GFP-based red-emissive fluorescent probes for dual imaging of ß-amyloid plaques and mitochondrial viscosity.

Alzheimer's disease (AD), with incurable neurodegenerative damage, has attracted growing interest in exploration of better AD biomarkers in its early diagnosis. Among various biomarkers, amyloid-ß (Aß) aggregates and mitochondrial viscosity are closely related to AD and their dual imaging might provide a potential and feasible strategy. In this work, five GFP-based red-emissive fluorescent probes were rationally designed and synthesized for selective detection of ß-amyloid plaques and viscosity, among which C25e exhibited superior properties and could successfully image ß-amyloid plaques and mitochondrial viscosity with different fluorescence wavelength signals "turn-on" at around 624 and 640 nm, respectively. Moreover, the staining of brain sections from a transgenic AD mouse showed that probe C25e showed higher selectivity and signal-to-noise ratio towards Aß plaques than commercially-available Thio-S. In addition, the probe C25e was, for the first time, employed for monitoring amyloid-ß induced mitochondrial viscosity changes. Therefore, this GFP-based red-emissive fluorescent probe C25e could serve as a dual-functional tool for imaging ß-amyloid plaques and mitochondrial viscosity, which might provide a unique strategy for the early diagnosis of Alzheimer's disease.

Facile Fabrication of Highly Quantum Dot/AuNP-Loaded Tags for a Dual-Modal Colorimetric/Reversed Ratiometric Fluorescence Immunochromatographic Assay.

Developing an easily-prepared, sensitive, and accurate point-of-need immunochromatographic assay (ICA) is significant in food safety screening, clinical diagnosis, and environmental monitoring. However, the current single-modal ICAs are limited in certain instinct drawbacks that restrict analytical performances. Herein, we introduce an ultrasensitive dual-modal colorimetric/reversed ratiometric fluorescence ICA based on facilely prepared immunoprobes with a high loading capacity of red quantum dots and AuNPs. By smartly integrating these red-colored/fluorescent signal probes with an immobilized green quantum dot antigen on the test lines, discrete "turn-on" visual inspection and reversed ratiometric quantification via a portable smartphone-based analyzer were accomplished. As an application, this method was employed to detect 11 phosphodiesterase-5 inhibitors in health foods with ultralow detection limits (0.0028-0.045 ng/mL), high repeatability (coefficient of variations of 0.3-1.91%), and reasonable accuracy (recoveries of 86.6-107%). The proposed method was further validated by the authorized liquid chromatography with tandem mass spectrometry method in actual sample detection. This new assay format can be extended to ultrasensitive flexible detection of other food contaminants, environmental pollutants, or tumor biomarkers within minutes, and it just requires simply prepared signal reporters, easy-to-operate procedures, and a low-cost miniaturized analyzer.

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch.

BACKGROUND: G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied. METHODS: Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation. RESULTS: G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle. CONCLUSIONS: Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch. GENERAL SIGNIFICANCE: These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.

Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter: implications for epigenetic regulation.

The C-5-methylation of cytosine in the CpG islands is an important pattern for epigenetic modification of gene, which plays a key role in regulating gene transcription. G-quadruplex is an unusual DNA secondary structure formed in G-rich regions and is identified as a transcription repressor in some oncogenes, such as c-myc and bcl-2. In the present study, the results from CD spectrum and FRET assay showed that the methylation of cytosine in the CpG islands could induce a conformational change of the G-quadruplex in the P1 promoter of bcl-2, and greatly increase the thermal-stability of this DNA oligomer. Moreover, the methylation of cytosine in the G-quadruplex could protect the structure from the disruption by the complementary strand, showing with the increasing ability to arrest the polymerase in PCR stop assay. This data indicated that the stabilization of the G-quadruplex structure in the CpG islands might be involved in the epigenetical transcriptional regulation for specific genes through the C-5-methylation modification pattern.

Au-Ag OHCs-based SERS sensor coupled with deep learning CNN algorithm to quantify thiram and pymetrozine in tea.

Pesticide residue detection in food has become increasingly important. Herein, surface-enhanced Raman scattering (SERS) coupled with an intelligent algorithm was developed for the rapid and sensitive detection of pesticide residues in tea. By employing octahedral Cu2O templates, Au-Ag octahedral hollow cages (Au-Ag OHCs) were developed, which improved the surface plasma effect via rough edges and hollow inner structure, amplifying the Raman signals of pesticide molecules. Afterward, convolutional neural network (CNN), partial least squares (PLS), and extreme learning machine (ELM) algorithms were applied for the quantitative prediction of thiram and pymetrozine. CNN algorithms performed optimally for thiram and pymetrozine, with correlation values of 0.995 and 0.977 and detection limits (LOD) of 0.286 and 29 ppb, respectively. Accordingly, no significant difference (P greater than 0.05) was observed between the developed approach and HPLC in detecting tea samples. Hence, the proposed Au-Ag OHCs-based SERS technique could be utilized for quantifying thiram and pymetrozine in tea.

Deep learning enabled miniature mass spectrometer for rapid qualitative and quantitative analysis of pesticides on vegetable surfaces.

Excessive pesticide use poses a significant threat to food safety. Rapid on-site detection of multi-target pesticide residues in vegetables is crucial due to their widespread distribution and limited shelf life. In this study, a rapid on-site screening method for pesticide residues on vegetable surfaces was developed by employing a miniature mass spectrometer. A direct pretreatment method involves placing vegetables and elution solution into a customized flexible ziplock bag, allowing thorough mixing, washing, and filtration. This process effectively removes pesticide residues from vegetable surfaces with minimal organic solvent usage and can be completed within 2 min. Moreover, this study introduced a deep learning algorithm based on a one-dimensional convolutional neural network, coupled with a feature database, to autonomously discriminate detection outcomes. By combining full scan MS and tandem MS analysis methods, the proposed method achieved a qualitative recognition accuracy of 99.62%. Following the qualitative discrimination stage, the target pesticide residue and internal standard can be simultaneously isolated and fragmented in the ion trap, thus enabling on-site quantitative analysis and warning. This method achieved a quantitative detection limit of 10 µg/kg for carbendazim in cowpea. These results demonstrate the feasibility of the proposed analytical system and strategy in food safety applications.

A sensitive and quantitative immunochromatographic assay for simultaneous detection of three stimulant laxatives in slimming food.

Bisacodyl, sodium picosulfate and their metabolite bis-(p-hydroxyphenyl)-pyridyl-2-methane (BHPM) are clinically used to treat constipation. In this study, with the rational hapten design, a broad-specific and highly sensitive monoclonal antibody (mAb) was obtained with half of inhibitory concentrations of 0.16, 0.18 and 0.65 ng/mL for bisacodyl, sodium picosulfate and BHPM, respectively. Based on this mAb, a rapid and sensitive lateral flow immunochromatographic assay toward bisacodyl, sodium picosulfate and BHPM in slimming foods was developed. This method can qualitatively and quantitatively screen three stimulant laxatives with cut-off values of 3-6 ng/mL by naked eye and quantitative detection limits of 0.14-0.41 ng/mL by reader. The acceptable recoveries of spiked samples (78.00 %-120.12 %) and consistent results with liquid chromatography with tandem mass spectrometry (LC-MS/MS) in real sample detection confirmed the accuracy of the method. The established method provides a technique for multiplex, sensitive, fast, and on-site detection of three stimulant laxatives in slimming food.

Novel Dual-Color Immunochromatographic Assay Based on Chrysanthemum-like Au@polydopamine and Colloidal Gold for Simultaneous Sensitive Detection of Paclobutrazol and Carbofuran in Fruits and Vegetables.

To ensure food safety and prevent the toxic effects of paclobutrazol (PBZ) and carbofuran (CAR) on humans, a sensitive and rapid method for the detection of PBZ and CAR in fruits and vegetables is required. Herein, a highly sensitive PBZ monoclonal antibody (PBZ mAb) and CAR monoclonal antibody (CAR mAb) with half-inhibitory concentrations (IC50) at 0.77 and 0.82 ng mL-1 were prepared, respectively. We proposed a novel dual-color immunochromatographic assay (ICA) with two test lines (T1 and T2) and an independent control line (C) based on chrysanthemum-like Au@Polydopamine (AuNC@PDA) and colloidal gold (AuNPs) for the simultaneous and sensitive detection of PBZ and CAR with naked-eye detection limits of 10 and 5 µg kg-1, respectively. The limits of detection (LOD) for PBZ and CAR were 0.117 and 0.087 µg kg-1 in orange, 0.109 and 0.056 µg kg-1 in grape, and 0.131 and 0.094 µg kg-1 in cabbage mustard, respectively. The average recoveries of PBZ and CAR in orange, grape, and cabbage mustard were 97.86-102.83%, with coefficients of variation from 8.94 to 11.05. The detection results of this method for 30 samples (orange, grapes, and cabbage mustard) agreed well with those of liquid chromatography-tandem mass spectrometry. The novel dual-color ICA was sensitive, rapid, and accurate for the simultaneous detection of PBZ and CAR in real samples.

LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus.

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a loop-mediated isothermal amplification (LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was established in one tube for the detection of the African swine fever virus (ASFV) p72 gene. The single-stranded DNA-fluorophore quencher reporter and CRISPR-derived RNA were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/µl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples; a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Highly selective monoclonal antibody-based lateral flow immunoassay for visual and sensitive determination of conazole fungicides propiconazole in vegetables.

The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g-1 and a linear range from 0.5 to 80 ng g-1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g-1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry.

Selective recognition of oncogene promoter G-quadruplexes by Mg2+.

