CN114561803B - 一种病毒捕集材料及生产工艺 - Google Patents
一种病毒捕集材料及生产工艺 Download PDFInfo
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Abstract
本申请公开了一种病毒捕集材料及生产工艺，包括支撑材料层、涂覆层以及接枝修饰层，支撑材料层是医用无纺布空气过滤材料；在支撑材料的外层上包覆厚度不大于50nm的涂覆层；所述接枝修饰层包覆在涂覆层远离支撑材料层的一侧，所述接枝修饰层厚度不大于10nm；所述涂覆层按重量份计包括，明胶1000份，Cu/Cu2+0.5‑3份，纤维素40‑200份，甘草酸50‑800；接枝修饰层按重量份计，纤连蛋白1份，RNaseA酶0.05‑3份；接枝修饰层用于特异性亲和吸附并捕捉经过微孔附近的病毒粒子然后协同涂覆层灭活病毒。该材料能够有有效捕集病毒，结合铜离子能够同时灭活病毒。
Description
技术领域
本发明涉及一种病毒的捕集材料及其用途，尤其涉及特异性捕获去除病毒的表面改性与涂层的材料，以及其捕集功能可用于检测病毒的用途。
背景技术
现有的空气过滤材料，例如各种无纺布材料，以及抗菌抗病毒的材料，如氧化铜无纺布，其截留气溶胶中悬浮颗粒的主要原理是， 通过撞击沉降等物理或者动力学方式发挥深层过滤功能，所以对于不同粒度大小的悬浮粒子的截留效果并不是线性的，例如使用N95外科手术口罩的无纺布滤片进行测试，对于微米级的细菌类颗粒的截留率要大于粒度在60-150nm左右的球形粒子，例如流感病毒，二者的截留率比值大于1。 所以使用这样的空气过滤材料，即便是国标或者ASTM标准合格的膜材，对于阻断具有传染性的病毒粒子，例如冠状病毒类的Cov229E,Cov19等就存在潜在的风险，因为这种单纯的物理截留并不具备针对某些危险的病原体的生物学特性的选择性截留以及灭活作用。
所以需要对上述材料进一步改性增加对特定病原体选择性吸附捕集的能力，常规采用的单抗表面接枝工艺不仅原材料昂贵，同时需要考虑很多因素，为了保留单抗的活性需要小心设计固定化工艺流程；另外的一个选择是采用廉价的明胶涂层或者几种赖氨酸聚合物涂层的方式，工艺操作中需要使用一些有毒性的双功能交联试剂，并且涂层厚薄在实际操作中并不易控制也是一个主要问题，在追求合格的涂层比表面积时往往出现滤膜的开孔率，通气参数等下降的缺点，所以需要专门开发较为精密的制膜设备以及操作人员技能培训，均造成生产成本过高。
另外，在滤过材料中使用直接涂覆的氧化铜虽然可以提高病原体杀灭的效率，但同时带来了Cu2+离子的解离释放，例如饮用水游离的Cu2+离子超过2ppm就会带来安全隐患。假如口罩生产时纳米铜未有紧密依附于口罩纤维，佩戴时便有机会直接吸入。尤其是当氧化铜粒子小到纳米单位，其穿透力会更强，吸入后纳米粒子或会产生基因毒性，为健康带来危机；氧化铜纳米颗粒悬浮在空气中将有机会被人体吸入，若数量较大外来物体已可破坏肺部组织，导致肺部纤维化。
另外，直接涂覆氧化铜涂层中Cu2+杀灭病原体利用的原理是，氧化铜尤其是Cu2+容易被病原体结构蛋白通过螯合配位吸附，当病原体吸附进去的Cu2+浓度达到1-10ppm以上可以在数分钟到几十分钟的时间内造成病原体的膜结构破坏。 同时为了实现该功能，过滤材料还必须能满足两个条件：首先病原体与过滤材料先要有紧密的接触以保证有实质性的Cu2+离子传质，同时还需要有足够的Cu2+传质时间。就目前报道的氧化铜空气过滤材料以及工艺均没有实现这些特点。
