Journal of Pharmaceutical and Biomedical Analysis Open最新文献_第4页

Comparative analysis of MS/MS search algorithms in label-free shotgun proteomics for monitoring host-cell proteins using trapped ion mobility and ddaPASEF 利用捕获离子迁移率和ddaPASEF监测宿主细胞蛋白的无标记鸟枪蛋白组学中MS/MS搜索算法的比较分析

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-07-21 DOI: 10.1016/j.jpbao.2025.100082

Somar Khalil, Michel Plisnier

Host cell proteins (HCPs) are critical quality attributes that can impact the safety, efficacy, and quality of biotherapeutics. Label-free shotgun proteomics is a vital approach for HCP monitoring, yet the choice of tandem mass spectrometry (MS/MS) search algorithms directly influences identification depth and quantification reliability. In this study, six prominent MS/MS search tools (Mascot, MaxQuant, SpectroMine, FragPipe, Byos, and PEAKS) were systematically benchmarked for their performance on complex samples spiked with isotopically labeled proteins from Chinese hamster ovary cells. The data were acquired using trapped ion mobility spectrometry and parallel accumulation–serial fragmentation in data-dependent acquisition mode. Key performance metrics, including peptide and protein identifications, data extraction precision, fold-change (FC) accuracy, linearity, and measurement trueness, were evaluated. A Bayesian modeling framework with Hamiltonian Monte Carlo sampling was employed to robustly estimate FC means and variances, alongside local false discovery rates through posterior probability calibration. Bayesian decision theory, implemented via expected utility maximization, was used to balance accuracy against posterior uncertainty and provide a probabilistic assessment of each tool’s performance. Through this cumulative analysis, variability across tools was observed: Byos and SpectroMine excelled in quantitative accuracy with minimal bias, FragPipe provided high precision and quantifiability, PEAKS offered deep protein coverage, Mascot showed strong trueness, and MaxQuant exhibited moderate identification performance with greater variability at lower spike levels. This study establishes a rigorous, data-driven framework for tool benchmarking and offers guidance for selecting MS/MS tools suited to HCP monitoring in biopharmaceutical development.

宿主细胞蛋白（HCPs）是影响生物治疗药物安全性、有效性和质量的关键质量属性。无标签霰弹枪蛋白质组学是监测HCP的重要方法，但串联质谱（MS/MS）搜索算法的选择直接影响鉴定深度和定量可靠性。在这项研究中，系统地对六个著名的MS/MS搜索工具（Mascot、MaxQuant、SpectroMine、FragPipe、Byos和PEAKS）在含有中国仓鼠卵巢细胞同位素标记蛋白的复杂样品上的性能进行了基准测试。数据采用捕获离子迁移率光谱法和数据依赖获取模式的平行累积-序列破碎法获取。关键性能指标，包括肽和蛋白质鉴定，数据提取精度，折叠变化（FC）准确性，线性度和测量准确性进行了评估。采用哈密顿蒙特卡罗抽样的贝叶斯建模框架稳健地估计FC均值和方差，并通过后验概率校准局部错误发现率。贝叶斯决策理论通过期望效用最大化实现，用于平衡准确性和后验不确定性，并提供每种工具性能的概率评估。通过这种累积分析，观察到不同工具之间的差异：Byos和SpectroMine在最小偏差的定量准确性方面表现出色，FragPipe提供高精度和可量化性，PEAKS提供深度蛋白质覆盖，Mascot具有很强的真实性，MaxQuant具有中等鉴定性能，在较低的峰值水平上具有较大的可变性。本研究建立了一个严格的、数据驱动的工具基准框架，并为选择适合生物制药开发中HCP监测的质谱/质谱工具提供指导。

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引用次数: 0

Design of experiments-assisted UPLC method for quantification of nitrosamine impurities in glimepiride and lobeglitazone sulfate: A green chemistry approach 格列美脲和硫酸洛贝列酮亚硝胺杂质定量的实验辅助超高效液相色谱法设计：绿色化学方法

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-05-05 DOI: 10.1016/j.jpbao.2025.100078

