Integrated microfluidic system for electrochemical sensing of glycosylated hemoglobin

This article reports a new miniature electrochemical detection system integrating a sample pretreatment device for fast detection of glycosylated hemoglobin (HbA_1C), which is a common indicator for diabetes mellitus. In this system, circular micropumps, normally closed microvalves, dielectrophoretic (DEP) electrodes, and electrochemical sensing electrode are integrated to perform several crucial processes. These processes include separation of red blood cells (RBCs), sample/reagent transportation, mixing, cell lysis, and electrochemical sensing. For the HbA_1C measurement, the RBCs are separated and are collected from whole human blood by using a positive DEP force generated by the DEP electrodes. The collected RBCs are then lysed to release HbA_1C for the subsequent electrochemical detection processes. Experimental data show that the RBCs are successfully separated and are collected using the developed system with a RBCs capture rate of 84.2%. The subsequent detection of HbA_1C is automatically completed by utilizing electrochemical sensing electrode. The microfluidic system only consumes a sample volume of 200 μl. The entire process is automatically performed within a short period of time (10 min). The development of this integrated microfluidic system may be promising for the clinical monitoring of diabetes mellitus.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

Microfluidic platforms for discovery and detection of molecular biomarkers

Microfluidics has emerged as a promising platform for discovery and detection of molecular biomarkers recently. With this approach, the discovery of these biomarkers could be more efficient in time and consumes less samples and reagents. Furthermore, the entire discovery process could be automated since all the functional microfluidic devices such as micropumps and microvalves could be integrated on a single chip. Similarly, the detection of the discovered molecular biomarkers is also promising. Detection of nucleic acid biomarkers, protein biomarkers, and metabolite biomarkers has been demonstrated on microfluidic platforms recently. When compared with their large-scale counterparts, the miniature system can perform the detection of these biomarkers within less analysis time while a multiplexed detection scheme could be easily achieved. Furthermore, the entire detection process could be automated on the single chip as well. This review paper is therefore to review the recent development of microfluidic devices and systems for the discovery and detection of the molecular biomarker. Techniques for biomarker discovery, verification, and detection that have been adapted into microfluidics were first reviewed, and their advantages were highlighted. The new approach of biomarker screening based on in vitro-generated affinity reagents such as nucleic acid aptamers and peptide affinity reagents was then reviewed. Finally, in the biomarker detection section, this review placed a special emphasis on commercialized microfluidic-based diagnostics for molecular biomarkers.     

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

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

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

An automatic microfluidic system that continuously performs the systematic evolution of ligands by exponential enrichment

The systematic evolution of ligands by exponential enrichment (SELEX) technique has been extensively used to screen molecule-specific aptamers from combinatorial libraries of synthetic nucleic acids. Aptamers are single-stranded DNA or RNA, which have a high affinity to a large variety of molecules ranging from small drugs or metabolites to cells. Therefore, they have a variety of promising applications such as for diagnostics and targeted therapeutics. In this study, a new microfluidic chip was developed to perform continuous screening of DNA-based aptamers in an automatic format. When compared with the existing manual procedure, the developed microfluidic chip has several advantages including a rapid and efficient screening process, automation, and less consumption of samples/reagents. Experimental data showed that an aptamer specific to alpha-fetoprotein was successfully screened from a random DNA pool. The entire screening process (five continuous, repetitive rounds) can be completed within 6?h, which is much faster than the traditional methods (more than 15?h). An automatic, rapid and efficient SELEX process was performed by this developed microfluidic chip, which may enable a generalized platform for the fast screening of DNA-based biomarkers in the future.     

A micropump using amplified deformation of resilient membranes through oil hydraulics

Toward the development of micropumps that operate under low external air pressures, a new polydimethylsiloxane (PDMS), pneumatic micropump using amplified deformation of resilient PDMS membranes through oil hydraulics was presented in this study. The new micropump employed oil-hydraulic chambers with pre-filled mineral oil to amplify the deformation of flexible PDMS membranes; it therefore delivered a higher pumping rate and withstood a greater back pressure while requiring a significantly lower external air pressure for actuation. The optimized pumping rate and back pressure of the oil-hydraulic micropump compared favorably to previous pneumatic micropumps. Characterization of the micropump revealed that the oil hydraulics amplified the deformation of PDMS membranes by approximately threefold and improved the pumping rate and the back pressure by 77 and 21 %, respectively. With high pumping performances and the capability to be driven with only a low air pressure, this new micropump may therefore become a key component in future microfluidic devices and lab-on-a-chip systems.     

