Analytical biochemistry最新文献_第8页

Uracil base PCR implemented for reliable DNA walking 实施尿嘧啶碱基 PCR，实现可靠的 DNA 走查。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-22 DOI: 10.1016/j.ab.2024.115697

Rongrong Wang , Yinwei Gu , Hong Chen , Bingkun Tian , Haixing Li

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

基于 PCR 的 DNA 走查对于捕获未知的侧翼基因组序列非常有效。在此，我们设计了一种具有良好特异性的尿嘧啶碱基 PCR（UB-PCR）来进行 DNA 扩增。初级 UB-PCR 将胸腺嘧啶碱基替换为尿嘧啶碱基，从而产生由 U-DNA 组成的初级 PCR 产物。然后，以四个正常碱基（腺嘌呤、胸腺嘧啶、胞嘧啶和鸟嘌呤）为底物，对主 PCR 产物进行单引物（主嵌套序列特异性引物）单循环扩增。显然，只有那些至少一边以主嵌套序列特异性引物为末端的 U-DNA 才能产生相应的正常单链。接下来，单循环产物经过尿嘧啶-DNA 糖基化酶处理，以破坏 U-DNA，而正常的单链则不受影响。之后，进行二次甚至三次 PCR，以专门富集目标产物。通过获得与三个选定基因位点相邻的未知序列，检验了 UB-PCR 的可行性。

引用次数: 0

Electrochemical biosensor based on copper sulfide/reduced graphene oxide/glucose oxidase construct for glucose detection 基于硫化铜/还原氧化石墨烯/葡萄糖氧化酶构建的电化学生物传感器用于葡萄糖检测。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-22 DOI: 10.1016/j.ab.2024.115696

Yuchen Zhang , Jiangnan Chen , Huifang Wang , Xianghua Gao , Baolong Niu , Wenfeng Li , Hong Wang

Due to the current increase in the number of people suffering from diabetes worldwide, how to monitor the blood glucose level in the human body has become an urgent problem to be solved nowadays. The electrochemical sensor method can be used for real-time glucose monitoring due to its advantages of real-time monitoring capability and high sensitivity. Reduced graphene oxide (rGO) has great potential for application in the field of sensors due to its advantages of large specific surface area, high stability, and good electrical and thermal conductivity. Meanwhile, the synergistic effect between two-dimensional transition metal sulfides and graphene can improve the electrochemical performance of materials due to their similar mechanical flexibility and strength. This article uses flake graphite, copper sulfate, and glucose oxidase (GOx) as raw materials to prepare CuS/rGO/GOx/GCE electrodes, and explores the performance of electrode electrocatalysis for glucose. The results showed that the prepared sensor was characterized by a low detection limit (1.75 nM) and a wide linear range (0.1–100 mM) for glucose detection, displaying a good overall detection performance, and its sensing mechanism and dynamic process were also investigated. In addition, the sensor has outstanding selectivity, anti-interference, repeatability, reproducibility and practicality.

由于目前全球糖尿病患者人数的增加，如何监测人体血糖水平已成为当前亟待解决的问题。电化学传感器方法具有实时监测能力强、灵敏度高等优点，可用于葡萄糖的实时监测。还原氧化石墨烯（rGO）具有比表面积大、稳定性高、导电导热性好等优点，在传感器领域具有很大的应用潜力。同时，由于二维过渡金属硫化物与石墨烯具有相似的机械柔韧性和强度，二者之间的协同效应可以提高材料的电化学性能。本文以鳞片石墨、硫酸铜和葡萄糖氧化酶（GOx）为原料制备了 CuS/rGO/GOx/GCE 电极，并探讨了电极对葡萄糖的电催化性能。结果表明，所制备的传感器对葡萄糖的检测具有检出限低（1.75 nM）、线性范围宽（0.1-100 mM）的特点，显示出良好的整体检测性能，并对其传感机理和动态过程进行了研究。此外，该传感器还具有突出的选择性、抗干扰性、重复性、再现性和实用性。

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引用次数: 0

Impact of Lectin biotinylation for surface plasmon resonance and enzyme-linked Lectin assays for protein glycosylation 表面等离子共振和酶联凝集素检测蛋白质糖基化的凝集素生物素化的影响。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-19 DOI: 10.1016/j.ab.2024.115693

