CN116425781A - 碱性pH值荧光探针、制备方法、检测方法及应用 - Google Patents
碱性pH值荧光探针、制备方法、检测方法及应用 Download PDFInfo
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Abstract
本发明涉及生物pH值检测领域,具体提供碱性pH值荧光探针、制备方法、检测方法及应用,本发明基于硼酸与羟基基团随碱性pH的可逆转换,利用荧光信号和紫外吸收变化建立了可逆的双重模式的碱性pH的检测方法。本方法操作简单、无需加热、反应迅速。且通过两种信号输出结果测定,减小了环境波动引起的误差,保证了检测结果的可靠性。
Description
技术领域
本发明涉及生物pH值检测领域,具体涉及碱性pH值荧光探针、制备方法、检测方法及应用。
背景技术
酸碱失调是临床上常见但易被忽视的问题,酸碱失衡在很大程度上影响细胞增殖、凋亡、内吞和离子传递等一系列重要功能。众所周知,酸碱平衡是由pH严格调控的。在正常生理条件下,动脉血pH值维持在7.36-7.44之间。线粒体的pH接近8.0。大肠和胰腺的pH值为碱性(pH≥8),中性粒细胞中的碱性磷酸酶pH稳定在8.0~10.0之间。然而,引起代谢性碱中毒的因素很多,如胃液流失、氯离子流失、钾离子消耗、过量使用、药物积累等,容易破坏pH的稳定性。有研究指出代谢性碱中毒导致的危重症和死亡与pH值的升高有关。当血液pH值大于7.60时,死亡风险高达61.1%。对生物体内pH值进行检测和监测具有重要意义。目前临床上酸碱度的测定多是基于试纸进行检测,这种方法难以实现在体动态分析,且无法进行多次测定,消耗率高,不经济环保。因此,有必要构建生物体内碱性pH值的传感器。
现有pH传感器多为纳米材料,粒径较大,肾小管上皮细胞摄取量极低,而小分子光学探针具有生物膜渗透性好、灵敏度高、时空分辨率高、无创性好等优点,常被设计为pH值的传感器,用于生命系统pH值的检测和成像。由于,大多数小分子荧光探针荧光随pH值的增大而减弱,到pH为碱性时,很难实现荧光成像,因此,目前大多数pH检测器的pKa位于酸性到中性pH范围内。因此,建立碱性范围内pH值的传感方法仍然是一项具有挑战性的任务。
发明内容
本发明提供了碱性pH值荧光探针、制备方法、检测方法及应用,其目的在于缓解现有技术中存在的碱性pH探针紧缺、检测耗材用量大、难以实现可视化成像等技术问题。
为实现上述目的,本发明的技术方案之一为:
碱性pH值荧光探针(DDPB),所述碱性pH值荧光探针结构式为
本发明的第二技术方案为:将(3,5,5-三甲基环己-2-烯亚基)丙二腈(与4-甲酰苯硼酸偶联制备所述碱性pH值荧光探针,所述(3,5,5-三甲基环己-2-烯亚基)丙二腈来源于双氰异佛尔酮染料,碱性pH值荧光探针是双氰异佛尔酮的衍生物。
具体反应流程如下:
进一步地,包括以下步骤:
S1、将(3,5,5-三甲基环己-2-烯亚基)丙二腈和4-甲酰苯硼酸中室温加入无水乙醇,
S2、加热后,加入催化剂,回流,
S3、蒸发除去溶剂,洗涤,提纯。
进一步地,所述S1的步骤中,所述(3,5,5-三甲基环己-2-烯亚基)丙二腈、所述4-甲酰苯硼酸和所述无水乙醇的摩尔比为:(3,5,5-三甲基环己-2-烯亚基)丙二腈:4-甲酰苯硼酸:无水乙醇=1mmol:1mmol:10mL~1mmol:2mmol:10mL。催化剂:1~1.5N。
进一步地,所述S2中的催化剂为醋酸铵、哌啶、氢氧化钾、1,8-二氮杂双环[5.4.0]十一碳-7-烯或三乙胺。
进一步地,所述S3中提纯为用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯。
