A signal-amplified whole-cell biosensor for sensitive detection of Hg2+ based on Hg2+-enhanced reporter module.

A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg2+ as the reporter module. The developed biosensor showed response for Hg2+ in a relatively wide range of 1-10,000 nmol/L, and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs. In addition, the biosensor could distinguish Hg2+ easily from multiple metal ions and displayed strong adaptability to extensive pH conditions (pH 4.0-10.0). More importantly, the developed biosensor was able to provide an initial assessment of Hg2+ spiked in the environmental water with the recoveries between 85.70% and 112.50%. The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other whole-cell biosensors, meeting the practical requirements with sufficient sensing performance.

Identification and Analysis of Components in Yizhi Granule and Cynomolgus Monkey Plasma after Oral Administration by UPLC/ESI-Q-TOF MS and Their Protective Effects on PC12 Cells.

Yizhi Granule (YZG) is a health food containing six traditional Chinese medicines (TCMs). It improves memory barriers in rat experiments. Here, we describe the first fast and sensitive ultraperformance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF MS) method for analyzing YZG in plasma. We used this technique for studies in cynomolgus monkey plasma. By comparing retention time, MS, and MS/MS data of reference compounds, 70 compounds were detected in YZG. Of these, 63 were identified including 60 saponins, 2 flavones, and 1 methyl ester. There were 33 saponins, 1 flavone, and 1 methyl ester in the plasma. Next, to study the therapeutic properties of YZG, the neuroprotective effect of some of the absorbed components was evaluated using PC12 cell damage caused by the Aß 25-35 model. The results showed that 9 compounds protect PC12 cells from Aß 25-35 with cell viability (%) of 111.00 ± 8.12 (G-Rb1), 102.20 ± 4.22 (G-Rb2), 100.34 ± 6.47 (G-Rd), 102.83 ± 2.10 (G-Re), 101.68 ± 7.64 (NG-Fa), 101.19 ± 7.83 (NG-R1), 102.53 ± 0.55 (NG-R2), 106.88 ± 4.95 (gypenoside A), and 103.95 ± 4.11 (gypenoside XLIX), respectively, versus the control group (87.51 ± 6.59). These results can reveal the real pharmacodynamic basis of YZG and provide a theoretical basis for subsequent studies. It can also provide some references for the research of Alzheimer's disease.

Visual detection of Hg2+ by manipulation of pyocyanin biosynthesis through the Hg2+-dependent transcriptional activator MerR in microbial cells.

Mercury pollution has always been a huge threat to human health due to its significant toxicity. Thus, it's the continuing goal to obtain new mercury detection techniques that are cost-effective, operational stable, performance efficient, and applicable to the environmental and biological milieus. In this research, the soluble pigment pyocyanin with anti-bacterial and anti-fungal activities, the biosynthesis pathway of which was engineered under the regulation of Hg2+-dependent transcriptional activator MerR, was firstly used as the visual detection signal in the whole-cell biosensor. The engineered biosensor displayed optical sensing window and a good linearity for Hg2+ in the range of 25-1000 nM, and the detection limit could reach as low as 10 nM. It permitted on-site detection of bioavailable Hg2+ with extraordinary selectivity and could resist the interferences of extra metal ions. What's more, the developed biosensor performed function well in a wide pH range (pH 4-10) as well as the environmental water. By fully imitating and utilizing the biosystems from nature, the engineered colorimetric biosensor has great economic and performance advantages over most chemosensors as well as whole-cell biosensors in the practical application of detecting Hg2+ in the contaminated aquatic systems.

Method Development and Validation of Indaziflam and Its Five Metabolites in Soil, Water, and Fruits by Modified QuEChERS and UHPLC-MS/MS.

A method for simultaneously determining indaziflam and its five metabolites in soil, water, and fruits using ultraperformance liquid chromatography/tandem mass spectrometry was established. The analytes were eluted in <4.5 min. Positive electrospray ionization mode was used. The analytes were extracted using acetonitrile containing 1% ammonium hydroxide, and then the extracts were purified using octadecylsilane and PRiME HLB cartridges. The quantification limits were 0.01-1.01 µg kg-1. The linearities of the calibrations for all analytes were excellent ( R2 > 0.9952). The recoveries at spike concentrations of 0.01, 0.1, and 1 mg kg-1 were 81.3-112.1%. The intraday and interday relative standard deviations were <13.5% and <12.3%, respectively. The method was successfully used to determine indaziflam and its five metabolites in samples from markets and fields. The results confirmed that the method is an effective and robust procedure for routinely determining indaziflam and its metabolites in soil, water, and fruit samples.

[Simultaneous determination of ten constituents in the Chinese medicinal preparation fufangxingxiangtu' erfeng capsules by ultra performance liquid chromatography with tandem mass spectrometry].

Using caffeic acid and icariin as internal standards, a method for the simultaneous determination of protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, scutellarin, isochlorogenic acid C, baicalin, luteolin, apigenin, atractylenolide III and atractylenolide I in Fufangxingxiangtu' erfeng capsules were established by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The separation was performed on a ZORBAX RRHD Eclipse Plus C18 column (50 mm x 2.1 mm, 1.8 microm) by using water containing 0.3% formic acid and methanol as mobile phases with the gradient elution at a flow rate of 0.3 mL/min. The analytes were detected by a tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via the switching of positive electrospray ionization (ESI(+)) and negative electrospray ionization (ESI(-)). Under optimum conditions, the calibration curves were linear in the range of 0.003 00-24.0 mg/L for protocatechuic acid, 0.017 0-2.00 mg/L for protocatechuic aldehyde, 0.015 0-30.0 mg/L for chlorogenic acid, 0.004 00-30.0 mg/L for scutellarin, 0.010 5-24.0 mg/L for isochlorogenic acid C, 0.003 00-30.0 mg/L for baicalin, 0.003 00-5.0 mg/L for luteolin, 0.006 00-1.50 mg/L for apigenin, 0.001 50-4.00 mg/L for atractylenolide III, and 0.000 600-0.900 mg/L for atractylenolide I with the detection limits of 1.0, 11, 5.0, 1.5, 3.5, 1.0, 1.0, 2.0, 0.50, 0.20 microg/L, respectively. The average recoveries of the ten effective components were between 92.5% and 106% with all relative standard deviations not more than 3.2%. The developed method was rapid, simple, accurate, reproducible, and suitable for the quality control of the Fufangxingxiangtu' erfeng capsules.

[Simultaneous determination of five alkaloids in Niuhuang Shangqing Tablets by micellar electrokinetic chromatography-mass spectrometry using laurel acyl malic acid ester].

A micellar electrokinetic chromatography-mass spectrometric method based on laurel acyl malic acid ester (LMAE) for the separation and determination of coptisine, berberine, jatrorrhizine, phellodendrine and ligustrazine in Niuhuang Shangqing Tablets was established. The baseline separation of the five compounds was attained within 18 min by an uncoated capillary (88 cm x 50 microm) on the operating voltage of 25 kV using 7.5 mmol/L LMAE-15 mmol/L ammonia-50 mmol/L ammonium acetate mixture (pH = 7.0) containing 12.5% (v/v) acetonitrile as the electrophoretic medium and 50% 2-propanol aqueous solution (containing 3 mmol/L acetic acid) as the sheath liquid. The peak area of each component to its concentration showed a good linear relationship. The relative standard deviations of migration times and peak areas of the five components were less than 5% and the recoveries were between 96.0% and 105%. The developed method is simple, rapid, accurate and is suitable for the routine analysis of the five alkaloid components in Niuhuang Shangqing Tablets.