药学学报

Experimental method	Advantage	Disadvantage	Ref.
Cell experiment	Simulate inflammatory response	In vitro experiments cannot reflect the effectiveness in vivo, long cycle	[14]
Animal model	Similar genetic background and immune system to humans	High cost, time-consuming, complicated operation	[15, 16]
Enzyme activity inhibition assay	Complete and reproducible method system	Difficult probe synthesis, long cycle, high cost, cumbersome operation	[17]
Surface plasmon resonance (SPR)	Real-time detection, label-free, high sensitivity	Interference from complex samples, high equipment cost	[18]
Localized surface plasmon resonance (LSPR)	Real-time and label-free detection, high sensitivity, strong specificity		[19-22]

Experimental method	Advantage	Disadvantage	Ref.
Cell experiment	Simulate inflammatory response	In vitro experiments cannot reflect the effectiveness in vivo, long cycle	[14]
Animal model	Similar genetic background and immune system to humans	High cost, time-consuming, complicated operation	[15, 16]
Enzyme activity inhibition assay	Complete and reproducible method system	Difficult probe synthesis, long cycle, high cost, cumbersome operation	[17]
Surface plasmon resonance (SPR)	Real-time detection, label-free, high sensitivity	Interference from complex samples, high equipment cost	[18]
Localized surface plasmon resonance (LSPR)	Real-time and label-free detection, high sensitivity, strong specificity		[19-22]

Sample group / nmol·L^-1	Measured value (radian)	RSD/%
50	0.214 4	0.223 3	0.215 8	3.45
	0.232 9	0.231 2	0.220 5
100	0.319 0	0.318 4	0.310 1	3.69
	0.303 6	0.337 5	0.311 7
500	1.474 1	1.467 6	1.628 8	5.73
	1.690 2	1.512 5	1.552 5

Sample group / nmol·L^-1	Measured value (radian)	RSD/%
50	0.214 4	0.223 3	0.215 8	3.45
	0.232 9	0.231 2	0.220 5
100	0.319 0	0.318 4	0.310 1	3.69
	0.303 6	0.337 5	0.311 7
500	1.474 1	1.467 6	1.628 8	5.73
	1.690 2	1.512 5	1.552 5

Method	Detection principle	LOD	LOQ	Detection range	Ref.
Detection method based on manganese-doped zinc sulfide quantum dots	Mn-doped ZnS phosphorescent QDs-based phosphorescence signal enhancement for rapid PMB detection and dosage calibration	0.03 μg·mL^-1	-	0.1-8.0 μg·mL^-1	[50]
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	PMB (B1/B2) in protein-precipitated BALF (IS: polymyxin E1) was detected by ESI-MRM MS after XB-C18 separation, with ELF concentration calibrated by urea for intrapulmonary pharmacokinetic analysis of aerosolized PMB	-	-	0.030 0/0.003 06-10.0/1.02 mg·L^-1	[51]
Based on the optically active supramolecular aggregates of water-soluble perylene diimide	Water-soluble PDI derivatives served as fluorescent probes; PMB triggered fluorescence quenching of PDI via non-covalent interaction-driven supramolecular aggregation in aqueous media, with the quenching degree correlated to PMB concentration for quantitative detection	18.5 nmol·L^-1	-	1-2 000 nmol·L^-1	[52]
LPS@AuNIs	LSPR-based LPS@AuNIs sensor: Specific PB-LPS binding changes the local refractive index of AuNIs, causing LSPR phase response variation for label-free, highly sensitive quantitative detection of PB via the phase-PB concentration dose-effect relationship	6.34 ± 0.002 3 nmol·L^-1	61.7 ± 0.001 735 nmol·L^-1	10-5 000 nmol·L^-1	This work

Method	Detection principle	LOD	LOQ	Detection range	Ref.
Detection method based on manganese-doped zinc sulfide quantum dots	Mn-doped ZnS phosphorescent QDs-based phosphorescence signal enhancement for rapid PMB detection and dosage calibration	0.03 μg·mL^-1	-	0.1-8.0 μg·mL^-1	[50]
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	PMB (B1/B2) in protein-precipitated BALF (IS: polymyxin E1) was detected by ESI-MRM MS after XB-C18 separation, with ELF concentration calibrated by urea for intrapulmonary pharmacokinetic analysis of aerosolized PMB	-	-	0.030 0/0.003 06-10.0/1.02 mg·L^-1	[51]
Based on the optically active supramolecular aggregates of water-soluble perylene diimide	Water-soluble PDI derivatives served as fluorescent probes; PMB triggered fluorescence quenching of PDI via non-covalent interaction-driven supramolecular aggregation in aqueous media, with the quenching degree correlated to PMB concentration for quantitative detection	18.5 nmol·L^-1	-	1-2 000 nmol·L^-1	[52]
LPS@AuNIs	LSPR-based LPS@AuNIs sensor: Specific PB-LPS binding changes the local refractive index of AuNIs, causing LSPR phase response variation for label-free, highly sensitive quantitative detection of PB via the phase-PB concentration dose-effect relationship	6.34 ± 0.002 3 nmol·L^-1	61.7 ± 0.001 735 nmol·L^-1	10-5 000 nmol·L^-1	This work

基于等离子体近场的LPS@AuNIs生物传感器从中药中筛选天然抗菌活性成分

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侯慧楠 ¹ , 徐斌 ¹ , 岳浩然 ¹ , 高文浩 ¹ , 孙璐 ¹ , 张子璇 ¹ , 刘泽林 ² , 赵颖 ³ , 辛军红 ¹ , 桂婷 ¹ , 王信 ⁴ , 盖秩舶 ⁵^,⁶^,^*

药学学报 | 研究论文 2026,61(8): 3074-3084

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药学学报 |研究论文 2026 , 61 (8) : 3074 -3084

基于等离子体近场的LPS@AuNIs生物传感器从中药中筛选天然抗菌活性成分

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侯慧楠¹, 徐斌¹, 岳浩然¹, 高文浩¹, 孙璐¹, 张子璇¹, 刘泽林², 赵颖³, 辛军红¹, 桂婷¹, 王信⁴, 盖秩舶⁵^,⁶^,^*

作者信息

¹山东中医药大学中医药创新研究院, 山东济南 250355

²山东中医药大学医学院, 山东济南 250355

³济宁医学院生命科学学院, 山东日照 276827

⁴山东中医药大学附属医院, 山东济南 250014

⁵山东中医药大学实验中心, 山东济南 250355

⁶中医药经典理论教育部重点实验室, 山东济南 250355

通讯作者:

盖秩舶, E-mail: zhibo.gai@sdutcm.edu.cn

作者简介:

共同第一作者.

