Analysis of Metabolic Components of JUNCAO Wine Based on GC-QTOF-MS.

JUNCAO wine fermentation metabolites are closely related to the final quality of the product. Currently, there are no studies of dynamic metabolite changes during fermentation of JUNCAO wine. Here, we used gas chromatography quadrupole time-of-flight mass spectrometry (GC-QTOF-MS) metabolomics and multivariate statistical analysis to explore the relationship between metabolites and fermentation time. A total of 189 metabolites were annotated throughout the fermentation process. The principal component analysis (PCA) revealed a clear separation between the samples in the early and late stages of fermentation. A total of 60 metabolites were annotated as differential during the fermentation (variable importance in the projection, VIP > 1, and p < 0.05), including 21 organic acids, 10 amino acids, 15 sugars and sugar alcohols, and 14 other metabolites. Pathway analysis showed that the most commonly influenced pathways (impact value > 0.1 and p < 0.05) were tricarboxylic acid cycle, alanine, aspartic acid and glutamic acid metabolism, pyrimidine metabolism, and other 10 metabolic pathways. Moreover, integrated metabolic pathways are generated to understand the conversion and accumulation of differential metabolites. Overall, these results provide a comprehensive overview of metabolite changes during fermentation of JUNCAO wine.

Comparison of DNA-Gold Nanoparticle Conjugation Methods: Application in Lateral Flow Nucleic Acid Biosensors.

Lateral flow nucleic acid biosensors (LFNABs) have attracted extensive attention due to their rapid turnaround time, low cost, and results that are visible to the naked eye. One of the key steps to develop LFNABs is to prepare DNA-gold nanoparticle (DNA-AuNP) conjugates, which affect the sensitivity of LFNABs significantly. To date, various conjugation methods-including the salt-aging method, microwave-assisted dry heating method, freeze-thaw method, low-pH method, and butanol dehydration method-have been reported to prepare DNA-AuNP conjugates. In this study, we conducted a comparative analysis of the analytical performances of LFNABs prepared with the above five conjugation methods, and we found that the butanol dehydration method gave the lowest detection limit. After systematic optimization, the LFNAB prepared with the butanol dehydration method had a detection limit of 5 pM for single-strand DNA, which is 100 times lower than that of the salt-aging method. The as-prepared LFNAB was applied to detect miRNA-21 in human serum, with satisfactory results. The butanol dehydration method thus offers a rapid conjugation approach to prepare DNA-AuNP conjugates for LFNABs, and it can also be extended to other types of DNA biosensors and biomedical applications.

AuPt nanoalloy with dual functionalities for sensitive detection of HPV16 DNA.

Human papillomavirus type 16 (HPV16), one of the high-risk types, is responsible for 53% of cervical cancers. The development of an early diagnostic approach with high sensitivity, low-cost, point-of-care testing (POCT) for HPV16 is urgent. In our work, a novel dual-functional AuPt nanoalloy-based lateral flow nucleic acid biosensor (AuPt nanoalloy-based LFNAB) was established with excellent sensitivity for detecting HPV16 DNA for the first time. The AuPt nanoalloy particles were prepared by a one-step reduction method, which was simple, rapid, and green. The AuPt nanoalloy particles retained the performance of initial Au nanoparticles owing to the catalytic activity enabled by Pt. Such dual-functionalities offered two kinds of detection alternatives (i.e., normal mode and amplification mode, respectively). The former is produced just by the black color from the AuPt nanoalloy material itself, and the latter is more color sensitive from its superior catalytic activity. The optimized AuPt nanoalloy-based LFNAB exhibited satisfactory quantitative ability in detecting the target HPV16 DNA in the range of 5-200 pM with a LOD of 0.8 pM at the "amplification mode". The proposed dual-functional AuPt nanoalloy-based LFNAB displayed great potential and promising opportunity in POCT clinical diagnostics.

Ultrasensitive lateral flow biosensor based on PtAu@CNTs nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid.

