IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-31 DOI: 10.1039/d4an00576g

Yiming Wang, JinTao He, Rong Feng, Jingwen Chen, Gege Xie, Shengrong Yu, Yongxiang Wu, Keqi Tang

Persistent luminescent nanoparticles (PLNPs) are an excellent luminescent material, and near-infrared PLNPs are efficiently applied for biosensing and bioimaging due to its advantages of no excitation, excellent light stability and long afterglow. However, due to interference from the complex environment within organisms, single-mode imaging methods often face limitations in selectivity, sensitivity, and accuracy. Therefore, it is desirable to construct a dual-mode imaging probe strategy with higher specificity and sensitivity for bioimaging. Magnetic resonance imaging (MRI) has been widely used in the field of bioimaging due to its advantages of high resolution, non-radiation and non-invasive. Here, by combing the near-infrared PLNPs and manganese dioxide (MnO2) nanosheets, a sensitive and convenient dual-mode “turn on” bioimaging nanoprobe ZGC@MnO2 has been developed for long afterglow imaging and MRI of endogenous hydrogen peroxide (H2O2) in the tumor microenvironment (TME). The monitoring of H2O2 has garnered significant attention due to its crucial role in human pathologies. For the dual-mode “turn on” bioimaging nanoprobe, the near-infrared PLNPs of quasi-spherical ZnGa2O4: Cr (ZGC) nanoparticles were synthesized as luminophores, and MnO2 nanosheets were utilized as fluorescent quencher, carrier and H2O2 recognizer. H2O2 in the TME could reduce MnO2 nanosheets to Mn2+ for MRI, and ZGC nanoparticles was released for long afterglow imaging. Finally, ZGC@MnO2 nanoprobe demonstrated rapid response, excellent signal-to-noise ratio and a limit of detection of 3.67 nM for endogenous H2O2 in the TME. This dual-mode approach enhances the detection sensitivity of endogenous H2O2, thereby facilitating the research of endogenous H2O2-associated diseases and clinical diagnostics.

持久性发光纳米粒子（PLNPs）是一种优良的发光材料，近红外PLNPs因其无需激发、光稳定性好、余辉长等优点被有效地应用于生物传感和生物成像。然而，由于受到生物体内复杂环境的干扰，单模成像方法往往在选择性、灵敏度和准确性方面受到限制。因此，人们希望构建一种特异性和灵敏度更高的双模成像探针策略，用于生物成像。磁共振成像（MRI）具有高分辨率、无辐射、无创伤等优点，已被广泛应用于生物成像领域。本文将近红外聚合纳米粒子（PLNPs）与二氧化锰（MnO2）纳米片相结合，开发出一种灵敏、便捷的双模式 "开启 "生物成像纳米探针 ZGC@MnO2，用于肿瘤微环境（TME）中内源性过氧化氢（H2O2）的长余辉成像和磁共振成像。由于 H2O2 在人类病理中的关键作用，对其进行监测已引起了广泛关注。为了实现双模式 "开启 "生物成像纳米探针，研究人员合成了近红外PLNPs准球形ZnGa2O4：Cr（ZGC）纳米颗粒作为发光体，并利用MnO2纳米片作为荧光淬灭剂、载体和H2O2识别器。TME中的H2O2可将MnO2纳米片还原成Mn2+，用于核磁共振成像，而ZGC纳米颗粒则被释放出来，用于长余辉成像。最后，ZGC@MnO2 纳米探针反应迅速，信噪比极佳，对 TME 中内源性 H2O2 的检测限为 3.67 nM。这种双模式方法提高了内源性 H2O2 的检测灵敏度，从而促进了内源性 H2O2 相关疾病的研究和临床诊断。

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引用次数: 0

Identification of new AAV vectors with enhanced blood-brain barrier penetration efficiency via organ-on-a-chip 通过器官芯片鉴定可提高血脑屏障穿透效率的新型 AAV 向量

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-30 DOI: 10.1039/d4an00404c

Mengmeng Li, Yujian Zhong, Mingyang Zhu, Chunjin Pang, Lu Xiao, Ye Bu, Huapeng Li, Yong Diao, Chaoyong Yang, Dan Liu

To overcome limitations in generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, that showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to 5 times higher titer, while AAV-M8 reached 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. Transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell type specificity of virus vectors.