作者贡献:

侯慧楠、徐斌负责实验方案设计与实施、数据处理与分析、文章撰写等工作; 岳浩然、高文浩、孙璐负责实验操作、数据处理及文章撰写; 张子璇、刘泽林、赵颖负责文献检索、图形和文章修改等工作; 辛军红、王信协助实验内容完成, 并修改完善文章; 桂婷、盖秩舶负责课题总体设计、实验技术指导及文章修改。

Screening of natural antibacterial active components from traditional Chinese medicines based on LPS@AuNIs biosensor driven by plasmonic near-field

Hui-nan HOU¹, Bin XU¹, Hao-ran YUE¹, Wen-hao GAO¹, Lu SUN¹, Zi-xuan ZHANG¹, Ze-lin LIU², Ying ZHAO³, Jun-hong XIN¹, Ting GUI¹, Xin WANG⁴, Zhi-bo GAI⁵^,⁶^,^*

Affiliations

¹Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

²School of Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

³School of Life Sciences, Jining Medical University, Rizhao 276827, China

⁴Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, China

⁵Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

⁶Key Laboratory of Traditional Chinese Medicine Classical Theory, Ministry of Education, Jinan 250355, China

出版时间: 2026-08-12 doi: 10.16438/j.0513-4870.2026-0178

文章导航

摘要

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通过等离子体近场传感对中药单体成分的检测, 快速筛选作用于脂多糖(lipopolysaccharide, LPS) 靶点的天然抗菌活性成分。本研究构建了一种基于金纳米粒子(gold nanoislands, AuNIs) 和LPS的等离子体近场生物传感器(LPS@AuNIs生物传感器), 并利用相位响应变化评估中药来源天然产物的LPS拮抗活性。LPS@AuNIs生物传感器对能与LPS特异性结合的多黏菌素B (polymyxin B, PB) 的检测限(limit of detection, LOD) 为6.34 ± 0.002 374 nmol·L^-1、定量限(limit of quantification, LOQ) 为61.7 ± 0.001 735 nmol·L^-1。对14种中药单体的检测结果显示, LPS@AuNIs生物传感器对β-五没食子酰葡萄糖(1, 2, 3, 4, 6-O-pentagalloylglucose, β-PGG) 和京尼平苷(geniposide, GP) 均呈现良好的浓度依赖性响应。对比发现, β-PGG表现出强效结合作用, β-PGG的k_a优于PB。通过最小抑菌试验(minimum inhibitory concentration, MIC) 与BODIPY TR cadaverine (BC) 置换实验进一步验证发现, β-PGG表现出与PB相同的强结合亲和力。本传感器可以实现LPS与待测成分的特异性结合, 抗干扰能力强, 兼具高灵敏度与高检测效率, 实现高精度定量检测。该研究创新性融合纳米光子学与天然药物研究, 建立了“药物筛选-靶点验证”一体化体系, 为天然抗菌活性成分的快速有效筛选提供了高效技术平台。

关键词

金纳米粒子 / 等离子体近场传感 / 快速检测 / 脂多糖 / 天然药物 / 靶向

Abstract

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To rapidly screen natural antibacterial active components targeting lipopolysaccharide (LPS) through the detection of traditional Chinese medicine (TCM) monomers via plasmonic near-field sensing. In this study, a plasmonic near-field biosensor based on gold nanoislands (AuNIs) and LPS (LPS@AuNIs biosensor) was constructed, and the LPS antagonistic activity of natural products derived from TCM was evaluated by the change in phase response. The limit of detection (LOD) and limit of quantification (LOQ) of the LPS@AuNIs biosensor for polymyxin B (PB), which specifically binds to LPS, were 6.34 ± 0.002 374 and 61.7 ± 0.001 735 nmol·L^-1, respectively. Detection results of 14 TCM monomers showed that the LPS@AuNIs biosensor exhibited excellent concentration-dependent responses to both 1, 2, 3, 4, 6-O-pentagalloylglucose (β-PGG) and geniposide (GP). Comparative analysis revealed that β-PGG displayed potent binding activity, with a dissociation constant (k_a) superior to that of PB. Further verification via minimum inhibitory concentration (MIC) and displacement assays confirmed that β-PGG possessed strong binding affinity comparable to PB. The developed biosensor enables specific binding between LPS and analytes, featuring high sensitivity and detection efficiency for high-precision quantitative analysis. This study innovatively integrates nanophotonics with natural medicine research, establishing an integrated system of "drug screening-target validation" and providing a high-efficiency technical platform for the rapid and effective screening of natural antibacterial active components.

