Highly sensitive serological approaches for Pepino mosaic virus detection.

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.

The NIa-Protease Protein Encoded by the Pepper Mottle Virus Is a Pathogenicity Determinant and Releases DNA Methylation of Nicotiana benthamiana.

It is well documented that the canonical function of NIa-protease (NIa-Pro) of the potyviruses is responsible for cleaving the viral polyprotein into functional proteins. Although NIa-Pro is vital for the infection cycle of potyviruses, the function of NIa-Pro in the interaction of the potyvirus host is not clear. In this study, NIa-Pro is ectopically expressed from a potato virus X (PVX) vector and infiltrates Nicotiana benthamiana wild type and 16-TGS. The pathogenicity and inhibition of host transcriptional gene silencing (TGS) are characterized. Ectopic expression of NIa-Pro from a PVX vector resulted in severe mosaic symptoms followed by a hypersensitive-like response in N. benthamiana. Furthermore, PepMoV NIa-Pro was able to reverse established TGS of a green fluorescent protein transgene by reducing methylation of promoter sequences in N. benthamiana and possessed the capacity to interfere with the global methylation of N. benthamiana. Taken together, the results of this study likely suggest that PepMoV NIa-Pro is a pathogenicity determinant and a potent suppressor of host TGS and suggest that NIa-Pro may employ novel mechanisms to suppress host antiviral defenses. To the best of our knowledge, this is the first report of a plant RNA virus modulating host TGS in a novel manner by interfering with the establishment of the methylation step of the plant DNA methylation pathway.

Adaptation mechanism and tolerance of Rhodopseudomonas palustris PSB-S under pyrazosulfuron-ethyl stress.

BACKGROUND: Pyrazosulfuron-ethyl is a long lasting herbicide in the agro-ecosystem and its residue is toxic to crops and other non-target organisms. A better understanding of molecular basis in pyrazosulfuron-ethyl tolerant organisms will shed light on the adaptive mechanisms to this herbicide. RESULTS: Pyrazosulfuron-ethyl inhibited biomass production in Rhodopseudomonas palustris PSB-S, altered cell morphology, suppressed flagella formation, and reduced pigment biosynthesis through significant suppression of carotenoids biosynthesis. A total of 1127 protein spots were detected in the two-dimensional gel electrophoresis. Among them, 72 spots representing 56 different proteins were found to be differently expressed using MALDI-TOF/TOF-MS, including 26 up- and 30 down-regulated proteins in the pyrazosulfuron-ethyl-treated PSB-S cells. The up-regulated proteins were involved predominantly in oxidative stress or energy generation pathways, while most of the down-regulated proteins were involved in the biomass biosynthesis pathway. The protein expression profiles suggested that the elongation factor G, cell division protein FtsZ, and proteins associated with the ABC transporters were crucial for R. palustris PSB-S tolerance against pyrazosulfuron-ethyl. CONCLUSION: Up-regulated proteins, including elongation factor G, cell division FtsZ, ATP synthase, and superoxide dismutase, and down-regulated proteins, including ALS III and ABC transporters, as well as some unknown proteins might play roles in R. palustris PSB-S adaptation to pyrazosulfuron-ethyl induced stresses. Functional validations of these candidate proteins should help to develope transgenic crops resistant to pyrazosulfuron-ethyl.

Selective and Sensitive Detection of Silver(I) Ion Based on Tetracationic Complex and TGA/GSH Co-capped Quantum Dots as an Effective Fluorescent Sensing Platform.

CdTe quantum dots capped with glutathione (GSH) and thioglycolic acid (TGA) were synthesized and the interaction between QDs and tetracationic Fe complex was investigated. Based on the specific interaction between Ag+ and cytosine bases (C), we designed a label-free DNA sensor for the detection of Ag+ in aqueous solution. Furthermore, tetracationic Fe complex with a higher positive charge is demonstrated to improve the sensitivity of the sensor. A detection limit of 3.3 nmol dm-3 was obtained, which was lower than in previous reports. This sensor also exhibits promising potential for real sample analysis.