为了克服目前AAV载体在通用性和效率方面的局限性，在本研究中，我们通过在AAV9囊膜基因的受体结合域插入随机七肽序列，构建了一个AAV变体库。然后，我们应用最近开发的人体血脑屏障（BBB）体外器官芯片模型，反复富集能有效穿越BBB并转导星形胶质细胞的变体。通过多轮筛选，我们获得了两个候选 AAV 变体：AAV-M6 和 AAV-M8，它们的 BBB 穿透效率明显高于 AAV9 或 AAV-PHP.eB。定量 PCR（qPCR）检测显示，AAV-M6 在模型神经室内的累积滴度是 AAV-PHP.eB 的 5 倍，而 AAV-M8 则是 AAV-PHP.eB 的 3 倍。转导实验进一步验证了AAV-M6候选载体能够在穿过BBB后感染HA-1800细胞，这表明它有可能在穿过hCMEC/D3层后以比AAV-PHP.eB更高的效率转导脑实质细胞。分子模拟表明，人类受体蛋白 LY6D 和 M6PR 可以高亲和力结合 AAV-M6 七肽插入物。这项研究提供了两种有希望的候选 AAV 载体，并证明了这种体外 BBB 模型可用于基因疗法的可扩展高通量筛选。这些工具可以推动对 BBB 通透性和病毒载体的细胞类型特异性机制的研究。

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引用次数: 0

Study of a Microwave Induced Plasma as a Universal Ion Source for Inorganic and Organic Mass Spectrometry 微波诱导等离子体作为无机和有机质谱分析通用离子源的研究

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-30 DOI: 10.1039/d4an00356j

Marc-Aurèle Boillat, Peter C Hauser

A proof-of-concept study of the utilization of a single mass spectrometer for qualitative molecular analysis as well as for quantitative metal determination is described. This was performed with an argon microwave plasma as ion source coupled to an ion trap mass spectrometer. A microwave induced plasma with tuneable power and gas flow rate was loaded with dried nebulized sample solutions. The commercially available plasma torch was slightly modified to allow the introduction of the sample in different regions of the plasma. Using soft settings, organic compounds introduced in the plasma plume could be detected as protonated molecular ions. Under harsher conditions, elemental analysis was demonstrated for several metals. Lead could be determined with a limit of detection of 150 nM. Minor on-the-fly adjustments of the power, flow rate and sampling distance allowed a mild fragmentation of organic compounds. Ionization differences observed were rationalized by emission spectroscopy and excitation and rotational temperatures were determined. Simultaneous determination of elemental and molecular information was demonstrated for a rubidium-crown-ether complex. The maximum argon gas consumption for these tasks was below 2 L·min-1 and the maximal power used was 90 W.

介绍了利用单一质谱仪进行定性分子分析和定量金属测定的概念验证研究。该研究使用氩微波等离子体作为离子源，并将其与离子阱质谱仪耦合。在功率和气体流速可调的微波诱导等离子体中装入干燥的雾化样品溶液。对市场上销售的等离子体炬稍作改动，以便在等离子体的不同区域引入样品。使用软设置时，等离子体羽流中引入的有机化合物可作为质子化分子离子被检测到。在更苛刻的条件下，对几种金属进行了元素分析。铅的检测限为 150 nM。对功率、流速和取样距离进行微小的即时调整，可对有机化合物进行温和的破碎。所观察到的电离差异通过发射光谱进行了合理解释，并确定了激发和旋转温度。对铷-冠-醚复合物进行了元素和分子信息的同步测定。这些任务的最大氩气消耗量低于 2 L-min-1，使用的最大功率为 90 W。

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引用次数: 0

Forced intercalation-induced light-up peptides as fluorogenic indicators for HIV-1 TAR RNA-ligand assay 将强制插层诱导发光肽作为 HIV-1 TAR RNA 配体检测的荧光指示剂

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-29 DOI: 10.1039/d4an00530a

En Ting Tabitha Lee, Yusuke Sato, Akunna F Ujuagu, Seiichi Nishizawa

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP keeps strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between “competitive” and “noncompetitive” inhibitors when used in fluorescence indicator displacement (FID) assay. The FID assay at stringent screening condition is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