本发明的第三技术方案为:提供细胞待检样品,用碱性pH值荧光探针进行检测,获得检测结果。
本发明的第四技术方案为:碱性pH值荧光探针的应用,在碱性pH下的传感响应的应用。
发明人发现,硼酸(B(OH)3)是一种方便和具有潜力的pH敏感剂,当pH低于或高于pKa(大多数硼酸衍生物的pKa在6.4-10.7之间)时,可以转换为不同形式的三角硼酸[RB(OH)2]或四面体硼酸离子[RB(OH)3ˉ]。到目前为止,许多硼酸基水凝胶被设计并应用于生物愈合领域。此外,硼酸与二醇基团之间的pH控制相互作用为糖类和核酸的传感提供了极大便利。据我们所知,大多数荧光pH有机探针构建是基于酚、奎宁、吡啶、荧光素等基团。而目前基于硼酸基团的光学探针在碱性环境下的传感和成像研究还未见报道。
基于硼酸功能化光学探针,开发了一个简单的紫外、荧光双模式pH传感平台。探针的双氰异佛尔酮基团作为光信号基团,硼酸基团作为pH响应位点。该传感器在pH值范围为7.00至10.23(pKa=9.33)具有pH敏感响应和良好的可逆性。如图7-图10所示,质谱和核磁氢谱准确验证了探针中硼酸和羟基的可逆转换的pH监测机理(图2、图3、图12、图13)。
本发明的第五技术方案为:碱性pH值荧光探针应用于制备碱性纸基pH传感器,具体方法为:将滤纸在0.2-2mM碱性pH值荧光探针乙醇溶液中浸泡均匀后风干。
基于分子内电荷转移机理(ICT),制备的碱性纸基pH传感器,便捷,灵敏,实现了快速pH可视化检测。
此外,基于肾脏是维持机体酸碱波动平衡的关键器官,通过激光共聚焦技术,该传感器也有效应用于碱中毒HK-2细胞中的pH监测。
与现有技术相比,本发明有以下优点:
1、本发明的探针分子合成路线简单,原料易得,碱性条件下灵敏度较高,响应速度快,生物相容性良好。
2、该探针可适用于近红外荧光和比色双重检测分析模式,具有循环性、可逆性,可反复使用等优点,可缓解现有技术中存在的碱性pH探针紧缺、检测耗材用量大、难以实现可视化成像等技术问题。
3、该探针制成的可视化的碱性纸基pH传感器,具有反应灵敏、易携带、操作简单等优点。
4、该探针可应用于监测HK-2细胞(人肾皮质近曲小管上皮细胞)碱中毒,以克服现有技术中抗光漂白性能差、生物毒性高、选择性差等弊端,在生物医学检测领域中具有很好的应用前景,动物医学和人体医学领域均可适用。
附图说明
图1是本发明碱性pH探针合成机制图。
图2是本发明碱性pH探针在DMSO-d6,400MHz条件下的核磁氢谱图。
图3是本发明碱性pH探针在阴离子模式下的高分辨质谱图。
图4是本发明碱性pH探针的(A)TG-DSC曲线和(B)FT-IR光谱。
图5是本发明碱性pH探针(溶解在20%乙醇溶液中)的紫外可见光谱和荧光发射光谱。激发波长为430nm。
图6是本发明碱性pH探针(在pH为10.23的20mM磷酸盐缓冲溶液-20%乙醇溶液中孵育)的吸光度光谱和荧光发射光谱。激发波长为490nm
图7是本发明(A)碱性pH探针(30μM)在不同pH值(7.00~10.23)的20%乙醇20mM磷酸盐缓冲溶液中的紫外可见光谱变化和(B)发射光谱响应。荧光激发波长为430nm,狭缝宽度为5/10nm。(C)在环境光和365nm紫外光持续照射下碱性pH探针(30μM)在含20%乙醇的不同pH值的20mM磷酸盐缓冲溶液中的图片。
图8是本发明碱性pH探针(30μM)在不同pH值(7.00~10.23)的20%乙醇20mM磷酸盐缓冲溶液中孵卵后荧光光谱响应。激发波长为490nm,狭缝宽度为5/10nm。
图9是本发明碱性pH探针基于比率荧光发射强度的pH滴定曲线(I667nm/I590nm)。激发波长分别为490nm(A)和430nm(B),狭缝宽度为5/10nm。(C)吸收强度比(A510nm/A420nm)随pH值变化的ExpDec1曲线拟合。
