基于近红外量子点的荧光共振能量转移生物探针构建及应用

本论文构建了基于近红外量子点In P/Zn S和Cy7(C45H44K3N3O16S4)的荧光共振能量转移(FRET)体系,完成了不同p H值和不同浓度下的FRET体系转换效率的检测。检测结果显示:当量子点浓度保持不变时,随着染料浓度的增加,体系转换效率也随之增加,当In P/Zn S量子点与Cy7浓度比为1∶250时,转换效率高达68%。细胞测试结果表明,FRET体系对p H值有较高敏感度,对细胞微环境p H值的检测精度可达0.1,该体系可以作为敏感型FRET探针用于生物微环境检测。     

量子点尺寸对量子点和金纳米粒子之间的荧光共振能量转移的影响

量子点（QDs）所具有的一元激发多元发射的特点预示着QDs在高通量的多元量化生物信息处理领域具有应用前景。采用柠檬酸钠直接还原法制备了粒径为15 nm的Au纳米粒子,利用静电自组装的方法构建了Au与QDs的荧光共振能量转移（FRET）系统。采用该系统研究了QDs的尺寸变化对FRET效率的影响,获得了FRET效率和给体与受体之间交叠积分的关系。结果证明,QDs是构建多元均相免疫检测技术的一个有效给体。     

水溶性Sm:ZnO-NH2量子点荧光探针制备及其用于选择性检测多巴胺

采用3-氨丙基三乙氧基硅烷(APTEs)封端氧化锌量子点(ZnO QDs)的方式,制备了一种荧光性能稳定的水溶性Sm:ZnO-NH₂ QDs荧光探针。通过透射电子显微镜(TEM)、X射线衍射(XRD)、X射线光电子能谱(XPS)、傅里叶变换红外光谱仪(FT-IR)、纳米粒度和Zeta电位仪(DLS)、稳瞬态荧光光谱仪(PL)等对材料进行表征。最优化条件下制备的该探针在水中具有良好的荧光性能,在4℃下贮存10 d后其荧光强度仍保留92%。基于该探针的荧光可以直接被多巴胺(DA)选择性猝灭,建立了一种选择性检测DA含量的荧光分析新方法,在50～1 600 ng/mL浓度内,该探针呈现良好的线性关系(y=0.4639x+0.008530,R²=0.996 3),检出限(LOD)为1.08 ng/mL,具有较好的稳定性和重现性,并成功应用于人体尿液样品的检测,回收率为98.18%～107.84%。     

荧光免疫及磁免疫层析法检测莱克多巴胺的研究

分别将量子点和超顺纳米磁珠作为荧光探针和磁信号探针应用于免疫反应中,构建了检测莱克多巴胺的荧光免疫和磁免疫层析的分析方法,并成功应用于尿液中莱克多巴胺的检测。两种方法均基于免疫竞争模式,在荧光免疫分析方法中,量子点偶联上识别莱克多巴胺的抗体,样品中莱克多巴胺和包被在ELISA板上莱克多巴胺的完全抗原竞争结合量子点,样品中莱克多巴胺的浓度越高,ELISA板上吸附的量子点越少,所测荧光强度值越低,该方法的检出限为1 ng·mL-1,检测时间为4 h。在磁免疫层析方法中,检测线上特异性捕获的纳米磁珠颜色的深浅和尿液中莱克多巴胺浓度成反比例关系,即莱克多巴胺的浓度越高,检测线的颜色越浅,该方法的定性检出限为10 ng·mL-1,检测时间为15 min。两种方法各有优缺点,基于量子点的荧光免疫分析法在痕量检测和定量分析方面具有优势,而磁免疫层析法更适合于现场快速检测。     

