微液滴高通量筛选方法的研究与应用进展

doi:10.12211/2096-8280.2023-033

摘要/Abstract

摘要：

在单细胞层面对生物功能进行高通量的分析和分选是对关键基因、元件、途径与细胞工厂进行优化的重要技术。基于微液滴的筛选方法因其低成本、超高通量等优势，已被广泛应用于生物、医药、食品和工业等各个领域。本文针对目前主流的荧光激活的液滴分选、吸光度激活的液滴分选，以及无标记液滴分选等微液滴筛选设备的进展进行综述，主要包括基于质谱、拉曼、核磁共振、电化学、图像识别等。并总结了近年来微液滴筛选设备在酶进化、微生物育种等领域应用成功的案例。此外还对不同的微液滴筛选设备的优势与面临的挑战进行了讨论，未来各种新的荧光探针的开发以及质谱等非标记检测方法的进一步发展，将是微液滴筛选设备的主要发展方向，在蛋白质工程、抗体工程、细胞分选及临床研究等方面具有重要的应用潜力。

关键词: 高通量筛选, 微流控, 分选方法, 微液滴, 检测信号

Abstract:

High throughput analysis and sorting of biological functions at the single-cell level is an important technology for optimizing the performance key genes, elements, pathways, and cell factories. The screening method based on microdroplet has been widely applied in various fields such as biology, medicine, food, and industry due to its advantages of low cost and ultra-high throughput. Traditional droplet sorting includes droplet generation, incubation, operation, and sorting. For the first three steps, there have been many technological advances in the last decade. The major limitation is in the sorting step, whose frequency and diversity restrict the sorting efficiency and target scope. According to the different principles of detection signals, droplet sorting technology is mainly divided into labeled and unlabeled sorting method. This article mainly reviews the progress of microdroplet screening equipment based on mainstream fluorescence-activated droplet sorting (FADS) to detect fluorescence signals, absorbance-activated droplet sorting (AADS) to detect UV/visible light absorption changes, and unlabeled droplet sorting, such as mass spectrometry, Raman spectrometry, nuclear magnetic resonance, electrochemistry, and image recognition. This article summarized the successful cases of microdroplet screening equipment applied in the fields of enzyme evolution and microbial breeding in the past 5 years. In addition, we also discussed the advantages and challenges faced by different microdroplet screening devices, and pointed out that the development of various new fluorescent probes and the further development of unlabeled detection methods such as mass spectrometry in the future will be the main development direction of microdroplet screening equipment. Although FADS remains the primary choice for cell sorting, the development of other microfluidic sorting devices has further expanded the application range of microfluidic sorting devices. Its high screening throughput and independent reaction environment provide a new technological platform for research in different fields, including protein engineering, antibody screening, sorting of different types of cells, and clinical directions.

Key words: high-throughput screening, microfluidics, sorting method, microdroplet, detection signal

中图分类号:

Q818

秦伟彤, 杨广宇. 微液滴高通量筛选方法的研究与应用进展[J]. 合成生物学, 2023, 4(5): 966-979.

Weitong QIN, Guangyu YANG. Research and application progress of microdroplets high throughput screening methods[J]. Synthetic Biology Journal, 2023, 4(5): 966-979.

图/表 7

图1 传统的微流控分选流程示意图

Fig. 1 The scheme of traditional microdroplets sorting devices

图2 标记液滴分选技术分选原理FADS—荧光激活的液滴分选技术；AADS—吸光度激活的液滴分选技术；S—底物；P—产物；E—酶分子；Ex—发射光谱；Em—吸收光谱

Fig. 2 The principle of labeled droplet sorting technologyFADS—Fluorescence activated droplet sorting technology; AADS—Absorbance activated droplet separation technology; S—Substrate; P—Product; E: Enzyme molecules; Ex—Emission spectrum; Em—Absorption spectrum

表1 不同微流控分选设备比较

Table 1 Comparison of different microfluidic sorting equipment

分选方法	最高分选效率	最高分选灵敏度	最小液滴体积	优点	缺点
FADS	5 kHz^[42]	2.5 nmol/L^[43]	2 pL^[43]	检测灵敏度高、分选速度快、平台发展成熟	大部分检测靶标缺乏适合的荧光耦联方法
AADS	1 kHz^[44]	10 μmol/L^[21]	100 pL^[45]	普适性较FADS高	检测灵敏度待提高
MADS	35 Hz^[46]	5 μmol/L^[46]	0.8 nL^[46]	无损伤、普适性高	分选速度慢、灵敏度待提高
RADS	4.3 Hz^[47]	50 μmol/L^[48]	65 pL^[47]	无损伤、普适性高	更适用于较大的细胞
NMR-ADS	—	1 mmol/L^[26]	130 pL^[49]	无损伤、提供信息广泛	NMR与液滴分选系统的整合较困难、检测灵敏度低
IBDS	10 Hz^[50]	—	35 pL^[50]	无损伤	适用范围窄、分选速度慢
EADS	10 Hz^[25]	1 μmol/L^[25]	30 nL^[25]	无损伤	适用范围窄、分选速度慢

图3 无标记液滴分选技术分选原理RE—参考电极；WE—工作电极；CE—对电极

Fig. 3 The principle of unlabeled droplet sorting technologyRE—Reference electrode; WE—Working electrode; CE—Counter electrode

表2 近五年微流控分选装置成功应用的案例

Table 2 Cases of successful application of microfluidic sorting devices in the past five years

发表时间	分选系统	目标酶	分选结果	参考文献
2018	FADS	酯酶	对S-布洛芬的对映选择性提高600倍	[17]
2019	FADS	硫酸酯酶	K_cat/K_m值提高30倍	[80]
2019	FADS	纤维素酶	筛选出产量提升46%的高纤维素酶菌株	[81]
2020	FADS	葡萄糖氧化酶	K_cat值比野生型高2.1倍	[82]
2020	AADS	胺脱氢酶	转化率提高3.3倍	[83]
2022	FADS	α-淀粉酶	产量提升50%的地衣芽孢杆菌突变株	[37]
2023	FADS	二乙酰壳二糖脱乙酰酶	催化效率提高1.8倍	[84]
2022	FADS	塑料降解酶	2株可降解塑料的菌株	[85]
2022	FADS	产鼠李糖脂的微生物	产量提升54%～208%的菌株	[86]
2022	AADS	葡萄糖脱氢酶	催化速度和效率提升10倍以上	[87]

图4 SNAPD工作流程图［96］（将单细胞和分析试剂共包裹到微滴中，收集并体外孵育，随后测量每个液滴的荧光以指示靶RNA的扩增）

Fig. 4 Schematic of the SNAPD workflow[96](Single cells are encapsulated into microdroplets with assay reagents, collected and incubated offline, and the fluorescence of each droplet is subsequently measured to indicate amplification of target RNAs)

图5 应用微流控分选设备进行抗体筛选的流程图［98］

Fig. 5 The scheme of antibody screening using microfluidic sorting device[98]

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