driven microfluidic oscillator and obtain a widely tunable flow rate and fluidic switching period. Modular fluidic resistors can be easily mounted onto and demounted from a main chip by means of plastic male connectors. The connectors enable a leak‐free connection between the modular resistors and main chip (leakage pressure > 140 kPa). With modular resistors, we show independent control of the flow rate and flow switching period of the oscillator system in a wide range (2.5 s–6.4 h and 2 μL/min–2 mL/min). This modular approach can be used to enhance the flexibility of instruction‐embedded microfluidic circuits in which their operational range is limited.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

Trapping and releasing of single microparticles and cells in a microfluidic chip

A microfluidic device was designed and fabricated to capture single microparticles and cells by using hydrodynamic force and selectively release the microparticles and cells of interest via negative dielectrophoresis by activating selected individual microelectrodes. The trap microstructure was optimized based on numerical simulation of the electric field as well as the flow field. The capture and selective release functions of the device were verified by multi-types microparticles with different diameters and K562 cells. The capture efficiencies/release efficiencies were 95.55% ± 0.43%/96.41% ± 1.08% and 91.34% ± 0.01%/93.67% ± 0.36% for microparticles and cells, respectively. By including more traps and microelectrodes, the device can achieve high throughput and realize the visual separation of microparticles/cells of interest in a large number of particle/cell groups.     

Fabrication of microfluidic chip using micro‐hot embossing with micro electrical discharge machining mold

This study develops an improved method for generating aluminum mold inserts used in the replication of polymer‐based microfluidic chip. Since molding masters that are suitable for microfluidic chip replication must have features whose dimensions are of the order of tens to hundreds of microns, micro electrical discharge machining is employed herein to fabricate an aluminum mold insert of a microfluidic chip. The width and depth of the aluminum mold insert for the microfluidic chip are 61.50 and 49.61 µm, respectively. The surface roughness values of the microchannel and the sample reservoir in aluminum mold insert for the microfluidic chip are 53.9 and 34.3 nm, respectively. PMMA material is adopted as the molded microfluidic chip that is produced by micro‐hot embossing molding. The PMMA material can replicate the microchannel and sample reservoir very well when the aluminum mold insert is used in micro‐hot embossing molding. The results indicate that the most important parameter in the replication of molded microfluidic chip is the embossing pressure, which is also the most important parameter in determining the surface roughness of the molded microfluidic chip. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.