摘要
胰蛋白酶生产障碍会阻碍消化过程,在胰腺组织以外产生胰蛋白酶可能涉及癌症过程。胰蛋白酶明显增高可能表明胰腺炎或者慢性肾功能衰竭等病症的发生,它的含量与生命活动息息相关,简单并及时监测胰蛋白酶含量对疾病的诊断具有重要的参考价值。因此,该研究构建氮化碳量子点和金纳米簇(CNQDs和AuNCs)的复合纳米探针检测尿液中胰蛋白酶含量。通过煅烧三聚氰胺获得氮化碳粉末,并将氮化碳粉末作为原材料通过溶剂热法合成了发射峰在440nm的类石墨相氮化碳量子点(CNQDs)。牛血清蛋白(BSA)和CNQDs两者同时作为还原剂和稳定剂合成了金纳米簇(AuNCs),且AuNCs吸附在氮化碳量子点表面形成具有双发射性质的CNQD-AuNCs复合荧光纳米材料,发射波长分别为440nm(CNQD的发射波长)和650nm(AuNC的发射波长)。由于胰蛋白酶能特异性的水解CNQD-AuNCs中的牛血清蛋白,导致牛血清蛋白结构被破坏,从而破坏AuNCs稳定的结构,使得其沉淀聚集,引起荧光猝灭。由于AuNCs产生的650nm处的荧光被猝灭,而CNQDs产生的440nm处的荧光不受影响,CNQD-AuNCs复合荧光纳米探针产生比率型荧光信号响应。利用比率型荧光信号的变化情况,可实现胰蛋白酶的定量检测。CNQD-AuNCs探针在650nm处的荧光强度随着胰蛋白酶浓度的增加而逐渐下降,而440nm处的荧光强度保持不变。胰蛋白酶在一定浓度下(10~400ng·mL^-1)与荧光强度比值(I650/I440)呈良好的线性关系,建立的线性方程为y=2.471-0.004x[y为荧光强度比值(I650/I440),x为胰蛋白酶的浓度(ng·mL^-1)],相关系数(R2)高达0.9976,检测限为1.5ng·mL^-1(3倍信噪比)。利用建立的荧光法检测尿液中胰蛋白酶(实际含量分别为50,100和150ng·mL^-1),检测得到的平均含量分别为52.41,103.25和154.39ng·mL^-1。尿液中胰蛋白酶的回收率和相对标准偏差范围分别为102.93%~104.82%和3.57%~4.16%。结果表明,利用荧光强度比值(I650/I440)作为胰蛋白酶定量检测的信号,能够校正外界影响因素的干扰,克服单一荧光信号易受光漂白、探针浓度、激发光强度以及光程等外界因素的影响的缺点。基于CNQD-AuNCs建立的比率型荧光分析方法能够实现尿液中胰蛋白酶的高灵敏度和高特异性检测,为实际样品中胰蛋白酶的检测提供科学依据。
Low level of trypsin has adverse impacts on digestion, and the obvious increase of trypsin may indicate the occurrence of pancreatitis or chronic renal failure . In addition, the secretory of trypsin outside of pancreatic tissue may invol ve a precursor to cancer. Trypsin concentration is closely related to life activities. Simple and timely monitoring of trypsin content can provide important r eference value for disease diagnosis. Therefore, a sensitive and rapid fluores cent method was developed for determination of trypsin in urine based on car bon nitride quantum dots (CNQDs) and gold nanoclusters (AuNCs). CNQDs was synthesize dvia solvothermal treatment of bulk carbon nitride (C 3N 4) powder which was obtained by calcining melamine. The CNQDs displayed blue emission under radia tion of UV light at 365 nm and the fluorescent band was at 440 nm. Albumin bo vine serum (BSA) and CNQDs were used as reducing agents and stabilizers to prepare AuNCs which absorbed on the surface of CNQDs forming CNQD-AuNCs. CNQD-AuNCs with dual emission wavelengths at 440 and 650 nm displayed red fluorescence under radiation of UV light at 365 nm. BSA and AuNCs structure can be destroyed leading to aggregation of AuNCs in the presence of trypsin owing to the hydrolysis of BSA catalyzed by trypsin. Emission at 650 attributed to AuNCs is quenched and emission at 440 nm produced by CNQDs remain unchanged. The detection of trypsin can be performed by using fluorescent responses of CNQD-AuNCs. Fluorescent intensity at 650 nm gradually decreased with increasing trypsin concentration, w hile fluorescent intensity at 450 nm stayed unchanged. The ratio of fluores cent intensities at 650 and 440 nm had a perfect linear correlation with the concent rations of trypsin in the range of 10~400 ng·mL^-1 with a good coefficient ( R 2=0.997 6). The linear regression equation was y=2.471~0.00 4x , where x was the concentration of trypsin (ng·mL^-1 ), and y rep rese nted ratio of intensity at 650 and 440 nm. Limit of detection (LOD) for tryps in was calculated to be 1.5 ng·mL^-1 at a signal-to-noise ratio of 3. The concentration of trypsin in urine (the actual concentration was 50, 100 and 150 ng·mL^-1) detected by this ratiometric method was 52.41, 103.25 an d 154.39 ng·mL^-1 , respectively. The recoveries of trypsin were 102.93 %~104.82% with relative standard deviations of 3.57%~4.16%. AuNC@CNQDs nan osensor provide build-in self-calibration for correction of a variety of unfav orable factors by using the ratiometric responses as signals to detect trypsin. The ratiometric method can overcome shortcomings of signal response which is su sceptible to effects of external factors such as light bleaching, nanosensor co ncentration, excitation light intensity and optical path, and so on. In summary, the developed method has been applied for detection of trypsin in urine with high sensitivity and selectivity, providing scientific basis for detection of trypsin in real application.
作者
胡雪桃
石吉勇
李艳肖
史永强
李文亭
邹小波
HU Xue-tao;SHI Ji-yong;LI Yan-xiao;SHI Yong-qiang;LI Wen-ting;ZOU Xiao-bo(School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China)
出处
《光谱学与光谱分析》
SCIE
EI
CAS
CSCD
北大核心
2019年第9期2901-2906,共6页
Spectroscopy and Spectral Analysis
基金
国家自然科学基金项目(31671844,31772073)
国家重点研发计划项目(2016YFD0401104)
江苏省重点研发计划项目(BE2016306)
江苏省六大人才高峰(GDZB-016)资助
