Glass-composite prototyping for flow PCR with <Emphasis Type="Italic">in situ</Emphasis> DNA analysis

In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 μl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 μl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (ΦX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.     

Temperature distribution effects on micro-CFPCR performance

Continuous flow polymerase chain reactors (CFPCRs) are BioMEMS devices that offer unique capabilities for the ultra-fast amplification of target DNA fragments using repeated thermal cycling, typically over the following temperature ranges: 90°C–95°C for denaturation, 50°C–70°C for renaturation, and 70°C–75°C for extension. In CFPCR, DNA cocktail is pumped through the constant temperature zones and reaches thermal equilibrium with the channel walls quickly due to its low thermal capacitance. In previous work, a polycarbonate CFPCR was designed with microchannels 150 μm deep, 50 μm wide, and 1.78 m long—including preheating and post-heating zones, fabricated with LIGA, and demonstrated. The high thermal resistance of the polycarbonate led to a high temperature gradient in the micro-device at steady-state and was partly responsible for the low amplification yield. Several steps were taken to ensure that there were three discrete, uniform temperature zones on the polycarbonate CFPCR device including: reducing the thickness of the CFPCR substrate to decrease thermal capacitance, using copper plates as heating elements to ensure a uniform temperature input, and making grooves between temperature zones to increase the resistance to lateral heat conduction between zones. Finite element analyses (FEA) were used to evaluate the macro temperature distribution in the CFPCR device and the micro temperature distribution along a single microchannel. At steady-state, the simulated CFPCR device had three discrete temperature zones, each with a uniform temperature distribution with a variation of ±0.3°C. An infrared (IR) camera was used to measure the steady-state temperature distribution in the prototype CFPCR and validated the simulation results. The temperature distributions along a microchannel at flow velocities from 0 mm/s to 6 mm/s were used to estimate the resulting temperatures of the DNA reagents in a single microchannel. A 500 bp DNA fragment was generated from a bacteriophage λ-DNA target using 20 cycles of PCR. The amplification efficiencies compared to a commercial thermal cycler were 72.7% (2 mm/s), 44% (3 mm/s), and 29.4% (4 mm/s). The amplification efficiency with the modified CFPCR device increased by 363% at 2 mm/s and 440% at 3 mm/s compared to amplification obtained using a CFPCR device with the same fluidic layout, (Hashimoto et al., Lab Chip 4:638, 2004) strictly due to the improved temperature distribution.     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

Continuous-flow thermal gradient PCR

Continuous-flow thermal gradient PCR is a new DNA amplification technique that is characterized by periodic temperature ramping with no cyclic hold times. The device reported in this article represents the first demonstration of hold-less thermocycling within continuous-flow PCR microfluidics. This is also the first design in which continuous-flow PCR is performed within a single steady-state temperature zone. This allows for straightforward miniaturization of the channel footprint, shown in this device which has a cycle length of just 2.1 cm. With a linear thermal gradient established across the glass device, the heating and cooling ramp rates are dictated by the fluid velocity relative to the temperature gradient. Local channel orientation and cross-sectional area regulate this velocity. Thus, rapid thermocycling occurs while the PCR chip is maintained at steady state temperatures and flow rates. Glass PCR chips (25 x 75 x 2 mm) of both 30 and 40 serpentine cycles have been fabricated, and were used to amplify a variety of targets, including a 181-bp segment of a viral phage DNA (PhiX174) and a 108-bp segment of the Y-chromosome, amplified from human genomic DNA. With this unique combination of hold-less cycling and gradient temperature ramping, a 40-cycle PCR requires less than 9 min, with the resulting amplicon having high yield and specificity.     

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1 ng/µL and 100 ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10⁶ of sample DNA through thermophillic helicase dependant amplification and 1×10⁵ copy numbers Chlamydia trachomatis genomic DNA within 10 min through recombinase polymerase nucleic acid amplification tests.     

Integrated polymerase chain reaction chips utilizing digital microfluidics

This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated, transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips, a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required an operation voltage of 12V_RMS at a frequency of 3 KHz for digital microfluidic actuation and 9V_DC for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip (LOC) by utilizing EWOD-based digital microfluidics.     

