Quartz Crystal Microbalance Biosensor for Saxitoxin Based on Immobilized Sodium Channel Receptors

This work explored the possibility of coupling the toxin receptor-binding principle with the piezoelectric transduction principle. The sensing component of the saxitoxin biosensor involves a piezoelectric quartz crystal that was coated with sodium channel receptors. The sodium channel receptors were isolated from the electroplax organ of Electrophorus electricus. Binding of the sodium channel extracts to the quartz crystal was facilitated by pre-coating the gold electrode with a hydrophobic self-assembled monolayer of dodecanethiol. The instrumentation system consisted of a flow cell that held the quartz crystal, an oscillator circuit, an injection port, and a frequency counter that was connected to a personal computer. The various immobilization and measurement parameters were optimized. Binding of saxitoxin standards with the immobilized sodium channels was monitored through the decrease in the crystal oscillation frequency readings (ΔF) upon the introduction of saxitoxin into the flow cell. A calibration curve for saxitoxin was constructed by plotting the ΔF values vs. saxitoxin concentrations in the range from 0.1 to 2.0 μg/mL. A correlation coefficient of 0.9653 was obtained. The saxitoxin biosensor developed has the potential to be applied to the rapid screening of total paralytic shellfish poisoning toxins.     

Synthetic studies towards zetekitoxin AB: preparation of 4,5-epi-11-hydroxy-saxitoxinol

A concise synthesis of 4,5-epi-11-hydroxy-saxitoxinol utilizing d-ribose to direct an asymmetric Mannich reaction. This approach allows many modes of reactivity, which can be used to access various analogs of saxitoxin.     

Synthesis and Identification of Key Biosynthetic Intermediates for the Formation of the Tricyclic Skeleton of Saxitoxin

Saxitoxin (STX) and its analogues are potent voltage‐gated sodium channel blockers biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified genetically predicted biosynthetic intermediates of STX at early stages, Int‐A′ and Int‐C′2, in these microorganisms. However, the mechanism to form the tricyclic skeleton of STX was unknown. To solve this problem, we screened for unidentified intermediates by analyzing the results from previous incorporation experiments with ¹⁵N‐labeled Int‐C′2. The presence of monohydroxy‐Int‐C′2 and possibly Int‐E′ was suggested, and 11‐hydroxy‐Int‐C′2 and Int‐E′ were identified from synthesized standards and LC‐MS. Furthermore, we observed that the hydroxy group at C11 of 11‐hydroxy‐Int‐C′2 was slowly replaced by CD₃O in CD₃OD. Based on this characteristic reactivity, we propose a possible mechanism to form the tricyclic skeleton of STX via a bicyclic intermediate from 11‐hydroxy‐Int‐C′2.     

Development of an Electrochemical Biosensor for the Rapid Detection of Saxitoxin Based on Air Stable Lipid Films with Incorporated Anti‐STX Using Graphene Electrodes

A miniaturized potentiometric saxitoxin sensor on graphene nanosheets with incorporated lipid films and Anti‐STX, the natural saxitoxin receptor, immobilized on the stabilized lipid films is described in the present paper. An adequate selectivity for detection over a wide range of toxin concentrations, fast response time of ca. 5–20 min, and detection limit of 1 nM have been achieved. 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 evaluated in lake water and shellfish samples. This novel ultrathin film technology is currently adapted to the rapid detection of other toxins that could be used in bioterrorism.     

Liquid chromatography/quadrupole time-of-flight mass spectrometry for determination of saxitoxin and decarbamoylsaxitoxin in shellfish

Saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) were determined by liquid chromatography with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A shellfish tissue was extracted with 0.1 mol/l HCl under ultrasonication, and cleanup of extract was accomplished by solid-phase extraction with a C18 cartridge. Chromatographic separation was carried out on a C18 column (150 mm x 2.1 mm, 3.5 microm) with gradient elution of MeOH-H2O (20:80) containing 0.05% heptafluorobutyric acid and MeOH-H2O (15:85) containing 0.05% acetic acid. The protonated molecule [M + H]+ ions at m/z 257 for dcSTX and 300 for STX were selected in precursor ion scanning for Q-TOF MS in the positive electrospray ionizaion mode. Average recoveries and relative standard deviations, by analyzing samples spiked at a level of 0.1, 0.8 or 1.6 microg/g, were 84-92 and 8-14%, respectively. Identification of the presence of the toxins in shellfish tissues was based on the structural information offered by Q-TOF MS.     

Capillary electrophoresis inductively coupled plasma mass spectrometry combined with metal tag for ultrasensitively determining trace saxitoxin in seafood

As one of paralytic shellfish toxins, the saxitoxin (STX) in the aqueous environment can be accumulated by most shellfish, and thus harms human health through the food chain. Therefore, it is crucial to determine trace STX in seafood samples in order to ensure the safety of seafood consumption. In this study, we developed a novel indirect method for ultrasensitively determining trace STX in seafood by using CE‐ICP‐MS together with Eu³⁺ chelate labeling. We demonstrated that diethylenetriamine‐N ,N ,N ′,N ″,N ″‐pentaacetic acid (DTPA) can couple with STX and simultaneously chelate with Eu³⁺ to realize metallic labeling of STX, and thus realize the ultrasensitive quantification of trace STX with CE‐ICP‐MS. The proposed method has strong antiinterference ability, good stability, and extremely high sensitivity. It could be used to determine trace STX in seafood samples with an extremely low detection limit of 0.38 fmol (3.8×10⁻⁹ M, 100 nL sample injection) and a relative standard deviation (RSD, n = 5) <7%. The success of this study provides an alternative to precise quantification of ultra‐trace STX in seafood samples, and further expands the application of ICP‐MS.     

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated μ-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n = 60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2 × 2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms.