发绿色荧光碳点的制备并用于Pb2+的灵敏检测和细胞成像

碳点作为一种新型的荧光纳米材料,与传统的半导体量子点相比具有优异的生物相容性、低毒性、易修饰等优点,使其在生物医学领域具有广泛的应用前景。本文以L-半胱氨酸和蔗糖为原料,在0.1 mol·L^-1的NaOH溶液中成功的制备出发绿色荧光的碳点(G-CDs),最佳发射波长是515 nm,经表征得出其表面主要还有氨基、羟基和羧基等基团,荧光量子产率高达33.4%,其荧光可被Pb^2+特异性猝灭,最终应用于Pb^2+的灵敏检测,检出限为0.025μmol·L^-1。经MTT法测定G-CDs对人肝癌细胞(SMMC-7721)为低毒性,最后应用到细胞荧光成像中,通过细胞成像技术成功检测细胞中Pb^2+的存在。     

基于上转换发光的碳量子点制备及应用研究

碳量子点具有易制备、低毒性、化学惰性高、荧光特性稳定等特点,和其他碳纳米材料(如富勒烯、碳纳米管和石墨烯等)一样引起了研究者的广泛关注。本文将从碳量子点的合成、特性、改性和应用等方面进行阐述,并对其受长波长光激发后可发出短波长光的这一上转换发光特性进行重点综述,为今后碳量子点的合成、改性以及应用提供一定的参考。     

碳纳米点@聚丙烯酸钠复合材料的制备及在发光二极管中的应用

将绿光碳纳米点(g-CDs)水溶液与聚丙烯酸钠(PAAS)复合, 制备了具有高荧光量子效率的(g-CDs@PAAS)复合材料. PAAS因具有大量羧基基团而具有强吸水性, 能与g-CDs表面大量的羧基、 羟基及氨基基团产生氢键作用, 使g-CDs在复合材料中维持单分散状态, 有效地克服了g-CDs在固态下因聚集而引发的荧光猝灭问题. 这种复合方法操作简单, 制备过程中原材料无损失, g-CDs负载量高达30%的发光复合材料的荧光量子效率为52%. 此外, g-CDs@PAAS复合材料具有良好的热稳定性、 光稳定性和耐有机溶剂等特点, 可作为颜色转换层应用于白光发光二极管.     

温敏性碳量子点的快速制备、荧光性质及细胞成像应用

以草酸和尿素为碳源,超纯水为溶剂,采用微波方法快速合成了具有热敏性的氮掺杂碳量子点,对其形貌、结构、荧光性质和荧光强度的影响因素,以及细胞毒性进行了测试和讨论,并用于小鼠结肠癌细胞(CT26.WT)成像。结果表明,制备的碳量子点近似球形,粒径约3.8 nm。碳量子点具有良好的温度响应的荧光性质。在10~80℃温度范围内,相对荧光强度与温度之间存在良好的线性关系。随着温度的增加,碳量子点的荧光强度减小,温度从10℃升至80℃,相对荧光强度减少了25%,当温度反向降低时,其荧光强度恢复到原始值。碳量子点温度与荧光强度之间的线性关系,使之在温度响应的荧光传感领域具有潜在的应用价值。对碳量子点进行氮掺杂能有效提高其荧光量子产率,当草酸和尿素质量比为3∶1时,荧光量子产率最高,为9.5%。另外,合成的碳量子点还具有优异的抗盐性能、强稳定性等特点,不仅能有效被CT26.WT细胞吞噬,还显示出低毒性和生物相容性,MTT分析结果表明,在1 000μg·mL~(-1)的碳量子点存在下,CT26.WT细胞存活率达85%,荧光碳量子点有望作为温敏性的荧光纳米探针在细胞研究领域得到应用。     

