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Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation 将深紫外激光烧蚀与纳米液滴样品制备结合起来进行空间蛋白质组学研究,提高亚细胞分辨率
Q3 Chemistry Pub Date : 2023-10-20 DOI: 10.1021/acsmeasuresciau.3c00033
Piliang Xiang, Andrey Liyu, Yumi Kwon, Dehong Hu, Sarah M. Williams, Dušan Veličković, Lye Meng Markillie, William B. Chrisler, Ljiljana Paša-Tolić* and Ying Zhu*, 

Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection. The two-step procedure also hinders the further improvement of spatial resolution to <10 μm as needed for subcellular proteomics. Herein, we developed a high-throughput and high-resolution spatial proteomics platform by seamlessly coupling deep ultraviolet (DUV) laser ablation (LA) with nanoPOTS (Nanodroplet Processing in One pot for Trace Samples)-based sample preparation. We demonstrated the DUV-LA system can quickly isolate and collect tissue samples at a throughput of ∼30 spots/min and a spatial resolution down to 2 μm from a 10 μm thick human pancreas tissue section. To improve sample recovery, we developed a proximity aerosol collection approach by placing DMSO droplets close to LA spots. We demonstrated the DUV-LA-nanoPOTS platform can detect an average of 1312, 1533, and 1966 proteins from ablation spots with diameters of 7, 13, and 19 μm, respectively. In a proof-of-concept study, we isolated and profiled two distinct subcellular regions of the pancreas tissue revealed by hematoxylin and eosin (H&E) staining. Quantitative proteomics revealed proteins specifically enriched to subcellular compartments.

组织微环境的多重分子图谱分析(或称空间 omics)可提供有关细胞功能和疾病病理的重要见解。将激光显微切割与基于质谱的蛋白质组学相结合,可以对 1000 种蛋白质进行深入而无偏见的绘制。然而,激光显微切割的通量往往受到繁琐的两步程序、连续激光切割和样品采集的限制。两步程序还阻碍了亚细胞蛋白质组学所需的空间分辨率进一步提高到 10 μm。在此,我们开发了一种高通量、高分辨率的空间蛋白质组学平台,将深紫外(DUV)激光烧蚀(LA)与基于样品制备的纳米POTS(痕量样品的纳米液滴一次性处理)无缝结合。我们展示了 DUV-LA 系统能以每分钟 ∼30 个点的吞吐量和低至 2 μm 的空间分辨率从 10 μm 厚的人体胰腺组织切片中快速分离和采集组织样本。为了提高样品回收率,我们开发了一种近距离气溶胶收集方法,将 DMSO 液滴放置在靠近 LA 点的位置。我们证明,DUV-LA-nanoPOTS 平台能从直径分别为 7、13 和 19 μm 的烧蚀点平均检测到 1312、1533 和 1966 个蛋白质。在概念验证研究中,我们分离并分析了苏木精和伊红(H&E)染色显示的胰腺组织的两个不同亚细胞区域。定量蛋白质组学揭示了特异性富集于亚细胞区的蛋白质。
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引用次数: 0
Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power 荧光素酶校准物可实现生物发光功率的绝对定量
Q3 Chemistry Pub Date : 2023-10-20 DOI: 10.1021/acsmeasuresciau.3c00036
Mark A. Klein*, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt and Rachel Friedman Ohana*, 

Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10–18 and 7.25 × 10–20 watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2–5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.

