CN111944656B - 一种微流控细胞磁捕获与检测系统及其捕获与检测方法 - Google Patents
一种微流控细胞磁捕获与检测系统及其捕获与检测方法 Download PDFInfo
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Abstract
本发明公开一种微流控细胞磁捕获与检测系统及其捕获与检测方法。将抗趋化因子受体CXCR4的抗体(12G5)、荧光分子Alexa Fluor 647(F647)修饰在磁性四氧化三铁纳米颗粒表面,构建磁性荧光纳米探针,并结合微流控芯片分选与磁捕获,以及荧光定量分析仪进行急性髓系白血病耐药细胞的检测。磁性荧光纳米探针能特异性靶向急性髓系白血病耐药细胞。将细胞与纳米探针共孵育后,以一恒速通过所述微流控芯片,在微流控芯片一端的细胞捕获区外加静磁场,被磁标记的细胞由于受到磁力作用滞留在微流控芯片的细胞捕获区内,而未被磁标记的阴性细胞以及溶液中多余的纳米探针会从微流控芯片的出口流出。
Description
技术领域
本发明属于生物与医学纳米材料及技术领域,具体涉及一种基于磁性荧光纳米探针结合微流控芯片与荧光定量分析仪实现细胞的捕获与检测。
背景技术
急性髓系白血病(AML)是造血系统一种常见的恶性肿瘤,主要表现为髓系祖细胞不同程度的分化成熟障碍,白血病细胞的恶性增殖和凋亡受阻,正常造血功能的紊乱。据2019年美国癌症数据统计,在所有类型的白血病中,急性髓系白血病(AML)患者新增人数与死亡人数均为第一,尤其是AML患者的死亡人数是其他类型白血病的2~10倍。目前AML诱导治疗的标准是阿糖胞苷与蒽环类药物(柔红霉素等)等广谱化疗药物联合化疗,容易发生耐药与复发。目前AML完全缓解率为60%~80%,然而50%~70%患者仍会产生耐药和复发,降低了AML生存期。急性髓系白血病的耐药和复发是目前AML的治疗瓶颈,因此解决AML耐药性的问题对于降低其死亡率具有重大意义,其中的关键技术问题之一是AML耐药细胞的捕获与检测。
趋化因子受体CXCR4可作为捕获AML耐药细胞的靶点。研究表明CXCR4与CXCL12生物轴是AML耐药的重要机制。CXCL12结合并激活其在白血病细胞上的同源受体CXCR4,诱导白血病细胞从血液循环迁移到骨髓,使得白血病细胞滞留在骨髓基质微环境中,导致白血病细胞存活增加。同时白血病细胞与造血干细胞竞争相同的骨髓微环境,导致正常造血功能紊乱。此外,白血病细胞通过细胞表面表达的相应受体(CD49d/VLA-4和CD44)与细胞外基质分子(如纤连蛋白和透明质酸)相互作用,不仅赋予细胞与骨髓微环境的黏附力,还通过激活下游信号通路,使得白血病细胞获得耐药和抗凋亡信号。临床数据统计显示CXCR4高表达的患者总生存期和无病生存期更短,耐药与复发的可能性更高。
目前主流的细胞分选方法主要有流式细胞分选和免疫磁珠细胞分选。流式细胞分选是用荧光染料标记的抗体通过受体-配体相互作用标记细胞,基于细胞的荧光和光散射特性实现细胞分选。虽然能获得较高的分离细胞纯度(大于90%),但其仪器昂贵、操作技术难度高,而且通常需要超过105个细胞作为起始上样量。免疫磁珠细胞分选是在磁珠表面偶联特异性抗体以标记靶细胞,随后通过施加磁场以分离磁标记的细胞。然而免疫磁珠细胞分选通常需要配合高梯度磁性细胞分离柱使用,且其达到的强磁场可能会吸引本身有磁性的细胞聚集在分离柱中,导致细胞的非特异性捕获。
因此,发明一种特异性强、准确度高的细胞捕获与检测方法,克服目前细胞分选方法的局限性,能够高效捕获并定量出急性髓系白血病病人外周血中CXCR4阳性的耐药细胞数量,对解决AML耐药性问题、降低其死亡率具有重大意义,具有广泛的临床应用前景。
发明内容
