Development of an Electrochemical Biosensor for the Rapid Detection of Cholera Toxin Based on Air Stable Lipid Films with Incorporated Ganglioside GM1 Using Graphene Electrodes

The present work describes a miniaturized potentiometric cholera toxin sensor on graphene nanosheets with incorporated lipid films. Ganglioside GM1, the natural cholera toxin receptor, immobilized on the stabilized lipid films, provided adequate selectivity for detection over a wide range of toxin concentrations, fast response time of ca. 5 min, and detection limit of 1 nM. The proposed sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, long shelf life and high sensitivity of ca. 60 mV/decade of toxin concentration. The method was implemented and validated in lake water samples. This novel ultrathin film technology is currently adapted to the rapid detection of other toxins that could be used in bioterrorism.     

Electrochemical Biosensor for Naphthalene Acetic Acid in Fruits and Vegetables Based on Lipid Films with Incorporated Auxin‐binding Protein Receptor Using Graphene Electrodes

The present work describes a miniaturized potentiometric naphthalene acetic acid (NAA) sensor on graphene nanosheets with incorporated lipid films. Auxin‐binding protein 1 receptor immobilized on the stabilized lipid films provided adequate selectivity for detection over a wide range of hormone concentrations, fast response time of ca. 5 min, and detection limit of 10 nM. The proposed sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, long shelf life and high sensitivity of ca. 56 mV/decade of hormone concentration. The reliability of the biosensor was successfully evaluated using a wide range of NAA‐spiked fruits and vegetables.     

Development of a Potentiometric Chemical Sensor for the Rapid Detection of Carbofuran Based on Air Stable Lipid Films with Incorporated Calix[4]arene Phosphoryl Receptor Using Graphene Electrodes

The present article describes a miniaturized potentiometric carbofuran chemical sensor on graphene nanosheets with incorporated lipid films. The graphene electrode was used for the development of a very selective and sensitive chemical sensor for the detection of carbofuran by immobilizing an artificial selective receptor on stabilized lipid films. The artificial receptor was synthesized by transformation of the hydroxyl groups of resorcin[4]arene receptor into phosphoryl groups. This chemical sensor responded for the wide range of carbofuran concentrations with fast response time of ca. 20 s. The presented potentiometric carbofuran chemical sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, rapid response times, long shelf life and high sensitivity of ca. 59 mV/decade over the carbofuran logarithmic concentration range from 10^?6 to 10^?3 M.     

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.     

A Selective Immunosensor for D‐dimer Based on Antibody Immobilized on a Graphene Electrode with Incorporated Lipid Films

The present article describes a miniaturized potentiometric D‐dimer biosensor on graphene nanosheets with incorporated lipid films. The graphene electrode was used for the development of a very selective and sensitive immunosensor for the detection of D‐dimer by immobilizing the mouse anti human D‐dimer antibody on stabilized lipid films. The immunosensor responded for the wide range of D‐dimer concentrations with fast response time of ca. 15 s. The presented potentiometric D‐dimer biosensor is easy to construct and exhibits good reproducibility, reusability, selectivity, rapid response times, long shelf life and high sensitivity of ca. 59 mV/decade over the D‐dimer logarithmic concentration range from 10^?6 μg/L to 10^?3 μg/L.     

Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively.The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.     

An electrochemical sensor concept for the detection of bacterial virulence factors from Staphylococcus aureus and Pseudomonas aeruginosa

This work presents an electrochemical sensor concept for bacterial toxin detection using biomimetic lipid vesicles containing potassium ferricyanide. The toxin mediated release of redox couples from the vesicles was quantified by measuring the redox current on screen printed electrodes, and the detection limits to rhamnolipid, delta toxin and bacterial supernatants from clinical wound pathogens were examined. Overall detection limits of both redox and carboxyfluorescein containing vesicles were one order of magnitude lower than the cytotoxic dose of studied toxins to T lymphocytes.     

