基于微流控芯片的抗恶臭假单胞菌生物膜活性天然产物筛选

doi:10.3877/cma.j.issn.1674-1366.2026.05.002

目的

针对恶臭假单胞菌（P. putida）在根管治疗失败及难治性根尖周炎中日益凸显的致病性及其生物膜耐药问题，解决天然产物因样品量极低而难以开展系统活性评估的瓶颈，探讨基于微流控芯片的微量化、可视化高通量药物筛选平台的构建。

方法

选用P. putida KT2440作为模式菌株，构建绿色荧光蛋白标记菌株并建立微流控生物膜模型。针对珍稀天然产物，确立了以200 μg/mL为固定阈值的"定性快筛"策略，利用荧光线段长度作为量化指标，对40种天然产物进行抗生物膜活性盲筛。以氢氧化钙和环丙沙星分别作为效力下限与上限，界定有效筛选区间，采用t检验比较与传统96孔板微量肉汤稀释法的组间差异，对比两种方法的样品消耗。

结果

微流控筛选平台成功将40种化合物区分为强效、中效和无效3个梯队，最终鉴定出6种具有显著抗P. putida生物膜活性的先导化合物（阳性检出率15%），其中包括新型大环肽类及部分单萜类化合物。微流控芯片筛选结果与传统方法具有极高的总体一致性。样品消耗方面，该平台单次测试仅需纳升（nL）级体积，单化合物消耗量低至8 ng，相比传统96孔板法节约样品约15 000倍。

结论

本研究构建的微流控高通量筛选平台，通过固定浓度定性筛查策略，有效克服了天然产物样品稀缺带来的筛选难题。该平台具有样品消耗低、分析通量高和结果准确可靠等优势，为快速锁定抗耐药菌生物膜的活性先导化合物提供了强有力的技术支撑。

关键词: 微流控芯片, 恶臭假单胞菌, 高通量筛选, 生物膜, 天然产物

Objective

To address the growing pathogenicity and biofilm-mediated drug resistance of Pseudomonas putida (P. putida) in root canal therapy failure and refractory apical periodontitis, and to overcome the bottleneck of "extremely low sample quantity" that hinders the systemic activity evaluation of natural products. This study aims to establish a micro-scale, visual, and high-throughput drug screening platform based on microfluidic chips.

Methods

P. putida KT2440 was selected as the model strain, and a green fluorescent protein (GFP) -labeled strain was constructed to establish a microfluidic biofilm model. To accommodate rare natural products, a "qualitative rapid screening" strategy with a fixed threshold of 200 μg/mL was established. Using the length of the fluorescent line segment as a quantitative metric, 40 natural products derived from myxobacteria and plants were blindly screened for anti-biofilm activity. Calcium hydroxide and Ciprofloxacin served as the lower and upper limits of efficacy, respectively, to define the effective screening range. A t-test was used to compare results with the traditional 96-well plate micro-broth dilution method; consistency between the two methods was verified, and sample consumption was compared.

Results

The microfluidic screening platform successfully categorized the 40 compounds into three tiers: Strong, moderate, and ineffective. Ultimately, 6 lead compounds with significant anti-P. putida biofilm activity were identified (positive detection rate of 15%), including novel macrocyclic peptides and certain monoterpenoids. Methodological validation showed high qualitative consistency between the microfluidic chip screening and traditional methods. regarding sample consumption, the platform required only nanoliter (nL) -level volumes per test, with a single compound consumption as low as 8 ng. This represented a sample saving of approximately 15 000-fold compared to the traditional 96-well plate method, significantly reducing the demand for fermentation products.

Conclusions

The microfluidic high-throughput screening platform constructed in this study effectively overcame the technical challenge of sample scarcity in natural product mining through a fixed-concentration qualitative screening strategy. The platform featured extremely low sample consumption, high analytical throughput, and accurate results, providing strong technical support for rapidly locking onto active lead compounds against drug-resistant bacterial biofilms.