Key words

gold nanoisland / plasmonic near-field sensing / rapid detection / lipopolysaccharide / natural medicine / target

引用本文

侯慧楠, 徐斌, 岳浩然, 高文浩, 孙璐, 张子璇, 刘泽林, 赵颖, 辛军红, 桂婷, 王信, 盖秩舶. 基于等离子体近场的LPS@AuNIs生物传感器从中药中筛选天然抗菌活性成分. 药学学报, 2026 , 61 (8) : 3074 -3084 . DOI: 10.16438/j.0513-4870.2026-0178

Hui-nan HOU, Bin XU, Hao-ran YUE, Wen-hao GAO, Lu SUN, Zi-xuan ZHANG, Ze-lin LIU, Ying ZHAO, Jun-hong XIN, Ting GUI, Xin WANG, Zhi-bo GAI. Screening of natural antibacterial active components from traditional Chinese medicines based on LPS@AuNIs biosensor driven by plasmonic near-field[J]. Acta Pharmaceutica Sinica, 2026 , 61 (8) : 3074 -3084 . DOI: 10.16438/j.0513-4870.2026-0178

正文

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脂多糖(lipopolysaccharide, LPS) 作为革兰阴性菌外膜的核心组分^[1], 脂质A是LPS发挥毒性的核心结构。LPS不仅是细菌生存的关键结构, 还可通过激活宿主Toll样受体4 (toll-like receptor 4, TLR4)/髓样分化受体2 (myeloid differentiation-2, MD-2) 复合物引发过度炎症反应, 导致脓毒症等致命并发症^[2]。因此, 靶向抑制细菌LPS的生物合成、运输或其本身, 成为克服耐药性(antimicrobial resistance, AMR) 的重要策略^[3-5]。基于此, 寻找一种有效筛选靶向LPS的天然产物的新方法, 以加快革兰阴性菌感染疾病治疗药物的研发是本研究工作的重点和焦点。

天然产物是由生物体(如植物、微生物、海洋生物等) 在其生命活动中合成的结构独特且生物活性显著的有机化合物^[6], 目前临床上使用的抗生素中绝大部分直接或间接来源于天然产物。多黏菌素最初从多黏类芽孢杆菌的发酵产物中发现, 经分离纯化、修饰改造后, 逐渐应用于临床治疗, 被誉为临床治疗多重耐药革兰阴性菌感染的最后一道防线。目前在临床上应用的产品主要是多黏菌素B (polymyxin B, PB) 和多黏菌素E (polymyxin E, PE) 的硫酸盐和甲磺酸盐。PB属于阳离子多肽抗生素, 具有双重作用机制, 既能靶向作用于革兰阴性菌外膜的LPS发挥抗菌功效, 又能中和内毒素^[7], 但神经毒性和肾毒性等不良反应也是不可忽视的问题。同时单味中药如金银花、黄芩、栀子、大青叶、板蓝根等及源自中药的单体成分如黄芩苷、京尼平苷(geniposide, GP)、姜黄素、虎杖苷、丹参素、β-五没食子酰葡萄糖(1, 2, 3, 4, 6-O-pentagalloylglucose, β-PGG)、白鲜皮生物碱类成分, 有直接对抗内毒素的作用^[8-12]。因此中药及复方制剂能提高机体防护能力、降低内毒素的活性, 减轻或者预防由LPS诱导的损伤。

传统天然药物筛选方法效率低下^[13], 需要通过大量的体外抑菌实验和动物模型验证活性, 耗时耗力, 难以满足高通量筛选需求。传统筛选方法(如细胞实验) 假阳性率高的同时周期长、干扰成分大、机制解析不足^[14], 严重制约了临床转化进程。动物模型(如小鼠模型、斑马鱼、果蝇) 尽管能提供整体药效数据, 但其高昂成本和较长的实验过程限制了大规模应用^{[15, 16]}。而探针合成、靶标垂钓^[17]也存在探针合成难度大、假阳性风险高、通量低、技术门槛高等局限。作为分子互作领域的“金标准”的表面等离子共振(surface plasmon resonance, SPR) 技术同样具有复杂样本易干扰、非特异性吸附难区分、设备成本高等缺点^[18]。本项目使用的局域表面等离子体共振(localized surface plasmon resonance, LSPR) 传感器通过局域表面等离子体共振效应, 具有灵敏度高、特异性强的优点^[19-22]。等离子体近场传感是一种基于LSPR、近场增强等特性的传感技术, 通过监测生物分子相互作用过程中传感器芯片表面折射率的细微变化, 实时反映分子结合的动力学特征, 直接反映药物与靶点的特异性结合^{[23, 24]}。同时兼具实时定量、样品用量少、可重复使用等特点, 为天然药物活性筛选和作用机制解析提供高效、可靠的技术支撑(表1)。

本文通过将LPS修饰在表面功能化后的金纳米粒子(gold nanoislands, AuNIs) 表面, 制成LPS@AuNIs等离子体近场生物传感器。采用通过该传感器对中药单体成分进行筛选, 同时, 验证不同浓度下潜在中药单体成分的拮抗能力, 为天然抗菌活性成分的快速有效筛选提供了高效技术平台, 推动了智能传感技术在抗感染治疗中的应用(图1)。

材料与方法

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药品与试剂 11-巯基十一烷酸(11-mercaptoundecanoic acid, MUA, 批号674427)、1-(3-二甲基氨基丙基)-3-乙基碳二亚胺[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC, 批号39391]、N-羟基丁二酰亚胺(N-ethylsuccinimide, NHS, 批号130672)、乙醇胺(ethanolamine, EA, 批号398136)、LPS (批号L2880) 购于美国Sigma-Aldrich公司; 2-吗啉乙磺酸缓冲液(2-morpholinoethanesulphonic acid buffer, MES, 批号pH176) 购于福州飞净生物科技公司; PB (批号A610318) 购于上海生工生物工程有限公司; 磷酸缓冲盐溶液(phosphate buffered saline, PBS, 批号CR0015) (10×) 购于山东思科捷生物技术有限公司; β-PGG (批号P421756)、GP (批号G422835)、万古霉素(vancomycin, 批号V301569)、红霉素(erythromycin, 批号E105345)、利福平(rifampicin, 批号R105455)、阿莫西林(amoxicillin, 批号L352648)、氟康唑(fluconazole, 批号E129360)、阿奇霉素(azithromycin, 批号A134451)、小檗碱(berberine, 批号B422528)、白藜芦醇(resveratrol, 批号R408711)、大黄素(emodin, 批号E409173)、葛根素(puerarin, 批号P408466)、丹参素(Danshensu, 批号D425886)、绿原酸(chlorogenic acid, 批号C422343)、哈巴苷(harpagide, 批号H425547)、龙胆苦苷(gentiopicroside, 批号G422524)、姜黄素(curcumin, 批号C408195)、黄柏碱(phellodendrine, 批号P425481)、土槿乙酸(pseudolaric acid B, 批号P426132)、穿心莲内酯(andrographolide, 批号A407762) 购于上海阿拉丁生化科技股份有限公司; 4-羟乙基哌嗪乙磺酸缓冲液(2-(4-(2-hydroxyethyl) piperazin-1-yl)ethanesulfonic acid, HEPES, 批号)、BODIPY TR cadaverine (BC, 批号D6251) 购于赛默飞世尔科技公司。