图10是本发明(A)45μM碱性pH值荧光探针碱性pH探针在20%乙醇20mM磷酸盐缓冲溶液中反复调节pH至7.2和9.9的可逆吸光度谱变化。(B)碱性pH值荧光探针在pH7.2~pH9.9之间的pH可逆性。(C)环境光下pH值7.2和9.9时探针溶液的颜色。(D)加入不同浓度HCl和NaOH时45μM碱性pH值荧光探针荧光强度对pH变化的响应时间扫描。Ex430nm,Em667nm,狭缝宽度5/3nm。
图11是本发明碱性pH探针选择性测定结果图。
图12是本发明碱性pH探针与200μMNaOH反应产物的阴离子ESI-MS图。
图13是本发明碱性pH探针与200μMNaOH反应产物的在DMSO-d6,400MHz条件下的核磁氢谱图。
图14是本发明碱性pH探针和产物的LUMO和HOMO之间的能隙计算。密度泛函理论(DFT)计算采用B3LYP/6-31G方法,采用高斯16程序。
图15是本发明碱性pH探针纸基传感器对不同pH值磷酸盐缓冲溶液的响应照片。
图16是本发明碱性pH探针试纸监测pH值的可逆性结果图。
图17是本发明用CCK-8法测定不同浓度碱性pH探针处理24和48小时后HK-2细胞的细胞活力图。
图18是本发明在用pH值分别为7.4、8.5、9.5和10.0的PBS缓冲液浸泡后,20μM碱性pH探针预处理HK-2细胞的共聚焦荧光显微镜图像。第一排照片在黄色通道(495-620nm),激发波长为533nm。第二排图像采集在红色通道(450-700nm),激发波长为674nm。最后一行显示黄色通道和红色通道叠加荧光。比例尺=20μm。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,通过实施例对本发明进行进一步详细阐述,但并不限制本发明。
碱性pH值荧光探针,所述碱性pH值荧光探针结构式为
优选的,将(3,5,5-三甲基环己-2-烯亚基)丙二腈与4-甲酰苯硼酸偶联制备所述碱性pH值荧光探针,所述(3,5,5-三甲基环己-2-烯亚基)丙二腈来源于双氰异佛尔酮染料,碱性pH值荧光探针是双氰异佛尔酮的衍生物。
具体反应流程如下:
优选的,包括以下步骤:
S1、将(3,5,5-三甲基环己-2-烯亚基)丙二腈和4-甲酰苯硼酸中室温加入无水乙醇,
S2、加热后,加入催化剂,回流,
S3、蒸发除去溶剂,洗涤,提纯。
优选的,所述S1的步骤中,所述(3,5,5-三甲基环己-2-烯亚基)丙二腈、所述4-甲酰苯硼酸和所述无水乙醇的摩尔比为:(3,5,5-三甲基环己-2-烯亚基)丙二腈:4-甲酰苯硼酸:无水乙醇=1mmol:1mmol:10mL~1mmol:2mmol:10mL。催化剂:1~1.5N。
优选的,所述S2中的催化剂为醋酸铵、哌啶、氢氧化钾、1,8-二氮杂双环[5.4.0]十一碳-7-烯或三乙胺。
优选的,提供细胞待检样品,用碱性pH值荧光探针进行检测,获得检测结果。
优选的,碱性pH值荧光探针的应用,在碱性pH下的传感响应的应用。
优选的,碱性pH值荧光探针应用于制备碱性纸基pH传感器,具体方法为:将滤纸在0.2-2mM碱性pH值荧光探针乙醇溶液中浸泡均匀后风干。
下面具体实施例用于进一步详细说明本发明,但实施例并不对本发明做任何形式的限定,本发明所使用的试剂和原料除自制外均为市售。
实施例1-6碱性PH探针的合成。
实施例1:
(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至80℃后,加入醋酸铵(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(70mg,22%)。
实施例2:
