Compact optical diagnostic device for isothermal nucleic acids amplification

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R² = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R² = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h.     

Development and application of a diaphragm micro-pump with piezoelectric device

In this study, a new type of thin, compact, and light weighed diaphragm micro-pump has been successfully developed to actuate liquid by the vibration of a diaphragm. The micro-diaphragm pump with two valves is fabricated in an aluminum case by using highly accurate CNC machine, and the cross-section dimension is 28 mm × 5 mm. Both valves and diaphragm are manufactured from PDMS. The amplitude of vibration by a piezoelectric device produces an oscillating flow and alters the chamber volume by the curvature change of a diaphragm. Several experimental set-ups for performance tests in a single micro-diaphragm pump, isothermal flow open system, and a closed liquid cooling system are designed and implemented. The performance of a one-side actuating micro-diaphragm pump is affected by the design of check valves, diaphragm, piezoelectric device, chamber volume, input voltage and frequency. The measured maximum flow rate of present design is 72 ml/min at zero total pump head in the range of operation frequency 70–180 Hz.     

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.     

Sub-microliter scale in-gel loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a common human disease that is prevalent in resource-deprived areas of the world. Current detection techniques for TB require expensive conventional instruments in a laboratory setting, preventing accessible and low cost diagnosis of the disease. Using a loop-mediated isothermal amplification (LAMP) assay, we have amplified and detected TB in a 6 × 8 semisolid polyacrylamide gel post array using an inexpensive prototype instrument. Each post contains 670 nL of volume, minimizing the need for large quantities of reagents. Amplified DNA is detected via fluorescence of the dye LCGreen Plus+, which is polymerized into the gel along with other reagents. The prototype device contains a Peltier element for heating, a diode laser as an excitation source, and a CCD camera for detecting fluorescence in real-time. About 12 Mycobacterium tuberculosis genomes per gel post can be detected within 75 min of amplification. This sensitivity is similar to that obtained by conventional methods using a commercial thermocycler. We achieved comparable LAMP amplification when the template is added externally or when the template is polymerized in the gel. This rapid isothermal amplification technology, with its simple thermal requirements, has the potential to be integrated into micro-devices and serves as a model for implementing future low-cost point of care diagnostics.     

Decreasing microfluidic evaporation loss using the HMDL method: open systems for nucleic acid amplification and analysis

Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss (V _loss/V _ini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures (T _HMDL and T _TR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the V _loss/V _ini within 60 min is low to 5% (T _HMDL = 105°C, T _TR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp fragment from Listeria monocytogenes genomic DNA.     

Surface plasmon resonance immunosensor using Au nanoparticle for detection of TNT

In order to develop the fully integrated portable surface plasmon resonance (SPR) system for detection of explosives, the amplification strategy of SPR signal was investigated. Indirect competitive inhibition method allowed the middle-sized SPR sensor to detect trinitrotoluene (TNT) at ppt level. However, this enhanced SPR signal was not high enough to detect TNT at ppt level by a miniaturized SPR sensor. Therefore, localized surface plasmon resonance (LSPR) effect using Au nanoparticle as further signal amplification approach was used. The amplification method of indirect competitive inhibition and LSPR were combined together for fabrication of the immunosurface using Au nanoparticle. TNT detectable range of this immunosurface was from 10 ppt (10 pg/ml) to 100 ppb (100 ng/ml), which was almost comparable to that without Au nanoparticle. The observed resonance angle change due to binding monoclonal TNT antibody (M-TNT Ab) with the immunosurface modified with Au nanoparticle was amplified to four times higher than that in absence of Au nanoparticle.     

Automating fluid delivery in a capillary microfluidic device using low-voltage electrowetting valves

A multilayer capillary polymeric microfluidic device integrated with three normally closed electrowetting valves for timed fluidic delivery was developed. The microfluidic channel consisted two flexible layers of poly (ethylene terephthalate) bonded by a pressure-sensitive adhesive spacer tape. Channels were patterned in the spacer tape using laser ablation. Each valve contained two inkjet-printed silver electrodes in series. Capillary flow within the microchannel was stopped at the second electrode which was modified with a hydrophobic monolayer (valve closed). When a potential was applied across the electrodes, the hydrophobic monolayer became hydrophilic and allowed flow to continue (valve opened). The relationship between the actuation voltage, the actuation time, and the distance between two electrodes was performed using a microfluidic chip containing a single microchannel design. The results showed that a low voltage (4.5 V) was able to open the valve within 1 s when the distance between two electrodes was 1 mm. Increased voltages were needed to open the valves when the distance between two electrodes was increased. Additionally, the actuation time required to open the valve increased when voltage was decreased. A multichannel device was fabricated to demonstrate timed fluid delivery between three solutions. Our electrowetting valve system was fabricated using low-cost materials and techniques, can be actuated by a battery, and can be integrated into portable microfluidic devices suitable for point-of-care analysis in resource-limited settings.     