Colorimetric detection and fingerprinting of bacteria by glass-supported lipid/polydiacetylene films

Glass-supported films of lipids and polydiacetylene were applied for visual detection and colorimetric fingerprinting of bacteria. The sensor films comprise polydiacetylene domains serving as the chromatic reporter interspersed within lipid monolayers that function as a biomimetic membrane platform. The detection schemes are based on either visible blue-red transitions or fluorescence transformations of polydiacetylene, induced by amphiphilic molecules secreted by proliferating bacteria. An important feature of the new film platform is the feasibility of either naked-eye detection of bacteria or color analysis using conventional scanners. Furthermore, we find that the degrees of bacterially induced color transformations depend both on the bacterial strains examined and the lipid compositions of the films. Accordingly, bacterial fingerprinting can be achieved through pattern recognition obtained by recording the chromatic transformations in an array of lipid/PDA films having different lipid components.     

Fluorescent strip sensor for rapid determination of toxins

Here, we report a simple fluorescent strip sensor based on aptamer-quantum dots technology that can meet toxin monitoring demands using ochratoxin A (OTA) as a model toxin. The limit of the detection (LOD) for the fluorescent strip was 1.9 ng mL(-1), while the time needed for the detection is only 10 min; this conforms to the standards of World Health Organization (WHO) or better. Overall functional parameters are also better than the analogous characteristics of gold nanoparticle strips. High selectivity was maintained as well, making them suitable for the samples with complex solution composition.     

Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk

This work describes the construction of a simple optical sensor for the rapid, selective and sensitive detection of urea in milk using air stable lipid films with incorporated urease. The lipid film is stabilized on a glass filter by polymerization using UV (ultra-violet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′-azobis-(2-methylpropionitrile) was the initiator. Urease is incorporated within this mixture prior to the polymerization. The presence of the enzyme in these films quenched this fluorescence and the colour became similar to that of the filters without the lipid films. A drop of aqueous solution of urea provided a “switching on” of the fluorescence which allows the rapid detection of this compound at the levels of 10⁻⁸ M concentrations. The investigation of the effect of potent interferences included a wide range of compounds usually found in foods and also of proteins and lipids. These lipid membranes were used for the rapid detection of urea in milk.     

On‐Chip Tailorability of Capacitive Gas Sensors Integrated with Metal–Organic Framework Films

Gas sensing technologies for smart cities require miniaturization, cost‐effectiveness, low power consumption, and outstanding sensitivity and selectivity. On‐chip, tailorable capacitive sensors integrated with metal–organic framework (MOF) films are presented, in which abundant coordinatively unsaturated metal sites are available for gas detection. The in situ growth of homogeneous Mg‐MOF‐74 films is realized with an appropriate metal‐to‐ligand ratio. The resultant sensors exhibit selective detection for benzene vapor and carbon dioxide (CO₂) at room temperature. Postsynthetic modification of Mg‐MOF‐74 films with ethylenediamine decreases sensitivity toward benzene but increases selectivity to CO₂. The reduced porosity and blocked open metal sites caused by amine coordination account for a deterioration in the sensing performance for benzene (by ca. 60 %). The enhanced sensitivity for CO₂ (by ca. 25 %) stems from a tailored amine–CO₂ interaction. This study demonstrates the feasibility of tuning gas sensing properties by adjusting MOF–analyte interactions, thereby offering new perspectives for the development of MOF‐based sensors.     

基于钒酸铋半导体材料的光电化学葡萄糖传感器的研究

葡萄糖浓度的检测在食品、医药、环境等领域的应用很广泛, 因此, 发展具有低成本、高灵敏度、高选择性的葡萄糖传感器具有重要意义. 光电化学传感器具有激发源与检测信号源相互独立的特点, 被认为是一种潜在的高灵敏的葡萄糖检测方法. 通过电化学沉积及高温热处理两步法在氟掺杂的SnO₂透明导电玻璃(FTO)基底上制备了BiVO₄薄膜. 通过X射线衍射分析(XRD)、拉曼光谱(Raman)、扫描电子显微镜分析(SEM)等表征技术对BiVO₄电极的结构组成和微观形貌等性质进行了表征. 结果表明, 所制得的BiVO₄为单斜晶系, 形貌为黏连的纳米颗粒. 进一步地, 对基于BiVO₄半导体构建的光电化学葡萄糖传感器性能进行了评价. 电化学测试表明, 葡萄糖在BiVO₄电极上的氧化电流与葡萄糖的浓度能够呈现出很好的线性关系, 在5~35 mmol/L浓度区间的检测灵敏度为17.38 μA/cm2/(mmol/L). 研究为葡萄糖提供了一种简易廉价的检测新策略.     

