Analytical biochemistry最新文献_第6页

qPCR-based method to measure plasmid internalization efficiency of non-viral nanoparticle carriers in adherent cell model 基于qpcr的非病毒纳米颗粒载体在贴壁细胞模型中质粒内化效率测定方法。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-15 DOI: 10.1016/j.ab.2025.115979

Priscila Tonon Baschirotto, Samara Valeria López Ramírez, Randall Loaiza-Montoya

Non-viral nanoparticle-mediated transfection systems have gained wide acceptance in modern pharmaceutical and biotechnological applications, including RNA delivery for COVID-19 vaccines. However, these systems may show low transfection and transcription efficiencies, potentially limiting the use of reporter molecules like GFP to estimate efficacy in early development stages. Moreover, the expression of reporter molecules may not reflect internalization efficiency, which can be pertinent information for the development of these systems. Therefore, as their popularity continues to rise, more sensitive and accurate tools will be necessary.

We validated a DNA extraction and qPCR assay to quantify the internalized plasmid copy number in NIH/3T3 cells transfected using LGA-PEI (lactic-co-glycolic acid-modified polyethylenimine) nanoparticles. Optimized qPCR and DNA extraction assays exhibited high linearity, sensitivity, and robustness. Additionally, we developed an appropriate extraction and extracellular plasmid digestion protocol that eliminated potential polymer interference with the qPCR reaction and increased specificity for internalized plasmid quantification.

Using these optimized protocols, we estimated that transfected NIH/3T3 cells incorporated approximately 9 500 to 150 000 plasmids/cell for transfection reactions that yielded as few as 4.1 %–18.2 % GFP-positive cells. Overall, these results indicate that our optimized method is reliable for quantifying transfected plasmids in context of low-efficiency nanoparticle carrier system.

非病毒纳米颗粒介导的转染系统已在现代制药和生物技术应用中得到广泛接受，包括用于COVID-19疫苗的RNA递送。然而，这些系统可能表现出较低的转染和转录效率，潜在地限制了报告分子（如GFP）在早期发育阶段评估疗效的使用。此外，报告分子的表达可能不能反映内化效率，这可以为这些系统的开发提供相关信息。因此，随着它们的普及程度不断提高，将需要更灵敏、更准确的工具。我们验证了DNA提取和qPCR检测，以量化使用LGA-PEI（乳酸-羟基乙酸修饰的聚乙烯亚胺）纳米颗粒转染的NIH/3T3细胞的内化质粒拷贝数。优化后的qPCR和DNA提取分析具有较高的线性、灵敏度和鲁棒性。此外，我们开发了一种合适的提取和细胞外质粒消化方案，消除了qPCR反应中潜在的聚合物干扰，提高了内化质粒定量的特异性。使用这些优化的方案，我们估计转染的NIH/3T3细胞含有大约9500至150000个质粒/细胞，转染反应产生的gfp阳性细胞仅为4.1%至18.2%。总之，这些结果表明，我们优化的方法是可靠的定量转染质粒在低效率的纳米颗粒载体系统。

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引用次数: 0

Biosynthesised reduced graphene oxide/CuO based nanocomposite using ‘Cordia dichotoma’ leaf extract for ‘nitrobenzene’ determination 生物合成还原氧化石墨烯/CuO基纳米复合材料，使用“Cordia dichotoma”叶提取物测定“硝基苯”

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-10 DOI: 10.1016/j.ab.2025.115972

Devkumari Patel , Robind Kumar , Sanju Yadav , Ankita Rai , Vijai K. Rai , Manorama Singh

Herein, plant-assisted synthesis of copper oxide nanoparticles (CuO NPs)_CD and reduced graphene oxide (CDrGO) was performed individually using the leaf extract of ‘Cordia dichotoma’ plant and they were associated with electrochemically active conducting clay ‘Sodium-Montmorillonite (Na-MT)’ leading to the synthesis of a new nanocomposite named ‘(CuO NPs)_CD@CDrGO-Na-MT’. The collaboration of CDrGO paired (CuO NPs)_CD and Na-MT enhanced the electrochemical reduction of noxious ‘Nitrobenzene (NB)’ at a lower potential. The prepared ternary nanocomposite was confirmed with characterisation techniques, Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Electrochemical properties and electrochemical reduction of ‘Nitrobenzene’ were inspected with cyclic voltammetry and differential pulse voltammetry techniques, respectively. The electro-reduction of NB was recorded in two wide linear ranges of 0.016–360 μM and 360–6980 μM with a detection limit of 15 nM and negligible effect of interferences.

