CN116410172B - 一种基于异氟尔酮-氧杂蒽的粘度荧光探针的制备和应用 - Google Patents
一种基于异氟尔酮-氧杂蒽的粘度荧光探针的制备和应用 Download PDFInfo
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Abstract
本发明涉及了一种基于异氟尔酮‑氧杂蒽的粘度荧光探针的制备和应用，该探针的结构为：
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种基于异氟尔酮-氧杂蒽的粘度荧光探针的制备和应用。
背景技术
粘度作为细胞内微环境的基本参数，与许多细胞生理过程相关。细胞粘度控制着细胞内物质的传输和扩散过程，对分子间相互作用和细胞间信号传导影响较大(Shi W J,Wei Y F,Yang J,et al.Novel Meso-trifluoromethyl BODIPY-based Near-infrared-emitting Fluorescent Probes for Organelle-specific Imaging ofCellularViscosity.Sensors andActuatorsB:Chemical,2022,359,131594.)。此外，各项生物活动，如蛋白质折叠、酶催化和信号传输，严重依赖于细胞环境中粘度的平衡(SongW,ZhangW,YueL,et al.Revealing the Effects of Endoplasmic Reticulum Stress on Ferroptosisby Two-Channel Real-Time Imaging of pH and Viscosity.Analytical Chemistry,2022,94,6557-6565；Wu Y,Yin C,Zhang W,et al.Mitochondrial-targeting Near-infrared Fluorescent Probe for Visualizing Viscosity in Drug-induced Cellsand a Fatty Liver Mouse Model.Analytical Chemistry,2022,94,5069-5074.)。最重要的是，粘度异常可能会破坏细胞功能，导致细胞功能紊乱，进而致使疾病的发生(HeY,ShinJ W,GongW J,et al.Dual-functional Fluorescent Molecular Rotor for EndoplasmicReticulum Microviscosity Imaging during Reticulophagy.ChemicalCommunications,2019,55,2453-2456.)。因此，找到能够实时灵敏的监测细胞粘度的方法对于生物医学研究具有重要意义。
传统测量粘度的方法包括：毛细管粘度计法，乌氏粘度计法，落球粘度计法等，此类测量方法操作复杂，且无法实现对细胞粘度的实时监测。(MaY,Chen Q,Pan XY,etal.Insight into Fluorescence Imaging and Bioorthogonal Reactions inBiologicalAnalysis.Topics in Current Chemistry,2021,379,1359-6446.)相较于传统的检测，近红外荧光探针具有特异性高、灵敏度好、穿透深度高、信噪比高等优点，(Yong H,Zhang P,Meng G,et al.Ratiometric Detection and Imaging of EndogenousHypochlorite in Live Cells and in Vivo Achieved by Using anAggregationInduced Emission(AIE)-based Nanoprobe.Chemical Communications,2016,52,7288-7291.)