发明内容
本申请实施例通过提供一种病毒捕集材料以及生产工艺，解决了现有技术中无纺布无法对病毒进行生物学特性截留和灭活的问题，实现了无纺布空气滤材能够对病毒进行生物学截留和灭活的效果。
本申请实施例提供了 一种病毒捕集材料，包括支撑材料层、涂覆层以及接枝修饰层，支撑材料层是医用无纺布空气过滤材料；
在支撑材料的外层上包覆厚度不大于50nm的涂覆层；
所述接枝修饰层包覆在涂覆层远离支撑材料层的一侧，所述接枝修饰层厚度不大于10nm；
所述涂覆层按重量份计包括，明胶1000份，Cu/Cu2+0.5-3份，纤维素40-200份，甘草酸50-800；
接枝修饰层按重量份计，纤连蛋白1份，RNaseA酶0.05-3份；
接枝修饰层用于特异性亲和吸附并捕捉经过微孔附近的病毒粒子然后协同涂覆层灭活病毒。
进一步的，所述涂覆层采用变性明胶蛋白、纤维素/硝化纤维素用过共价方式老化处理后与螯合的Cu/Cu2+、甘草酸发挥病毒灭活功能；
所述的接枝层的纤连蛋白、RNaseA通过自组装的方式与涂覆层的明胶蛋白和纤维素/硝化纤维素通过非共价方式连接组装，协同涂覆层对捕集到的病毒粒子进行灭活。
进一步的，制作涂覆层喷涂溶液浓度为明胶20-200mg/L,硫酸铜/氧化铜0.05-5mg/L,纤维素/硝化纤维素5mg-50mg/L,甘草酸5mg-80mg/L；
所述接枝层喷涂溶液浓度范围纤连蛋白1.0ug-2.0mg/L,RNase A 1.0ug-5.0mg/L。
进一步的，添加的铜或者铜离子不超过1毫摩尔每公斤无纺布。
进一步的，所构建的涂覆层中的Cu/Cu2+的单质形式包埋在明胶/甘草酸/纤维素涂覆层里面，Cu/Cu2+的离子形式全部以非游离的方式与氨基酸残基结合作为催化剂负责催化局部氧化反应；所构建的接枝层的纤连蛋白N端通过自组装的方式固定在底层的明胶蛋白上，纤连蛋白的RGD序列通过亲和吸附的方式与病毒粒子的S蛋白结合。
一种生产工艺，包括以下步骤：
步骤一、用含有Cu2+的（0.05-5ppm硫酸铜/氧化铜）明胶涂层液浸涂喷熔无纺布表面，压缩空气或者氢气离心脱去多余的浸涂液后老化,还原处理；
步骤二、用含（2ug/L-2mg/L）的纤连蛋白，RNase A,甘草酸，硝酸纤维素接枝液修饰，将配置的溶液喷涂至步骤一形成的涂覆层表面。
各组分之间通过自组装方式形成复合结构，其凝胶溶胶的组装顺序是：处于底层的明胶分子以共价方式在基质材料表面固化包埋硫酸铜，硝酸纤维素/纤维素与明胶分子以静电吸附方式组装成网孔结构，纤连蛋白的N端与明胶分子以亲和吸附的方式组装成复合体，纤连蛋白的C端处于游离状态，在N端和C端的中间结构与RNase A组合的方式完成接枝过程。。
申请实施例中提供的一个或多个技术方案，至少具有如下技术效果或优点：
通过采用了纤连蛋白复合病毒灭活成分，有效解决了现有技术中的无纺布无法有效对病毒进行选择性截留的问题，进而实现了无纺布能够截留并灭活病毒的效果。
