CN112694469A - 基于吡啰红肼的HOCl荧光探针、制备方法及应用 - Google Patents
基于吡啰红肼的HOCl荧光探针、制备方法及应用 Download PDFInfo
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Abstract
本发明提供了一种基于吡啰红肼的HOCl荧光探针、制备方法及应用，探针结构式如式Ⅰ和式Ⅱ所示：
或
。本发明是将肼反应位点与吡啰红荧光团相结合构建的探针，探针由于C=N异构化以及PET过程具有极低的背景荧光，且在红光区域对HClO显示出快速、显著的荧光off‑on响应、灵敏度极高，已成功应用于癌细胞/组织与正常细胞/组织的区分。
Description
技术领域
本发明涉及荧光探针领域，具体涉及一种基于吡啰红肼的HOCl荧光探针、制备方法及应用。
背景技术
鉴于高敏感性、可视化、生物兼容性、无辐射、实时检测等特点，荧光技术已经是生物学、医学领域不可或缺的研究手段。尤其近十年，随着生命科学的发展、荧光影像设备的不断突破，以及各种新型荧光染料和探针分子的相继开发，这一技术已从对离子、生物小分子、酶等的检测拓展到了对细胞、组织、活体动物的快速、三维显微成像。不仅如此，荧光技术近年来在癌症的诊断治疗方面也获得了重要进展，凭借荧光导航试剂，医生能在术中实时判断肿瘤边界和毫米级的微小肿瘤，从而完成肿瘤的精准切除；凭借靶向肿瘤的光动力治疗试剂，医生能用特定波长的激光高精度地杀死癌细胞，并降低了对正常组织的损伤。
癌症是一类细胞生长和细胞分裂失控的疾病，近年发病率和死亡率逐年升高，目前癌症发展和复发的机制尚不明确，因此对于现代医学技术来说癌症的治疗依然面临巨大挑战。由于癌症的传统诊断手段（如，血管造影、CT检查、磁共振成像、超声波检查等）缺乏充足的特异性及敏感性，肿瘤只有在发展到中期甚至到晚期发生转移扩散时，才能实现准确诊断。相比之下，荧光探针影像技术具有灵敏度高、成本低、分析时间短、操作简单、可视化、无电离辐射等优点，可以克服常规肿瘤检查手段中存在的诸多缺陷，为肿瘤精准诊断及治疗提供了一种手段。此类荧光探针最常见的设计策略是将配体（如小分子、多肽、蛋白质，抗体和核酸）与荧光团直接相连接，连接荧光团的配体能与癌细胞中过表达的受体高效配位，从而达到对癌细胞的影像目的。此外，由于癌细胞与正常细胞代谢方式不同，即有氧糖酵解（也称为Warburg效应），这种代谢会为癌细胞创造一个特殊的微环境，如缺氧、细胞内外pH值降低、活性硫（RSS）和活性氧（ROS）水平升高、极性降低等，其中癌细胞与正常细胞内的ROS（包括O2 -、H2O2、OH▪、ONOO-和ClO-等）浓度差异最大，癌细胞大约是正常细胞的10倍，因此，利用癌细胞与正常细胞内ROS浓度的不同，有望实现对二者的区分。
发明内容
本发明提出了一种基于吡啰红肼的HOCl荧光探针、制备方法及应用，该探针由于C=N异构化以及光诱导的电荷转移（PET）的双重作用导致极低的背景荧光，并在红光区域显示出对HOCl快速、高选择性和高灵敏的荧光响应。这些优异的特性使其在区分癌细胞和正常细胞/组织的实际应用中获得成功。
实现本发明的技术方案是：
一种基于吡啰红肼的HOCl荧光探针，探针结构式如式Ⅰ和式Ⅱ所示：
式Ⅰ 式Ⅱ
基于吡啰红肼的HOCl荧光探针的制备方法，步骤如下：
（1）在0 °C、氮气环境中，将氧杂蒽酮溶于无水四氢呋喃中，向上述反应液中缓慢加入甲基溴化镁的THF溶液，室温搅拌过夜，反应结束后用水猝灭、DCM萃取、减压去除溶剂，得到的产物重新溶解在乙腈和高氯酸的水溶液中，搅拌10分钟后再次用DCM萃取，有机相用无水Na2SO4干燥，过滤蒸发得到粗产品，柱色谱纯化得到中间体2；
（2）将步骤（1）得到的中间体2和I2溶解于CHCl3，回流反应0.5 h，向上述溶液中加入二甲亚砜继续回流48 h，反应结束后冷却至室温，混合物用饱和Na2S2O3淬灭、二氯甲烷萃取，有机相经无水Na2SO4干燥、过滤蒸发，柱色谱纯化得到中间体3；
（3）将步骤（2）得到的中间体3、肼基化合物和4Å分子筛加到DCM中，室温搅拌过夜，反应液经过滤、水洗、DCM萃取，有机相经干燥、过滤蒸发、柱色谱纯化得到探针。