Benjamin Serafin , Amine Kamen , Gregory De Crescenzo , Olivier Henry

Lectins are widely employed for the assessment of protein glycosylation as their carbohydrate binding specificities have been well characterized. In glycosylation assays, lectins are often conjugated with biotin tags, which interact with streptavidin to functionalize biosensing surfaces or recruit signal generating molecules, depending on the assay configuration. We here demonstrate that a high degree of biotin conjugation can limit total capture to streptavidin functionalized SPR surfaces due to multipoint binding, and can additionally bias the reported kinetic evaluations when measuring the interaction between lectins and glycoproteins by SPR. For microplate assays using different configurations, high biotinylation ratios can effectively amplify the signal obtained when using Streptavidin conjugates for detection, in some cases significantly lowering the limit of detection. The cumulative results express the importance of customizing the ligand biotinylation ratios for different assay configurations, as commercially obtained pre-biotinylated lectins are not necessarily optimized for different assay configurations.

凝集素被广泛用于评估蛋白质糖基化，因为它们与碳水化合物结合的特异性已经得到了很好的表征。在糖基化检测中，凝集素通常与生物素标签连接，生物素标签与链霉亲和素相互作用，使生物传感表面功能化，或根据检测配置吸附信号生成分子。我们在此证明，由于多点结合，高度的生物素共轭会限制链霉亲和素功能化 SPR 表面的总捕获量，而且在通过 SPR 测量凝集素和糖蛋白之间的相互作用时，还会使报告的动力学评估出现偏差。对于使用不同配置的微孔板检测，当使用链霉亲和素共轭物进行检测时，高生物素化比率可有效放大获得的信号，在某些情况下可显著降低检测限。累积的结果表明了针对不同的检测配置定制配体生物素化比率的重要性，因为市售的预生物素化凝集素并不一定针对不同的检测配置进行了优化。

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引用次数: 0

The downstream purification of bispecific antibodies 双特异性抗体的下游纯化。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-18 DOI: 10.1016/j.ab.2024.115692

Qian Li , Hongyang Zhao , Xiaoying Liang, Qingquan He, Zicheng Wang, Guohong Qin, GuoZhu Li, Dan Xu

Bispecific antibodies, a class of therapeutic antibodies, can simultaneously bind to two distinct targets. Compared with monospecific antibodies, bispecific antibodies offer advantages, including superior efficacy and reduced side effects. However, because of their structural complexity, the purification of bispecific antibodies is highly challenging. The purification process must strike a delicate balance between purity and productivity, eliminating a broad spectrum of contaminants, including product-related and process-related impurities, while also maximizing the yield wherever feasible. This review systematically describes the strategies for bispecific antibody capture, the elimination of product-related impurities, and the mitigation of the formation of process-related impurities, thereby, providing guidance for the development of downstream purification processes for bispecific antibodies.

双特异性抗体是一类治疗性抗体，可同时与两个不同的靶点结合。与单特异性抗体相比，双特异性抗体具有疗效更佳、副作用更小等优点。然而，由于其结构复杂，双特异性抗体的纯化极具挑战性。纯化过程必须在纯度和产量之间取得微妙的平衡，既要剔除各种杂质，包括与产品和工艺相关的杂质，又要尽可能提高产量。本综述系统地阐述了双特异性抗体捕获、消除产品相关杂质和减少工艺相关杂质形成的策略，从而为双特异性抗体下游纯化工艺的开发提供指导。

引用次数: 0

Decreased solubility and increased adsorptivity of a biotinylated humanized anti-cocaine mAb 生物素化人源化抗柯卡因 mAb 的溶解度降低，吸附性增强。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-18 DOI: 10.1016/j.ab.2024.115690

Terence L. Kirley, Andrew B. Norman

Biotinylation of proteins, including antibodies, is a very useful and important modification for a variety of biochemical characterizations, including anti-drug antibody (ADA) assays used to detect antibodies raised against therapeutic antibodies. We assessed different degrees of biotin labeling of an anti-cocaine mAb currently under development for treating cocaine use disorder. We noted that higher levels of biotin labeling dramatically decreased mAb solubility, and increased the tendency to bind to surfaces, complicating characterization of the biotinylated antibody. Specifically, in phosphate buffered saline, labeling stoichiometries of more than about 3 biotin/mAb resulted in decreased recoveries due to increased binding to surfaces and decreased mAb solubility. Native gel agarose electrophoresis, differential scanning fluorimetry, and isothermal titration calorimetry all demonstrated changes in the mAb which became more pronounced above a labeling ratio of 3 biotin/mAb. At 3.0 biotin/mAb, there were minimal changes in solubility, adsorptivity, exposure of hydrophobic dye-binding sites, heat stability, and cocaine binding, in stark contrast to labeling with 5.6 biotin/mAb. Thus, the degree of biotinylation should be kept at about 3 biotin/mAb to maintain antigen binding and general structure, solubility, and stability of this mAb, a finding which may be important for other similar mAbs currently in use or under development.