(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至80℃后,加入哌啶(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(149mg,47%)。
实施例3:
(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至80℃后,加入氢氧化钾(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(51mg,16%)。
实施例4:(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至100℃后,加入1,8-二氮杂双环[5.4.0]十一碳-7-烯(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(38mg,12%)。
实施例5:
(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至80℃后,加入三乙胺(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(61mg,19%)。
实施例6:
(3,5,5-三甲基环己烷-2-烯基)丙二腈(187mg,1mmol)和4-甲酰苯硼酸(150mg,1mmol)在室温下加入10mL无水乙醇。加热至100℃后,加入醋酸铵(1N),反应溶液回流12h。反应结束后,蒸发除去溶剂,用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯,得到鲜橙色粉末(73mg,23%)。
实施例7:pH光谱检测步骤
将碱性pH值荧光探针溶于乙醇中,得到贮备液(3mM)。将20μL贮备液加入2mL不同pH值(7.003~10.275)的20%乙醇20mM磷酸盐缓冲溶液中。随后测定溶液的紫外和荧光发射波谱。荧光光谱激发波长为390nm,狭缝宽度为5/10nm。分别在环境光和365nm紫外光照射下观察并拍摄溶液颜色变化。(时间优化:1-60min内变化趋势不明显,实验中选择配置好试剂立即测定实验结果。乙醇浓度优化:浓度升至20%后变化波动不明显,选取20%乙醇作为反应溶剂。)
碱性pH值荧光探针的表征和光学性质:碱性pH值荧光探针的1HNMR谱如图2所示。ESI-MS谱图(图3)进一步确认了碱性pH值荧光探针的化学结构,m/z=317.1470,与[DDPB]ˉ一致。为了评价碱性pH值荧光探针的光学性能,图4测量了碱性pH值荧光探针的紫外可见吸收光谱和荧光光谱。420nm处的主要吸收峰是碱性pH值荧光探针的最大吸收带。在390nm激发下,碱性pH值荧光探针的荧光光谱最大发射为590nm。
实施例8:pH可逆性检测步骤45μM碱性pH值荧光探针含20%乙醇20mM磷酸盐缓冲溶液,采用浓盐酸和浓氢氧化钠溶液调节溶液pH值,反复调节pH至7.2或9.9,测定溶液紫外光谱,并观察溶液颜色变化。采用时间扫描模式观察荧光可逆变化,首先将45μM碱性pH值荧光探针溶液用盐酸调节至酸性,随后逐滴加入氢氧化钠溶液,在激发光430nm、发射光667nm下观察溶液荧光恢复情况。
传感器的可逆性和选择性:可逆性是pH传感系统的关键性能之一。如图10所示,该探针在pH值在7.2到9.9之间来回调节至少三次,具有良好的可逆性,并且可以观察到A510/A420的可重复吸收衰减不显著(图10A、10B)。同时,溶液的颜色迅速从黄色切换到红色(图10C),很容易通过肉眼观察这种变化。在490nm连续照射下进一步进行可逆荧光响应,如图10D所示。可见,pH为9.9的探针溶液在加入H+(由HCl提供)后荧光强度迅速下降,而在逐渐加入OHˉ(由NaOH提供)后荧光强度恢复到接近初始值。此外,碱性pH值荧光探针不仅表现出良好的pH可逆性,而且在传感环境中表现出良好的光漂白抗性。实验结果表明,碱性pH值荧光探针适用于细胞连续成像,对实验室和细胞水平的pH变化均有敏感的识别能力。