由荧光素酶催化氧化荧光素而发出的生物发光已被广泛用于报告生物事件,主要是通过光输出的相对变化。蛋白质工程和合成化学领域的最新进展已经产生了亮度和生物利用率显著提高的生物发光系统。这些发展不仅使生物事件的检测表达水平大大降低,也为利用生物发光驱动细胞内的光化学提供了新的机会。无论是哪种应用,生物发光分析在很大程度上都依赖于使用光度计来测量系统的光输出。目前的光度计以相对单位报告样品的光输出,限制了仪器间数据比较的能力,也无法量化生物发光系统的绝对功率。由荧光酶及其同源荧光素组成的荧光溶液校准液具有绝对光输出的特征,可以校准任何特定的发光仪进行绝对光子计数。为此,我们定制了一种光检测仪器,并将其与波长匹配的 LED 光源(波长分别为 450 纳米和 561 纳米)一起使用,分别鉴定了一系列 NanoLuc 和萤火虫荧光素酶溶液的绝对功率。这种方法显示,这两种常见的荧光素酶产生的功率分别为 3.72 × 10-18 瓦特/分子和 7.25 × 10-20 瓦特/分子。这些发光溶液校准液的成分可在市场上买到,并能在 2-5 分钟内产生稳定的生物发光信号,因此任何发光仪都能对这两种荧光素酶发出的生物发光进行功率测量校准,单位为瓦特或光子/秒。
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引用次数: 0
Sensing Liquid- and Gas-Phase Hydrocarbons via Mid-Infrared Broadband Femtosecond Laser Source Spectroscopy 通过中红外宽带飞秒激光源光谱传感液相和气相碳氢化合物
Q3 Chemistry Pub Date : 2023-10-12 DOI: 10.1021/acsmeasuresciau.3c00026
Michael Hlavatsch, Andrea Teuber, Max Eisele and Boris Mizaikoff*, 

In this study, we demonstrate the combination of a tunable broadband mid-infrared (MIR) femtosecond laser source separately coupled to a ZnSe crystal horizontal attenuated total reflection (ATR) sensor cell for liquid phase samples and to a substrate-integrated hollow waveguide (iHWG) for gas phase samples. Utilizing this emerging light source technology as an alternative MIR radiation source for Fourier transform infrared (FTIR) spectroscopy opens interesting opportunities for analytical applications. In a first approach, we demonstrate the quantitative analysis of three individual samples, ethanol (liquid), methane (gas), and 2-methyl-1-propene (gas), with limits of detection of 0.3% (ethanol) and 22 ppmv and 74 ppmv (methane and isobutylene), respectively, determined at selected emission wavelengths of the MIR laser source (i.e., 890 cm–1, 1046 and 1305 cm–1). Hence, the applicability of a broadband MIR femtosecond laser source as a bright alternative light source for quantitative analysis via FTIR spectroscopy in various sensing configurations has been demonstrated.

在这项研究中,我们展示了将可调谐宽带中红外(MIR)飞秒激光光源分别与用于液相样品的 ZnSe 晶体水平衰减全反射(ATR)传感器单元和用于气相样品的基底集成空心波导(iHWG)相结合的方法。利用这种新兴光源技术作为傅立叶变换红外(FTIR)光谱的替代中红外辐射源,为分析应用带来了有趣的机遇。在第一种方法中,我们展示了对乙醇(液体)、甲烷(气体)和 2-甲基-1-丙烯(气体)这三种样品的定量分析,检测限分别为 0.3%(乙醇)、22 ppmv 和 74 ppmv(甲烷和异丁烯),是在选定的中红外激光源发射波长(即 890 cm-1、1046 和 1305 cm-1)下测定的。因此,宽带近红外飞秒激光源作为一种明亮的替代光源,适用于在各种传感配置中通过傅立叶变换红外光谱进行定量分析。
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引用次数: 0
Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers 评估成纤维细胞对ER+乳腺癌雌激素信号转导影响的纸基协同培养平台
Q3 Chemistry Pub Date : 2023-10-09 DOI: 10.1021/acsmeasuresciau.3c00032
Zachary R. Sitte, Abel Andre Miranda Buzetta, Sarina J. Jones, Zhi-Wei Lin, Nathan Ashbrook Whitman and Matthew R. Lockett*, 

