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.     

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.     

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.     

Design of novel camphane-based derivatives with antimycobacterial activity

Although tuberculosis (TB) continues to be one of the leading infectious disease killers globally, it is curable and preventable. Despite the existence of safe, well tolerated and effective drugs used in the TB treatment, the interest in new entities, combinations and regimens increases during the last 10 years. Recently, we reported for a new class of anti-TB agents – camphane-based derivatives with nanomolar activity against Mycobacterium tuberculosis strains. The quantitative structure–activity relationship (QSAR) study on 12 compounds revealed several structural requirements for antimycobacterial activity: two hydrogen bond donors, two or three rings and no large branched substituents. Here, we describe the design of a set of nine novel camphane-based derivatives following these requirements. The compounds were synthesized and tested against M. tuberculosis strain H37Rv. Four of them showed activities in the nanomolar range, significantly higher than the activities in the initial set. The QSAR study based on all 21 derivatives pointed to two main structural requirements for anti-TB activity: two hydrogen bond donors and a side chain with aromatic ring.     

A fast prototype reduction method based on template reduction and visualization-induced self-organizing map for nearest neighbor algorithm

The k nearest neighbor is a lazy learning algorithm that is inefficient in the classification phase because it needs to compare the query sample with all training samples. A template reduction method is recently proposed that uses only samples near the decision boundary for classification and removes those far from the decision boundary. However, when class distributions overlap, more border samples are retrained and it leads to inefficient performance in the classification phase. Because the number of reduced samples are limited, using an appropriate feature reduction method seems a logical choice to improve classification time. This paper proposes a new prototype reduction method for the k nearest neighbor algorithm, and it is based on template reduction and ViSOM. The potential property of ViSOM is displaying the topology of data on a two-dimensional feature map, it provides an intuitive way for users to observe and analyze data. An efficient classification framework is then presented, which combines the feature reduction method and the prototype selection algorithm. It needs a very small data size for classification while keeping recognition rate. In the experiments, both of synthetic and real datasets are used to evaluate the performance. Experimental results demonstrate that the proposed method obtains above 70 % speedup ratio and 90 % compression ratio while maintaining similar performance to kNN.     

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.     

Identification of novel inhibitors against Mycobacterium tuberculosis l-alanine dehydrogenase (MTB-AlaDH) through structure-based virtual screening

Mycobacterium tuberculosis (MTB) the etiological agent of tuberculosis (TB) survives in the human host for decades evading the immune system in a latent or persistent state. The Rv2780 (ald) gene that codes for l-alanine dehydrogenase (l-AlaDH) enzyme catalyzes reversible oxidative deamination of l-alanine to pyruvate and is overexpressed under hypoxic and nutrient starvation conditions in MTB. At present, as there is no suitable drug available to treat dormant tuberculosis; it is essential to identify drug candidates that could potentially treat dormant TB. Availability of crystal structure of MTB l-AlaDH bound with co-factor NAD⁺ facilitated us to employ structure-based virtual screening approach to obtain new hits from a commercial library of Asinex database using energy-optimized pharmacophore modeling. The resulting pharmacophore consisted of three hydrogen bond donor sites (D) and two hydrogen bond acceptor sites (A). The database compounds with a fitness score more than 1.0 were further subjected to Glide high-throughput virtual screening and docking. Thus, we report the identification of best five hits based on structure-based design and their in vitro enzymatic inhibition studies revealed IC₅₀ values in the range of 35–80 μM.     

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.     

NEWS SECTION

Abstract

Studies on brightness temperature (TB) data of 325 passes acquired by Bhaskara-II satellite microwave radiometers (SAMIR) at 19 [sdot] 35, 22 [sdot] 235 and 31 [sdot] 4 GHz indicate that certain passes have interesting TB data trends over land and sea in spite of coarse ground resolution. Here an attempt is made to analyse such passes in terms of the ground features and agrochmatic conditions. Certain passes have shown more or less constant TB values over a long stretch of the pass. Average TB data values have been extracted from such passes and are summarized.     