仪器光谱仪(Avaspec-ULS2048TEC) 购于北京爱万提斯(AVANTES) 科技有限公司; 马弗炉(SX2-4-10) 购于上海善志仪器设备有限公司; LED光源(LZ1-00W00)、BK7直角玻璃棱镜(32-336)、偏振片(LPVISC050) 购于索雷博光电科技(上海) 有限公司(Thorlabs); 酶标仪(Infinite^® 200 PRO) 购于瑞士TECAN。

光谱处理方法及检测原理 白光LED光源的可发射光谱范围为480~730 nm, 通过线性宽带偏振片(Edmund Optics, #89-602) 进行线性偏振后, 光线经过双折射晶体并产生p偏振光与s偏振光之间的光程差^[25]。采用BK7直角玻璃棱镜实现全内反射, 并在玻璃/分析物界面处激发自组装(self-assembly, SAM) AuNIs的LSPR效应, 所用入射角为65°^[26]。

当AuNIs受入射光激发产生LSPR电场时, 引发等离子体共振的局域电场增强效应, 显示出强近场捕获能力。当目标样品与AuNIs接触后, 近场光场与其发生相互作用, 通过共振耦合效应进一步增强局部电磁场。而局部折射率的变化会引起LSPR共振频率和相位的变化, 从而提供相关的分子生物物理特性(如折射率、分子质量等) 的局部变化信号。采用LSPR原理构建的等离子体近场生物传感器, 由于其同时具备了等离子体近场光学操控能力及平行原位检测能力^{[27, 28]}, 且具有高灵敏度、无标记和实时测量的优点^[29], 使得其在检测领域具有潜在的技术优势。

AuNIs的表面功能化及LPS@AuNIs生物传感器的建立 采用本课题组发表的热退火SAM方法^[23]进行在介电玻璃基底上制备AuNIs。即清洗过的BK7玻璃载玻片(35 mm × 25 mm × 2 mm) 与Au膜进行磁控溅射, 然后在550 ℃下在空气中退火3 h, 可得到AuNIs芯片。溅射后的Au膜厚度为5.5 nm, 其对应的AuNIs结构能兼顾灵敏度与稳定性, 后续多数传感实验基于该厚度制备, 退火温度升高速率固定为15 ℃·min^-1。

Xu等^[30]已完成功能化修饰的方法。将AuNIs芯片放入无水乙醇中超声1 min, 然后用无水乙醇冲洗20 min (流速20 μL·min^-1)。在AuNIs芯片上注入浓度为20 mmol·L^-1的MUA溶液30 min, 然后用无水乙醇冲洗20 min。接着, 注入固定比例的EDC/NHS混合物(0.4 mol·L^-1 EDC和0.15 mol·L^-1 NHS 1∶1混合), 实现偶联最优效果, 然后用PBS缓冲液冲洗10 min。注入LPS (1∶1 000) 溶液50 min (在每次修饰之前, 将LPS溶液超声处理20 min, 以分散LPS聚集物), 用PBS缓冲液冲洗10 min, 注入0.1 mol·L^-1的EA溶液3 min, 封闭非特异性结合位点, 用PBS缓冲液冲洗10 min。通过调节第二偏振片, 完成4次光路调节, 建立共光路干涉, 从而建立LPS@AuNIs生物传感器。

LPS@AuNIs生物传感器检测PB灵敏度 将表面功能化的AuNIs芯片与LPS标准品连接, 然后用EA阻断。接着采用PBS缓冲液冲洗20 min建立基线, 分别注入不同浓度的PB标准品溶液(分别为5 000、1 000、500、100、50、10 nmol·L^-1) 25 min, 最后采用PBS缓冲液冲洗30 min。采用LSPR光谱仪检测相位响应, 通过加窗傅里叶变换计算相位变化, 分析PB标准品各浓度的相位响应及光谱变化。检测原理如图2A所示。

LPS@AuNIs生物传感器检测PB精密度测试 配置高、中、低3个浓度梯度的PB标准品溶液, 进行精密度检测。对每个浓度的样本, 连续重复测定6次, 计算每个样本相对标准偏差(relative standard deviation, RSD)。

LPS@AuNIs生物传感器检测PB的抗干扰能力和特异性测试 取万古霉素、红霉素、利福平、阿莫西林、氟康唑、阿奇霉素及PB (浓度均为500 nmol·L^-1) 等样本, 通过对比不同样本的检测响应信号, 评估LPS@AuNIs传感器的抗干扰能力和特异性。

LPS@AuNIs生物传感器对靶向LPS天然药物的筛选 分别吸取20 μL 10 mmol·L^-1的中药单体原液(即β-PGG、GP、小檗碱、白藜芦醇、大黄素、葛根素、丹参素、绿原酸、哈巴苷、龙胆苦苷、姜黄素、黄柏碱、土槿乙酸、穿心莲内酯这14个中药单体) 溶于1 980 μL的PBS缓冲液, 配制得到标准品溶液。将表面功能化的AuNIs芯片与LPS标准品反应, 用EA阻断。接着采用PBS缓冲液冲洗20 min建立基线, 分别注入14种中药单体标准品溶液各25 min, 最后采用PBS缓冲液冲洗30 min。采用LSPR光谱仪检测相位响应, 通过加窗傅里叶变换计算相位变化, 分析其相位响应及光谱变化。检测原理如图2B所示。