KiranKumar Chagarlamudi , Venkata Kanaka Srivani Maddala , Kumaraswamy Gandla

A robust ultra-performance liquid chromatography (UPLC) method was developed and systematically optimized using a Design of Experiments (DoE) approach for the simultaneous quantification of glimepiride, lobeglitazone sulfate, nitrosamine impurity-3 (IMP-3), and impurity-1 (IMP-1) in the marketed LOBG-G1 formulation. Critical method parameters, including organic phase composition, flow rate, and mobile phase pH, were identified through a comprehensive risk assessment and subsequently optimized using a Box-Behnken design. The final chromatographic conditions—50 % organic phase composition, a flow rate of 0.2 mL/min, and a mobile phase pH of 2.6—ensured efficient separation and quantification of all four analytes. The method was validated in accordance with ICH guidelines, demonstrating excellent linearity (r² > 0.999), high accuracy, and precision, with low relative standard deviation values. Stability studies conducted under different stress conditions revealed significant degradation of all four compounds in acidic, alkaline, and oxidative environments. Degradation products were further characterized using LC-MS/MS analysis, confirming their structural identity. In addition to its analytical performance, the method's environmental sustainability was evaluated using multiple green analytical chemistry assessment tools. The DoE-guided UPLC method offers a highly sensitive, selective, and reproducible analytical platform for the detection of nitrosamine impurities in antidiabetic drugs, providing enhanced method understanding while aligning with sustainability principles.

建立了一种高效液相色谱（UPLC）方法，并采用实验设计（DoE）方法进行了系统优化，用于同时定量上市的LOBG-G1制剂中格列美脲、硫酸洛贝列酮、亚硝胺杂质-3 （IMP-3）和杂质-1 （IMP-1）。通过综合风险评估确定了关键方法参数，包括有机相组成、流速和流动相pH，随后使用Box-Behnken设计进行了优化。最终色谱条件为有机相组成为50% %，流速为0.2 mL/min，流动相pH为2.6，确保了四种分析物的有效分离和定量。该方法按照ICH指南进行了验证，显示出良好的线性(r²>；0.999)，准确度高，精密度高，相对标准偏差值低。在不同的应激条件下进行的稳定性研究表明，这四种化合物在酸性、碱性和氧化环境下都有显著的降解。利用LC-MS/MS对降解产物进行进一步表征，确定了降解产物的结构特征。除了分析性能外，还使用多种绿色分析化学评估工具对该方法的环境可持续性进行了评估。doe指导的UPLC方法为检测抗糖尿病药物中的亚硝胺杂质提供了一个高灵敏度、选择性和可重复性的分析平台，增强了对方法的理解，同时符合可持续性原则。

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引用次数: 0

Advancing biomedical analysis: Harnessing laser-induced graphene for next-gen of low-cost sensor technology 推进生物医学分析：利用激光诱导石墨烯用于下一代低成本传感器技术

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-04-26 DOI: 10.1016/j.jpbao.2025.100077

Elsa Maria Materón , Liana Melo Lins de Azevedo , Juliana Martins Dias , Ketley Caroline Rocha Pereira , Gustavo Miguel Sousa , Matheus Santos Dias , Camila Marchetti Maroneze , Daiane Dias , Cecilia de Carvalho Castro Silva

As biosensors and biomedical devices gain increasing importance in everyday diagnostics and health status monitoring, the need to develop and improve their reliability and versatility becomes more pronounced. In this context, the search for new materials for biosensors and biomedical devices has intensified, leading to the emergence of laser-induced graphene (LIG) as a promising candidate. LIG's environmentally sustainable nature, cost-effectiveness, and significant potential for large-scale graphene production and directed writing electronics circuits make it very interesting. In this review, we provide an overview of the mechanisms and precursor materials involved in LIG production, strategies to enhance graphene properties through the in-situ generation of hybrid materials via direct laser writing, and the crucial role of LIG in the development of cost-effective, point-of-care, and wearable devices for medical diagnosis and real-time health status monitoring.

