Hg2+-triggered cascade strand displacement assisted CRISPR-Cas12a for Hg2+ quantitative detection using a portable glucose meter.

CRISPR-Cas12a is a powerful and programmable tool that has revolutionized the field of biosensing. However, the construction of a CRISPR-Cas12a-mediated portable system for on-site and quantitative detection of mercury ion (Hg2+) has yet to be explored. By integrating a target-triggered cascade toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a, we herein construct a portable on-site biosensor for the quantitative, sensitive, and selective detection of Hg2+ with a glucose meter. The Hg2+ initiates two cascade TSDRs through the T-Hg2+-T interaction to produce multiple double-stranded DNAs that can activate Cas12a's trans-cleavage activity. The Cas12a cleaves the sucrase-modified DNA on the electrode, resulting in the liberation of sucrase into the solution. The freed sucrase can catalyze sucrose to generate glucose, which can be quantitatively monitored by a glucometer. The developed portable biosensor provides a dynamic range of 5 orders of magnitude with a detection limit of 40 fM. This biosensor also displays excellent selectivity and stability for detecting Hg2+. Moreover, environmental water samples are utilized to further verify the robustness and effectiveness of the developed biosensor, highlighting its potential application in environmental monitoring and food safety analysis.

PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA.

The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDR, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDR, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.

Investigations in enhancement biodesulfurization of model compounds by ultrasound pre-oxidation.

In this study, complicated model sulfur compounds in crude oil were biodesulfurized in a batch process by microbial consortium enriched from oil contaminated soil. Dibenzothiophene (DBT) was selected as model sulfur compounds. Ultrasonic radiation was used to pre-oxidize the model sulfur compounds before the biodesulfurization (BDS) process. The enhancement mechanism of ultrasound pre-oxidation (UPO) on the biodesulfurization of DBT was investigated. The effects of initial conditions on the biodesulfurization of DBT in UPO/BDS system such as solution initial pH, DBT initial concentration, sulfur source, biocatalyst initial concentration, and incubation temperature were discussed. The results show that the application of UPO before BDS procedure significantly improved the efficiency of the biodesulfurization and allowed sulfur removal in shorter time through oxidizing DBT to DBT sulfone, resulting in shortening the "4S" pathway for biodesulfurization from 4 steps to 2 steps, enhancement in reaction velocity and enzyme-substrate affinity as well as reduction in substrate inhibition. The concentration of 2-HBP increased fast with the use of ultrasound pre-oxidation, which was dependent on solution initial pH, DBT initial concentration, sulfur source, biocatalyst initial concentration, and incubation temperature.

Combinative Method Using Multi-components Quantitation and HPLC Fingerprint for Comprehensive Evaluation of Gentiana crassicaulis.

BACKGROUND: Gentiana crassicaulis () is an important traditional Chinese herb. Like other herbs, its chemical compounds vary greatly by the environmental and genetic factors, as a result, the quality is always different even from the same region, and therefore, the quality evaluation is necessary for its safety and effective use. In this study, a comprehensive method including HPLC quantitative analysis and fingerprints was developed to evaluate the quality of Cujingqinjiao and to classify the samples collected from Lijiang City of Yunnan province. A total of 30 common peaks including four identified peaks, were found, and were involved for further characterization and quality control of Cujingqinjiao. Twenty-one batches of samples from Lijiang City of Yunnan Province were evaluated by similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA) and factor analysis (FA) according to the characteristic of common peaks. RESULTS: The obtained data showed good stability and repeatability of the chromatographic fingerprint, similarity values were all more than 0.90. This study demonstrated that a combination of the chromatographic quantitative analysis and fingerprint offered an efficient way to quality consistency evaluation of Cujingqinjiao. Consistent results were obtained to show that samples from a same origin could be successfully classified into two groups. CONCLUSION: This study revealed that the combinative method was reliable, simple and sensitive for fingerprint analysis, moreover, for quality control and pattern recognition of Cujingqinjiao. SUMMARY: HPLC quantitative analysis and fingerprints was developed to evaluate the quality of Gentiana crassicaulisSimilarity analysis, hierarchical cluster analysis, principal component analysis and factor analysis were employed to analysis the chromatographic dataset.The results of multi-components quantitation analysis, similarity analysis, hierarchical cluster analysis, principal component analysis and factor analysis were consistent.All samples could be classified into two groups, which could to some extent reflect the quality differences of theses samples. Abbreviations used: SA: Similarity analysis, HCA: Hierarchical cluster analysis, PCA :Principal component Analysis, FA :Factor analysis.

[The Effection Study of Transplanting Techniques for Yield and Quality of Gentiana crassicaulis].

Objective: To study the difference in yield and quality of Gentiana crassicaulis with different transplanting period and transplanting methods, and to determine the optimum transplanting technique of Gentiana crassicaulis in Ludian. Methods: The variation in fresh weight,dry weight,dry discount rate, length, diameter, branches,the content of gentiopicroside,loganin acid,alcohol-soluble extract, total ash were measured, and made a comprehensive evaluation of yield and quality by gray relational distance ideal comprehensive evaluation method. Results: There was a big difference in yield and quality of Gentiana crassicaulis with different transplanting time and transplanting methods. Gentiana crassicaulis were transplanted in March 10 with the density of 25 cm × 24 cm,and overburden 1 cm and flat transplanting had the best comprehensive evaluation of yield and quality. Conclusion: The transplanting time of Gentiana crassicaulis in Ludian should begin at the end of early March,and reduce overburden soil on the traditional transplanting methods.

[Influences of Base Fertilizer Dosage and Root Size on Quality and Yield of Gentiana crassicaulis Root].

