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1.
Sci Total Environ ; 686: 538-545, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185401

RESUMO

The wetland-microbial fuel cell (MFC) is a novel electricity generating technology. However, these systems can generate only limited electric energy. Since nitrification is a key mechanism driving electrical power in wetland-MFC systems, an effective nitrifying bacteria, Bacillus thuringiensis, was used to inoculate a wetland-MFC to enhance the maximum power density of the system. B. thuringiensis effectively enhanced the maximum power density, producing about 20-35 mW m-2 of maximum power density. Interestingly, over the first 120 days of operation, the wetland-MFC system with only B. thuringiensis generated more power than a system containing an Echinodosus cordifolius plant in addition to B. thuringiensis, because E. cordifolius can took up nitrate (NO3-) and phosphate (PO43-) in system's solution. Nitrate and PO43- act as important anions driving electric current in the system. After 120 days of operation though, the combined E. cordifolius and B. thuringiensis system maintained 20-35 mW m-2 maximum power density and the maximum power density of the system only inoculated with B. thuringiensis decreased continuously. Gene (16S rRNA) copy numbers for B. thuringiensis showed that when E. cordifolius was presented, the bacterium was able to continue growing after 120 days of operation. B. thuringiensis did not grow as well after 120 days in the system that did not contain a plant. This study presents a strategy for enhancing electric power output from a wetland-MFC by inoculating the system with B. thuringiensis and maintaining the bacterium's population with the support of an E. cordifolius plant. The result clearly show that B. thuringiensis can enhance electric power generation in the presence of the plant and the system can self-sustain for longer than 180 days of operation while producing 20-35 mW m-2 maximum power density.


Assuntos
Alismataceae/fisiologia , Bacillus thuringiensis/fisiologia , Fontes de Energia Bioelétrica , Áreas Alagadas
2.
Biotechnol Appl Biochem ; 66(5): 842-849, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228877

RESUMO

Salmonella Typhimurium is a major cause of food poisoning. To solve the limitations of the routine enzyme linked immunosorbent assay such as laborious assay procedure, lack of long-term enzyme stability, and insufficient sensitivity, we provided a non-enzymatic colorimetric immunosorbent assay platform to overcome these problems. The highly photostable redox dye particles was constructed by silica particles (diameter = 598 ± 14.4 nm) loaded with methylene blue (Si-MB) and applied to be a label for immunoassay of S. Typhimurium. The sandwich assay format involved incubation of an analyte in a microplate wells modified with monoclonal anti-Salmonella, followed by exposure to a polyclonal anti-Salmonella/Si-MB bioconjugate and then measurement of absorbance at 598 nm. The platform had an assay time of 20 min, could detect heat-killed Salmonella with a limit of detection of 48 CFU mL-1 , and gave good recoveries in milk. The labels could be stored at 4 °C for 70 days without any deterioration.


Assuntos
Imunoensaio , Azul de Metileno/química , Salmonella typhimurium/isolamento & purificação , Dióxido de Silício/química , Tamanho da Partícula , Processos Fotoquímicos , Propriedades de Superfície
3.
Anal Chim Acta ; 1025: 108-117, 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-29801598

RESUMO

Drug susceptibility testing (DST) for Mycobacterium tuberculosis currently faces multiple challenges, including lengthy ineffective standard methods, the expensive cost of molecular tests, and large bulky diagnostic machines. In this work, a disposable MPT64 sensor was developed to rapidly determine drug susceptibility (DS-TB) and multidrug resistant tuberculosis (MDR-TB) using an electrochemical sandwich-immunosensor for the detection of MPT64 as an indicator of Mycobacterium tuberculosis growth. Anti-MPT64 (as capture antibody; cAb) was immobilised on screen printed carbon electrodes to specifically bind with MPT64 target protein. A reporter probe of anti-MPT64 conjugated with horseradish peroxidase (HRP labeled rAb) then completed the sandwich immunocomplex. The current signals were received from the catalytic reaction between 3,3'-5,5'-tetramethylbenzidine (TMB), H2O2 and HRP labeled rAb using the chronoamperometric mode on a portable potentiostat. Increasing MPT64 concentration was taken as an indicator of growth, which was measured by the disposable MPT64 sensor. This sensor shows the possibility of determining DST by comparing the signals of DS-TB and MDR-TB growth in drug-free and drug-containing liquid medium. The time required to identify DS-TB and MDR-TB in pure culture MTB and leftover sputum sediments from patients are 3 days and 4-6 days, respectively. Therefore, using this sensor is significantly rapid and inexpensive and has the potential to be used for drug susceptibility testing of tuberculosis in middle to low income countries.