pKa值的计算
配制一系列pH范围为7.00~10.23的20%乙醇20mM磷酸缓冲液,向上述溶液中加入30μM碱性pH值荧光探针,记录每种溶液的荧光强度和吸光度值。建立了信号变化(I667nm/I590nm,A510nm/A420nm)与pH的关系。pKa值由式(1)计算。R为在一定pH下,667nm和590nm波长的荧光强度之比或510nm和420nm波长的吸光度之比。Ra和Rb为R的中性和碱性限值,公式适用于碱性缓冲溶液。
荧光量子产率的计算
以罗丹明B(在乙醇中Фs=89%)为标准,通过式(2)计算碱性pH值荧光探针(乙醇中)和pH响应产物(HDM)(乙醇中)的荧光量子产率(Φ)。
Φ=Фs×(I/Is)×(As/A)×(η/ηs)^2(2)
其中:I表示的是样品和标准光谱的综合荧光强度区域;A是样品和标准品在激发波长处的吸光度;η表示样品和参考溶液的折射率。
实施例9:基于碱性pH值荧光探针构建纸基pH传感器
以普通滤纸制备了试验纸。首先,将滤纸在0.25mM碱性pH值荧光探针溶液中浸泡5分钟,风干后,将制备好的pH试纸切成直径为1.0cm的圆盘使用。如图15所示,随后在室温下添加不同pH(7.4、8.0、8.5、9.0、9.5和10.0)的磷酸盐缓冲溶液,在环境光下观察试纸变化。
在制备好的黄色pH试纸(图16A)上加入NaOH(pH=10)溶液后,颜色迅速变红(图16B)。随后,最后三张试纸用盐酸(pH=1)溶液中和,试纸颜色立即由红色变为黄色(图16C)。在此基础上,第5张试纸再次与NaOH(pH=10)溶液相互作用后,颜色又恢复成红色(图16D)。这些令人满意的检测性能表明,纸基传感器具有良好的可逆性和pH响应灵敏性。
实施例10:CCK-8试验
采用CCK-8法检测碱性pH值荧光探针对HK-2细胞的细胞毒性。将HK-2细胞以1×104细胞/孔(总体积200μL/孔)的密度接种于96孔检测板中,贴壁24h。随后,将细胞暴露于浓度为0~24μM的碱性pH值荧光探针中24h或48h。用PBS清洗液洗涤细胞三次后,在每个孔中加入10%的CCK-8培养基溶液,在37℃含5%CO2的潮湿环境中孵育3h。最后,用EponchBioTek微板阅读器在450nm处记录光密度(OD)。细胞活力计算公式为:细胞活力(%)=(ODtreated/ODcontrol)×100%,其中ODtreated和ODcontrol分别是在碱性pH值荧光探针处理/不处理的情况下获得的。
如图17所示,在较低的浓度下,24h和48h存活率都明显上升,当碱性pH值荧光探针达到6μM时,存活率开始下降。这可能与剂量-应激效应有关:即低浓度促进增殖,高剂量抑制增殖。当碱性pH值荧光探针浓度达到21μM时,24和48h细胞存活率仍均高于90%。表明,碱性pH值荧光探针生物毒性低,具有良好的生物相容性,可用于细胞成像和监测细胞内pH波动。
实施例11:HK-2细胞碱中毒成像
将约1×105个HK-2细胞接种于35mm玻底20mm宽的培养皿中,贴壁24h。PBS清洗液洗涤后,加入20μM碱性pH值荧光探针孵育4h。随后,将含有10μM制霉菌素的不同pH值PBS缓冲溶液(pH值分别为7.4、8.5、9.5、10.0)孵育HK-2细胞20min。最后,用PBS清洗液洗涤3次后,采用激光扫描共聚焦成像技术获取HK-2细胞pH变化的荧光图像,其中黄色通道观察波长范围为495-620nm,激发波长为533nm;红光通道观察波长范围为450-700nm,激发波长为674nm;叠加图像为黄色与红色通道叠加后呈现的图像。(注释:制霉菌素用于促进细胞质和培养基之间H+/OHˉ离子快速达到平衡)