Systematic review on the proteomic profile of Mycobacterium tuberculosis exposed to drugs

The authors present an overview about proteomics studies in Mycobacterium tuberculosis exposed to some anti‐tuberculosis drugs and new candidates, using two‐dimensional gel electrophoresis and mass spectrometry. To date, that the authors have knowledge, this is the first studies that was performed specifically in M. tuberculosis using systematic review on electronic literature conducted in three databases using the following search terms: tuberculosis OR mycobacterium tuberculosis, proteome OR proteomics, and mass spectrometry electrospray ionization OR matrix‐assisted laser desorption ionization OR two‐dimensional gel electrophoresis. By electronic search, 622 abstracts of the original articles published from November 2003 to March 2016 were selected. After the selection, four articles fulfill proposed criteria and were included in this study. The studies reported changes in the protein profile of M. tuberculosis after exposure to isoniazid, ethambutol, streptomycin, ofloxacin, moxifloxacin and two new drugs candidates, SQ109 and ATB107. In conclusion, the proteins changes were related to the synthesis of mycolic acids, cellular metabolism pathways, bacterial stress and starvation.     

Maximal Pairs of Computably Enumerable Sets in the Computably Lipschitz Degrees

A set A is computably Lipschitz or cl-reducible, for short, to a set B if A is Turing reducible to B by an oracle Turing machine with use function ? such that ? is bounded by the identity function up to an additive constant, i.e., ?(n)??n+O(1). In this paper we study maximal pairs of computably enumerable (c.e.) cl-degrees or maximal pairs, for short, i.e., pairs of c.e. cl-degrees such that there is no c.e. cl-degree that is above both cl-degrees in this pair. Our main results are as follows. (1) A c.e. Turing degree contains a c.e. cl-degree that is half of a maximal pair if and only if this Turing degree contains a maximal pair if and only if this Turing degree is array noncomputable. (2) The cl-degrees of all weak truth-table complete sets are halves of maximal pairs while there is a Turing complete set A such that the cl-degree of A is not half of any maximal pair. In fact, any high c.e. Turing degree contains a c.e. cl-degree that is not half of a maximal pair. (3) Above any c.e. cl-degree there is a maximal pair. (4) There is a maximal pair which at the same time is a minimal pair. (5) There is a pair of c.e. cl-degrees that is not maximal and does not possess a least upper bound. Moreover, we make some observations on the structure of the c.e. cl-degrees in general. For instance, we give a very simple proof of the fact that there are no maximal c.e. cl-degrees.     

Homology modelling,docking, pharmacophore and site directed mutagenesis analysis to identify the critical amino acid residue of PknI from Mycobacterium tuberculosis

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen. PknI is one of the 11 functional Serine/Threonine Protein Kinases which is predicted to regulate the cell division of M. tuberculosis. In order to find newer drugs and vaccine we need to understand the pathogenesis of the disease. We have used the bioinformatics approach to identify the functionally active residues of PknI and to confirm the same with wet lab experiments. In the current study, we have created homology model for PknI and have done comparative structural analysis of PknI with other kinases. Molecular docking studies were done with a library of kinase inhibitors and T95 was found as the potent inhibitor for PknI. Based on structure based pharmacophore analysis of kinase substrate complexes, Lys 41 along with Asp90, Val92 and Asp96 were identified as functionally important residues. Further, we used site directed mutagenesis technique to mutate Lys 41 to Met resulting in defective cell division of Mycobacterium smegmatis mc². Overall, the proposed model together with its binding features gained from pharmacophore docking studies helped in identifying ligand inhibitor specific to PknI which was confirmed by laboratory experiments.     

Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection

Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.     