已被广泛用于构建功能性探针，用于活细胞中的酶，蛋白质，气体信号分子和微环境的检测，在对于各项生理过程的研究具有重要的意义。然而，在检测细胞粘度的过程中，(FengYZ,Nie G,Liang W J,et al.Real-time Imaging of Acute Alcoholic LiverInjury in Vivo via a Robust Viscosity Probe with Aggregation-induced EmissionNature.Sensors andActuators B:Chemical,2022,355,131285.)。由于存在发射波长短，灵敏度低，容易受到自身背景荧光的干扰等不足，荧光探针在生物系统中的应用受到了阻碍。因此，发射波长长，灵敏度高，背景荧光干扰少的荧光探针有待开发。
研究发现，多数基于二氰基异氟尔酮的荧光染料，都具有制备简单，灵敏度高，斯托克斯位移大等优点(Hong J,Li Q,Xia Q,et al.Real-Time and High-fidelityTracking of Lysosomal Dynamics with aDicyanoisophorone-basedFluorescentProbe.Analytical Chemistry,2021,93,16956-16964.)其中，部分利用二氰基异氟尔酮衍生物制成的荧光探针已经成功应用于一些目标物的检测，如：H2S，Cys等(Yan L,Gu Q S,JiangW L,et al.Near-infrared Fluorescent Probe with Large Stokes Shift for Imagingof Hydrogen Sulfide in Tumor-bearing Mice.Analytical Chemistry,2022,94,5514-5520；Liu J,Wang Z Q,Mao G J,et al.ANear-infrared Fluorescent Probe with LargeStokes Shift for Imaging Cys in Tumor Mice.Analytica ChimicaActa,2021,1171,338655.)但是，迄今为止，在粘度检测的相关研究中还未采用此类探针。因此，本发明设计和合成一种基于异氟尔酮-氧杂蒽染料的粘度近红外荧光探针用于检测活体细胞粘度，该探针具有近红外发射，灵敏度高，较深的组织穿透深度，不易受到生物自体荧光的干扰等优点，在生物系统检测方面具备良好应用前景。
发明内容
根据所提出的要求，本发明人对此进行了深入研究，在付出了大量创造性劳动后，提出了一种基于异氟尔酮-氧杂蒽的粘度荧光探针
本发明的技术方案是，一种基于异氟尔酮-氧杂蒽的粘度荧光探针，其结构式如下：
一种基于异氟尔酮-氧杂蒽的粘度荧光探针的制备方法。步骤如下：
在25mL的圆底烧瓶中，将1.0当量的二氰基异氟尔酮和1.0～1.2当量的二乙氨基氧杂蒽溶解在10mL的乙腈中，并在搅拌和无氧的条件下，加入0.2～0.4mL的哌啶，70～90℃的油浴中回流14～18h，反应结束后，减压蒸馏除去溶剂，所得粗产物经柱层析纯化，以二氯甲烷:甲醇＝90:1～110:1作为洗脱剂，得到的蓝黑色固体IX-V，即为所述的荧光探针。
本发明的有益效果是，一种基于异氟尔酮-氧杂蒽的粘度荧光探针的良好光谱响应性能。首先，对该探针的荧光光谱性质进行分析。在水溶液中，探针没有出现明显的近红外发射峰；而当被加入到具有一定粘度的甘油和水的混合溶液中时，在693nm处出现了明显的近红外发射峰，且随着粘度的增大，其近红外荧光强度不断增强。在一定粘度范围内，该探针荧光强度的对数和粘度的对数成正比，表明其对粘度变化的检测具有较强的灵敏性。其次，对探针的紫外吸收光谱进行研究。在水溶液中，探针在620nm处有明显的吸收峰；随着溶液的粘度增加至1203.1cp，620nm处的吸收峰降低。