Acoustic quantification of ATP using a quartz crystal microbalance with dissipation

A quartz crystal microbalance with a dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of adenosine-5'-triphosphate (ATP) by using an aptamer. The binding of ATP molecules on the aptamer films could be calculated as accurate mass changes using multiple frequency and dissipation measurements. The detection is achieved by calculating the mass changes from conformational rearrangements of the sensor surface upon interaction with the target. The sensor was demonstrated to respond to changes in ATP concentrations in real time suitable for continuous monitoring applications. This sensor showed excellent selectivity toward ATP compared with other chemically similar nucleotide GTP. The feasibility of the sensor was demonstrated by analyzing ATP concentrations in cell culture media with serum. The maximum frequency change was about -2 Hz after injection of 500 μM ATP. The affinity constant of the aptamer was determined to be 49 ± 7.59 μM. The proposed sensor can extend the application of the QCM-D system in medical diagnosis, and could be adopted for the detection of other small molecules with the use of specific aptamers.     

A High Sensitivity Electrochemical Sensor Based on Fe3+‐Ion Molecularly Imprinted Film for the Detection of T‐2 Toxin

The excellent detection sensitivity in various matrices of T‐2 toxin (T‐2), which has cytotoxic and immunosuppressive effects in DNA and RNA synthesis, is a highly desirable characteristic. A sensitive molecularly imprinted electrochemical sensor was constructed for the selective detection of T‐2. In this study, iron ions (Fe³⁺) were introduced to increase the chelation of the metal ions and templates for preparing molecularly imprinted polymers (MIPs). With the increased chelation of the metal ions and templates, the selectivity and sensitivity of the MIPs were effectively improved. The imprinted sensor was successfully employed to detect T‐2 in cereals and human serum samples.     

Determination of Saxitoxin by Aptamer-Based Surface-Enhanced Raman Scattering

Saxitoxin is one of the most harmful paralytic shellfish toxins due to its high toxicity and adverse effects on the environment and human health. Aptasensors provide simple detection procedures because they have the advantages of chemical stability, easy synthesis and modification, and high convenience in signal transformation. Surface-enhanced Raman scattering (SERS) is an analytical technique that amplifies the analytical signals of molecules at extremely low concentrations, or even at the single molecule level, when the analyte is very close to rough metal surfaces or nanostructures. In this study, an SERS aptasensor is reported for the determination of saxitoxin for the first time. The optimized saxitoxin aptamer (M-30f) was modified on gold nanoparticles and served as the recognition element. Crystal violet was used as the Raman reporter without chemical bounding. The analytical principles of the aptasensor are that saxitoxin destabilized the conformations of the aptamer at high temperature conditions and altered the binding of crystal violet on the gold nanoparticles. In the presence of saxitoxin, the conformation of aptamer containing the G-quadruplex that selectively bound crystal violet unfolded to a large extent and hence the crystal violet molecules were released from gold nanoparticles with a reduced SERS signal. The effects of the gold nanoparticle size, the amount of DNA, aptamer density, sodium chloride concentration, and operation temperature upon the SERS determination were optimized. The resulting simple SERS aptasensor was developed with a satisfactory limit of detection (11.7?nM) and selectivity. The application for the analysis of real shellfish samples with simple procedures demonstrates that this SERS aptasensor is promising for on-site applications.     