A compact electromagnetic micro-actuator using the meander springs partially exposed to magnetic field

We present the design, fabrication, and testing of a compact electromagnetic micro-actuator using meander springs that are partially exposed to the magnetic field. The previous electromagnetic micro-actuators using cantilever springs require large electromagnetic force for large displacement actuation and therefore require high-current input and a large magnet. The previous electromagnetic actuators using low-stiffness meander springs are limited in large displacement actuation because actuation forces cancelled each other due to changes in the current path under the magnetic field. In this paper, we compare two prototypes: a conventional prototype F with the meander springs exposed fully to the magnetic field and the present prototype P with the meander springs partially exposed to the magnetic field. In the experimental study, the amplitude of prototype P is measured as 30.49 ± 0.36 µm at the 40 mA square input current, while the amplitude of prototype F is measured as 26.02 ± 0.65 µm. The amplitude of prototype P is 17.2 % larger than that of prototype F, verifying the effect of partial exposure of the meander springs to the magnetic field. Therefore, we experimentally demonstrate the large-displacement actuation performance of the present actuator using the partial exposure of the meander springs to the magnetic field.     

Multi-algorithmic cancelable fingerprint template generation based on weighted sum rule and <Emphasis Type="Italic">T</Emphasis>-operators

With the emergence of biometric-based authentication systems in real-world applications, template protection in biometrics is a significant issue to be considered in the recent years. This paper presents two feature set computation algorithms, namely nearest neighbor feature set (NNFS) and Delaunay triangle feature set (DTFS), for a fingerprint sample. Further, the match scores obtained from these algorithms are fused using weighted sum rule and T-operators (T-norms and T-conorms). The experimental evaluation done on FVC 2002 databases confirms the credibility of fusion method compared to each individual algorithm used for fusing. The EER obtained for proposed method is 0 %, 0.059 %, and 3.93 % for FVC 2002 DB1, DB2, and DB3 databases, respectively. This paper also aims to prove the effectiveness of applying T-operators for fusion at score level in fingerprint template protection.     

Analytical solution of plane Poiseuille flow within Burnett hydrodynamics

In the current paper, low-speed isothermal microscale gas flows have been investigated utilizing the augmented Burnett equations. There has been limited success to analytically solve the Burnett equations till date. We propose an analytical solution to Burnett equations, which is shown to satisfy the full set of augmented Burnett equations up to Kn of 2.2 with an error of 1 %. Detailed validation shows that the solution represents the integral flow parameters accurately up to Kn ~ 2.2 and local field properties up to Kn ~ 0.5. The range over which the proposed Burnett analytical solution is applicable is substantially better than existing analytical solutions, without incorporating any wall scaling functions into constitutive relations and variation of slip coefficients in the boundary conditions. Normalized mass flow rate, friction factor, and axial velocity profile results show very good agreement with the experimental and simulation data. The analytical solution is also able to predict the change in the curvature of streamwise pressure profile.     

Integration of ligand and structure based approaches for identification of novel MbtI inhibitors in Mycobacterium tuberculosis and molecular dynamics simulation studies

Mycobacterium tuberculosis is an obligate pathogen of mammals and is responsible for more than two million deaths annually. The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. M. tuberculosis acquires iron exclusively through the siderophores. Several lines of evidence suggest that siderophores have a critical role in bacterial growth and virulence. Hence, in the present study, we have used a combined ligand and structure-based drug design approach for identification of novel inhibitors against salicylate synthase MbtI, a unique and essential enzyme for the biosynthesis of siderophores in M. tuberculosis. We have generated the ligand based and structure based pharmacophores and validated exhaustively. From the validation results it was found that GH (Goodness of Hit) scores for the selected ligand based and structure based pharmacophore models were 0.89 and 0.97, respectively, which indicate that the quality of the pharmacophore models are acceptable as GH value is >0.7. The validated pharmacophores were used for screening the ZINC database. A total of 73 hits, obtained through various insilico screening techniques, were further enriched to 17 hits using docking studies. Molecular dynamics simulations were carried out to compare the binding mode and stability of complexes of MbtI bound with substrate, known inhibitors, and three top ranked hits. The results obtained in this study gave assurance about the identified hits as prospective inhibitors of MbtI.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

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).     

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.