如图18所示,发现碱性pH值荧光探针可穿透HK-2细胞膜,在细胞质中快速分布,并发出黄色荧光。随着培养基pH值的升高,黄色通道的荧光强度逐渐下降,而当pH值达到10.0时,红色通道的荧光强度显著增强。在中性和碱性条件下,HK-2细胞中黄通道和红通道之间出现的现象与在磷酸盐缓冲溶液中的信号响应相当一致。结果表明,基于碱性pH值荧光探针的pH传感器是一种理想的适用于监测HK-2细胞碱中毒的方法。
实施例12:碱性pH值荧光探针的pH传感响应:
将pH值荧光探针溶于乙醇中,得到贮备液(3mM)。将20μL贮备液加入2mL不同pH值(7.003~10.275)的20%乙醇20mM磷酸盐缓冲溶液中。测定溶液的紫外和荧光发射波谱。荧光光谱激发波长为390nm,狭缝宽度为5/10nm。分别在环境光和365nm紫外光照射下观察并拍摄溶液颜色变化。
碱性pH值荧光探针的光学性质在碱性pH下发生了变化,如图6所示。与图5相比,当碱性pH值荧光探针溶解在pH为10.23的PBS-乙醇溶液中时,在510nm处观察到新的吸收峰,在667nm处观察到荧光发射。从图7可以看出,随着pH值的增加(从中性7.003到碱性10.275),420nm处的吸光度下降,而510nm处的吸光度上升(图7A)。溶液颜色由黄色变红色,变化明显,波长位移为90nm,表明pH测定的分辨率极好,可肉眼分辨(图7C)。荧光特性与紫外吸收具有相同的响应趋势。随着碱性pH增加,在590nm处亮黄色荧光明显猝灭,而在667nm处,出现了一个新的近红外波段。较大的斯托克斯位移为荧光识别和成像提供了极大的便利(图7B、图8)。碱性pH值荧光探针的pKa值根据Henderson-Hasselbach方程和pH工作曲线计算为9.33(图9)。碱性pH值荧光探针的这种pKa值适合于从中性到碱性的pH监测,表明所提出传感器可在中性和碱性的生理或病理环境中应用。
实施例为了评价碱性pH值荧光探针平台在生物系统pH监测中的准确性,选择性是评价传感系统抗干扰能力的一个重要参数。本实验研究了生物分子、生物相关阳离子、恶名猝灭阴离子、必需金属离子和重金属离子等几种可能的干扰物。从图11可以看出,在干扰物存在的情况下,DDPB-pH传感系统的荧光强度比(I667/I590)变化不大。结果表明,该比率型pH传感器具有良好的特异性。
实施例13:碱性pH值荧光探针检测pH的机理:
采用氘代甲醇配制100mg/mL碱性pH值荧光探针溶液,取0.5mL该溶液加入0.125mL1MNaOHD2O溶液,制得碱性待测液。另取0.5mL该溶液加入0.125mLD2O溶液,制得探针待测液。为了阐明碱性pH值荧光探针对pH的传感机理,采用MS和1HNMR对碱性待测液与探针待测液进行测定。如图12所示,m/z=289.13[M–H]ˉ即等于(E)-2-(3-(4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile(HDM)的理论计算值。此外,进一步进行1HNMR以验证如图13所示的结果。显然,图2中作为B-OH中氢的δ8.17峰被图13中作为Ar-OH中氢的δ6.20(s,1H)峰所取代。结果表明,碱性pH值荧光探针传感器通过硼酸就地切换策略传感pH值。
