Development of a High-Throughput Candida albicans Biofilm Chip

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.本研究开发了一种由白色念珠菌（Candida albicans）纳米生物膜（nano-biofilms）构成的高密度微阵列平台。本研究采用自动化微阵列点样仪（robotic microarrayer），将封装于胶原基质中的白色念珠菌酵母细胞以低至50纳升的体积，点印至经表面修饰的显微镜载玻片上。经孵育后，这些细胞可生长为完整的“纳米生物膜”。 本研究通过扫描电子显微镜（scanning electron microscopy）与共聚焦扫描激光显微镜（confocal scanning laser microscopy），对该生物膜的形态与结构复杂度进行评估；并利用微阵列扫描仪，基于FUN 1代谢过程引发的荧光强度变化，定量评估生物膜的形成程度。该染色技术还可适配抗真菌药敏试验，结果显示：与常规生物膜一致，芯片上的纳米生物膜内的细胞对氟康唑（fluconazole）与两性霉素B（amphotericin B）两类抗真菌剂的耐药水平显著升高。 综上，结构分析与抗真菌药敏试验的结果表明：尽管经过微型化处理，这些生物膜仍展现出生物膜生长模式所特有的典型表型特性。 最终形态的白色念珠菌生物膜芯片（Candida albicans biofilm chip, CaBChip）在单张载玻片上集成了768个独立且空间分隔的等效纳米生物膜，且可同时完成多张芯片的点印与处理流程。相较于当前主流的微生物生物膜构建方法——96孔微孔板模型（96-well microtiter plate model），这款真菌生物膜芯片在微型化与自动化层面具备显著优势：可减少试剂消耗与分析时长，简化高人力投入的操作步骤，并大幅降低检测成本。该芯片可同时完成数百至数千种化合物的快速、便捷且低成本筛选，有望加速抗真菌药物的研发进程。