Assuntos
Antituberculosos/farmacologia , Técnicas Eletroquímicas/instrumentação , Testes de Sensibilidade Microbiana/instrumentação , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Técnicas Eletroquímicas/economia , Técnicas Eletroquímicas/métodos , Desenho de Equipamento , Humanos , Imunoensaio/economia , Imunoensaio/instrumentação , Imunoensaio/métodos , Testes de Sensibilidade Microbiana/economia , Testes de Sensibilidade Microbiana/métodos , Fatores de Tempo
4.
PLoS One ; 13(5): e0196383, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746494

RESUMO

Morphological transformations in primitive organisms have long been observed; however, its biomechanical roles are largely unexplored. In this study, we investigate the structural advantages of dimorphism in Arthrospira platensis, a filamentous multicellular cyanobacterium. We report that helical trichomes, the default shape, have a higher persistence length (Lp), indicating a higher resistance to bending or a large value of flexural rigidity (kf), the product of the local cell stiffness (E) and the moment of inertia of the trichomes' cross-section (I). Through Atomic Force Microscopy (AFM), we determined that the E of straight and helical trichomes were the same. In contrast, our computational model shows that I is greatly dependent on helical radii, implying that trichome morphology is the major contributor to kf variation. According to our estimation, increasing the helical radii alone can increase kf by 2 orders of magnitude. We also observe that straight trichomes have improved gliding ability, due to its structure and lower kf. Our study shows that dimorphism provides mechanical adjustability to the organism and may allow it to thrive in different environmental conditions. The higher kf provides helical trichomes a better nutrient uptake through advection in aquatic environments. On the other hand, the lower kf improves the gliding ability of straight trichomes in aquatic environments, enabling it to chemotactically relocate to more favorable territories when it encounters certain environmental stresses. When more optimal conditions are encountered, straight trichomes can revert to their original helical form. Our study is one of the first to highlight the biomechanical role of an overall-shape transformation in cyanobacteria.


Assuntos
Forma Celular/fisiologia , Spirulina/citologia , Spirulina/metabolismo , Fenômenos Bioquímicos , Transporte Biológico/fisiologia , Fenômenos Biomecânicos , Biofísica , Simulação por Computador , Cianobactérias/citologia , Cianobactérias/metabolismo , Cianobactérias/fisiologia , Tricomas/fisiologia
5.
ACS Sens ; 3(6): 1149-1155, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29808674

RESUMO

The detection and identification of multiple components in a complex sample such as food in a cost-effective way is an ongoing challenge. The development of on-site and rapid detection methods to ensure food quality and composition is of significant interest to the food industry. Here we report that an electrochemical method can be used with an unmodified glassy carbon electrode for the identification of the key ingredients found within Thai green curries. It was found that green curry presents a fingerprint electrochemical response that contains four distinct peaks when differential pulse voltammetry is performed. The reproducibility of the sensor is excellent as no surface modification is required and therefore storage is not an issue. By employing particle swarm optimization algorithms the identification of ingredients within a green curry could be obtained. In addition, the quality and freshness of the sample could be monitored by detecting a change in the intensity of the peaks in the fingerprint response.


