CN110951605B - 可用于2019-nCoV病毒高通量检测的阵列式纸基芯片及其制造方法 - Google Patents

可用于2019-nCoV病毒高通量检测的阵列式纸基芯片及其制造方法 Download PDF

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CN110951605B
CN110951605B CN202010083700.7A CN202010083700A CN110951605B CN 110951605 B CN110951605 B CN 110951605B CN 202010083700 A CN202010083700 A CN 202010083700A CN 110951605 B CN110951605 B CN 110951605B
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孙浩
东辉
贾原
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Fuzhou University
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Abstract

本发明涉及一种可用于2019‑nCoV病毒高通量检测的阵列式纸基芯片及其制造方法。所述阵列式纸基芯片，包括从下而上依次设置的玻璃基底层、纸基单元层和单元栅格层，所述包括阵列排列的N个直径为R的圆形纸基检测单元，所述单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元。本发明阵列式纸基芯片结构简单，且制作工艺简单、快速和成品稳定，对加工环境和条件要求很低，加工设备价格低廉；并且，加工过程不涉及任何化学试剂，比紫外光刻等方法更为环保。

Description

可用于2019-nCoV病毒高通量检测的阵列式纸基芯片及其制 造方法
技术领域
本发明涉及一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片及其制造方法。
背景技术
微流控芯片实验室可实现多种单元技术在微小可控平台上灵活组合和规模集成，具有操作简单、成本低廉等优点，可作为仪器类方法的重要补充。自上世纪90年代初期诞生以来，各国团队研发的微流控芯片已成功应用于SARS、H1N1、MERS、HIV、肝炎和埃博拉病毒检测及相关重大传染性疾病的诊治。纸基微流控芯片最初由美国哈佛大学G. Whitesides等学者在2007年提出，一般以常规滤纸为基体材料，借助其内部的毛细纤维结构实现流体的自驱动。
本发明提出了可兼容聚合酶链式反应（95oC~65 oC~25 oC三段循环温控模式）和环介导等温核酸扩增（65oC恒温模式）等多种反应的新型高通量阵列式纸基芯片。旨在为缓解我国2019-nCoV病毒检测压力提供新途径，支撑医疗设备欠缺地区的病毒检测。同时为未来重大公共卫生疾病诊治与防控储备技术。
发明内容
本发明的目的在于提供一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片及其制造方法，该阵列式纸基芯片结构简单，且制作工艺简单、快速和成品稳定，对加工环境和条件要求很低，加工设备价格低廉；并且，加工过程不涉及任何化学试剂，比紫外光刻等方法更为环保。
为实现上述目的，本发明的技术方案是：一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片，包括从下而上依次设置的玻璃基底层、纸基单元层和单元栅格层，所述包括阵列排列的N个直径为R的圆形纸基检测单元，所述单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元。
在本发明一实施例中，所述玻璃基底层与纸基单元层之间通过高透明胶层粘接。
在本发明一实施例中，所述高透明胶层为高透明亚克力双面胶。
在本发明一实施例中，所述纸基单元层采用Whatman Grade 1定性滤纸。
在本发明一实施例中，所述圆形纸基检测单元的容积为0.3-0.5微升。
本发明还提供了一种基于上述所述的可用于2019-nCoV病毒高通量检测的阵列式纸基芯片的制造方法，将纸基单元层、玻璃基底层通过高透明胶层粘接，而后采用数字化激光雕刻/切割机将滤纸激光切割与亚克力粘膜熔覆一体化加工，芯片设计由平面矢量图设计软件完成，并导入激光切割机用户界面，激光器的运动速度、工作功率、运动轨迹的工艺参数可根据设备和环境优化和确定，而后在玻璃基底层上获得阵列排列的N个直径为R的圆形纸基检测单元，同理采用同样加工方法加工单元栅格层，并将单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元，从而得到所需的阵列式纸基芯片。
相较于现有技术，本发明具有以下有益效果：
1. 提高确诊效率：阵列式纸基芯片滤纸内部纤维结构固有的较大比表面积为提高单位体积/时间内生化反应强度提供了自然条件，方法原理上有助于在更短时间内，甚至即时获取低丰度样本信息；同时，芯片检测通量可根据需求而灵活设计，单次分析即可提供数十至数千个样本信息，极大提升临床确诊效率；
2. 降低检测成本：在试剂消耗方面：滤纸芯片检测单元反应体积通常处于或低于亚微升级，远低于常规反应管（20微升以上）内试剂消耗；在仪器成本方面：纸基芯片成本可低至1元人民币以内，其检测技术可独立于商业设备，避免对贵重仪器尤其是价格数百万元进口设备的依赖；
3. 规避操作繁冗：滤纸芯片不引入仪器复杂的能源、光学、电子和机械系统，不涉及设备运维等专技要求，极大简化了实验流程和操作难度；