技术问题:本发明的目的是提供一种微流控细胞磁捕获与检测系统及其捕获与检测方法,针对目前急性髓系白血病的耐药与复发现状以及当前细胞分选方法存在的局限,以磁性四氧化三铁纳米颗粒作为载体,设计一种磁性荧光纳米探针,用于微流控实现细胞捕获,并利用荧光定量分析仪实现细胞数量的定量检测。
技术方案:本发明通过以下技术方案予以实现:
本发明的一种微流控细胞磁捕获与检测系统,由磁性荧光纳米探针、微流控芯片以及荧光定量分析仪三部分组成;所述磁性荧光纳米探针是表面具有特异性识别趋化因子受体CXCR4的抗体12G5的四氧化三铁纳米颗粒,所述磁性荧光纳米探针能够特异性靶向样本中的CXCR4阳性细胞;
所述微流控芯片的样品入口通过样品通道与微柱阵列的过滤区的一端相连,该过滤区另一端与样品出口1联通;所述过滤区一端与一段较长的蛇形非对称弯曲通道相连,所述蛇形非对称弯曲通道与纳米探针出口3、细胞捕获区汇合,形成一个通道交汇腔;所述细胞捕获区另一端与非特异性细胞出口2相连;所述微流控芯片整体采用PDMS-玻璃键合,赋予芯片一定硬度,便于插入荧光定量分析仪实现荧光定量检测。
所述细胞捕获区为4mm×1mm的长方形微腔,内部分布了三种不同开口方向的U型结构阵列组合,U型结构中间有一10μm宽的缝隙可以让流体和非特异性细胞通过。
所述微流控芯片,其主流道的深度为40μm。
本发明微流控细胞磁捕获与检测系统的微流控细胞捕获方法包括利用磁性荧光纳米探针特异性标记样本中白血病耐药细胞,然后通过微注射泵进样到微流控芯片,分选掉游离的纳米探针、不相关细胞并特异性将磁标记的白血病耐药细胞捕获到检测区域,最后将芯片插入到荧光定量分析仪读取荧光信号。
该捕获方法包括:
步骤1、在磁性纳米颗粒Fe3O4-PEG即羧基末端聚乙二醇修饰的Fe3O4磁性纳米颗粒溶液中加入抗CXCR4的抗体12G5,同时加入2-吗啉乙磺酸MES调节溶液pH至5.5~6.0,置于20~25℃摇床混匀吸附30~60min;
步骤2、在反应体系中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC,EDC与四氧化三铁纳米颗粒铁元素质量比为0.5~1,于20~25℃摇床进行交联反应得到反应液;
步骤3、将步骤2得到的反应液通过磁分离柱纯化,以除去游离的抗体,撤去磁场,收集磁性纳米探针Fe3O4-PEG-12G5;
步骤4、在步骤3得到的磁性纳米探针Fe3O4-PEG-12G5表面继续修饰AlexaFluorTM647NHS Ester荧光染料简称为F647;在磁性纳米探针Fe3O4-PEG-12G5溶液中,加入荧光染料F647、0.01~0.2M硼酸盐BB、缓冲液,调节溶液pH至8~8.5,20~25℃摇床1~2小时;
步骤5、过磁分离柱分离游离荧光染料,撤去磁场,收集磁性荧光纳米探针Fe3O4-PEG-12G5-F647。
其中,
在步骤1中,所述的磁性纳米颗粒与12G5抗体质量比为1~5,磁性纳米颗粒的质量浓度为0.5~1mg/mL。
在步骤1中,所述的2-吗啉乙磺酸MES为0.01~0.2M,pH 5.5~6.0。
在步骤2中所述的交联反应,反应时间为2~6小时。
在步骤4中,所述的荧光染料F647与磁性纳米探针Fe3O4-PEG-12G5中所含12G5抗体的摩尔比为30~50。
本发明的微流控细胞磁捕获与检测系统的微流控细胞的检测方法包括如下步骤:
步骤1、将样品中的细胞用4%多聚甲醛固定15~30min,0.01M PBS洗涤,质量分数为5%~10%的BSA溶液封闭30~60min,0.01M PBS洗涤,将细胞重悬于含0.5%~1%BSA的PBS溶液中;加入所述的Fe3O4-PEG-12G5-F647磁性荧光纳米探针,加入含有0.5%~1%BSA的PBS溶液,37℃孵育30~60min;