随着生物传感器和生物医学设备在日常诊断和健康状态监测中的重要性日益增加，开发和提高其可靠性和多功能性的需求变得更加明显。在这种背景下，对生物传感器和生物医学设备新材料的研究已经加强，导致激光诱导石墨烯（LIG）作为一个有前途的候选者出现。LIG的环境可持续性、成本效益以及大规模石墨烯生产和定向写入电子电路的巨大潜力使其非常有趣。在这篇综述中，我们概述了LIG生产的机制和前驱体材料，通过直接激光写入原位生成混合材料来增强石墨烯性能的策略，以及LIG在开发成本效益高、即时护理和可穿戴医疗诊断和实时健康状态监测设备中的关键作用。

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引用次数: 0

Determination of rapid and sensitive extraction of colchicine from Colchicum baytopiorum and Colchicum decaisnei leaves by HPLC-UV method 高效液相色谱-紫外分光光度法测定秋水仙叶和秋水仙叶中秋水仙碱的快速灵敏提取

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-04-22 DOI: 10.1016/j.jpbao.2025.100076

Efe Deniz , Ümit Babacan , Ceren Selim , Mehmet Fatih Cengiz

This study focuses on the extraction and analysis of colchicine, a bioactive alkaloid, from the leaves of two endemic species, Colchicum baytopiorum (n = 13) and Colchicum decaisnei (n = 11), collected from Antalya, Türkiye. Colchicine, known for its pharmacological applications for the treatment of gout and cancer, was extracted using ultrasonic-assisted extraction, a more environmentally friendly alternative to conventional methods like Soxhlet and solid-liquid extraction. High-performance liquid chromatography was used for the quantification of colchicine levels in the plant samples. The method was validated in terms of linear dynamic range, limit of detection (LOD), limit of quantification (LOQ) and relative standard deviation (RSD). Results showed that LOD, LOQ and RSD values were determined to be 0.210 ppm, 0.799 ppm and 1.420, respectively. C. baytopiorum had a higher colchicine content (206.24 ± 87.48 ppm) compared to C. decaisnei (11.23 ± 23.04 ppm). These findings are consistent with previous studies, which report varying colchicine concentrations across different Colchicum species. The study suggests that genetic research could help identify the specific genes responsible for colchicine production, which could enhance its availability in pharmaceutical applications. This research contributes to understanding colchicine extraction and its potential for medical use.

本研究对采自土耳其安塔利亚的两种特有植物秋水仙碱（Colchicum baytopiorum, n = 13）和秋水仙（Colchicum decaisnei, n = 11）的叶片进行了生物活性成分的提取和分析。秋水仙碱以其治疗痛风和癌症的药理应用而闻名，它是用超声波辅助提取的，这是一种比索氏提取和固液提取等传统方法更环保的选择。采用高效液相色谱法定量测定植物样品中秋水仙碱的含量。从线性动态范围、检出限（LOD）、定量限（LOQ）和相对标准偏差（RSD）等方面对该方法进行了验证。结果表明，测定的LOD、LOQ和RSD分别为0.210 ppm、0.799 ppm和1.420 ppm。秋水仙碱含量（206.24 ± 87.48 ppm）高于秋水仙碱含量（11.23 ± 23.04 ppm）。这些发现与先前的研究一致，这些研究报告了不同秋水仙属植物的秋水仙碱浓度不同。该研究表明，基因研究可以帮助确定负责秋水仙碱生产的特定基因，从而可以提高其在制药应用中的可用性。本研究有助于了解秋水仙碱的提取及其潜在的医学用途。

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引用次数: 0

Magnetic solid-phase extraction of Sudan dyes from beverages-coated magnetite/silica materials 饮料包覆磁铁矿/二氧化硅材料中苏丹红染料的磁固相萃取

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-12 DOI: 10.1016/j.jpbao.2025.100073

Yugao Guo , Xiaoxiao Niu , Boyu Li , Pei Liu , Youqing Sun , Sumin Lu

In this study, a polydopamine-coated magnetite/silica composite material (Fe₃O₄@SiO₂/PDA) was successfully synthesized and characterized using Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). By integrating EM-MSPE with high-performance liquid chromatography (HPLC), a novel EM-MSPE-HPLC method was established for the sensitive and accurate determination of Sudan I-IV dyes. Key experimental parameters, such as adsorbent dosage, pH, inorganic salt concentration, adsorption time, voltage, eluent type, eluent volume, and desorption time, were systematically investigated and optimized. Under optimal conditions, the method demonstrated excellent linearity (R² > 0.999) within a concentration range of 5–1000 μg L⁻¹, with limits of detection (LODs) ranging from 0.11 to 0.17 μg L⁻¹. The recoveries of Sudan dyes in real samples ranged from 89.1 % to 101.9 %, with relative standard deviations (RSDs) between 0.3 % and 3.6 %. Furthermore, the Fe₃O₄@SiO₂/PDA adsorbent exhibited consistent performance over ten consecutive extraction cycles without significant loss in recovery efficiency. These findings demonstrate that the proposed method is accurate, reliable, and reproducible for the simultaneous determination of Sudan dyes in complex beverage matrices, offering a robust analytical approach for food safety applications.

