Weiming Tan

Development of Protein A Functionalized Microcantilever Immunosensors for the Analyses of Small Molecules at Parts per Trillion Levels

Development of microcantilever biosensors for small molecules was exemplified with the beta-adrenergic agonist clenbuterol and the antibiotic chloramphenicol. In this paper, antibody sulfhydrylation and protein A were used to modify the microcantilever Au surface, and the antibody activities on the microcantilever were evaluated with direct competitive enzyme-linked immunosorbent assay (dcELISA). The activity of the antibodies immobilized on the microcantilever via protein A was 1.7-fold of that via the sulfhydrylation reagent 2-iminothiolane hydrochloride. A microcantilever immunosensor method with protein A as the functionalization reagent was established to detect the residues of clenbuterol and chloramphenicol at limits of detection (LOD) of approximately 0.1 and 0.2 ng/mL, respectively. Such LODs were better than that of the corresponding dcELISAs. The concentration of clenbuterol in a fortified feed sample detected with the microcantilever immunosensor after thorough extraction and purification agreed well with that detected with the dcELISA. Protein A showed to be simple and reproducible for functionalization of the antibodies on the Au surface and, thus, has common application values in microcantilever immunosensor development. The results suggest that microcantilever immunosensors be suitable for detection of small molecules, and the assay sensitivity is mainly related to the quality and activities of the antibodies.

Improved biological effects of uniconazole using porous hollow silica nanoparticles as carriers.

BACKGROUND The aim of this work is to prepare a controlled-release formulation of uniconazole using porous hollow silica nanoparticles (PHSNs) as carrier, and to investigate the biological effects on rice growth. RESULTS PHSNs with a shell thickness of ~15 nm and a particle size of 80-100 nm were synthesised through a sol-gel route using nanosized calcium carbonate particles as templates. Simple immersing (SI) and supercritical fluid drug loading (SFDL) technologies were employed to load uniconazole into PHSNs with loading efficiencies of ~22 and ~26% respectively. The prepared uniconazole-loaded PHSNs (UCZ-PHSNs) by SI and SFDL both demonstrated sustained release properties, and the latter showed better controlled release ability with a slower release rate. Compared with free uniconazole, UCZ-PHSNs exhibited a weaker growth retardation effect in the early stage but more significant retardation ability in later stages for agar-cultured rice seedlings. For the rice that grew in clay, UCZ-PHSNs demonstrated a weaker plant height retardation effect than free uniconazole at the early jointing stage by foliar spraying, but exhibited a stronger retardation capacity than free uniconazole by being applied into soil before seedling transplantation. CONCLUSION The results indicated that the prepared UCZ-PHSNs possessed good controlled-release properties and had improved retardation effects on rice growth. It is recommended that UCZ-PHSNs be applied into soil before seedling transplantation rather than administered by foliar spraying at the early jointing stage.

Development of a Secondary Antibody Thio-Functionalized Microcantilever Immunosensor and an ELISA for Measuring Ginsenoside Re Content in the Herb Ginseng

Ginsenoside Re (GRe) is a major active component of the Chinese medicinal herb ginseng, Panax ginseng . A sensitive and specific monoclonal antibody (mAb), designated as mAb3D6, was generated with a GRe-bovine serum albumin conjugate as an immunogen. Microcantilever immunosensors (MCS), one modified with thiolated anti-GRe antibody and one modified with thiolated goat antimouse immunoglobulin G (IgG), were developed to detect the content of ginsenoside. The MCS immobilized with thiolated goat antimouse IgG had a better sensitivity than the MCS modified with thiolated anti-GRe antibody. The advantage of a secondary antibody thio-functionalized MCS was verified with the anti-paclitaxel mAb. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was also established with mAb3D6. The concentration of analyte producing 50% inhibition and the working range of icELISA were 1.20 and 0.15-16.1 ng/mL, respectively. The icELISA had a cross-reactivity of 89% with ginsenoside Rg1 and less than 3% with other ginsenosides. The icELISA and MCS with thiolated secondary antibody were applied for the determination of GRe in ginseng samples, and the results agreed well with those determined by high-performance liquid chromatography.