所述工艺制备的修饰层结合牢固，螯合Cu2+离子主要以单质Cu形式被明胶包埋储存，只有在捕集获得病原体后才产生较大量的活性Cu2+,所以不会存在潜在的毒性，由于本发明是通过了一个独特设计的电化学机制进行消毒，所以需要的Cu2+的涂覆浓度降低到与配基相结合的范围以结合态存在；正是由于此安全浓度特点，在Cu2+产生消毒的整个过程中，Cu2+都是处于和涂层上的氨基酸残基结构相互螯合的结合态，所以完全没有游离的离子释放。
所述工艺制备的修饰层捕捉气相中的病原体病毒后，为后续的Cu2+传质以及灭活过程提供了足够的反应时间，病毒被Fn固定后互相连接成为电子导体，病毒细菌本身具备的过电势让病原体阳极自主触发了电化学反应进行灭活过程；如果病原体不存在就没有明显的阳极反应，材料表面的Cu2+浓度仍然保持低于安全浓度的状态，所以此方案下毒性游离态Cu2+就完全不会解离到液态或者气态，不会流失到环境或者人体内。
附图说明
图1 为恒温微压差测试夹具：
图 2 为涂层液处理膜片的压降变化测试结果；
图 3 为涂层液处理前、后的膜片的透过率Rp（ % ）的定性比较；
图 4 Cov229E捕获膜片的循环伏安扫描曲线；
图 5 为接枝液3修饰后膜片S蛋白吸附载量测试结果。
图中，1.不锈钢盘片式滤器进口；2. 测试膜片；3. 承压微孔板；4.不锈钢盘片式滤器出口；5. 恒温水浴外套。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本申请进行更全面的描述；附图中给出了本发明的较佳实施方式，但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施方式；相反地，提供这些实施方式的目的是使对本发明的公开内容理解的更加透彻全面。
需要说明的是，本文所使用的术语“垂直”、“水平”、“上”、“下”、“左”、“右”以及类似的表述只是为了说明的目的，并不表示是唯一的实施方式。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同；本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本发明；本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
纤连蛋白的相对分子量约450KD，其由通过链间二硫键连接的约230KD的两个亚基形成。在许多代谢途径中，纤连蛋白与细胞粘附，扩散，增殖，迁移和胚胎发育密切相关。它还参与组织的多种病理过程，例如伤口愈合，炎症，纤维化和免疫细胞的调节作用，例如捕获和调理冠状病毒，HIV，狂犬病病毒和多种细菌。
纤连蛋白的主要功能之一是介导细胞粘附。纤连蛋白通过细胞信号转导途径帮助调节细胞形状和细胞骨架组织，并促进细胞扩散。大量研究和临床应用证明，FN在介导细胞免疫和体液免疫中起着非常重要的作用。根据其HIII区的结构，FN分子可以介导CD4阳性T细胞与gp41/gp120或类似位点在T细胞免疫活性中的复杂配位。