LPS@AuNIs生物传感器对β-PGG靶向结合LPS能力的检测 将表面功能化的AuNIs芯片与LPS标准品连接, 然后用EA阻断。接着采用PBS缓冲液冲洗20 min建立基线, 分别注入不同浓度的β-PGG标准品溶液(50、10、5、1 μmol·L^-1) 25 min, 采用PBS缓冲液冲洗30 min。采用LSPR光谱仪检测相位响应, 通过加窗傅里叶变换计算相位变化, 分析其相位响应及光谱变化。

LPS@AuNIs生物传感器对GP靶向结合LPS能力的检测 将表面功能化的AuNIs芯片与LPS标准品连接, 然后用EA阻断。接着采用PBS缓冲液冲洗20 min建立基线。然后, 分别注入不同浓度的GP溶液(500、300、100、50、10 μmol·L^-1) 25 min。最后, 采用PBS缓冲液冲洗30 min, 分析其相位响应及光谱变化。

最小抑菌浓度测定 最小抑菌浓度(minimum inhibitory concentration, MIC) 测定遵循临床和实验室标准协会(Clinical and Laboratory Standards Institute, CLSI) 推荐的方案进行。所有化合物均溶于无菌纯水中, 配制成12.8 mg·mL^-1的储存液。实验中, PB用作革兰阴性菌的阳性对照。将化合物储存液在96孔板中进行连续稀释, 采用2倍稀释法, 使每孔终体积为50 μL, 最高质量浓度可达64 μg·mL^-1。将测试菌株的过夜培养物用新鲜培养基稀释5 000倍, 然后取50 μL稀释菌液加入各孔中。培养16 h后, 以肉眼观察不到细菌生长的最低化合物浓度记录为MIC值。所有测定均平行重复两次, 并独立重复实验3次。

BC置换测定 通过评估待测药物从LPS-BC混合物中置换BC的能力, 来测定其与LPS的亲和力。将20 μg·mL^-1 LPS与20 μmol·L^-1 BC在5 mmol·L^-1 HEPES缓冲液(pH 7.4) 中预混合, 并于37 ℃温育30 min。随后, 将混合物转移至96孔平底黑色微孔板中(每孔50 μL)。加入50 μL不同质量浓度的β-PGG (溶解于5 mmol·L^-1 HEPES中), 使其终质量浓度范围为0.0~2.0 μg·mL^-1。以PB作为阳性对照。在避光条件下温育5 min后, 使用Infinite 200 Pro酶标仪测定荧光强度(激发/发射波长= 580/620 nm)。

数据处理与分析 使用MATLAB软件中的WFT方法提取不同浓度的β-PGG、PB和GP标准品溶液注入前后光谱的提取相位变化。提取的数据采用GraphPad Prism 9.5.0软件绘制时间-相位变化图及标准曲线。

结果

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1　LPS@AuNIs生物传感器功能化结果

在已发表文章中^[30], 已完成该传感器对梯度浓度乙醇溶液的相位响应, 从而改变AuNIs芯片上的环境介质, 检查AuNIs芯片的可重复使用性, 结果表明本研究使用的AuNIs芯片具有良好的灵敏度及可逆性。同时完成生物传感器的功能化, 确定了最佳反应条件(图3): 以20 mmol·L^-1 MUA、0.4 mol·L^-1 EDC和0.15 mol·L^-1 NHS混合物、pH 4.7的MES缓冲液。在这一条件下进行的AuNIs表面修饰不仅表现出较高的表面覆盖率和良好的稳定性, 还具有优异的生物相容性。接着将LPS修饰在表面功能化后的AuNIs芯片表面, 成功构建等离子体近场生物传感器(LPS@AuNIs生物传感器)。

2　LPS@AuNIs生物传感器对PB靶向结合LPS能力检测及其灵敏度、精密度和特异性研究

PB分子的疏水端可以与LPS的脂质A部分紧密结合, 并且通过静电作用中和脂质A所带的负电荷。这就有效地阻断了LPS与宿主TLR4/MD2受体复合物的结合, 从而避免过度的炎症反应(如细胞因子风暴) 出现, 抑制脓毒症的进一步发展。本研究建立LPS@AuNIS生物传感器, 使用PB为阳性对照, 首先利用LPS与PB相互作用机制, 检测LPS@AuNIs生物传感器对PB靶向结合LPS能力的灵敏度、精密度和特异性研究。

不同浓度PB溶液的相位响应如图4所示。通过检测浓度范围为10~5 000 nmol·L^-1的PB标准品溶液, LPS@AuNIs生物传感器展现出显著的浓度依赖性响应特征。实验结果表明(图4A~C), 随着PB浓度的逐步降低, 传感器的相位响应呈现指数衰减趋势, 并在低浓度区间趋于稳定。具体而言, 在空白PBS缓冲液条件下, 传感器的相位响应基线为-0.009 748 ± 0.029 96 rad, 为信号分析提供了稳定的基准参考。通过对实验数据的非线性拟合分析(图4D), 建立了描述相位响应与PB浓度对数值之间关系的数学模型: Y = 0.028 22e^1.301X, R² = 0.991。其中X表示PB浓度, 单位为log₁₀ (nmol·L^-1), Y表示系统的相位响应。该模型表明实验数据与理论曲线高度吻合, 验证了传感器在PB检测中的灵敏度和重现性。计算得出, LPS@AuNIs生物传感器检测PB的LOD为6.34 ± 0.002 374 nmol·L^-1, LOQ为61.7 ± 0.001 735 nmol·L^-1。且在本LPS@AuNIs生物传感器实验条件下, PB与LPS的结合速率常数(association rate constant, k_a) 为4.6×10⁴ L·mol^-1·s^-1, 解离速率常数(dissociation rate constant, k_d) 为1.3×10³ s^-1, 表明其可在10~5 000 nmol·L^-1范围内实现PB的高精度定量检测。同时重复性实验结果显示(表2), LPS@AuNIs生物传感器在PB浓度为50~500 nmol·L^-1范围内, RSD值为3.45%~5.73%, 表明本生物传感器具有较好的精密度。

