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.     

A disposable,integrated loop-mediated isothermal amplification cassette with thermally actuated valves

An inexpensive, disposable, integrated, polymer-based cassette for loop-mediated isothermal amplification (LAMP) of target nucleic acids was designed, fabricated, and tested. The LAMP chamber was equipped with single-use, thermally actuated valves made with a composite consisting of a mixture of PDMS and expandable microspheres. The effect of the composite composition on its expansion was investigated, and the valve’s performance was evaluated. In its closed state, the valve can hold pressures as high as 200 kPa without any significant leakage. Both the LAMP chamber and the valves were actuated with thin film heaters. The utility of the cassette was demonstrated by carrying out LAMP of Escherichia coli DNA target and reverse transcribed loop meditated isothermal amplification (RT-LAMP) of RNA targets. The amplicons were detected in real time with a portable, compact detector. The system was capable of detecting as few as 10 target molecules per sample in well under 1 h. The portable, integrated cassette system described here is particularly suited for applications at the point of care and in resource-poor countries, where funds and trained personnel are in short supply.     

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.     

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.     

Rapid glucose concentration detection utilizing disposable integrated microfluidic chip

A novel three-dimensional (3D) disposable glucose concentration detection chip is presented. The chip comprises a four-layer polymethyl methacrylate (PMMA) structure and is fabricated using a commercial CO₂ laser and a hot-press bonding technique. In the proposed device, the glucose solution is injected into a double parallel connection micromixer (DPCM) and is mixed with DNS reagent by means of a self-rotation effect. An experimental platform has been created for multiple reaction process by integrating chip and micro-heater. The fluid streams exiting the two circular mixing chambers of the DPCM are then combined and mixed further at a T-type microchannel outlet before passing to a collection chamber. Numerical simulations are performed to analyze the vortex streamline distribution within the DPCM and to estimate the mixing performance. The numerical results show that a mixing efficiency as high as 92.5% can be obtained at low Reynolds numbers (Re = 12). It is found a good linear relation of R ² = 0.9953 from the chip detection method comparing to the traditional method of R ² = 0.9976 at DNS reagent and glucose solution volume ratio of 1:1. In addition, the experimental results show that the accuracy of the glucose concentration measurements obtained using the proposed microfluidic chip is comparable with that of the measurements obtained using a conventional large-scale detection method. Overall, the results presented in this study indicate that the DPCM chip provides a rapid and low-cost means of detecting the concentration of glucose solutions.     

Automatic detection of Salmonella enterica in sprout irrigation water using a nucleic acid sensor

A nucleic acid sensor capable of automated sample and reagent loading, real-time PCR, automated detection, and sample line cleaning was tested. Real-time PCR reactions were performed with Salmonella enterica in autoclaved and spent alfalfa sprout irrigation water. S. enterica boiled cells were detected over a range of approximately 10⁴ to 10⁸ CFU/reaction (rxn). It was possible to generate enough PCR product to visualize a band on a gel at the expected size over approximately five orders of magnitude from 3.2 × 10³ to 10⁸ CFU/rxn. Automated detection experiments yielded correct identification of 9/9 positive control reactions over a range of 10⁴ to 10⁸ CFU/rxn, correctly identified a negative control reaction, and a sample of 3.2 × 10³ CFU/rxn was incorrectly identified as negative. Primer dimers were not seen in positive or negative control reactions with sprout irrigation water, suggesting that it may be possible to improve the detection limit simply by increasing the number of thermal cycles or by lowering the annealing temperature. The system required no interpretation of real-time PCR data by the operator. The entire process of loading, running the PCR, automated data interpretation, and sample line cleaning was completed in under 2 h and 20 min, significantly faster than it would take to ship a sample and have it tested by an independent laboratory.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-κ-NN′]dichlormanganous and DNA