基于无标记荧光共振能量转移的microRNA检测方法研究

利用以阳离子共轭聚合物为能量供体的荧光共振能量转移(FRET)策略和滚环扩增放大技术,建立了一种新型的microRNA(miRNA)检测方法。阳离子共轭聚合物采用聚[(9,9-双(6’-N,N,N-三乙基铵)己基)亚芴基亚苯基二溴化物](PFP)。PFP是一种由大量吸光单元共轭而成的阳离子聚合物,具有独特的光捕获和荧光增强性能,可以和带有负电荷的DNA通过静电作用相互结合。SG是一种能够结合于所有双链DNA双螺旋小沟区域的染料,其在游离状态下,荧光微弱,但一旦与双链DNA结合后,荧光会大大的增强。首先,设计了一条可与目标分子特异性杂交的锁式探针和与RCA产物序列互补的DNA链。当体系中存在miRNA时,在T4 DNA连接酶作用下,锁式探针连接成环;随后,在phi29 DNA聚合酶和dNTPs共同作用下,在miRNA的3’端滚环扩增出一条与锁式探针序列互补的长单链DNA,所得产物与互补DNA链杂交形成双链DNA(dsDNA)。此时SG作为FRET受体掺入其中,形成SG-dsDNA共同体。随后, SG-dsDNA与PFP因静电相互作用而紧密接近,由于PFP的发射光谱与SG的激发光谱有重叠,因此二者之间可以发生FRET现象。反之,当体系中不存在miRNA时,挂锁探针则无法连接成环,阻止了扩增反应的进行及其产物与互补DNA链的杂交反应。加入SG后,由于SG与单链DNA的结合能力很弱, SG则游离于溶液中,不会与PFP发生有效的FRET。因此目标分子的浓度与体系的FRET效率直接相关。以let 7a作为待测miRNA分子,在0.05~5 nmol·L-1的范围内, let 7a的浓度与从反应体系测得的FRET效率(I520/I423)成正比。同时以无PFP参加的检测方案作为对比实验,证明了PFP确实具有提高灵敏度的作用。另外,以四种同族miRNA分子及两种其他miRNA分子作为干扰物质对方法的特异性进行了考察,发现除了两种与目标分子序列高度相似的物质存在干扰外,其他物质几乎不产生信号。利用该方法对细胞总RNA提取液中let 7a的含量及其加标含量进行了检测,测量所得回收率基本令人满意。所建立的方案不需要荧光标记探针,有效降低了检测成本,简化了操作步骤,在与miRNA相关的疾病诊断领域具有一定的应用前景。     

基于双荧光中心碳量子点的比率型荧光探针快速检测乐果

环境和农产品中乐果的残留,对人体和生态环境都造成了巨大的威胁。急需构建一种高效、简便及廉价的检测方法用于乐果的快速检测。通过对木糖和碳酸氢铵进行简单的水热处理,成功制备了掺杂氮型碳量子点(N-CQDs)。N-CQDs的激发光谱表明,其在238和330 nm处各有一个荧光中心,它们的荧光发射中心均位于402 nm左右。随着乐果的不断加入,238 nm激发的发射光谱能在1 min内被有效猝灭,而330 nm激发的发射光谱荧光变化不明显,基于此构建了比率型荧光探针用于乐果的快速检测。在最佳反应条件下,比率型荧光探针对乐果的检测范围为2～100和100～180 ng·mL^-1,检出限为0.67 ng·mL^-1。常见的离子和农药对乐果的干扰不大,表明该比率型荧光探针无需特异性酶的标记即可对乐果显示出良好的选择性。该方法用于实际火龙果样品中乐果的检测,并与国家标准规定的气相色谱-质谱法(GC-MS)比较。所建立的方法在实际火龙果样品检测中回收率在92.55%～102.24%之间,其相对标准偏差(RSD)小于3.62%(n=3)。而国标规定的GC-MS法的...     

