RSC Advances
·
2014
·Vol. 4
·期 56
·29925-29932
·
IF 3.708
Y. L. Li,X. L. Yang,C. X. He,X. L. Hu,J. Pu,L. Liu,G. B. Long,F. Liao
The maximum activities of fusion-tagged enzyme/mutants from cell lysates adsorbed by an anti-tag antibody immobilized in microplate wells were predicted to serve as equivalents of their specific activities for comparison with a six-histidine (6His)-tagged esterase and its tagged mutant as models. In brief, (a) a fixed quantity of a monoclonal anti-6His antibody was immobilized in microplate wells; (b) the maximum activity of a tagged enzyme/mutant from a cell lysate for saturation binding to the immobilized antibody (Vs) was predicted from the response of activities of the adsorbed tagged enzyme/mutant to quantities of total proteins in wells from the same lysate; and (c) Vs of tagged enzyme/mutants in lysates served as equivalents of their specific activities for comparison. Prediction of Vs of a tagged enzyme needed initial rates for absorbance changes of over 0.090 in 30 min, the highest occupancy of over 40% of binding sites of the immobilized antibody, and sufficient abundance of the tagged enzyme in lysates. With 0.6 μg antibody for immobilization in wells and total proteins of 10.0 to 128 μg from cell lysates, Vs of the tagged esterase had a coefficient of variation below 10% when the apparent specific activities in lysates varied more than four times. The ratio of Vs of the tagged esterase to the tagged mutant had higher precision and consistency with the ratio of their apparent specific activities from a large number of independent lysates. Hence, Vs predicted for tagged enzyme/mutants in cell lysates was suitable for comparison and may be applicable to verify positive mutants in a library.
研究领域:
生物化学
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分子生物学
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蛋白质工程
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DOI
高影响因子
ANALYTICAL BIOCHEMISTRY
·
2018
·
IF 2.275
Jianyong Zheng,Wei Wei,Xing Lan,Yinjun Zhang,Zhao Wang
This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert -butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant ( K m) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.
研究领域:
生物化学
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酶学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2013
·
IF 4.387
Yufeng Zhang,Shun Xiao,Lijuan Sun,Zhiwei Ge,Fengkai Fang,Wen Zhang,Yi Wang,Yiyu Cheng
A high throughput method was developed for rapid screening and identification of bioactive compounds from traditional Chinese medicine, marine products and other natural products . The system, integrated with five-channel chromatographic separation and dual UV–MS detection, is compatible with in vitro 96-well microplate based bioassays. The stability and applicability of the proposed method was validated by testing radical scavenging capability of a mixture of seven known compounds (rutin, dihydroquercetin, salvianolic acid A , salvianolic acid B , glycyrrhizic acid, rubescensin A and tangeretin). Moreover, the proposed method was successfully applied to the crude extracts of traditional Chinese medicine and a marine sponge from which 12 bioactive compounds were screened and characterized based on their anti-oxidative or anti-tumor activities. In particular, two diterpenoid derivatives, agelasine B and (−)-agelasine D, were identified for the first time as anti-tumor compounds from the sponge Agelas mauritiana , showing a considerable activity toward MCF-7 cells (IC 50 values of 7.84 ± 0.65 and 10.48 ± 0.84 μM, respectively). Our findings suggested that the integrated system of 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays can be a versatile and high efficient approach for the discovery of active compounds from natural products.
研究领域:
中药学
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分析化学
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药理学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL LETTERS
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2020
·
IF 1.467
Jiajia Hong,Xiaotang Hao,Tiantian Liu,Weiting Liu,Meiyi Xie,Mei Wang,Qian Xu,Bingyi Yang
A sensitive method termed rapid synergistic cloud point extraction (RS-CPE) combined with partial least squares (PLS) was developed for the extraction and simultaneous determination of trace amounts of phenol and three detrimental chlorophenols (CPs) via a microplate assay (MPA). In this study, n-pentanol was used as a novel synergistic inducer and the linear nonionic surfactant Tergitol 15-S-7 was used as an extractant. The whole process of RS-CPE was accomplished in a few minutes without the need for the time-consuming water- or ice-bath steps used in traditional cloud point extraction. An automated and high-throughput assay using a 96-well plate allowed for the simultaneous processing of multiple samples with low sample volume requirements (approximately 200 µL). However, synchronized determination of several CPs using MPA method was problematic because of spectral interferences. Fortunately, a multivariate calibration method using PLS was applied to circumvent these limitations. Under the optimum conditions, the linear dynamic concentration ranges were from 0.5 to 15 µg/mL for 2-chlorophenol and 0.5 to 20 µg/mL for the other analytes. The detection limit range was from 0.275 to 0.419 µg/mL. The enrichment factor was up to 15.4 fold. The developed methodology was successfully applied to quantitative analysis of phenol and three CPs in water samples with recoveries from 90.0% to 109%.
