CN106619720A - 一种荧光碳颗粒的生物酶切法制备方法 - Google Patents
一种荧光碳颗粒的生物酶切法制备方法 Download PDFInfo
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Abstract
发明公开了一种荧光碳颗粒的制备方法，这种荧光碳颗粒的制备方法，其包括如下步骤：①黑冰片的炮制；②生物酶切法制备荧光碳颗粒。通过胃液消化和肠液消化把大的碳颗粒消化成纳米级的碳颗粒，然后被肠道吸收进入血液发挥药理作用，即利用生物体制备碳点的一种方法‑生物酶切法。本发明的优点在于：攻克了价格昂贵，成本高的局面，无需高温高压条件，不需要氧化剂和钝化剂，不使用强酸或具有强腐蚀性液体，反应在生物体内缓慢进行且符合环境友好的要求，容易控制产物成分，杂质少，纯化过程比较简单等优点。
Description
技术领域
本发明涉及一种荧光碳颗粒的制备方法，特别是涉及一种生物酶切制备方法。
背景技术
目前科学家们已经开发了多种制备碳颗粒(碳点)的方法，如合成法、电化学法、激光消融法、强酸溶解法、水热一步法等，但是这些制备方法价格昂贵，成本高，需要高温高压条件，需要氧化剂和钝化剂，使用强酸或具有强腐蚀性液体，反应剧烈且不符合环境友好的要求，不易控制产物成分，杂质多，纯化过程繁琐困难，产量低等缺点。
然而一种重要的制备法——生物酶切法却尚未应用于碳点的制备，更重要的是：此法与蒙药和中药中的传统炭药在人体中的消化和吸收具有密切的相关性，甚至会直接影响其药理作用——即真正发挥药效的应该是经酶切消化后的“纳米碳点”。经我们做实验发现：通过该方法制备的碳点，其表面富含官能性有机基团，使得其不仅具有良好的分散性和水溶性(传统碳点难溶于水)，而且易于功能化修饰和生物偶联标记，同时还具有很好的生物相容性。
发明内容
本发明的目的在于提供一种荧光碳颗粒的制备方法。本发明中的荧光碳颗粒的制备方法是模拟蒙药炭药黑冰片在人体胃肠道里的药理作用：通过胃液消化和肠液消化把大的碳颗粒消化成纳米级的碳颗粒，然后被肠道吸收进入血液发挥药理作用。
本发明的目的由如下技术方案实施：
第一步：模拟黑冰片碳颗粒在人体内的消化过程并制备荧光碳颗粒。1.黑冰片的炮制；2.生物酶切法制备荧光颗粒；
1.黑冰片的炮制
选用马弗炉对黑冰片进行充分煅烧成生物炭。没有选用传统方法炮制是因为传统方法不能把黑冰片完全碳化。
2.生物酶切法制备荧光颗粒
利用模拟黑冰片在胃中和肠道中的消化吸收过程，制备出模拟胃液和肠液把黑冰片大的炭颗粒逐步在胃液中、肠液中消化成纳米级的小颗粒，然后利用微波法和超声法激发荧光制备荧光颗粒。
第二步：黑冰片荧光颗粒的动态光散射测量
1.荧光碳颗粒形貌表征
利用原子力显微镜(AFM)技术检测获得黑冰片荧光颗粒的粒度大小及三维图像，根据图像分析可以直接得到颗粒的三维尺寸。
2.光谱性质的表征
紫外吸收光谱性质的表征使用U-3010型紫外可见光谱仪测定。采用Zata电位仪测试碳点的表面电位。为了确定制备出的聚合物纳米颗粒的结构，使用美国Perkin Elmer公司的FT-IR Spectrometer(Spectrum one)傅立叶变换红外光谱仪，采用KBr压片法，在波数500～4000cm-1对黑冰片碳点样品进行红外光谱测试分析。荧光光谱性质用F-45000荧光光谱仪进行表征。
附图概要说明
附图1：黑冰片荧光颗粒的制备及细胞吞噬示意图.