能够与人类免疫缺陷病毒-1（HIV-1）反式激活应答（TAR）RNA结合的荧光指示剂是探索性研究抗 HIV 候选药物鉴定的有力工具。本研究基于噻唑橙（TO）染料（FIT）的强制插层，提出了一种利用转录激活因子（Tat）衍生多肽设计荧光指示剂的新策略。所开发的 9 聚体 FIT 肽（RKKRR-TO-RRR：命名为 FiLuP）的特点是将 TO 单元集成到 Tat 肽序列中间的 Dap（2,3-二氨基丙酸）残基上；Tat 肽（RKKRR-Q-RRR）中的 Q（谷氨酸）残基被 TO 取代，就像氨基酸替代物一样。这有助于在与 TAR RNA 结合时产生明显的光亮反应（λem = 541 nm 时为 450 倍，Φfree = 0.0057，Φbound = 0.61）。与其他非识别 RNA 相比，FiLuP 对 TAR RNA 的反应具有高度选择性，并且 FiLuP 保持着很强的结合亲和力（Kd = 1.0 ± 0.6 nM）。值得注意的是，与之前开发的基于 Tat 肽的 FRET 探针相比，FiLuP 在荧光指示剂位移（FID）测定中能够区分 "竞争性 "和 "非竞争性 "抑制剂。在严格的筛选条件下进行 FID 检测，还能筛选出对 TAR RNA 具有超强竞争性的结合剂。

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引用次数: 0

Magneto-Electrochemical Method for Chiral Recognition of Amino Acid Enantiomers 手性识别氨基酸对映体的磁电化学方法

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-27 DOI: 10.1039/d4an00547c

Yueqian Jia, Wubin Wu, Rui Chen, Hong Wang, Chuang Zhang, Lili Chen, Jiannian Yao

Chiral recognition of enantiomers with identical mirror-symmetric molecular structures is important for the analysis of biomolecules, and it conventionally relies on stereoselective interactions in chiral chemical environments. Here, we develop a magneto-electrochemical method for the enhanced detection of chiral amino acids (AAs), that combines the advantages of the high sensitivity of electrochemiluminescence (ECL) biosensors and chirality-induced effects under magnetic field. The ECL difference between L- and D-enantiomers can be amplified by over 35-fold under a field of 3.5 kG, and the chiral discrimination can be achieved in dilute AA solutions down to nM level. The field-dependent ECL and chronocoulometry measurements suggest that chiral AAs can lock the spins on their radicals and thus enlarge the ECL change under applied magnetic field (magneto-ECL, MECL), which explains the field-enhanced chiral discrimination of AA enantiomers. Finally, a detailed protocol is demonstrated for the identification of unknown AA solutions, in which the species, chirality and concentration of AAs can be determined simultaneously from the 2D plots of the ECL and MECL results. This work benefits the development of field-assisted detection methods and represents a promising and universal strategy for the comprehensive analysis of chiral biomolecules.

对具有相同镜像对称分子结构的对映体进行手性识别对于生物大分子的分析非常重要，而这通常依赖于手性化学环境中的立体选择性相互作用。在这里，我们开发了一种增强检测手性氨基酸（AAs）的磁电化学方法，该方法结合了电化学发光（ECL）生物传感器的高灵敏度和磁场下手性诱导效应的优点。在 3.5 kG 的磁场下，L-对映体和 D-对映体之间的 ECL 差异可放大 35 倍以上，在稀释 AA 溶液中可实现低至 nM 水平的手性鉴别。与磁场相关的 ECL 和计时器测量结果表明，手性 AA 可将自旋锁定在其自由基上，从而扩大了外加磁场下的 ECL 变化（magneto-ECL，MECL），这就解释了为什么 AA 对映体的手性鉴别在磁场中得到了增强。最后，还展示了用于鉴定未知 AA 溶液的详细方案，其中 AA 的种类、手性和浓度可同时从 ECL 和 MECL 结果的二维图中确定。这项工作有利于现场辅助检测方法的发展，是全面分析手性生物大分子的一种前景广阔的通用策略。

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引用次数: 0

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii† 在 Komagataella Phaffii 中表达的重组人乳铁蛋白的表征

IF 3.6 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-27 DOI: 10.1039/D4AN00333K

Xiaoning Lu, Chad Cummings, Udodili A. Osuala, Neela H. Yennawar, Kevin E. W. Namitz, Brittney Hellner, Pamela B. Besada-Lombana, Ross D. Peterson and Anthony J. Clark