DNA Interaction, Photocleavage and Topoisomerase I Inhibition by Ru(II) Complex with a New Ligand Possessing Phenazine Unit.

A new ruthenium complex with a dppz-like ligand pyidppz, [Ru(bpy)2(pyidppz)](2+) (pyidppz = 2-(pyridine-2-yl)imidazo-[4,5-b]dipyrido-[3,2-a:2',3'-c]phenazine) has been synthesized and characterized by ES-MS, elemental analysis, (1)H NMR. Intercalative mode of the complex bound to calf thymus DNA has been supported by different spectroscopic methods and viscosity measurements. The introduction of phenazine unit may be one of the main reasons for the weak emission of Ru(II) complex in aqueous solution. Under irradiation, this complex can efficiently cleave DNA. And the photocleavage reaction of the complex is found to be inhibited in the presence of singlet oxygen scavenger. Topoisomerase inhibition and DNA strand passage assay demonstrated that [Ru(bpy)2(pyidppz)](2+) and its parent complex [Ru(bpy)2(pyip)](2+) (pyip = 2-(pyridine-2-yl)imidazo[4,5-f][1,10]phenanthroline) can act as efficient catalytic inhibitor of DNA topoisomerase I.

Study on DNA binding behavior and light switch effect of new coumarin-derived Ru(II) complexes.

A new ligand mhcip (mhcip=2-(4-methyl-7-hydroxyl-8-coumarinyl)imidazo[4,5-f]-[1,10]phenanthroline) and its ruthenium complexes, [Ru(L)2mhcip](2+) (L=bpy (2,2'-bipyridine), phen (1,10-phenanthroline)), have been synthesized and characterized. The introduction of coumarin ring may play an important role in the strong fluorescence of the complexes. Intercalative binding mode between both complexes and CT-DNA was determined by UV-visible spectroscopy, fluorescence spectroscopy and viscosity measurements. The two complexes show efficient DNA photocleavage under irradiation at 365 nm. The cycling of light switch off and on has been achieved for both complexes through the introduction of Cu(2+) and EDTA in the absence or presence of DNA.

Isothermal amplification system based on template-dependent extension.

A novel template-dependent extension based isothermal amplification (TEIA) system with high single-base discrimination capability is developed, where the interference caused by non-specific reaction in isothermal strand displacement amplification (SDA) technique is substantially avoided via using a functionalized template probe, showing potential value in the development and application of SDA based detection devices.

Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction.

In order to develop a highly sensitive and selective piezoelectric transducer for the detection of DNA, the bio-recognizing probe is for the first time designed by introducing a hairpin structure and a recognition site for EcoRI into an oligonucleotide sequence and signal amplifiers are prepared by modifying gold nanoparticles (GNPs) with biomolecules, deepening the application and understanding of biomaterials. The piezoelectric transducer is prepared by immobilizing designed hairpin recognition probe onto the quartz-crystal-microbalance (QCM). In the absence of target DNA, the hairpin probe is removed from the QCM surface after exposure to endonuclease, inhibiting the subsequent signaling reaction. In contrast, introduction of target DNA can open the hairpin probe due to the probe/target hybridization, dissociating the cleavable double-stranded portion. In this case, even if being treated with endonuclease, the integrated hairpin probe is maintained. Subsequent introduction of GNPs modified with detection probes that can hybridize to the terminal sequence of hairpin probe results in a many-folds increase of the frequency response. Utilizing the proposed transduction scheme, the reliable target DNA detection can be accomplished. The detection limit of 2 pM and dynamic response range for target DNA from 2 to 300 pM are obtained. Furthermore, single-base mismatched DNAs can be easily identified. The developed proof-of-principle of a novel piezoelectric transduction scheme is expected to establish a potential platform for the disease-associated mutation analysis and DNA hybridization detection in biotechnology and medical diagnostics.