与碱性pH值荧光探针相比,观察到HDM在吸收和荧光方面产生了显著的红移(图5、图6),这可能归因于ICT过程。为了证明这一观点,利用密度泛函理论(DFT)计算(使用B3LYP/6-31G方法和高斯16程序执行)来说明分子水平上的响应机制。由图14可知,DDPB与对应产物HDM的能量差分别为3.13eV和3.06eV。HDM在LOMO和HOMO之间的能量差略小,与上述实验结果保持一致。
探针的双氰异佛尔酮基团作为光信号基团,硼酸作为pH响应位点。该传感器在pH值范围为7.00至10.23(pKa=9.33)具有pH敏感响应和良好的可逆性。用质谱和核磁氢谱准确验证了探针中硼酸和羟基的可逆转换的pH监测机理。基于分子内电荷转移机理(ICT),成功制备了一种快速、灵敏的碱性pH分析光学试纸,实现了快速pH可视化检测。此外,基于肾脏是维持机体酸碱波动平衡的关键器官,通过激光共聚焦技术,该传感器也有效应用于碱中毒HK-2细胞中的pH监测。
本发明基于硼酸与羟基基团随碱性pH的可逆转换,利用荧光信号和紫外吸收变化建立了可逆的双重模式的碱性pH的检测方法。本方法操作简单、无需加热、反应迅速。且通过两种信号输出结果测定,减小了环境波动引起的误差,保证了检测结果的可靠性。
通过碱性pH检测体系构建的可逆型碱性纸基pH传感器,具有节约环保、反应灵敏、易携带、操作简单等优点。
通过碱性pH检测体系对HK-2细胞(人肾皮质近曲小管上皮细胞)碱中毒进行监测,检测体系生物相容性好、抗干扰能力强、抗光漂白性能好、近红外信号易穿透等优势,可实现肾脏细胞pH活体成像监测。
本发明拓展硼酸光学探针在检测碱性pH中的应用,为碱性pH探针的设计提供了新的策略;硼酸与羟基相互转换的pH响应策略为设计碱性pH探针以探索pH在生物和病理环境中的作用提供更多思路。
以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,且本发明不限于上述的实施例,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
Claims (10)
2.碱性pH值荧光探针的制备方法,其特征在于:将(3,5,5-三甲基环己-2-烯亚基)丙二腈与4-甲酰苯硼酸偶联制备得所述碱性pH值荧光探针。
3.根据权利要求2所述的碱性pH值荧光探针的制备方法,其特征在于:包括以下步骤:
S1、将(3,5,5-三甲基环己-2-烯亚基)丙二腈和4-甲酰苯硼酸中室温加入无水乙醇,
S2、加热后,加入催化剂,回流,
S3、蒸发出去溶剂,洗涤,提纯。
4.根据权利要求3所述的碱性pH值荧光探针的制备方法,其特征在于:所述S1的步骤中,所述(3,5,5-三甲基环己-2-烯亚基)丙二腈:4-甲酰苯硼酸:无水乙醇=1mmol:1mmol:10mL~1mmol:2mmol:10mL,所述催化剂为1~1.5N。
5.根据权利要求3所述的碱性pH值荧光探针的制备方法,其特征在于:所述S2中的加热温度为80-100℃,所述回流时间为10-12h。
6.根据权利要求3所述的碱性pH值荧光探针的制备方法,其特征在于:所述S2中的催化剂为醋酸铵、哌啶、氢氧化钾、1,8-二氮杂双环[5.4.0]十一碳-7-烯或三乙胺。
7.根据权利要求3所述的碱性pH值荧光探针的制备方法,其特征在于:所述S3中提纯为用去离水洗涤三次,在硅胶柱上按石油醚-乙酸乙酯(1:1)等度提纯。
8.碱性pH值荧光探针的检测方法,其特征在于:提供细胞待检样品,用碱性pH值荧光探针进行检测,获得检测结果。
9.碱性pH值荧光探针的应用,其特征在于:在碱性pH下的传感响应的应用。
10.碱性pH值荧光探针的应用,其特征在于,所述碱性pH值荧光探针应用于制备碱性纸基pH传感器,具体方法为:将滤纸在0.2-2mM碱性pH值荧光探针乙醇溶液中浸泡均匀后风干。
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