A rapid and ultrasensitive lateral flow biosensor was developed, which based on gold and platinum nanoparticles-decorated carbon nanotubes (PtAu@CNTs) nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid. Independent platinum and gold nanoparticles modified functional carbon nanotubes (PtAu@CNTs) were prepared by in-situ reduction. Sandwich-type hybridization reaction occurred between PtAu@CNTs-labeled DNA probe, target DNA and Biotin-modified DNA probes, which was captured on test zone of the strip. Accumulation of PtAu@CNTs nano-labels formed a characteristic colored band. After systematic optimization and catalytic chromogen, the naked eye detection limit of PtAu@CNTs-LFA was about 2 pM, and the theoretical detection limit of target DNA is calculated to be 0.43 pM according to the standard curve. The results indicates a rapid, sensitive and specific methods for DNA detection in biological samples, showing great promise for biomedical diagnosis in some malignant diseases in clinical application.

Gold nanorods-based lateral flow biosensors for sensitive detection of nucleic acids.

A gold nanorod (AuNR)-based lateral flow nucleic acid biosensor (LFNAB) is reported for visual detection of DNA with a short test time and high sensitivity. AuNRs with an approximate length of 60 nm were utilized as a colored tag to label the detection DNA probe (Det-DNA). The capture DNA probe (Cap-DNA) was immobilized on the test region of LFNAB. Sandwich-type complex was formed among the AuNR-Det-DNA, target DNA (Tar-DNA), and Cap-DNA on the LFNAB by Watson-Crick base pairing. In the presence of Tar-DNA, AuNRs were thus seized on the test region of LFNAB, and the accumulation of AuNRs subsequently produced a characteristic colored band. The optimized LFNAB was able to detect 10 pM Tar-DNA without instrumentation. Quantitative analysis could be established by measuring the intensity of test band using a portable strip reader, and the detection limit of 2 pM target DNA was achieved on the LFNAB without signal amplification. The detection limit of the AuNR-based LFNAB is 250-fold lower than that of gold nanoparticle (AuNP)-based LFNABs. This work unveiled a sensitive, rapid, and economical strategy for the detection of nucleic acids, and simultaneously opening new promising routes for disease diagnosis and clinical applications. Gold nanorods are used as colored tags for lateral flow nucleic acid biosensor.

Sensitive detection of microRNA-21 in cancer cells and human serum with Au@Si nanocomposite and lateral flow assay.

We report a highly sensitive approach for detecting microRNA-21 (miR-21) in cancer cells and human serum by using Au@Si nanocomposite labeled lateral flow assay. The Au@Si nanocomposite was prepared by coating numerous 3-5 nm gold nanoparticles (GNP) on a silica nanoparticle (SiNP) with a diameter of 150 nm and used as colored label on the lateral flow assay for signal amplification. TEM results show there are around 1000 GNPs coated on the SiNP surface. The principle of miR-21 detection is based on on-strip DNA-microRNA hybridization reactions to form DNA-miR-21-DNA-Au@Si complexes, which are captured on the test zone of the lateral flow test strip and produce a visible red band. A thiol-modified detecting DNA probe (Det-DNA) and a biotin-modified capturing DNA probe (Cap-DNA), which are complementary to miR-21, were used to prepare the lateral flow test strips. After systematic optimization, the method can detect a minimum concentration of 1.0 pM miR-21, which is 60 times lower than that of the GNP-based lateral flow assay (Gao et al. Biosens & Bioelectro, 2014, 54, 578-584). The method was applied to detect miR-21 in cancer cells and spiked human serum with satisfactory results.

A terbium(III)-functionalized zinc(II)-organic framework for fluorometric determination of phosphate.