多种病毒，例如HIV，重症急性呼吸***综合症冠状病毒（SARS-CoV）和中东呼吸综合征冠状病毒（MERS-CoV），它们均可通过共同的受体CD4分子以及gp120/gp41阳性的膜蛋白受体进入细胞，而CD4广泛存在于T细胞的膜表面，这些类型的病毒颗粒可以以类似受体的方式进入并进入宿主细胞。这些通路模式通常与与内皮细胞的紧密相互作用有关，内皮细胞具有支持T细胞增殖和增加Treg抑制功能的作用。这种内皮增强Treg功能的能力可以为免疫分子提供靶标，以增强炎症性疾病期间Treg的活性。许多的证据表明使用FN或类似RGD/RGDS肽的片段可以通过Spacer-Arm配体样方式干扰阻断病毒的感染。这源于FN与病毒外壳或者外膜的多种蛋白质分子具有特异的亲和能力，换而言之得到的推论之一为，使用FN作为材料表面结构有可能可以捕集这一类病毒。
新冠病毒的是2019-nCoV以及Covid19或者SARS-CoV的中文称谓，2019-nCoV具有含有gp41相似基序的S蛋白的包膜。其颗粒为圆形或椭圆形，通常为多角形，直径为60nm至140nm。它与SARS-CoV的同源性超过85％。体外分离和培养96小时后，可在人呼吸道上皮细胞中发现2019-nCoV，这包含有FN密切参与的过程。并且已有证据，证明2019-nCoV的感染也是CD4介导的途径。
以上背景材料表明多种病毒包括新冠病毒，其S蛋白首先发挥定位结合、踹开宿主细胞的大门的那部分功能都基于其拥有的RGD氨基酸基序结合结构域，而RGD结构大量存在于Fibronectin（FN）分子结构中；天然来源的纤连蛋白或其酶消化的片段具有一个潜在的功能，有可能对组织损伤修复，中和和固定、调理病毒、细菌有效，同时具有天然产物无毒性、无残留的特点，以及经过普遍认可的环境、生物安全性和高度的稳定性以及经济性。
基于此原理关于治疗与病毒预防都有报道和专利申请，但是利用此特性制作病毒捕集与灭杀的空气过滤材料并没有涉及。
所以本发明内容的核心是，利用这种天然互补特性，制备可以高效特异性地捕集这一类病毒的表面修饰材料。本发明的特征在于，利用天然、经济的Fibronectin分子与便宜、无毒的甘草酸，RNaseA，纤维素或硝化纤维素衍生物等天然产物组合物，在纺织品，例如具有消毒杀菌作用的氧化铜无纺布，纳米银无纺布表面制备一层纳米级别的涂层，起到捕获、过滤、调理特定病毒、延长病毒灭活的有效时间；这种设计的溶液组合物具有层次结构，可以在材料表面形成一定强度的超分子复合结构，协同作用后，使活性成分在低剂量状态下发挥高效灭活病毒的效能；此外，包埋层会大大降低原来基材表面的低毒成分释出，例如防止氧化铜颗粒，纳米银，纳米金纤维颗粒从无纺布材料，电极材料表面剥脱流失。
实施例一
一种病毒捕集材料，包括支撑材料层、涂覆层以及接枝修饰层，支撑材料层是医用无纺布空气过滤材料；
在支撑材料的外层上包覆厚度不大于50nm的涂覆层；
所述接枝修饰层包覆在涂覆层远离支撑材料层的一侧，所述接枝修饰层厚度不大于10nm；
所述涂覆层按重量份计包括，明胶1000份，Cu/Cu2+0.5-3份，纤维素40-200份，甘草酸50-800；
接枝修饰层按重量份计，纤连蛋白1份，RNaseA酶0.05-1份；
接枝修饰层用于特异性亲和吸附并捕捉经过微孔附近的病毒粒子然后协同涂覆层灭活病毒。
制膜步骤： 市售的N95聚丙烯喷熔无纺布（比表面积>12000cm2/g）用含0.1%TritonX100,pH3.0预处理液浸泡清洗2小时，用图1所述的装置或者类似原理的更大尺寸的夹具，安装固定一片单层的无纺布膜片（直径90mm）,用蠕动泵将基础涂层配比液送入夹具反复过滤15分钟，连接压缩空气反复吹扫3分钟； 取出膜片放入湿度60%，温度50℃的恒温恒湿老化箱老化24小时后通入3%氢气，湿度调到低于40%，温度降到室温处理6小时，接着用压缩空气流吹干作为基础涂层膜片备用,微天平称重法测得涂层厚度均值31.3nm。