本研究成功合成了一种聚多巴胺包覆磁铁矿/二氧化硅复合材料（Fe3O4@SiO2/PDA），并利用傅里叶变换红外光谱（FT-IR）、x射线衍射（XRD）和x射线光电子能谱（XPS）对其进行了表征。将EM-MSPE与高效液相色谱（HPLC）相结合，建立了灵敏、准确测定苏丹红I-IV染料的EM-MSPE-HPLC新方法。对吸附剂用量、pH、无机盐浓度、吸附时间、电压、洗脱液类型、洗脱液体积、脱附时间等关键实验参数进行了系统的考察和优化。在最佳条件下，该方法具有良好的线性(R²>；0.999)，浓度范围为5-1000 μg L -⁻¹，检测限（lod）范围为0.11至0.17 μg L -⁻¹。苏丹红染料在实际样品中的回收率为89.1 % ~ 101.9 %，相对标准偏差（rsd）为0.3 % ~ 3.6 %。此外，Fe3O4@SiO2/PDA吸附剂在连续10次萃取循环中表现出一致的性能，且回收率没有明显下降。这些结果表明，该方法准确、可靠、可重复性好，可用于复杂饮料基质中苏丹红染料的同时测定，为食品安全应用提供了可靠的分析方法。

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引用次数: 0

Direct sample introduction in high-performance liquid chromatography analysis of oily medicinal cannabis samples using surfactant-free microemulsion 使用无表面活性剂微乳液对含油药用大麻样品进行高效液相色谱分析，直接进样

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-11 DOI: 10.1016/j.jpbao.2025.100075

Ana Paula Lamounier, Bárbara da Conceição Coelho Cotta Cardoso, Alessandra Licursi Maia Cerqueira da Cunha

The therapeutic use of medicinal Cannabis has raised challenges in quality control and cannabinoid quantification in oil extracts, primarily due to incompatibility with liquid chromatography systems. Surfactant-free microemulsion (SFME) systems offer a promising, efficient, and sustainable solution, enabling direct analysis by high-performance liquid chromatography. These microemulsions are dispersions of two immiscible liquids, typically oil and water, made compatible by an amphiphilic substance that interacts with both polar and nonpolar fractions. After SFME testing and critical analysis of the results, clear and homogeneous microemulsions were observed regardless of the oil-to-water ratio studied, provided that sufficient quantities of 1-octanol:1-propanol (3:10 v/v) were added. Based on the established robust working range, the 1:2 (oil:ultrapure water) ratio (% w/w) was chosen as the compromise condition, minimizing medicinal oil use while ensuring compatibility with the chromatographic system. Univariate optimization of chromatographic conditions enabled simultaneous analysis of five cannabinoids, including cannabidiol (CBD), cannabidiolic acid (CBDA), tetrahydrocannabutol (THCB), cannabinol (CBN), and tetrahydrocannabinol (Δ9-THC) using a ZORBAX Eclipse Plus C18 column at 35 °C. The method employed isocratic elution with a mobile phase of 82:18 %v/v methanol:ultrapure water containing 0.15 ± 0.05 % v/v formic acid, a 1.0 mL min⁻¹ flow rate, and a 20 μL injection volume, using the fluorescence and UV absorption detectors. This proposal offers sensitivity (LOD in the ng mL⁻¹), selectivity, and separation efficiency, associated with low cost, minimal waste generation, and low energy demand, aligning with Green Chemistry principles.