步骤2、将细胞悬液加入注射器,用微注射泵控制5~15μL/min流速通过微流控芯片,微流控芯片捕获区下方平行于流动方向固定一磁铁,以吸引被磁性荧光纳米探针标记的细胞;
步骤3、将芯片插入到与之匹配的荧光定量分析仪,激发波长为610nm,检测波长为690nm,检测出滞留在微流控芯片捕获区的靶细胞的荧光信号。
有益效果:与现有技术相比,本发明具有如下有益效果
本发明与现有细胞捕获与检测方法不同,该方法提出了基于磁性荧光纳米探针的微流控细胞捕获与检测方法,突破了现有细胞分选方法的局限性。目前主流细胞分选方法是流式细胞分选和免疫磁珠细胞分选。然而流式细胞分选仪器昂贵、操作技术难度高,通常需要超过105个细胞作为起始上样量。免疫磁珠细胞分选通常需要配合高梯度磁性细胞分离柱使用才能实现细胞分离,且其达到的强磁场可能会吸引本身有磁性的细胞聚集在分离柱中,导致细胞的非特异性捕获。本发明开发的基于磁性荧光纳米探针的微流控细胞捕获与检测方法结合了微流控和传统的磁分选的优点,在维持高的细胞捕获效率以及分选纯度的同时,具有成本低、操作简单、不需要强梯度磁场、仪器尺寸小、可便携、所需试剂量少、处理少量细胞、分选过程自动化的优势,具有便携性和临床应用的潜力。
附图说明
图1是磁性荧光纳米探针的制备过程示意图。
图2(A)是高温热解法制备的油酸修饰的Fe3O4纳米颗粒的透射电镜图;图2(B)为配体添加法制备的水溶性二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)修饰的Fe3O4纳米颗粒(Fe3O4-PEG)的磁滞回线;图2(C)为磁性荧光纳米探针(Fe3O4-PEG-12G5-F647)的荧光发射谱(激发波长640nm)。
图3是微流控芯片设计图。
图4是基于磁性荧光纳米探针的微流控细胞捕获与检测方法过程示意图。
图5是微流控芯片的细胞捕获情况。(A)捕获区上端的荧光图;(B)捕获区中部的荧光图;(C)捕获区下端的荧光图;(D)通向出口的支路处的荧光图;(E-J)捕获区上端、中部、下端的放大图;(H)实验装置现场图。A-D的标尺为200μm,E-J的标尺为50μm。
图6是微流控芯片的细胞捕获与检测的性能评估。(A)线性范围;(B)检测限;(C)检测细胞数与真实细胞数之间的拟合曲线;(D)检测细胞数与真实细胞数之间的相对偏差。
具体实施方式
下面结合附图和具体实施例,进一步阐明本发明,应理解这些实施方式仅用于说明本发明而不用于限制本发明的范围,在阅读本发明之后,本领域的技术人员对本发明的各种等价形式的修改均落于本申请的权利要求所限定的范围。
本发明所述一种基于磁性荧光纳米探针的微流控细胞捕获与检测系统由磁性荧光纳米探针、微流控芯片以及荧光定量分析仪三部分组成;检测方法包括利用磁性荧光纳米探针特异性标记样本中白血病耐药细胞,然后通过微注射泵进样到微流控芯片,分选掉游离的纳米探针、不相关细胞并特异性将磁标记的白血病耐药细胞捕获到检测区域,最后将芯片插入到荧光定量分析仪读取荧光信号。
本发明所述磁性荧光纳米探针的构建如图1所示,核心部分是DSPE-PEG修饰的磁性纳米颗粒,分别将抗体12G5、荧光分子F647修饰在Fe3O4-PEG纳米颗粒,构建用于细胞捕获的磁性荧光纳米探针(Fe3O4-PEG-12G5-F647)。该磁性荧光纳米探针具有特异性靶向CXCR4阳性细胞的能力。
具体实施例如下:
实施例1
高温热解法制备油相尺寸为20nm的Fe3O4纳米颗粒