Molecularly Imprinted Polymer Thin Film Based Surface Plasmon Resonance Sensor to Detect Hemoglobin

Molecularly imprinted polymer（MIP） films for hemoglobin detection were prepared onto the Au/Cr coated surface plasmon resonance（SPR） sensor chips by the in situ electropolymerization of 3-aminophenylboronic acid（3-APBA）.The formation of the films and rebinding processes of hemoglobin were monitored by in situ electrochemical-SPR（EC-SPR） spectroscopy,with allowed real-time observation of the simultaneous changes in electrochemical and optical properties of the films.Scanning electron microscopy（SEM） and atomic force microscopy（AFM）were used to characterize the surface morphologies of the MIP films.The effects of pH,ion strength,different metal ions on rebinding Hb,the specific binding and the selective recognition were investigated.The results obtained with the molecular imprinted SPR chips indicate a good adsorption of Hb in a range of 0.0005-5 mg/mL in 0.05 mol/L sodium phosphate buffer at pH=7.0.A linear calibration curve（R2=0.94） of the SPR sensor for Hb detection was obtained in a range of 0.05-5 mg/mL.The detection limit for hemoglobin by this method was 0.000435 mg/mL（S/N=3）.Interference studies indicate that the MIP films have a good selectivity compared with the referenced proteins.The stability of the sensor was also established.Results indicate that the SPR sensor chip keeps 87.6％ of its original response after 14 d of storage under dry and ambient conditions.     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: interlaboratory study

An interlaboratory study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3 together), gonyautoxins 1 and 4 (GTX1,4 together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2 together), and C-3 and C-4 (C3,4 together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Samples of mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with samples of clams, oysters, and scallops. Twenty-one samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries.     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: collaborative study

A collaborative study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3; together), gonyautoxins 1 and 4 (GTX1,4; together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2; together), and C-3 and C-4 (C3,4; together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with clams, oysters, and scallops. Twenty-one test samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL.     

Stripping voltammetric detection of mercury(II) based on a surface ion imprinting strategy in electropolymerized microporous poly(2-mercaptobenzothiazole) films modified glassy carbon electrode

This work reports a surface ion imprinting strategy in electropolymerized microporous poly(2-mercaptobenzothiazole) (MPMBT) films at the surface of glassy carbon electrode (GCE) for the electrochemical detection of Hg(II). The Hg(II)-imprinted MPMBT/GCE exhibits larger binding to functionalized capacity, faster binding kinetics and higher selectivity to template Hg(II) due to their high ratio of surface-imprinted sites, larger surface-to-volume ratios, the complete removal of Hg(II) templates and larger affinity to Hg(II). The square wave anodic stripping voltammetry (SW ASV) response of the Hg(II)-imprinted MPMBT/GCE to Hg(II) is ca. 3.0 and 5.9 times larger than that at the direct imprinted poly(2-mercaptobenzothiazole) modified GCE and non-imprinted MPMBT/GCE sensor, respectively; and the detection limit for Hg(II) is 0.1 nM (which is well below the guideline value given by the World Health Organization). Excellent wide linear range (1.0–160.0 nM) and good repeatability (relative standard deviation of 2.5%) were obtained for Hg(II). The interference experiments showed that mercury signal was not interfered in the presence of Pb(II), Cd(II), Zn(II), Cu(II) and Ag(I), respectively. These values, particularly the high sensitivity and excellent selectivity compared favorably with previously reported methods in the area of electrochemical Hg(II) detection, demonstrate the feasibility of using the prepared Hg(II)-imprinted MPMBT/GCE for efficient determination of Hg(II) in aqueous environmental samples.     

Preparation and Application of Urea Electrochemical Sensor Based on Chitosan Molecularly Imprinted Films

A novel urea electrochemical sensor was constructed based on chitosan molecularly imprinted films which were prepared by potentiostatic electrodeposition of chitosan in the presence of urea followed by eluting with 0.1 M KCl. Various techniques were carried out to investigate the formation of molecularly imprinted polymer (MIP) films and the performance of the sensor. According to our expectation, the urea MIP electrochemical sensor showed excellent selectivity to urea among the structural similarities and co‐existences, high linear sensitivity to urea in the range from 1.0×10^?8 to 4.0×10^?5 M with a detection limit of 5.0×10^?9 M. Furthermore, the recovery ranged from 96.3 % to 103.3 % and therefore offered great potential for clinical diagnosis applications.