A terbium(III)-functionalized zinc(II)-organic framework (Tb-MOF-Zn) is shown to be a viable fluorescent probe for phosphate. The organic ligands 4,4',4″-[((2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene))tris(oxy)]tribenzoic acid (H3L3) contains multiple carboxyl groups that can react with zinc(II) to yield tubular MOF-Zn. The MOF-Zn was further functionalized with Tb(III) to produce a lanthanide composite of type Tb-MOF-Zn which displays strong fluorescence with excitation/emission maxima at 285/544 nm. Fluorescence is quenched by phosphate because of the specific interaction with Tb(III) in Tb-MOF-Zn. The concentration of Tb-MOF-Zn, reaction time and pH value of the solution were optimized. Fluorescence drops linearly in the 0.01 to 200.0 µM phosphate concentration range, and the detection limit is 4.0 nM. The fluorescent probe was also used to prepare a microdot array on a glass slide for visual detection of phosphate under illumination with UV light. Graphical abstractA terbium(III) functionalized zinc(II)-organic framework was synthesized and used as fluorescent probe for determination of phosphate ions.

Biosensors for early diagnosis of pancreatic cancer: a review.

Pancreatic cancer is characterized by extremely high mortality and poor prognosis and is projected to be the leading cause of cancer deaths by 2030. Due to the lack of early symptoms and appropriate methods to detect pancreatic carcinoma at an early stage as well as its aggressive progression, the disease is often quite advanced by the time a definite diagnosis is established. The 5-year relative survival rate for all stages is approximately 8%. Therefore, detection of pancreatic cancer at an early surgically resectable stage is the key to decrease mortality and to improve survival. The traditional methods for diagnosing pancreatic cancer involve an imaging test, such as ultrasound or magnetic resonance imaging, paired with a biopsy of the mass in question. These methods are often expensive, time consuming, and require trained professionals to use the instruments and analyze the imaging. To overcome these issues, biosensors have been proposed as a promising tool for the early diagnosis of pancreatic cancer. The present review critically discusses the latest developments in biosensors for the early diagnosis of pancreatic cancer. Protein and microRNA biomarkers of pancreatic cancer and corresponding biosensors for pancreatic cancer diagnosis have been reviewed, and all these cases demonstrate that the emerging biosensors are becoming an increasingly relevant alternative to traditional techniques. In addition, we discuss the existing problems in biosensors and future challenges.

Carbon nanotube-based lateral flow immunoassay for ultrasensitive detection of proteins: application to the determination of IgG.

Authors report on a carbon nanotube (CNT)-based lateral flow immunoassay (LFI) for ultrasensitive detection of proteins. Shortened multiwalled CNTs were used as a colored (black) tag. The detection antibody was covalently immobilized on the CNT surface via diimide-activated conjugation between the carboxyl groups on the CNT surface and amino groups of antibodies. The assay involved the capture of target protein in a sandwich-type format between an immobilized capture antibody on the test zone of LFI and a CNT-labelled detection antibody. CNTs were thus captured on the test zone of the LFI and gave a black colored line to enable visual detection of protein. Quantitative results were obtained by reading the test line intensities with a portable strip reader. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. Combining the advantages of lateral flow assay with the unique physical properties of CNT (color, high aspect-to-size ratio and ease of surface modification), the optimized LFI can detect of 1.3 pg mL-1 of rabbit IgG (S/N = 3). This is three orders lower than that of gold nanoparticle-based LFI. Rabbit IgG spiked into human plasma samples was successfully detected with this LFI. Conceivably, this method can be extended to various other proteins for which adequate antibodies do exist. Graphical abstract Carbon nanotubes are used as black tags in an ultrasensitive lateral flow immunoassay (LFI). The LFI was applied to the determination of rabbit IgG. The detection limit is more than 3 orders of magnitude lower than that of the conventional gold nanopaticle-based LFI.

Gold-platinum nanoflowers as a label and as an enzyme mimic for use in highly sensitive lateral flow immunoassays: application to detection of rabbit IgG.