实际制备中，可在配比中添加辅助性组分：
涂覆层配比： 明胶 100mg ，甘草酸 50mg ,硝化纤维素20mg, 磷酸氢二钠0.28g，磷酸二氢钠 1.5g，氯化钠 5.6g，硫酸铜0.15mg,丙二醛1mg,室温溶解在注射用水1000ml里面。
接枝液配比1：FN 2mg，RNase A 0.1mg ，磷酸氢二钠 0.28g，磷酸二氢钠 1.5g，氯化钠 5.6g， 注射用水 1000ml。
制膜步骤： 用图1所述的装置或者类似原理的更大尺寸的夹具，安装固定一片单层的基础涂层膜片,用蠕动泵将接枝液配比1送入夹具反复过滤3分钟，接压缩空气反复吹扫10分钟吹干后作为接枝液配比1修饰膜片备用,微天平称重法测得涂层厚度均值增加了1.2nm。
接枝液配比2：FN 20mg ，RNase A 10mg ，粒径5nm纳米金颗粒100mg ，磷酸氢二钠0.28g ，磷酸二氢钠 1.5g ，氯化钠 5. 6g ，氯化钾 1.4g ，注射用水 1000ml。
制膜步骤： 用图1所述的装置或者类似原理的更大尺寸的夹具，安装固定一片单层的基础涂层膜片,用蠕动泵将接枝液配比2送入夹具反复过滤3分钟，接压缩空气反复吹扫10分钟吹干后作为接枝液配比2修饰膜片备用,微天平称重法测得涂层厚度增加值3.7nm。
接枝液配比3：Fn 2ug，RNase A 5ug，酸氢二钠0.28g，磷酸二氢钠1.5g溶解于注射用水1000ml。
制膜步骤： 用图1所述的装置或者类似原理的更大尺寸的夹具，安装固定一片单层的基础涂层膜片,用蠕动泵将接枝液配比3送入夹具反复过滤3分钟，接压缩空气反复吹扫10分钟吹干后作为接枝液配比3修饰膜片备用,微天平称重法测得涂层厚度均值增加了0.7nm。
实施例二
如图1表示的， 网购的医用无纺布样品（G1）作为对照，用实施例一中涂覆层处理的起始膜片开始，然后分别用接枝液配比1涂层液（G2），接枝液配比2涂层液（G3） 接枝液配比3涂层液（G4）涂层处理后制备各种样片10片，分别取1片，2片，3片试样（图1的2）装夹在图1的不锈钢盘片式滤器中，滤器进口（图 1的1）用PEEK管与恒压空气气源相连，同时并接到微压差计（量程0-60pa）的一个端口；不锈钢盘片式滤器的出口端（图 1的4）通过PEEK管与微压差计（量程0-60pa）的另一端连接，并将整个不锈钢盘片式滤器浸没于图1的5所示的恒温水浴箱内（恒温30℃±1℃）。具体测试时慢慢打开出气阀，将气源端与大气的压差调节到50pa±5pa 左右，然后关闭放气阀，慢慢开启进气阀向滤器内通气，等压差表稳定后读取结果。
如图2表示的， 各种涂层液处理的无纺布材料的单层，双层通气静压差均小于5Pa, 填装三层的情形下压差达到8-15个帕斯卡； 如图2表示的，各种涂层液处理的无纺布材料的单层，双层通气静压差小于5Pa, 填装三层的情形下压差达到8-15个帕斯卡，三层填装的无涂层的压差为40.5 ±1.2 pa; 涂层1为40.5±0.8Pa; 涂层2为40.7±2.0Pa; 涂层3为37.6±1.8Pa.