选择不靶向结合LPS、对PB的作用靶点无交叉, 且在实验体系中无相互干扰的抗生素, 即万古霉素、红霉素、利福平、阿莫西林、氟康唑、阿奇霉素, 分别注入LPS@AuNIS生物传感器。将上述样品的响应信号与PB的响应信号进行了对比。检测结果(图5A) 可以显示, 在相同浓度下(500 nmol·L^-1) LPS@AuNIs生物传感器对PB的响应信号(1.520 6 ± 0.04 rad) 是对其他抗生素的响应信号的29~45倍, 表明该生物传感器对靶向结合LPS具有极好的特异性、抗干扰能力强, 为后续实验提供稳定的检测特性。

3　中药单体的筛选

抗菌中草药临床使用历史悠久, 对机体各器官不良反应小, 可在菌体内作用途径多样化, 通常表现为整体量效作用, 能产生多方面药理效应, 被认为极具潜力成为替代抗生素的抗菌药之一, 其单体成分同样具有抗内毒素作用, 且物质基础和作用机制明确。通过查询文献^[31-44]及TCM-ID数据库, 从具有清热解毒功效的中药中搜索出多种可能与LPS发生拮抗作用的单体成分, 即β-PGG、GP、小檗碱、白藜芦醇、大黄素、葛根素、丹参素、绿原酸、哈巴苷、龙胆苦苷、姜黄素、黄柏碱、土槿乙酸、穿心莲内酯。采用构建的LPS@AuNIs生物传感器, 对固定浓度(100 nmol·L^-1) 的单体成分与阳性药物PB进行对比检测, 比较结合效率、浓度依赖性响应等关键指标。

结果如图5B所示, 100 nmol·L^-1浓度的β-PGG、GP的相位响应分别为2.42 ± 0.02和0.148 9 ± 0.01 rad, 表明其具有较高的相位响应。说明β-PGG、GP具有较强靶向结合LPS的作用。而小檗碱等中药单体的相位响应范围为0.082 91~0.033 06 rad, 接近传感器的相位响应基线(-0.009 748 ± 0.029 96 rad), 表明小檗碱等中药单体没有靶向结合LPS的作用。接下来, 着重对β-PGG、GP浓度对靶向结合LPS的能力进行探究。

4　 β-PGG靶向结合LPS能力探究结果

研究发现, PGG能够抑制经LPS刺激的腹膜巨噬细胞内IL-1β、TNF-α和IL-6等促炎因子的产生, 同时增加具有抗炎作用的细胞因子IL-10的表达水平^[45]。为进一步明确β-PGG对LPS的靶向结合能力, 开展了梯度浓度β-PGG与LPS的相互作用实验。LPS@AuNIs生物传感器对不同浓度β-PGG溶液的相位响应如图6A所示。通过检测浓度范围为1~100 μmol·L^-1的β-PGG标准品溶液, LPS@AuNIs传感器展现出显著的浓度依赖性响应特征。随着β-PGG浓度的逐步降低, 传感器的相位响应呈现指数衰减趋势, 并在低浓度区间(< 5 μmol·L^-1) 趋于稳定。趋势与PB相似, 提示其具有强效的LPS结合能力。通过对实验数据的非线性拟合分析(图6B), 建立了描述相位响应与β-PGG浓度对数值之间关系的数学模型: Y = 0.061 59e^1.843X (R² = 0.994)。计算得出LOD为0.062 ± 0.001 660 μmol·L^-1。同时计算得出β-PGG与LPS靶向结合的k_a为2.5×10⁵ L·mol^-1·s^-1, 可以实现与LPS的靶向结合。

5　GP靶向结合LPS能力探究结果

相关研究表明^[46], GP可以通过抑制导致NF-κB激活的信号通路, 减少LPS诱导的巨噬细胞中促炎细胞因子(如TNF-α、IL-6) 的产生。鉴于初步筛选已证实GP具有一定的LPS靶向结合潜力, 为深入明确其靶向结合能力与浓度的量化关系, 开展梯度浓度GP与LPS的相互作用验证实验。LPS@AuNIs生物传感器对不同浓度GP溶液的相位响应如图6C所示。通过检测浓度范围为10~500 μmol·L^-1的GP标准品溶液, LPS@AuNIs传感器展现出显著的浓度依赖性响应特征。随着GP浓度的逐步降低, 传感器的相位响应呈现指数衰减趋势, 并在低浓度区间(< 10 μmol·L^-1) 趋于稳定, 变化趋势与PB具有较大的相似性, 说明GP同样能与LPS进行强效结合。通过对实验数据的非线性拟合分析(图6D), 建立了描述相位响应与GP浓度对数值之间关系的数学模型: Y = 0.005 680e^1.557X (R² = 0.983), 计算得出LOD为5.71 ± 0.017 03 μmol·L^-1, GP的k_a为5.0×10³ L·mol^-1·s^-1, 可以实现与LPS的靶向结合。

6　最小抑菌浓度与BC置换结果

经对比, β-PGG与LPS结合的k_a值(2.5×10⁵ L·mol^-1·s^-1), 优于PB (4.6×10⁴ L·mol^-1·s^-1) 及GP (5.0×10³ L·mol^-1·s^-1), 这与β-PGG的靶向性强具有密切关系。因此作者通过一系列实验对β-PGG的潜在靶点作进一步的验证。

