Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml⁻¹ while 0.05 U ml⁻¹ was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml⁻¹ and 0.05–40 U ml⁻¹ for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P < 0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae isolated from the rearing water of shrimp (Penaeus vannamei) postlarvae

Shrimp farming has experienced rising costs as a result of disease outbreaks associated with Vibrio spp. Suitable strategies for disease prevention and control are therefore urgently needed. This study aimed to evaluate the antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae in the rearing water of Pacific white shrimp (Penaeus vannamei) postlarvae. In vitro assays included the determination of minimum inhibitory concentration (MIC) of M. oleifera seed powder against V. cholerae, whereas in vivo assays included the effect of M. oleifera seed powder on bacterial load and water quality parameters in the rearing tanks, as well as its effect on shrimp postlarvae survival. M. oleifera seed powder inhibited the growth of V. cholerae with MIC values of 62·5 µg ml⁻¹. Moreover, seawater pH of treated tanks (8·66) was significantly lower (P < 0·01) than pH of the control tanks (9·02), whereas the visibility of treated tanks (37·08 cm) was significantly higher (P < 0·01) as compared to control tanks (35·37 cm). Likewise, V. cholerae load was significantly reduced (P < 0·01) from 4·7 × 10⁴ to 3·1 × 10³ CFU per ml in tanks treated with M. oleifera seed powder. Altogether, this study demonstrates the antimicrobial activity of M. oleifera against V. cholerae in shrimp culture.     

A Speckle-Free Angular Interrogation SPR Imaging Sensor Based on Galvanometer Scan and Laser Excitation

A speckle-free fast angular interrogation surface plasmon resonance imaging (SPRi) sensor based on a diode-pumped all-solid-state laser and galvanometer is reported in this work. A bidirectional scan using a galvanometer realizes the fast scanning of the incidence angle. The experimental results showed that the time needed for completing an SPR dip measurement was decreased to 0.5 s. And through cascading an immovable diffuser and two diffusers rotating in opposite directions, laser speckle was eliminated. The dynamic detection range and the sensitivity reached 4.6 × 10⁻² and 1.52 × 10⁻⁶ refractive index unit (RIU), respectively, in a 2D array sensor when the angle scanning range was set as 7.5°. More importantly, the results demonstrated that the angular interrogation SPR imaging sensor scheme had the capability to perform fast and high-throughput detection of biomolecular interactions at 2D sensor arrays.

     

Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Summary Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 × 10⁴ V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c.f.u./ml in ground water, 1.2 × 10² c.f.u. ml⁻¹ in sewer water and 1.2 × 10³c.f.u. ml⁻¹ in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.     

Successful Application of Enzyme-Labeled Oligonucleotide Probe for Rapid and Accurate Cholera Diagnosis in a Clinical Laboratory

Two cholera cases were diagnosed using an enzyme-labeled oligonucleotide probe (ELONP) hybridization test for detection of cholera toxin gene (ctx) in a clinical laboratory at Osaka Airport Quarantine Station. The ELONP test with suspicious colonies of Vibrio cholerae O1 grown on TCBS or Vibrio agar plates gave positive result for ctx within 3 hr. We also tried to apply the ELONP test for direct detection of ctx in their stool and their non-selective culture. Specimens from Case #1, which contained 5.9 × 10⁵ CFU/g of V. cholerae O1 in the stool, cultured for 7–8 hr or longer in alkaline peptone water or Marine broth at 37C, became positive for ctx. On the other hand, specimens from Case #2, which contained 8.7 × 10⁸ CFU/ml (of V. cholerae O1 in the stool), gave positive result in this stool itself without any further culture. These data suggest that the ELONP test provides successfully a more rapid and accurate means of identifying “toxigenic” V. cholerae O1 in a clinical laboratory.     

Tannase enzyme production by entrapped cells of Aspergillus niger FETL FT3 in submerged culture system

The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of 1 × 10⁶ spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL). It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated runs.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Determination of free cholesterol based on a novel flow‐injection chemiluminescence method by immobilizing enzyme

A novel flow injection chemiluminescence method is proposed for determination of cholesterol in this paper. The cholesterol oxidase was immobilized onto sol–gel and prepared as an enzymatic reaction column. The determination of cholesterol was performed by quantitative determination of hydrogen peroxide produced from an enzymatic reaction. The luminol–H₂O₂–metal chelate diperiodatocuprate(III) system ensured that the method was highly sensitive and selective. Free cholesterol was determined over the range 5.0 × 10^–8 mol/L–5.0 × 10^–7 mol/L, with a limit of detection (3σ) of 1.9 × 10^–8 mol/L. The relative standard deviation (RSD) for 2.5 × 10^–7 mol/L was 2.7% (n = 7). The proposed method offered the advantages of sensitivity, selectivity, simplicity and rapidity for free cholesterol determination, and was successfully applied to the direct determination of free cholesterol in serum. Copyright © 2008 John Wiley & Sons, Ltd.     

