Nanobiotechnology: quantum dots in bioimaging

Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots.     

Aqueous synthesis of highly stable CdTe/ZnS Core/Shell quantum dots for bioimaging

In this work, we report the synthesis, characterization and biological application of highly stable CdTe/ZnS (cadmium tellurite/zinc sulphide) Core/Shell (CS) quantum dots (QDs) capped with mercaptosuccinic acid (MSA). The CS QDs were synthesized using a simple one‐pot aqueous method. The synthesized CdTe/ZnS CS QDs were found to exhibit excellent stability even 100 days after preparation and also showed better photoluminescence quantum yield (PLQY) of about 50% compared with that of only CdTe QDs which was nearly 12%. The formation of the CdTe/ZnS CS was confirmed by high‐resolution transmission electron microscopy (HR‐TEM), and Fourier transform infra‐red (FTIR) and X‐ray diffraction (XRD) analyses. Further, on extending our study towards bioimaging of E. coli cells using the QDs samples, we found that CdTe/ZnS CS QDs showed better results compared with CdTe QDs.     

Cu‐doped quantum dots: a new class of near‐infrared emitting fluorophores for bioanalysis and bioimaging

Transition metal ion‐doped quantum dots (QDs) exhibit unique optical and photophysical properties that offer significant advantages over undoped QDs, such as larger Stokes shift to avoid self‐absorption/energy transfer, longer excited‐state lifetimes, wider spectral window, and improved chemical and thermal stability. Among the doped QDs emitters, Cu is widely introduced into the doped QDs as novel, efficient, stable, and tunable optical materials that span a wide spectrum from blue to near‐infrared (NIR) light. Their unique physical and chemical characteristics enable the use of Cu‐doped QDs as NIR labels for bioanalysis and bioimaging. In this review, we discuss doping mechanisms and optical properties of Cu‐doped QDs that are capable of NIR emission. Applications of Cu‐doped QDs in in vitro biosensing and in in vivo bioimaging are highlighted. Moreover, a prospect of the future of Cu‐doped QDs for bioanalysis and bioimaging are also summarized.     

Mercaptopropionic acid–capped CdTe quantum dots as fluorescence probe for the determination of salicylic acid in pharmaceutical products

Mercaptopropionic acid (MPA)–capped cadmium telluride (CdTe) quantum dot (QDs) fluorescent probes were synthesized in aqueous solution and used for the determination of salicylic acid. The interaction between the MPA–capped CdTe QDs and salicylic acid was studied using fluorescence spectroscopy and some parameters that could modify the fluorescence were investigated to optimize the measurements. Under optimum conditions, the quenched fluorescence intensity of MPA–capped CdTe QDs was linearly proportional to the concentration of salicylic acid in the range of 0.5–40 µg mL^–1 with a coefficient of determination of 0.998, and the limit of detection was 0.15 µg mL^–1. The method was successfully applied to the determination of salicylic acid in pharmaceutical products, and satisfactory results were obtained that were in agreement with both the high pressure liquid chromatography (HPLC) method and the claimed values. The recovery of the method was in the range 99 ± 3% to 105 ± 9%. The proposed method is simple, rapid, cost effective, highly sensitivity and eminently suitable for the quality control of pharmaceutical preparation. The possible mechanisms for the observed quenching reaction was also discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Modification of hyaluronic acid with aromatic amino acids

Hyaluronic acid was modified with aromatic amino acids (5-aminosalicylic, 4-aminosalicylic, anthranilic, and p-aminobenzoic) in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The modified glycans contained 9–43% of arylamide groups and 10–33% of isoureidocarbonyl groups depending on the nature of the amino acid. Reduction with sodium borohydride allowed the conversion of isoureidocarbonyl groups into hydroxymethyl groups.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 90–95.Original Russian Text Copyright © 2005 by Ponedelkina, Odinokov, Vakhrusheva, Golikova, Khalilov, Dzhemilev.     