实施例三
在超净台内操作下述过程。 用接枝层厚度最小的膜片，即接枝配比液3制作的无纺布膜片装入图1的压差检测夹具中，气源接入口连接气溶胶发生器，用于制备气溶胶的样品液体为检测用的金葡菌细菌培养物。夹具出口通过一个三角形漏斗接60mm琼脂培养平板。 金葡菌细菌培养物母液浓度为6x106CFU/ml，检测时用1xPBS稀释50倍,调节雾化发生器风机速度，保持每只样品杯内的25ml样品大约在60分钟内耗尽即可。 每次雾化喷施完毕换一个新样片以及一只琼脂检测平板。
反复重复上述过程分别对10只外购的无纺布膜片，以及10只用配比4涂层液制作的膜片进行测试，最终用于检查膜片漏过率的细菌培养计数的结果件表1：
表1
未处理的膜片对金葡菌的透过率为56.2%±15.4%；相比经过接枝配比液3涂层液处理的膜片的金葡菌的透过率为37.9%±8.9% 。
实施例四
同前述的实施例，在超净台内操作下述过程。 用接枝配比3制作的无纺布膜片装入图1的压差检测夹具中，气源接入口接气溶胶发生器，用于制备气溶胶的样品液体为检测用的普通感冒病毒Cov229E细胞裂解液。夹具出口通过一个三角形漏斗接60mm 培养干平板，使用之前用0.1% 明胶铺板消毒处理。 Cov229E裂解液母液浓度为4.5x105CFU/ml，检测时用1xPBS稀释50倍,调节雾化发生器风机速度，保持每只样品杯内的25ml样品大约在60分钟内耗尽即可。 每次雾化喷施完毕换一个新样片以及一只琼脂平板。 具体的病毒感染检测方法为，对每只平板接入1x105MDCK细胞，2小时后加入含1%小牛血清的DMEM培养基培养48小时，接着用1xPBS清洗、含0.15%Triton的破膜液处理，分别用S蛋白一抗以及HRP酶标二抗显色读板,Karber法转换读出每只膜片相关的病毒粒子含量的TCID50取值。
反复重复上述过程分别对3只外购的无纺布膜片，以及6只用配比3涂层液制作的膜片进行测试得到的膜片漏过的病毒粒子的TCID50的结果如见表2：
表2
将上表TCID50结果转换为经过每只膜片的病毒粒子的透过率Rp，3只市购的膜片的Rp为67.4%±19.9%； 经过配比3涂层液处理的膜片的Rp 为34.7%±12.41% ，这个结果具体对涂层修饰膜材的特性的影响由图3 进行说明。
通常，气溶胶粒子经过空气过滤材料时，部分粒子通过各种机制被阻截，部分粒子可以透过，透过部分的比例Rp可以表述为：。
剩余部分（1-Rp）被截留于深度滤材中，要完全回收这部分粒子进行检查往往有很大的误差，而评价透过率Rp的方法相对简单可靠，在实际工艺开发中进一步简化为只需要考察材料的厚度 th以及检测用的气溶胶目标粒子的粒径大小 df 的倒数，ASTM标准方法就是这样采用Staphylococcus aureus评价滤材的除菌效率的。但在需要横向评价不同性质的目标粒子的过滤效能时，系数η需要重新被定义为向量,这需要大量的测定分析和计算，加入了η之后可以定性认为，过滤材料的过滤效能Rp与气溶胶粒子尺度 df并不是线性的关系，例如可以列举的一个事实，即常规的医用级别的外科口罩材料无纺布在滤除小于50纳米到150纳米的气溶胶粒子时一般是基于不出现与与线性公式反常的假设的前提下，所以出于安全考虑实际操作中常常选用ΦX174噬菌体代替冠状病毒进行评价。但在滤除小于150纳米的气溶胶粒子时，测得的Rp值实际是大于在滤除小于150纳米大于50纳米的气溶胶粒子的线性理论Rp值 。