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

本研究首次对在酵母系统中以工业规模表达的 Helaina 重组人乳铁蛋白（rhLF，Effera™）进行了全面鉴定。蛋白质组分析证实，其氨基酸序列与原生人乳铁蛋白相同。在三个已知的糖基化位点（即天冬酰胺-156、-497 和 -642）检测到了 N-连接的聚糖，这些聚糖主要是具有五到九个甘露糖的低聚甘露糖结构。微流体调制光谱显示，Helaina rhLF 的蛋白质二级结构与人乳乳铁蛋白（hmLF）几乎相同。小角 X 射线散射（SAXS）和分析超速离心分析的结果证实，与 hmLF 一样，海拉娜 rhLF 在溶液中也显示出折叠良好的球状结构。通过SAXS分析获得的海莲娜rhLF的重建溶剂包膜与所报道的铁结合原生hmLF的晶体结构非常吻合。通过差示扫描量热法研究海拉娜 rhLF 的热稳定性，发现其变性温度分别为 68.7±0.9 ℃ 和 91.9±0.5 ℃，与在 apo- 和 holo-hmLF 中观察到的变性温度一致。总体而言，海莲娜rhLF与hmLF所拥有的N-聚糖不同；然而，表征结果证实，海莲娜rhLF纯度很高，其球状结构与hmLF十分相似。

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引用次数: 0

Label-Free Liquid Crystal-Based Optical Detection of Norfloxacin using an Aptamer Recognition Probe in Soil and Lake Water 基于无标签液晶的诺氟沙星光学检测--使用土壤和湖水中的色素识别探针

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-25 DOI: 10.1039/d4an00236a

SAYANI DAS, Soma Sil, Santanu Kumar Pal, Przemysław Kula, Susanta Sinha Roy

Norfloxacin(NOX),a broad spectrum fluoroquinolone (FQ) antibiotic, is commonly detected in environmental residues, potentially contributing to biological drug resistance. In this paper, an aptamer recognition probe has been used to develop a label-free liquid crystal-based biosensor for simple and robust optical detection of NOX in aqueous solutions. Stimuli-receptive liquid crystals (LCs) have been employed to report aptamer-target binding events at the LC-aqueous interface. The homeotropic alignment of LCs at aqueous-LC interface is due to the self-assembly of the cationic surfactant Cetyltrimethylammonium bromide (CTAB). In the presence of a negatively charged NOX aptamer, the ordering changes to planar/tilted. On addition of NOX, the aptamer-NOX binding causes redistribution of CTAB at LC-aqueous interface and the homeotropic orientation is restored. This results in a bright-to-dark optical transition under a polarized optical microscope (POM). This optical transition serves as a visual indicator to mark the presence of NOX. The devised aptasensor demonstrates high specificity with a minimum detection limit of 5 nM (1.596 ppb). Moreover, the application of the developed aptasensor for the detection of NOX in freshwater and soil samples underscores its practical utility in environmental monitoring. This proposed LC-based method offers several advantages over conventional detection techniques for a rapid, feasible and convenient way to detect Norfloxacin.

诺氟沙星（NOX）是一种广谱氟喹诺酮类（FQ）抗生素，通常在环境残留物中检出，有可能导致生物耐药性。本文利用一种适配体识别探针开发了一种基于液晶的无标记生物传感器，用于对水溶液中的 NOX 进行简单、稳健的光学检测。采用刺激感受性液晶（LC）来报告液晶-水界面上的适配体-目标结合事件。液晶在水-液晶界面的各向同性排列是由于阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）的自组装。在带负电荷的 NOX 合体存在的情况下，排序变为平面/倾斜。加入 NOX 后，aptamer-NOX 结合会导致 CTAB 在 LC-水界面重新分布，从而恢复顺向取向。这导致在偏振光学显微镜（POM）下出现由亮到暗的光学转变。这种光学转变可作为标记 NOX 存在的视觉指标。所设计的适配传感器具有很高的特异性，最低检测限为 5 nM（1.596 ppb）。此外，将所开发的灵敏传感器用于淡水和土壤样品中 NOX 的检测，也凸显了其在环境监测中的实用性。与传统的检测技术相比，这种基于液相色谱法的方法具有多种优势，可快速、可行、方便地检测诺氟沙星。