药用大麻的治疗用途在油提取物的质量控制和大麻素定量方面提出了挑战，主要是由于与液相色谱系统不相容。无表面活性剂微乳液（SFME）系统提供了一种有前途的、高效的、可持续的解决方案，可以通过高效液相色谱进行直接分析。这些微乳液是两种不相容的液体（通常是油和水）的分散体，通过与极性和非极性组分相互作用的两亲性物质使其相容。在SFME测试和结果的临界分析后，只要加入足够数量的1-辛醇：1-丙醇（3:10 v/v），无论所研究的油水比如何，都可以观察到清晰均匀的微乳液。基于已建立的稳健工作范围，选择1:2（油：超纯水）比（% w/w）作为折衷条件，在保证与色谱系统相容性的同时，最大限度地减少药用油的使用。采用ZORBAX Eclipse Plus C18色谱柱，对色谱条件进行单因素优化，可在35℃条件下同时分析五种大麻素，包括大麻二酚（CBD）、大麻二酸（CBDA）、四氢大麻酚（THCB）、大麻酚（CBN）和四氢大麻酚（Δ9-THC）。方法采用等容洗脱，流动相为82:18 %v/v甲醇：超纯水（含0.15 ± 0.05 % v/v甲酸），流速1.0 mL min−1，进样量20 μL，荧光和紫外吸收检测器。该方案提供了灵敏度（LOD在ng mL−1），选择性和分离效率，与低成本，最小的废物产生和低能源需求相关联，符合绿色化学原则。

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引用次数: 0

Interstitial fluid-based optical biosensors 基于间隙流体的光学生物传感器

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-10 DOI: 10.1016/j.jpbao.2025.100074

Atefeh Rahimzadeh , Abrisham Arjomandkhah , Mohammad Ali Kiani, Hamed Golmohammadi

The high reproducibility and reliability of optical biosensors, alongside the extraordinarily features of interstitial fluid (ISF) as a promising biological fluid with blood-like composition and yet noninvasive or minimally invasive sampling, have led to the development of a variety of the ISF-based optical biosensors for diagnostic and health monitoring applications. In the present review, while introducing ISF, its characteristics, and the methods developed for its sampling, various types of optical biosensors developed so far for the colorimetric, fluorometric, and surface-enhanced Raman spectroscopy determination of (bio)chemical compounds in ISF are reviewed. Lastly, future prospects and views on the main challenges facing the further development of ISF-based optical biosensors are delineated. Building upon the extraordinary features of ISF-based optical biosensors as highly promising and potential biosensors, we anticipate that they will be greatly welcomed and many of the existing blood-based optical biosensors will be replaced by ISF-based ones in the near future.

光学生物传感器的高重复性和可靠性，以及间质液（ISF）作为一种具有血液样成分的有前途的生物液体的非凡特征，以及非侵入性或微创性采样，导致了各种基于ISF的光学生物传感器的发展，用于诊断和健康监测应用。在本文中，介绍了生物纤维、生物纤维的特性和开发的采样方法，综述了迄今为止开发的用于比色法、荧光法和表面增强拉曼光谱法测定生物纤维中（生物）化合物的各种类型的光学生物传感器。最后，对基于isf的光学生物传感器进一步发展面临的主要挑战进行了展望和展望。鉴于基于isf的光学生物传感器作为一种非常有前途和潜力的生物传感器的非凡特性，我们预计它们将受到极大的欢迎，并且在不久的将来，许多现有的基于血液的光学生物传感器将被基于isf的光学生物传感器所取代。

引用次数: 0

How does HSA's antioxidant activity change under the influence of lincomycin and spectinomycin mixture? – A spectroscopic study 林可霉素与大观霉素混合作用对HSA的抗氧化活性有何影响？-光谱研究

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-08 DOI: 10.1016/j.jpbao.2025.100070

Wojciech Rogóż, Aleksandra Owczarzy, Karolina Kulig, Małgorzata Maciążek-Jurczyk

Lincomycin (LIN) belongs to the lincosamides group and is used in bacterial infections of soft tissues, the respiratory system, bone marrow, and bones, while spectinomycin (SPE) belongs to the group of aminoglycoside antibiotics and is used for the treatment of gonorrhoea. Both antibiotics are commonly used to treat humans separately while their mixture is used in veterinary preparations (SPE-LIN), and the combination of these antibiotics has not yet been used for human treatment. The aim of this study was to search for modulators of human serum albumin (HSA) antioxidant activity among antibacterial drugs, lincomycin (LIN) and spectinomycin (SPE). In order to study the effect of LIN and SPE on HSA antioxidant properties the DPPH and ABTS tests were used. UV-Vis spectrophotometry and circular dichroism (CD) spectroscopic techniques were used to find the cause of LIN and SPE modulatory activity related to HSA antiradical potential. Both LIN and SPE, as well as their mixture, did not show significant antioxidant activity, while in combination with HSA, they stimulated its antioxidant properties. CD measurements determined that the dominant secondary structure of HSA, regardless of the presence of ligands, was α-helix, and its percentage share slightly increased under the influence of LIN and SPE. The obtained results confirm the viability of further studies on the potential application of the combination of SPE-LIN in the treatment, not only in breeding animals. The proposed concept of research may be a response to the future needs in medicine. The assumed goal, which was the search for HSA modulators, has been achieved.