Assuntos
Técnicas Eletroquímicas , Qualidade dos Alimentos , Especiarias/análise , Algoritmos , Carbono/química , Eletrodos , Tailândia
6.
Nanoscale ; 10(12): 5466-5473, 2018 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-29445795

RESUMO

Zwitterionic nanoparticles are typically utilized as nanoprobes and delivery vehicles in nanomedicine and therapeutics due to their resistance to interferences. Their high stability also shows great potential to be applied in sensing applications. Here, we report a selective, sensitive and rapid colorimetric sensing of nickel ions (Ni2+) using zwitterionic polypeptide, EKEKEKPPPPC (EK)3, capped gold nanoparticles (AuNP-(EK)3). By taking advantage of the alternate carboxylic (-COOH)/amine (-NH2) groups, the zwitterionic peptide can function dually by being able to sense metal ions and maintain colloidal stability. Ni2+ can trigger the aggregation of the AuNP-(EK)3 nanoprobe, which results in a red-to-purple color change of the AuNP-(EK)3 solution. Our 40 nm AuNP-(EK)3 nanoprobe can detect Ni2+ as low as 34 nM within 15 min with a linear range of 60-160 nM, and is stable in soil, urine and water samples. We demonstrate that the aggregation mechanism of the nanoprobe is due to the interactions between the -NH2 group of glutamic acid at the N-terminus of the peptide and Ni2+, and the aggregation process is reversible. Furthermore, the slight modification of two amino acid sequences at the N-terminus allows the nanoprobe to retain its stability, even in a high ionic strength medium. We believe that by adjusting or extending the peptide sequences, new metal ion selective peptides could be created.

7.
Anal Chim Acta ; 990: 67-77, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29029744

RESUMO

Conducting polymers with graphene/graphene oxide hydrogels represent a unique class of electrode materials for sensors and energy storage applications. In this article, we report a facile in situ method for the polymerisation of aniline resulting in the decoration of 1D conducting polyaniline (PANI) nanofibers onto the surface of 2D graphene oxide (GO) nanosheets followed by hydrogel formation at elevated temperature. The synthesized nanomaterial exhibits significant properties for the highly sensitive electrochemical determination as well as removal of environmentally harmful lead (Pb2+) ions. The square wave anodic stripping voltammetry (SWASV) determination of Pb2+ ions showed good electroanalytical performance with two linear ranges in 0.2-250 nM (correlation coefficient = 0.996) and 250-3500 nM (correlation coefficient = 0.998). The developed protocol has shown a limit of detection (LOD) of about 0.04 nM, which is much lower than that of the World Health Organization (WHO) threshold limits. The prepared electrode showed an average of ∼99.4% removal of Pb2+ ions with a relative standard deviation (RSD) of 3.4%. Selectivity of the electrode towards Pb2+ ions were tested in presence of potential interferences such as Na+, K+, Ca2+, Mg2+, Cu2+, Cd2+, Hg2+, Zn2+, Co2+, Ni2+, Fe2+ and Fe3+ of similar and higher concentrations. The sensor showed good repeatability and reproducibility. The developed protocol was used to analyse samples from industrial effluents and natural water samples. The results obtained were correlated with atomic absorption spectroscopy (AAS).

8.
Talanta ; 167: 14-20, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28340704

RESUMO

We have constructed biobarcode labels based on 468nm diameter latex spheres. Modification with polyallylamine and then glutaraldehyde was used to attach a high DNA loading, consisting of aminated probe DNA (approx. 1.01×102 molecules per sphere) and biobarcode DNA (approx. 1.66×104 molecules per sphere). Detection of the biobarcodes was performed by application of a Ag enhancer solution, causing association of the Ag+ ions with the phosphate groups of the DNA. The deposited Ag was detected by differential pulse voltammetry. A 30 mer sequence from the BL21 strain of E. coli was detected with an LOD of 2.6fM (calibration range 10 aM to 0.1pM, r2=0.91, n=45). The LOD was lowered to 0.56aM (calibration range 100zM to 0.1nM, r2=0.991, n=50) by utilizing a sandwich assay with PNA-modified screen printed electrodes, which lowered the Ag background current. The sandwich assay platform was used to calibrate E. coli strain BL2(DE3) with an LOD of 17.0 CFU mL-1 (calibration range 10 to 106 CFU mL-1, r2=0.99, n=33) with good discrimination against Salmonella.