当步骤（3）中肼基化合物为2-肼基苯并噻唑，得到式Ⅰ结构荧光探针。
当所述步骤（3）中肼基化合物为2-肼吡啶，将中间体3、2-肼吡啶、分子筛和浓盐酸溶解于DCM中，室温搅拌过夜，过滤洗涤后利用二氯甲烷提取，旋转蒸发除去溶剂，色谱纯化得到式Ⅱ结构荧光探针。
所述步骤（1）中氧杂蒽酮化合物的结构式如下：
所述中间体2的结构式如下：
所述中间体3的结构式如下：
所述步骤（1）中氧杂蒽酮化合物和甲基溴化镁的摩尔比为1:1.25；步骤（2）中间体2和I2的摩尔比为1:1，步骤（3）中间体3和肼基化合物的摩尔比为1：（1-2）。
所述的荧光探针在红区检测HOCl的应用，式Ⅰ结构荧光探针的检出限为5.4 nm，式Ⅱ结构荧光探针的检出限为0.9nm。
优选地，制备的荧光探针通过检测细胞内ROS水平区分癌细胞和正常细胞。
本发明的探针的合成步骤如下：
本发明的有益效果是：本发明是将肼反应位点与吡啰红荧光团相结合构建的探针，探针由于C=N异构化以及PET过程具有极低的背景荧光，且在红光区域对HClO显示出快速、显著的荧光off-on响应、灵敏度极高，已成功应用于癌细胞/组织与正常细胞/组织的区分。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例中间产物2的1H NMR图；
图2为实施例中间产物2的13C NMR图；
图3为实施例中间产物2的HRMS图；
图4为实施例中间产物3的1H NMR图；
图5为实施例中间产物3的13C NMR图；
图6为实施例中间产物3的HRMS图；
图7为式Ⅰ的1H NMR图；
图8为式Ⅰ的13C NMR；
图9为式Ⅰ的HRMS图；
图10为式Ⅱ的1H NMR图；
图11为式Ⅱ的13C NMR；
图12为式Ⅱ的HRMS图；
图13（A）为式Ⅰ结构荧光探针（2 μM）在PBS (20 mM, pH 7.4)体系中紫外吸收光谱及荧光光谱随时间的变化；（B）为式Ⅱ结构荧光探针（2 μM）在PBS (20 mM, pH 7.4) /CH3CN (v/v, 7/3)体系中紫外吸收光谱及荧光光谱随时间的变化；
图14（A）为式Ⅰ结构荧光探针（2 μM）加入HClO后632 nm处荧光光谱随时间的变化；（B）为式Ⅱ结构荧光探针（2 μM）加入HClO后628 nm处荧光光谱随时间的变化；
图15（A）为随着ClOˉ用量的增加，式Ⅰ结构荧光探针（2 μM）荧光光谱的变化；（B）为随着ClOˉ用量的增加，式Ⅱ结构荧光探针（2 μM）荧光光谱的变化；
图16（A）为式Ⅰ结构荧光探针（2 μM）经不同物质处理后的荧光光谱变化；（B）为式Ⅱ结构荧光探针（2 μM）经不同物质处理后的荧光光谱变化；
图17为不同条件下Cos7细胞荧光成像图。（A）中(i)为仅被式Ⅱ结构荧光探针（2 μM，10 min）处理的Cos7细胞，(ii-viii)为预先被式Ⅱ结构荧光探针（2 μM，10 min）处理、随后被2 μM ClO-、SIN-1、NOC-9、H2O2、1O2、HO·和KO2处理的RAW264.7细胞，收集波长为650-750nm（λex = 633 nm）；（B）为（A）的定量数据；
图18为不同条件下RAW264.7细胞荧光成像图。（A）中(i)为仅被式Ⅱ结构荧光探针（2 μM，10 min）处理的RAW264.7细胞，(ii)为预先被式Ⅱ结构荧光探针（2 μM，10 min）处理、随后被2 μM ClO-处理的RAW264.7细胞；(iii)为预先用LPS（1 mg/mL) /IFN-γ（50 ng/mL）培养4 h、随后经式Ⅱ结构荧光探针（2 μM，10 min）处理的RAW264.7细胞；(iv)为预先用抑制剂ABAH（300 μM）、LPS （1 μg/mL）/IFN-γ（50 ng/mL）培养4 h、随后经式Ⅱ结构荧光探针（2 μM，10 min）处理的RAW264.7细胞，收集波长为650-750nm（λex = 633 nm）；（B）为（A）的定量数据；
图19（A）为预先用式Ⅱ结构荧光探针（2 μM，10 min）处理后正常细胞和癌细胞的荧光成像图；（B）为（A）的定量数据；