本研究中，植物辅助合成氧化铜纳米颗粒（CuO NPs）CD和还原性氧化石墨烯（CDrGO）分别使用“Cordia dichotoma”植物的叶提取物进行，并将它们与电化学活性导电粘土“钠蒙脱土（Na-MT）”相结合，合成了一种新的纳米复合材料“（CuO NPs）CD@CDrGO-Na-MT”。CDrGO对（CuO NPs）CD和Na-MT的协同作用在较低电位下增强了有害的“硝基苯（NB）”的电化学还原。采用表征技术、透射电子显微镜（TEM）、傅里叶变换红外光谱（FTIR）、x射线衍射（XRD）和x射线光电子能谱（XPS）对所制备的三元纳米复合材料进行了表征。采用循环伏安法和差分脉冲伏安法分别考察了硝基苯的电化学性能和电化学还原性能。在0.016 ~ 360 μM和360 ~ 6980 μM的宽线性范围内记录了NB的电还原，检测限为15 nM，干扰影响可以忽略不计。

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引用次数: 0

Mapping transposon insertion sites within bacterial genomes by direct Sanger sequencing 通过直接Sanger测序在细菌基因组中定位转座子插入位点。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-10 DOI: 10.1016/j.ab.2025.115971

Scott W. Herke , Linda M. Heffernan , William N. Beavers , Basel H. Abuaita

Microbial biomedical research frequently involves mutagenesis by insertion of transposons into the genome. Currently, transposon insertion locations are typically elucidated by Next Generation Sequencing or by Sanger sequencing of PCR products. Here, transposons were located by direct Sanger sequencing of bacterial genomic DNA from both Salmonella enterica (Gram-negative) and Staphylococcus aureus (Gram-positive) cultures. DNA was prepared by shearing to a modal size of ∼2 kb followed by purification by paramagnetic beads. Sequencing reactions involved relatively minor modifications of standard protocols (e.g., extra sequencing polymerase, 75–100 PCR cycles); completed reactions were cleaned by ethanol-EDTA precipitation. Reads were generated on the ABI 3130xl Genetic Analyzer using 50-cm capillary arrays and a run protocol modified for extra sample injection time. Good quality reads of ∼500–800 nt were routinely generated; BLAST results returned nearly 100% matches to genomes in the NCBI database. As implemented, the optimized protocol (post-DNA extraction) could be performed within an 8-h workday (with sequencing results the following day) for ∼$10 (USD) per sequencing reaction. Although this method was developed to locate transposons inserted into bacterial genomes, it seems likely that it can be extended to generate sequence data from even native single-copy genes from small genomes (e.g., <5 Mb).

微生物生物医学研究经常涉及将转座子插入基因组的诱变。目前，转座子插入位置通常是通过下一代测序或PCR产物的Sanger测序来确定的。在这里，转座子是通过直接桑格测序从肠炎沙门氏菌（革兰氏阴性）和金黄色葡萄球菌（革兰氏阳性）培养的细菌基因组DNA定位。通过剪切至约2kb的模态大小制备DNA，然后用顺磁珠纯化。测序反应涉及对标准方案相对较小的修改（例如，额外的测序聚合酶，75-100个PCR循环）；完成的反应用乙醇- edta沉淀法清洗。在ABI 3130xl遗传分析仪上使用50厘米毛细管阵列和修改的运行方案以增加样品注射时间。常规生成约500-800 nt的高质量读数；BLAST结果与NCBI数据库中的基因组几乎100%匹配。优化后的方案（dna后提取）可以在8小时工作日内完成（第二天有测序结果），每个测序反应约10美元。虽然这种方法是为了定位插入细菌基因组的转座子而开发的，但它似乎可以扩展到从小基因组(例如，

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引用次数: 0

Easy reference-guided assembly of nanopore whole plasmid sequencing datasets 易于参考引导组装纳米孔全质粒测序数据集。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-05 DOI: 10.1016/j.ab.2025.115970

Vinita Sharma , Naiyu Jiang , Yukihiro Nagashima , Hisashi Koiwa

Whole plasmid sequencing (WPS) using Nanopore long read sequencing has emerged as a cost-effective alternative for dideoxy sequencing methods. De novo sequence assembly for large plasmids, however, are not always successful and may produce large assembly gaps. Here we streamlined a reference-guided alignment of WPS nanopore reads using galaxy platform. The process is straightforward and facilitate introduction of WPS as an alternative for researchers who are more used to dideoxy sequencing platform. We demonstrate that our procedure can assemble nanopore sequence reads with different quality datasets and identify variations from reference sequence.