3-巯基异丁酸衍生物对碲化镉量子点水相合成的影响

为探索适宜制备粒径分布窄、荧光量子产率高、发光区域在红光至近红外光区段的量子点的巯基酸的条件, 我们研究了三类3-巯基异丁酸(MIBA)衍生物在以亚碲酸钠为碲源的水相法水热制备CdTe量子点中的影响. 这三类衍生物包括设计合成的主链亲水性衍生物3-巯基异丁酰甘氨酸(MIBGly)、3-巯基异丁酰-3-氨基丙酸(MIBAPA)和3-巯基异丁酰天冬氨酸(MIBAsp), 支链亲水性衍生物N-乙酰基半胱氨酸(ACys)和支链疏水性衍生物3-巯基-2-甲基丁酸(MMBA)和3-巯基-2,2-二甲基丙酸(MDMPA). 以这三类MIBA 衍生物为修饰剂时,CdTe量子点均能够保持MIBA体系较窄的荧光半峰宽. 采用亲水性的MIBA衍生物(MIBGly、MIBAPA、MIBAsp和ACys)比采用疏水性的MIBA衍生物(MMBA和MDMPA)更有利于制备发射波长较长的量子点. 采用支链亲水性的MIBA衍生物((ACys)比采用主链亲水性的MIBA衍生物(MIBGly、MIBAPA和MIBAsp)更容易获得高荧光量子产率的量子点.     

水溶性量子点标记狂犬病P蛋白单克隆抗体的研究

利用共价偶联的方式,在水溶性缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(Sulfo-NHS)促进作用下,将400 μL的2 g/L狂犬病P蛋白抗体与适量的聚丙烯酸修饰后的水溶性硫脲修饰ZnO掺Cd量子点进行共价偶联反应,经磷酸盐缓冲液(PBS,0.01 mol/L,pH 7.4)透析纯化得到目标偶联物,采用荧光发射光谱、生物质谱、酶联免疫法等对偶联物进行表征.结果表明:偶联后的量子点荧光最大发射波长红移了10 nm,荧光强度随着狂犬病P蛋白抗原浓度的增加而逐渐增强;量子点标记狂犬病P蛋白抗体后的分子离子峰在m/z 67580处,比狂犬病P蛋白抗体分子离子峰增大了1453.由此证实狂犬病P蛋白抗体成功偶联到水溶性量子点上,且结构未受破坏.     

由蚕砂制备的碳量子点在不同激发、pH、金属离子、温度及极性环境下的荧光性质研究

碳量子点作为一种新兴的荧光纳米材料,具有粒径分布均匀、光稳定性好、激发-发射波长可调控、表面可修饰等优良的性质,兼具低毒性、生物相容性好等优点,在分析检测和生物成像等领域展现出广阔的应用前景。而蚕砂是家蚕的干燥粪便,简单易得。利用蚕砂作为碳量子点制备原料,采用微波合成的方法制备得到了一种平均水合粒径为4.86 nm,含氮、硫修饰的碳量子点材料,可作为针对激发波长、pH、金属离子浓度、温度及溶剂极性的变化有着显著响应特性的碳量子点型荧光探针。该探针的荧光最大发射波长随激发波长或pH的增加而红移;荧光强度随温度或pH的降低而增加;随着金属离子,特别是铜离子的加入而逐渐降低,并随着EDTA络离子的加入而逐渐回复。在多种溶剂中该探针均具有较好的溶解度,当换用不同极性的溶剂时,随着溶剂极性的增加荧光发射波长逐渐红移。荧光性质随多重环境参数变化为该碳量子点在未来的生物检测和成像领域提供了广阔的应用前景。     

发光碳量子点的合成、性质和应用

基于碳量子点具有良好的水溶性、化学惰性、低毒性、易于功能化和抗光漂白性等优异性能,碳量子点和其它的碳纳米材料（如富勒烯、碳纳米管和石墨烯等）同样引起了研究者广泛的关注。 碳量子点可以通过很多较为廉价的一步法进行大规模的制备,包括化学氧化法、超声法、微波法和激光烧蚀法等。 本文主要介绍了不同碳量子点的合成方法,以及依赖于碳量子点尺寸和波长等性质的发光性能,并且讨论了碳量子点在生物成像、光催化、能量转换/储存、光电子、光限幅和传感器等方面的应用。     

CdSe量子点修饰电极电化学发光法测定叶酸

制备了水溶性的CdSe量子点,用紫外光谱和荧光光谱对其进行了表征.并将其修饰到金电极的表面,得到了CdSe量子点修饰电极(CdSe/GE),研究了其电化学发光性质.结果表明:在强碱介质中,CdSe/GE对鲁米诺电化学发光具有增敏作用,在此发光体系中加入叶酸后,会产生进一步增强的电化学发光信号,由此建立了电化学发光检测叶酸的新方法.考察了缓冲溶液pH值、鲁米诺的浓度和扫速等条件对电化学发光强度的影响.在优化的实验条件下,叶酸在1×10~(-13)~1.1×10~(-4) mol/L浓度范围内与相对发光强度(ΔI)呈现良好的线性关系,检测限为6.0×10~(-14) mol/L(S/N=3),并用于市售叶酸片剂中叶酸的测定,得到令人满意的实验结果.     