The effect of phosphate buffer solutions on uniconazole complexation with hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin

The inclusion complexes of uniconazole [(E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)-1-penten-3-ol, UCZ] with two cyclodextrin derivatives, hydroxypropyl-β-cyclodextrin (HP-β-CD) and methylated-β-cyclodextrin (Me-β-CD), were prepared and characterized by 1H NMR and FT-IR. The phase solubility of UCZ and HP-β-CD, UCZ and Me-β-CD, which displays the ability of CDs complexation and solubilization, was studied in aqueous solutions and phosphate buffer solutions (PBS) with different property pH values (6.2, 7.2, 8.0). The solubility results indicated that the pH of PBS showed more enhancement on the interaction of HP-β-CD and UCZ than Me-β-CD with the increasing pH value, and the optimal pH value for complexation of UCZ and HP-β-CD, UCZ and Me-β-CD was at 8.0 and at 7.2, respectively. These were also determined by UCZ release behavior and dissolution studies of the complexes in solid state.

Photoprotectant improves photostability and bioactivity of abscisic acid under UV radiation.

Photosensitivity causes serious drawback for abscisic acid (ABA) application, but preferable methods to stabilize the compound were not found yet. To select an efficient photoprotectant for the improvement of photostability and bioactivity of ABA when exposed to UV light, we tested the effects of a photostabilizer bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (HS-770) and two UV absorbers 2-hydroxy-4-n-octoxy-benzophenone (UV-531) and 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid (BP-4) with or without HS-770 on the photodegradation of ABA. Water soluble UV absorber BP-4 and oil soluble UV absorber UV-531 showed significant photo-stabilizing capability on ABA, possibly due to competitive energy absorption of UVB by the UV absorbers. The two absorbers showed no significant difference. Photostabilizer HS-770 accelerated the photodegradation of ABA and did not improve the photo-stabilizing capability of BP-4, likely due to no absorption in UVB region and salt formation with ABA and BP-4. Approximately 26% more ABA was kept when 280mg/l ABA aqueous solution was irradiated by UV light for 2h in the presence of 200mg/l BP-4. Whats more, its left bioactivity on wheat seed (JIMAI 22) germination was greatly kept by BP-4, comparing to that of ABA alone. The 300 times diluent of 280mg/l ABA plus 200mg/l BP-4 after 2h irradiation showed more than 13% inhibition on shoot and root growth of wheat seed than that of ABA diluent alone. We concluded that water soluble UV absorber BP-4 was an efficient agent to keep ABA activity under UV radiation. The results could be used to produce photostable products of ABA compound or other water soluble agrichemicals which are sensitive to UV radiation. The frequencies and amounts of the agrichemicals application could be thereafter reduced.

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

The Phytotoxin Coronatine Induces Abscission-Related Gene Expression and Boll Ripening during Defoliation of Cotton

Defoliants can increase machine harvest efficiency of cotton (Gossypium hirusutum L.), prevent lodging and reduce the time from defoliation to harvest. Coronatine (COR) is a chlorosis-inducing non-host-specific phytotoxin that induces leaf and/or fruit abscission in some crops. The present study investigates how COR might induce cotton leaf abscission by modulating genes involved in cell wall hydrolases and ACC (ethylene precursor) in various cotton tissues. The effects of COR on cotton boll ripening, seedcotton yield, and seed development were also studied. After 14 d of treatment with COR, cells within the leaf abscission zone (AZ) showed marked differentiation. Elevated transcripts of GhCEL1, GhPG and GhACS were observed in the AZs treated with COR and Thidiazuron (TDZ). The relative expression of GhCEL1 and GhACS in TDZ treated plants was approximately twice that in plants treated with COR for 12 h. However, only GhACS expression increased in leaf blade and petiole. There was a continuous increase in the activity of hydrolytic enzymes such as cellulase (CEL) and polygalacturonase (PG), and ACC accumulation in AZs following COR and TDZ treatments, but there was greater increase in ACC activity of COR treated boll crust, indicating that COR had greater ripening effect than TDZ. Coronatine significantly enhanced boll opening without affecting boll weight, lint percentage and seed quality. Therefore, COR can be a potential cotton defoliant with different physiological mechanism of action from the currently used TDZ.

