The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.     

A review on immobilised aptamers for high throughput biomolecular detection and screening

The discovery of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has led to the generation of aptamers from libraries of nucleic acids. Concomitantly, aptamer-target recognition and its potential biomedical applications have become a major research endeavour. Aptamers possess unique properties that make them superior biological receptors to antibodies with a plethora of target molecules. Some specific areas of opportunities explored for aptamer-target interactions include biochemical analysis, cell signalling and targeting, biomolecular purification processes, pathogen detection and, clinical diagnosis and therapy. Most of these potential applications rely on the effective immobilisation of aptamers on support systems to probe target species. Hence, recent research focus is geared towards immobilising aptamers as oligosorbents for biodetection and bioscreening. This article seeks to review advances in immobilised aptameric binding with associated successful milestones and respective limitations. A proposal for high throughput bioscreening using continuous polymeric adsorbents is also presented.     

食品中农药残留的高通量分析之展望(英文)

The screening of pesticide residues plays a vital role in food safety.Applications of high throughput analytical procedures are desirable for screening a large number of pesticides and food samples in a time-efficient and cost-effective manner.This review discusses how sample throughput of pesticide analysis could be improved with an emphasis on sample preparation,instrumentation and data analysis.     

基于报告基因技术的MDR1转录抑制剂高通量筛选模型的建立和应用

MDR1基因是引起肿瘤多药耐药的主要基因,其编码的P-gp蛋白可持续将药物由胞内排出胞外以降低胞内药物浓度导致多药耐药,MDR1基因的转录抑制剂可抑制MDR1基因在癌细胞中的表达,从而逆转肿瘤多药耐药.通过克隆MDR1基因的启动子,将其插入pGL3-basic质粒构建MDR1-luc＋报告基因载体,再将重组载体转染入HepG2肝癌细胞并筛选单克隆细胞株,构建了MDR1启动子的高通量筛选模型,Z′因子为0.75;通过对中药样品库的筛选,得到两种中药提取物高良姜水提物、红豆蔻醇提物有明显耐药逆转效果,EC50值分别为高良姜水提物16.37mgL-1和红豆蔻醇提物14.96mgL-1,RT-PCR验证上述两种阳性样品具有明显的抑制MDR1基因表达的作用.以上结果为MDR1基因的转录抑制剂高通量筛选奠定了基础.     

高通量高效液相色谱法快速检测西红花中15种非法添加色素

建立高通量的高效液相色谱法同时检测15种西红花中非法添加色素,对于阳性样品采用液相色谱–质谱联用法确证。采用Poroshell 120 EC–C18色谱柱(100 mm×4.6 mm,2.7 μm)分离,以甲醇–0.02 mol/L乙酸铵溶液(用氨水调pH至5.9±0.05)进行梯度洗脱,流量为1.2 mL/min,柱温为35 ℃,以450 nm和500 nm双波长检测。缓冲盐pH值和流动相流量对色谱条件影响大,需严格控制。该方法重现性好,各物质分离度均大于1.5,标准曲线的线性相关系数r为0.998~1.000,样品高、中、低浓度的加标回收率为70.1%~93.1%,测定结果的相对标准偏差为1.2%~6.6%(n=6),检出限为0.01~0.05 mg/kg。该方法精密度良好,分析时间短,分析结果稳定,可用于中药材西红花中15种非法添加色素的快速筛查和含量测定,同时能为相关部门制定监督抽检中药材染色掺伪的补充检验方法。     

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user‐defined goal) coupled with droplet‐based microfluidics allows the detailed analysis of millions of enzyme variants on ultra‐short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet‐based microfluidics, made in this context towards the high‐throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell‐based assays, as in the case of directed evolution experimentations.     

基于微流控技术的细胞水平高通量药物筛选系统的研究进展

药物筛选是新药研发的关键步骤,创新药物的发现需要采用适当的药物作用靶点对大量化合物样品进行筛选.高通量筛选系统能够实现数千个反应同时测试和分析,大大提高了药物筛选的实验规模和效率.其中基于细胞水平的高通量药物筛选系统因为更加接近人体生理条件,成为主要的筛选模式.而目前发展成熟的高通量细胞筛选系统主要基于多孔板,存在细胞...     

