Magnetic-based purification system with simultaneous sample washing and concentration

Simultaneous washing and concentration of magnetic microparticles was demonstrated using a rotational magnetic system under a continuous-flow condition. The rotation of periodically arranged permanent magnets close to a fluidic channel carrying a suspension of magnetic particles allows the trapping and releasing of particles along the fluidic channel in a periodic manner. Each trapping and releasing event resembles one washing cycle in conventional biological assays. Concentration efficiencies of 99.75?±?0.083% at a flow rate of 200 µl/min and 88.10?±?3.17% at a flow rate of 1,000 µl/min and a purification efficiency of 99.10?±?4.3% at a flow rate of 900 µl/min were achieved.     

Magnetic core shell nanoparticles trapping in a microdevice generating high magnetic gradient

Magnetic core shell nanoparticles (MCSNPs) 30 nm diameter with a magnetic weight of 10% are usually much too small to be trapped in microfluidic systems using classical external magnets. Here, a simple microchip for efficient MCSNPs trapping and release is presented. It comprises a bed of micrometric iron beads (6-8 μm diameter) packed in a microchannel against a physical restriction and presenting a low dead volume of 0.8 nL. These beads of high magnetic permeability are used to focus magnetic field lines from an external permanent magnet and generate local high magnetic gradients. The nanoparticles magnetic trap has been characterised both by numerical simulations and fluorescent MCSNPs imaging. Numerical simulations have been performed to map both the magnetic flux density and the magnetic force, and showed that MCSNPs are preferentially trapped at the iron bead magnetic poles where the magnetic force is increased by 3 orders of magnitude. The trapping efficiency was experimentally determined using fluorescent MCSNPs for different flow rates, different iron beads and permanent magnet positions. At a flow rate of 100 μL h(-1), the nanoparticles trapping/release can be achieved within 20 s with a preconcentration factor of 4000.     

Flow-through immunomagnetic separation system for waterborne pathogen isolation and detection: Application to Giardia and Cryptosporidium cell isolation

Simultaneous sample washing and concentration of two waterborne pathogen samples were demonstrated using a rotational magnetic system under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to a fluidic channel carrying magnetic particle suspension allows the trapping and release of particles along the fluidic channel in a periodic manner. Each trapping and release event resembles one washing cycle.The performance of the magnetic separation system (MSS) was evaluated in order to test its functionality to isolate magnetic-labelled protozoan cells from filtered, concentrated tap water, secondary effluent water, and purified water. Experimental protocols described in US Environmental Protection Agency method 1623 which rely on the use of a magnetic particle concentrator, were applied to test and compare our continuous flow cell separation system to the standard magnetic bead-based isolation instruments. The recovery efficiencies for Giardia cysts using the magnetic tube holder and our magnetic separation system were 90.5% and 90.1%, respectively, from a tap water matrix and about 31% and 18.5%, respectively, from a spiked secondary effluent matrix. The recovery efficiencies for Cryptosporidium cells using the magnetic tube holder and our magnetic separation system were 90% and 83.3%, respectively, from a tap water matrix and about 38% and 36%, respectively, from a spiked secondary effluent matrix. Recoveries from all matrices with the continuous flow system were typically higher in glass tubing conduits than in molded plastic conduits.     

Continuous microfluidic DNA extraction using phase-transfer magnetophoresis

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 μl s(-1) and an eluate flow rate of 0.35 μl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.     

Magneto-mechanical trapping systems for biological target detection

We demonstrate a magnetic microsystem capable of detecting nucleic acids via the size difference between bare magnetic beads and bead compounds. The bead compounds are formed through linking nonmagnetic beads and magnetic beads by the target nucleic acids. The system comprises a tunnel magneto-resistive (TMR) sensor, a trapping well, and a bead-concentrator. The TMR sensor detects the stray field of magnetic beads inside the trapping well, while the sensor output depends on the number of beads. The size of the bead compounds is larger than that of bare magnetic beads, and fewer magnetic beads are required to fill the trapping well. The bead-concentrator, in turn, is capable of filling the trap in a controlled fashion and so to shorten the assay time. The bead-concentrator includes conducting loops surrounding the trapping well and a conducting line underneath. The central conducting line serves to attract magnetic beads in the trapping well and provides a magnetic field to magnetize them so to make them detectable by the TMR sensor. This system excels by its simplicity in that the DNA is incubated with magnetic and nonmagnetic beads, and the solution is then applied to the chip and analyzed in a single step. In current experiments, a signal-to-noise ratio of 40.3 dB was obtained for a solution containing 20.8 nM of DNA. The sensitivity and applicability of this method can be controlled by the size or concentration of the nonmagnetic bead, or by the dimension of the trapping well.