Fast detection of genetic information by an optimized PCR in an interchangeable chip

In this paper, we report the construction of a polymerase chain reaction (PCR) device for fast amplification and detection of DNA. This device consists of an interchangeable PCR chamber, a temperature control component as well as an optical detection system. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 μl, while the heating and cooling rate was as fast as 12.7°C/second ensuring that the total time needed of only 25 min to complete the 35 cycle PCR amplification. An optimized PCR with two-temperature approach for denaturing and annealing (T_d and T_a) of DNA was also formulated with the PCR chip, with which the amplification of male-specific sex determining region Y (SRY) gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system.     

Analytical study of a microfludic DNA amplification chip using water cooling effect

A novel continuous-flow polymerase chain reaction (PCR) chip has been analyzed in our work. Two temperature zones are controlled by two external controllers and the other temperature zone at the chip center is controlled by the flow rate of the fluid inside a channel under the glass chip. By employing a water cooling channel at the chip center, the sequence of denaturation, annealing, and extension can be created due to the forced convection effect. The required annealing temperature of PCR less than 313 K can also be demonstrated in this chip. The Poly(methyl methacrylate) (PMMA) cooling channel with the thin aluminum cover is utilized to enhance the temperature uniformity. The size of this chip is 76 mm?×?26 mm?×?3 mm. This device represents the first demonstration of water cooling thermocycling within continuous-flow PCR microfluidics. The commercial software CFD-ACE+^TM is utilized to determine the distances between the heating assemblies within the chip. We investigate the influences of various chip materials, operational parameters of the cooling channel and geometric parameters of the chip on the temperature uniformity on the chip surface. Concerning the temperature uniformity of the working zones and the lowest temperature at the annealing zone, the air gap spacing of 1 mm and the cooling channel thicknesses of 1 mm of the PMMA channel with an aluminum cover are recommended in our design. The hydrophobic surface of the PDMS channel was modified by filling it with 20 % Tween 20 solution and then adding bovine serum albumin (BSA) solution to the PCR mixture. DNA fragments with different lengths (372 bp and 478 bp) are successfully amplified with the device.     

Effects of a novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional stabilizing reagents

Stabilization of nucleated blood cells by cell stabilizing reagent (BCT reagent) present in the Cell-Free DNA BCT™ blood collection device and consequent prevention of cell-free DNA contamination by cellular DNA during sample storage and shipping have previously been reported. This study was conducted to investigate the effect of this novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional cell stabilizing reagents, formaldehyde and glutaraldehyde. A 787 bp long DNA fragment from human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified by PCR and used as model system. DNA samples and blood samples were treated with BCT reagent, 0.1% formaldehyde or 0.1% glutaraldehyde at room temperature. DNA amplification was studied using conventional and real-time quantitative PCR. Results indicate that exposure of DNA to the BCT reagent for up to 14 days had no effect on DNA amplification by PCR as compared to the untreated control DNA. However, there was statistically significant decrease in DNA amplification in the DNA samples treated with formaldehyde and glutaraldehyde. We conclude that the BCT reagent used in Cell-Free DNA BCT blood collection device to prevent cell-free DNA contamination by cellular DNA had no effect on DNA amplification by PCR.     

Design Considerations for a Microfluidic Device to Quantify the Platelet Adhesion to Collagen at Physiological Shear Rates

Accurate assessment of blood platelet function is essential in understanding thrombus formation which plays a central role in cardiovascular disease. Parallel plate flow chambers have been widely used as they allow for platelet adhesion on a collagen surface at physiologically relevant fluid mechanical forces. Standard parallel plate flow chambers typically need several milliliters of blood, which is substantially more than can be obtained from small animals. We designed, fabricated, and assessed the functionality of a microfluidic channel with a width of 500 μm and a height of 50 μm in which a wall shear rate of 1000 s⁻¹ can be achieved with a flow rate of 15 μL/min. The velocity distribution in the microchannel predicted from the equations of motion was compared to experimentally measured velocities of fluorescent beads. This analysis showed that the motion of beads was quite similar to the predicted motion. Adhesion of platelets from whole blood at a shear rate of 1000 s⁻¹ onto a collagen surface using the microfluidic flow channel was qualitatively similar to platelet adhesion observed with a standard sized parallel plate flow chamber. After 5 min flow the surface coverage of platelets in the microfluidic device was about 55% while in a traditional size flow chamber the surface coverage was about 75%. This suggests that the microfluidic flow chamber can be used to quantify platelet adhesion for system where only very small amounts of blood are available.     