SPR Label-Free Biosensor with Oxide-Metal-Oxide-Coated D-Typed Optical Fiber: a Theoretical Study

In this article, a surface plasmon resonance (SPR) biosensor based on D-typed optical fiber coated by Al₂O₃/Ag/Al₂O₃ film is investigated numerically. Resonance in near infrared with an optimized architecture is achieved. Refractive index sensitivity of 6558 nm/RIU (refractive index unit) and detection limit of 1.5 × 10⁻⁶ RIU, corresponding to 0.4357 nm/μM and detection limit of 23 nM in BSA (bovine serum albumin) concentration sensing, are obtained. The analysis of the performance of the sensor in gaseous sensing indicates that this proposed SPR sensor is much suitable for label-free biosensing in aqueous media.

     

Efficacy of cocktail phage therapy in treating Vibrio cholerae infection in rabbit model

Ability of a cocktail of five lytic vibriophages to combatting Vibrio cholerae O1 infection in rabbit model was examined. In one group, rabbits were administered 1 × 10⁸ plaque forming unit of phage cocktail 6 and 12 h prior to the administration of V. cholerae O1, while in the other group, same procedure was applied 6 and 12 h post infection. It was observed that oral administration of the phage cocktail after oral bacterial challenge lowered the shedding of bacteria significantly (p < 0.01). In contrast phage treatment prior to bacterial challenge had no such effect (p > 0.05). Results suggest that oral administration of phage subsequent to V. cholerae challenge could provide a possible means of combatting V. cholerae infection.     

Toxigenic Vibrio cholerae from environmental sources associated with the cholera outbreak after ‘AILA’ cyclone in West Bengal,India

Aims: West Bengal experienced a devastating storm named ‘AILA’ in its coastal and southern districts. We attempted to understand the transmission dynamics emphasizing on potable water to detect the presence of toxigenic strains of Vibrio cholerae, followed by the natural devastation. Methods and Results: A total of 33 water samples (from tap, tube well and ponds) were analysed. From them, 11 (33·3%) samples were found to be contaminated with V. cholerae, among which 5 (45%) isolates were V. cholerae O1 biotype Ogawa. Antibiogram profile reveals that most of the V. cholerae O1 isolates were highly sensitive against fluroquinolone group of antibiotics, but less sensitive against cefuroxime (50%), cefotaxime (40·9%), ceftriaxone (38·63%), trimethoprim (37·3%), streptomycin (29·2%) and furazolidon (4·54%). Three (36%) V. cholerae isolates were found to be ctxB positive (2 ctxB classical). Conclusions: Potable water plays a crucial role in cholera transmission. Natural disasters like the reported one aided with feacal–oral contamination enhance the possibilities of drinking water contamination. Significance and Impact of the Study: The application of the modified technique, making use of the enrichment subsequently followed by culture and PCR, will help us to detect the presence of toxigenic V. cholerae contamination in different aquatic environment. Moreover, natural extremes have a direct role in increase of salinity level, followed by higher predominance of V. cholerae along with their toxicity development in terms of genetic modification.     

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. Methods and Results: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 × 10⁴ CFU ml^?1 (c. 200 CFU per PCR) to 2 × 10³ CFU ml^?1 (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. Conclusions: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. Significance and Impact of the Study: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.     

Nuclear fast red‐based colorimetric sensors for sensitive and selective detection of Ag ions

The reduction of nuclear fast red (NFR) stain by sodium tetrahydroboron was catalyzed in the presence of silver ions (Ag⁺). The fluorescence properties of reduced NFR differed from that of NFR. The product showed fluorescence emission at 480 nm with excitation at 369 nm. Furthermore, the fluorescence intensity of the mixture increased strongly in the presence of Ag⁺ and Britton–Robinson buffer at pH 4.78. There was a good linear relationship between increased fluorescence intensity (ΔI) and Ag⁺ concentration in the range 5.0 × 10^?9 to 5.0 × 10^?8 M. The correlation coefficient was 0.998, and the detection limit (3σ/k) was 1.5 × 10^?9 M. The colour of the reaction system changed with variation in Ag⁺ concentration over a wide range. Based on the colour change, a visual semiquantitative detection method for recognition and sensing of Ag⁺ was developed for the range 1.0 × 10^?8 to 5.0 × 10^?4 M, with an indicator that was visible to the naked eye. Therefore, a sensitive, simple method for determination of Ag⁺ was developed. Optimum conditions for Ag⁺ detection, the effect of other ions and the analytical application of Ag⁺ detection of synthesized sample were investigated.     