Assuntos
DNA Bacteriano/análise , DNA Bacteriano/química , Eletroquímica/instrumentação , Látex/química , Ácidos Nucleicos Peptídicos/química , DNA Bacteriano/genética , Eletrodos , Escherichia coli/genética , Hibridização de Ácido Nucleico , Impressão
9.
ACS Nano ; 10(2): 2243-50, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26766635

RESUMO

DNA nanotechnology offers precise geometrical control of the positioning of materials, and it is increasingly also being used in the development of nanomechanical devices. Here we describe the development of a nanomechanical device that allows switching of the position of a single-molecule conjugated polymer. The polymer is functionalized with short single-stranded (ss) DNA strands that extend from the backbone of the polymer and serve as handles. The DNA polymer conjugate can be aligned on DNA origami in three well-defined geometries (straight line, left-turned, and right-turned pattern) by DNA hybridization directed by single-stranded guiding strands and ssDNA tracks extending from the origami surface and polymer handle. We demonstrate switching of a conjugated organic polymer conformation between left- and right-turned conformations of the polymer on DNA origami based on toehold-mediated strand displacement. The switching is observed by atomic force microscopy and by Förster resonance energy transfer between the polymer and two different organic dyes positioned in close proximity to the respective patterns. Using this method, the polymer conformation can be switched six times successively. This controlled nanomechanical switching of conjugated organic polymer conformation demonstrates unique control of the shape of a single polymer molecule, and it may constitute a new component for the development of reconfigurable nanophotonic and nanoelectronic devices.


Assuntos
DNA de Cadeia Simples/química , Nanoconjugados/química , Transferência Ressonante de Energia de Fluorescência
10.
Biosens Bioelectron ; 77: 805-11, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26513287

RESUMO

The ability of a diagnostic test to detect multiple pathogens simultaneously is useful to obtain meaningful information for clinical treatment and preventive measures. We report a highly sensitive and specific electrochemical biosensor assay for simultaneous detection of three gene targets using quantum dots (QDs). The targets are novel non-protein coding RNA (npcRNA) sequences of Vibrio cholerae, Salmonella sp. and Shigella sp., which cause diarrheal diseases. QDs (PbS, CdS, ZnS) were synthesized and functionalized with DNA probes that were specific to each pathogen. Electrochemical detection of QDs was performed using square wave anodic stripping voltammetry (SWASV). The QDs gave distinct peaks at 0.5 V (PbS), 0.75 V (CdS) and 1.1 V (ZnS). There was no interference in signal response when all three QDs were mixed and detected simultaneously. The detection limits of single and multiplex assays with linear targets and PCR products were in the attomolar ranges. The high assay sensitivity, in combination with specific npcRNA sequences as novel diagnostic targets, makes it a viable tool for detecting pathogens from food, environment and clinical samples.


Assuntos
Condutometria/instrumentação , Microquímica/instrumentação , Pontos Quânticos , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA não Traduzido/química , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem
11.
Anal Chim Acta ; 896: 152-9, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26481999

RESUMO

A facile and simple paper-based scanometric assay was developed to detect Pb(2+) using GR5-DNAzyme. Magnetic beads (MBs) and gold nanoparticles (AuNPs) were used as a signal collector and a signal indicator, respectively. They were linked together by GR5-DNAzyme, comprising an enzyme and a substrate strand pairing up with each other. In the presence of Pb(2+), the substrate strand is cut into two pieces, resulting in the disassembly of AuNPs from the MBs. These AuNPs were spotted on predefined areas on a chromatography paper, where signal is amplified through silver reduction. This sensing platform exhibits high sensitivity and selectivity toward Pb(2+), giving a detection limit of 0.3 nM and a linear fitting range from 0.1 to 1000 nM. Testing of this biosensor in river water and synthetic urine samples also showed satisfying results. Besides offering simultaneous and multi-sample analysis, this paper-based sensing platform presented here could be potentially applied and served as a general platform for on-site, naked eyes, and low-cost monitoring of other heavy metal ions in environmental and body fluid samples.