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

以细胞为基础的化验可以在分子水平上研究细胞对候选药物或潜在毒素的反应。核受体是哺乳动物细胞中基因靶标的关键转录调控因子,转录激活测定可量化核受体的激活或抑制情况。其中一种检测方法将荧光素酶的表达与雌激素受体-α(ERα)的转录活性结合起来。虽然这种检测方法经常用于筛选雌激素信号通路的激动剂和拮抗剂,但其设置依赖于单层培养,在单层培养中,细胞被直接移植到与细胞兼容的塑料容器表面。 肿瘤微环境不仅仅是癌细胞的集合,还受到组织结构、细胞外基质的存在以及邻近非癌细胞(如成纤维细胞)产生的细胞间信号分子的深刻影响。目前需要一种三维培养平台,既能快速制作原型以评估新的配置,又能随时大量生产,满足转化研究和筛选应用的需要。 在这里,我们展示了纸基培养平台在探究两种细胞类型之间的细胞间信号转导效应方面的实用性。我们使用纸质支架生成类似肿瘤的环境,形成悬浮在胶原蛋白中的乳腺癌细胞的确定体积。通过将纸支架置于商用 96 孔板中,我们比较了仅有乳腺癌细胞的单培养与在不同位置含有成纤维细胞的共培养配置,后者模拟了乳腺癌的进展阶段。我们发现,T47D-KBluc 细胞系中的 ERα 转录活化受成纤维细胞的存在、数量和邻近程度的影响,是细胞间信号分子的结果。在筛选了一小部分成纤维细胞分泌的信号分子后,我们发现白细胞介素-6(IL-6)是降低雌二醇敏感性的主要驱动因素。在共培养配置中加入 IL-6 中和抗体可减轻这些影响。我们还评估了雌激素受体的表达和转录调控,进一步证明了纸质平台在详细机理研究中的实用性。
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引用次数: 0
Screening of Indoor Transformation Products of Organophosphates and Organophosphites with an in Silico Spectral Database 利用硅谱数据库筛选有机磷和有机膦的室内转化产物
Q3 Chemistry Pub Date : 2023-10-05 DOI: 10.1021/acsmeasuresciau.3c00039
Steven Kutarna, Wanzhen Chen, Ying Xiong, Runzeng Liu, Yufeng Gong and Hui Peng*, 

Numerous transformation products are formed indoors, but they are outside the scope of current chemical databases. In this study, an in silico spectral database was established to screen previously unknown indoor transformation products of organophosphorus compounds (OPCs). An R package was developed that incorporated four indoor reactions to predict the transformation products of 712 seed OPCs. By further predicting MS2 fragments, an in silico spectral database was established consisting of 3509 OPCs and 28,812 MS2 fragments. With this database, 40 OPCs were tentatively detected in 23 indoor dust samples. This is the greatest number of OPCs reported to date indoors, among which two novel phosphonates were validated using standards. Twenty-four of the detected OPCs were predicted transformation products in which oxidation from organophosphites plays a major role. To confirm this, the in silico spectral database was expanded to include organophosphites for suspect screening in five types of preproduction plastics. A broad spectrum of 14 organophosphites was detected, with a particularly high abundance in polyvinyl chloride plastics and indoor end-user goods. This demonstrated the significant contribution of organophosphites to indoor organophosphates via oxidation, highlighting the strength of in silico spectral databases for the screening of unknown indoor transformation products.

在室内会形成许多转化产物,但这些产物不属于现有化学数据库的范围。本研究建立了一个硅学光谱数据库,用于筛选以前未知的有机磷化合物(OPCs)室内转化产物。开发的 R 软件包结合了四种室内反应,预测了 712 种 OPC 的转化产物。通过进一步预测 MS2 片段,建立了一个由 3509 种 OPC 和 28,812 个 MS2 片段组成的硅谱数据库。利用该数据库,在 23 份室内灰尘样本中初步检测出 40 种 OPC。这是迄今为止在室内报告的最多的 OPCs,其中两种新型膦酸盐已通过标准验证。在检测到的 OPC 中,有 24 种是预测的转化产物,其中有机膦酸盐的氧化作用发挥了主要作用。为了证实这一点,我们对硅学光谱数据库进行了扩展,将有机膦酸盐纳入其中,以便对五种生产前塑料中的可疑物质进行筛选。结果检测到 14 种有机膦酸盐,其中聚氯乙烯塑料和室内最终用户产品中的有机膦酸盐含量尤其高。这表明有机膦酸盐通过氧化作用对室内有机膦酸盐的贡献很大,突出了硅谱数据库在筛选未知室内转化产品方面的优势。
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引用次数: 0
Use of 3D Printing Techniques to Fabricate Implantable Microelectrodes for Electrochemical Detection of Biomarkers in the Early Diagnosis of Cardiovascular and Neurodegenerative Diseases 使用3D打印技术制造可植入微电极,用于心血管和神经退行性疾病早期诊断中生物标记物的电化学检测。
Q3 Chemistry Pub Date : 2023-09-20 DOI: 10.1021/acsmeasuresciau.3c00028
Nemira Zilinskaite, Rajendra P. Shukla and Ausra Baradoke*, 

