Detection Of Cholera Toxin Research Articles

Detection of Cholera Toxin in Culture Medium by Dot-Immunoassay on Dipsticks

Detection of Cholera Toxin in Culture Medium by Dot-Immunoassay on Dipsticks

Advances in phage display based nano immunosensors for cholera toxin.

Cholera, a persistent global public health concern, continues to cause outbreaks in approximately 30 countries and territories this year. The imperative to safeguard water sources and food from Vibrio cholerae, the causative pathogen, remains urgent. The bacterium is mainly disseminated via ingestion of contaminated water or food. Despite the plate method's gold standard status for detection, its time-consuming nature, taking several days to provide results, remains a challenge. The emergence of novel virulence serotypes raises public health concerns, potentially compromising existing detection methods. Hence, exploiting Vibrio cholerae toxin testing holds promise due to its inherent stability. Immunobiosensors, leveraging antibody specificity and sensitivity, present formidable tools for detecting diverse small molecules, encompassing drugs, hormones, toxins, and environmental pollutants. This review explores cholera toxin detection, highlighting phage display-based nano immunosensors' potential. Engineered bacteriophages exhibit exceptional cholera toxin affinity, through specific antibody fragments or mimotopes, enabling precise quantification. This innovative approach promises to reshape cholera toxin detection, offering an alternative to animal-derived methods. Harnessing engineered bacteriophages aligns with ethical detection and emphasizes sensitivity and accuracy, a pivotal stride in the evolution of detection strategies. This review primarily introduces recent advancements in phage display-based nano immunosensors for cholera toxin, encompassing technical aspects, current challenges, and future prospects.

Caco-2 cell-derived biomimetic electrochemical biosensor for cholera toxin detection

Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of ∼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.

Amine Functionalized Gadolinium Oxide Nanoparticles-Based Electrochemical Immunosensor for Cholera.

Herein, we report the synthesis and functionalization of gadolinium oxide nanoparticles (Gd2O3 NPs) to fabricate a highly efficient immunosensor for the detection of Vibrio cholera toxin (CT). Gd2O3 NPs were produced in a straightforward manner utilizing the microwave irradiation technique using a domestic microwave oven. X-ray diffraction, transmission electron microscopy, and spectroscopic techniques were used to characterize the structural and physical aspects of Gd2O3 NPs. The Gd2O3 NPs were then functionalized with 3-(Aminopropyl) triethoxysilane (APTES) and electrophoretically deposited onto an ITO-coated glass substrate. The anti-CT monoclonal antibodies were covalently attached to the APTES-Gd2O3/ITO electrode via EDC-NHS chemistry, followed by bovine serum albumin (BSA). For CT detection, electrochemical response experiments using BSA/anti-CT/APTES-Gd2O3/ITO immunoelectrodes were carried out (5-700 ng mL-1). The immunoelectrode demonstrated an outstanding electrochemical reaction against CT, with a sensitivity of 8.37 mA ng-1 mL cm-2 and a detection limit of 1.48 ng mL-1.

Polimeric Immunoglobulin Diagnosticum for Detection of Cholera Toxin and Assessing the Level of Its Production by Vibrios

A marker of the epidemic significance of Vibrio cholerae is their toxigenicity. Therefore, much attention is currently paid to the creation of diagnostic preparations for the detection of cholera toxin and assessment of the level of its production. The volumetric immunosuspension agglomeration reaction, carried out with the help of latex diagnosticums, is an analogue of the indirect hemagglutination reaction, an affordable and technically simple method, since it does not require special equipment and can be used when conducting research in the field. The aim of the study was to design a polymeric immunoglobulin diagnosticum for determining cholera toxin and the level of its production by vibrio strains. Materials and methods. Cholera toxin was obtained from the producer strain Vibrio cholerae Classical 569 B. Rabbit serum to the toxin was obtained according to the method selected by the authors. A polymeric diagnostic immunoglobulin antitoxic drug was obtained through sensitizing immunoglobulins from cholera antitoxic rabbit serum on the surface of polyacrolein microspheres with a size of (1±0.1) μm. Results and discussion. The analytical sensitivity of the developed diagnostic preparation with control cholera toxin is 100 ng/ml. It detects cholera toxin in toxigenic strains of Vibrio cholerae in a titer of 1:16 – 1:512, gives negative results with non-toxigenic strains of V. cholerae O1, V. Cholerae nonO1/nonO139, with samples of heterologous cultures, LPS preparations, liquid nutrient medium used for the cultivation of V. cholerae. Thus, a polymeric immunoglobulin diagnosticum has been constructed to detect and quantify the production of cholera toxin by vibrio strains, and its analytical sensitivity and specificity have been established.