Assuntos
Técnicas Biossensoriais , DNA Catalítico/metabolismo , Chumbo/análise , Papel , Ouro/química , Ouro/metabolismo , Íons/análise , Íons/metabolismo , Chumbo/metabolismo , Nanopartículas Metálicas/química
12.
J Biomed Nanotechnol ; 11(4): 702-10, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26310076

RESUMO

An ultrasensitive electrochemical genosensing assay was developed for the sequence-specific detection of Vibrio cholerae DNA using magnetic beads as the biorecognition surface and gold nanoparticle-loaded latex microspheres (latex-AuNPs) as a signal-amplified hybridization tag. This biorecognition surface was prepared by immobilizing specific biotinylated capturing probes onto the streptavidin-coupled magnetic beads. Fabricating a hybridization tag capable of amplifying the electrochemical signal involved loading multiple AuNPs onto polyelectrolyte multilayer film-coated poly(styrene-co-acrylic acid) latex microspheres as carrier particles. The detection targets, single-stranded 224-bp asymmetric PCR amplicons of the V. cholerae lolB gene, were sandwich-hybridized to magnetic bead-functionalized capturing probes and fluorescein-labeled detection probes and tagged with latex-AuNPs. The subsequent electrochemical stripping analysis of chemically dissolved AuNPs loaded onto the latex microspheres allowed for the quantification of the target amplicons. The high-loading capacity of the AuNPs on the latex microspheres for sandwich-type dual-hybridization genosensing provided eminent signal amplification. The genosensing variables were optimized, and the assay specificity was demonstrated. The clinical applicability of the assay was evaluated using spiked stool specimens. The current signal responded linearly to the different V. cholerae concentrations spiked into stool specimens with a detection limit of 2 colony-forming units (CFU)/ml. The proposed latex-AuNP-based magnetogenosensing platform is promising, exhibits an effective amplification performance, and offers new opportunities for the ultrasensitive detection of other microbial pathogens.


Assuntos
Técnicas Biossensoriais , Eletroquímica/métodos , Ouro/química , Nanopartículas Metálicas/química , Vibrio cholerae/metabolismo , DNA/química , Desenho de Equipamento , Látex , Magnetismo , Microscopia Eletrônica de Transmissão , Microesferas , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Células-Tronco
13.
Biosens Bioelectron ; 73: 181-187, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26067330

RESUMO

We report a highly sensitive method for the electrochemical detection of genomic DNA, based on the employment of two sub-micron oligonucleotide labels - one for magnetic collection and the other for voltammetric detection - and their incorporation onto a stem loop DNA probe. The magnetic label consists of a latex particle of mean diameter 441±6 nm, bearing magnetic Fe3O4 particles and approx. 3.5×10(5) anti-DIG antibodies. The voltammetric label is a hollow polyelectrolyte shell containing approx. 1.0×10(11) Au atoms in the form of well dispersed Au nanoparticles. A DIG tag on one arm of the stem loop enables binding to the magnetic label, while a thiol tag on the other arm enables attachment to the Au nanoparticles. Due to steric hindrance from the two relatively large labels, attachment of both moieties is dependant on target-probe hybridisation straightening the loop. Once attached, sensitive DNA measurement is facilitated by magnetic collection of the DNA into a small volume and by the high quantity of Au atoms available for detection. Using differential pulse anodic stripping voltammetry we calibrated a 30 mer sequence common to 71 strains of Escherichia coli across the concentration range from 0.1 aM to 100 pM with a LOD of 1.8 aM. Three strains of E. coli, BL 21, ATCC8739, O157:H7, when spiked into UHT milk and fermented palm juice, could be detected with LODs of approx. 2-4 CFU mL(-1) in an assay time of approx. 140 min.


Assuntos
Sondas de DNA , DNA/análise , DNA/genética , Técnicas Eletroquímicas/métodos , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/estatística & dados numéricos , Eletrólitos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Microbiologia de Alimentos , Ouro , Limite de Detecção , Magnetismo , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Polímeros
14.
Talanta ; 139: 167-73, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25882423

RESUMO

Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs-PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs-PSA-avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1aM to 1fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50ngµl(-1). The novel lyophilized AuNPs-PSA-avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.