图20（A）为荷瘤小鼠经尾静脉注射式Ⅱ结构荧光探针（1.5 mg/kg）的活体成像图；(B) 为（A）中荷瘤小鼠经解剖获得的肿瘤及主要器官的荧光成像图；采用630 nm的激发滤光片和700 nm的发射滤光片。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
（1）在0 °C、氮气环境中，将氧杂蒽酮（1.01 g, 3 mmol）溶于无水四氢呋喃（30mL）中，向上述反应液中缓慢加入甲基溴化镁（3.75 mmol）的THF溶液，室温搅拌过夜，反应结束后用水猝灭（50 mL）、DCM萃取（3×50 mL）、减压去除溶剂，得到的产物重新溶解在乙腈和高氯酸的水溶液中，搅拌10分钟后再次用DCM萃取（3×50 mL），有机相用无水Na2SO4干燥，过滤蒸发得到粗产品，柱色谱纯化得到中间体2（1.05 g, 80%）。
1H NMR (600 MHz, CDCl3) δ 7.96 (d, J = 9.6 Hz, 2H), 7.03 (d, J = 9.6Hz, 2H), 6.70 (s, 2H), 3.63 (q, J1 = 7.2 Hz, J2 = 6.6 Hz, 8H), 2.88 (s, 3H),1.34 (t, J = 6.6 Hz, 12H); 13C NMR (150 MHz, CDCl3) δ 152.39, 140.597,123.53, 123.06, 115.71, 115.02, 66.78, 49.03, 21.55; ESI-MS [M+H]+: calcd for337.2274, Found 337.2276.
（2）将步骤（1）得到的中间体2（1.5 g, 3.43 mmol）和I2（0.87 g, 3.44 mmol）溶解于CHCl3，回流反应0.5 h，向上述溶液中加入二甲亚砜（8 mL）继续回流48 h，反应结束后冷却至室温，混合物用饱和Na2S2O3（50 mL）淬灭、二氯甲烷萃取（3×50 mL），有机相经无水Na2SO4干燥、过滤蒸发，柱色谱纯化得到中间体3（0.81 g, 52%）；
1H NMR (600 MHz, CDCl3) δ 10.98 (s, 1H), 8.20 (d, J= 9.6 Hz, 2H), 7.12(d, J= 9.6 Hz, 2H), 6.90 (s, 2H), 3.70 (q, J 1 = 7.2 Hz, J 2 = 6.6 Hz, 8H), 1.38(t, J= 6.6 Hz, 12H); 13C NMR (150 MHz, CDCl3) δ 191.35, 158.42, 155.45,140.42, 129.47, 115.69, 111.87, 97.55, 46.43, 12.69; ESI-MS [M+H]+: calcd for351.2067, Found 351.2068.
（3）将步骤（2）得到的中间体3（67 mg, 0.15 mmol）、2-肼基苯并噻唑（28 mg,0.17 mmol）和4Å分子筛（5粒）加到DCM（10 mL）中，室温搅拌过夜，反应液经过滤、水洗、DCM萃取（3×50 mL），有机相经干燥、过滤蒸发，柱色谱纯化（DCM/ EA= 5/1）得到深绿色固体为式Ⅰ结构荧光探针（49 mg, 55%）。
1H NMR (600 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.51 (s, 2H), 7.86 (d, J =7.2 Hz, 1H), 7.55 (t , J = 0.6 Hz, 1H), 7.42 (t, J = 6.6 Hz, 1H), 7.26 (t, J= 8.4, 3H), 6.88 (s, 2H), 3.69 (t, J = 6.6 Hz, 8H), 1.25 (t, J = 8.4 Hz,12H); 13C NMR (150 MHz, DMSO-d6) δ 157.60, 157.38, 155.56, 154.62, 143.11,130.94, 127.21, 123.70, 122.65, 114.82, 114.63, 112.058, 111.14, 96.73,96.60, 45.60, 13.06; ESI-MS [M]+: calcd for 498.2322, Found 498.2319.