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引用次数: 0

Super-assembled periodic mesoporous organosilica membranes with hierarchical channels for efficient glutathione sensing† 具有分层通道的超组装周期性介孔有机硅膜，用于高效谷胱甘肽传感

IF 3.6 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-24 DOI: 10.1039/D4AN00559G

Hui Zeng, Shan Zhou, Xin Zhang, Qirui Liang, Miao Yan, Yeqing Xu, Yaxin Guo, Xiaomeng Hu, Lei Jiang and Biao Kong

Bioinspired nanochannel-based sensors have elicited significant interest because of their excellent sensing performance, and robust mechanical and tunable chemical properties. However, the existing designs face limitations due to material constraints, which hamper broader application possibilities. Herein, a heteromembrane system composed of a periodic mesoporous organosilica (PMO) layer with three-dimensional (3D) network nanochannels is constructed for glutathione (GSH) detection. The unique hierarchical pore architecture provides a large surface area, abundant reaction sites and plentiful interconnected pathways for rapid ionic transport, contributing to efficient and sensitive detection. Moreover, the thioether groups in nanochannels can be selectively cleaved by GSH to generate hydrophilic thiol groups. Benefiting from the increased hydrophilic surface, the proposed sensor achieves efficient GSH detection with a detection limit of 1.2 μM by monitoring the transmembrane ionic current and shows good recovery ranges in fetal bovine serum sample detection. This work paves an avenue for designing and fabricating nanofluidic sensing systems for practical and biosensing applications.

基于生物启发的纳米通道传感器因其卓越的传感性能、坚固的机械性能和可调化学特性而备受关注。然而，由于材料的限制，现有的设计面临着局限性，阻碍了更广泛的应用可能性。在此，我们构建了一种由周期性介孔有机硅（PMO）层和三维（3D）网络纳米通道组成的异膜系统，用于谷胱甘肽（GSH）的检测。独特的分层孔隙结构提供了大表面积、丰富的反应位点和大量相互连接的通道，有利于离子的快速传输，从而实现高效、灵敏的检测。此外，纳米通道中的硫醚基团可被 GSH 选择性地裂解，生成亲水性硫醇基团。得益于亲水性表面的增加，所提出的传感器通过监测跨膜离子电流实现了高效的 GSH 检测，检测限为 1.2 μM，并在胎牛血清样品检测中显示出良好的回收范围。这项工作为设计和制造用于实际生物传感应用的纳米流体传感系统铺平了道路。

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引用次数: 0

Mobility capillary electrophoresis-native mass spectrometry reveals dynamic conformational equilibrium of calmodulin and its complexes 移动毛细管电泳-原位质谱法揭示钙调蛋白及其复合物的动态构象平衡

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-24 DOI: 10.1039/d4an00378k

Yi Zhao, Wenjing Zhang, Jie Hong, Lei Yang, Yuanyuan Wang, Feng Qu, Wei Xu

Benefitting from the rapid evolution of artificial intelligence and structural biology, an expanding collection of high-resolution protein structures has greatly improved our understanding of protein functions. Yet, proteins are inherently flexible, these static structures can only offer limited snapshots of their true dynamic nature. The conformational and functional changes of calmodulin(CaM) induced by Ca2+ binding have always been a focus of research. In this study, conformational dynamics of CaM and its complexes were investigated using a mobility capillary electrophoresis (MCE) and native mass spectrometry (native MS) based method. By analyzing the ellipsoidal geometries of CaM in solution phase at different Ca2+ concentrations, it is interesting to discover that CaM molecules, whether bound to Ca2+ or not, possess both a closed and open conformation. Moreover, each individual CaM molecule actively “jumps” (equilibrium exchange) between these two distinct conformations on a timescale ranging from milli- to micro-seconds. The binding of Ca2+ ions did not affect the structure dynamics of CaM; while the binding of a peptide ligand would stabilize CaM, leading to the observation of a single, compact conformation of the resulting protein complex. A target recognition mechanism was also proposed based on these new findings, suggesting that CaM's interaction with targets may favor a conformational selection model. This enriches our understanding of the binding principles between CaM and its numerous targets.