利用纳米孔长读测序的全质粒测序（WPS）已成为二脱氧测序方法的一种具有成本效益的替代方法。然而，大型质粒的从头序列组装并不总是成功的，并且可能产生较大的组装间隙。本研究利用星系平台简化了WPS纳米孔reads的参考导向比对。该过程简单明了，便于引入WPS作为更习惯于二脱氧测序平台的研究人员的替代方案。我们证明了我们的程序可以用不同质量的数据集组装纳米孔序列，并识别参考序列的差异。

引用次数: 0

PreRBP: Interpretable deep learning for RNA-protein binding site prediction with attention mechanism PreRBP：基于注意机制的rna -蛋白结合位点预测的可解释深度学习。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115968

Huixian Chen , Yun Zuo , Xiangrong Liu , Xiangxiang Zeng , Zhaohong Deng , Jiasong Wu

In the complex process of gene expression and regulation, RNA-binding proteins occupy a pivotal position for RNA. Accurate prediction of RNA-protein binding sites can help researchers better understand RNA-binding proteins and their related mechanisms. And prediction techniques based on machine learning algorithms are both cost-effective and efficient in identifying these binding sites. However, there are some shortcomings in the currently available machine learning methods, such as the input features of the model only consider RNA sequence features, and most of the datasets suffer from class imbalance. To address these issues, this study first uses the publicly available 27 RNA-protein binding site datasets to construct a benchmark dataset. Then, we use RNAshapes and EDeN to obtain the secondary structure of RNA. Higher-order encoding method is used to extract the key information hidden in the RNA sequences and structures. In order to solve the class imbalance problem existing in the dataset, this study utilizes four undersampling algorithms, namely, random undersampling, NearMiss, ENN, and one-sided selection, to remove redundant samples in the negative samples, and lastly, based on Convolutional Neural Network, Bidirectional Long and Short Term Memory Network, this study constructs model PreRBP to predict RNA-protein binding sites.

The experimental results show that the model used in this study has an average AUC of 0.88, which is higher than other existing RNA-protein binding site prediction methods. Also, for the convenience of prediction, an online predictor is developed in this study. The predictor and experimental codes are available at https://github.com/B12-Comet/RBPPrediction.

在复杂的基因表达和调控过程中，RNA结合蛋白对RNA起着举足轻重的作用。准确预测rna -蛋白结合位点有助于研究人员更好地了解rna -蛋白结合及其相关机制。而基于机器学习算法的预测技术在识别这些结合位点方面既经济又有效。然而，目前可用的机器学习方法存在一些不足，例如模型的输入特征只考虑RNA序列特征，大多数数据集存在类不平衡。为了解决这些问题，本研究首先使用公开可用的27个rna -蛋白结合位点数据集构建基准数据集。然后，我们使用RNAshapes和EDeN来获得RNA的二级结构。采用高阶编码方法提取隐藏在RNA序列和结构中的关键信息。为了解决数据集中存在的类不平衡问题，本研究利用随机欠采样、NearMiss、ENN和片面选择四种欠采样算法去除负样本中的冗余样本，最后基于卷积神经网络、双向长短期记忆网络构建PreRBP模型预测rna -蛋白结合位点。实验结果表明，本研究使用的模型的平均AUC为0.88，高于现有的其他rna -蛋白结合位点预测方法。此外，为了便于预测，本研究还开发了一种在线预测器。预测器和实验代码可在https://github.com/B12-Comet/RBPPrediction上获得。

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引用次数: 0

Exploration of endoplasmic reticulum stress-related gene markers in amyotrophic lateral sclerosis: a comprehensive analysis of bioinformatics and machine learning 肌萎缩侧索硬化症内质网应激相关基因标记的探索：生物信息学和机器学习的综合分析

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115969

Jing Wang , Xinmin Li , Fangjie Yang , Pengxue Guo , Chunlin Ren , Zhengfei Duan , Mengyao Bi , Yuting Kong , Yasu Zhang , Jianwei Lu

