微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

doi:10.3724/SP.J.1123.2021.07009

摘要/Abstract

摘要：

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关。然而人体血液中CTCs的含量非常低,通常来说仅有0~10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集。微流控芯片技术凭借样品消耗少,分离效率高,易于自动化和集成化等特点,在CTCs分离分析研究中具有诸多优势。近年来,利用微流控芯片开展CTCs分离检测的研究进展迅速,多种技术原理和检测方法相继出现。从技术原理角度进行区分,可分为生物亲和法和物理筛选法。生物亲和法主要依赖抗原抗体相互作用,或核酸适配体与靶标的特异性结合,该方法选择性高,但效率和捕获率偏低。物理筛选法则主要依据细胞本身的物理性质,诸如尺寸、密度和介电性质等差异实现分离。例如,可通过芯片微结构对CTCs进行阻隔或捕获,通过外加物理场(声、电、磁)辅助分选,也可以利用微观尺度流体力学作用对混合细胞进行筛分。物理筛选法一般通量较高,但往往分离纯度较低。同时,利用微流控芯片的集成优势将两种方法相结合,往往能得到更好的分离效果。除了以CTCs作为直接目标的正向富集外,还可以采取反向富集的策略,通过将作为干扰项的白细胞等作为靶标进行选择性地去除,可以避免直接筛选方式对CTCs细胞活性产生的影响。该文概述性介绍了利用微流控芯片开展循环肿瘤细胞分离检测的技术原理、芯片原位检测方法和研究进展,并结合现阶段存在的问题对其未来发展趋势予以展望。

关键词: 微流控芯片, 循环肿瘤细胞, 分离检测

Abstract:

The isolation and analysis of circulating tumor cells (CTCs) is an important issue in tumor research. CTCs in peripheral blood, which are important biomarkers of liquid biopsy, are closely related to the occurrence of cancer and are used to monitor the effect of treatment on cancer patients. However, the number of CTCs in the blood samples of cancer patients is very low, usually being present at only 0-10 CTCs/mL. Therefore, prior to the detection of CTCs, it is important to preprocess clinical blood samples for efficient separation and enrichment. With the advantages of low sample consumption, high separation efficiency, ease of automation and integration, microfluidic chips can be a suitable platform for the isolation of CTCs. In the last few years, CTC separation and detection using microfluidic chips have developed rapidly, and a variety of detection methods have been developed. According to the technical principle used, microfluidics for CTC separation can be divided into biological property-based methods and physical property-based methods. The biological property-based methods mainly depend on the interaction between the antigen and antibody, or the specific binding of the aptamer and target. These methods have high selectivity but low efficiency and recovery rates. Physical separation is based on the physical properties of CTCs such as their size, density, and dielectric properties. For example, CTCs can be blocked or captured by the microstructure in the channels of microfluidic chips, sorted by external physical fields (acoustic, electrical, magnetic), or screened by micro-scale hydrodynamics. Physical property-based methods generally have a higher flux but lower separation purity. However, the advantages of biological property-based methods and physical property-based methods can be integrated to provide microfluidic chips having better separation performance. In addition to the direct positive enrichment of CTCs, a negative enrichment strategy can also be adopted. The influence of direct screening on the activity of CTCs can be avoided by selectively removing white blood cells. In this paper, recent advances in microfluidics utilized in the isolation of CTCs, including physical and immune methods and positive and negative enrichment, are reviewed. We summarized the technical principles, detection methods, and research progress in CTC separation and detection using microfluidic chips. Developing trends in microfluidics for CTC separation and analysis are also discussed.

Key words: microfluidics, circulating tumor cells (CTCs), separation and detection

中图分类号:

O658

曹荣凯, 张敏, 于浩, 秦建华. 微流控芯片系统在循环肿瘤细胞分离检测中的应用进展[J]. 色谱, 2022, 40(3): 213-223.

CAO Rongkai, ZHANG Min, YU Hao, QIN Jianhua. Recent advances in isolation and detection of circulating tumor cells with a microfluidic system[J]. Chinese Journal of Chromatography, 2022, 40(3): 213-223.