为研究探针的选择性，考察了探针在各种金属离子(Ca2+,Mg2+,K+,Fe3+,Cu2+)，阴离子(Cl-,Br-,I-,HS-)，活性氧(ClO-,H2O2,ONOO-)，生物硫醇(GSH,Cys,Hcy)，氨基酸(Trp,Met,Leu,Phe,Lys,Val,Lle,Thr)和甘油中的荧光响应情况。结果显示，仅粘度高的甘油能引起荧光光谱的改变，其荧光强度较在水溶液中增强131倍，且探针的荧光光谱受其他干扰物影响不明显，表明探针具有较高的选择性和灵敏度。通过分析pH值对探针粘度测定的影响可知，当pH值在5到11之间时，水和40％的甘油中的探针荧光基本不变，说明pH不影响其对粘度的测定，所以该探针可以用于生理条件下粘度的测定。此外，在连续2h的激发光照射下，该荧光探针的发射强度保持稳定，证明探针具有优异的光稳定性，确保了它的实用性。
一种基于异氟尔酮-氧杂蒽的粘度荧光探针的应用。采用HeLa细胞进行实验，当细胞中加入荧光探针后孵育30分钟，通过荧光成像可以观察到微弱的荧光，当加入脂多糖(LPS)诱导细胞粘度增大，荧光增强，这说明探针能在活细胞中对粘度的变化产生响应。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针在不同粘度溶液中的荧光光谱图。
横坐标为波长，纵坐标为荧光强度。荧光探针的浓度为10μM，甘油在水中的占比分别为：0,10％,20％,30％,40％,50％,60％,70％,80％,85％,90％,95％。对应的粘度分别为1.6cp,2.0cp,2.9cp,4.5cp,6.9cp,12.8cp,28.9cp,75.5cp,200.0cp,390.4cp,903.2cp,1203.1cp。发射波长为630-800nm，对应的激发波长为620nm。
图3为荧光探针荧光强度对数与粘度对数的关系图。
横坐标为粘度的对数，纵坐标为荧光强度的对数。荧光探针的浓度为10μM。
图4为荧光探针在不同粘度溶液中的紫外可见吸收光谱图。
图5为荧光探针的选择性图。
荧光探针的浓度为10μM，其它分析物浓度均为200μM。
图6为pH对荧光探针的影响图。
图7为荧光探针的光稳定性图。
图8为细胞毒性实验图。横坐标为荧光探针的浓度，纵坐标为细胞的存活率。
图9荧光探针的细胞成像图。
图10相对荧光强度图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。
实施例1：
荧光探针的合成
合成路线如图1。在25mL的圆底烧瓶中，将二氰基异氟尔酮(204mg,1.2mmol)和二乙氨基氧杂蒽(283mg,1mmol)溶解在10mL的乙腈中，并在搅拌和无氧的条件下，加入0.3mL的哌啶，70～90℃的油浴中回流18h，反应结束后，减压蒸馏除去溶剂，所得粗产品经柱层析纯化，以二氯甲烷:甲醇＝110:1作为洗脱剂，得到的蓝黑色固体IX-V(0.3g)，即为所述的荧光探针，产率65％。1H NMR(400MHz,Chloroform-d)δ7.65(d,J＝15.5Hz,1H),6.93(d,J＝8.5Hz,1H),6.69(s,1H),6.42(s,1H),6.38–6.27(m,3H),3.39(q,J＝7.1Hz,4H),2.54(s,2H),2.51(s,4H),2.45(t,J＝5.9Hz,2H),1.78(t,J＝6.2Hz,2H),1.20(t,J＝7.0Hz,6H),1.07(s,6H).
实施例2：
荧光探针和不同比例甘油溶液配制及粘度的测定
探针溶液的制备：称取一定量探针溶解在二甲基亚砜中，配成1×10-4M的备用溶液。将1.0mL探针的备用溶液加入到10mL的容量瓶中，用Tris-HCl缓冲溶液定容后，得到浓度为1.0×10-5mol/L的荧光探针溶液。分别配制甘油和水比例不同的溶液(0,10％,20％,30％,40％,50％,60％,70％,80％,85％,90％,95％)，并用NDJ-1指针数显旋转粘度计测量其粘度，获得对应的粘度分别为1.6cp,2.0cp,2.9cp,4.5cp,6.9cp,12.8cp,28.9cp,75.5cp,200.0cp,390.4cp,903.2cp,1203.1cp。
实施例3：
荧光探针在不同粘度溶液中荧光光谱的测定