A new Mn(II) complex of MnL₂Cl₂ (L = azino-di(5,6-azafluorene)-κ²-NN′) was synthesized and utilized as an electrochemical indicator for the determination of hepatitis B virus (HBV) based on its interaction with MnL₂Cl₂. The electrochemical behavior of interaction of MnL₂Cl₂ with salmon sperm DNA was investigated on glassy carbon electrode (GCE). In the presence of salmon sperm DNA, the peak current of [MnL₂]²⁺ was decreased and the peak potential was shifted positively without appearance of new peaks. The binding ratio between [MnL₂]²⁺ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 3.72 × 10⁸ mol² L⁻². The extent of hybridization was evaluated on the basis of the difference between signals of [MnL₂]²⁺ with probe DNA before and after hybridization with complementary sequence. Control experiments performed with non-complementary and mismatch sequence demonstrated the good selectivity of the biosensor. With this approach, a sequence of the HBV could be quantified over the range from 1.76 × 10⁻⁸ to 1.07 × 10⁻⁶ mol L⁻¹, with a linear correlation of r = 0.9904 and a detection limit of 6.80 × 10⁻⁹ mol L⁻¹. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between MnL₂Cl₂ and DNA was found to be primary intercalation binding.     

Environmentally friendly disposable sensors with microfabricated on-chip planar bismuth electrode for in situ heavy metal ions measurement

This paper presents an environmentally friendly disposable heavy metal ion sensor for in situ and online monitoring in the nature and physiological systems. The miniaturized sensor chip consists of a non-toxic microfabricated bismuth (Bi) working electrode that replaces the conventional mercury electrodes, an integrated Ag/AgCl reference electrode, a gold counter electrode, and microfluidic channels. In this work, the electrochemical behavior of the Bi working electrode was characterized in several non-deaerated buffer solutions using cyclic voltammetry. The detection and quantification of Pb (II) and Cd (II) were statically performed using anodic stripping voltammetry inside the microchannels, in the Pb (II) concentration range of 25–400 ppb (R² = 0.991) with limit of detection of 8 ppb for 60 s deposition, and in the Cd (II) concentration range of 28–280 ppb (R² = 0.986) with limit of detection of 9.3 ppb for 90 s deposition. Particularly, the applications of this sensor chip have been reported with the examples of in situ measurement of Cd (II) concentration in soil pore and ground water and online direct measurement of Cd (II) concentration in cell culture media in its native environment.     

Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

Microfluidics is an emerging technology enabling the development of lab-on-a-chip systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. Currently, available nucleic acid diagnostic tests take advantage of polymerase chain reaction that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. At the same time, isothermal methods for DNA amplification are being developed and are preferred for their simplified protocols and the elimination of thermocycling. Here, we present a low-cost and fast DNA amplification device for isothermal helicase dependent amplification implemented in the detection of mutations related to breast cancer as well as the detection of Salmonella pathogens. The device is fabricated by mass production amenable technologies on printed circuit board substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zone necessary for isothermal amplification methods.     

A rapid and selective method for monitoring the growth of coliforms in milk using the combination of amperometric sensor and reducing of methylene blue

A novel method for monitoring the growth of coliforms in milk was developed based on measuring the current change in an amperometric sensor. The sensor consists of a circuit with a homemade potentiostat and a pair of electrodes. The electrode was immersed in milk samples containing methylene blue with various concentrations of bacterial inoculums. The microbial metabolism led to the reduction of methylene blue resulting in a change of current. The time required to identify readily detectable change (detection time, DT) provided an approximate measurement of the amount of microorganisms in the initial inoculums. The sensor system used in this study has the selectivity towards coliform bacteria such as Escherichia coli and Enterobacter aerogenes. The calibration curve of DT against concentration of coliform showed a linear correlation coefficient (R² = 0.9192) over the range of 10²–10⁸ CFU/mL. The sensor was able to detect the coliform bacteria at initial concentrations of 10⁵ CFU/mL within 6 h, making it suitable for use in real-time monitoring of bacterial growth. This system has potential application in the detection of coliform concentration in milk at dairy farms when a proper selective media is designed.     