氨基化单分散量子点/二氧化硅核壳纳米粒子的制备及其细胞标记

通过反向微乳液法, 在油溶性量子点表面包裹二氧化硅外壳, 使油溶性量子点水溶性化, 再利用3-氨丙基三乙氧基硅烷(APTES)在已形成的二氧化硅纳米颗粒表面进行氨基化改性, 制备富含氨基的二氧化硅包裹的量子点荧光纳米球. 通过透射电子显微镜(TEM)、粒径分析、zeta电位检测、紫外-可见分光光度、荧光分光光度和红外光谱等手段对产品进行了表征. 结果表明, 所制备的二氧化硅量子点纳米球(45 nm)具有单分散性、水溶性好及光化学稳定性强等优点. 通过静电作用, 所制备的单分散氨基化二氧化硅量子点对肿瘤细胞表面膜电荷进行了初步标记显像.     

Combined ATRP and 'click' chemistry for designing stable tumor-targeting superparamagnetic iron oxide nanoparticles

Important issues in the design of superparamagnetic iron oxide nanoparticles (SPIONs) for cancer diagnosis include stability under physiological conditions and specificity in targeting the cancer cells. In the present study, atom transfer radical polymerization (ATRP) was used to graft SPIONs with poly(glycidyl methacrylate-co-poly(ethylene glycol) methyl ether methacrylate) (SPIONs-P(GMA-co-PEGMA)). The PEGMA in the copolymer chain confers high stability to the nanoparticles in aqueous medium, and prevents recognition by macrophages with the aim of prolonging their in vivo circulation time. The GMA groups were used for conjugating the cancer targeting ligand, folic acid (FA), via 'click' chemistry. Using this method, the amount of FA conjugated to the nanoparticles (SPIONs-P(GMA-co-PEGMA)-FA) can be readily controlled. The specificity of cellular uptake of the nanoparticles by three different cell lines was investigated. The cellular iron uptake by KB cells (human epidermoid carcinoma) after 24 h of incubation is about thirteen and five times higher than those by 3T3 fibroblasts and macrophages, respectively. No significant cytotoxicity was observed with these three types of cells. The high targeting efficiency and biocompatibility of these nanoparticles are promising features for in vivo specific targeting and detection of tumor cells which overexpress the folate receptor.     

增强CT联合血清指标诊断结直肠癌的价值

本文探讨癌胚抗原(CEA)、糖类抗原19-9(CA19-9)、胰岛素样生长因子结合蛋白-3(IGFBP-3)及增强CT在结直肠癌中的联合诊断效果。选取100例结直肠癌患作为观察组,同时选取结直肠良性肿瘤患者60例作为对照组,检测血清CEA、CA19-9、IGFBP-3,同时给予增强CT检查。观察组血清CEA和CA19-9高于对照组(P<0.05),而IGFBP-3低于对照组(P<0.05);TNM分期Ⅲ~Ⅳ和Ⅰ~Ⅱ患者血清CEA、CA19-9和IGFBP-3差异比较有统计学意义(P<0.05);增强CT联合血清指标联合诊断灵敏性为89.00%、特异性为90.00%;增强CT诊断结直肠癌T分期、N分期与病理结果Kappa值分别为0.696和0.790(P<0.05)。CEA、CA19-9、IGFBP-3联合增强CT是一种具有较高效能的结直肠癌诊断方法。     

DNA aptamer selected for specific recognition of prostate cancer cells and clinical tissues

Prostate cancer is the most common malignancy in men lack of efficient early diagnosis and therapeutics, calling for effective molecular probes. Herein, we performed cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to obtain specific recognition of human prostate cancer cells PC- 3M. Four aptamers were successfully obtained that can bind to target cells with high affinity and specificity. A 51-nt truncated sequence named Xq-2-C1 was identified after further elaborative analysis on the secondary structure. More importantly, the achieved aptamer Xq-2-C1 not only demonstrated excellent specific to target cells, but also revealed specific recognition to clinical prostate cancer tissue. The tissue imaging results showed that Xq-2-C1 had better recognition ratio for clinical prostate cancer tissue samples (85%) compared to the random sequence (9%). These results demonstrate that these newly generated aptamers would furnish potential applications in the early diagnosis and clinical treatment of prostate cancer.     

