A novel micro-fluidic biosensor for the rapid and sequence-specific detection of DNA with electrophoretic driving mode and laser-induced fluorescence detector

A novel DNA biosensor, which combines the merits of micro-fluidic chips, the electrophoretic driving mode, paramagnetic beads amplification, and laser-induced fluorescence detection was developed for the rapid and sequence-specific detection of DNA in this study. The proposed DNA biosensor has much higher discrimination ability for the detection of single-base mismatch and much stronger resistibility to the complex matrixes of real samples in comparison with previous biosensors. These features, as well as its ease of fabrication (the fabrication of the sensor takes only 10 min except the fabrication of micro-fluidic chip), operation convenience, stability, better re-usability (micro-fluidic chip can be reused without any extra treatment) and short analysis time (one determination only takes 15 min), make it a promising alternative to rapid detection of DNA in clinical diagnosis. With the help of the biosensor, we successfully determined DNA, which related to oral cancer, in a saliva sample without any pre-separation or dilution with a detection limit of 4.2 × 10^?11 M and a relative standard deviation (n = 5) <5 %. The success in the present biosensor served as a significant step toward the practical application of the biosensor in clinical diagnosis.     

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.     

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.     

On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10³ and 1.1 × 10³ CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.     

An effective PDMS microfluidic chip for chemiluminescence detection of cobalt (II) in water

A microfluidic chip for the chemiluminescence detection of cobalt (II) in water samples, based on the measurement of light emitted from the cobalt (II) catalysed oxidation of luminol by hydrogen peroxide in basic aqueous solution, is presented. The microfluidic chip was designed and fabricated from polydimethylsiloxane using micro-molding method. Optimized reagents conditions were found to be 5.0 × 10^?4 mol/L luminol, 1.0 × 10^?2 mol/L hydrogen peroxide, and 8.0 × 10^?2 mol/L sodium hydroxide. The system can perform fully automated detection with a reagent consumption of only 2.4 μL each time. The linear range of the cobalt (II) ions concentration was 1.0 × 10^?10–1.0 × 10^?3 mol/L and the detection limit was 5.6 × 10^?11 mol/L with the S/N ratio of 3. The relative standard deviation was 4.6 % for 1.0 × 10^?5 mol/L cobalt (II) ions (n = 10).     

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10⁻⁷ ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe.     

Measurement of dissolved oxygen diffusion coefficient in a microchannel using UV-LED induced fluorescence method

The diffusion coefficient of dissolved oxygen (DO) was measured in a microchannel using the UV-LED induced fluorescence method. Mass transfer between oxic and anoxic de-ionized (DI) water was quantitatively visualized in a Y-shaped microchannel. Oxygen-sensitive ruthenium (tris (2,2′-bipyridine) ruthenium (II) chloride hexahydrate] and a 450-nm UV-LED were used for the optical measurement of a DO concentration field. In situ pixel-by-pixel calibration was carried out to obtain Stern–Volmer equations to measure the DO concentration field with a spatial resolution of 0.625 μm/pixel. The diffusion layers are successfully acquired for different Reynolds numbers (Re = 0.14, 1.4, and 14). The DO diffusion coefficient is calculated by both the constant-assumed and the concentration-dependent diffusion coefficient methods. The measured DO diffusion coefficient, 2.32 × 10^?9 m²/s, is very close to that of the theoretical prediction of the oxygen gas diffusion coefficient, 2.16 × 10^?9 m²/s.     

Development of a capillary flow microfluidic Escherichia coli biosensor with on-chip reagent delivery using water-soluble nanofibers

Although the potential role of microfluidics in point of care diagnostics is widely acknowledged, the practical limitations to their use still limit deployment. Here, we developed a capillary flow microfluidic with on-chip reagent delivery which combines a lateral flow assay with microfluidic technology. The horseradish peroxidase tagged antibody was electrospun in a water-soluble polyvinylpyrrolidone nanofibers and stored in a microfluidic poly(methyl methacrylate) chip. During the assay, the sample containing Escherichia coli on immunomagnetic beads came in contact with the nanofibers causing them to dissolve and release the reagents for binding. Following hybridization, the solution moved by capillary flow toward a detection zone where the analyte was quantified using chemiluminescence. The limit of detection was found to be approximately 10⁶ CFU/mL of E. coli O157. More importantly, the ability to store sensitive reagents within a microfluidic as nanofibers was demonstrated. The fibers showed almost instant hydration and dissemination within the sample solution.     