Deep flexion helps to avoid popliteal artery injury during all-inside lateral meniscal repair: A cadaveric study

BackgroundArthroscopic meniscus repair rarely leads to major complications such as popliteal artery injury. The distance between the suturing device and the popliteal artery, and the risk of popliteal artery injury at different knee flexion angles during all-inside lateral meniscal repair remain unclear.MethodsAll-inside devices were inserted into 10 human cadaveric knees at the posterior horn of the lateral meniscus through the anterolateral portal at 60°, 90°, and 120° knee flexion; posterior segment of the lateral meniscus through the anterolateral portal at 60°, 90°, and 120°; and anteromedial portal at 90°. Distance and positional relationship between the device and popliteal artery were measured radiographically.ResultsIn posterior horn repair through the anterolateral portal, the median distance increased from 5.7 mm at 60° to 9.1 mm at 90° (P = 0.63) and 18.0 mm at 120° (P = 0.02). The device pushed the wire at 60° in three cases, 90° in one case, and 120° in 0 cases. In posterior segment repair through the anterolateral portal, the median distance was 12.6 mm at 60°, 10.4 mm at 90°, and 18.3 mm at 120° (P = 0.08). The median distance at 90° was 18.1 mm through the anteromedial portal, the same as that at 120° through the anterolateral portal (P = 0.43), but greater than that at 90° through the anterolateral portal (P = 0.04). The wire was not pushed in any case.ConclusionAlthough all-inside repair of the posterior part of the lateral meniscus through the anterolateral portal is risky, deeper knee flexion reduces the risk of popliteal artery injury.     

A Novel Shape Memory Alloy Annuloplasty Ring for Minimally Invasive Surgery: Design,Fabrication, and Evaluation

A novel annuloplasty ring with a shape memory alloy core has been developed to facilitate minimally invasive mitral valve repair. In its activated (austenitic) phase, this prototype ring has comparable mechanical properties to commercial semi-rigid rings. In its pre-activated (martensitic) phase, this ring is flexible enough to be introduced through an 8-mm trocar and easily manipulated with robotic instruments within the confines of a left atrial model. The core is constructed of 0.50 mm diameter NiTi, which is maintained below its martensitic transition temperature (24 °C) during deployment and suturing. After suturing, the ring is heated above its austenitic transition temperature (37 °C, normal human body temperature) enabling the NiTi core to attain its optimal geometry and stiffness characteristics indefinitely. This article summarizes the design, fabrication, and evaluation of this prototype ring. Experimental results suggest that the NiTi core ring could be a viable alternative to flexible bands in robot-assisted minimally invasive mitral valve repair.     

A simple device using magnetic transportation for droplet-based PCR

The Polymerase chain reaction (PCR) was successfully and rapidly performed in a simple reaction device devoid of channels, pumps, valves, or other control elements used in conventional lab-on-a-chip technology. The basic concept of this device is the transportation of aqueous droplets containing hydrophilic magnetic beads in a flat-bottomed, tray-type reactor filled with silicone oil. The whole droplets sink to the bottom of the reactor because their specific gravity is greater than that of the silicone oil used here. The droplets follow the movement of a magnet located underneath the reactor. The notable advantage of the droplet-based PCR is the ability to switch rapidly the proposed reaction temperature by moving the droplets to the required temperature zones in the temperature gradient. The droplet-based reciprocative thermal cycling was performed by moving the droplets composed of PCR reaction mixture to the designated temperature zones on a linear temperature gradient from 50°C to 94°C generated on the flat bottom plate of the tray reactor. Using human-derived DNA containing the mitochondria genes as the amplification targets, the droplet-based PCR with magnetic reciprocative thermal cycling successfully provided the five PCR products ranging from 126 to 1,219 bp in 11 min with 30 cycles. More remarkably, the human genomic gene amplification targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was accomplished rapidly in 3.6 min with 40 cycles.     

Performance of nanoliter-sized droplet-based microfluidic PCR

A microfluidic device was used to characterize PCR in aqueous-in-oil droplets for potential point-of-care applications. Droplets with a volume range of 5–250 nL can be formed on-chip reproducibly, and PCR in the droplets shows amplification efficiencies comparable to benchtop reactions with no evaporation loss. A higher polymerase concentration is required in the reaction droplet while the optimal Magnesium ion concentration is the same for both on-chip and benchtop systems. The optimal hold time is 9 s and 30 s for denaturation and annealing/extension in thermal cycling, respectively. With the optimized cycling parameters, the total reaction time is reduced to half of that required for benchtop PCR. For the droplets containing the same quantity of template DNA, the PCR yield is approximately the same with either fixed droplet size or fixed template DNA concentration. The droplet-based PCR can be monitored in real time with FRET probes, and provide amplification with a cycle threshold of ~10 cycles earlier than the benchtop instruments.     