首先, 检测了β-PGG的MIC^[47], 将10 μL稀释后的大肠杆菌(ATCC 25922) 悬液依次加入浓度梯度的β-PGG药物孔(0.25~256 μg·mL^-1) 中, 置于37 ℃细菌培养箱中培养16~18 h。相同浓度梯度的PB作为阳性对照。3次检测结果显示, β-PGG的最低抑菌浓度(MIC) 值为2 μg·mL^-1, 而PB的MIC为0.25 μg·mL^-1。

然后, 评估了在大肠杆菌细菌成分存在下的抗菌活性, 选取的大肠杆菌细菌成分包括总基因组DNA、总蛋白质及细胞壁成分, 如肽聚糖(peptidoglycan) 和LPS (图7A)。虽然添加DNA或蛋白质并未显著改变β-PGG的MIC, 但仍有可能在提取过程中某些DNA或蛋白质复合物被解散或部分变性, 从而阻碍β-PGG与大肠杆菌的相互作用。相比之下, LPS以剂量依赖的方式抑制了β-PGG的抗菌活性(图7B), 这表明LPS可能是β-PGG的主要靶点。此外, β-PGG竞争性地排除了脂质A特异性的荧光染料BODIPY-TR, 这进一步确认了其与LPS之间的强结合亲和力(图7C)。以上结果与传感实验的结果一致表明, β-PGG通过特异性结合细胞壁成分LPS发挥拮抗作用。

讨论

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细菌耐药性的全球蔓延已成为21世纪公共卫生领域的重大挑战。中草药因其多靶点、整体调节的特性, 在抗感染领域展现出独特潜力。例如, 金银花提取物绿原酸、连翘提取物连翘苷等^{[48, 49]}不仅能直接抑制细菌生长, 还能通过调控炎症信号通路(如NF-κB) 减轻LPS诱导的损伤。

本文构建了一种基于等离子体近场的LPS@AuNIs生物传感器, 实现天然产物抗菌活性的高通量筛选与快速检测。通过AuNIs芯片表面功能化策略(MUA/EDC/NHS偶联体系), 偶联LPS标准品, 开发兼具高灵敏度与检测效率的LPS@AuNIs生物传感器。将本方法与已报道的检测PB的方法进行对比发现(表3^[50-52]), 基于水溶性苝酰亚胺光学活性超分子聚集体及基于锰掺杂硫化锌量子点检测方法的LOD较高, 且该技术需要通过化学发光试剂放大信号, 可能会引入干扰或误差。本传感器无需标记、灵敏度高(传感器的LOD为6.34 ± 0.002 374 nmol·L^-1, LOQ为61.7 ± 0.001 735 nmol·L^-1), 且在10~5 000 nmol·L^-1范围内具有显著的非线性相关性(相关系数为0.991), 可以实现PB的高精度定量检测, 且抗干扰能力强, 可以直接对样品进行检测, 避免了外源试剂可能带来的干扰, 初步确定了传感器的传感特性。

为了确定拮抗LPS的中药单体, 通过对文献及数据库筛选出的中药单体进行初步筛选, 并通过LPS@AuNIs生物传感平台检测, 结果发现β-PGG与GP的相位响应较高, 具有一定的靶向结合LPS的能力。接着系统评估β-PGG、GP等天然产物的LPS拮抗活性, 可以发现β-PGG和GP在本传感器中表现出显著的浓度依赖性响应, 进一步的研究证实, β-PGG的k_a为2.5×10⁵ L·mol^-1·s^-1, 优于PB (k_a = 4.6×10⁴ L·mol^-1·s^-1)。且通过MIC与BC置换实验进一步验证发现, β-PGG表现出与PB类似的强结合亲和力。

本研究构建的LPS@AuNIs生物传感器, 与传统的基于SPR技术的抗菌药物筛选方法相比, 通过不同的修饰策略, 建立LPS的检测传感器, 具有无标记、灵敏检测, 特异性强的优点, 同时可以实现定时定量、节约成本、重复使用等, 为天然药物活性成分的筛选与作用机制的系统解析, 搭建高效稳定的技术平台, 为相关研究提供新的技术思路。

在LPS检测上, 目前的检测方法主要包括鲎试剂法、细胞生物学方法、免疫学方法、色谱质谱联用法及生物传感技术。其中, 鲎试剂法(limulus amebocyte lysate, LAL)^[53]是公认的LPS检测标准, 具有灵敏度高、操作简便等优势, 但易受样品基质干扰, 且无法对LPS结构进行精准区分; 而细胞生物学方法^[54]可以反映LPS的体内生物活性, 但检测周期长、重复性较差; 色谱质谱技术^[55]也因仪器昂贵、前处理繁琐, 难以满足现场快速检测需求无法实现检测。而本研究构建的检测传感器在检测LPS过程中, 具有快速、实时、无标记、灵敏等特点, 可以为复杂基质中LPS的高效、灵敏检测提供新的检测方案。

与传统的最小抑菌浓度检测、BC荧光染料置换测定相比, 本传感器具有以下优点: ①实现直接检测LPS与待测成分的特异性结合; ②检测时间缩短至1~2 h; ③检测灵敏度高, 实现高精度定量检测, 突破传统方法效率低下、耗时耗力、灵敏度低、定量精度不足的缺点^{[56, 57]}。但本研究仅模拟了LPS与拮抗剂的体外结合过程, 并对已知天然产物单体成分进行了筛选, 未涉及体内复杂的微环境(如血清蛋白干扰、代谢稳定性等), 以及药材提取物等复杂样本的检测。利用分子间相互作用原理建立药材提取物中活性分子靶向垂钓系统, 是目前中药现代化研究中一个非常热门的领域, 后续将通过建立“LSPR分子垂钓”系统, 从中药提取物中筛选靶向结合目标物质(如LPS) 的单体成分, 提高LPS@AuNIs生物传感器应用价值。