林可霉素（Lincomycin， LIN）属于林可胺类药物，用于软组织、呼吸系统、骨髓和骨骼的细菌感染，而大观霉素（spectinomycin， SPE）属于氨基糖苷类抗生素，用于治疗淋病。这两种抗生素通常用于单独治疗人类，而它们的混合物用于兽医制剂（SPE-LIN），这些抗生素的组合尚未用于人类治疗。本研究的目的是在抗菌药物林可霉素（LIN）和大观霉素（SPE）中寻找人血清白蛋白（HSA）抗氧化活性的调节剂。为了研究LIN和SPE对HSA抗氧化性能的影响，采用DPPH和ABTS试验。利用紫外-可见分光光度法和圆二色（CD）光谱技术研究了LIN和SPE调节HSA抗自由基电位的原因。LIN和SPE及其混合物均不表现出显著的抗氧化活性，而与HSA结合后，则能激发其抗氧化性能。CD测定表明，无论配体是否存在，HSA的优势二级结构都是α-螺旋结构，在LIN和SPE的影响下，其百分比略有增加。所获得的结果证实了进一步研究SPE-LIN联合应用于治疗的可行性，而不仅仅是在育种动物中。提出的研究概念可能是对未来医学需求的回应。假设的目标，即寻找HSA调制器，已经实现。

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引用次数: 0

“Quartz crystal microbalance-based biosensor for rapid and ultrasensitive SARS-CoV-2 detection" 用于快速超灵敏检测SARS-CoV-2的石英晶体微天平生物传感器

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-07 DOI: 10.1016/j.jpbao.2025.100071

Sahera Saleh , Habib Alkalamouni , Karen Antar , Joe Rahme , Michel Kazan , Pierre Karam , Jit Muthuswamy , Hassan Zaraket , Massoud L. Khraiche

The COVID-19 pandemic highlighted the urgent need for rapid, sensitive, and affordable diagnostic tests, especially in resource-limited settings. While RT-qPCR remains the gold standard for SARS-CoV-2 detection, it is expensive and requires specialized equipment. Antigen-based tests, though faster, lack sufficient sensitivity. Therefore, there is a pressing need for a platform that combines the rapidity of antigen tests with the sensitivity of molecular tests. In this work, we address this problem by developing a Quartz Crystal Microbalance (QCM) biosensor for the rapid detection of SARS-CoV-2 nucleocapsid proteins. We designed a QCM biosensor with polyethylene glycol (PEG)-based surface functionalization, which significantly improves sensitivity and specificity. The platform achieved a detection limit of 53.3 TCID₅₀/mL and a sensitivity of 0.263 Hz/ TCID₅₀/mL, with results available in approximately 15 min. Cross-reactivity tests with Influenza A demonstrated its high specificity for SARS-CoV-2. Comprehensive surface characterization using Scanning Electron Microscopy (SEM), Digital Holographic Microscopy, and Raman Spectroscopy confirmed the stability and integrity of the functionalized sensor surface. The platform is cost-effective, scalable, and designed for ease of use in resource-limited settings. This work presents the first open-source QCM biosensing platform for SARS-CoV-2 detection that combines high sensitivity, rapid results, and affordability. It offers a deployable solution for COVID-19 diagnostics, particularly in underserved regions, and is adaptable for future pandemic preparedness.