A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system

We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.     

Surface acoustic wave induced particle manipulation in a PDMS channel—principle concepts for continuous flow applications

A device for acoustic particle manipulation in the 40 MHz range for continuous-flow operation in a 50 μm wide PDMS channel has been evaluated. Unidirectional interdigital transducers on a Y-cut Z-propagation lithium nixobate wafer were used to excite a surface acoustic wave that generated an acoustic standing wave inside the microfluidic channel. It was shown that particle alignment nodes with different inter-node spacing could be obtained, depending on device design and driving frequency. The observed inter-node spacing differed from the standard half-wavelength inter-node spacing generally employed in bulk acoustic transducer excited resonant systems. This effect and the related issue of acoustic node positions relative the channel walls, which is fundamental for most continuous flow particle manipulation operations in channels, was evaluated in measurements and simulations. Specific applications of particle separation and alignment where these systems can offer benefits relative state-of the art designs were identified.     

A micro circulating PCR chip using a suction-type membrane for fluidic transport

A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 μL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1°C was over 90% for a denaturing temperature of 94°C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10⁵ to 10²copies/μL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.     

Vortical flow in human elbow joints: a three‐dimensional computed tomography modeling study

The human elbow joint has been regarded as a loose hinge joint, with a unique helical motion of the axis during extension–flexion. This study was designed to identify the helical axis in the ulnohumeral joint during elbow extension–flexion by tracking the midpoint between the coronoid tip and the olecranon tip of the proximal ulna in a three‐dimensional (3D) computed tomography (CT) image model. The elbows of four volunteers were CT‐scanned at four flexion angles (0°, 45°, 90°, and 130°) at neutral rotation with a custom‐made holding device to control any motion during scanning. Three‐dimensional models of each elbow were reconstructed and a 3D ulnohumeral joint at 45°, 90°, and 130° was superimposed onto a fully extended joint (0°) by rotating and translating each 3D ulnohumeral joint along the axes. The midpoints of the olecranon and coronoid tips were interpolated using cubic spline technique and the dynamic elbow motion was plotted to determine the motion of the helical axis. The means and standard deviations were subsequently calculated. The average midpoint pattern of joint motion from extension to flexion was elliptical‐orbit‐like when projected onto a sagittal plane and continuously translated a mean 2.14 ± 0.34 mm (range, 1.83–2.52 mm) to the lateral side during elbow extension–flexion. In 3D space, the average midpoint pattern of the ulnohumeral joint resembles a vortical flow, spinning along an imaginary axis, with an inconsistent radius from 0° to 130° flexion. The ulnohumeral joint axis both rotates and translates during elbow extension–flexion, with a vortex‐flow motion occurring during flexion in 3D model analysis. This motion should be considered when performing hinged external fixation, total elbow replacement and medial collateral ligament reconstruction surgery.     

An automated micro-solid phase extraction device involving integrated \high-pressure microvalves for genetic sample preparation

This paper presents an automated micro-SPE device for DNA extraction using monolithically integrated high-pressure microvalves. The automated micro-SPE device was fabricated through glass-to-glass thermal bonding and microfluidic system interface technologies. To increase the DNA extraction efficiency, silica beads were packed in the extraction microchannel involving two weir structures. Experimental results show that the DNA extraction efficiency using the automated micro-SPE device containing bare silica beads was 75.87% in the first 8 μl of solution eluted by automated SPE procedure. In addition, the reproducibility of the DNA extraction was evaluated by ten successive measurements. Genomic DNA extracted from human WBCs had an absorbance ratio of DNA to protein (A₂₆₀/A₂₈₀) of 1.56. The applicability of this automated micro-SPE device to genetic sample preparation was verified by PCR amplification of a β-globulin gene using the genomic DNA extracted from WBCs. Consequently, we demonstrated that the proposed automatic micro-SPE device can extract nucleic acids from biological samples, thereby facilitating its integration with downstream genetic analyses in a micro format.