This Review provides a comprehensive overview of 3D printing techniques to fabricate implantable microelectrodes for the electrochemical detection of biomarkers in the early diagnosis of cardiovascular and neurodegenerative diseases. Early diagnosis of these diseases is crucial to improving patient outcomes and reducing healthcare systems' burden. Biomarkers serve as measurable indicators of these diseases, and implantable microelectrodes offer a promising tool for their electrochemical detection. Here, we discuss various 3D printing techniques, including stereolithography (SLA), digital light processing (DLP), fused deposition modeling (FDM), selective laser sintering (SLS), and two-photon polymerization (2PP), highlighting their advantages and limitations in microelectrode fabrication. We also explore the materials used in constructing implantable microelectrodes, emphasizing their biocompatibility and biodegradation properties. The principles of electrochemical detection and the types of sensors utilized are examined, with a focus on their applications in detecting biomarkers for cardiovascular and neurodegenerative diseases. Finally, we address the current challenges and future perspectives in the field of 3D-printed implantable microelectrodes, emphasizing their potential for improving early diagnosis and personalized treatment strategies.

这篇综述全面概述了3D打印技术,以制造可植入微电极,用于电化学检测心血管和神经退行性疾病早期诊断中的生物标志物。这些疾病的早期诊断对于改善患者预后和减轻医疗系统负担至关重要。生物标记物是这些疾病的可测量指标,可植入微电极为其电化学检测提供了一种很有前途的工具。在这里,我们讨论了各种3D打印技术,包括立体光刻(SLA)、数字光处理(DLP)、熔融沉积建模(FDM)、选择性激光烧结(SLS)和双光子聚合(2PP),强调了它们在微电极制造中的优势和局限性。我们还探索了用于构建可植入微电极的材料,强调了它们的生物相容性和生物降解特性。研究了电化学检测的原理和所用传感器的类型,重点介绍了它们在检测心血管和神经退行性疾病生物标志物中的应用。最后,我们讨论了3D打印植入式微电极领域的当前挑战和未来前景,强调了它们在改善早期诊断和个性化治疗策略方面的潜力。
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引用次数: 0
Evaluation of Pure PFAS Decrease in Controlled Settings 控制设置中纯PFAS降低的评估
Q3 Chemistry Pub Date : 2023-09-07 DOI: 10.1021/acsmeasuresciau.3c00027
Marco Mancini, Valentina Gioia, Federica Simonetti, Alessandro Frugis and Stefano Cinti*, 

Since 1940, poly- or perfluorinated alkyl substances (PFAS) have been largely used in many applications, including paints, fire foaming, household items, product packaging, and fabrics. Because of their extremely high persistency, they have been defined as “forever chemicals”. Although the EU is taking action to reduce their use, their widespread occurrence in environmental matrices and their harmful effects on human health require the use of highly performing analytical methods for efficient monitoring. Furthermore, novel PFAS are constantly revealed by both EU and National environmental agencies. The objective of this work is to investigate the cause of the signal decrease during the analysis of a standard PFAS mixture in water-based matrices, by proposing an efficient technical procedure for laboratory specialists. The analyses were carried out on a mixture of 30 PFAS, including both regulated and unknown substances (which are expected to be introduced in the guidelines), characterized by different chemical features, using LC-vials of two different materials, namely, glass and polypropylene, and dissolved in two solvents, namely, water and water–methanol. The temperature of analysis and the concentration of PFAS were also considered through LC-MS analyses at different times, in the 0–15 h range. Depending on the chemical structure and length of the PFAS, sampling and treatment procedures may be adopted to tackle the decrease and the release from the containers, reducing the risk of underestimating PFAS also in real water matrices.

