Construction of eco-biosensor and its potential application for highly selective, sensitive and fast detection of viscumin.

Viscum album lectin 1 (Viscumin) is one of the most important plant-based protein of potential adjuvant in cancer treatment. Therefore, the use of nano-biosensor technology as a novel emerges of biosensors is crucial to detect this modal agent in pharmacological study. Molecular imprinted polymer using 9-mer peptides sequence (epitope) was applied as a template. Using ultraviolet light, hydrogen bonding attained between the functional monomer and epitope, leading to the formation of a molecularly imprinted polymer. In the following, the epitope was derived from the surface of the polymer by sodium dodecyl sulfate (SDS) 2.5% and acetic acid 0.6% w/w. Finally, the designed nano-biosensor was exposed to different concentrations of the epitope. The selectivity of the nano-biosensor was tested in complex matrices such as blood plasma and urine. The scatchard analysis was covered for a consequence of the dissociation constants and the numbers of binding sites. Based on the results, the designed nano-biosensor has a limit of detection of 0.117 ng/µl and limit of quantification of 0.517 ng/µl in PBS buffer, respectively. These amounts stood 0.5 ng/µl and 0.8 ng/µl for urine environment and 1.25 ng/µl and 5 ng/µl for human blood fresh frozen plasma in the presence of ricin as the most homologue of viscumin (ML1) in fixed concentration (12:1), respectively. The time of detection and optimum pH was 8.0 min and 7.4, respectively. Designed and synthesized nano-biosensor is adequately qualified to be used in diverse complex areas, due to good efficiency.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Single-drop optimization of protein crystallization.

A completely new crystal-growth device has been developed that permits charting a course across the phase diagram to produce crystalline samples optimized for diffraction experiments. The utility of the device is demonstrated for the production of crystals for the traditional X-ray diffraction data-collection experiment, of microcrystals optimal for data-collection experiments at a modern microbeam insertion-device synchrotron beamline and of nanocrystals required for data collection on an X-ray laser beamline.

Identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry.

Ricin is a toxic protein produced in the seeds of the castor bean plant. The toxicity of the protein and the ease in which it can be extracted from the seeds makes it a potential biological warfare agent. There has been extensive work in the development of analytical techniques that can identify the protein robustly and rapidly. On-target tryptic digestion and MALDI MS was used to distinguish ricin from bovine serum albumin and three other type 2 ribsome-inactivating proteins (RIPs), abrin, agglutinin (RCA(120)), and viscumin, using the peptide mass fingerprint. The sequence coverage obtained was enhanced using methanol-assisted tryptic digestion and was particularly useful for the detection of these toxins in complex matrixes. When used in conjunction with intact protein MALDI mass measurement, a positive identification of ricin (or any of the other RIPs) was achieved including confirmation of the integrity of the disulfide bond between the A and B chains. This applicability of this methodology was demonstrated by the identification of ricin in a typical "crude white powder" that may be illicitly produced in a clandestine lab. The analysis on the solubilized sample using this method can be undertaken in around an hour with minimal sample preparation.