庆大霉素与水溶性CdTe量子点的荧光共振能量转移作用

以巯基丙酸(mercaptopropionic acid,MPA)为稳定剂合成水溶性CdTe最子点(quantum dots,QDs),以CdTe QDs作为能量供体.庆大霉素(Gentamycin,GT)作为能垦受体,建立了荧光共振能量转移(fluorescence resonance energy transfer,FRET)体系.在690 nm处可见发射峰,半峰宽约10 nm,在一定范围内荧光强度与GT的含量旱线性关系,线性范围为2～20 mg·L-1,相关系数r=0.986 7.优化了不同的激发波长、pH、离子强度、时间和温度等凼素对反应的影响,并应用傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)和高效液相色谱(high-performance liquid chromatography,HPLC)分别表征了化学结构和相对专一性.结果表明巯基丙酸的巯基中S原子和羧基中氧原子与纳米微粒表面的富Cd离子发生了配位作用,CdTe QDs与GT的耦合主要是通过量子点周围巯基丙酸羧基(-COOH)中的氧原子与GT的胺基(-NH2)形成分子问氧键实现的;GT与CdTe QDs的结合率为0.35:1.研究表明GT可以作为检测CdTe QDs标记牛血清白蛋白(bovine serum albumin,BSA)的荧光增敏剂,荧光强度值增强6倍,应用前景广阔.     

基于MPA包覆的Mn掺杂ZnS量子点/米托蒽醌复合体系对DNA的检测

利用3-羟基丙酸(MPA)包覆的Mn掺杂Zn S量子点与盐酸米托蒽醌(MTX)构建了一种可简单测定DNA的磷光复合体系。该体系以MTX作为良好的电子受体,通过光诱导电子转移(PIET)原理猝灭Mn掺杂Zn S量子点的室温磷光(RTP)。当体系中加入DNA后,DNA通过静电和嵌插作用与MTX结合,形成更稳定的复合物,并使MTX从Mn掺杂Zn S量子点表面脱离,Mn掺杂Zn S量子点的RTP恢复,从而实现了对DNA的痕量检测。该体系检测DNA的线性范围为0.1~20 mg·L~(-1),检出限为0.07 mg·L~(-1)。该方法可有效避免其他共存物质的干扰并可应用于实际生物样品中DNA含量的快捷测定。     

Goldview标记的DNA荧光毛细生物传感器的研究

对Goldview(GV)作为荧光标记物的DNA荧光毛细生物传感器进行了研究。以荧光毛细分析法(fluorescence capillary analysis, FCA)为基础, 在毛细管内壁通过Poly-l-lysine将20-mer-ssDNA探针固定, 制成DNA荧光毛细生物传感器(DNA fluorescence capillary biosensor, DNA-FCB),DNA-FCB与互补靶DNA杂交,通过GV染色后,检测杂交产物的荧光强度,实现对靶DNA的定性和定量分析。样品用量12 μL, 靶DNA的浓度在0.4～4 μmol·L-1(2.4～24 mg·L-1)范围内和荧光强度有良好的线性关系(y=65.911x+3.994 4,r=0.998 9);RSD<3.5%,检出限0.39 μmol·L-1(2.2 mg·L-1),能达到定量检测靶DNA的目的。用DNA-FCB测定靶DNA操作简便, 试样、试剂用量少,测定成本极低, 能大大减少环境污染。     

碳点嫁接海藻酸钙复合结构的制备及其对Cu~(2+)的检测

基于铜离子与碳点的荧光猝灭作用,建立了用碳点作为荧光探针来检测铜离子的新方法。该方法将碳点还原后再嫁接于海藻酸钙,从而得到一种新型的含还原碳点的海藻酸钙薄膜荧光探针。用荧光分光光度计和紫外-可见分光光度计对探针的荧光特性以及探针与金属离子的相互作用进行了研究。研究结果表明:改性后的荧光探针具有很高的荧光强度,因此可以根据探针荧光强度的变化实现对铜离子的检测,并通过乙二胺四乙酸二钠(EDTA)的作用实现对铜离子的重复检测。铜离子浓度在5×10-6~100×10-6mol·L-1范围内与该荧光探针的荧光猝灭强度呈良好的线性关系。该方法不仅可以对铜离子检测,更实现了对碳点的固载,该技术有望实现荧光探针的回收再利用。     