Environmental drivers on seasonal abundance of riverine-estuarine V. cholerae in the Indian Sundarban mangrove

Gangetic delta is considered as the homeland of cholera, which is thought to be influenced by changes in populations of estuarine Vibrio cholerae. We aim to identify the environmental, biotic and abiotic driving forces influencing the V. cholerae dynamics in riverine-estuarine environment of southern deltaic Bengal. Cultivable Vibrio count (CVC) ranged between 1 and 10³ colony forming units (CFU)/mL at a salinity gradient of 1.9–30 practical salinity unit (PSU). Increased water temperatrure during summer influences the higher CVC followed by a sudden fall along with the onset of monsoon upto winter. While summer V. cholerae O1 peak (50–100 CFU/mL) can be associated with higher water temperature (P < 0.05) and higher turbidity (P < 0.005); sharp fall during monsoon (15–45 CFU/mL) is attributed to reduced salinity (25–2.5 PSU). Plankton attached V. cholerae O1 varied between 10 and 1000 CFU/mL with a highest peak at winter followed by summer and monsoon. Prevalence of toxigenic V. cholerae O1 in low salinity (2–7.5 PSU) during monsoon identifies that high water temperature (>25 °C), higher turbidity (>100 NTU) and lower salinity plays the pivotal role in toxicity acquisition. Present investigation establishes the role of Sundarban mangrove, where V. cholerae exist in an avirulent condition. During migration towards low saline inland system, V. cholerae pool possibly acquires toxin genes under the influence of environmental factors. Planktonic attachment is possibly a survival strategy at adverse condition, when they do not acquire any toxin gene. Seasonal V. cholerae dynamics has been thoroughly established in environmental settings of high saline mangrove and brackish water flowing to inland low saline condition.     

Surface plasmon resonance immunosensor for detection of<Emphasis Type="Italic">Legionella pneumophila</Emphasis>

An immunosensor based on surface plasmon resonance (SPR) onto a protein G layer by self-assembly technique was developed for detection ofLegionella pneumophila. The protein G layer by self-assembly technique was fabricated on a gold (Au) surface by adsorbing the 11-mercaptoundecanoic acid (MUA) and an activation process for the chemical binding of the free amine (-NH₂) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of the protein G layer by self-assembly technique on the Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface topographies of the fabricated thin films on an Au substrate were also analyzed by using an atomic force microscope (AFM). Consequently, an immunosensor for the detection ofL. pneumophila using SPR was developed with a detection limit of up to 10² CFU per mL.     

A sensitive fluorescence method for detection of E. Coli using rhodamine 6G dyeing

Negatively charged bacteria combined with positively charged alkaline dye rhodamine 6G (Rh6G) in NaH₂PO₄–Na₂HPO₄ buffer solution pH 7.4, by electrostatic interaction. The dyed bacteria exhibited a strong fluorescence peak at 552 nm and fluorescence intensity was directly linear to Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) concentrations in the range of 7.06 × 10⁴ to 3.53 × 10⁷, 4.95 × 10⁵ to 2.475 × 10⁸ and 32.5 to 16250 colony forming unit/mL (cfu/mL) respectively, with detection limits of 3.2 × 10⁴ cfu/mL E. coli, 2.3 × 10⁵ cfu/mL B. subtilis and 16 cfu/mL S. aureus, respectively. Samples were cultured for 12 h, after which the linear detection range for E. coli was 2 to 88 cfu/mL. This simple, rapid and sensitive method was used for the analysis of water and drinking samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Highly sensitive spectrofluorimetric determination of trace amounts of prulifloxacin using the aluminium(III)–prulifloxacin system

The fluorescence of the prulifloxacin (PUFX)–Al(III) system was investigated . Experiments indicated that the fluorescence intensity of prulifloxacin could be greatly enhanced by Al(III) and sensitized by sodium dodecylbenzene sulphonate (SDBS). Accordingly, a sensitive spectrofluorimetric method for the determination of prulifloxacin was established. While excited at 275 nm, the enhanced fluorescence intensity at 412 nm of the system (ΔF) showed a good linear relationship with the concentration of prulifloxacin within the range 4.0 × 10^–8–3.0 × 10^–6 mol/L. The regression equation was ΔF = 9.83 + 10.8 × 10⁷c (mol/L); the correlation coefficient and detection limit (3σ/k) were 0.99901 and 2.0 × 10^–8 mol/L, respectively. The proposed method has been successfully applied to determine prulifloxacin in real pharmaceutical samples. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

Electrochemical nitrite biosensor based on the immobilization of hemoglobin on an electrode modified by multiwall carbon nanotubes and positively charged gold nanoparticle

The report is on an electrochemical biosensor with remarkably improved sensitivity toward nitrite. In this strategy, positively charged gold nanoparticle (PCNA) is used in combination with multiwall carbon nanotubes (MWCNT) by electrostatic adsorption for fabricating PCNA/MWCNT films. Then hemoglobin (Hb) biocatalyst will easily be attached to the surface of the combination films aforementioned. After that, the Hb/PCNA films are immobilized onto the Hb/PCNA/MWCNT films through layer-by-layer assembly technique. The (Hb/PCNA)₂/MWNT/GC electrode thus prepared exhibits enhanced electrocatalytic behavior to the reduction of nitrite at −0.10 V versus SCE in 0.05 M H₂SO₄ solution_. On condition of the low detecting potential and low pH, interference caused by direct electrochemical oxidation or oxidizable substances can be prevented. Therefore, the modified electrode shows fast response time, very high sensitivity, good selectivity and stability. The current response of the sensor increases linearly with nitrite concentration from a range of 3.6 × 10⁻⁶ to 3.0 × 10⁻³ M with a detection limit(S /N = 3) of 9.6 × 10⁻⁷ M.