High throughput screening and measurements of proton conductivity of newly developed PEM materials based on proton transport visualization

An electrochemical method for proton transport visualization was developed and applied to the investigation of proton-conducting membrane materials. The method employs the change in the visual appearance of chemo-chromic tungsten oxide WO₃ in the presence of atomic hydrogen. An all-solid electrochemical cell arranged by substituting a fuel cell cathode with a thin film of WO₃-electrode was built and shown to generate both optical and electrical response to hydrogen gas exposure. The design of the cell was extended to a high throughput screening system that was utilized to characterize proton transport properties of samples, including a number of new compounds synthesized in-house by sol–gel wet chemistry. Non-destructive introduction of superacidic groups promoting proton hopping in the membrane materials was achieved by photodecomposition of a photoacid generator just after membrane casting. A model quantitatively describing current–voltage relation in the cell was developed and successfully applied to derive area-specific resistance of proton-conductive membranes from the experimental results. Area-specific resistances of membranes are derived from the slopes of optically reconstructed voltage–current curves. Sensitivity and dynamic range of the screening method are discussed.     

Structural models in the assessment of protein druggability based on HTS data

Insights on the potential of target proteins to bind small molecules with high affinity can be derived from the knowledge of their three-dimensional structural details especially of their binding pockets. The present study uses high-throughput screening (HTS) results on various targets, to obtain mathematical predictive models in which a minimal set of structural parameters significantly contributing to the hit rates or the affinity of the protein binding pockets for small molecular entities, is identified. An emphasis is given to focus on target variation aspect of the data by consideration of commonly tested compounds against the HTS targets. We identify ‘four-parameter’ models with R ², , SEE, and LOO q ² values of 0.70, 0.60, 0.27 and 0.50, respectively, or better. We demonstrate through cross-validation exercises that our regression models apply well on varied data sets. Thus we can use these models to estimate hit rates for HTS campaigns and thereby assign priority to drug targets before they undergo such resource intense experimental screening and follow-up.

Highly selective molecular recognition and high throughput detection of melamine based on molecularly imprinted sol-gel film

Multimode reader has been generally applied in immunoassay, and in the proposed paper, the 96 well micro-plate was modified with molecularly imprinted melamine sol-gel film, based on which the highly selective and high throughput detection of melamine was achieved. Melamine was imprinted into silica sol-gel films directly using phenyltrimethoxysilane and methyltrimethoxysilane as functionalized organosilicon precursors. The binding characteristic of the imprinted film to melamine was evaluated by equilibrium binding experiments and the morphology was studied by scanning electronic microscope (SEM). Scatchard analysis was carried out to estimate the binding parameters of the imprinted film. The proposed method exhibited excellent selectivity because of specific recognition of MM by molecularly imprinted film. Under the optimum conditions, the chemiluminescence (CL) intensity had a linear relationship against the concentration of melamine over the range of 0.1-50 μg mL⁻¹ with a lower detection limit of 0.02 μg mL⁻¹.     

High‐throughput polymer tip‐electrospray ionization mass spectrometry for enhanced detection of neopterin and biopterin in clinical urine samples

Urinary biopterin (Bio) and neopterin (Neo) are important markers for clinical diagnosis of hyperphenylalaninemia. Herein, we developed a high‐throughput analysis method based on electrospray ionization mass spectrometry (ESI‐MS) with polymer tips for the rapid quantitative detection of Bio and Neo in clinical urine samples. Different polymer tips were investigated. It is found that the best detection sensitivity was achieved with hydrophobic polymer tip, ie, polyethylene tips. The high‐throughput polymer tip‐ESI‐MS method allowed a rapid analysis speed at ~40 seconds per sample. The limits of quantification (LOQ) (S/N ≥ 10) for the detection of Bio and Neo were improved to be 5.0 ng/mL. Acceptable relative standard deviation (RSD) values for Neo and Bio were measured to be 12.2% and 13.4% for direct measurement of Bio and Neo in raw urine samples, respectively. Furthermore, Bio and Neo were directly quantified from 18 clinical urine samples by presented method. The ratios of urinary Bio‐to‐Neo were analyzed for diagnosis of hyperphenylalaninemia. The results demonstrated that the present polymer tip‐ESI‐MS method is a promising strategy for the rapid analysis of clinical samples.     

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

A homogeneous phase protein-based assay for the high throughput screening of drugs was developed using enhanced green fluorescent protein (EGFP) as the reporter. For that, a fusion protein between calmodulin (CaM) and EGFP was constructed in order to monitor the conformational changes induced in CaM upon binding to tricyclic anti-depressant drugs. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. Further, the conformational changes induced in CaM upon binding to Ca²⁺ and the target analyte drug, leads to a change in the microenvironment of EGFP concomitant with a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Further, the response of CaM–EGFP fusion protein in the presence of Ca²⁺ to increasing concentrations of phenothiazines and structurally related tricyclic anti-depressants was investigated. Dose-response curves for various tricyclic anti-depressants were prepared. Moreover, this assay can serve as a model system for other homogeneous binding assays for pharmaceuticals employing genetically fused binding proteins with reporter proteins and may find applications in the high throughput screening of tricyclic anti-depressants.