4. 简化反应维度：精确的温度控制是体外核酸扩增反应的必备条件，常规PCR或LAMP温控一般由桌面级温控箱或内置加热/冷却池实现，传热过程在多方向，核酸扩增在三维体积内，反应管材料一般为导热性能普通的聚丙烯；纸基芯片传热过程是单方向，核酸扩增可视作在二维纸层平面内，导热材料可灵活装配，温控响应更精确。
附图说明
图1为本发明纸基芯片结构。
图2是本发明纸基芯片结构设计示意图。
图3是本发明纸基芯片温控反应试剂蒸发抑制测试；其中，A) 室温中芯片上纯水与3种亲水性墨水液滴；B) 室温中液封后的纯水与3种亲水性墨水溶液；C) 65 oC热板加热测试中的纸基芯片；D) 98 oC热板加热测试中的纸基芯片。
图4是核酸反应测试中的芯片样机。
具体实施方式
下面结合附图，对本发明的技术方案进行具体说明。
本发明提供了一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片，包括从下而上依次设置的玻璃基底层、纸基单元层和单元栅格层，所述包括阵列排列的N个直径为R的圆形纸基检测单元，所述单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元。所述玻璃基底层与纸基单元层之间通过高透明胶层粘接。所述高透明胶层为高透明亚克力双面胶。所述纸基单元层采用Whatman Grade 1定性滤纸。所述圆形纸基检测单元的容积为0.3-0.5微升。
本发明还提供了一种基于上述所述的可用于2019-nCoV病毒高通量检测的阵列式纸基芯片的制造方法，将纸基单元层、玻璃基底层通过高透明胶层粘接，而后采用数字化激光雕刻/切割机将滤纸激光切割与亚克力粘膜熔覆一体化加工，芯片设计由平面矢量图设计软件完成，并导入激光切割机用户界面，激光器的运动速度、工作功率、运动轨迹的工艺参数可根据设备和环境优化和确定，而后在玻璃基底层上获得阵列排列的N个直径为R的圆形纸基检测单元，同理采用同样加工方法加工单元栅格层，并将单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元，从而得到所需的阵列式纸基芯片。
以下为本发明的具体实现过程。
本发明提供了一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片，其为可兼容聚合酶链式反应（95oC~65 oC~25 oC三段循环温控模式）和环介导等温核酸扩增（65oC恒温模式）等多种反应的新型高通量阵列式纸基芯片。
本发明芯片设计过程如下：
考虑到手持操作与观测的便携性，以及后续包装运输，芯片基底选择通用载玻片，整体外观尺寸为25×75 mm。纸片基体材料选择Whatman Grade 1定性滤纸，32个直径3 mm圆形纸基检测单元以4行8列形式布列，纸基单元与玻璃基底由高透明亚克力双面胶（厚度0.1 mm）粘接。反应单元理论容积约0.67微升，实际可用容积为0.3~0.5微升。采用相同设计理念，芯片检测通量可进一步拓展。这种设计方案大幅提升了现有同类纸基芯片检测能力，首次将纸基芯片检测通量与常规多孔板阵列匹配，是领域内首次。圆形纸基检测单元间由黑色栅格结构隔断，该结构可以有效阻拦单元间样本交叉干扰并降低荧光观测时背景噪声。芯片结构如图1、2所示。
本发明芯片制造如下：
针对本发明阵列式芯片设计，在此自主提出一种新型工艺，采用滤纸激光切割与亚克力粘膜熔覆一体化加工，在常规载玻片上一次性获得数十个检测单元。单块纸基芯片耗材成本低至0.8元人民币，加工周期约3分钟/片。经加工参数调试，芯片良品率接近100%。本工艺具有简单、快速和成品稳定等优点，对加工环境和条件要求很低，设备价格低廉。并且，加工过程不涉及任何化学试剂，比紫外光刻等方法更为环保。
本发明芯片反应区液封：
温度影响核酸反应试剂中酶活性，直接决定核酸扩增是否有效。微流控技术液滴式数字化PCR或连续载流式PCR均需在实验前验证高温加热条件下的试剂蒸发和聚合物结构表面分子吸附等问题。本发明采用无色透明且化学惰性极强的石蜡液滴封装圆形纸基反应单元，测试了液封效果（图3）。试验表明：未做液封处理的纸基单元内墨水溶液或纯水（0.5 微升）经65℃热板加热20分钟后完全蒸发；石蜡液封处理（虚线框）后反应单元内的纯水或墨水经65℃热板加热60分钟（或95℃热板加热45分钟）后，仍保持液态，证明了芯片反应区液封方法的工程可靠性。
核酸反应测试中的芯片样机如图4所示。
本发明阵列式纸基芯片具有以下优点：
1. 提高确诊效率。阵列式纸基芯片滤纸内部纤维结构固有的较大比表面积为提高单位体积/时间内生化反应强度提供了自然条件，方法原理上有助于在更短时间内，甚至即时获取低丰度样本信息；同时，芯片检测通量可根据需求而灵活设计，单次分析即可提供数十至数千个样本信息，极大提升临床确诊效率。
2. 降低检测成本。在试剂消耗方面：滤纸芯片检测单元反应体积通常处于或低于亚微升级，远低于常规反应管（20微升以上）内试剂消耗；在仪器成本方面：纸基芯片成本可低至1元人民币以内，其检测技术可独立于商业设备，避免对贵重仪器尤其是价格数百万元进口设备的依赖。
3. 规避操作繁冗。滤纸芯片不引入仪器复杂的能源、光学、电子和机械系统，不涉及设备运维等专技要求，极大简化了实验流程和操作难度。
4. 简化反应维度。精确的温度控制是体外核酸扩增反应的必备条件，常规PCR或LAMP温控一般由桌面级温控箱或内置加热/冷却池实现，传热过程在多方向，核酸扩增在三维体积内，反应管材料一般为导热性能普通的聚丙烯；纸基芯片传热过程是单方向，核酸扩增可视作在二维纸层平面内，导热材料可灵活装配，温控响应更精确。
以上是本发明的较佳实施例，凡依本发明技术方案所作的改变，所产生的功能作用未超出本发明技术方案的范围时，均属于本发明的保护范围。