High quality factor silicon cantilever driven by piezoelectric thin film actuator for resonant based mass detection

A high quality factor (Q-factor) piezoelectric lead zirconate titanate (PZT) thin film actuated single crystal silicon cantilever was proposed in this paper for resonant based ultra sensitive mass detection. Intrinsic energy dissipation and other negative effects from PZT-electrode stack were successfully compressed by separating the PZT actuator from the resonant structure. Excellent Q-factor, which is comparable to silicon cantilever (without actuator) and several times larger than that of latest reported other integrated cantilevers, was successfully obtained under both atmospheric pressure and reduced pressures. For a 30 μm-wide 100 μm-long cantilever, the Q-factor was measured as 1,113 and 7,279 under the pressure of 101.2 KPa and 35 Pa, respectively. Besides, it was found that greater Q-factor can be achieved at high vibration mode by the proposed structure. However, support loss became significant because of the increased actuator’s vibration amplitude which in turn leads to unexpected energy dissipation. Therefore, an optimized structure using node-point actuation was suggested and discussed in last section of this paper.     

Design and analysis of novel micro displacement amplification mechanism actuated by chevron shaped thermal actuators

This paper presents design and analysis of microelectromechanical system (MEMS) based displacement amplification mechanism actuated using thermal actuators with enhanced performance. The proposed model consists of chevron shaped thermal actuators, an amplification mechanism capable of amplifying displacement 20 times and an electrostatic comb drives for sensing displacements. When voltage is applied to thermal chevrons, displacement is produced which is then amplified 20 times. Steady state static thermal electrical analysis is performed under variable resistivity and voltage bias of 2 V. In-plane reaction forces of magnitude 194.2 and 150.91 µN along X and Y-axis, respectively, thus producing displacement of 0.11 and 2.22 µm along X and Y-axis, respectively. Time domain simulations of device are carried with constant electrical resistivity, variable voltage and convective boundary conditions. Modal analysis of the mechanism is carried out to predict the natural frequencies and associated mode shapes of mechanism during free vibrations. The desired mode is at frequency of 286.160 kHz. Dynamic simulations including direct integration-transient, transient modal and steady state modal analysis are performed on the device for time span of 0.0006 s, under application of 25 g and frequency range of 200–300 kHz. Simulation results prove the viability of the mechanism as an amplification device with enhanced voltage–stroke ratio.

     

Exploration on relationship between flow rate and sound pressure level of piezoelectric pump

This study presents the relationship between flow rate and sound pressure level of piezoelectric pump. It also presents the analysis and theoretical calculation for umbrella valve piezoelectric pump on the sound pressure level. Sound pressure level of umbrella valve piezoelectric pump in composite field (medium are air and water) was simulated by software Actran. Simulation results reveal that: The sound pressure level of Piezoelectric actuator in air field is bigger than that in water field. This result means that pump chamber with air and water has a higher sound pressure level than pump chamber with water. The flow rate and sound pressure level characteristics of umbrella valve piezoelectric pump with different pump chamber depths (d = 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm) is obtained by experiments in a voltage range of 160–220 V and a frequency range of 160–340 Hz. Experimental results show that: At different pump chamber depth (d), the range of flow rate is from 173.4 to 271.0 mL/min at 0.5 mm chamber depth, 75.0 to 149.0 mL/min at 1.0 mm chamber depth, 3.4 to 57.0 mL/min at 1.5 mm chamber depth, 2.8 to 32.8 mL/min at 2.0 mm chamber depth, the sound pressure level is from 57.52 to 65.14 dB, 54.54 to 60.05 dB, 55.26 to 69.24 dB, 53.39 to 61.19 dB. For piezoelectric pump with different pump chamber depths (d), flow rate and sound pressure level of piezoelectric pump increase as voltage increases. It is a great method to monitoring flow rate by monitoring the sound pressure level.