Assuntos
Técnicas Biossensoriais/métodos , Cólera/diagnóstico , DNA Bacteriano/análise , Técnicas Eletroquímicas/métodos , Ouro/química , Nanopartículas Metálicas/química , Vibrio cholerae/genética , Bioensaio , Cólera/microbiologia , Liofilização , Humanos , Látex , Limite de Detecção , Microesferas , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos
15.
Biomicrofluidics ; 8(3): 034108, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-25379069

RESUMO

A simple microwell-based microfluidic chip for microalgal cells trapping was fabricated. An electrostaticcell trapping mechanism, enabled by a positively charged glass surface, was used. The chip was capable of capturing multiple algal cell types. In the case of filamentous Spirulina platensis, we observed single filament occupancy of up to ∼30% available wells, as high as some previously proposed methods. Captured filaments were not of any preferential size, suggesting well randomized cell trapping. It was found that the electrostatic attraction did not affect the cell growth. Total replacement of liquid inside the wells could be achieved by pumping new solutions via the inlet, making single cell experiments in controlled chemical conditions possible. After the top layer of the chip was removed, cells in the wells could be simply transferred using a micropipette, turning the chip into a platform for strain selection.

16.
Analyst ; 139(22): 5740-6, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25262699

RESUMO

We have described a highly sensitive method for detecting DNA hybridisation using a redox-labeled stem loop probe. The redox labels were poly(styrene-co-acrylic) (PSA) spheres of 454 nm diameter, modified by methylene blue (MB) deposited alternatively with poly(sodium 4-styrene sulphonate) (PSS) in a layer-by-layer process. Each PSA sphere carried approx. 3.7 × 10(5) molecules of MB, as determined optically. DIG-tagged stem loop probes were immobilised on screen printed electrodes bearing anti-DIG antibodies. Binding with the target enabled straightening of the stem loop, which made attachment to the MB-coated PSA spheres possible. For measuring the current from the direct reduction of MB by differential pulse voltammetry, a 30 mer DNA target common to 70 strains of Escherichia coli was calibrated across the range 1.0 fM to 100 pM (gradient = 3.2 × 10(-8) A (log fM)(-1), r(2) = 0.95, n = 60), with an LOD of ∼58 fM. By using Fe(CN)6(3-/4-) as a solution phase mediator for the MB reduction, we were able to lower the LOD to ∼39 aM (gradient = 5.95 × 10(-8) A (log aM)(-1), r(2) = 0.96, n = 30), which corresponds to the detection of 0.76 ag (∼50 molecules) in the 2 µL analyte sample. We hypothesise that the lowering of the LOD was due to the fact that not all the MB labels were able to contact the electrode surface.


Assuntos
DNA Bacteriano/química , Técnicas Eletroquímicas/normas , Eletrodos , Hibridização de Ácido Nucleico , Sequência de Bases , Calibragem , Escherichia coli/genética , Limite de Detecção , Oxirredução , Espectrofotometria Ultravioleta
17.
Talanta ; 117: 312-7, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24209346

RESUMO

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.


Assuntos
Aphanomyces/isolamento & purificação , DNA Intergênico/genética , Ouro/química , Nanopartículas Metálicas/química , RNA Ribossômico 18S/genética , Animais , Aphanomyces/genética , Primers do DNA/química , Técnicas Eletroquímicas , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Limite de Detecção , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
18.
Analyst ; 138(17): 5011-8, 2013 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-23833764

RESUMO

We describe a sensitive electrochemical immunoassay for Salmonella enterica serovar Typhimurium, a common foodborne pathogen which can cause infection at extremely small doses. The assay is based on the recognition of DNA biobarcode labels by differential pulse anodic stripping voltammetry (DPASV), following Ag enhancement. The biobarcodes consist of latex spheres (mean diameter 506 nm ± 22 nm) modified by ferromagnetic Fe3O4 particles. Each biobarcode is loaded by adsorption with approx. 27 molecules of mouse monoclonal antibody against S. Typhimurium and 3.5 × 10(5) molecules of 12 mer ssDNA. The assay is performed by adding the biobarcode, S. Typhimurium cells, and biotin-conjugated rabbit polyclonal antibody against Salmonella into well plates. After antigen-antibody binding, magnetic collection enables the excess polyclonal antibody to be washed off. Exposure to avidin-coated screen printed electrodes, and formation of the avidin-biotin bond, then enables the excess biobarcode to be removed. The biobarcode remaining on the electrode is quantified by DPASV measurement of Ag(+) ions following catalytic Ag deposition. The assay showed a negligible response to 10(7) CFU mL(-1)E. coli and had a limit of detection of 12 CFU mL(-1) in buffer, and 13 to 26 CFU mL(-1) for heat-killed and whole cell S. Typhimurium in plain milk, green bean sprouts and raw eggs. To the best of our knowledge, this is the lowest reported limit of detection for Salmonella by an electrochemical immunoassay not requiring sample pre-enrichment.