研究领域:
分析化学
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环境科学
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DOI
高影响因子
Analytical Methods
·
2019
·Vol. 11
·期 48
·6084-6091
·
IF 2.378
He-Ye Wang,Ya-Li Yang,Yue Wang,Shuai-Feng Zhang,Li Li
Organic polymer monoliths have been used extensively for separation and purification of molecules because of their excellent mass transfer properties and versatile surface modification. However, organic polymer monolithic materials have significant UV absorption, which blocked UV-visible absorbance detection. Thus, we developed novel disk-shaped mycotoxin immune-affinity monolithic microplates for quantitative detection of mycotoxins. The concentric disk-shaped format allows the obscure organic monoliths to capture mycotoxins and unobstructed UV-visible absorbance measurement was performed in the same microplates. The limits of detection of 96 disk-shaped mycotoxin immune-affinity monolithic microplates were 161.73, 22.52, 0.14, and 0.21 μg kg−1 for Deoxynivalenol (DON), Zearalenone (ZEN), Aflatoxin B1 (AFB1), and Ochratoxin A (OTA), respectively, which meet well the requirements for detection of foodstuffs. The recovery rate ranges of the four mycotoxins were 87–92%, 82–89%, 78–116%, and 83–87% for DON, ZEN, AFB1, and OTA disk-shaped microplates, respectively, which proved that the disk-shaped monolithic microplates have good reproducibility for mycotoxin detection. In summary, 96 disk-shaped mycotoxin immune-affinity monolithic microplates are a sensitive, accurate, stable, and convenient platform for detection of mycotoxins in agro-food.
研究领域:
分析化学
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食品安全
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
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2025
·
IF 6.7
Shuai Guo,Zhizhong Wen,Long Chen,Mengchen Jia,Yajing Wang,Jingfang Sun
The development of biocatalysis depends heavily on high-throughput screening (HTS) approaches to uncover engineered enzymes with superior biocatalytic activity. Recent advances in mass spectrometry (MS) enable label-free, high-speed analysis of biocatalytic samples in standard microplates by omitting the chromatographic separation. However, most MS methods need a complicated sampling interface system and face challenges due to biological matrix interferences, which can diminish the sensitivity and reliability. Herein, we established a direct microplate sampling (DMS)-MS technique for HTS of the enzymatic activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) in steroid biocatalysis. The implementation of an open port interface (OPI) probe with a customized ion source for microwell direct insertion sampling and ionization has streamlined the setup, reduced operational complexity, and shortened the analysis time to 0.9–6.5 s/sample. A sample preparation strategy involving derivatization, followed by high-ratio dilution (e.g., 10,000-fold), was further integrated prior to DMS-MS for mitigating ion suppression effects across diverse biological matrices. Ultimately, this workflow, when applied for monitoring multiple steroids in 17β-HSD biocatalytic processes, demonstrated high stability (peak area RSDs < 7%), minimal carryover (<1%), and quantitative accuracy comparable to conventional liquid chromatography (LC) experiments while providing a >150-fold throughput enhancement. After screening a total of 5760 17β-HSD mutants across three different steroid conversion reactions, we identified and verified two new variants with enhanced biocatalytic activities. This study successfully established a DMS-MS-based HTS workflow for microplate screening, providing a simplified, robust, and highly reliable analytical platform for microbial or enzyme engineering projects involving complex biological matrices.