     

Coupled flow and reaction during natural convection PCR

Polymerase chain reaction (PCR) in a microfluidic Rayleigh–Benard convection cell represents a promising route towards portable PCR for point-of-care uses. In the present contribution, the coupled fluid mechanics and heat transport processes are solved numerically for a 2-D flow cell. The resultant velocity and temperature fields serve as the inputs to a convection-diffusion-reaction model for the DNA amplification, wherein the reaction kinetics are modeled by Gaussian distributions around the conventional bulk PCR reaction temperatures. These evolution equations are integrated to determine the exponential growth rate of the double-stranded DNA concentration. The predicted doubling time is approximately 10–25 s, increasing with the Péclet number. This effect is attributed to low velocity, slow kinetics “dead zones” located at the center of the reactor. The latter observation provides an alternative rationalization for the use of loop-based natural convection PCR systems.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

Magnetic active-valve micropump actuated by a rotating magnetic assembly

We report on a high-efficiency and self-priming active-valve micropump consisting of a microfluidic chamber structure in glass that is assembled with a polydimethylsiloxane (PDMS) elastic sheet. The latter comprises two valving membranes and a central pumping chamber actuation membrane, having each an integrated permanent magnet that is magnetically actuated by arc-shaped NdFeB permanent magnets mounted on the rotation axis of a DC minimotor. The choice of this actuation principle allows very low-voltage (0.7 V) and low power (a few 10 mW) operation of the micropump. For the realisation, we use affordable powder blasting glass micropatterning and PDMS molding technologies. A flow rate of 2.4 mL/min and up to 70 mbar backpressure are obtained at the micropump resonance frequency of around 12 Hz, values that are much higher than reported so far for such type of micropump.     

Pre-programmable polymer transformers as on-chip microfluidic vacuum generators

This paper develops novel polymer transformers using thermally actuated shape memory polymer (SMP) materials. This paper applies SMPs with thermally induced shape memory effect to the proposed novel polymer transformers as on-chip microfluidic vacuum generators. In this type of SMPs, the morphology of the materials changes when the temperature of materials reaches its glass transition temperature (T _g). The structure of the polymer transformer can be pre-programmed to define its functions, which the structure is reset to the temporary shape, using shape memory effects. When subjected to heat, the polymer transformer returns to its pre-memory morphology. The morphological change can produce a vacuum generation function in microfluidic channels. Vacuum pressure is generated to suck liquids into the microfluidic chip from fluidic inlets and drive liquids in the microchannel due to the morphological change of the polymer transformer. This study adopts a new smart polymer with high shape memory effects to achieve fluid movement using an on-chip vacuum generation source. Experimental measurements show that the polymer transformer, which uses SMP with a T _g of 40°C, can deform 310 μm (recover to the permanent shape from the temporary shape) within 40 s at 65°C. The polymer transformer with an effective cavity volume of 155 μl achieved negative pressures of −0.98 psi. The maximum negative up to −1.8 psi can be achieved with an effective cavity volume of 268 μl. A maximum flow rate of 24 μl/min was produced in the microfluidic chip with a 180 mm long channel using this technique. The response times of the polymer transformers presented here are within 36 s for driving liquids to the end of the detection chamber. The proposed design has the advantages of compact size, ease of fabrication and integration, ease of actuation, and on-demand negative pressure generation. Thus, this design is suitable for disposable biochips that need two liquid samples control. The polymer transformer presented in this study is applicable to numerous disposable microfluidic biochips.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

Small volume PCR in PDMS biochips with integrated fluid control and vapour barrier

In this paper we demonstrate a new method for microfabricating PDMS devices that controls vapour diffusion, thereby reducing water loss at elevated temperatures and greatly increasing the reliability of the PCR. In the past, the vapour and liquid diffusion properties of the PDMS material in microfluidic devices have impaired performance. We show that this water loss is primarily due to vapour diffusion from the PDMS biochip and by implanting a polyethylene vapour barrier layer in the PDMS, the overall fluid loss was almost eliminated (reduced by a factor of 3). We have also developed a procedure to ensure irreversible bonding between the PDMS and the implant. With this improved microfabrication method we demonstrate the feasibility and advantages of performing small volume PCR genetic amplification (i.e. with less than 2 μl of PCR sample) within a PDMS–glass hybrid biochip. Diaphragm pumps and pinch-off valves were integrated in the system and these enabled fluid retention during the amplification stage and will facilitate higher levels of on-chip automation.     