Unsymmetrically Substituted Nickel Triazatetra-Benzcorrole and Phthalocynanine Complexes: Conjugation to Quantum Dots and Applications as Fluorescent “Turn ON” Sensors

We report on the design and application of fluorescent nanoprobes based on the covalent linking of _L-glutathione-capped CdSe@ZnS quantum dots (QDs) to newly synthesized unsymmetrically substituted nickel mercaptosuccinic acid triazatetra-benzcorrole (3) and phthalocyanine (4) complexes. Fluorescence quenching of the QDs occurred on conjugation to complexes 3 or 4. The nanoprobes were selectively screened in the presence of different cations and Hg²⁺ showed excellent affinity in “turning ON” the fluorescence of the nanoprobes. Experimental results showed that the sensitivity of QDs-4 towards Hg²⁺ was much higher than that of QDs-3 nanoprobe. The mechanism of reaction has been elucidated based on the ability of Hg²⁺ to coordinate with the sulphur atom of the Ni complex ring and apparently “turn ON” the fluorescence of the linked QDs.     

Dual-Color Quantum Dot–Encoded Nanoprobe for DNA Assays and Cell Imaging

In this article, a novel dual-color quantum dot–encoded fluorescent nanoprobe was prepared by the reverse microemulsion method and layer-by-layer assembly method. First, red fluorescence–emitting CdTe quantum dots were encapsulated in silica nanoparticles by the reverse microemulsion method. Yellow fluorescence–emitting quantum dots were deposited on the surface of silica nanoparticles to form a dual-color quantum dot@silica beads/quantum dot nanoprobe. Then capture DNA was linked to a QSQ nanoprobe via covalent bonding. We utilized the quantum dot@silica beads/quantum dot nanoprobe to capture and detect the mutant BRAF DNA sequence through the competitive immunoassay method. The resulting quantum dot@silica beads/quantum dot nanoprobe-capture DNA conjugates showed sequence-specific hybridization with target DNA. Furthermore, a multispectral imaging system was utilized to distinguish the quantum dot optical code in the quantum dot@silica beads/quantum dot nanoprobe. The quantum dot@silica beads/quantum dot nanoprobe was used in human osteoblast-like HepG2 cell imaging. The proposed quantum dot@silica beads/quantum dot nanoprobe and decoding analysis method could be used for targeting imaging, biological assays, and early detection of cancer.     

基于石墨相氮化碳量子点直接荧光猝灭法检测碘离子的研究

石墨相氮化碳(g-C₃N₄)荧光纳米材料具有原料便宜、制备容易、荧光量子产率高、光学稳定性好、毒性低等优点,并且避免有机荧光染料复杂的合成步骤或者金属半导体量子点对环境潜在的危害,这些优点使得g-C₃N₄纳米材料成为新兴的荧光探针用于检测金属离子。最近,已有文献报道重金属汞离子能够高灵敏高选择性地猝灭g-C₃N₄量子点的荧光,加入碘离子能够提取被键合的汞离子形成碘化汞(HgI₂)进而恢复g-C₃N₄量子点的荧光,从而建立一种高灵敏检测碘离子的荧光传感器。然而,该方法依然需要重金属汞离子的参与,限制了该方法的推广应用。通过硝酸氧化块体g-C₃N₄并结合水热法处理制备了一种水溶性好、荧光强度高的g-C₃N₄量子点。该量子点的荧光发射波长位于368 nm,且其荧光发射波长不随激发波长的改变而改变,表明该量子点的尺寸比较均一。笔者发现碘离子在220 nm处有一个较强的吸收峰,与该量子点的激发光谱(中心波长245 nm)具有较大的重叠,从而产生内滤效应引起该量子点的荧光发生猝灭。利用这一性质,构建了一种选择性检测碘离子的新型荧光传感器。在最优检测条件下,g-C₃N₄量子点的荧光猝灭强度(ΔF)与碘离子浓度(X,μmol·L-1)在10～400 μmol·L-1之间具有良好的线性关系,线性方程为ΔF=0.325 79X+6.039 05(R2=0.999 5),检出限为5.0 μmol·L-1。通过“混合即检测”并且不需要借助与重金属离子的配位作用就能够检测碘离子,因此该方法具有快速、环保以及操作简便等优点。     