取7.06g乙酰丙酮铁(Fe(acac)3)粉末于500mL三颈烧瓶中,然后加入100mL二苄醚和23mL油酸,混合均匀,在130mL/min的氮气保护及冷凝回流的条件下,进行程序升温。调节控温装置设置成两个阶段的程序升温过程,第一阶段为从室温升至220℃后温度恒定维持1小时,升温速度为3.3℃/min;第二阶段为从220℃继续升温至290℃后维持恒定温度30分钟,升温速度为3.3℃/min。反应结束后,溶液自然冷却至室温,所得产物倾倒入250mL烧杯中,加入无水乙醇洗涤,将烧杯置于磁铁上,待反应产物完全沉淀后,进行磁分离,重复上述洗涤操作,直至上清液呈无色透明状态,完成洗涤。将黑色反应产物加入100mL三氯甲烷,超声溶解,将氧化铁纳米颗粒分散于三氯甲烷中室温保存(浓度为10mg/mL[Fe])。
图2(A)是高温热解法制备的油酸修饰的Fe3O4纳米颗粒的透射电镜图,纳米颗粒平均尺寸为20.26±1.68nm,制得的油溶性Fe3O4纳米颗粒形貌规则、分散均匀、粒径分布较均一。在这一方法中,最终得到的Fe3O4纳米颗粒的所含的铁元素含量为1g,产率大于89%。
实施例2
配体添加法制备水溶性磁性纳米颗粒Fe3O4-PEG
分别称取150mg DSPE-MPEG2000、50mg DSPE-PEG2000-COOH,充分溶解于4mL三氯甲烷中;加入铁含量为10mg的油相Fe3O4纳米颗粒,超声以充分混合;加入4mL去离子水,并超声混合;在70℃水浴锅中旋转蒸发10min,充分除去三氯甲烷,得到透亮的Fe3O4-PEG纳米颗粒水溶液。过220nm滤膜除去团聚体,将获得的纳米颗粒水溶液于4℃冰箱中保存待用。
图2(B)是Fe3O4-PEG纳米颗粒的磁滞回线,水溶性Fe3O4-PEG的矫顽力和剩磁均近似等于0,表明纳米颗粒具有良好的超顺磁性。20nm Fe3O4纳米颗粒的饱和磁化强度为93emu/g[Fe]。
实施例3
化学偶联法制备磁性纳米探针Fe3O4-PEG-12G5
取0.5mg Fe3O4-PEG(以Fe计)溶液,加入100μg 12G5抗体,同时加入100μL MES(0.01M pH 5.5)调节溶液pH至5.5,铁浓度为0.5mg/mL,置于25℃摇床混匀吸附30min(摇速为120rpm)。在反应体系中加入0.5mg EDC进行交联,25℃摇床反应4.5h(摇速为120rpm)。反应结束后,反应液通过磁分离柱纯化,以除去游离的抗体,撤去磁场,收集得到磁性纳米探针Fe3O4-PEG-12G5,于4℃冰箱中保存待用。
实施例4
制备磁性荧光纳米探针Fe3O4-PEG-12G5-F647
在0.5mg Fe3O4-PEG-12G5中按照n(F647/12G5)=30加入荧光分子F647,加入0.2MBB缓冲液(pH 8)调节溶液pH至8,铁浓度为0.5mg/mL,25℃摇床1h(摇速为120rpm)。反应结束后,过磁分离柱分离游离荧光染料,撤去磁场,收集磁性荧光纳米探针Fe3O4-PEG-12G5-F647,于4℃冰箱中保存待用。
图2(C)是磁性荧光纳米探针Fe3O4-PEG-12G5-F647的荧光发射谱,其在670nm处有最大荧光强度。
实施例5
微流控设计
采用Auto CAD设计芯片结构,联系专业公司定制加工PDMS微流控芯片。首先加工5英寸铬版掩膜;加工4英寸纯硅模具,高度为40μm,制备过程包括涂胶、曝光、显影、坚膜、腐蚀和去胶等基本制作过程;制备好的纯硅模具上浇注PDMS,PDMS注塑厚度为3mm,进出口打孔直径为0.6mm,配标准接头导管。微流控芯片为PDMS(3mm)与玻璃(l mm)键合,采用激光切割玻璃,玻璃尺寸与PDMS尺寸相同。