图/表 6

图1 基于生物亲和原理的循环肿瘤细胞分离芯片

Fig. 1 Microfluidics for separation of circulating tumor cells (CTCs) with biological property-based methods a. microfluidics with epithelial cell adhesion molecule (EpCAM) antibody-modified nanofibers[38]; b. microfluidics with cancer cell-replicated surface[40]; c. microfluidics with fluidic multivalent membrane nanointerface[41]. PLGA: poly lactic-co-glycolic acid; PEG: polyethylene glycol; CellRePDMS: cell-replicated topological structure on the surface of polydimethylsiloxane; PBMC: peripheral blood mononuclear cell.

图2 基于物理筛选方法的循环肿瘤细胞分离芯片

Fig. 2 Microfluidics for separation of circulating tumor cells with physical property-based methods a. acoustic CTC separation chip[47]; b. sequential size-based chip[50]; c. inertial force-based straight chip[52]. WBC: white blood cell; SAW: surface acoustic wave; PDMS: polydimethylsiloxane.

图3 基于综合作用的循环肿瘤细胞分离芯片

Fig. 3 Microfluidics for separation of circulating tumor cells using integrated methods a. microfluidics integrating lateral filter arrays with immunoaffinity[62]; b. antibody-functional microsphere-integrated filter chip with inertial microflow[63]; c. deterministic lateral displacement (DLD)-patterned chip modified with multivalent aptamer-functionalized nanospheres[65]. AP: aptamer; GSH: glutathione.

图4 基于反向富集策略的循环肿瘤细胞分离芯片

Fig. 4 Microfluidics for negative enrichment of circulating tumor cells a. 3D-printed microfluidic device with immunocapture channels and microfiltration[66]; b. microfluidic chip integrated with DLD arrays and magnetic field[68]; c. CD45 antibody-based MACS combined with an inertial focusing chip[69]. MACS: magnetic activated cell sorting; RBC: red blood cell; PLT: platelet.

表1 基于微流控技术的循环肿瘤细胞分离方法

Table 1 Separation methods of circulating tumor cells with microfluidics

Methods	Sample	Throughput/ (mL/h)	Recovery/ %	Depletion of WBCs/%	Viability/ %	Ref.
Biological property-based methods
Antigen modified microstructure	PC3 cells spiked into whole blood	1.2	~92	14 (purity)	~95	[36]
Aptamer functionalized nanointerface	Cancer cells suspended in buffer	0.35	~91	>99.9	~98	[41]
Antigen coated 3D scaffold	MCF-7 cells and WBCs suspended	6	~93	N/A	~91	[42]
	in PBS
Physical property-based methods
Dielectrophoretic field-flow	MDA-MB-435 and PBMN cells	90	~92	>95	~90	[46]
fractionation	suspended in buffer
Surface acoustic wave	Cancer cells and WBCs suspended	7.5	>86	>97	N/A	[47]
	in PBS
Biomimetic filtration membranes	MDA-MB-231 cells spiked into diluted	30	~90	>45 (purity)	~91	[49]
	blood
Deterministic lateral displacement	A549 and K562 cells spiked into	60	>96	>99.99	>98	[51]
	diluted blood
Inertial focusing	A549 cells spiked into lysed blood	180	~94	~79 (purity)	N/A	[56]
Integrated methods
Lateral filter arrays with immunoaffinity	L3.6pl cells spiked into diluted blood	3.6	~95	>99.5	~84	[62]
Immunobeads integrated filter chip	MCF-7 cells spiked into lysed whole	3	>85	~40 (purity)	N/A	[63]
with inertial flow	blood
Multivalent aptamer modified DLD-array	Cancer cells spiked into whole blood	1	~84	~99.99	~96	[65]
Negative enrichment strategy
Immunocapture channels with	Cancer cells spiked into whole blood	0.5	>90	~96	>90	[66]
microfiltration
DLD arrays integrated with MACS	Cancer cells spiked into whole blood	8	~97	>99.9	N/A	[67]
and inertial focusing
MACS combined with inertial focusing	Cancer cells spiked into whole blood	168	~86	~99.97	N/A	[69]

图5 芯片原位检测循环肿瘤细胞方法

Fig. 5 On-chip analysis of circulating tumor cells a. optically combined encoding of single CTC on chip[76]; b. visual quantifiable detection of CTCs in a volumetric bar-chart chip[83]; c. electrochemical determination of iron ions leaking from CTCs by DPV technique[84]. BTC: bis(trichloromethyl)carbonate; magMOF: magnetic metal-organic framework; FITC-GOD: fluorescein isothiocyanate-glucose oxidase; CB: conduction band; VB: valence band.

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