Feasibility of PSO–ANFIS model to estimate rock fragmentation produced by mine blasting

Desired rock fragmentation is the main goal of the blasting operation in surface mines, civil and tunneling works. Therefore, precise prediction of rock fragmentation is very important to achieve an economically successful outcome. The primary objective of this article is to propose a new model for forecasting the rock fragmentation using adaptive neuro-fuzzy inference system (ANFIS) in combination with particle swarm optimization (PSO). The proposed PSO–ANFIS model has been compared with support vector machines (SVM), ANFIS and nonlinear multiple regression (MR) models. To construct the predictive models, 72 blasting events were investigated, and the values of rock fragmentation as well as five effective parameters on rock fragmentation, i.e., specific charge, stemming, spacing, burden and maximum charge used per delay were measured. Based on several statistical functions [e.g., coefficient of correlation (R ²) and root-mean-square error (RMSE)], it was found that the PSO–ANFIS (with R ² = 0.89 and RMSE = 1.31) performs better than the SVM (with R ² = 0.83 and RMSE = 1.66), ANFIS (with R ² = 0.81 and RMSE = 1.78) and nonlinear MR (with R ² = 0.57 and RMSE = 3.93) models. Finally, the sensitivity analysis shows that the burden and maximum charge used per delay have the least and the most effects on the rock fragmentation, respectively.

     

Applications of soft computing techniques for prediction of energy dissipation on stepped spillways

In this study, numbers type of soft computing including artificial neural network (ANN), support vector machine (SVM), multivariate adaptive regression splines (MARS), and group method of data handling (GMDH) were applied to model and predict energy dissipation of flow over stepped spillways. Results of ANN indicated that this model including hyperbolic tangent sigmoid as transfer function obtained coefficient of determination (R ² = 0.917) and root-mean-square error (RMSE = 6.927) in testing stage. Results of development of SVM showed that developed model consists of radial basis function as kernel function achieved R ² = 0.98 and RMSE = 2.61 in validation stage. Developed MARS model with R ² = 0.99 and RMSE = 0.65 has suitable performance for predicating the energy dissipation. Results of developed GMDH model show with R ² = 0.95 and RMSE = 5.4 has suitable performance for modeling energy dispersion. Reviewing of results of prepared models showed that all of them have suitable performance to predict the energy dissipation. However, MARS and SVM are more accurate than the others. Attention to structures of GMDH and MARS models declared that Froude number, drop number, and ratio of critical depth to height of step are the most important parameters for modeling energy dissipation. The best radial basis function was found that as best kernel function in developing the SVM.

     

A Better Constant-Factor Approximation for Selected-Internal Steiner Minimum Tree

The selected-internal Steiner minimum tree problem is a generalization of original Steiner minimum tree problem. Given a weighted complete graph G=(V,E) with weight function c, and two subsets R ^′ ⊊ R⊆V with |R−R ^′|≥2, selected-internal Steiner minimum tree problem is to find a minimum subtree T of G interconnecting R such that any leaf of T does not belong to R ^′. In this paper, suppose c is metric, we obtain a (1+ρ)-approximation algorithm for this problem, where ρ is the best-known approximation ratio for the Steiner minimum tree problem.     

Evaluation of peach quality indices using an electronic nose by MLR, QPST and BP network

In this paper, responses of a gas sensor array were employed to establish a quality indices model evaluating the peach quality indices. The relationship between sensor signals and the firmness, the content of sugar (CS) and acidity of “Dabai” peach were developed using multiple linear regressions with stepwise procedure, quadratic polynomial step regression (QPST) and back-propagation network. The results showed that the multiple linear regression model represented good ability in predicting of quality indices, with high correlation coefficients (R² = 0.87 for penetrating force CF; R² = 0.79 for content of sugar CS; R² = 0.81 for pH) and relatively low average percent errors (ERR) (9.66%, 7.68% and 3.6% for CF, CS and pH, respectively). The quadratic polynomial step regression provides an accurate quality indices model, with high correlation (R² = 0.92, 0.87, 0.83 for CF, CS and pH, respectively) between predicted and measured values and a relatively low error (5.47%, 3.45%, 2.57% for CF, CS and pH, respectively) for prediction. The feed-forward neural network also provides an accurate quality indices model with a high correlation (R² = 0.90, 0.81, 0.87 for CF, CS and pH, respectively) between predicted and measured values and a relatively low average percent error (6.39%, 6.21%, 3.13% for CF, CS and pH, respectively) for prediction. These results prove that the electronic nose has the potential of becoming a reliable instrument to assess the peach quality indices.     