This study aimed to investigate potential biomarkers related to Endoplasmic reticulum (ER) stress in Amyotrophic lateral sclerosis (ALS) through a comprehensive bioinformatic approach. The gene expression profiles of ALS patients and healthy controls were downloaded from the Gene Expression Omnibus (GEO) database. ER stress-related genes were collected from the MSigDB databases and document literature. The “limma” R package was employed to detect the differentially expressed ER stress-related genes (DE-ERSGs). Three methods of machine learning were applied to select the hub DE-ERSGs. ROC curves were conducted to evaluate model performance. An external dataset was chosen to evaluate the diagnostic capability of hub genes. The CIBERSORT algorithm was used to evaluate the immune cell infiltration characteristics. Additionally, we constructed a systematic ceRNA regulatory network using Cytoscape software and predicted the possible drug candidates using the Enrichr platform. Molecular docking analysis was used to further validate the binding ability of the candidate drug molecules to the hub genes. Six hub DE-ERSGs (ABCA1, CKAP4, TOR1AIP1, MMP9, EDC4, and ALPP) were identified, and the related models performed well. These hub genes were concentrated in multiple pathways and related to various immune cells. Drugs such as nitroglycerin, diazepam, FENRETINIDE, and edaravone exhibited good binding affinity to the hub genes, indicating that they may be promising drugs for the management of ALS. This study revealed the essential role of ER stress in the pathogenesis of ALS from an integrative perspective, providing guidance for the development of new therapeutic targets and diagnostic strategies.

本研究旨在通过综合生物信息学方法研究肌萎缩侧索硬化症（ALS）内质网（ER）应激相关的潜在生物标志物。从gene expression Omnibus （GEO）数据库下载ALS患者和健康对照者的基因表达谱。从MSigDB数据库和文献文献中收集内质网应激相关基因。“limma”R包检测内质网应激相关基因（de - ergs）的差异表达。采用三种机器学习方法选择轮毂de - ergs。采用ROC曲线评价模型的性能。选择一个外部数据集来评估枢纽基因的诊断能力。采用CIBERSORT算法评价免疫细胞浸润特性。此外，我们使用Cytoscape软件构建了一个系统的ceRNA调控网络，并使用enrichment平台预测了可能的候选药物。通过分子对接分析进一步验证候选药物分子与枢纽基因的结合能力。确定了6个hub de - ergs (ABCA1、CKAP4、TOR1AIP1、MMP9、EDC4和ALPP)，相关模型表现良好。这些枢纽基因集中在多种途径中，与多种免疫细胞有关。硝酸甘油、地西泮、芬拉啶和依达拉奉等药物与中枢基因表现出良好的结合亲和力，表明它们可能是治疗ALS的有希望的药物。本研究从综合角度揭示了内质网应激在ALS发病机制中的重要作用，为开发新的治疗靶点和诊断策略提供指导。

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引用次数: 0

Mismatch-sensitive DNA hybridization controlled by inchworm-type peptide nucleic acid–PEG conjugates 尺蠖型肽核酸- peg偶联物控制错配敏感DNA杂交

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115967

Toshihiko Sakurai , Yusuke Hamashita , Takahiro Shibata

The duplex-forming behavior of an inchworm-type PNA–PEG conjugate (i-PPc), engineered for the selective recognition of point mutations in DNA, was assessed through thermodynamic analysis employing UV melting curves and circular dichroism spectroscopy. The i-PPc demonstrated the ability to form stable duplexes exclusively with fully complementary DNA sequences, while no hybridization with single-base mismatched sequences. This binary on/off hybridization behavior was maintained even under physiologically relevant conditions (37 °C), thereby illustrating the exceptional point mutation discrimination capability of i-PPc. The behavior observed can be ascribed to the distinctive structure of i-PPc, wherein two PNA segments, possessing intrinsically different duplex-forming stabilities—high and low—are covalently linked via a flexible PEG linker. The high-stability PNA segment functions as the primary recognition domain for point mutations, thereby defining the sequence specificity of duplex formation. Conversely, the low-stability segment contributes cooperatively to the overall duplex stabilization only when the high-stability segment successfully hybridizes with the target DNA. This cooperative mechanism underlies the sequence-selective duplex formation of i-PPc, highlighting its potential as a highly specific probe for DNA mutation diagnostics. These findings indicate that i-PPc represents a promising platform for point mutation detection and nucleic acid-based molecular diagnostics grounded in DNA hybridization under physiological conditions.