Detection of melanoma using antibody-conjugated quantum dots in a coculture model for high-throughput screening system

We proposed an effective strategy for evaluating the targeting specificity of an antibody-conjugated quantum dot (QD) nanoprobe in a coculture system mimicking an in vivo-like tumor microenvironment in which cancer cells grow with normal cells. Analysis of the images was performed with automated confocal microscopy. We have employed a melanoma-melanocyte coculture model to assess the specific binding of QDs conjugated with melanoma antibodies. Conjugation of antibodies to the QD significantly improved the melanoma specificity, while unconjugated antibody alone suffered from non-specific binding to melanocytes. Concentration-dependent binding and competitive inhibition studies with QD-antibody conjugates reproducibly proved the specificity to melanoma cells against melanocytes. The specificity and targeting efficiency of nanoprobes evaluated in a simple coculture model may provide a reasonable assessment for the in vitro diagnosis of early stage melanoma development before in vivo studies. Further, a rapid and sensitive cancer cell detection system demonstrated herein may allow for the development of high-throughput screening platforms for early cancer diagnosis and anti-cancer therapeutics.     

Cell microarray chip system for accurate,rapid diagnosis and target treatment of breast cancer cells SK-BR-3

Biometrics probe is a molecule that specifically interacts with a specific target molecule and can be detected by a specific method. Three-dimensional (3D) embedded cell scaffold in the cell array chip can affect culture cancer cells in a 3D environment with continuous medium supplementary and help controlling the diffusion of small molecules drugs. Based on modification of DNA segment, this type of cell micro-array chip is a new biochip technology with convenient focusing and high throughput screening.     

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (K_d = 38.71 nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (K_d ~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.     

Dual-targeting conjugates designed to improve the efficacy of radiolabeled peptides

Radiolabeled regulatory peptides are useful tools in nuclear medicine for the diagnosis (imaging) and therapy of cancer. The specificity of the peptides towards GPC receptors, which are overexpressed by cancer cells, and their favorable pharmacokinetic profile make them ideal vectors to transport conjugated radionuclides to tumors and metastases. However, after internalization of the radiopeptide into cancer cells and tumors, a rapid washout of a substantial fraction of the delivered radioactivity is often observed. This phenomenon may represent a limitation of radiopeptides for clinical applications. Here, we report the synthesis, radiolabeling, stability, and in vitro evaluation of a novel, dual-targeting peptide radioconjugate designed to enhance the cellular retention of radioactivity. The described trifunctional conjugate is comprised of a Tc-99m SPECT reporter probe, a cell membrane receptor-specific peptide, and a second targeting entity directed towards mitochondria. While the specificity of the first generation of dual-targeting conjugates towards its extracellular target was demonstrated, intracellular targeting could not be confirmed probably due to non-specific binding or hindered passage through the membrane of the organelle. The work presented describes a novel approach with potential to improve the efficacy of radiopharmaceuticals by enhancing the intracellular retention of radioactivity.     

Gd3+与RGD共修饰量子点用于胰腺癌细胞的荧光及MR双模态成像

结合磁共振成像(MRI)和荧光成像技术,以钆离子(Gd3+)、量子点及精氨酸(R)-甘氨酸(G)-天冬氨酸(D)(RGD)多肽等为功能单元,采用纳米载体组装技术构建了MRI弛豫率/荧光效率高和靶向性强的Gd3+与RGD共修饰的量子点双模态纳米探针(QDs@Gd3+-RGD),并将其用于胰腺癌细胞的双模态成像.实验结果表明,QDs@Gd3+-RGD双模态纳米探针具有较高的弛豫率,且能对胰腺癌patu8988细胞进行荧光和T1-weighted MR成像.     

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.     

Exponential isothermal amplification coupled MALDI-TOF MS for microRNAs detection

MicroRNAs (miRNAs) have attracted significant attention in biomedical research and clinical diagnosis. However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple miRNAs at low abundance is still a challenge. In this work, a method coupling exponential amplification reaction (EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple miRNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for miRNAs detection. It also shows high specificity to distinguish miRNAs at single-nucleotide resolution. We used the method to detect the miRNA-21, let-7a, miRNA-100, and miRNA-125b in samples of spiked human serum and breast cancer cells (i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR. Consequently, the label-free mass-spectrometric platform could be a potential tool for miRNAs analysis in complex biological samples, and may be used for clinical diagnosis.