蛋白质（包括抗体）的生物素化是一种非常有用和重要的修饰，可用于各种生化鉴定，包括用于检测针对治疗性抗体的抗药物抗体（ADA）检测。我们评估了目前正在开发用于治疗可卡因使用障碍的抗可卡因 mAb 的不同生物素标记程度。我们注意到，生物素标记程度越高，mAb 的溶解度就越低，与表面结合的倾向就越大，从而使生物素化抗体的表征变得更加复杂。具体来说，在磷酸盐缓冲盐水中，生物素/mAb 的标记比例超过 3 时，由于与表面的结合增加和 mAb 溶解度降低，回收率会下降。原生凝胶琼脂糖电泳、差示扫描荧光测定法和等温滴定量热法都显示了 mAb 的变化，当生物素/mAb 的标记比率超过 3 时，这种变化会更加明显。在生物素/mAb 为 3.0 时，溶解度、吸附性、疏水性染料结合位点的暴露、热稳定性和可卡因结合力的变化都很小，这与生物素/mAb 为 5.6 时的标记形成了鲜明对比。因此，生物素化程度应保持在 3 生物素/mAb 左右，以维持这种 mAb 的抗原结合力、一般结构、溶解性和稳定性。

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引用次数: 0

Analysis of human milk oligosaccharides from women with gestational diabetes mellitus 分析妊娠糖尿病妇女的母乳低聚糖。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-18 DOI: 10.1016/j.ab.2024.115689

Xinyue Ma , Yue Lu , Chuncui Huang , Zhendong Guo , Zheng Xiang , Huanyu Gao , Keli Zhao , Yao Zhao , Yan Li

Human milk oligosaccharides (HMOs) are bioactive components which play an important role in infant health. HMO composition is vulnerable to changes of maternal conditions including lactation stages and maternal phenotypes. Pregnant diseases such as gestational diabetes mellitus (GDM) are commonly found in women during pragnancy, and may cause disorder in maternal physiological metabolism which is harmful to infants. Unfortunately, anlysis of oligosaccharides from women with GDM is limited. To address this issue, we analyzed HMO compositions and profiles in breast milk from women with GDM using an established 96-well plate permethylation platform and MALDI-TOF-MS. We enrolled 127 women with GDM, and investigated HMO abundances in colostrum, transition milk, and mature milk respectively. We found that GDM affected HMO compositions in breast milk, and the level of fucosylation became higher over the course of lactation for all the women with GDM. Interestingly, the relative abundances of fucosylated HMOs in different lactation stages were affected differentially by GDM, with the most pronounced effect in colostrum. In particular, the relative abundances of H3N1F1 and H3N1F2 sharply decreased over time, showing very low levels in late lactation. These differences in our findings need further investigation to develop optimal feeding for mothers with GDM.

母乳低聚糖（HMO）是一种生物活性成分，对婴儿健康起着重要作用。人乳寡糖的组成易受母体状况（包括哺乳阶段和母体表型）变化的影响。妊娠期疾病如妊娠糖尿病（GDM）常见于孕产妇，可能导致母体生理代谢紊乱，对婴儿造成危害。遗憾的是，对妊娠期糖尿病妇女体内低聚糖的分析十分有限。为了解决这个问题，我们使用已建立的 96 孔板过甲基化平台和 MALDI-TOF-MS 分析了 GDM 妇女母乳中的 HMO 成分和特征。我们招募了 127 名 GDM 妇女，分别研究了初乳、过渡乳和成熟乳中的 HMO 丰度。我们发现，GDM 影响了母乳中的 HMO 组成，所有 GDM 妇女在哺乳期的岩藻糖基化水平都会升高。有趣的是，GDM 对不同泌乳阶段的岩藻糖基化 HMO 的相对丰度有不同的影响，其中初乳的影响最为明显。特别是，H3N1F1 和 H3N1F2 的相对丰度随着时间的推移急剧下降，在泌乳后期的水平非常低。我们的研究结果中的这些差异需要进一步研究，以便为患有 GDM 的母亲制定最佳喂养方式。