采用紫外熔化曲线和圆二色光谱热力学分析评估了一种用于选择性识别DNA点突变的尺蠖型PNA-PEG共轭物（i-PPc）的双工形成行为。i-PPc能够与完全互补的DNA序列形成稳定的双链，而与单碱基不匹配的序列不杂交。即使在生理相关条件下（37°C），这种二元开/关杂交行为也能保持，从而说明i-PPc具有特殊的点突变识别能力。观察到的行为可以归因于i-PPc的独特结构，其中两个具有本质上不同的双工形成稳定性的PNA片段-高和低-通过柔性PEG连接共价连接。高稳定性的PNA片段作为点突变的主要识别域，从而定义了双链形成的序列特异性。相反，只有当高稳定性片段成功地与目标DNA杂交时，低稳定性片段才有助于整体双工稳定。这种合作机制是i-PPc序列选择性双工形成的基础，突出了其作为DNA突变诊断的高度特异性探针的潜力。这些发现表明，在生理条件下，i-PPc为点突变检测和基于DNA杂交的核酸分子诊断提供了一个很有前景的平台。

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引用次数: 0

Continuous assay for the dNTP triphosphohydrolase of activated SAMHD1 活化SAMHD1的dNTP三磷酸水解酶连续测定。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-01 DOI: 10.1016/j.ab.2025.115966

Roozbeh Eskandari, Daniel P. Groom, Ryo Tamura, Vern L. Schramm

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is the only member of the triphosphoric monoester hydrolase family in humans (dNTP + H₂O → dN + PPPi). The dNTPase activity of SAMHD1 inhibits DNA synthesis, resulting in cell-cycle arrest and restricting viral replication. The complex allosteric regulation mechanism of SAMHD1 and a reaction that lacks a direct spectroscopic signal make its kinetic analysis and inhibitor discovery challenging. We describe a continuous assay for monitoring SAMHD1 phosphatase activity in its activated physiological state. The assay uses a sequential assembly to generate the active tetrameric form of the enzyme. Two phosphatases convert inorganic triphosphate (PPPi) to inorganic phosphate (Pi). The released Pi reacts with the 7-methyl-6-thioguanosine and purine nucleoside phosphorylase to provide a sensitive continuous spectrophotometric assay. The assay is suitable for 96-microwell plate formats to provide a continuous measurement of SAMHD1 activity. The assay is benchmarked with inhibitors of SAMHD1. With a Z-prime value > 0.90, the assay can be used for high-throughput screening of inhibitors for SAMHD1 and characterizing the allosteric or catalytic activity of the new inhibitors.

无菌α基序和组氨酸-天冬氨酸结构域蛋白1 （SAMHD1）是人类三磷酸单酯水解酶家族（dNTP + H2O→dN + PPPi）中唯一的成员。SAMHD1的dNTPase活性抑制DNA合成，导致细胞周期阻滞，限制病毒复制。SAMHD1复杂的变构调节机制和缺乏直接光谱信号的反应给其动力学分析和抑制剂的发现带来了挑战。我们描述了一个连续监测SAMHD1磷酸酶活性在其激活的生理状态。该分析使用顺序组装来产生活性四聚体形式的酶。两种磷酸酶将无机三磷酸（PPPi）转化为无机磷酸（Pi）。释放的Pi与7-甲基-6-硫鸟嘌呤核苷磷酸化酶反应，提供灵敏的连续分光光度测定。该分析适用于96微孔板格式，以提供SAMHD1活性的连续测量。该试验以SAMHD1抑制剂为基准。该试验的Z-prime值为> 0.90，可用于高通量筛选SAMHD1抑制剂，并表征新抑制剂的变构或催化活性。

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引用次数: 0

Boron nitride quantum dot-based fluorescent sensor for carbamazepine determination in exhaled breath condensate 氮化硼量子点荧光传感器测定呼出液中卡马西平

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-26 DOI: 10.1016/j.ab.2025.115964

Saba Ershadi , Maryam Khoubnasabjafari , Vahid Jouyban-Gharamaleki , Elaheh Rahimpour , Abolghasem Jouyban

Carbamazepine is a widely prescribed antiepileptic drug with a narrow therapeutic index, necessitating precise monitoring to avoid toxicity and ensure therapeutic efficacy. This study presents a fluorescence-based nanosensor using boron nitride quantum dots (BNQDs) for the rapid and sensitive detection of carbamazepine in exhaled breath condensate (EBC). BNQDs were prepared via a simple hydrothermal technique and characterized using transmission electron microscopy, dynamic light scattering, energy-dispersive X-ray, and attenuated total reflectance-Fourier transform infrared techniques. The sensor exhibited a concentration-dependent quenching of BNQD fluorescence upon carbamazepine addition, with a linear response range of 0.2–2.4 μg mL⁻¹ and a low detection limit of 0.05 μg mL⁻¹. Stern–Volmer analysis confirmed a dynamic quenching mechanism. The nanosensor also demonstrated high selectivity against common co-administered drugs and was successfully applied to real EBC samples collected from patients receiving carbamazepine therapy. This BNQD-based sensing platform offered a rapid, cost-effective, and user-friendly approach for the non-invasive therapeutic drug monitoring of carbamazepine.