Key words: Microfluidic chip, Pseudomonas putida, High-throughput screening, Biofilm, Natural products

图1 光刻工艺制备高通量筛选微流控芯片　A：光刻工艺制备流程；B：芯片实物图；C：芯片微流道扫描电镜（SEM）图像；PDMS：聚二甲基硅氧烷。

图2 表达绿色荧光蛋白的恶臭假单胞菌（P. putida）在微流道中形成的生物膜与常规培养条件下菌体的形态及荧光特征　A：微流道中生物膜的扫描电镜（SEM）图像；B：正常生长的SEM图像；C：微流道中生物膜的荧光显微镜图像；D：正常生长的荧光显微镜图像。

图3 微流控芯片中不同浓度环丙沙星和氢氧化钙的剩余荧光线段长度　A：荧光显微镜图像；B：百分比统计分布图；图B横坐标采用以2为底的对数刻度；横坐标各点对应的具体浓度顺序与图A中各处理组自上而下的浓度排列一致；横坐标左侧（紫色）与右侧（蓝色）并无连续关系。

表1 6种显著抗恶臭假单胞菌（P. putida）生物膜活性的天然产物及其潜在机制

序号	分子式	剩余荧光线段长度占比（%， ± s，n = 60）	化学分类	结构特点	潜在机制	参考文献
1	C₅₃H₆₅N₁₅O₁₁	13.09 ± 1.82	大环肽	刚性双环骨架，七肽	靶向外膜BamA蛋白	[20，21]
2	C₂₁H₂₀O₆	22.22 ± 2.92	多酚	双酮结构，共轭双键	抑制生物膜黏附和群体感应	[22，23]
3	C₂₀H₁₈NO₄⁺	27.78± 2.13	生物碱	异喹啉骨架，季铵盐	嵌入DNA，抑制FtsZ细胞分裂	[24]
4	C₁₀H₁₄O	43.44 ± 2.53	单萜酚	酚羟基，异丙基	破坏细胞膜完整性	[25，26，27]
5	C₁₀H₁₄O	55.55 ± 2.81	单萜酚	酚羟基，异丙基	破坏细胞膜完整性	[28，29]
6	C₁₀H₁₈O	78.84 ± 2.01	单萜醇	长链醇，无苯环	增加膜通透性	[30，31]

表1 6种显著抗恶臭假单胞菌（P. putida）生物膜活性的天然产物及其潜在机制

序号	分子式	剩余荧光线段长度占比（%， ± s，n = 60）	化学分类	结构特点	潜在机制	参考文献
1	C₅₃H₆₅N₁₅O₁₁	13.09 ± 1.82	大环肽	刚性双环骨架，七肽	靶向外膜BamA蛋白	[20，21]
2	C₂₁H₂₀O₆	22.22 ± 2.92	多酚	双酮结构，共轭双键	抑制生物膜黏附和群体感应	[22，23]
3	C₂₀H₁₈NO₄⁺	27.78± 2.13	生物碱	异喹啉骨架，季铵盐	嵌入DNA，抑制FtsZ细胞分裂	[24]
4	C₁₀H₁₄O	43.44 ± 2.53	单萜酚	酚羟基，异丙基	破坏细胞膜完整性	[25，26，27]
5	C₁₀H₁₄O	55.55 ± 2.81	单萜酚	酚羟基，异丙基	破坏细胞膜完整性	[28，29]
6	C₁₀H₁₈O	78.84 ± 2.01	单萜醇	长链醇，无苯环	增加膜通透性	[30，31]

表2 微流控芯片法与传统96孔板法在天然产物筛选中的样品消耗对比

筛选方法	单次测试体积	单化合物消耗量	所需发酵液当量	加样时间
96孔板微量稀释法	100 ~ 200 μL/孔	约120 μg（重复3次）	约60 L发酵液	10 ~ 40 min
微流控芯片法	~ 2 nL/通道	8 ng（重复60次）	约0.06 L发酵液	<5 min
差异倍数	100倍	15 000倍	100倍	2 ~ 8倍

表2 微流控芯片法与传统96孔板法在天然产物筛选中的样品消耗对比

筛选方法	单次测试体积	单化合物消耗量	所需发酵液当量	加样时间
96孔板微量稀释法	100 ~ 200 μL/孔	约120 μg（重复3次）	约60 L发酵液	10 ~ 40 min
微流控芯片法	~ 2 nL/通道	8 ng（重复60次）	约0.06 L发酵液	<5 min
差异倍数	100倍	15 000倍	100倍	2 ~ 8倍

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Microfluidic chip-based screening of natural products with activity against Pseudomonas putida biofilms

模态框（Modal）标题

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Microfluidic chip-based screening of natural products with activity against Pseudomonas putida biofilms