Conductimetric immunosensor for atrazine detection based on antibodies labelled with gold nanoparticles

A novel conductimetric immunosensor for atrazine detection has been designed and developed. This immunosensor is mainly based on antibodies labelled with gold nanoparticles. Additionally, the immunosensor consists of an array of two coplanar non-passivated interdigitated metallic μ-electrodes (IDμE) and immunoreagents specifically developed to detect this pesticide. The chemical recognition layer was covalent immobilized on the interdigital space. Immunochemical detection of the concentration of atrazine is achieved by a competitive reaction that occurs before the inclusion of the labelled antibodies. It is shown that the gold nanoparticles provide an amplification of the conductive signal and hence makes possible to detect atrazine by means of simple DC measurements.The conductimetric immunosensor and its biofunctionalization steps have been characterized by chemical affinity methods and impedance spectroscopy.This work describes the immunosensor structure, fabrication, physico-chemical and analytical characterization, and the immunosensor response using conductivity measurements. The immunosensor developed detects atrazine with limits of detection in the order of 0.1–1 μg L⁻¹, far below the maximum residue level (MRL) (100 μg L⁻¹) established by European Union (EU) for residues of this herbicide in the wine.Although in this paper the competitive reaction occurs in buffer, an initial study of the wine matrix effect is also described.     

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10² cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.     

Uq(sl(2)) Satisfies a Bernstein Duality

It is well known that in some cases the functorExt_R^μ( − , R) defines a duality between module categories. In earlier papers we studied when this duality can be represented by a bimodule and have characterized when this happens. In this paper, using some computational methods of noncommutative Gröbner bases in the construction of projective resolutions of irreducible finite-dimensional representations, we show new examples of algebras satisfying this property.     

Investigation on nanocrystalline copper-doped zirconia thin films for optical sensing of carbon monoxide at high temperature

Nanocrystalline copper-doped zirconia (CDZ; Cu:Zr = 16:84) thin films have been synthesized on long-period fiber gratings (CDZ-LPFG) by a polymeric precursor method. The CDZ-LPFG device was demonstrated to have high sensitivity and good reversibility for low-concentration CO sensing at high temperatures. The CDZ-LPFG responds with red shifts of its resonant wavelength (λ_R) to CO-containing gases and the λ_R shift reverses when it is exposed to air. The optical response of the CDZ-LPFG to CO is due primarily to the CDZ refractive index variations resulted from the reversible redox reactions (i.e. Cu²⁺ ⇔ Cu⁺) in reducing and oxidizing atmospheres. The magnitude of the λ_R shift exhibited a strong dependence on CO concentration in a range from 0 to 1000 ppm that is potentially useful for quantitative measurement.     

pH-ChemFET-based analysis devices for the bacterial activity monitoring

Silicon and polymer microtechnologies have been developed in order to integrate pH-metry techniques in the frame of medical diagnosis. Thus, a fluidic analysis device has been designed and realised in order to monitor pH-related bacterial activities. It includes a SiO₂/Si₃N₄ pH-sensitive chemical field effect transistor (pH-ChemFET), its titanium/gold pseudo-reference gate electrode and a poly-dimethylsiloxane (PDMS) integrated flow-cell (total volume: ≤2 mm³). The whole analysis device has been used to detect the biological activities of the Lactobacillus crispatus bacteria and to estimate its sensitivity to antibiotics. Results demonstrate the detection of pH-related bacterial metabolisms in microvolumes, enabling to reduce significantly the analysis response time with regards to standard procedures and to the development of pH-ChemFET-metry for medical analysis.