Assuntos
DNA/química , Imunoensaio/métodos , Fenômenos Magnéticos , Salmonella typhimurium/isolamento & purificação , Prata/química , Animais , Eletroquímica , Camundongos
19.
Chem Cent J ; 7: 102, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23764320

RESUMO

BACKGROUND: A label-free immunosensor from as-grown double wall carbon nanotubes (DW) bundles was developed for detecting Salmonella typhimurium. The immunosensor was fabricated by using the as-grown DW bundles as an electrode material with an anti-Salmonella impregnated on the surface. The immunosensor was electrochemically characterized by cyclic voltammetry. The working potential (100, 200, 300 and 400 mV vs. Ag/AgCl) and the anti-Salmonella concentration (10, 25, 50, 75, and 100 µg/mL) at the electrode were subsequently optimized. Then, chronoamperometry was used with the optimum potential of 100 mV vs. Ag/AgCl) and the optimum impregnated anti-Salmonella of 10 µg/mL to detect S. typhimurium cells (0-10(9) CFU/mL). RESULTS: The DW immunosensor exhibited a detection range of 10(2) to 10(7) CFU/mL for the bacteria with a limit of detection of 8.9 CFU/mL according to the IUPAC recommendation. The electrode also showed specificity to S. typhimurium but no current response to Escherichia coli. CONCLUSIONS: These findings suggest that the use of a label-free DW immunosensor is promising for detecting S. typhimurium.

20.
Anal Bioanal Chem ; 405(18): 5965-74, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23681202

RESUMO

A novel multi-channel poly(methyl methacrylate) (PMMA) microfluidic biosensor with interdigitated ultramicroelectrode arrays (IDUAs) for electrochemical detection was developed. The focus of the development was a simple fabrication procedure and the realization of a reliable large IDUA that can provide detection simultaneously to several microchannels. As proof of concept, five microchannels are positioned over a large single IDUA where the channels are parallel with the length of the electrode finger. The IDUAs were fabricated on the PMMA cover piece and bonded to a PMMA substrate containing the microfluidic channels using UV/ozone-assisted thermal bonding. Conditions of device fabrication were optimized realizing a rugged large IDUA within a bonded PMMA device. Gold adhesion to the PMMA, protective coatings, and pressure during bonding were optimized. Its electrochemical performance was studied using amperometric detection of potassium ferri and ferro hexacyanide. Cumulative signals within the same chip showed very good linearity over a range of 0-38 µM (R(2) = 0.98) and a limit of detection of 3.48 µM. The bonding of the device was optimized so that no cross talk between the channels was observed which otherwise would have resulted in unreliable electrochemical responses. The highly reproducible signals achieved were comparable to those obtained with separate single-channel devices. Subsequently, the multi-channel microfluidic chip was applied to a model bioanalytical detection strategy, i.e., the quantification of specific nucleic acid sequences using a sandwich approach. Here, probe-coated paramagnetic beads and probe-tagged liposomes entrapping ferri/ferro hexacyanide as the redox marker were used to bind to a single-stranded DNA sequence. Flow rates of the non-ionic detergent n-octyl-ß-D-glucopyranoside for liposome lysis were optimized, and the detection of the target sequences was carried out coulometrically within 250 s and with a limit of detection of 12.5 µM. The robustness of the design and the reliability of the results obtained in comparison to previously published single-channel designs suggest that the multi-channel device offers an excellent opportunity for bioanalytical applications that require multianalyte detection and high-throughput assays.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Técnicas Analíticas Microfluídicas/métodos , Sequência de Bases , Cryptosporidium parvum/genética , Detergentes/química , Desenho de Equipamento , Ferrocianetos/química , Glucosídeos , Ouro , Limite de Detecção , Lipossomos , Microeletrodos , Técnicas Analíticas Microfluídicas/instrumentação , Dados de Sequência Molecular , Ozônio/química , Polimetil Metacrilato , RNA de Protozoário/análise , Reprodutibilidade dos Testes , Raios Ultravioleta
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