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引用次数: 0

Chemical composition alterations in rat brain hypothalamus induced by irisin administration using spectroscopic and machine learning techniques 利用光谱学和机器学习技术研究施用鸢尾素诱导的大鼠大脑下丘脑化学成分变化

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-16 DOI: 10.1016/j.ab.2024.115687

Zozan Guleken , Huri Dedeakayoğulları , Esra Kutlu , Zeynep Ceylan , Joseph Cebulski , Joanna Depciuch

This study employed Fourier transform infrared (FTIR) spectroscopy to determine the chemical composition of brain tissues and the changes induced by irisin at doses of 50 mg and 100 mg. Brain tissues were collected from control rats and those administered with irisin, and key vibrational peaks were analyzed. In the 50 mg irisin group, all described vibrations decreased compared to control tissues, while the 100 mg group showed a decrease only in lipid vibrations. Comparatively, the 50 mg group had lower absorbance of phospholipids, amides, and lipid functional groups than the 100 mg group. Lower amounts of these compounds were found in treated tissues compared to controls, with higher levels in the 100 mg group. Ratios between amide peaks revealed significant differences between groups. Principal component analysis (PCA) differentiated control and irisin-treated tissues, primarily using PC1 and PC3. The decision tree model exhibited high classification accuracy, especially in the 800–1800 cm⁻¹ range, with high sensitivity and specificity. FTIR spectroscopy effectively highlighted chemical changes in brain tissues due to irisin, demonstrating dose-dependent variations. The combination of PCA, ROC analysis, and decision tree modeling underscored the potential of FTIR spectroscopy for studying the biochemical effects of compounds like irisin.

本研究采用傅立叶变换红外光谱（FTIR）测定脑组织的化学成分以及鸢尾素在 50 毫克和 100 毫克剂量下引起的变化。采集了对照组大鼠和服用鸢尾素组大鼠的脑组织，并对关键振动峰进行了分析。与对照组相比，50 毫克鸢尾素组的所有描述振动都有所下降，而 100 毫克组只显示脂质振动有所下降。与 100 毫克组相比，50 毫克组的磷脂、酰胺和脂质官能团的吸光度较低。与对照组相比，处理组组织中这些化合物的含量较低，而 100 毫克组的含量较高。酰胺峰之间的比率显示组间存在显著差异。主成分分析（PCA）主要利用 PC1 和 PC3 对对照组和鸢尾素处理组的组织进行区分。决策树模型显示出较高的分类准确性，尤其是在 800-1800 cm-1 范围内，具有较高的灵敏度和特异性。傅立叶变换红外光谱有效地突出了鸢尾素引起的脑组织化学变化，并显示出剂量依赖性变化。PCA、ROC分析和决策树模型的结合强调了傅立叶红外光谱在研究鸢尾素等化合物的生化效应方面的潜力。

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引用次数: 0

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time 用于实时监测蛋白质水解的新型简易荧光蛋白酶测定法

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-16 DOI: 10.1016/j.ab.2024.115688

Miha Bahun , Nataša Poklar Ulrih

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

测量蛋白酶的活性对于研究这些酶的生理功能和商业应用至关重要。与众多基于发色性或荧光性肽底物的蛋白酶检测方法相比，目前还缺乏实时监测蛋白质降解的方法。在这里，我们报告了一种蛋白酶检测方法，它采用 SYPRO Orange 作为荧光探针来跟踪蛋白水解过程。我们使用四种不同的蛋白质底物，对两种热稳定性不同的亚铁酶进行了测定，证明了该测定法的功能。该检测方法与实时 PCR 仪器兼容，即使在高温条件下也能对低容量样品进行连续荧光测量。因此，该检测方法特别适用于对热稳定性蛋白酶进行高通量表征。

引用次数: 0

Photoelectrochemical biosensors: Prospects of graphite carbon nitride-based sensors in prostate-specific antigen diagnosis 光电化学生物传感器：基于氮化石墨的传感器在前列腺特异性抗原诊断中的应用前景。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-10 DOI: 10.1016/j.ab.2024.115686

