Detection of Listeria monocytogenes Using Luminol-Functionalized AuNF-Labeled Aptamer Recognition and Magnetic Separation.

A capture probe was constructed using a combination of magnetic Fe3O4 nanoparticles and an aptamer directed towardListeria monocytogenes. A signal probe was prepared by combining luminol-functionalized flowerlike gold nanoparticles, obtained by combining luminol with chitosan bearing a complementary sequence of the aptamer. The complex consisting of the capture probe and signal probe could be removed through magnetic separation. Where the target was present within a sample, it competed with the complementary sequence for binding to the aptamer, causing a change of the chemiluminescent signal. The results indicated that a good linear relationship existed over the concentration range 1.0 × 101-1.0 × 105 CFU·mL-1. It was established that it was feasible to use this approach to detect L. monocytogenes at levels as low as 6 CFU·mL-1 in milk samples.

Molecular AND logic gate for multiple single-nucleotide mutations detection based on CRISPR/Cas9n system-trigged signal amplification.

Precise detection of single-nucleotide mutations (SNMs) is extremely important in various biomedical applications, but the simultaneous detection of multiple SNMs remains a great challenge. Herein, we developed a new method based on CRISPR/Cas9 system for multiple SNMs detection. The CRISPR/Cas9 system transduces the nucleic acid into an intermediate trigger to initiate the isothermal amplification reaction and further form fluorescence signals. According to this strategy, we established nucleic acid bio-computing operations-molecule logic gate in simultaneous distinction of the genetic locus. We demonstrate that the fluorescence signals generated from different input combinations can be used to discriminate the multiple genetic locus, and the molecular logic gate has great potential in single-base mismatch detection. In addition, the successful assay of real samples indicates that the novel strategy could further adapt for the pathogenic monitoring and biomedical research.

Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification.

A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA. Graphical abstract Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.

Double-integrated mimic enzymes for the visual screening of Microcystin-LR: Copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme.

Recently, mimic enzymes have obtained particular interest by their high activity, stability, and biocompatibility. In this work, by coupling copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme to form a double-integrated mimic enzyme, a visual, sensitive and selective immunosensor was established to detect microcystin-LR (MC-LR). In this immunoassay, the microplates were modified with core-shell silica/nickel silicate as the substrate to capture MC-LR antigens. Then, Cu(OH)2 nanocages with fine regulation were used as the label to capture the secondary antibody for immunoreaction and the DNA primer for propagation, followed by using hybridization chain reaction to amplify the DNA primer, thus numerous DNAzymes (G-quadruplex/hemin) can be formed on the surface of Cu(OH)2 nanocages with the aid of hemin. Such double-integrated mimic enzyme including Cu(OH)2 nanozymes and DNAzymes showed excellent peroxidase activity for the chromogenic reaction of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which realized the visual detection of MC-LR in the range from 0.007 to 75 µg/L with the detection limit of 6 ng/mL, and thus provided the probability for the portable assessment of MC-LR in real sample.

Dual-Modal Split-Type Immunosensor for Sensitive Detection of Microcystin-LR: Enzyme-Induced Photoelectrochemistry and Colorimetry.

Microcystins, the lethal cyanotoxins from Microcystis aeruginosa, can inhibit the activity of protein phosphatase and promote liver tumors. Herein, a dual-modal split-type immunosensor was constructed to detect microcystin-LR (MC-LR), based on the photocurrent change of CdS/ZnO hollow nanorod arrays (HNRs) and the blue shift of the surface plasmon resonance peak from Au nanobipyramids@Ag. By using mesoporous silica nanospheres as the carrier to immobilize secondary antibody and DNA primer, a hybridization chain reaction was adopted to capture alkaline phosphatase, while its catalytic reaction product, ascorbic acid, exhibited dual functions. The detailed mechanism was investigated, showing that ascorbic acid can not only act as the electron donor to capture the holes in CdS/ZnO-HNRs, leading to the increase photocurrent, but also as the reductant to form silver shells on Au nanobipyramids, generating multiply vivid color variations and blue shifts. Compared with the traditional photoelectrochemical immunosensor or colorimetric method for MC-LR, a more accurate and reliable result can be obtained, due to different mechanisms and independent signal transduction. Therefore, this work can not only propose a new dual-modal immunosensor for MC-LR detection but also provide innovative inspiration for constructing sensitive, accurate, and visual analysis for toxins.

Metal Contents, Bioaccumulation, and Health Risk Assessment in Wild Edible Boletaceae Mushrooms.

Eight wild edible Boletaceae mushrooms (227 samples) and their soils were collected from 40 locations, Yunnan province, China. Four essential metals (Fe, Mg, Zn, and Cu) and 2 toxic metals (Pb and Cd) were determined. The results showed that Boletaceae mushrooms have abundance of 4 essential metals. The highest Pb mean value was 0.70 mg/kg DW, lower than legal limits, but Cd contents significantly exceeded legal limits. Generally, bioconcentration factor (BCF) indicated that Zn and Cu were easily bioaccumulated by mushrooms. However, the BCFCd of Boletus griseus reached to 6.40. Target hazard quotients showed Cd was the main risk metal in Boletaceae mushrooms. The metal compositional variability and the similarity of metal contents were further determined by principal component analysis. Regression model analysis indicated that Cd contents in mushrooms were positively correlated with soil Cd contents, and negatively correlated with soil pH, except for the samples of Boletus bicolor.

Purification of Antioxidant Peptides by High Resolution Mass Spectrometry from Simulated Gastrointestinal Digestion Hydrolysates of Alaska Pollock (Theragra chalcogramma) Skin Collagen.

In this study, the stable collagen hydrolysate was prepared by alcalase hydrolysis and twice simulated gastrointestinal digestion from Alaska pollock skin. The characteristics of hydrolysates and antioxidant activities in vitro, including 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS•+) scavenging activity, ferric-reducing antioxidant power (FRAP) and hydroxyl radical (OH·) scavenging activity, were determined. After twice simulated gastrointestinal digestion of skin collagen (SGI-2), the degree of hydrolysis (DH) reached 26.17%. The main molecular weight fractions of SGI-2 were 1026.26 and 640.53 Da, accounting for 59.49% and 18.34%, respectively. Amino acid composition analysis showed that SGI-2 had high content of total hydrophobic amino acid (307.98/1000). With the simulated gastrointestinal digestion progressing, the antioxidant activities increased significantly (p < 0.05). SGI-2 was further purified by gel filtration chromatography, ion exchange chromatography and high performance liquid chromatography, and the A1a3c-p fraction with high hydroxyl radical scavenging activity (IC50 = 7.63 µg/mL) was obtained. The molecular weights and amino acid sequences of key peptides of A1a3c-p were analyzed using high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) combined with de novo software and UniProt of MaxQuant software. Four peptides were identified from A1a3c-p, including YGCC (444.1137 Da) and DSSCSG (554.1642 Da) identified by de novo software and NNAEYYK (900.3978 Da) and PAGNVR (612.3344 Da) identified by UniProt of MaxQuant software. The molecular weights and amino acid sequences of four peptides were in accordance with the features of antioxidant peptides. The results indicated that different peptides were identified by different data analysis software according to spectrometry mass data. Considering the complexity of LC-ESI-LTQ-Orbitrap-MS, it was necessary to use the different methods to identify the key peptides from protein hydrolysates.