反复强调这一点的原因在于，悬浮于气溶胶中的粒径大小在50-150纳米左右的颗粒的生物学性质往往是更加具有环境安全意义的一个类别， 因为这一类颗粒大多数情况下是包括了各种冠状病毒在内的传染性病毒粒子，也例如一些空气传染的支原体等。 如果所生产的气溶胶过滤材料对于粒径在50-150纳米区段具有低于产品所标识的粒子捕集与消除能力，将会形成容易被忽视的漏洞，这种违反直觉的漏洞往往难以被重视，所以往往会造成难以弥补的损失； 反方向而言，如果所设计的过滤材料对于粒径在50-150纳米区段具有选择性的捕集与消除作用，在评价其 Rp比值时应该出现与线性公式相翻转的现象，即如果在评价的过程中可以找到这样的例证就可以直接说明其存在上述的选择性截留能力，而不需要经过大量繁琐的参数测算以及各种数值模拟的过程去刻画向量η以及其它材料特性相关的系数 。 简而言之，如果通过简易的气溶胶释放阻截测试，测量得到上述阳性结果时推论就成立。
附图3的1表示的是用粒径大约在300-400nm附近的金葡菌对市售的医用无纺布做气溶胶的透过率测试的结果，这个Rp是56.2%±15.4%；附图3的3表示的是再用粒径大约在60-120nm附近的Cov229E对市售的医用无纺布做气溶胶的透过率测试的结果，这个Rp是67.4%±19.9%；Rp(SA)/Rp（Cov）<1; 附图3的2表示的是用金葡菌对配比4涂层液无纺布做气溶胶的透过率测试的结果，这个Rp是37.9%±8.9%；附图3的4表示的用Cov229E对配比4涂层液无纺布做气溶胶的透过率测试的结果，这个Rp是 34.7%±12.4%，这个比例Rp(SA)/Rp（Cov）>= 1。
实施例五
用打孔器截取经过接枝配比3涂层液处理过的CuO无纺布膜片圆片（直径3.5mm）贴在BST154裸金电极的WE上，用Ag/Cl2做对电极和参比电极，恒电位仪采用EmStat-Pico模块连接一只Stm32f103单片机作为串口通讯单元，用Script CV 指令以及DPV指令做循环伏安扫描和峰值检测。扫描电位范围从1.0V-（-0.35V），扫描速度50mV/S,电解质溶液是在 0.1M Na2SO4/0.1xPBS (pH 7.2)。通氮气条件下预扫描做电极清洗后进行伏安检测， 图4 的曲线1是使用捕获Cov229E的膜材样片扫描的结果，图4的曲线2是使用没有经过病毒气溶胶过滤的接枝无纺布膜材样片扫描的结果，曲线1，曲线2连续扫描5次后平均的氧化还原电位大约处在0.1-0.2V之间，属于主要形态接近为单个电子转移反应，曲线2 出现的氧化反应阳性的氧化还原曲线在连续扫描5-10个循环后逐渐回落，向接近曲线2的方向下降。曲线1，2以及各种变形的过程均为接近单个电子转移反应，特征为属于结合态的 Cu2+ 或者Cu+所参与的反应，说明固相催化剂Cu+离子没有被解离释放到电解质液相里面。
实施例六
用膜片打孔器制取接枝配比3涂层液处理过的CuO无纺布膜片，装入可以反复使用的ф35mm滤头，确认不漏液后接微量注射泵进液，对含有0.5ng/ml S蛋白，0.45 % NaCl/0.05x PBS，pH6.5的测试液用1um孔径的醋酸纤维素滤头预滤后，用1ml/分钟的速度向过滤头缓慢打入30ml测试液，每进液1分钟间歇停止1分钟，每次过滤用于S蛋白吸附的时间控制为1分钟；接着分别用1ml A,B,C 3种洗脱液分3次洗脱膜片，离心收集合并洗脱液并测试S蛋白含量。其中3种洗脱液的成分为：A: 0.45 % NaCl/ 0.01x PBS，pH6.5; B: 0.9 %NaCl/ 0.01x PBS，pH6.5; C: 0.5 M NaCl/ 0.01x PBS，pH6.5 。
将S蛋白标准品和洗涤液样品100ul, 移入包埋了二抗的测试板孔，加入100ul洗脱缓冲液A洗涤3遍，用生物素化一抗识别。 洗去未结合抗体，HRP 偶联、TMB显色，在 450nm 处测定。S蛋白吸附能力结果换算方法： 单位吸附能力Ad(0.1ng/cm2）= (150 - 100 x洗脱液S蛋白浓度（ng/ml）)/9.0 。
