A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods.

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.

Femtomolar endogenous adenosine triphosphate-responded photoelectrochemical biosensor based on Au@Cu2O core-shell nanocubes for the ultrasensitive determination of Escherichia coli O157:H7 in foods.

Sensitive and precise determination of virulent foodborne pathogens is significant for food safety. Herein, an ultrasensitive photoelectrochemical (PEC) bioanalysis was developed using the endogenous adenosine triphosphate (ATP)-responded Au@Cu2O core-shell nanocubes (Au@Cu2O NCs) to measure Escherichia coli O157: H7 (E. coli O157:H7) in food. Briefly, the phage-functionalized gold wire was used to specifically recognize the target pathogen. With the bacteriolysis of lysozyme, the endogenous ATP molecules were emitted from the captured target bacteria and enriched by another ATP aptamer-modified gold wire. Following the exchange with complementary DNA (cDNA) chains, the bonded ATP would be released. It could simultaneously etch the Au@Cu2O NCs and compete with external circuit electrons to combine photogenerated holes on the Au@Cu2O NCs-modified screen-printed electrode. With the synergy of the two signal amplification mechanisms, a significant attenuation of photocurrent signal appeared even with femtomolar ATP. Therefore, the purpose of ultrasensitive determination of E. coli O157:H7 was realized, which depended on the endogenous ATP rather than exogenous signal probes. The proposed biosensor presented a good analysis performance within 10-106 CFU/mL with a detection limit of 5 CFU/mL. Besides, its specificity, repeatability, and stability were also investigated and acceptable. The detection results for food samples matched well with the results detected by the plate counting method. This work gives an innovative and sensitive signal amplification strategy for PEC bioassays in foodborne pathogens detection.

High-speed identification system for fresh tea leaves based on phenotypic characteristics utilizing an improved genetic algorithm.

BACKGROUND: High-quality tea requires leaves of similar size and tenderness. The grade of the fresh leaves determines the quality of the tea. The automated classification of fresh tea leaves improves resource utilization and reduces manual picking costs. The present study proposes a method based on an improved genetic algorithm for identifying fresh tea leaves in high-speed parabolic motion using the phenotypic characteristics of the leaves. During parabolic flight, light is transmitted through the tea leaves, and six types of fresh tea leaves can be quickly identified by a camera. RESULTS: The influence of combinations of morphology, color, and custom corner-point morphological features on the classification results were investigated, and the necessary dimensionality of the model was tested. After feature selection and combination, the classification performance of the Naive Bayes, k-nearest neighbor, and support vector machine algorithms were compared. The recognition time of Naive Bayes was the shortest, whereas the accuracy of support vector machine had the best classification accuracy at approximately 97%. The support vector machine algorithm with only three feature dimensions (equivalent diameter, circularity, and skeleton endpoints) can meet production requirements with an accuracy rate reaching 92.5%. The proposed algorithm was tested by using the Swedish leaf and Flavia data sets, on which it achieved accuracies of 99.57% and 99.44%, respectively, demonstrating the flexibility and efficiency of the recognition scheme detailed in the present study. CONCLUSION: This research provides an efficient tea leaves recognition system that can be applied to production lines to reduce manual picking costs. © 2022 Society of Chemical Industry.

A Multicolor Fluorescence Nanoprobe Platform Using Two-Dimensional Metal Organic Framework Nanosheets and Double Stirring Bar Assisted Target Replacement for Multiple Bioanalytical Applications.

Multicolor fluorescence probes can show fluorescence of different colors when detecting different targets, and the excellent feature can create a highly differentiated multicolor sensing platform. However, most of the previously reported multicolor luminescent materials usually suffer from high toxicity and photobleaching, complex preparation procedures, and poor water solubility, which may not be conducive to bioanalytical applications. Two-dimensional metal organic frameworks (2D MOFs), which have large specific surface areas with long-range fluorescence quenching coupled with biomolecular recognition events, have encouraged innovation in biomolecular probing. Here, we propose a 2D-MOF-based multicolor fluorescent aptamer nanoprobe using a double stirring bar assisted target replacement system for enzyme-free signal amplification. It utilizes the interaction between 2D MOFs and DNA molecules to detect multiple antibiotics quickly, sensitively, and selectively. Since 2D MOFs have excellent quenching efficiency for luminescence of fluorescent-dye-labeled single-strand DNA (ssDNA), the background fluorescence can be largely reduced and the signal-to-noise ratio can be improved. When the adsorbed ssDNA formed double helix double-stranded DNA with its complementary ssDNA, its fluorescence can be almost fully recovered. The assay was tested by detecting chloramphenicol (CAP), oxytocin (OTC), and kanamycin (KANA) in biological samples. The developed aptasensor was sufficiently sensitive to detect the antibiotic residues as low as 1.5 pM CAP, 2.4 pM OTC, and 1 pM KANA (S/N = 3). It has been preliminarily used for multicolor imaging of three different antibiotics in fish tissue slices with satisfactory results.