研究领域:
生物化学
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分析化学
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酶工程
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产品关联
DOI
PubMed
高影响因子
ACS Omega
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2017
·
IF 0
Jiqing Yang,Xiaoxia Liu,Shucheng Sun,Xin Liu,Li Yang
A facile, cost-effective, and high-throughput screening method was developed for enzyme-based assays based on Robolid/Microplate (RLMP) platform. The RLMP platform is constructed by immobilizing enzyme on commercial robolids and combining it with a standard 96-well microplate to achieve high-throughput analysis. The initiation and quenching of enzymatic reaction can be performed by simply sandwiching or unsealing the enzyme-immobilized robolids and the sample-containing microplate. This platform enables measurements of multiple target analytes simultaneously based on immobilized enzymatic reactions, with analysis time independent of the number of wells in the microplate. Using urea as the model analyte, we have shown that the RLMP platform exhibits large linear detection range of up to 10 mM, fast analysis time of 30 min/96 samples, as well as good reproducibility and stability. Measurements of urea in human urine and serum samples were performed using the RLMP platform and were compared with the commercial urea test kit. A good correlation was found between the two methods. This study shows that the present RLMP platform has promising prospects for detection of clinical markers and application in disease diagnosis and biochemical analysis.
研究领域:
生物技术
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分析化学
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临床诊断
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DOI
PubMed
SENSORS AND ACTUATORS B-CHEMICAL
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2024
·
IF 8
Ziyi Tong,Shengyan Hou,Zhenkun Zhang,Zhen Liu,Yifei Zhang
Molecular data storage is an emerging supplement to conventional information storage methods. In addition to the information storage with DNA or synthetic polymers, a recently proposed strategy that uses small molecule mixtures as information carriers has sparked interest since it is a synthesis-free method. Here, we demonstrate two methods for decoding digital information stored in two sets of small-molecule metabolites. The information is stored as binary code, 0 or 1, based on the absence or presence of each particular metabolite in the mixture. In the first approach, the information was encoded by five metabolites (sarcosine, cholesterol, xanthine, glucose, and galactose) and stored as mixture solutions in a 384-well microplate. The information was read out by cascade enzymatic reactions that can specifically convert the metabolites to a colorimetric product, which was then assayed by a microplate reader. We stored a 60×32-pixel picture (1920 bits) with 384 mixtures of small molecules with a reading accuracy of 96.68 %, and also demonstrated the encoding of a 220-bit text message. Alternatively, another set of six metabolites (bilirubin, uric acid, urea, creatinine, glucose, and triglyceride) was deployed to encode a 78×102-pixel picture (7956 bits) with 1326 mixtures, which was decoded automatically by a biochemistry analyzer with a 98.96 % accuracy. This work showcases that common metabolites can be used as digital molecules for information storage, and the information decoding can be done by accessible instruments in an ordinary biochemical laboratory.
研究领域:
生物化学
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信息技术
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分子生物学
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
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2025
·
IF 5.1
Tiantian Wang,Yanli Zhou,Lantao Liu,Hui Dong,Yinghan Xu,Yidan Sun,Yaocun Yue,Jiaqing Tian,Maotian Xu
Abnormal Tumor necrosis factor-α (TNF-α) levels in serum are closely linked to Alzheimer's disease onset, progression, and severity. Highly sensitive and specific detection of TNF-α offers a key biomarker for early diagnosis and disease evaluation, providing significant clinical value for improved disease management. In this study, an iridium complex [(piq)₂Ir(dpppy)] containing a cyclometalated ligand was used as a novel and highly efficient electrochemiluminescence (ECL) luminophore to construct an ultrasensitive electrochemical biosensor for the detection of TNF-α. First, the TNF-α antibody was immobilized in the microplate reactor, and a quasi-specific sandwich immunoassay was performed. Subsequently, aptamers modified with silver nanoparticles-which were in-situ generated on the C-rich sequence-modified aptamer, captured the TNF-α antigen. After dissociation with nitric acid, the solution containing Ag + was transferred to the electrode surface modified with the cyclometalated iridium complex for ECL signal detection. The constructed immunosensor not only exhibited a wide linear range of 0.001 ng mL −1 –100 ng mL −1 , but also showed excellent recovery in human serum samples, ranging from 98.6 % to 101.7 %, with a relative standard deviation of 0.66 %–3.8 %. This strategy not only enhanced the detection sensitivity due to the efficient performance of the iridium complex in the 3 LCπ − π* state, but also this strategy of this biosensor effectively maintained the specificity and stability.