（4）将步骤（2）得到的中间体3（45 mg, 0.1 mmol）、2-肼基吡啶（22 mg, 0.2mmol），浓盐酸（8 μL）和4Å分子筛（5粒）加到DCM（10 mL）中，室温搅拌过夜，反应液经过滤、水洗、DCM萃取（3×50 mL），有机相经干燥、过滤蒸发，柱色谱纯化（DCM/乙醇= 50/1）得到深绿色固体为式Ⅱ结构荧光探针（30.4 mg, 56%）。
1H NMR (600 MHz, CDCl3) δ 9.05 (s, 1H), 8.70 (d, J = 9.6 Hz, 2H),8.37 (d, J = 7.2 Hz, 1H), 7.71 (t , J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.00 (q,J1 = 1.8 Hz, J2 = 1.8 Hz, 2H), 6.96 (t , J = 6.0 Hz, 2H), 6.88 (t , J = 1.8Hz, 2H), 3.59 (t, J = 7.2 Hz, 8H), 1.34 (t, J = 7.2 Hz, 12H); 13C NMR (150MHz, CDCl3) δ157.49, 154.43, 154.13, 148.15, 145.76, 138.66, 131.62, 118.14,113.67, 111.34, 110.02, 110.00, 96.01, 45.72, 12.77; ESI-MS [M]+: calcd for442.2601, Found 442.2604.
性能测试
1. 溶液配制
将式Ⅰ和式Ⅱ结构荧光探针用乙腈配成2 mM的储液，随后用20 mM的PBS（pH 7.4）稀释至相应浓度。
次氯酸盐溶液（ClO-）用商业化NaClO溶液在去离子水中稀释制备，其浓度通过测量溶液在292 nm处的吸收来确定（ClO-在去离子水中的摩尔消光系数为350 M-1 cm-1）；过氧亚硝酸盐溶液（ONOO-）根据文献报道制备（R.M. Uppu, W.A. Pryor, Synthesis ofperoxynitrite in a two-phase system using isoamyl nitrite and hydrogenperoxide, Anal. Biochem. 1996, 236, 242-249.），其浓度通过测量溶液在302 nm处的吸收来确定（ONOO-溶液在0.1 M NaOH中的摩尔消光系数为1670 M-1 cm-1)；过氧化氢溶液（H2O2）用商业化H2O2溶液在去离子水中稀释制备。
2. 细胞培养和荧光成像
所有细胞系均购自GeneFull生物技术有限公司(中国)。
所有细胞均培养在37℃、含5%的二氧化碳的培养箱中，其中Raw 264.7细胞和Cos-7细胞培养在含有10%的胎牛血清、100 U /mL青霉素G钠和100 μg/ mL链霉素的RPMI 1640培养基中；A549细胞、HeLa细胞、HepG2细胞、T98G细胞、BEAS-2B细胞和HUCEC细胞培养在含有10%的胎牛血清、100 U /mL青霉素G钠和100 μg/ mL链霉素的DMEM（高糖）培养基中；进行细胞影像实验前，预先将细胞置于30 mm的玻底细胞培养皿上，静置12小时待细胞贴壁，用磷酸缓冲液（PBS）洗涤细胞3次，然后使用Ceiss LMS 880共聚焦显微镜进行荧光成像，收集波长为640-740 nm (λex = 633 nm)。
3. 活细胞中HClO成像
在影像细胞中外源性HClO的实验中，Cos7细胞首先用式Ⅱ结构荧光探针（2 μM）预处理10分钟，然后分别用ClO- (20 μM)、HO▪ (Fe2+/H2O2: 100 μM/200 μM)、NO (NOC-9: 200μM)、SIN-1 (200 μM)、1O2 (ClO-/H2O2: 20 μM/200 μM)和O2▪- (xanthine/ xanthineoxidase: 200 μM/0.01 U/mL)处理30分钟；在影像细胞中内源性HClO的实验中，Raw 264.7细胞用LPS (1 μg/mL)/IFN-γ (50 ng/mL)预处理4 h，然后用式Ⅱ结构荧光探针（2 