OBJECTIVE: In order to optimize the base fertilizer dosage and root size of Gentiana crassicaulis in Ludian,Yulong, Yunnan,the differences in yield and quality of Gentiana crassicaulis caused by different base fertilizer dosage and root size were investigated. METHODS: The variation in fresh weight, dry weight, drying rate, length, diameter, branches as well as the contents of gentiopicroside, loganin acid, alcohol-soluble extract and total ash were determined. The ideal distance grey relational degree comprehensive evaluation method was applied to make a comprehensive evaluation on the quality and yield of Gentiana crassicaulis. RESULTS: There were big differences in yield and quality of Gentiana crassicaulis among different base fertilizer dosage and root size. 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate, 100 kg/667 m2 of plant ash and mix size of transplanting roots showed the best comprehensive quality and the highest yield. CONCLUSION: 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate,100 kg/667 m2 of plant ash, high quality transplanting roots and stagger certain depth to transplant are highly recommended to produce high quality and high yield of Gentiana crassicaulis in Ludian, Yulong, Yunnan.

[Optimum harvest study of Gentiana crassicaulis in Ludian].

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.

[Evaluation of Bupleuri Radix resources in Qingchuan based on DTOPSIS and grey related degree].

In order to select high quality and suitable Bupleuri Radix varieties in Qingchuan, and establish a new comprehensive method to evaluation the quality of Bupleuri Radix, 12 characters of 14 samples were evaluated by DTOPSIS and grey related degree. The results showed that varieties No. 8 and No. 10 had high quality. DTOPSIS and grey related degree gave the uniformity result, and the biggest difference of value of Ci in DTOPSIS method was 46. 33% , but the biggest difference of the weighting correlation number( r (i)) in grey related degree was only 13.10%. The DTOPSIS combined with grey related degree can evaluate the quality of Bupleuri Radix comprehensively and objectively.

[Effect of combined application of zinc, boron and molybdenum on yield and saikosaponin a, saikosaponin d contents of Bupleurum chinense].

This research use "3414" fertilizer effect experiments to handle zinc, boron and molybdenum trace element fertilizer, determined the dry matter accumulation and content of saikosaponion a and d, to investigate the different ratio of zinc, boron and molybdenum on yield and saikosaponin a, saikosaponin d contents of Bupleurum chinense. Found The suitable ratio of zinc, boron and molybdenum play an active role on dry matter accumulation and distribution, the treatment Zn2B2Mo3 is the best one to promote the dry matter accumulation and transfer to the underground part; in a certain range, only use zinc or molybdenum can promote the yield of B. chinense, the yield of treatment Zn2B2Mo1 is the highest one. According to the results of regression analysis: in accordance with Zn 48.45 g x hm(-2), B 355.05 g x hm(-2), Mo 86.40 g x hm(-2), can obtain the yield with 3313.05 kg x hm(-2); the treatment Zn2BMo2 is most effective to promote the total saikosaponin a and d accumulated, according to the results of regression analysis: in accordance with Zn 36.15 g x hm(-2), B 343.05 g x hm(-2), Mo 106.35 g x hm(-2), the content of total saikosaponin a and d can reach 1.23%. This research first discovered the suitable ratio of zinc, boron and molybdenum can promote the yield and saikosaponin a, saikosaponin d contents on B. chinense.

Gas chromatography-mass spectrometry analysis of essential oils from five parts of Chaihu (Radix Bupleuri Chinensis).

OBJECTIVE: To analyze the essential oils from flowers, leaves, stems, roots, and fruits of Chaihu (Radix Bupleuri Chinensis). METHODS: We extracted essential oils from different parts of Chaihu (Radix Bupleuri Chinensis) using a steam distillation method. The essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS). Data were collected in full scan mode (m/z 60-600). Volatile components were identified based on their retention indices and by comparing their mass spectra with those in the National Institute of Standards and Technology 2005 database, assisted by tandem mass spectrometry information. The relative content of each constituent was determined by area normalization. RESULTS: We identified 111 components, of which 12 were common to all 5 parts, 30 were found only in roots, 14 were found only in flowers, 6 were found only in leaves, 4 were found only in stems, and 17 were found only in fruits. CONCLUSION: Our results show that the stems, flowers, leaves, and fruits of Chaihu (Radix Bupleuri Chinensis) contain a high concentration of essential oils, and that the exact composition of the essential oils differs among the plant parts. To develop new medicines and make full use of the Chaihu (Radix Bupleuri Chinensis) resource, it is important to characterize the essential oils from different parts of the plant. In future research, it will be important to determine the pharmacological effects of the various components and the essential oil mixtures.

[Stability evaluation of chemical compositions of Rhizoma gastrodiae with 60Co-γ irradiation by high performance liquid chromatography].

The main chromatographic fingerprint peaks of Rhizoma gastrodiae were established for evaluating the stability of chemical compositions of Rhizoma gastrodiae with 60Co-gamma irradiation to control the dosage of 60Co-gamma irradiation sterilization by high performance liquid chromatography (HPLC). Eight Rhizoma gastrodiae samples were analyzed. The chromatographic fingerprints of Rhizoma gastrodiae were constructed with 14 common fingerprint peaks, and its 10 main peaks were identified using the similarity evaluation system of chromatographic fingerprints of traditional Chinese medicine (version 2004 A) recommended by State Food and Drug Administration of China. The relative standard deviations of the peak areas of the 10 main peaks were used to evaluate the stability of corresponding chemical compositions of Rhizoma gastrodiae with 60Co-gamma irradiation. The results showed that the compositions of 3 of the 10 main peaks were influenced and a good relationship was obtained between the influence and 60Co-gamma irradiation doses for the eight Rhizoma gastrodiae samples. Some chemical components of Rhizoma gastrodiae weren't steady after 60Co-gamma irradiation, so a low dosage should be selected for 60Co-gamma irradiation sterilization.