3D-printed peristaltic microfluidic systems fabricated from thermoplastic elastomer

Recent advancements in 3D printing technology have provided a potential low-cost and time-saving alternative to conventional PDMS (polydimethylsiloxane)-based microfabrication for microfluidic systems. In addition to reducing the complexity of the fabrication procedure by eliminating such intermediate steps as molding and bonding, 3D printing also offers more flexibility in terms of structural design than the PDMS micromolding process. At present, 3D-printed microfluidic systems typically utilize a relatively ‘stiff’ printing material such as ABS (acrylonitrile butadiene styrene copolymers), which limits the implementation of large mechanical actuation for active pumping and mixing as routinely carried out in a PDMS system. In this paper, we report the development of an active 3D-printed microfluidic system with moving parts fabricated from a flexible thermoplastic elastomer (TPE). The 3D-printed microfluidic system consists of two pneumatically actuated micropumps and one micromixer. The completed system was successfully applied to the detection of low-level insulin concentration using a chemiluminescence immunoassay, and the test result compares favorably with a similarly designed PDMS microfluidic system. Prior to system fabrication and testing, the material properties of TPE were extensively evaluated. The result indicated that TPE is compatible with biological materials and its 3D-printed surface is hydrophilic as opposed to hydrophobic for a molded PDMS surface. The Young’s modulus of TPE is measured to be 16 MPa, which is approximately eight times higher than that of PDMS, but over one hundred times lower than that of ABS.     

无透镜微流控成像流动细胞检测与计数系统

在未来面向个人化的生物医疗诊断中,实时的细胞检测与计数具有重要需求.现有的细胞检测和计数系统例如流式细胞仪和血细胞计数器不适用于小型化流动细胞实时检测和计数.通过将CMOS图像传感器芯片和微流控芯片结合,提出了一种用于流动细胞检测和计数的无透镜微流控成像系统,与用于计数静态细胞的其它无透镜微流控成像系统不同,该系统可以通过基于时域差分的运动检测算法检测和计数微流体通道中连续流动的细胞样本.测试结果表明:该系统可以对微流控通道中流动的人体骨髓基质细胞实现自动检测和计数,并具有-6.53％的低统计错误率.该系统提供了面向未来即时应用的细胞检测和计数解决方案.     

Programmable autonomous micromixers and micropumps

Programmable autonomous micromixers and micropumps have been designed and realized via a merger between MEMS and microfluidic tectonics (/spl mu/FT). Advantages leveraged from both fabrication platforms allow for relatively simple and rapid fabrication of these microfluidic components. Nickel (Ni) microstructures, driven by an external rotating magnetic field, are patterned in situ and serve as the microactuators in the devices. /spl mu/FT permits in situ patterning through the use of a step-and-repeat fabrication process known as liquid-phase photopolymerization (LP/sup 3/). LP/sup 3/ is a polymer-based fabrication process tool that offers additional fabrication materials, including responsive hydrogels that expand and contract under different stimuli. Using pH- and temperature-sensitive hydrogels as clutches, autonomous micromixers and micropumps have been fabricated and tested that perform as closed-loop microsystems. The step-and-repeat fabrication process allows pre-programming of the system, like a programmable read-only memory chip, to be sensitive to a desired stimulus. Different Ni blade designs, and pH-sensitive hydrogel geometries and dimensions have been designed and tested to better ascertain their effects on micromixing efficiency and response times of hydrogels (related to the autonomous functionality), respectively. Temperature-responsive hydrogels have allowed for development of temperature-sensitive micromixers and micropumps with applications in areas demanding temperature control. [1498].     

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD_15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.     

Microfluidic aptameric affinity sensing of vasopressin for clinical diagnostic and therapeutic applications

We present a microfluidic aptameric biosensor, or aptasensor, for selective detection of clinically relevant analytes with integrated analyte enrichment, isocratic elution and label-free detection by mass spectrometry. Using a microfluidic platform that is coupled to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), we demonstrate specific purification, enrichment, and label-free detection of trace amounts of arginine vasopressin (AVP), a peptide hormone that is responsible for arterial vasoconstriction. During extreme physical trauma, in particular immunological shock or congestive heart failure, AVP is excreted abnormally and is hence a biomarker for such conditions. The device uses an aptamer, i.e., an oligonucleotide that binds specifically to an analyte via affinity interactions, to achieve highly selective analyte capture and enrichment. In addition, via thermally induced reversible disruption of the aptamer-analyte binding, the device can be easily regenerated for reuse and allows isocratic analyte elution, i.e., release and collection of analytes using a single aqueous solution. Furthermore, the device is coupled to MALDI-MS using a microfluidic flow gate, which directs the eluted analyte onto a MALDI sample plate for mass spectrometry. We first perform systematic characterization of kinetic and thermal release properties, as well as the overall timescale of the assay, using fluorescently labeled AVP. We then demonstrate MALDI-MS detection of unlabeled AVP at clinically relevant concentrations approaching 1 pM.     