Условия и сроки хранения лиофилизированных поли- и моноклональных антитоксических пероксидазных конъюгатов

The article discusses the selection of optimal conditions for the lyophilization of poly- and monoclonal peroxidase conjugates used in the direct version of the enzyme immunoassay intended for the cholera toxin detection in Vibrio cholerae O1, O139 strains. Effective stabilizers included in protective media comprise 1 % sucrose, 1 % sodium thiosulfate, 0.5 % egg albumin, or 0.5 % bovine serum albumin. Their use increases the shelf life, increases stability, and contributes to the preservation of high sensitivity and specificity of poly- and monoclonal peroxidase conjugates. Evaluation of the serological activity of antitoxic conjugates in ELISA after lyophilization in a protective medium with stabilizers showed that it remains at the initial level. The results of testing lyophilized conjugates during storage allow us to speak about the possibility of their use for two years without changing the main indicators.

Detection of cholera toxin with surface plasmon field-enhanced fluorescent spectroscopy.

In this work, a biosensor based on surface plasmon field-enhanced florescence spectroscopy (SPFS) method was successfully constructed to detect the truncated form of cholera toxin, that is, its beta subunit (CTX-B). CTX-B is a relatively small molecule (12kDa) and it was chosen as model analyte for the detection of protein toxins originated from waterborne pathogens. Recognition layer was prepared on gold-coated LaSFN9 glasses modified with 11-mercaptoundecanoic acid (11-MUA). Biotin-conjugated anti-CTX-B polyclonal antibody (B-Ab) was immobilized on streptavidin (SA) layer constructed on the 11-MUA-modified surface. CTX-B amount was determined with direct assay using B-Ab in surface plasmon resonance (SPR) mode and with sandwich assay in SPFS mode using Cy5-conjugated anti-CTX-B polyclonal antibody. Minimum detected CTX-B concentrations were 10 and 0.01μg/ml with SPR and SPFS, respectively, showing the sensitivity of the SPFS system over the conventional one. The detection was done in 2-6h, which was faster than both culture and polymerase chain reaction (PCR)-based methods. Stability tests were performed with SA-coated sensors (excluding B-Ab). In this form, the layer was stable after 30days of storage in phosphate-buffered saline (PBS; 0.01M, pH = 7.4) at +4°C. B-Ab layer was formed immediately on them before each measurement.

Detection of cholera toxin (ctxA and ctxAB) genes in Vibrio cholerae isolated from clinical and environmental samples in Limbang Sarawak by multiplex polymerase chain reaction (PCR)

Detection of cholera toxin (ctxA and ctxAB) genes in Vibrio cholerae isolated from clinical and environmental samples in Limbang Sarawak by multiplex polymerase chain reaction (PCR)

Graphdiyne Micromotors in Living Biomedia.

Graphdiyne (GDY), a new kind of two-dimensional (2D) material, was combined with micromotor technology for "on-the-fly" operations in complex biomedia. Microtubular structures were prepared by template deposition on membrane templates, resulting in functional structures rich in sp and sp2 carbons with highly conjugated π networks. This resulted in a highly increased surface area for a higher loading of anticancer drugs or enhanced quenching ability over other 2D based micromotors, such as graphene oxide (GO) or smooth tubular micromotors. High biocompatibility with almost 100 % cell viability was observed in cytotoxicity assays with moving micromotors in the presence of HeLa cells. On a first example, GDY micromotors loaded with doxorubicin (DOX) were used for pH responsive release and HeLa cancer cells killing. The use of affinity peptide engineered GDY micromotors was also illustrated for highly sensitive and selective fluorescent OFF-ON detection of cholera toxin B through specific recognition of the subunit B region of the target toxin. The new developments illustrated here offer considerable promise for the use of GDY as part of micromotors in living biosystems.

A universal ultrasensitive platform for enzyme-linked immunoassay based on responsive surface-enhanced Raman scattering

Enzyme-linked immunosorbent assay (ELISA) is the current gold standard assay for biomarkers. However, its detection sensitivity is greatly limited by the colorimetric readout. Herein, we developed a high-performance immunoassay through the combination of traditional ELISA and ultrasensitive surface-enhanced Raman scattering (SERS) assay using a responsive SERS probe. The probe is a Au nanoplate constituted cabbage-like microparticle functionalized with synthesized phenol-responsive Raman reporter 4, 4’-dithiodibenzyldiazonium (DTDBD). The new SERS probe can detect trace amount of phenol with a limit of detection (LOD) of 0.4 nM, and alkaline phosphatase (LOD of 0.04 mU/L) with phenol as an enzymatic product. More important, the ultrasensitive detection of cholera toxin (CT) in real serum samples indicates such ELISA-SERS assay promising for practical applications using alkaline phosphatase as an enzyme labeling and SERS probe as a signal readout. We envision that this new method can be employed as a universal platform for the profiling of any antigen using alkaline phosphatase as the enzymatic label to its corresponding antibody.