磁免疫和荧光免疫传感器的构建及其在检测甲胎蛋白中的应用

甲胎蛋白(AFP)是常见的肝癌肿瘤标志物,在早期诊断方面起到重要作用。分别设计构建了磁免疫和荧光免疫传感器并将其应用于AFP的定量检测。在磁免疫传感器中,采用免疫磁珠代替传统固相载体,实现了目标物的快速分离;利用标记抗体上的辣根过氧化物酶(HRP)催化底物显色,根据底物吸光度值的大小进行定量检测。构建的AFP检测方法的检出限为3.6 ng·mL-1,线性范围为10~80 ng·mL-1。在荧光免疫传感器中,将碲化镉量子点(CdTe QDs)的荧光作为信号输出,并通过同时将多个CdTe QDs连接在纳米硅球表面实现信号放大,通过测量量子点荧光强度实现定量检测。该方法AFP检出限为4.2 ng·mL-1,线性范围为5~150 ng·mL-1。所设计的两种传感器均具有特异性强、灵敏度高的特点,为AFP的检测提供了新的思路和方法。     

Sensitive Detection of Luteolin Using Thioglycolic Acid–Capped Cadmium Telluride/Cadmium Sulphide Quantum Dots as the Fluorescent Probe

ABSTRACT

A sensitive and simple method for the determination of luteolin (LTL) was developed based on the fluorescence quenching effect of LTL for thioglycolic acid–capped (TGA-capped) CdTe/CdS quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 0.3 to 20.0 µg · mL^?1 with a correlation coefficient of 0.9972, and the detection limit was 7.2 ng · mL^?1. The fluorescence quenching mechanism has been proposed on the basis of electron transfer supported by ultraviolet-visible (UV-Vis) absorption, fluorescence (FL) spectroscopy. The proposed method was successfully applied to the determination of LTL in commercial capsules and human urine samples. It manifested several advantages such as high sensitivity, short analysis time, low cost, and ease of operation.     

Fluorescence “Switch on” of Conjugates of CdTe@ZnS Quantum Dots with Al, Ni and Zn Tetraamino-Phthalocyanines by Hydrogen Peroxide: Characterization and Applications as Luminescent Nanosensors

In this study, we have developed a novel nanoprobe for H₂O₂ based on the conjugation of CdTe@ZnS quantum dots (QDs) to different metal tetraamino-phthalocyanine (MTAPc): (M?=?(OAc)Al, {OAc?=?acetate}, Ni and Zn). Chemical coordination of the QDs to the MTAPc resulted in the fluorescence “switch off” of the linked QDs which was associated with Förster resonance energy transfer (FRET). In the presence of varying concentration of H₂O₂, the fluorescence of the linked QDs was progressively “switched on” and the FRET mechanism between the QDs and the MTAPc was disrupted. The sensitivity/limit of detection of the nanoprobe followed the order: QDs-ZnTAPc (2.2 μM)?>?QDs-NiTAPc (4.4 μM)?>?QDs-AlTAPc (9.8 μM) while the selectivity followed the order: QDs-NiTAPc?>?QDs-AlTAPc?>?QDs-ZnTAPc. The varying degree of sensitivity/selectivity and mechanism of detection is discussed in detail.     