Mg(2+) is one of the most important cations in cells, affecting the structures and functions of the proteins and nucleic acids. It should be noted that Mg(2+) is indispensable in DNA transcription, where G-quadruplex is believed to be actively involved. Therefore, it is important to investigate the influence of Mg(2+) on G-quadruplex. Here we studied the effect of Mg(2+) on G-quadruplex DNA with CD, FRET, EMSA, and PCR-stop assay. We found that various G-quadruplexes could be differentiated through simultaneous addition of both K(+) and Mg(2+), which could be used for selective identification of G-quadruplexes in promoter oncogene but not in telomere. Mg(2+) at physiological relevant concentration not only greatly enhanced the thermostability of oncogene G-quadruplexes but also efficiently protected them from unfolding by their complementary strands, which revealed the great impact of Mg(2+) on the equilibrium between promoter G-quadruplex and duplex DNA. The PCR-stop assay further confirmed that Mg(2+) could affect gene transcription by stabilizing promoter G-quadruplex. The above studies were carried out for various G-quadruplexes of varying sequences in promoter oncogenes and telomeric region. Our results suggest that Mg(2+) may be a key regulator for G-quadruplexes of oncogene promoter, which can subsequently affect the expression of related genes.

Effective detection and separation method for G-quadruplex DNA based on its specific precipitation with Mg(2+).

G-quadruplex is a type of DNA secondary structure formed by specific guanine-rich sequences. Because of their enrichment in functional genomic regions and their biological significance, G-quadruplexes are recognized as significant drug targets for cancer and other diseases. Here, we tested the precipitation efficiency of Mg(2+) for various DNA oligomers, including single-stranded, double-stranded, triplex, hairpin, i-motif, and some reported G-quadruplex DNA. It was found that Mg(2+) could specifically recognize and precipitate G-quadruplex DNA with a particularly high efficiency of close to 100% for G-quadruplex structures with parallel conformation, which provided an inexpensive and convenient method for detecting and separating G-quadruplex DNA from other DNA structures as well as identifying parallel G-quadruplex from other conformational G-quadruplexes. Further experiments with both CD and gel electrophoresis validated the effectiveness of this approach. The structure of the precipitate was characterized using transmission electron microscopy (TEM), and the observed linear precipitate suggested that a polymerization of G-quadruplex DNA was formed through π-π stacking of end to end by the unique large aromatic surface of G-quadruplex.

A G-quadruplex DNA-based, label-free and ultrasensitive strategy for microRNA detection.

MicroRNAs (miRNAs) have been considered to be potent biomarkers for early disease diagnosis and for cancer therapy. The rapid and selective detection of miRNAs without reverse transcription and labelling is highly desired. Herein, we report a simple and label-free miRNA detection method that is based on the Duplex-Specific Nuclease (DSN)-Assisted simple target miRNA recycling procedure. The interaction of the G-quadruplex DNA structure with N-methyl mesoporphyrin IX (NMM) led to a label-free signal output. Under the optimised conditions, this method allowed for simple, rapid, and sequence-specific detection of miR-141 over a dynamic range from 1 fM to 100 nM with a linear range from 1 pM to 100 nM. Moreover, our method offered an excellent capacity to discriminate between miRNA family members with just one mismatched nucleotide. This simple and label-free strategy holds great potential in applications in biomedical research and in early clinical diagnostics.

DNA G-quadruplex binders: a patent review.

INTRODUCTION: DNA G-quadruplexes are four-stranded DNA structures and are widely distributed in functional regions of the human genome, such as telomeres and gene promoter regions. G-quadruplex structures can play important roles, including in immunoglobulin gene rearrangements, DNA replication, gene transcription, and are viewed as valid therapeutic targets in human cancer diseases. Design of G-quadruplex binders that target these structures and regulate related gene functions through stabilization of these structures are emerging as an exciting new class of potential anticancer agents. Besides as drug candidate, DNA G-quadruplex binders can also serve as excellent probes, helping the further exploration of biological functions of G-quadruplex and early diagnosis of G-quadruplex-related disease. AREAS COVERED: This review provides an overview of current knowledge on patents of DNA G-quadruplex binders from 2008 to 2013. Information is collected from an extensive search, covering Derwent Innovations Index, Espacenet, SciFinder, and Google patent search. EXPERT OPINION: With the accumulating evidence of G-quadruplex as an effective drug target, an increasing number of DNA G-quadruplex binders with diverse structural features were developed. These binders are either used as drug candidates targeting G-quadruplex, or as probes for diagnostic purpose in genomic study. This review would mainly focus on the patents published after 2008 (including 2008). In order to cover all the diverse structural types of DNA G-quadruplex binders, some patents published before 2008 would be mentioned as well.

Synthesis and evaluation of graveoline and graveolinine derivatives with potent anti-angiogenesis activities.

A series of graveoline and graveolinine derivatives were synthesized. The biological results showed that most of graveoline derivatives possessed higher cytotoxicity and better inhibitive effect against the adhesion and migration of human umbilical vein endothelial cell (HUVEC) than graveolinine derivatives. Among these compounds, 8d was the most potent agents that also showed significant anti-angiogenesis activities in chick embryo chorioallantoic membrane (CAM) assay.