A thermo-responsive supramolecular hydrogel that senses cholera toxin via color-changing response.

A Pyrene-based amphiphile with C4-alkanoyl spacer and lactose (PyLac) self-assembles in the aqueous media to form an injectable hydrogel. It shows preferential binding with Cholera Toxin (CT) via its terminal galactose residue, and hence can be employed for the selective detection of CT via color-changing response.

Real-Time Detection of Markers in Blood.

The real-time selective detection of disease-related markers in blood using biosensors has great potential for use in the early diagnosis of diseases and infections. However, this potential has not been realized thus far due to difficulties in interfacing the sensor with blood and achieving transparent circuits that are essential for detecting of target markers (e.g., protein, ions, etc.) in a complex blood environment. Herein, we demonstrate the real-time detection of a specific protein and ion in blood without a skin incision. Complementary metal-oxide-semiconductor technology was used to fabricate silicon micropillar array (SiMPA) electrodes with a height greater than 600 μm, and the surface of the SiMPA electrodes was functionalized with a self-assembling artificial peptide (SAP) as a receptor for target markers in blood, i.e., cholera toxin (CTX) and mercury(II) ions (Hg). The detection of CTX was investigated in both in vitro (phosphate-buffered saline and human blood serum, HBO model) and in vivo (mouse model) modes via impedance analysis. In the in vivo mode, the SiMPA pierces the skin, comes into contact with the blood system, and creates comprehensive circuits that include all the elements such as electrodes, blood, and receptors. The SiMPA achieves electrically transparent circuits and, thus, can selectively detect CTX in the blood in real time with a high sensitivity of 50 pM and 5 nM in the in vitro and in vivo modes, respectively. Mercury(II) ions can also be detected in both the in vitro and the in vivo modes by changing the SAP. The results illustrate that a robust sensor that can detect a variety of molecular species in the blood system in real time that will be helpful for the early diagnosis of disease and infections.

Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin.

Aptamers are envisioned to serve as powerful synthetic substitutes to antibodies in a variety of bioanalytical assay formats. However, lateral flow assays (LFAs) remain dominated by antibody-based strategies. In this study, a LFA for the detection of cholera toxin as a model analyte is developed and optimized using a synthetic aptamer and a naturally occurring receptor as biorecognition elements and directly compared with solely aptamer and aptamer and antibody-based alternative approaches. The aptamer (CT916) recently selected by our group, GM1 receptors and an anti-cholera toxin antibody were evaluated. Relying solely on molecules that can easily be synthesized while aiming for high sensitivity, we applied a novel combination of capture aptamer and GM1 cell receptor-labeled liposomes for cholera toxin detection, achieving a limit of detection (LOD) of 2 ng ml-1 (3σ)/10 ng ml-1 (visual) in ∼15 min. To put our novel aptasensor into perspective, we developed a competitive lateral flow assay, exploiting the competition of cholera toxin in solution with immobilized cholera toxin for binding of aptamer-coated gold nanoparticles (AuNPs) (LOD = 51 ng ml-1 (3σ)/100 ng ml-1 (visual), assay time ∼10 min). As dual simultaneously binding aptamers were not available, we designed aptamer antibody pair-based lateral flow assays using aptamer-coated AuNPs which yielded a LOD of 5 ng ml-1 (by the 3σ rule)/10 ng ml-1 (visual) in a 10 min assay and an even better LOD of 0.6 ng ml-1 (3σ)/1 ng ml-1 (visual), with an ∼20 min total assay time. All set-ups are highly specific and provide an excellent alternative for cholera toxin detection in places where professional knowledge and sophisticated equipment are not readily available and cost efficient, simple, and rapid tests are needed, while the combination of GM1 cell receptor-labeled liposomes and aptamers is clearly the most promising.