Design,optimization and simulation of a low-voltage shunt capacitive RF-MEMS switch

This paper presents the design, optimization and simulation of a radio frequency (RF) micro-electromechanical system (MEMS) switch. The capacitive RF-MEMS switch is electrostatically actuated. The structure contains a coplanar waveguide, a big suspended membrane, four folded beams to support the membrane and four straight beams to provide the bias voltage. The switch is designed in standard 0.35 µm complementary metal oxide semiconductor process and has a very low pull-in voltage of 3.04 V. Taguchi method and weighted principal component analysis is employed to optimize the geometric parameters of the beams, in order to obtain a low spring constant, low pull-in voltage, and a robust design. The optimized parameters were obtained as w = 2.5 µm, L1 = 30 µm, L2 = 30 µm and L3 = 65 µm. The mechanical and electrical behaviours of the RF-MEMS switch were simulated by the finite element modeling in software of COMSOL Multiphysics 4.3^® and IntelliSuite v8.7^®. RF performance of the switch was obtained by simulation results, which are insertion loss of ?5.65 dB and isolation of ?24.38 dB at 40 GHz.     

The use of autonomous vehicles for spatially measuring mean velocity profiles in rivers and estuaries

Autonomous vehicles (AVs) are commonly used in oceanic and more recently estuarine and riverine environments because they are small, versatile, efficient, moving platforms equipped with a suite of instruments for measuring environmental conditions. However, moving vessel observations, particularly those associated with Acoustic Doppler Current Profiler (ADCP) measurements, can be problematic owing to instrument noise, flow fluctuations, and spatial variability. A range of ADCPs manufactured by different companies were integrated on to an Unmanned Surface Vehicle (USV), an Unmanned Underwater Vehicle (UUV), and some additional stationary platforms and were deployed in a number of natural riverine and estuarine environments to evaluate the quality of the velocity profile over the depth, minimum averaging time interval requirements, and AV mission planning considerations. Measurements were obtained at fixed locations to eliminate any spatial variations in the mean flow characteristics. The USV has the unique capability to station-keep to within 1 m owing to its dual-propeller design, providing the best setup for spatially mapping velocity profiles. Single-propeller UUVs can perform a quasi-station-keeping (< 10 m) operation, but are designed for traveling underwater at speeds > 1 m/s. An appropriate averaging window, T _*, was determined using the Kalman Algorithm with a Kalman gain equal to 1%. T _* was found to be independent of depth, flow velocity, and environment. There was no correlation (R ² = 0.18) for T _* between flow magnitude and direction. Results from all measurements had a similar T _* of approximately 3 min. Based on this, an averaging window of 4 min is conservatively suggested to obtain a statistically confident measure of the mean velocity profile.     

Detection of mosaic virus disease in cassava plants by sunlight-induced fluorescence imaging: a pilot study for proximal sensing

Cassava mosaic disease (CMD) is a prominent virus infection that causes considerable crop damage and yield reduction. Early detection of crop damage by remote sensing could be a useful tool for initiating remedial measures to reduce further crop damage. This article presents a non-destructive method for detection and classification of CMD infection, based on the red:far-red chlorophyll (chl) fluorescence image ratio. This pilot study was carried out in 14 varieties of potted cassava plants (Manihot esculenta Crantz) with a multispectral imaging system (MSIS) consisting of an electron multiplying charge coupled device (EMCCD) camera. Sunlight-induced chl fluorescence (SICF) images of plant leaves were recorded using the MSIS at the Fraunhofer lines of O₂-B at 687 nm and O₂-A at 759.5 nm and their off-lines at 684 and 757.5 nm. The recorded images were analysed using the Fraunhofer line discrimination (FLD) technique to extract the SICF from the solar reflectance in the recorded images. The chl fluorescence image ratio (red:far-red, F₆₈₇:F₇₆₀) was computed and correlated with the laser-induced chl fluorescence (LICF) ratio (F₆₈₅:F₇₃₅) determined by point monitoring, chl content variation, and the net photosynthetic rate (P_n). The scatter plot of the F₆₈₇:F₇₆₀ image ratio showed good discrimination between different levels of CMD infection as evidenced by the high sensitivity and specificity values. It is observed that the fluorescence image ratio (F₆₈₇:F₇₆₀) has a good correlation with P_n (coefficient of determination (R²) = 0.85), chl content (R² = 0.82), and the LICF ratio (F₆₈₅:F₇₃₅) (R² = 0.80), thereby highlighting the potential of the SICF image ratio in the discrimination of CMD infection. The results clearly indicate that changes in the red:far-red fluorescence image ratio due to CMD stress can easily be detected at an early stage and the technique has great potential for monitoring the health of crops and vegetation from proximal sensing platforms.     