Microfluidic chips for capillary electrophoresis with integrated electrodes for capacitively coupled conductivity detection based on printed circuit board technology

A simple and low budget microfabrication method compatible with mass production was developed for the integration of electrodes for capacitively coupled contactless conductivity detection (C4D) in Lab on a Chip devices. Electrodes were patterned on a printed circuit board using standard processing. This was followed by lamination-photolithography-lamination to cover the electrodes in dry film photoresist (DFR) using an office laminator. This resulted in a flush, smooth surface on top of the detection electrodes, enabling subsequent integration of a microfluidic network at a distance dictated by the thickness of the DFR (17 μm, 30 μm and 60 μm were used in this work). This process was applied to create two types of detectors, re-usable detectors to be used in combination with a separate microfluidic network and integrated detectors where the microfluidic network is irreversibly sealed to the detector. A poly(dimethylsiloxane) (PDMS) slab containing the microfluidic network was positioned on top of the re-usable detectors to create the PDMS hybrid devices. The integrated DFR devices were created by patterning and sealing the microchannel in DFR using subsequent lamination and lithographic steps. The sensitivity of the C4D made using this new technology for small inorganic cations was between 6 and 20 μM, which is comparable with devices made using significantly more advanced technologies. Where the 17 μm film slightly improved the sensitivity, the use of 30 μm thick insulating films was preferred as these did not impose significant restrictions on the applicable field strengths.     

Demonstration and characterization of biomolecular enrichment on microfluidic aptamer-functionalized surfaces

This paper demonstrates and systematically characterizes the enrichment of biomolecular compounds using aptamer-functionalized surfaces within a microfluidic device. The device consists of a microchamber packed with aptamer-functionalized microbeads and integrated with a microheater and temperature sensor to enable thermally controlled binding and release of biomolecules by the aptamer. We first present an equilibrium binding-based analytical model to understand the enrichment process. The characteristics of the aptamer-analyte binding and enrichment are then experimentally studied, using adenosine monophosphate (AMP) and a specific RNA aptamer as a model system. The temporal process of AMP binding to the aptamer is found to be primarily determined by the aptamer-AMP binding kinetics. The temporal process of aptamer-AMP dissociation at varying temperatures is also obtained and observed to occur relatively rapidly (<2 s). The specificity of the enrichment is next confirmed by performing selective enrichment of AMP from a sample containing biomolecular impurities. Finally, we investigate the enrichment of AMP by either discrete or continuous introduction of a dilute sample into the microchamber, demonstrating enrichment factors ranging from 566 to 686×, which agree with predictions of the analytical model.     

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

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

A multifunction and bidirectional valve-less rectification micropump based on bifurcation geometry

In this paper, we introduce a novel valve-less rectification micropump based on bifurcation geometry. Three micropumps based on three different bifurcation configurations were designed, fabricated and experimentally investigated. These designs demonstrate the potentials of developing bidirectional micropumps and multifunction microfluidic devices (combined functions of micro pumping and mixing). Polydimethylsiloxane (PDMS) was employed to fabricate the micropumps. Circular piezoelectric transducers (PZT) were used as flow actuators. Detailed fabrication procedures are illustrated. The micropumps were tested against two ranges of actuator frequencies. The first test was conducted in a frequency range between 0 and 100 Hz with small increments of 5 Hz, while the second test was conducted in a frequency range between 0 and 300 Hz with increments of 50 Hz. Ethanol was used as the working fluid in all experiments. A new dimensionless parameter was introduced to evaluate the efficiency of valve-less rectification micropumps and determine the optimum operational frequency. The flow rate and maximum back pressure were measured. Results of experiments confirmed and demonstrated the feasibility of valve-less rectification micropumps based on bifurcation geometry at a low frequency range. Additionally, results showed the potentials of multifunctional, bidirectional, and self-priming micropumps.