If biological targets are present, they link magnetic beads and fluorescent beads. This results in less magnetic beads to be on the surface of magnetic sensor, causing a smaller signal, thus biological targets are detected.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

Development and characterization of "push-pull" sampling device with fast reaction quenching coupled to high-performance liquid chromatography for pharmaceutical process analytical technologies

A push-pull sampling system interfaced on-line to high-performance liquid chromatography (HPLC) was developed for micro-volume real-time monitoring of reaction mixtures. The device consists of concentric tubes wherein sample was continuously withdrawn through the outer tube and reaction quenchant continuously delivered through a recessed inner tube. The device allowed sampling rates of 0.1-6.0 μL/min from a reaction vessel and stopped the reaction by passive mixing with quenchant to preserve the conditions observed in the reaction vessel. A finite element model of the system showed that reaction mixtures could be completely mixed with quenchant within 4.3s at a flow rate of 1.0 μL/min. The model also showed that an offset distance of 1mm between the push capillary and sample capillary tips is sufficient to avoid leakage of quenchant/diluent into the bulk sample for push flow rates up to 95% of the pull flow rate. The maximum relative push flow rate was determined to be 90% of the pull flow rate experimentally. Delay between sampling and delivery to the HPLC was from 111±3s to 317±9s for pull flow rates from 1.0 to 3.0 μL/min in agreement with expected delays based on tubing volume. Response times were from 27±1s to 52±6s over the same flow rate range. The sampler was tested to determine the effects of sample viscosity. The sampler was also used to demonstrate periodic sampling capabilities. As a test of the system, it was used to monitor the base-catalyzed hydrolysis of aspirin for 1.5h, demonstrating its utility for monitoring an ongoing reaction.     

Three‐dimensional printed magnetophoretic system for the continuous flow separation of avian influenza H5N1 viruses

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three‐dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer‐modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle–virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three‐dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate‐buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus–nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 μL.     

Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 μL or 20 μL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system.     

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500 ppb) under optimum conditions. The time required for analysis is less than 15 min.     

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.     

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

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.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

A Bead Injection System for Copper Determination

ABSTRACT

A bead injection system for copper determination is proposed. Chelex-100 resin beads are introduced in the system as a suspension that is trapped in the Jet Ring Cell. The passage of the sample zone by the beads promotes the sorption of Cu(II). When the colorimetric reagent (APDC) perfuses the beads it reacts with copper ions, forming a colored complex that is monitored at 436 nm. After the measurement, the spent beads are sent to waste and a new portion of fresh beads is trapped in the system. The bead injection system is versatile and can be used to concentrate analyte in different sample volumes, permitting determinations of a wide range of copper concentrations. The detection limit is 0.5 μg l^-1 with a 500 μl sample, and 1.2 μg l^-1 with a 100 μ1 sample.     

A microfluidic magnetic bead impact generator for physical stimulation of osteoblast cell

We developed a novel microfluidic cell culture device in which magnetic beads repetitively collide with osteoblast cells, MC3T3‐E1, owing to attractive forces generated by pulsed electromagnetic fields and consequently the cells were physically stimulated by bead impacts. Our device consists of an on‐chip microelectromagnet and a microfluidic channel which were fabricated by a microelectromechanical system technique. The impact forces and stresses acting on a cell were numerically analyzed and experimentally generated with different sizes of bead (4.5, 7.6 and 8.4 μm) and at various pulse frequencies (60 Hz, 1 kHz and 1 MHz). Cells were synchronized at each specific phase of the cell cycle before stimulation in order to determine the most susceptible phase against bead impacts. The cells were stimulated with different sizes of bead at various pulse frequencies for 1 min at G1, S and G2 phases, respectively, and then counted immediately after one doubling time. The growth rate of cells was highly accelerated when they were stimulated with 4.5 μm beads at G1 phase and a pulse frequency of 1 MHz. Almost all of the cells were viable after stimulation, indicating that our cell stimulator did not cause any cellular damage and is suitable for use in new physical stimulus modalities.     

Analysis of herbicides on a single C30 bead via a microfluidic device combined with electrospray ionization quadrupole time-of-flight mass spectrometer

An integrated system combining microfluidic device with electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF-MS) was developed for detecting a series of herbicides on a single C₃₀ bead. We presented single C₃₀ beads manipulation which had absorbed sequential herbicides, based on the precise control of the concentration and absorption time. The simple microchip consisting of a straight channel and a hole in the middle enabled single C₃₀ bead introduction, transformation and location. ESI-Q-TOF-MS was employed to realize highly sensitive qualitative analysis and implement semi-quantitative analysis. Once the C₃₀ bead contacted with eluting solution, the solute transferred into eluting and would be detected by ESI-Q-TOF-MS. Depending on desorption curves obtained, accurate and clear characteristic peaks for each analysts were carried out. Our investigations on single-particle analysis showed that the combination of microfluidic device and mass spectrum could reduce the analysis time to 5 min and the solvent consumption to 2.5 μL, realize high sensitivity detection, and avoid the complex sample pretreatment. The significant potential on analysis of environmental samples on this combination system by single particle was demonstrated.     

Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100 ms using standard APnEOs sample solutions at various concentrations (0–1000 ppb) under optimum conditions. The lower detection limit defined as IC₈₀ is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.     

A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads

We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure.     

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.