Osthole improves therapy for osteoporosis through increasing autophagy of mesenchymal stem cells.

Osteoporosis is a common skeletal disorder resulting in elevated fracture risk. Improvement of osteogenic differentiation is thought to be the top priority in osteoporosis treatment projects. Significant characteristics of bone marrow mesenchymal stem cells (BMMSCs), especially attractive ability to differentiate into osteoblasts, have made them alternatives for osteoporosis treatment. However, therapeutic effect with BMMSCs remains to be improved. Here, osthole, a bioactive simple coumarin derivative extracted from many medicinal plants, was introduced to pre-stimulate BMMSCs and then applied in osteoporosis therapy. The results showed that osthole-treated-BMMSCs (OBMMSCs) brought a better outcome than BMMSCs alone in estrogen deficiency-induced osteoporosis model. And elevated autophagy level was suggested to be the underlying mechanism of the ability of osthole to promote osteoblast differentiation, which is indicated by the upregulation of protein and mRNA expression level of autophagy-associated genes, Beclin1 and LC3. We concluded from these experiments that OBMMSCs are more effective than BMMSCs in osteoporosis treatment maybe through upregulation level of autophagy level induced by osthole.

Metabolic Flux Enhancement and Transcriptomic Analysis Displayed the Changes of Catechins Following Long-Term Pruning in Tea Trees ( Camellia sinensis).

The tea tree is a perennial woody plant, and pruning is one of the most crucial cultivation measurements for tea plantation management. To date, the relationship between long-term pruning and metabolic flux enhancement in tea trees has not been studied. In this research, 11-year-old pruned tea trees from four different cultivars were randomly selected for transcriptome analysis and characteristic secondary metabolite analysis together with controls. The findings revealed that epigallocatechin gallate (EGCG) accumulation in pruned tea trees was significantly higher than that in unpruned tea trees. SCPL1A expression (encoding a class of serine carboxypeptidase), which has been reported to have a catalytic ability during EGCG biosynthesis, together with LAR, encoding leucoanthocyanidin reductase, was upregulated in the pruned tea trees. Moreover, metabolic flux enhancement and transcriptome analysis revealed low EGCG accumulation in the leaves of unpruned tea trees. Because of the bitter and astringent taste of EGCG, these results provide a certain understanding to the lower bitterness and astringency in teas from "ancient tea trees", growing in the wild with no trimming, than teas produced from pruned plantation trees.

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL-1 to 10ngmL-1, with the detection limits of 0.7pgmL-1 and 0.9pgmL-1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.

A triple-amplification colorimetric assay for antibiotics based on magnetic aptamer-enzyme co-immobilized platinum nanoprobes and exonuclease-assisted target recycling.

Herein, an ultrasensitive and selective colorimetric assay for antibiotics, using chloramphenicol (CAP) as the model analyte, was developed based on magnetic aptamer-HRP-platinum composite probes and exonuclease-assisted target recycling. The composite probes were prepared through immunoreactions between the double stranded DNA antibody (anti-DNA) labeled on core-shell Fe3O4@Au nanoparticles (AuMNP-anti-DNA) as the capture probe, and the double stranded aptamer (aptamer hybrid with its complementary oligonucleotides) labeled on Pt@HRP nanoparticles as the nanotracer (ds-Apt-HRP-PtNPs). When the CAP samples were incubated with the probes for 30 min at room temperature, they could be captured by the aptamer to form a nanotracer-CAP complex, which was then released into the supernatant after magnetic separation. This is because the anti-DNA on the capture probes cannot recognize the single strand aptamer-CAP complex. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer-CAP from the 3'-end of the aptamer and the CAP in the aptamer-CAP complex can be released again, which can further participate in a new cycling process to react with the probes. Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system. Moreover, Exo I can assist the target recycling, which can further amplify the signal. Thus, the triple amplified signal can be quantified by ultraviolet-visible spectroscopy. The experimental results showed that the CAP detection possessed a linear range of 0.001-10 ng mL(-1) and a detection limit of 0.0003 ng mL(-1) (S/N = 3). The assay was successfully employed to detect CAP in milk, which is much more facile, time saving, and sensitive than the commercial ELISA kits.

A "signal-on'' aptasensor for simultaneous detection of chloramphenicol and polychlorinated biphenyls using multi-metal ions encoded nanospherical brushes as tracers.