卡马西平是一种广泛使用的抗癫痫药物，治疗指标较窄，需要精确监测，以避免毒性，确保治疗效果。本研究提出了一种利用氮化硼量子点（BNQDs）的荧光纳米传感器，用于快速灵敏地检测呼出液（EBC）中的卡马西平。采用简单的水热法制备了BNQDs，并利用透射电子显微镜、动态光散射、能量色散x射线和衰减全反射-傅里叶变换红外技术对其进行了表征。该传感器在卡马西平的作用下BNQD荧光猝灭具有浓度依赖性，线性响应范围为0.2 ~ 2.4 μ mL−1，低检出限为0.05 μ mL−1。Stern-Volmer分析证实了动态淬火机制。该纳米传感器还显示出对常见药物的高选择性，并成功应用于卡马西平治疗患者的真实EBC样本。这种基于bnqd的传感平台为卡马西平的无创治疗药物监测提供了一种快速、经济、用户友好的方法。

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引用次数: 0

ITGB3 as a promising non-invasive biomarker for type 2 diabetes and diabetic nephropathy ITGB3有望成为2型糖尿病和糖尿病肾病的无创生物标志物

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-25 DOI: 10.1016/j.ab.2025.115965

Seyed Amirhossein Hosseini , Parisa Ajorlou , Hasti Haddadian , Shahla Sohrabipour

The prevalence of Type 2 diabetes mellitus (T2DM) is increasing worldwide and represents a major risk factor for the development of diabetic nephropathy (DN), a severe microvascular complication. Chronic hyperglycemia activates inflammatory and fibrotic signaling pathways, which contribute to kidney damage. Integrins, as transmembrane adhesion receptors, play pivotal roles in regulating inflammation, immune cell trafficking, and insulin resistance. This research focused on identifying non-invasive biomarkers for T2DM and DN using PBMCs. Differentially expressed genes related to diabetes were identified through the analysis of multiple datasets retrieved from the Gene Expression Omnibus, including GSE95849, GSE9006, GSE25724, and GSE159984. ITGB3 was identified as a common gene across these datasets, and its expression in DN was further examined using the GSE142025 dataset. Real-time PCR analysis of PBMC samples revealed a significant upregulation of ITGB3 expression in individuals with DN and T2DM compared to healthy controls. The TF2DNA and miRNASNPv3 databases identified 10 transcription factors and 10 variants of ITGB3 involved in 60 miRNA interactions. Additionally, the DGIdb database revealed 15 drugs potentially regulating ITGB3 expression. These findings underscore the importance of integrin-related pathways in diabetes and suggest ITGB3 as a promising target for future research and therapeutic development.

2型糖尿病（T2DM）的患病率在全球范围内呈上升趋势，是糖尿病肾病（DN）发展的主要危险因素，是一种严重的微血管并发症。慢性高血糖会激活炎症和纤维化信号通路，从而导致肾脏损伤。整合素作为跨膜粘附受体，在调节炎症、免疫细胞运输和胰岛素抵抗中起关键作用。本研究的重点是利用pbmc识别T2DM和DN的非侵入性生物标志物。通过分析从Gene Expression Omnibus检索到的多个数据集，包括GSE95849、GSE9006、GSE25724和GSE159984，鉴定出与糖尿病相关的差异表达基因。ITGB3被鉴定为这些数据集中的共同基因，并使用GSE142025数据集进一步检测其在DN中的表达。PBMC样本的实时PCR分析显示，与健康对照相比，DN和T2DM患者的ITGB3表达显著上调。TF2DNA和miRNASNPv3数据库鉴定了参与60种miRNA相互作用的10个转录因子和10个ITGB3变体。此外，DGIdb数据库还发现了15种可能调节ITGB3表达的药物。这些发现强调了整合素相关通路在糖尿病中的重要性，并表明ITGB3是未来研究和治疗开发的有希望的靶点。

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引用次数: 0