A projective general linear group based algorithm for the construction of substitution box for block ciphers

The substitution boxes are used in block ciphers with the purpose to induce confusion in data. The design of a substitution box determines the confusion ability of the cipher; therefore, many different types of boxes have been proposed by various authors in literature. In this paper, we present a novel method to design a new substitution box and compare its characteristics with some prevailing boxes used in cryptography. The algorithm proposed in this paper apply the action of projective linear group PGL(2, GF(2⁸)) on Galois field GF(2⁸). The new substitution box corresponds to a particular type of linear fractional transformation (35z + 15)/(9z + 5). In order to test the strength of the proposed substitution box, we apply non-linearity test, bit independence criterion, linear approximation probability method, differential approximation probability method, strict avalanche criterion, and majority logic criterion. This new technique to synthesize a substitution box offers a powerful algebraic complexity while keeping the software/hardware complexity within manageable parameters.     

Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via online pre-concentration of field-amplified sample stacking

In this paper, polydopamine/gold nanoparticles (PDA/Au NPs) were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis (MCE) for the separation of reduced glutathione (GSH) and oxidized glutathione (GSSH). The formation of the PDA/Au NPs was characterized by scanning electron microscopy, transmission electron microscope, UV–Vis spectra and ATR-FTIR. An online pre-concentration strategy involving field-amplified sample stacking was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria (Escherichia coli, Staphylococcus aureus and Salmonella enterica serovar Typhimurium) and HaCaT cells samples by MCE with laser-induced fluorescence detection. The influences of the separation voltage, the concentration of the running buffer and the pH value of running buffer, were carefully investigated. Using this studied method, GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s. The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM, respectively (S/N = 3), which corresponded to approximately 180–301-fold improvements in peak height. Moreover, this method was successfully applied to the analysis of bacteria (E. coli, S. aureus and S. typhimurium) and HaCaT cell samples with a satisfactory recovery rate.     

Maximal and sub-maximal functional lifting performance at different platform heights

Introducing valid physical employment tests requires identifying and developing a small number of practical tests that provide broad coverage of physical performance across the full range of job tasks. This study investigated discrete lifting performance across various platform heights reflective of common military lifting tasks. Sixteen Australian Army personnel performed a discrete lifting assessment to maximal lifting capacity (MLC) and maximal acceptable weight of lift (MAWL) at four platform heights between 1.30 and 1.70 m. There were strong correlations between platform height and normalised lifting performance for MLC (R² = 0.76 ± 0.18, p < 0.05) and MAWL (R² = 0.73 ± 0.21, p < 0.05). The developed relationship allowed prediction of lifting capacity at one platform height based on lifting capacity at any of the three other heights, with a standard error of < 4.5 kg and < 2.0 kg for MLC and MAWL, respectively.     

Two-Color Capillary Electrophoresis with On-Column Excitation and Wave-Guide Based Fluorescent Detection

An optical wave-guide based two-color capillary electrophoresis laser induced fluorescence (CE-LIF) instrument is described. The wave-guide based approach allows for on column excitation and detection with two-color discrimination. The instrument is designed to allow either electrokinetic or hydrodynamic injections. In its present configuration, the attainable limit of detection (LOD, S/N = 3) was 50 X 10⁻²¹ moles of fluorescein with a 488-nm excitation source. This study was designed to test the instrument design for applications in protein analyses. Fluorescent dyes with two different wavelengths were simultaneously separated and detected as were complexes formed by labeled antibodies to NFκB p65 and cdc2p34. Quantification of both proteins in THP-1 cell lysates performed using this approach illustrates a rapid screening application of this instrument.     

Electrochemical determination of glutamic pyruvic transaminase using a microfluidic chip

The activity of glutamic pyruvic transaminase (GPT) is an important clinical evidence for some acute diseases such as acute hepatopathy and myocardial infarction. Thus, there is a demand for rapid determination of GPT in small formats at point-of-need. Herein, we describe a novel method of electrochemical determination of GPT with microfluidic technique. GPT activity was indirectly determined via the electrochemical (EC) detection of nicotinamide adenine dinucleotide (NADH) produced from the GPT transdeamination reaction. A type of microfluidic chip was developed, in which a passive mixer comprising 100 sub-ribs and a three-electrode strip for EC were integrated. To verify the response to NADH, a series of NADH concentrations varying from 19 µM to 5 mM were calibrated with cyclic voltammetry within the microfluidic chip. And a linear relationship with R ² 0.9982 between the peak current and the concentration of NADH was obtained. Then, the GPT activity was determined using the chips containing and not containing a ribs-type mixer. And a linear relationship which contained two sections between the GPT activity and the peak current was obtained. The chip with a ribs-type mixer exhibited the sensitivity of 0.0341 μA U^?1 L in the range of 10–50 U L^?1 and 0.0236 μA U^?1 L in the range of 50–250 U L^?1. And the detection limit of the chip with a ribs-type mixer was 9.25 U L^?1. The complete detection process of GPT activity within the microfluidic chip was realized, and the time-consuming problem was remarkably improved too.     