图3是微流控芯片设计图。设计思路是:细胞流体从芯片入口进入后,首先通过微柱阵列的过滤区,以阻挡住大的细胞团块或污染物,防止堵塞微流控芯片。之后细胞经过一段较长的蛇形非对称弯曲通道后,进入细胞捕获区。细胞捕获区为4mm×1mm的长方形微腔,内部分布了三种不同开口方向的U型结构阵列组合,U型结构中间有一10μm宽的缝隙可以让流体和非特异性细胞通过。在细胞捕获区下方放置一恒磁场,被磁标记的细胞由于受到磁吸引而滞留在捕获区的U型区域内,而未被磁标记的细胞则可流出芯片。
实施例5
基于磁性荧光纳米探针的微流控细胞捕获与检测方法
图4是基于磁性荧光纳米探针的微流控细胞捕获与检测方法过程示意图。将磁性荧光纳米探针Fe3O4-PEG-12G5-F647与细胞共孵育后,采用注射泵控制流速,以一恒速通过微流控芯片,在微流控芯片一端的细胞捕获区外加静磁场,被磁标记的细胞由于受到磁力作用滞留在长条形细胞捕获区内,而未被磁标记的CXCR4阴性细胞以及溶液中多余的纳米探针会从出口流出。将芯片细胞捕获区一端插入到与之匹配的荧光定量分析仪,荧光定量分析仪读出捕获区荧光信号值,实现AML耐药细胞的捕获与定量检测。
图5为微流控芯片不同区域的细胞捕获情况,A、B、C分别为捕获区上、中、下端的细胞捕获荧光显微镜图像,D为通向出口的支路处的荧光显微镜图像,E、F、J分别为捕获区上、中、下端的细胞捕获放大图像,H为实验装置现场图。结果表明该微流控芯片能够有效地截留磁标记的靶细胞,细胞进入微流控芯片后,绝大部分进入捕获区,通向出口的支路很少有细胞流入,且细胞主要集中在捕获区的中上端部分。
图6为基于磁性荧光纳米探针的微流控细胞捕获与检测方法的性能评估结果。A为所述方法的线性范围。在阳性HL-60细胞数为104~5*105范围内,微流控细胞捕获与检测的荧光强度与HL-60细胞数之间被验证具有良好的线性关系,R2为0.990,微流控细胞数量检测的标准曲线为y=47.566x+15720.126。B为所述方法的检测限。取细胞数为零的样本重复测定20次,得到相应的信号平均值(M)和标准差(SD),计算M+2SD,然后将M+2SD的结果代入标准曲线方程中,求出对应的细胞数,计算得检测限为1.53*103个。C、D为所述方法的准确度评估,C为检测细胞数与真实细胞数之间的拟合曲线,D为检测细胞数与真实细胞数之间的相对偏差。在106个MS-5细胞中分别加入105、2*105、3*105、4*105、5*105个HL-60细胞(总体系50μL),作为模拟样本,加入Fe3O4-PEG-12G5-F647纳米探针捕获后,采用微流控芯片进行细胞捕获,并采用荧光定量分析仪测定荧光强度,根据标准曲线计算样本中的靶细胞数量,并计算检测值与真实值之间的相对偏差。结果表明检测到的靶细胞数量与真实的靶细胞数量之间具有很好的一致性(R2大于0.99),且检测细胞数量与真实细胞数量之间的相对偏差均小于10%,证明了本发明所述的基于磁性荧光纳米探针的微流控细胞捕获与检测方法具有较高的准确度。
以上详细描述了本发明的实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种等同变换,这些等同变换均属于本发明的保护范围。
Claims (9)
1.一种微流控细胞磁捕获与检测系统,其特征在于,该系统由磁性荧光纳米探针、微流控芯片以及荧光定量分析仪三部分组成;所述磁性荧光纳米探针是表面具有特异性识别趋化因子受体CXCR4的抗体12G5的四氧化三铁纳米颗粒,所述磁性荧光纳米探针能够特异性靶向样本中的CXCR4阳性细胞;
所述微流控芯片的样品入口通过样品通道与微柱阵列的过滤区的一端相连,该过滤区另一端与样品出口1联通;所述过滤区一端与一段较长的蛇形非对称弯曲通道相连,所述蛇形非对称弯曲通道与纳米探针出口3、细胞捕获区汇合,形成一个通道交汇腔;所述细胞捕获区另一端与非特异性细胞出口2相连;所述微流控芯片整体采用PDMS-玻璃键合,赋予芯片一定硬度,便于插入荧光定量分析仪实现荧光定量检测;