μM）处理10分钟，PBS洗涤三次后，进行荧光影像；在抑制实验中，细胞用MPO特异性抑制剂ABAH（300 μM）、LPS (1 μg/mL)/IFN-γ (50 ng/mL) 预处理4 h，然后用式Ⅱ结构荧光探针（2 μM）处理10分钟，PBS洗涤三次后，进行荧光影像；在区分正常细胞和癌细胞的实验中，BEAS-2B细胞、HUCEC细胞、Raw 264.7细胞、Cos-7细胞（正常细胞），A549细胞、HeLa细胞、HepG2细胞、T98G细胞（癌细胞）分别用式Ⅱ结构荧光探针（2 μM）处理10分钟，PBS洗涤三次后，进行荧光影像。
4. 用式Ⅱ结构荧光探针对荷瘤鼠模型进行肿瘤成像
所有的动物实验都是按照山西大学伦理委员会颁布的相关法律和指南进行的。BALB/c雄性裸鼠（6-8周龄）购自北京维通利华实验动物技术有限公司。将A549细胞（1×106个细胞）经皮下注射于裸鼠左腋处，接种15天后，荷瘤小鼠经尾静脉注射式Ⅱ结构荧光探针（1.5 mg/kg）。活体动物成像是在Bruker多模式活体成像系统中进行的，选择630 nm的激发滤光片和700 nm的发射滤光片。
测试结果
1. 光谱研究
由于式Ⅰ和式Ⅱ结构荧光探针的溶解性不同，故二者分别选取PBS (20 mM, pH7.4) /CH3CN (v/v, 7/3)以及PBS（20 mM, pH 7.4）反应体系进行体外研究。首先研究式Ⅰ以及式Ⅱ结构荧光探针（2 μM）的稳定性，紫外吸收光谱及荧光光谱显示，式Ⅰ结构荧光探针最大吸收峰值和发射峰值分别为602 nm和632 nm，式Ⅱ结构荧光探针最大吸收峰值和发射峰值分别为596 nm和628 nm，且30分钟内二者均表现出好的稳定性（如图13）；动力学研究表明，式Ⅰ和式Ⅱ结构荧光探针与HOCl反应极快，反应时间分别为5 s和10 s（如图14）；荧光滴定实验表明，如图15所示，由于C=N异构化导致的非辐射跃迁过程以及PET过程，两个探针均在各自的反应体系中几乎没有荧光，而向溶液中加入ClO-后，在红光区引起了显著的荧光增强，最大发射峰分别位于632 nm和628 nm处，且ClO-的浓度分别与632 nm和628 nm处的荧光强度呈良好的线性关系，检测限分别为5.4 nM和0.9 nM；选择性实验证实，该探针对ClO-的选择性高于其他ROS/RNS以及生物相关阳离子、阴离子和生物硫醇。包括，还原物质：(1) Cys, (2) Hcy, (3) GSH, (4) SH‾, (5) HSO3‾ , (6) NADH, (7) VC；阴阳离子：(8) Cu2+ , (9) Ca2+ , (10) Zn2+ (11) Fe2+, (12) Fe3+ , (13) Mg2+, (14) K+, (15)Na+, (16) SO4 2‾, (17) SCN‾, (18) HSO4‾, (19) HCO3‾, (20) AcO‾, (21) NO2‾,(22) Cl‾；活性氧化物: (23) 30 μM ONOO‾, (24) 100 μM H2O2, (25) 100 μM KO2,(26) 100 μM NO, (27) 50 μM 1O2, (28) 50 μM HO ▪, (20) 20 μM HClO（图16）。
2. 细胞成像研究
考虑到式Ⅱ结构荧光探针具有更加优良的水溶性，故选取式Ⅱ结构荧光探针进行以下的生物影像研究。
首先，在Cos7细胞中检测了式Ⅱ结构荧光探针对HClO的影像能力。如图17所示，预先孵化式Ⅱ结构荧光探针（2 μM，10 min）的Cos7细胞用633 nm的激光激发时，红色荧光几乎可以忽略；当预先孵化式Ⅱ结构荧光探针（2 μM，10 min）的Cos7细胞继续孵化几种具有代表性的ROS时，只有HClO能诱导细胞发出明亮的红色荧光。接下来在RAW264.7细胞中进行内生HClO影像实验，如图18所示，当RAW264.7细胞预先孵化式Ⅱ结构荧光探针（2 μM，10min）后，在细胞内几乎观察不到荧光信号；当RAW264.7细胞预先被LPS (1 μg/mL)/IFN-γ(50 ng/mL) 预处理4 h，继续孵化式Ⅱ结构荧光探针（2 μM，10 min）后，可以观察到明亮的红色荧光；当细胞用MPO特异性抑制剂ABAH（300 μM）、LPS (1 μg/mL)/IFN-γ (50 ng/mL)预处理4 h，然后用式Ⅱ结构荧光探针（2 μM）处理10分钟，几乎观察不到明显的荧光信号。这些结果表明该探针具有良好的细胞膜通透性，并且可以选择性影像细胞内源性及外源性的HClO。