Claims (1)

1.一种可用于2019-nCoV病毒高通量检测的阵列式纸基芯片，其特征在于，包括从下而上依次设置的玻璃基底层、纸基单元层和单元栅格层，所述纸基单元层包括阵列排列的N个直径为R的圆形纸基检测单元，所述单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元；所述玻璃基底层与纸基单元层之间通过高透明胶层粘接；所述高透明胶层为高透明亚克力双面胶；所述纸基单元层采用Whatman Grade 1定性滤纸；所述圆形纸基检测单元的容积为0.3-0.5微升；所述可用于2019-nCoV病毒高通量检测的阵列式纸基芯片的制造方法为：将纸基单元层、玻璃基底层通过高透明胶层粘接，而后采用数字化激光雕刻/切割机将滤纸激光切割与亚克力粘膜熔覆一体化加工，芯片设计由平面矢量图设计软件完成，并导入激光切割机用户界面，激光器的运动速度、工作功率、运动轨迹的工艺参数可根据设备和环境优化和确定，而后在玻璃基底层上获得阵列排列的N个直径为R的圆形纸基检测单元，同理采用同样加工方法加工单元栅格层，并将单元栅格层的单元栅格与纸基检测单元一一对应，以隔开各纸基检测单元，从而得到所需的阵列式纸基芯片。
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