所述细胞捕获区为4 mm ×1 mm的长方形微腔,内部分布了三种不同开口方向的U型结构阵列组合,U型结构中间有一10 μm宽的缝隙可以让流体和非特异性细胞通过。
2.根据权利要求1所述的一种微流控细胞磁捕获与检测系统,其特征在于,所述微流控芯片,其主流道的深度为40 μm。
3.一种采用权利要求1所述微流控细胞磁捕获与检测系统的微流控细胞捕获方法,其特征在于,该捕获方法包括利用磁性荧光纳米探针特异性标记样本中白血病耐药细胞,然后通过微注射泵进样到微流控芯片,分选掉游离的纳米探针、不相关细胞并特异性将磁标记的白血病耐药细胞捕获到检测区域,最后将芯片插入到荧光定量分析仪读取荧光信号。
4.根据如权利要求3所述微流控细胞捕获方法,其特征在于,该捕获方法包括:
步骤1、在磁性纳米颗粒Fe3O4-PEG即羧基末端聚乙二醇修饰的Fe3O4磁性纳米颗粒溶液中加入抗CXCR4的抗体12G5,同时加入2-吗啉乙磺酸MES调节溶液pH至5.5~6.0,置于20~25℃摇床混匀吸附30~60 min;
步骤2、在反应体系中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC,EDC与四氧化三铁纳米颗粒铁元素质量比为0.5~1,于20~25℃摇床进行交联反应得到反应液;
步骤3、将步骤2得到的反应液通过磁分离柱纯化,以除去游离的抗体,撤去磁场,收集磁性纳米探针Fe3O4-PEG-12G5;
步骤4、在步骤3得到的磁性纳米探针Fe3O4-PEG-12G5表面继续修饰Alexa Fluor™ 647NHS Ester荧光染料简称为F647;在磁性纳米探针Fe3O4-PEG-12G5溶液中,加入荧光染料F647、0.01~0.2 M 硼酸盐BB、缓冲液,调节溶液pH至8~8.5,20~25℃摇床1~2小时;
步骤5、过磁分离柱分离游离荧光染料,撤去磁场,收集磁性荧光纳米探针Fe3O4-PEG-12G5-F647。
5.如权利要求4所述的微流控细胞捕获方法,其特征在于,在步骤1中,所述的磁性纳米颗粒与12G5抗体质量比为1~5,磁性纳米颗粒的质量浓度为0.5~1 mg/mL。
6.如权利要求4所述的微流控细胞捕获方法,其特征在于,在步骤1中,所述的2-吗啉乙磺酸MES为0.01~0.2 M ,pH 5.5~6.0。
7.如权利要求4所述的微流控细胞捕获方法,其特征在于,在步骤2中所述的交联反应,反应时间为2~6小时。
8.如权利要求4所述的微流控细胞捕获方法,其特征在于,在步骤4中,所述的荧光染料F647与磁性纳米探针Fe3O4-PEG-12G5中所含12G5抗体的摩尔比为30~50。
9.一种采用权利要求1所述微流控细胞磁捕获与检测系统的微流控细胞的检测方法,其特征在于,所述检测方法包括如下步骤:
步骤1、将样品中的细胞用4%多聚甲醛固定15~30 min,0.01 M PBS洗涤,质量分数为5%~10%的BSA溶液封闭30~60 min,0.01 M PBS洗涤,将细胞重悬于含0.5%~1% BSA的PBS溶液中;加入所述的Fe3O4-PEG-12G5-F647磁性荧光纳米探针,加入含有0.5%~1% BSA的PBS溶液,37℃孵育30~60 min;
步骤2、将细胞悬液加入注射器,用微注射泵控制5~15 μL/min流速通过微流控芯片,微流控芯片捕获区下方平行于流动方向固定一磁铁,以吸引被磁性荧光纳米探针标记的细胞;
步骤3、将芯片插入到与之匹配的荧光定量分析仪,激发波长为610 nm,检测波长为690nm,检测出滞留在微流控芯片捕获区的靶细胞的荧光信号。
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