Evaluation of Harvest Aid Chemicals for the Cotton-Winter Wheat Double Cropping System

Abstract Timing of harvest is critical for mechanical picking in cotton production, especially in those regions with double cropping system. Appropriate and safe harvest aids will improve timing and facilitate harvest of cotton in the double cropping system. Three defoliants (dimethipin, thidiazuron, and thidiazuron-diuron) and one boll opener (ethephon) were included in this research. They were evaluated for their effects on defoliation, boll opening, seedcotton yield, seed quality, and fiber quality of field grown cotton when used alone or as a mixture in 2009 and 2010. Defoliation and/or boll opening were increased by all three defoliants and ethephon, especially by mixtures of a defoliant and ethephon. First harvest of seedcotton was significantly increased with defoliant-ethephon mixtures. No significant adverse effects were observed on boll weight, lint percentage, seed quality, and fiber properties. It was estimated that tank mixes of ethephon and one of the three defoliants can improve the adjusted gross revenue. Boll opening can be used as an alternative indicator for the adjusted gross revenue, because, it was linearly and positively correlated with the relative adjusted gross revenue and convenient in measurements. Wheat seedling growth was not affected by thidiazuron, whereas its seedling emergence, root dry weight, relative water content, and electrolyte leakage were adversely affected by dimethipin and thidiazuron-diuron when concentration was above 340 and 100 g (a.i.) ha−1, respectively. 90% defoliation and 80% boll opening were observed with the high rate of thidiazuron-ethephon mixture, but no adverse effects on winter wheat. The results suggested that tank mixes of ethephon with thidiazuron can be used effectively and safely in the cotton-winter wheat double cropping system to improve yield without adverse effects on seed quality and fiber quality.

Lignosulfonate Improves Photostability and Bioactivity of Abscisic Acid under Ultraviolet Radiation

Abscisic acid (ABA), as a commonly used plant growth regulator, is easy to be degraded and lose its bioactivity under sunshine. To select an eco-friendly and efficient photoprotectant for the improvement of photostability and bioactivity of ABA when exposed to ultraviolet (UV) light, we tested the effects of three biodegradable natural-derived high polymers, sodium lignosulfonates 3A [molecular weight (MW) > 50000, with degree of sulfonation (DS) of 0.48] and NA (20000 < MW < 50000, with DS of 0.7) and calcium lignosulfonate CASA (MW < 20000, with DS of 0.7), on the photodegradation of ABA. Lignosulfonates 3A, NA, and CASA showed significant photostabilizing capability on ABA. Lignosulfonate 3A showed preferable photostabilizing effects on ABA compared to CASA, while NA showed an intermediate effect. That indicated that lignosulfonate with a high MW and low DS had a stronger UV absorption and the hollow aggregate micelles formatted by lignosulfonate protect ABA from UV damage. Approximately 50% more ABA was kept when 280 mg/L ABA aqueous solution was irradiated by UV light for 2 h in the presence of 2000 mg/L lignosulfonate 3A. The bioactivity on wheat (JIMAI 22) seed germination was greatly kept by 3A in comparison to that of ABA alone. The 300 times diluent of 280 mg/L ABA plus 2000 mg/L 3A after 2 h of irradiation showed 20.8, 19.3, and 9.3% more inhibition on shoot growth, root growth, and root numbers of wheat seed, separately, in comparison to ABA diluent alone. We conclude that lignosulfonate 3A was an eco-friendly and efficient agent to keep ABA activity under UV radiation. This research could be used in UV-sensitive and water-soluble agrichemicals and to optimize the application times and dosages of ABA products.

A Novel Bikinin Analogue for Arabidopsis and Rice with Superior Plant Growth-Promoting Activity

Bikinin was previously identified in a chemical genetics screening as one of the most active brassinolide (BL) mimetics. To determine the effects of different heterocyclic groups replacing the pyridine ring on activity and to isolate novel highly active BL mimetics, a series of bikinin analogues bearing an ethyl ester in the carboxyl group attached on various heterocyclic moieties were synthesized for investigation of bioactivity on Arabidopsis and rice. The results showed that methylthiazole amide displayed better growth-promoting activity and greater efficacy than bikinin.