自 1940 年以来,多氟烷基或全氟烷基物质 (PFAS) 已广泛应用于涂料、防火发泡、家居用品、产品包装和织物等诸多领域。由于它们具有极高的持久性,因此被定义为 "永远的化学品"。尽管欧盟正在采取行动减少它们的使用,但它们在环境基质中的广泛存在及其对人类健康的有害影响要求使用高性能的分析方法进行有效监测。此外,欧盟和各国的环保机构也不断发现新型 PFAS。这项工作的目的是通过为实验室专家提出一种有效的技术程序,研究在分析水基基质中标准全氟辛烷磺酸混合物时信号下降的原因。分析对象是 30 种 PFAS 混合物,包括受管制物质和未知物质(预计将在指南中引入),它们具有不同的化学特征,使用两种不同材料(玻璃和聚丙烯)的液相色谱仪,溶解在两种溶剂(水和水甲醇)中。分析温度和全氟辛烷磺酸的浓度也通过 0-15 小时范围内不同时间的 LC-MS 分析加以考虑。根据全氟辛烷磺酸的化学结构和长度,可采用采样和处理程序来解决容器中全氟辛烷磺酸的减少和释放问题,从而降低低估实际水基质中全氟辛烷磺酸的风险。
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引用次数: 0
Evaluation of Surface Treatments of PDMS Microfluidic Devices for Improving Small-Molecule Recovery with Application to Monitoring Metabolites Secreted from Islets of Langerhans PDMS微流控装置表面处理提高小分子回收率的评价及其在朗格汉斯胰岛分泌代谢物监测中的应用
Q3 Chemistry Pub Date : 2023-08-24 DOI: 10.1021/acsmeasuresciau.3c00025
Ashley E. Lenhart,  and , Robert T. Kennedy*, 

Microfluidic devices are becoming an important tool for bioanalysis with applications including studying cell secretion, cell growth, and drug delivery. Small molecules such as drugs, cell products, or nutrients may partition into polydimethylsiloxane (PDMS), a commonly used material for microfluidic devices, potentially leading to poor recovery or inaccurate delivery of such chemicals. To decrease small-molecule partitioning, surface and bulk PDMS treatments have been developed; however, these have been tested on few analytes, or their biocompatibility are unknown. Studies often focus on one analyte, whereas a diversity of chemicals are of interest and possibly affected. In this study, 11 device treatments are tested and applied to 21 biologically relevant small molecules with a variety of chemical structures. Device treatments are characterized using water contact angle measurements and evaluated by measuring recovery of the 21 target analytes using liquid chromatography–mass spectrometry. 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene), a positively charged polymer, produced the least hydrophilic surface and was found to provide the best recovery with most of the analytes having >50% recovery and up to 92% recovery; however, recovery varied by analyte highlighting the importance of analyte diversity rather than targeting a single analyte in evaluating treatments. A polybrene-treated device was applied to investigate secretion from pancreatic islets, which are micro-organs involved in glucose homeostasis and diabetes. Islets secrete small molecules that have been shown to modulate the secretion of islets’ main functional products, glucose-regulating hormones. The polybrene treatment enabled the detection of 20 target analytes from islets-on-chip during isosmotic and hypo-osmotic glucose perfusions and resulted in detection of more significant secretion changes compared to untreated PDMS.