A "signal-on'' aptasensor was developed for simultaneous detection of chloramphenicols (CAP) and polychlorinated biphenyl-72 (PCB72) with a novel multi-metal ions encoded nanospherical brushes as nanotracers. To construct the assay, the respective aptamer of CAP and PCB72 labeled magnetic gold nanoparticles as capture probes (aptamer-MGPs), and their complementary single strand DNA (s-DNA) encoded metal ions (Cd(2+) and Pb(2+)) on nanospherical branched polyethylene imine brushes as tracers (s-DNA-MSPEIs), were simultaneously synthesized. After that, the capture probe and tracers were connected through a hybridization reaction between s-DNA and aptamers. In the presence of CAP and PCB72, the analytes could react with the aptamers on capture probes and release the tracers into supernatant after magnetic separation. The released tracers with metal ions (Cd(2+) and Pb(2+)) could be simultaneously detected through the square wave voltammetry (SWV) without acid dissolution, which can switch the signals of the biosensor to "on'' state. Under optimal conditions, the assay could detect CAP and PCB72 as low as 0.3 pg mL(-1) with the dynamitic range from 0.001 to 100 ng mL(-1) and exhibited excellent selectivity. More importantly, the strategy can be extended easily to other targets after changing the corresponding aptamers and other metal ions tracers, which provides a promising and facile approach in multiplex detection of ultra-trace level of pollutants in food safety without more complex separation and washing steps.

A novel "dual-potential" electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol.

A novel type of "dual-potential" electrochemiluminescence (ECL) aptasensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for simultaneous detection of malachite green (MG) and chloramphenicol (CAP) in one single assay. The SPCE substrate consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). In the system, CdS quantum dots (QDs) were modified on WE1 as cathode ECL emitters and luminol-gold nanoparticles (L-Au NPs) were modified on WE2 as anode ECL emitters. Then the MG aptamer complementary strand (MG cDNA) and CAP aptamer complementary strand (CAP cDNA) were attached on CdS QDs and L-Au NPs, respectively. The cDNA would hybridize with corresponding aptamer that was respectively tagged with cyanine dye (Cy5) (as quenchers of CdS QDs) and chlorogenic acid (CA) (as quenchers of l-Au NPs) using poly(ethylenimine) (PEI) as a bridging agent. PEI could lead to a large number of quenchers on the aptamer, which increased the quenching efficiency. Upon MG and CAP adding, the targets could induce strand release due to the highly affinity of analytes toward aptamers. Meanwhile, it could release the Cy5 and CA, which recovered cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. This "dual-potential" ECL strategy could be used to detect MG and CAP with the linear ranges of 0.1-100 nM and 0.2-150 nM, with detection limits of 0.03 nM and 0.07 nM (at 3sB), respectively. More importantly, this designed method was successfully applied to determine MG and CAP in real fish samples and held great potential in the food analysis.

[Studies on phenanthrene constituents from stems of Dendrobium candidum].

OBJECTIVE: To investigate chemical constituents from the stems of Dendrobium candidum. METHODS: The constituents were isolated by various column chromatography methods, and their structures were elucidated by spectral analysis (IR, UV, NMR and MS). RESULTS: Six compounds were isolated from ethanol extract and their structures were identified as 2,3,4,7-tetramethoxyphenanthrene (I), nakaharain (II), 2,5-dihydroxy-3,4-dimethoxyphenanthrene (III), confusarin (IV), nudol (V) and bulbophyllanthrin (VI). CONCLUSION: Among these compounds, compounds II, III, VI are isolated from Dendrobium candidum for the first time.

[Studies on constituents of rootsanel leaves from Desmodium blandum and their cytotoxic activity against growth of several tumor cells].

OBJECTIVE: To investigate the chemical constituents of Desmodium blandum and their cytotoxic activity against the growth of several tumor cells. METHOD: Various chromatographic techniques including silica gel, Sephadex LH-20 column chromatography were employed for the isolation and purification of the constituents. The structures of compounds were elucidated by spectral analyses (IR, UV, NMR, MS). Their cytotoxic activity was then studied. RESULT: Eight compounds were isolated from the stems of D. blandum and identified as N, N-dimethyltryptamine (1), 5-methoxy-N, N-dimethyltryptamine (2), citrusinol (3), yukovanol (4), (Z)-1-(4-hydroxy-2, 3-dimethoxyphenyl)-3-(4-hydroxyphenyl) propene (5), (Z)-1-(3-hydroxy-2, 4-dimethoxy-phenyl)-3-(4-hydroxy-3-methoxy-phenyl) propene (6), methylprotocatechuate (7), katuranin (8). CONCLUSION: Among these compounds, compound 6 was isolated from D. blandum for the first time. In the MTT test, compounds 2 and 6 exhibit cytotoxic activities against the KB cell, and compounds 3 and 6 exhibit the same activities against the HepG2 cell.