图2为荧光探针在不同粘度溶液中的荧光光谱，荧光探针的浓度为10μM，溶液的甘油的占比依次为0,10％,20％,30％,40％,50％,60％,70％,80％,85％,90％,95％。实验所用激发波长为620nm，发射波长范围为630-800nm。狭缝宽度为5.0nm/10.0nm，所用的荧光测定仪器为日立F4600荧光分光光度计。由图可知，当探针在水溶液中，发射峰并不明显；随着甘油的占比不断增加，即溶液粘度的增大，荧光逐渐增强。图3为探针对不同粘度对数的线性响应图。荧光强度的对数值和粘度的对数值呈线性关系，线性范围是1.6cP-1203.1cP。该探针具有较宽的检测范围，能满足对细胞粘度的检测要求。
实施例4：
荧光探针在不同粘度溶液中紫外可见吸收光谱的测定
图4为荧光探针在不同粘度下的紫外可见吸收光谱图，荧光探针的浓度为10μM，溶液的甘油的占比依次为0和95％，紫外可见吸收光谱测定用的仪器为安捷伦Cary60紫外可见分光光度计。从图4可以看出，水溶液中，探针在620nm处有明显的吸收峰；溶液的粘度增加至1203.1cp时，620nm处的吸收峰降低。
实施例5：
荧光探针对粘度测定的选择性
图5为荧光探针对粘度的选择性，探针与各种金属阳离子(Ca2+,Mg2+,K+,Fe3+,Cu2+)和阴离子(Cl-,Br-,I-,HS-)，活性氧(ClO-,H2O2,ONOO-),生物硫醇(GSH,Cys,Hcy)以及氨基酸(Trp,Met,Leu,Phe,Lys,Val,Lle,Thr)，以及对粘度的荧光响应情况。结果显示，只有粘度能引起荧光光谱的改变，其他干扰物对探针的荧光光谱没有明显的影响。说明该荧光探针对粘度的响应具有很好的选择性。
实施例6：
溶液pH值对荧光探针测定粘度的荧光性质的影响
考察pH值对荧光探针测定粘度的荧光光谱的影响，其结果如图6所示。我们研究的pH范围为5-11，荧光探针的浓度为10μM，溶液的粘度分别为1.6cp(水溶液)和6.9cp(40％甘油)。从图中可以看出，水溶液中，探针荧光强度基本不受pH值变化的影响。在40％的甘油中时，溶液的粘度增加，荧光强度也基本稳定，说明在生理环境的pH范围内，探针仍保持对粘度的良好响应，不影响探针对粘度的测定，表明该探针在生物样品的检测中的应用具备良好的前景。
实施例7：
荧光探针在甘油中光稳定性的测定
通过为研究荧光探针在水和95％甘油溶液中的光稳定性，将探针加入上述两种粘度的溶液中开始测量、计时，直至测量时间为两小时。其结果如图7所示，在水和95％甘油中，2h内该探针仍可保持对粘度良好的响应，说明其具有良好的光稳定性，这能够满足在实际样品中进行监测的要求。
实施例8：
荧光探针在活细胞中的应用
首先，对细胞进行毒性试验，如图8所示。当加入0～30μM探针时，HeLa细胞的存活率超过90％。由此可知，该荧光探针毒性较小，可应用于活细胞内粘度监测。其次，研究该探针在活细胞中的应用，选择HeLa细胞进行共聚焦显微成像，结果如图9所示。在加入探针后，可观察到微弱的荧光，通过脂多糖(LPS)诱导使细胞粘度增大，荧光也随之明显增强。图10显示了两组细胞相对荧光强度，结果说明该探针可以高灵敏性检测细胞内的粘度。
Claims (3)
1.一种基于异氟尔酮-氧杂蒽的粘度荧光探针，即IX-V，其特征在于，其结构如下：
2.根据权利要求1所述的基于异氟尔酮-氧杂蒽的粘度荧光探针的制备方法，其特征在于，反应步骤如下：
在25mL的圆底烧瓶中，将1.0当量的化合物1和1.0～1.2当量的化合物2溶解在10mL的乙腈中，并在搅拌和无氧的条件下，加入0.2～0.4mL的哌啶，70～90℃的油浴中回流14～18h，反应结束后，减压蒸馏除去溶剂，所得粗产品经柱层析纯化，以二氯甲烷:甲醇＝90:1～110:1作为洗脱剂，得到的蓝黑色固体IX-V，即为所述的荧光探针。
3.根据权利要求1所述的一种基于异氟尔酮-氧杂蒽的粘度荧光探针的应用，其特征在于，所述荧光探针应用于非疾病诊断与治疗目的的活细胞中粘度的检测。
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