微流控装置正在成为生物分析的重要工具,其应用包括研究细胞分泌、细胞生长和药物传递。药物、细胞产物或营养物质等小分子可能分裂成聚二甲基硅氧烷(PDMS),这是一种微流体装置常用的材料,可能导致这些化学物质的回收率低或输送不准确。为了减少小分子的分裂,人们开发了表面和块状PDMS处理方法;然而,这些已经在很少的分析物上进行了测试,或者它们的生物相容性是未知的。研究通常集中在一种分析物上,而对多种化学物质感兴趣并可能受到影响。在本研究中,测试了11种器件处理方法,并将其应用于21种具有多种化学结构的生物学相关小分子。设备处理使用水接触角测量来表征,并通过使用液相色谱-质谱法测量21种目标分析物的回收率来评估。1,5-二甲基-1,5-重氮十一亚甲基聚甲溴(聚苯乙烯)是一种带正电的聚合物,其表面亲水性最低,回收率最高,大多数分析物的回收率为50%,最高可达92%;然而,回收率因分析物而异,强调了分析物多样性的重要性,而不是针对单一分析物来评估治疗。应用聚苯乙烯处理的装置研究胰岛的分泌,胰岛是参与葡萄糖稳态和糖尿病的微器官。胰岛分泌的小分子已被证明可以调节胰岛的主要功能产物——血糖调节激素的分泌。在等渗和低渗葡萄糖灌注期间,聚苯乙烯处理能够从芯片上的胰岛中检测到20种目标分析物,与未处理的PDMS相比,检测到更显著的分泌变化。
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引用次数: 0
Legion: An Instrument for High-Throughput Electrochemistry 军团:高通量电化学仪器
Q3 Chemistry Pub Date : 2023-07-13 DOI: 10.1021/acsmeasuresciau.3c00022
Benjamin H. R. Gerroll, Krista M. Kulesa, Charles A. Ault and Lane A. Baker*, 

Electrochemical arrays promise utility for accelerated hypothesis testing and breakthrough discoveries. Herein, we report a new high-throughput electrochemistry platform, colloquially called “Legion,” for applications in electroanalysis and electrosynthesis. Legion consists of 96 electrochemical cells dimensioned to match common 96-well plates that are independently controlled with a field-programmable gate array. We demonstrate the utility of Legion by measuring model electrochemical probes, pH-dependent electron transfers, and electrocatalytic dehalogenation reactions. We consider advantages and disadvantages of this new instrumentation, with the hope of expanding the electrochemical toolbox.

电化学阵列有望加速假设检验和突破性发现。在此,我们报告了一种新的高通量电化学平台,俗称“军团”,用于电分析和电合成。Legion由96个电化学电池组成,尺寸与普通的96孔板相匹配,这些板由现场可编程门阵列独立控制。我们通过测量模型电化学探针、ph依赖的电子转移和电催化脱卤反应来证明Legion的实用性。我们考虑了这种新仪器的优点和缺点,希望扩大电化学工具箱。
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引用次数: 1
Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)? 扫描电化学显微镜(SECM)能测量单细胞呼吸吗?
Q3 Chemistry Pub Date : 2023-07-10 DOI: 10.1021/acsmeasuresciau.3c00019
Kelsey Cremin, Gabriel N. Meloni, Dimitrios Valavanis, Orkun S. Soyer* and Patrick R. Unwin*, 

Ultramicroelectrode (UME), or, equivalently, microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates via the oxygen consumption rate (OCR) in single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potential is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced) and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 1 × 10–18 to 1 × 10–16 mol s–1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at, or below, the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized.

超微电极(UME)或等效微电极探针越来越多地用于单细胞测量细胞特性和过程,包括生理活动,如代谢通量和呼吸率。这种测量灵敏度的主要挑战包括:(i)细胞和UME通量的相对大小(表现在电流中);以及(ii)UME响应随时间变化的稳定性问题。为了探索这些因素对电化学细胞测量精度的影响程度,我们对测量条件和实验参数进行了系统分析,以通过单HeLa细胞中的耗氧率(OCR)确定单细胞呼吸率。使用以铂UME为探针的扫描电化学显微镜(SECM),我们采用了一种自参考测量协议,该协议在SECM中很少使用,通过该协议,UME从本体溶液到电池重复接近,并在电池附近和本体溶液中进行短脉冲氧还原反应(ORR)电位。这种方法能够周期性地跟踪近电池响应与之重复比较(参考)的批量UME响应,并且还确保仅短暂地执行电池附近的ORR,从而最大限度地减少电化学过程对电池的影响。SECM实验与有限元法(FEM)建模框架相结合,以模拟氧气扩散和UME响应。将单细胞OCR的实际范围设为1×10-18至1×10-16mol s-1,FEM模拟和自参考SECM测量相结合的结果表明,这些OCR值等于或低于该技术目前的检测灵敏度。我们为未来改进这些测量提供了一组基于模型的建议,但强调如果要实现单细胞OCR测量,就需要在SECM测量的稳定性和精度方面进行非凡的改进。
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ACS Measurement Science Au
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