研究领域:
生物化学
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生物传感器
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免疫学
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电化学
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医学诊断
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产品关联
DOI
高影响因子
ENVIRONMENTAL SCIENCE & TECHNOLOGY
·
2021
·
IF 9.028
Xiao Juan Du,Yang Chen,Ling Yun Qin,Hong Qun Luo,Nian Bing Li,Bang Lin Li
The hybrid of l-cysteine and agarose can reduce HAuCl4 and support the rapid growth of plasmonic gold nanoparticles (Au NPs) in the hydrogel phase. The l-cysteine-doped agarose hydrogel (C-AGH) not only offers the substrate the capacity to reduce Au(III) ions but also stabilizes and precisely modulates the in situ grown Au NPs with high repeatability, easy operation, and anti-interference performance. Herein, before the incubation of HAuCl4, the improved hydrogel is preincubated in the aqueous solution containing mercury ions, and the cysteine can specifically conjugate with mercury via the thiol groups. Subsequently, the responsive allochroic bands from dark blue to red can be identified in the solid hydrogel after the incubation of HAuCl4, which is attributed to the formation of regulated Au–Hg nanoamalgams. As a proof-of-concept, toxic Hg2+ ions are exploited as targets for constructing novel sensing assays based on the improved C-AGH protocol. Based on naked-eye recognition, Hg2+ could be rapidly and simply measured. Additionally, the high-throughput and trace analysis with a low limit of detection (3.7 nM) is performed using a microplate reader. On the basis of the filtering technique and remodeling of hydrogels, C-AGH working as the filtering membrane can even achieve the integration of enrichment and measurement with enhanced sensitivity. Significantly, the strategy of using an allochroic hydrogel with the staining of Au NPs can promote the rapid and primary assessment of water quality in environmental analysis.
研究领域:
纳米技术
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环境科学
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分析化学
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2016
·Vol. 8
·期 35
·6560-6565
·
IF 1.915
Changqing Zhu,Ying Hong,Zhen Xiao,Yang Zhou,Yuan Jiang,Ming Huang,Xinglian Xu,Guanghong Zhou
A sensitive method for the colorimetric determination of Salmonella typhimurium (S. typhimurium) was developed based on aptamer recognition and gold nanoparticles. First, a biotinylated specific aptamer for S. typhimurium was immobilized on avidin-coated microplate wells. Then, target bacteria, a biotinylated aptamer and avidin-catalase were successively introduced into the microplate wells. Finally, hydrogen peroxide and freshly prepared gold(III) chloride trihydrate were added, and the absorbance of the reaction product was measured with a plate reader. Under optimized conditions, there was a linear relationship between the absorbance (A550 nm) and the concentration of S. typhimurium over the range of 101 to 106 cfu mL−1 (R2 = 0.9920), with a detection limit of 10 cfu mL−1. This simple rapid method provided a promising tool for on-site screening of S. typhimurium in raw chicken samples.
研究领域:
微生物学
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分析化学
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食品安全
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产品关联
DOI
高影响因子
ENVIRONMENTAL GEOCHEMISTRY AND HEALTH
·
2023
·Vol. 45
·期 7
·4779-4793
·
IF 4.2
Zhang Wenjie,Wang Jinhua,Zhu Lusheng,Wang Jun,Mao Shushuai,Yan Xiaojing,Wen Shengfang,Wang Lanjun,Dong Zikun,Kim Young Mo
Residual antibiotics (ABs) and heavy metals (HMs) are continuously released from soil, reflecting their intensive use and contamination of water and soil, posing an environmental problem of great concern. Relatively few studies exist of the functional diversity of soil microorganisms under the combined action of ABs and HMs. To address this deficiency, BIOLOG ECO microplates and the Integrated Biological Responses version 2 (IBRv2) method were used to comprehensively explore the effects of single and combined actions of copper (Cu) and enrofloxacin (ENR), oxytetracycline (OTC), and sulfadimidine (SM2) on the soil microbial community. The results showed that the high concentration (0.80 mmol/kg) compound group had a significant effect on average well color development (AWCD) and OTC showed a dose–response relationship. The results of IBRv2 analysis showed that the single treatment group of ENR or SM2 had a significant effect on soil microbial communities, and the IBRv2 of E1 was 5.432. Microbes under ENR, SM2, and Cu stress had more types of available carbon sources, and all treatment groups were significantly more enriched with microorganisms having D-mannitol and L-asparagine as carbon sources. This study confirms that the combined effects of ABs and HMs can inhibit or promote the function of soil microbial communities. In addition, this paper will provide new insights into IBRv2 as an effective method to evaluate the impacts of contaminants on soil health.
研究领域:
环境科学
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微生物学
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土壤学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）