The authors describe the preparation of gold-platinum nanoflower (AuPt NFs) and show that they can be simultaneously used as a label and as an enzyme mimic in lateral flow immunoassays (LFIs). The AuPt NFs were prepared by growing Pt nanowires on the surface of gold nanoparticle. The assay involves the capture of target proteins (here: rabbit IgG as a model analyte) by the immobilized capture antibody, and by using AuPt NF-labeled secondary antibody. The AuPt NFs are thus captured by the test zone and produce a characteristic black band for visual detection of the antigen (IgG). The coloration of the test line can be further enhanced by addition of the chromogenic substrate 3-amino-9-ethyl-carbazole which is catalytically oxidized by the captured Pt nanowires on the AuPt NF and produce a red coloration. Quantitative results were obtained by reading the test line intensities with a portable strip reader. The LFI has a 5 pg mL-1 detection limit for IgG under optimized experimental conditions. This is 100 times lower than that of the conventional AuNP-based LFI. Conceivably, this assay has a wide scope in that it may be applied to numerous other targets for which appropriate antibodies are available. Graphical abstract Gold-platinum nanoflowers are used as a label and as an enzyme mimic in a highly sensitive lateral flow immunoassay for IgG. The detection limit of gold-platinum nanoflower-based lateral flow assay is 100 times lower than that of the conventional gold nanopaticle-based lateral flow assay.

Microflora of fresh white button mushrooms (Agaricus bisporus) during cold storage revealed by high-throughput sequencing and MALDI-TOF mass spectrometry fingerprinting.

BACKGROUND: Fresh Agaricus bisporus is popular and consumed throughout the world because of its taste, as well as its nutritional and medicinal properties, but it is also prone to microbial growth. There is very limited information about the dynamic changes of microbial communities during storage. The present study aimed to analyze the microbial diversity of button mushroom during cold storage using Illumina HiSeq sequencing. Bacteria isolated from the later storage period were identified by MALDI-TOF mass spectrometry and a bioassay of pathogenicity was carried out. RESULTS: High-throughput sequencing showed that Pseudomonas was the predominant genus throughout the storage period. Pedobacter and Flavobacterium grew prolifically on the eighth day, while the relative abundance of Oscillospira continued to decrease. Pseudomonas, Ewingella and Chryseobacterium were isolated at the later period of mushroom storage. A pathogenicity bioassay on the cap of mushrooms showing brown blotch indicated an infection by Pseudomonas tolaasii. However, Ewingella americana did not have a pathogenic effect in our study. CONCLUSION: Bacterial communities of fresh Agaricus bisporus during cold storage were characterized by high-throughput sequencing. MALDI-TOF MS provides a good analytical procedure, in addition to 16S rRNA gene sequencing. © 2019 Society of Chemical Industry.

Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin.

Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.

Lateral flow test for visual detection of silver (I) based on cytosine-Ag(I)-cytosine interaction in C-rich oligonucleotides.

The authors describe an oligonucleotide-based lateral flow test for visual detection of Ag(I). The assay is based on cytosine-Ag(I)-cytosine [C-Ag(I)-C] coordination chemistry to capture gold nanoparticle (AuNP) tags in the test zone. A thiolated C-rich oligonucleotide probe was immobilized on the AuNPs via gold-thiol chemistry, and a biotinylated C-rich oligonucleotide probe was immobilized on the test zone. The AuNPs labelled with C-rich oligonucleotides are captured by Ag(I) ions in the test zone through the C-Ag(I)-C coordination. The resulting accumulation of AuNPs produces a readily visible red band in the test zone. Under optimized conditions, the test is capable of visually detecting 1.0 ppb of Ag(I) which is 50 times lower than the maximum allowable concentration as defined by the US Environmental Protection Agency for drinking water. Hence, the test is inexpensive and highly sensitive. It was applied to the detection of Ag(I) in spiked samples of tap water and river water. In our perception, the test is a particularly valuable tool in limited resource settings.

Gold Nanoparticle Coated Silica Nanorods for Sensitive Visual Detection of microRNA on a Lateral Flow Strip Biosensor.

We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor.

Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence.

In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1 nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40 pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5 nM, Mao et al. Anal. Chem. 2009, 81, 1660-1668). Another important feature is that the preparation of MWCNT-DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT-antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.