Synthesis of water-soluble fluorescent polymeric glycoconjugate for the detection of cholera toxin

ABSTRACTConsidering inherence optical properties of adjoint polyfluorenes and special functions of water-soluble conjugated glycopolymers, a triazole chain glycoconjugate via one-pot method were rapidly synthesized to prepare a lactate ligand polyfluorene with a clear fluorescent label by a nickel-catalyzed Yamamoto coupling polymerization. The water solubility and biocompatibility of the glycoconjugated polymer were ameliorated when the lactose group introduced as the side chain of the conjugated polymer. As a fluorescent multivalent system of glycoconjugates containing pyranogalactose groups, the interaction between pyranogalactose group and cholera toxin B subunit was studied by fluorescence spectrophotometric titration. PF-Lac has a broad application prospect in the check of cholera toxin and the study of glycoprotein interaction.

Ultrasensitive Detection of Bacterial Protein Toxins on Patterned Microarray via Surface Plasmon Resonance Imaging with Signal Amplification by Conjugate Nanoparticle Clusters.

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20 nm AuNP via streptavidin bridge, followed by attachment of 5 nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently sized conjugated nanoparticles effectively increases their packing density on surface and significantly improves SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 × 10-16 to 2.51 × 10-13 mol/cm2 with the limit of detection of 5.01 × 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.

Application of Biosensors Based on Lipid Membranes for the Rapid Detection of Toxins.

Lipid assemblies in the form of two dimensional films have been used extensively as biosensing platforms. These films exhibit certain similarities with cell membranes, thus providing a suitable means for the immobilization of proteinaceous moieties and, further, a number of intrinsic signal amplification mechanisms. Their implementation in the detection of toxins yielded reliable and fast detectors for in field analyses of environmental and clinical samples. Some examples are presented herein, including aflatoxin and cholera toxin detection. The conditions and parameters that determine the analytical specifications of the lipid membrane sensors are discussed, advantages and technology bottlenecks are reviewed, and possible further developments are highlighted.

Highly affine and selective aptamers against cholera toxin as capture elements in magnetic bead-based sandwich ELAA

Aptamers are single-stranded DNA or RNA oligonucleotides, which have been emerging as recognition elements in disease diagnostics and food control, including the detection of bacterial toxins. In this study, we employed the semi-automated just in time-selection to identify aptamers that bind to cholera toxin (CT) with high affinity and specificity. CT is the main virulence factor of Vibrio cholerae and the causative agent of the eponymous disease. For the selected aptamers, dissociation constants in the low nanomolar range (23–56 nM) were determined by fluorescence-based affinity chromatography and cross-reactivity against related proteins was evaluated by direct enzyme-linked aptamer assay (ELAA). Aptamer CT916 has a dissociation constant of 48.5 ± 0.5 nM and shows negligible binding to Shiga-like toxin 1B, protein A and BSA. This aptamer was chosen to develop a sandwich ELAA for the detection of CT from binding buffer and local tap water. Amine-C6- or biotin-modified CT916 was coupled to magnetic beads to serve as the capture element. Using an anti-CT polyclonal antibody as the reporter, detection limits of 2.1 ng/ml in buffer and 2.4 ng/ml in tap water, with a wide log-linear dynamic range from 1 ng/ml to 1000 ng/ml and 500 ng/ml, respectively, were achieved.

A Nanosensor for the Rapid Detection of Cholera Toxin Using Graphene Electrodes with Incorporated Polymer Lipid Films

A Nanosensor for the Rapid Detection of Cholera Toxin Using Graphene Electrodes with Incorporated Polymer Lipid Films

Label-free impedimetric glycosensor based on β-galactose-functionalized gold electrode for the determination of cholera toxin

A label-free impedimetric glycosensor has been developed for the direct detection of cholera toxin (CT). The glycosensor has been fabricated by forming the self-assembled monolayers of β-galactose derivatives containing poly-ethyleneglycol (PEG) spacer on gold electrode surfaces. The selective binding of CT to the β-galactose receptors on the fabricated glycosensor has been directly monitored by electrochemical impedance spectroscopy in the presence of 5.0mM K3Fe(CN)6/K4Fe(CN)6 (1:1, v/v) redox couple. gmonolayers on gold electrode surface has been determined to be ca. 2.53×1010M−1, indicating that the present glycosensor can selectively capture the target CT from sample solutions. Therefore, the present monosaccharide β-galactose derivative can be used as a receptor for CT as an alternative to CT-specific antibodies or pentasaccharide ganglioside GM1, most widely used in conventional CT assays. The present glycosensor can detect CT in the concentration range from 1.18×10−12M to 1.18×10−9M (r2=0.963) with a limit of detection (S/N=3) down to 7.83×10−13M, which is much lower than those obtained with other detection methods. Since the present glycosensor-based CT assay does not require complex and time-consuming signal amplification steps generally employed in sandwich-type immunoassays, it enables the simple and rapid detection of CT.

Selection of affinity peptides for interference-free detection of cholera toxin

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5.