COVID-19大流行凸显了对快速、敏感和负担得起的诊断检测的迫切需求，特别是在资源有限的环境中。虽然RT-qPCR仍然是检测SARS-CoV-2的金标准，但它很昂贵，需要专门的设备。基于抗原的测试虽然更快，但缺乏足够的灵敏度。因此，迫切需要一种结合抗原检测的快速性和分子检测的敏感性的平台。在这项工作中，我们通过开发一种用于快速检测SARS-CoV-2核衣壳蛋白的石英晶体微天平（QCM）生物传感器来解决这一问题。我们设计了一种基于聚乙二醇（PEG）表面功能化的QCM生物传感器，该传感器显著提高了灵敏度和特异性。该平台的检出限为53.3 TCID50/mL，灵敏度为0.263 Hz/ TCID50/mL，可在约15 min内获得结果。与甲型流感病毒的交叉反应试验表明其对SARS-CoV-2具有高特异性。利用扫描电子显微镜（SEM）、数字全息显微镜和拉曼光谱进行了全面的表面表征，证实了功能化传感器表面的稳定性和完整性。该平台具有成本效益，可扩展，并且在资源有限的环境中易于使用。本工作提出了首个开源的QCM生物传感平台，用于检测SARS-CoV-2，该平台兼具高灵敏度、快速结果和可负担性。它为COVID-19诊断提供了可部署的解决方案，特别是在服务不足的地区，并可适用于未来的大流行防范。

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引用次数: 0

Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography 使用离子对反相色谱法定量脂质纳米颗粒中的治疗性siRNA

Journal of Pharmaceutical and Biomedical Analysis Open

Pub Date : 2025-03-03 DOI: 10.1016/j.jpbao.2025.100072

Ludivine Ferey, Sandy Al Bardawil, Emilie Dols, Aurore Guédin, Viet-Ha Phan, Philippe Barthélémy, Jeanne Leblond Chain

The clinical development of lipid nanoparticles encapsulating small interfering RNA (siRNA-LNP) requires robust and efficient analytical methods for quality assessment. In this study, we investigated the chromatographic profiles of siRNAs using Ion-Pair Reversed-Phase (IP-RP) HPLC at six different temperatures, including the melting temperature of duplexes, on two RP columns. The column temperature was identified as a critical parameter influencing chromatographic profiles, with characteristic profiles evolving in a temperature-dependent manner, corresponding to the melting temperatures (T_m) of the two siRNA duplexes analyzed, across both tested columns. Moreover, the siRNA dissociation observed on the columns was consistent with the melting temperatures of the duplexes measured by UV-Vis spectroscopy under HPLC mobile phase conditions. An IP-RPHPLC method using 100 mM TEAA with ACN and a column temperature of 80 °C, above the T_m of the analyzed duplexes, was successfully applied for the quantitative determination of siRNAs in LNP formulations. A simple, one-step sample preparation was employed by directly adding a neutral surfactant to the LNP samples before injection. This method proved highly accurate, as shown by a 100 % recovery of siRNA immediately following microfluidic formulation. Additionally, the impact of the dialysis purification process on siRNA recovery was assessed, revealing a 30 % loss of siRNA. Importantly, the results obtained from this method were consistent with those from the fluorescence reference method. This method helps improve the quality control of siRNA-LNP therapeutics, thereby enhancing their clinical translation potential and supporting the development of next-generation RNA-based therapies.

脂质纳米颗粒封装小干扰RNA （siRNA-LNP）的临床开发需要可靠和有效的分析方法来进行质量评估。在本研究中，我们使用离子对反相（IP-RP）高效液相色谱法研究了sirna在六种不同温度下的色谱图谱，包括双相物的熔化温度，在两根RP柱上。柱温被确定为影响色谱图谱的关键参数，其特征图谱以温度依赖的方式演变，对应于两种测试柱上分析的两种siRNA双链物的熔化温度（Tm）。此外，柱上观察到的siRNA解离与HPLC流动相条件下紫外可见光谱测量的双相物的熔化温度一致。采用100 mM TEAA加ACN，柱温80°C，高于分析的双链物的Tm， IP-RPHPLC方法成功地用于LNP制剂中sirna的定量测定。通过在LNP样品中直接加入中性表面活性剂进行简单的一步样品制备。该方法被证明是高度准确的，正如在微流体配方后立即回收100 %的siRNA所示。此外，我们还评估了透析纯化过程对siRNA恢复的影响，结果显示siRNA损失了30% %。重要的是，该方法得到的结果与荧光参比法一致。该方法有助于提高siRNA-LNP疗法的质量控制，从而增强其临床翻译潜力，并支持下一代rna疗法的发展。

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引用次数: 0