如图19所示，用探针处理癌细胞，包括A549细胞、HepG2细胞和T98G细胞10分钟后影像，所有细胞均有明亮的红色荧光；而用探针处理正常细胞，包括BEAS-2B细胞、HUVEC细胞和COS-7细胞10分钟后影像，所有细胞均显示微弱的荧光。荧光定量分析显示，癌细胞的荧光增强约为正常细胞的6倍，这表明该探针有可能根据细胞内ROS水平的差异区分癌细胞和正常细胞。
肿瘤影像研究
将式Ⅱ结构荧光探针（1.5 mg/kg）经尾静脉注射到A549荷瘤鼠体内，30分钟后在小动物活体成像系统下成像。如图20所示，肿瘤区域显示出明显的荧光信号，而正常组织区域显示出微弱的荧光信号。这些结果表明，由于癌细胞本底的ROS水平高于正常细胞，该探针可以选择性影像荷瘤鼠的肿瘤部位。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种基于吡啰红肼的HOCl荧光探针，其特征在于，探针结构式如式Ⅰ和式Ⅱ所示：
2.权利要求1所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于步骤如下：
（1）在0 ℃、氮气环境中，将氧杂蒽酮溶于无水四氢呋喃中，向上述反应液中缓慢加入甲基溴化镁的THF溶液，室温搅拌过夜，反应结束后用水猝灭、DCM萃取、减压去除溶剂，得到的产物重新溶解在乙腈和高氯酸的水溶液中，搅拌10分钟后再次用DCM萃取，有机相用无水Na2SO4干燥，过滤蒸发得到粗产品，柱色谱纯化得到中间体2；
（2）将步骤（1）得到的中间体2和I2溶解于CHCl3，回流反应0.5 h，向上述溶液中加入二甲亚砜继续回流48 h，反应结束后冷却至室温，混合物用饱和Na2S2O3淬灭、二氯甲烷萃取，有机相经无水Na2SO4干燥、过滤蒸发，柱色谱纯化得到中间体3；
（3）将步骤（2）得到的中间体3、肼基化合物和4Å分子筛溶解于DCM中，室温搅拌过夜，反应液经过滤、水洗、DCM萃取，有机相经干燥、过滤蒸发、柱色谱纯化得到探针。
3.根据权利要求2所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述步骤（3）中肼基化合物为2-肼基苯并噻唑，得到式Ⅰ结构荧光探针。
4.根据权利要求2所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述步骤（3）中肼基化合物为2-肼吡啶，将中间体3、2-肼吡啶、分子筛和浓盐酸溶解于DCM中，室温搅拌过夜，过滤洗涤后利用二氯甲烷提取，旋转蒸发除去溶剂，色谱纯化得到式Ⅱ结构荧光探针。
5.根据权利要求2所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述步骤（1）中氧杂蒽酮的结构式如下：
6.根据权利要求2所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述中间体2的结构式如下：
7.根据权利要求2所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述中间体3的结构式如下：
8.根据权利要求2-7任一项所述的基于吡啰红肼的HOCl荧光探针的制备方法，其特征在于：所述步骤（1）中氧杂蒽酮化合物和甲基溴化镁的摩尔比为1:1.25；步骤（2）中间体2和I2的摩尔比为1:1，步骤（3）中间体3和肼基化合物的摩尔比为1：（1-2）。
9.权利要求1所述的荧光探针在红区检测HOCl的应用，其中式Ⅰ结构荧光探针的检出限为5.4 nm，式Ⅱ结构荧光探针的检出限为0.9nm。
10.权利要求1所述的荧光探针在区分癌细胞和正常细胞中的应用，其特征在于：通过检测细胞内ROS水平区分癌细胞和正常细胞。
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