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection.

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34nM of HNE, and the response is linear in the tested concentration range of 0.34-68nM with the detection limit of 47pM.

Novel fluorescence enhancement IgE assay using a DNA aptamer.

In this paper, we demonstrate a fluorescence immunoglobulin E (IgE) assay probe based on a DNA aptamer. A Texas red-labeled short DNA strand (T-DNA) complementary with part of the IgE aptamer sequence was used to produce the fluorescence enhancement effected upon the binding of IgE to the aptamer. Another short DNA strand labeled with dabcyl quencher (Q-DNA) complementary with part of the aptamer sequence nearby the T-DNA location was used to lower the background fluorescence. The IgE can be detected in the concentration range from 9.2 x 10(-11) to 3.7 x 10(-8) mol L(-1) with a detection limit of 5.7 x 10(-11) mol L(-1).

Reusable electrochemical sensing platform for highly sensitive detection of small molecules based on structure-switching signaling aptamers.

Aptamers are nucleic acids that have high affinity and selectivity for their target molecules. A target may induce the structure switching from a DNA/DNA duplex to a DNA/target complex. In the present study, a reusable electrochemical sensing platform based on structure-switching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is first modified with polytyramine and gold nanoparticles. Then, thiolated capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on the electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of the aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability, and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for an adenosine assay.

Homogeneous, unmodified gold nanoparticle-based colorimetric assay of hydrogen peroxide.

An unmodified gold nanoparticle-based colorimetric assay system in homogeneous format has been developed using hydrogen peroxide (H(2)O(2)) as a model analyte. H(2)O(2) is added to o-phenylenediamine/horseradish peroxidase solution, and allowed to react for 10 min. Then, unmodified gold nanoparticles that serve as "reaction indicators" are added to the reaction solution. The resulting mixture color changes dramatically from red to blue. The reason is that azoaniline, a horseradish peroxidase-catalyzed oxidation product, induces the nanoparticle aggregation. Using this approach, H(2)O(2) can be semiquantitatively determined over the concentration range of approximately 4 orders of magnitude by the naked eye. If the observed peak intensity at 420 nm is used for the construction of the calibration plot, hydrogen peroxide can be accurately determined down to concentration levels of 1.3 x 10(-6) M. Compared with the conventional electrochemical protocol, this sensing system offers several important advantages: (1) ability to be monitored by the naked eye, (2) avoiding the need of surface modification of electrodes or gold nanoparticles and (3) detection in homogeneous solution. It is worthy of note that this efficient and convenient strategy is also suitable for the detection of other species, such as glucose and cholesterol.

A novel immunoassay strategy based on combination of chitosan and a gold nanoparticle label.

A novel immunoassay strategy based on combination of chitosan (CHIT) and a gold nanoparticle (GNP) label has been developed. The susceptibility of CHIT to further chemical modifications due to the abundant amino groups is explored in order to covalently immobilize antibody (Ab) onto the (3-aminopropyl) triethoxysilane derivatized glass slide by cross-linking with glutaraldehyde (GA). After incubating in antigen (Ag) solution, the obtained substrate is immersed in GNP labeled antibody solution for signal generation. The two steps were repeated alternatively for three times, forming multilayer of gold nanoparticles via antigen-antibody specific reaction. Ultraviolet-visible (UV-vis) absorption spectrum is recorded to obtain quantitative information about the specific antigen. The presented immunoassay strategy is applied for determination of human serum albumin (HSA) as a model analyte. The immunoassay of HSA is specific. Compared to previous correlative work, the proposed immunosensing strategy shows some advantages, such as improved sensitivity as much more gold nanoparticles can be coupled to the functionalized surface making use of the abundant amino groups of CHIT. Moreover, a significantly extended linear detection range of 8.0-512.0mug/mL is gained under the optimized experimental conditions. In particular, the presented biosensing method shows low cost and simplicity, and only a conventional UV-vis detector is involved.