在使用洗脱液A的条件下： CuO无纺布膜片的S蛋白吸附能力Ad(n=8)为3.5±2.0(0.1ng/cm2）; 配比3涂层液处理过的CuO无纺布膜的 S蛋白吸附能力Ad(n=8)为13.8±3.9(0.1ng/cm2）；
在使用洗脱液B的条件下： CuO无纺布膜片的S蛋白吸附能力Ad(n=8)为2.1±3.3(0.1ng/cm2）; 配比3涂层液处理过的CuO无纺布膜的 S蛋白吸附能力Ad(n=8)为13.2±4.7(0.1ng/cm2）；
在使用洗脱液C的条件下： CuO无纺布膜片的S蛋白吸附能力Ad(n=8)为0.5±1.8(0.1ng/cm2）; 配比3涂层液处理过的CuO无纺布膜的 S蛋白吸附能力Ad(n=8)为13.1±3.9(0.1ng/cm2）。
Claims (7)
1.一种病毒捕集材料，其特征在于，包括支撑材料层、涂覆层以及接枝修饰层，支撑材料层是医用无纺布空气过滤材料；
在支撑材料的外层上包覆厚度不大于50nm的涂覆层；
所述接枝修饰层包覆在涂覆层远离支撑材料层的一侧，所述接枝修饰层厚度不大于10nm；
所述涂覆层按重量份计包括，明胶1000份，Cu/Cu 2+ 0.5-3份，纤维素40-200份，甘草酸50-800份；
接枝修饰层按重量份计，纤连蛋白1份，RNaseA酶0.05-3份；
接枝修饰层用于特异性亲和吸附并捕捉经过微孔附近的病毒粒子然后协同涂覆层灭活病毒。
2.根据权利要求1所述的病毒捕集材料，其特征在于，所述涂覆层采用变性明胶蛋白、纤维素/硝化纤维素用过共价方式老化处理后与螯合的Cu/Cu2+、甘草酸发挥病毒灭活功能；
所述的接枝层的纤连蛋白、RNaseA通过自组装的方式与涂覆层的明胶蛋白和纤维素/硝化纤维素通过非共价方式连接组装，协同涂覆层对捕集到的病毒粒子进行灭活。
3.根据权利要求2所述的病毒捕集材料，其特征在于，制作涂覆层喷涂溶液浓度为明胶20-200mg/L,硫酸铜/氧化铜0.05-5mg/L,纤维素/硝化纤维素5mg-50mg/L,甘草酸5mg-80mg/L；
所述接枝层喷涂溶液浓度范围纤连蛋白1.0ug-2.0mg/L,RNase A 1.0ug-5.0mg/L。
4.根据权利要求3所述的病毒捕集材料，其特征在于，添加的铜或者铜离子不超过1毫摩尔每公斤无纺布。
5.根据权利要求1，2，4任一项所述的病毒捕集灭活材料，其特征在于，所构建的涂覆层中的Cu/Cu 2+ 的单质形式包埋在明胶/甘草酸/纤维素涂覆层里面，Cu/Cu 2+ 的离子形式全部以非游离的方式与氨基酸残基结合作为催化剂负责催化局部氧化反应；所构建的接枝层的纤连蛋白N端通过自组装的方式固定在底层的明胶蛋白上，纤连蛋白的RGD序列通过亲和吸附的方式与病毒粒子的S蛋白结合。
6.一种生产权利要求1所述的病毒捕集材料的工艺，其特征在于，包括以下步骤：
步骤一、用含有Cu2 + 的0.05-5ppm硫酸铜/氧化铜明胶，甘草酸，硝酸纤维素涂层液浸涂喷熔无纺布表面，压缩空气或者氢气离心脱去多余的浸涂液后老化,还原处理；
步骤二、用含2ug/L-2mg/L的纤连蛋白，RNase A,接枝液修饰，将配置的溶液喷涂至步骤一形成的涂覆层表面。
7.一种根据权利要求 6 所述的生产工艺，其特征在于，各组分之间通过自组装方式形成复合结构，其凝胶溶胶的组装顺序是：处于底层的明胶分子以共价方式在基质材料表面固化包埋硫酸铜，硝酸纤维素/纤维素与明胶分子以静电吸附方式组装成网孔结构，纤连蛋白的N端与明胶分子以亲和吸附的方式组装成复合体，纤连蛋白的C端处于游离状态，在N端和C端的中间结构与RNase A组合的方式完成接枝过程。
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