Microfluidic Evolution‐On‐A‐Chip Reveals New Mutations that Cause Antibiotic Resistance

Abstract

Microfluidic devices can mimic naturally occurring microenvironments and create microbial population heterogeneities ranging from planktonic cells to biofilm states. The exposure of such populations to spatially organized stress gradients can promote their adaptation into complex phenotypes, which are otherwise difficult to achieve with conventional experimental setups. Here a microfluidic chip that employs precise chemical gradients in consecutive microcompartments to perform microbial adaptive laboratory evolution (ALE), a key tool to study evolution in fundamental and applied contexts is described. In the chip developed here, microbial cells can be exposed to a defined profile of stressors such as antibiotics. By modulating this profile, stress adaptation in the chip through resistance or persistence can be specifically controlled. Importantly, chip‐based ALE leads to the discovery of previously unknown mutations in Escherichia coli that confer resistance to nalidixic acid. The microfluidic device presented here can enhance the occurrence of mutations employing defined micro‐environmental conditions to generate data to better understand the parameters that influence the mechanisms of antibiotic resistance.

Emerging microfluidic chips on medical application

“…，⼜称芯⽚实验室(Lab-on-a Chip)或者⽣物芯⽚。微流控技术 [2] 由微 机电加⼯系统(Micro-Electro-Mechanical System，MEMS)发展⽽来，它是⼀种在微⽶级微管 [3] 中精确操纵 [4] 微量流体的技术⼿段，具有将⽣物、化学等实验室微缩到⼀个⼏平⽅厘⽶芯⽚中的基本功能(样品的制备、 分离、反应、检测等)。随着材料科学的发展，作为微流控芯⽚载体的材料也层出不穷，从硅、玻璃到纸基 [5] 、 ⽔凝胶 [6] 以及各类聚合物和纳⽶材料 [7,8] 。与此同时，微流控芯⽚的制备技术也蓬勃发展如丝⽹印刷，喷墨打 印、3D 打印 [9]， [10] 等，⼀些⾼精尖的加⼯技术如⻜秒激光加⼯技术、双光⼦ 3D 打印技术也为⾼精密度的微流 控芯⽚的制作提供了更多可能性。 微流控芯⽚有着微型化、⾼灵敏度、⾼集成、⾼通量、反应快、检测时间短等技术优势，在⽣物医学研 究，合成分析 [11] ，司法鉴定等众多领域有着⼴泛的应⽤(可穿戴微流控设备 [12] 、体外医疗诊断 [13] 、仿⽣⽪肤 [14] 组织器官 [15] ，⽣化 [16] 与环境分析、单细胞分析 [17] 、核酸分析、药物筛选递送 [18] )。迄今为⽌，微型化、集成 化和智能化已经成为现代科技⼿段的⼀个重要趋势，在这样的⼤环境下微流控芯⽚的发展⼗分迅速，它的分 类⽅法丰富多样，可根据不同的分析检测⽅法可以将微流控芯⽚分为电化学检测法 [19] 、光谱分析法(核磁共 振 [20] 、化学发光分析法 [21] )、⾮标记检测法 [22] ，分离富集 [8] 等。 微流控芯⽚是医学领域新⼀代床旁诊断(Point of care testing,POCT) [23,24] 主流技术。POCT 可直接在被检 者身边提供快捷有效的⽣化指标，现场指导⽤药，使检测、诊断、治疗成为⼀个连续过程，对于疾病的早期 发现和治疗具有突破性的意义。基于微流控芯⽚技术的 POCT 仪器发展趋势是⼩型化、操作便捷，直接输⼊ 体液样本 [25] ，即可迅速得到诊断结果 [26] ，由医⽣指导⽤药。⽬前，市场上有多种即时诊断⽅法，仅有简单地 流动测试⼯作没有流体管理技术，⽽当测试复杂性增加时，微流控技术是⼀⻔不可或缺的技术。在治疗肿瘤 癌症的医疗领域⽅⾯，微流控技术同样⼤放异彩，如何使药物特异性杀死肿瘤细胞⽽不伤害正常细胞，这⼀ 直是个业界难题，代尔夫特理⼯⼤学 Jan H. van Esch 的课题组 [27] 将微流控设备中搭载⼀种具有双重功能的新 ⽉形微凝胶，研究发现这些微凝胶对肺癌细胞有⾼亲和、⾼选择性，可以达到肺癌化疗的效果并且减轻副作 ⽤对⼈体的伤害。微流控细胞/组织/器官操作芯⽚是哺乳动物细胞以及微环境操作最重要的技术平台，渴望 应⽤于代替各类动物模型，模拟⼈体内环境(⼈体、组织芯⽚ [28] )，⽤于药物筛查递送，研究药物毒理和药 理作⽤，⼈体耐药性 [29] [30] . (b)基于激光打印技术制备 3D 纳⽶等离⼦体的示意图 [31] .…”

“…(f)Schematic diagram of perfusion of a new artificial placental microfluidic oxygenator (APMBO) for blood analysis [49] . [56] . (b)应⽤于模拟微流控肿瘤切 ⽚模型研究代谢细胞⾏为的示意图 [57] .…”

“…Figure 6 Related applications of microfluidic chips in medical field. (a)Schematic diagram of the mechanism of antibiotic resistance [56] . (b )Schematic diagram of metabolic cell behavior in a simulated microfluidic tumor section model [57] .…”

“…微流控芯⽚技术在药物递送、药物筛选 [60] ⽅式上提供了新的可能，如结合微针达到⽆痛缓控透⽪给药， 再如图 6(a)所示，Ahmed 等⼈ [56] 发明了⼀种微流控芯⽚，它在连续的微室中使⽤精确的化学梯度来执⾏微 there is still a lack of reports on the latest applications of microfluidic chips especially in the medical field. Therefore, we summarize the progress of medical microfluidics research in a comprehensive manner, so that readers can fully understand the field.…”

A Microfluidic Chip for Studies of the Dynamics of Antibiotic Resistance Selection in Bacterial Biofilms

“…These flow systems suffer from randomness with respect to the location for biofilm formation in the devices, and difficulty in harvesting biofilms with precision. Specifically, there are several microfluidic chips developed for antibiotic resistance studies ( Kim et al., 2010 ; Straub et al., 2020 ; Zoheir et al., 2021 ). However, the current devices are either sensitive towards formation of air bubbles which disrupt the cultivation of biofilms on chip or could be sensitive towards changes in the antibiotic concentration gradient generated as a result of biofilm cultivation.…”