Seyed Saman Nemati, Gholamreza Dehghan

Prostate cancer (PC) is very common in old age and causes many deaths. Early diagnosis and monitoring of the progress of the disease and the effectiveness of PC treatment are critical. On the other hand, choosing a specific biomarker for PCs is essential. Prostate-specific antigen (PSA) is a specific biomarker secreted in the prostate epithelial cells, which increases in cancer cells. Between all employed sensing mechanism, electrochemical sensors based on nanomaterials have created many hopes. Meanwhile, graphite carbon nitride (g-C₃N₄) is interested in developing photoelectrochemical sensors due to its large surface area, stability, easy modification, and good photoelectronic properties. In this review, electrochemical sensors based on nanocomposites containing g-C₃N₄ have been investigated in PSA detection. After providing an overview of the characteristics of g-C₃N₄ and cancer biomarkers, it reviews the strategies and mechanisms involved in identifying PSA. Different approaches to photoelectrochemistry, impedimetric immunosensors, photocatalysis, and luminescence have been used in diagnostic mechanisms. Then, challenges and prospects for electrochemical sensors based on nanocomposites containing g-C₃N₄ in PSA detection have been analyzed. The recent review generally opens an efficient view in PSA diagnosis and the application of g–C₃N₄–based electrochemical sensors in personalized medicine diagnosis and treatment.

前列腺癌（PCs）在老年人中非常常见，并导致许多人死亡。早期诊断、监测疾病进展和癌症治疗效果至关重要。另一方面，选择前列腺癌的特异性生物标记物也至关重要。前列腺特异性抗原（PSA）是前列腺上皮细胞分泌的一种特异性生物标志物，在癌细胞中会增加。在所有采用的传感机制中，基于纳米材料的电化学传感器给人们带来了许多希望。与此同时，氮化石墨（g-C3N4）因其比表面积大、稳定性好、易于改性和良好的光电特性而在光电化学传感器的开发中备受关注。本综述研究了基于含 g-C3N4 纳米复合材料的电化学传感器在 PSA 检测中的应用。在概述了 g-C3N4 和癌症生物标记物的特性之后，本综述回顾了识别 PSA 所涉及的策略和机制。诊断机制采用了光电化学、阻抗免疫传感器、光催化和发光等不同方法。然后，分析了基于含 g-C3N4 纳米复合材料的电化学传感器在 PSA 检测中面临的挑战和前景。最近的综述总体上为 PSA 诊断以及基于 g-C3N4 的电化学传感器在个性化医学诊断和治疗中的应用开辟了一条有效的途径。

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引用次数: 0

Urea biosensors based in zeolites and chalcogenide-oxide semiconductor thin films as active materials: A review 以沸石和氧化镓半导体薄膜为活性材料的尿素生物传感器：综述。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-10-05 DOI: 10.1016/j.ab.2024.115685

Manuel A.Chairez Ortega , Rafael E. González Landaeta , Francisco S. Aguirre Tostado , Soledad V. Torres Argüelles , Amanda Carrillo Castillo

Diagnosis of renal failure by measuring urea levels has been a topic of intense study in recent years. A major focus has been on improving the sensitivity, linearity, precision, accuracy, and selectivity of biosensors for measuring urea. Although various materials have been used in the fabrication of urea biosensors, ceramics, and chalcogenides have been less explored in this field. Recently, the use of ceramics such as zeolite has been investigated to improve enzyme immobilization methods in urea biosensors and their application in ion-selective membranes, to increase the specificity of the devices. While oxides have been widely used as transducers in urea biosensors, chalcogenide semiconductor materials from Group VI of the periodic table also show promising properties, such as chemical stability, to signal transduction capability, and improved electrical measurements. This review provides a comprehensive overview of recent research in urea biosensors, with a special emphasis on the use of ceramics for enzyme immobilization and chalcogenides as transducers and how these materials contribute to improving the performance of these devices.

近年来，通过测量尿素水平来诊断肾功能衰竭一直是一个热门研究课题。研究的重点是提高尿素生物传感器的灵敏度、线性度、精确度、准确性和选择性。虽然尿素生物传感器的制造中使用了多种材料，但陶瓷和钙化物在这一领域的应用较少。最近，人们研究了陶瓷（如沸石）的使用，以改进尿素生物传感器中的酶固定方法及其在离子选择膜中的应用，从而提高设备的特异性。虽然氧化物已被广泛用作尿素生物传感器中的传感器，但元素周期表第 VI 族的铬化半导体材料也显示出良好的性能，如化学稳定性、信号传导能力和改进的电学测量。本综述全面概述了尿素生物传感器的最新研究成果，特别强调了使用陶瓷固定酶和铬化物作为换能器的情况，以及这些材料如何有助于提高这些设备的性能。

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引用次数: 0