Neon-, krypton-, and xenon-broadened spectral lines of water vapor

Photoluminescent semiconductor quantum dots (QDs) are widely used in many branches of diagnostics and biomedicine. Using ultrasmall QDs for designing fluorescent nanoprobes increases their capacity for penetrating through cell membranes, which allows one to use them for tracking intracellular processes at the molecular level. Obtaining small-size QDs is usually impeded due to the fast kinetics of reactions of their formation and growth in a colloidal medium. We propose a method of synthesizing defectless CdSe QDs with a diameter of 1.5 nm based on the injection reaction in an organic medium, with superfast termination of the growth of QDs at the early stage of their formation. Separation of QDs by means of gel-permeation chromatography allows one to completely remove the excess of cadmium precursors not entering the reaction, which ensures the subsequent obtaining of QDs with a controllable fluorescence wavelength and high quantum yield in the process of depositing protective epitaxial shells of different compositions. The obtained ultrasmall QDs may find application in developing photoluminescent nanoprobes for visualizing intranuclear processes in cells.     

A Simple QD–FRET Bioprobe for Sensitive and Specific Detection of Hepatitis B Virus DNA

We report here a simple quantum dot-FRET (QD-FRET) bioprobe based on fluorescence resonance energy transfer (FRET) for the sensitive and specific detection of hepatitis B virus DNA (HBV DNA). The proposed one-pot HBV DNA detection method is very simple, rapid and convenient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide on the surface of QDs with mercaptoacetic acid (MAA). Subsequently, DNA was attached to QDs surface to form the functional QD-DNA bioconjugates by simple surface ligand exchange. After adding 6-carboxy-X-rhodamine (ROX)-modified HBV DNA (ROX-DNA) into the QD-DNA bioconjugates solution, DNA hybridization between QD-DNA bioconjugates and ROX-DNA was formed. The resulting hybridization brought the ROX fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to energy transfer from QDs to ROX. When ROX-DNA was displaced by the unlabeled HBV DNA, the efficiency of FRET was dramatically decreased. Based on the changes of both fluorescence intensities of QDs and ROX, HBV DNA could be detected with high sensitivity and specificity. Under the optimized conditions, the linear range of HBV DNA determination was 2.5 – 30 nmol L^?1, with a correlation coefficient (R) of 0.9929 and a limit of detection (3σ black) of 1.5 nmol L^?1. The relative standard deviation (R.S.D.) for 12 nmol L^?1 HBV DNA was 0.9 % (n?=?5). There was no interference to non-complementary DNA. Time-resolved fluorescence spectra and fluorescence images were performed to verify the validity of this method and the results were satisfying.     

Label-free tungsten disulfide quantum dots as a fluorescent sensing platform for highly efficient detection of copper(Ⅱ) ions

A fluorescent probe for the sensitive and selective determination of copper ion(Cu~(2+)) is presented. It is based on the use of tungsten disulfide quantum dots(WS_2 QDs) which is independent of the p H of solution and emits strong blue fluorescence. Copper ions could cause aggregation of the WS_2 QDs and lead to fluorescence quenching of WS_2 QDs. The change of fluorescence intensity is proportional to the concentration of Cu~(2+), and the limit of detection is 0.4 μM. The fluorescent probe is highly selective for Cu~(2+) over some potentially interfering ions. These results indicate that WS_2 QDs,as a fluorescent sensing platform, can meet the selective requirements for biomedical and environmental application.     

CdTe量子点-罗丹明B荧光共振能量转移法测定溶菌酶

合成了以硫代乙醇酸为稳定剂的CdTe量子点,以发射波长为530 nm的量子点为供体,罗丹明B为受体,建立一种以十六烷基三甲基溴化铵为介质的荧光共振能量体系检测溶菌酶含片中溶菌酶含量的方法。结果表明:在pH=5.0时,溶菌酶的浓度与共振能量转移效率降低值在2×10^-7~ 8×10^-6 mol·L^-1范围内呈线性关系,其线性方程为Y=306.07-13.85X,相关系数为0.991 0,检出限为2×10^-8 mol·L^-1,RSD为5.8%,平均回收率为101%(n=5)。