Optimization design of multi-material micropump using finite element method

This paper presents a micropump fabricated from low cost materials with specific goal of cost reduction. The micropump does not require any valve flap and comprises one plastic pump polyether–ether–ketone (PEEK) body, one metal diaphragm, and three piezoelectric ceramics to form piezoelectrically actuated diaphragm valves. The valve actuation simplifies micropump structural designs and assembly processes to make the pump attractive for low cost bio-medical drug delivery applications. A detailed optimization design of geometric parameters of the piezoelectrically actuated diaphragm is undertaken by use of 3D finite element method (FEM) to maximize piezoelectric actuation capability and ensure actuation reliability. An optimized geometric dimensional design: the ratio of thicknesses between the piezoelectric ceramics and the metal diaphragm, and the lateral dimension of the piezoelectric ceramic, is obtained through simulations. Based on the optimized design, a good agreement has been reached between simulated and measured strokes of the micropumps. The tested results show that the micropump has a high pump flow rate for air, up to 39 ml/min, and for water, up to 1.8 ml/min, and is capable of ensuring diaphragm’s maximum stress and strain is within material strength for reliable work.     

Polymeric (SU-8) optical microscanner driven by electrostatic actuation

A torsional micromechanical scanner was fabricated using photosensitive polymer (SU-8). The proposed polymer-based optical microscanner with reduced torsional stiffness offers a new approach to increase scanning angles. The scanner consists of two parts; the top layer (micro mirror and electrodes) and the bottom layer (anchors and electrodes). The SU-8 scanner is actuated by electrostatic force generated by gap-closing electrodes. For the fabricated optical scanner with the mirror size of 3 × 3 mm², the experimentally obtained scanning angles were 0.43° for 60 Hz (non-resonant) and 1.54° for 1.13 kHz (resonant) at the input voltage of 160 V. This paper also proposes a simple and new fabrication method, which can effectively control the stiffness of the torsional springs by molding SU-8 photoresist through V-groove on the silicon substrate, thereby increasing the scanning angles.     

Design and characterization of a platform for thermal actuation of up to 588 microfluidic valves

In this paper, we describe a large-scale microfluidic valve platform for thermally actuated phase change (PC) microvalves. PC microvalves can be actuated by heat sources such as ohmic resistors, which can be highly integrated resulting in dense arrays of individually addressable microfluidic valves. We present a custom-made electronic platform with custom-written control software that allows controlling a total of 588 individually addressable resistors each of which can be used as the actuator for a separate PC valve. The platform is demonstrated with direct PC microvalve (the simplest example of a PC valve) where working fluid and phase change material are the same media. We present experimental results for single valve setups as well as for a 24 microvalve setup showing the scalability of the system. Furthermore, we demonstrate that precise and individual ‘per-resistor’ temperature profiles are required for valve actuation in order to decrease thermal latency and ensure that the time required for switching the valve state is independent from the “thermal history” (i.e. the duration of the previous valve state) of the valve. To the best of our knowledge, there is no such platform described in the literature, which offers an equal potential for individual valve operation (potentially up to 588 individual valves) as presented in this work.     

A biomechanical assessment of hand/arm force with pneumatic nail gun actuation systems

A biomechanical model is presented to estimate user hand/arm force exertion with two pneumatic nail gun trigger systems. The sequential actuation trigger (SAT) is safer than the contact actuation trigger (CAT) but increases the user's exertion of force because the trigger must be actuated after the safety tip is held pressed against the workpiece. Time integrated hand force was calculated for a single user based on direct measurement of nail gun tip force against the workpiece (tip contact) and from estimated force to support the tool weight during transfer between nails and during idle holding. The model shows that hand/arm force increases when nailing with the SAT (relative to CAT) and with a vertically-oriented workpiece (relative to horizontal). Expressed per nail fired, the user exerted 0.13 Ns (horizontal orientation) and 2.88 Ns (vertical orientation) integrated hand force during tip contact with CAT compared to 26.15 Ns (horizontal) and 46.08 Ns (vertical) with SAT. Depending upon idle holding duration, integrated hand force during tip contact was estimated to have been 1–3% of 48–132 Ns total hand force with CAT and 21–44% of 83–167 Ns total hand force with SAT (average of horizontal and vertical orientations). Based on standard time allowances from work measurement systems it is proposed that efficient application of hand force during tip contact with SAT can reduce this contribution to 6–15% of 55–139 Ns total hand force. The model is useful for considering differences in hand/arm force exertion between the SAT and CAT systems