总体而言, 本研究为天然抗菌活性成分的快速有效筛选提供创新性解决方案, 为中药微生物及细菌活性成分的精准鉴定开辟了新路径。

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2026年第61卷第8期

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doi: 10.16438/j.0513-4870.2026-0178

接收时间：2026-02-10
首发时间：2026-09-21
出版时间：2026-08-12

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收稿日期：2026-02-10
修回日期：2026-03-12

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¹山东中医药大学中医药创新研究院, 山东济南 250355

²山东中医药大学医学院, 山东济南 250355

³济宁医学院生命科学学院, 山东日照 276827

⁴山东中医药大学附属医院, 山东济南 250014

⁵山东中医药大学实验中心, 山东济南 250355

⁶中医药经典理论教育部重点实验室, 山东济南 250355

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盖秩舶, E-mail: zhibo.gai@sdutcm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Experimental method	Advantage	Disadvantage	Ref.
Cell experiment	Simulate inflammatory response	In vitro experiments cannot reflect the effectiveness in vivo, long cycle	[14]
Animal model	Similar genetic background and immune system to humans	High cost, time-consuming, complicated operation	[15, 16]
Enzyme activity inhibition assay	Complete and reproducible method system	Difficult probe synthesis, long cycle, high cost, cumbersome operation	[17]
Surface plasmon resonance (SPR)	Real-time detection, label-free, high sensitivity	Interference from complex samples, high equipment cost	[18]
Localized surface plasmon resonance (LSPR)	Real-time and label-free detection, high sensitivity, strong specificity		[19-22]

Experimental method

Advantage

Disadvantage

Ref.

Cell experiment

Simulate inflammatory response

In vitro experiments cannot reflect the effectiveness in vivo, long cycle

[14]

Animal model

Similar genetic background and immune system to humans

High cost, time-consuming, complicated operation

[15, 16]

Enzyme activity inhibition assay

Complete and reproducible method system

Difficult probe synthesis, long cycle, high cost, cumbersome operation

[17]

Surface plasmon resonance (SPR)

Real-time detection, label-free, high sensitivity

Interference from complex samples, high equipment cost

[18]

Localized surface plasmon resonance (LSPR)

Real-time and label-free detection, high sensitivity, strong specificity

[19-22]

Sample group / nmol·L^-1	Measured value (radian)	RSD/%
50	0.214 4	0.223 3	0.215 8	3.45
	0.232 9	0.231 2	0.220 5
100	0.319 0	0.318 4	0.310 1	3.69
	0.303 6	0.337 5	0.311 7
500	1.474 1	1.467 6	1.628 8	5.73
	1.690 2	1.512 5	1.552 5

Sample group / nmol·L^-1

Measured value (radian)

RSD/%

0.214 4

0.223 3

0.215 8

3.45

0.232 9

0.231 2

0.220 5

100

0.319 0

0.318 4

0.310 1

3.69

0.303 6

0.337 5

0.311 7

500

1.474 1

1.467 6

1.628 8

5.73

1.690 2

1.512 5

1.552 5

Method	Detection principle	LOD	LOQ	Detection range	Ref.
Detection method based on manganese-doped zinc sulfide quantum dots	Mn-doped ZnS phosphorescent QDs-based phosphorescence signal enhancement for rapid PMB detection and dosage calibration	0.03 μg·mL^-1	-	0.1-8.0 μg·mL^-1	[50]
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	PMB (B1/B2) in protein-precipitated BALF (IS: polymyxin E1) was detected by ESI-MRM MS after XB-C18 separation, with ELF concentration calibrated by urea for intrapulmonary pharmacokinetic analysis of aerosolized PMB	-	-	0.030 0/0.003 06-10.0/1.02 mg·L^-1	[51]
Based on the optically active supramolecular aggregates of water-soluble perylene diimide	Water-soluble PDI derivatives served as fluorescent probes; PMB triggered fluorescence quenching of PDI via non-covalent interaction-driven supramolecular aggregation in aqueous media, with the quenching degree correlated to PMB concentration for quantitative detection	18.5 nmol·L^-1	-	1-2 000 nmol·L^-1	[52]
LPS@AuNIs	LSPR-based LPS@AuNIs sensor: Specific PB-LPS binding changes the local refractive index of AuNIs, causing LSPR phase response variation for label-free, highly sensitive quantitative detection of PB via the phase-PB concentration dose-effect relationship	6.34 ± 0.002 3 nmol·L^-1	61.7 ± 0.001 735 nmol·L^-1	10-5 000 nmol·L^-1	This work

Method

Detection principle

LOD

LOQ

Detection range

Ref.

Detection method based on manganese-doped zinc sulfide quantum dots

Mn-doped ZnS phosphorescent QDs-based phosphorescence signal enhancement for rapid PMB detection and dosage calibration

0.03 μg·mL^-1

0.1-8.0 μg·mL^-1

[50]

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

PMB (B1/B2) in protein-precipitated BALF (IS: polymyxin E1) was detected by ESI-MRM MS after XB-C18 separation, with ELF concentration calibrated by urea for intrapulmonary pharmacokinetic analysis of aerosolized PMB

0.030 0/0.003 06-10.0/1.02 mg·L^-1

[51]

Based on the optically active supramolecular aggregates of water-soluble perylene diimide

Water-soluble PDI derivatives served as fluorescent probes; PMB triggered fluorescence quenching of PDI via non-covalent interaction-driven supramolecular aggregation in aqueous media, with the quenching degree correlated to PMB concentration for quantitative detection

18.5 nmol·L^-1

1-2 000 nmol·L^-1

[52]

LPS@AuNIs

LSPR-based LPS@AuNIs sensor: Specific PB-LPS binding changes the local refractive index of AuNIs, causing LSPR phase response variation for label-free, highly sensitive quantitative detection of PB via the phase-PB concentration dose-effect relationship

6.34 ± 0.002 3
nmol·L^-1

61.7 ± 0.001 735
nmol·L^-1

10-5 000 nmol·L^-1

This work