Chemiluminescence detection for Cu (II) based on 1,10-Phenanthroline-hydrogen peroxide reaction on a PDMS microfluidic chip

A simple, rapid and effective method for the determination of copper (II) in water on a PDMS microfluidic chip with chemiluminescence (CL) detection is presented. The CL reaction was based on oxidation of 1,10-phenanthroline by hydrogen peroxide in basic aqueous solution. Polydimethylsiloxane (PDMS) was chosen as material for fabricating the microfluidic chip with two steps lithography method. Optimized reagents conditions were found to be 6.0 × 10^?5 mol/L 1,10-phenanthroline, 1.2 × 10^?3 mol/L hydrogen peroxide, 6.5 × 10^?2 mol/L sodium hydroxide and 2.0 × 10^?3 mol/L Hexadecyl trimethyl ammonium Bromide (CTMAB). In the continuous flow injection mode the system can perform fully automated detection with a reagent consumption of only 3.4 μL each time. The linear range of the Cu (II) ions concentration was 1.0 × 10^?8 mol/L to 1.0 × 10^?4 mol/L, and the detection limit was 9.2 × 10^?9 mol/L with the S/N ratio of 3. The relative standard deviation was 2.8 % for 1.0 × 10^?6 mol/L Cu (II) ions (n = 8). The most notable features of the detection method are simple operation, rapid detection and easy fabrication of the microfluidic chip.     

Spin coating of hydrophilic polymeric films for enhanced centrifugal flow control by serial siphoning

In this paper, we implement rotational flow control on a polymeric microfluidic “lab-on-a-disc” platform by combining serial siphoning and capillary valving for sequential release of a set of on-board stored liquid reagents into a common (assay) channel. The functionality of this integrated, multi-step, multi-reagent centrifugal assay platform critically depends on the capability to establish very reproducible, capillary-driven priming of the innately only weakly hydrophilic siphon microchannels made from common poly(methyl methacrylate) (PMMA) substrates. Due to the relatively high contact angle of the native PMMA substrate, it was practically impossible to ensure sequential release of on-board stored reagents using the capillary-driven serial siphon valves. In this work, we demonstrate that spin-coated hydrophilic films of poly(vinyl alcohol) (PVA) and (hydroxypropyl)methyl cellulose (HPMC) provide stable contact angles on PMMA substrates for more than 60 days. The deposited films were characterized using contact angle measurements, surface energy calculations and X-ray photoelectron spectroscopy spectra. The PVA and HPMC films reduced the water contact angle of the PMMA substrate from 68° to 22° and 27° while increasing their surface energies from 47 to 62 and 57 mN m^?1, respectively. On the centrifugal microfluidic platform, the films were validated to enable the effective and reproducible priming of the serial siphon microchannels at low rotational frequencies while ensuring that the in-line capillary valves are not opened until their respective burst frequencies are passed. Furthermore, the biocompatibility of the proposed surface modification method was examined, and the platform was used to run a sandwich immunoassay for the detection of human immunoglobulin G, and its performance was proven to be comparable to dynamic coating using surfactants.     

Probe recording technology using novel MEMS devices

In this paper, we present novel micro-electro-mechanical systems (MEMS) devices for unique probe recording technology, where the 1-D cantilever probe array approach requires a small number of cantilever probe tips for a large media platform and hence has higher reliability. The probe storage system is composed of three key MEMS devices: MEMS XY-stage, linear motor and 1-D cantilever probe array with integrated heater. The design and fabrication process of three MEMS devices are given with prototypes. Their performances are discussed with the experimental results. The compact MEMS XY-stage device can be driven with ±20 μm movement, in X- and Y-directions. The miniature linear motor is smoothly driven to move back and forth at the speed of 20 mm/s and step of 150 μm by 150 mA pulse driving current. The indented (writing) bit size of 100 nm on polymer media is achieved by the prototyped cantilever probe tip with integrated heater.