得益于人工智能和结构生物学的快速发展，高分辨率蛋白质结构的收集范围不断扩大，大大提高了我们对蛋白质功能的认识。然而，蛋白质本身是灵活的，这些静态结构只能提供其真实动态性质的有限快照。钙调蛋白（CaM）在 Ca2+ 结合下的构象和功能变化一直是研究的重点。本研究采用基于迁移率毛细管电泳（MCE）和原生质谱（native MS）的方法研究了 CaM 及其复合物的构象动态。通过分析不同 Ca2+ 浓度下 CaM 在溶液相中的椭圆形几何结构，我们发现 CaM 分子无论是否与 Ca2+ 结合，都同时具有封闭和开放两种构象。此外，每个 CaM 分子都会在这两种不同构象之间主动 "跳跃"（平衡交换），时间尺度从毫秒到微秒不等。Ca2+ 离子的结合不会影响 CaM 的结构动态；而肽配体的结合则会稳定 CaM，从而观察到由此产生的蛋白质复合物具有单一、紧凑的构象。根据这些新发现还提出了一种目标识别机制，表明 CaM 与目标的相互作用可能有利于构象选择模型。这丰富了我们对 CaM 与其众多靶标之间结合原理的理解。

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引用次数: 0

Reagentless molecularly imprinted polymers-based electrochemical biosensor for single-step detection of Troponin I in biofluids 基于无残留分子印迹聚合物的电化学生物传感器，用于单步检测生物流体中的肌钙蛋白 I

IF 4.2 3区化学 Q1 Chemistry

Analyst

Pub Date : 2024-05-23 DOI: 10.1039/d4an00247d

Mahmoud Ayman Saleh, Arash Khorrami Jahromi, Hamed Shieh, Roozbeh Siavash Moakhar, Carolina del Real Mata, Sara Mahshid

Reagentless molecular-imprinted polymer (MIP) electrochemical biosensors can offer the next generation of biosensing platforms for the detection of biomarkers owing to their simplicity, cost-efficacy, tunability, robustness, and accuracy. In this work, a novel combination of Prussian blue (PB), coated as an embedded redox probe on a gold working electrode (GWE), and a signal-off MIP assay has been proposed in an electrochemical format for the detection of troponin I (TnI) in biofluids. TnI is a variant exclusive to the heart muscles, and its elevated level in the bloodstream is indicative of acute myocardial infarction (AMI). The proposed lab-manufactured PB/MIP electrochemical biosensor, consisting of the simple signal-off MIP assay and the PB redox probe embedded on the GWE surface, is the first of its kind that allows for reagentless, label-free, and single-step electrochemical biosensing of proteins. The preparation steps of the biosensor were fully characterized by cyclic voltammetry (CV), atomic force microscopy (AFM), and Raman spectroscopy. Finally, the performance of the optimized biosensor was investigated through the determination of various concentrations of TnI, ranging from 10 to 100 pg/ml within 5 min, in serum and plasma with limits of detection less than 3.6 pg/ml, and evaluating the selectivity towards TnI using some relevant proteins that exist in the biofluids with higher concentrations.

无残留分子印迹聚合物（MIP）电化学生物传感器因其简便性、成本效益、可调性、稳健性和准确性，可为生物标记物的检测提供新一代生物传感平台。在这项工作中，我们提出了一种新颖的普鲁士蓝（PB）组合，在金工作电极（GWE）上涂覆作为嵌入式氧化还原探针，并以电化学形式进行信号关断 MIP 分析，用于检测生物流体中的肌钙蛋白 I（TnI）。TnI 是心肌独有的一种变体，其在血液中的水平升高表明急性心肌梗死（AMI）。实验室制造的 PB/MIP 电化学生物传感器由简单的信号关 MIP 检测和嵌入 GWE 表面的 PB 氧化还原探针组成，是首款可对蛋白质进行无试剂、无标记和单步电化学生物传感的生物传感器。该生物传感器的制备步骤通过循环伏安法（CV）、原子力显微镜（AFM）和拉曼光谱进行了全面表征。最后，通过在 5 分钟内测定血清和血浆中 10 至 100 pg/ml 不同浓度的 TnI（检测限小于 3.6 pg/ml），研究了优化生物传感器的性能，并利用生物流体中浓度较高的一些相关蛋白质评估了对 TnI 的选择性。

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引用次数: 0

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