附图2：用原子力显微镜观察黑冰片荧光颗里的尺寸大小。
附图3：荧光光谱扫描图。
具体实施方式
第一步：模拟黑冰片碳颗粒在人体内的消化过程并制备荧光碳颗粒。1.黑冰片的炮制；2.生物酶切法制备荧光颗粒。其中，
1.黑冰片的炮制实验：称取1公斤净制的野猪成型干燥粪，置瓷碗中加盖，中间缝隙用盐水泥密封(水盐比5∶1)，晾干后置于马弗炉内，于500℃煅烧2小时，取出放凉，利用研磨器研磨后过200目的筛子，称重保存干燥处备用。
2.生物酶切法制备荧光颗粒：
(1)模拟人胃、肠液的配制：A胃液的配制：0.2g NaCl，0.32g胃蛋白酶，0.7ml浓盐酸，双蒸馏水定容于100ml。pH约为1.2。(胃蛋白酶购自于Sigma公司。胃蛋白酶的活力为2330U/mg pro)。B、模拟肠液的配制：将0.68g KH2PO4溶于25ml双蒸水，振荡，完全溶解后加19ml 0.2mo l/LNaOH和40ml的双蒸水。加胰蛋白酶1.0g，混匀，用0.2mol/L NaOH调pH到7.5±0.1，双蒸水定容于100ml。(胰蛋白酶购自于Sigma公司。胰蛋白酶的活力为10010U/mgprot。)
(2)黑冰片荧光颗粒的制备实验
本次荧光颗粒的制备通过三步进行：首先，将1g黑冰片碳粉投入到在预先加入模拟人胃液的圆底烧瓶中，置于油浴锅中37℃、磁力搅拌回流12h。反应完后，离心分离除去未反应物，取上面黄色上清液，转入到透析袋(MWC01000)在超纯水中透析2～3天(期间不断换水)，直到pH值接近中性。透析处理后的黄色液体再逐级离心分离提纯，最后以16000转/秒离心30min后提取上清液4℃保存。其次，将胃液消化离心沉淀的黑冰片碳颗粒回收，并倒入适量超纯水轻轻混匀然后16000转/秒离心10min。以上方法重复两次，最后把沉淀物溶于肠液置于油浴锅中37℃、磁力搅拌回流12h。反应完后，离心分离除去未反应物，取上面黄色上清液，转入到透析袋(MWC01000)在超纯水中透析2～3天(期间不断换水)，直到pH值接近中性。最后以16000转/秒离心30min后提取上清液4℃保存。然后，两次提取的黄色上清液混合后经旋转蒸发浓缩至25mL左右。接着把浓缩的液体放入微波炉以50Hz微波5min后放入冷冻干燥机干燥，然后加入适量(例如1ml)超纯水到干燥碳颗粒里，超声1min后回收，得到碳颗粒溶液，可直接在荧光检测仪下检测发荧光等情况后4℃保存，或通过冷冻干燥获得荧光碳颗粒。
第二步：黑冰片荧光颗粒的动态光散射
1.碳点形貌表征
利用原子力显微镜(Atomic Force Microscopy，AFM)技术可以直接获得样品的三维图像，根据图像分析可以直接得到颗粒的三维尺寸。取0.1μg/ml碳点溶液滴于新解离的白云母片上，待溶液完全展开，放置在真空干燥箱内，80℃干燥保持2h后，取出测试。
附图1显示了模拟炭药在人体里消化吸收及激发荧光过程。
2.黑冰片荧光颗粒表型分析
附图2显示了用原子力显微镜观察黑冰片荧光颗里的尺寸大小。说明所制备的碳颗粒大小均匀。碳颗粒大小尺寸在15nm以下。
3.光谱性质的表征
紫外吸收光谱性质的表征使用U-3010型紫外可见光谱仪测定。采用Zata电位仪测试碳点的表面电位。为了确定制备出的聚合物纳米颗粒的结构，我们使用美国PerkinElmer公司的FT-IR Spectrometer(Spectrum one)傅立叶变换红外光谱仪，采用KBr压片法，在波数500～4000cm-1对黑冰片碳点样品进行红外光谱测试分析。荧光光谱性质用F-45000荧光光谱仪进行表征。
附图3显示了当激发波长从280到440nm时荧光强度逐渐增加。说明样品碳颗粒本身发出荧光。
Claims (1)
1.一种荧光碳颗粒的制备方法，其特征在于模拟黑冰片碳颗粒在人体内的消化过程并制备荧光碳颗粒，其包括如下步骤：①黑冰片的炮制；②生物酶切法制备荧光颗粒，具体为：
①黑冰片的炮制：称取1公斤净制的野猪成型干燥粪，置瓷碗中加盖，中间缝隙用盐水泥密封(水盐比5∶1)，晾干后置于马弗炉内，于500℃煅烧2小时，取出放凉，利用研磨器研磨后过200目的筛子，称重保存干燥处备用。
②生物酶切法制备荧光颗粒：
(1)模拟人胃、肠液的配制：A、胃液的配制：0.2g NaCl，0.32g胃蛋白酶，0.7ml浓盐酸，双蒸馏水定容于100ml。pH约为1.2。B、模拟肠液的配制：将0.68g KH2PO4溶于25ml双蒸水，振荡，完全溶解后加19ml0.2mol/LNaOH和40ml的双蒸水。加胰蛋白酶1.0g，混匀，用0.2mol/L NaOH调pH到7.5±0.1，双蒸水定容于100ml。
(2)黑冰片荧光颗粒的制备：
首先，将1g黑冰片碳粉投入到在预先加入模拟人胃液的圆底烧瓶中，置于油浴锅中37℃、磁力搅拌回流12h。反应完后，离心分离除去未反应物，取上面黄色上清液，转入到透析袋在超纯水中透析2～3天(期间不断换水)，直到pH值接近中性。透析处理后的黄色液体再逐级离心分离提纯，最后以16000转/秒离心30min后提取上清液4℃保存。其次，将胃液消化离心沉淀的黑冰片碳颗粒回收，并倒入适量超纯水轻轻混匀然后16000转/秒离心10min，以上方法重复两次，最后把沉淀物溶于肠液置于油浴锅中37℃、磁力搅拌回流12h，反应完后，离心分离除去未反应物，取上面黄色上清液，转入到透析袋在超纯水中透析2～3天(期间不断换水)，直到pH值接近中性。最后以16000转/秒离心30min后提取上清液4℃保存。最后，两次提取的黄色上清液混合后经旋转蒸发浓缩至25mL左右，接着把浓缩的液体放入微波炉以50Hz微波5min后放入冷冻干燥机干燥，然后加入适量超纯水到干燥碳颗粒里，超声1min后回收，得到碳颗粒溶液，可通过冷冻干燥获得在荧光碳颗粒。
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