Cytotoxicity of CdSe-based quantum dots incorporated in glass nanoparticles evaluated using human keratinocyte HaCaT cells

CdSe quantum dots (QDs) are potential fluorescent reagents, but leakage of Cd and Se often induces cytotoxicity. Here we prepared CdSe-based QDs with glass to reduce their leakage and examined their cytotoxicity using keratinocyte cells. The cytotoxicity of the QDs with glass was obviously lower than that of the commercial QDs with polymer, suggesting their safety for biological applications.     

Graphene quantum dots as additives in capillary electrophoresis for separation cinnamic acid and its derivatives

A facile capillary electrophoresis (CE) method for the separation of cinnamic acid and its derivatives (3,4-dimethoxycinnamic acid, 4-methoxycinnamic acid, isoferulic acid, sinapic acid, cinnamic acid, ferulic acid, and trans-4-hydroxycinnamic acid) using graphene quantum dots (GQDs) as additives with direct ultraviolet (UV) detection is reported. GQDs were synthesized by chemical oxidization and further purified by a macroporous resin column to remove salts (Na₂SO₄ and NaNO₃) and other impurities. Transmission electron microscopy (TEM) indicated that GQDs have a relatively uniform particle size (2.3 nm). Taking into account the structural features of GQDs, cinnamic acid and its derivatives were adopted as model compounds to investigate whether GQDs can be used to improve CE separations. The separation performance of GQDs used as additives in CE was studied through variations of pH, concentration of the background electrolyte (BGE), and contents of GQDs. The results indicated that excellent separation can be achieved in less than 18 min, which is mainly attributed to the interaction between the analytes and GQDs, especially isoferulic acid, sinapic acid, and cinnamic acid.     

离子交换层析纯化透明质酸

考察6种离子交换树脂的静态吸附解析效果,选出201＊7阴离子交换树脂填柱,确定洗脱流速为0.6mL/min,40mL0.3mol/LNaCl和50mL0.5mol/L NaCl双浓度洗脱,实现透明质酸和杂蛋白的分离。制得透明质酸产品蛋白含量为0.057%,葡萄糖醛酸含量为43%,平均相对分子质量大于1.1×10^6,收率为54%,符合医用级透明质酸行业标准的要求。     

Potential clinical applications of quantum dots

The use of luminescent colloidal quantum dots in biological investigations has increased dramatically over the past several years due to their unique size-dependent optical properties and recent advances in biofunctionalization. In this review, we describe the methods for generating high-quality nanocrystals and report on current and potential uses of these versatile materials. Numerous examples are provided in several key areas including cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. We also explore toxicity issues surrounding these materials and speculate about the future uses of quantum dots in a clinical setting.     

Relaxation time measurements of hyaluronic acid

The N-acetyl-groups of hyaluronic acid and its sodium salts are shown to have pH-dependent T₁, T₂ and NOE values, unlike all other carbonatoms and protons. This behaviour at high temperatures can be explained by assuming that parts of the helical structure are converted into random coil by H-bond breaking. Moreover, the concentration of Na⁺, Mg²⁺, or Ca²⁺ is shown to have no influence on the relaxational behaviour of HA.     

量子点在新药开发中的应用

由于具有优异的光学特性,量子点在生物医学领域内的研究和应用取得了一些有意义的进展,同时也引起了新药开发人员的兴趣.本文概述了量子点在新药开发中所具有的优势,分析了量子点在药物传输、药物筛选和药靶确证方面的潜在应用,进一步讨论了当前量子点应用于新药开发存在的问题和不足.     

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

发酵透明质酸寡糖兽疫链球菌工程菌株的构建

透明质酸(HA)广泛应用于医学、化妆品、食品等领域。HA的生物活性取决于其分子量(M_w)。透明质酸寡糖由于具有重要的生理活性与特殊生理功能,在医药领域具有重要的应用前景。兽疫链球菌因其发酵周期短、生产强度较强的特点,在商业生产HA上具有广泛的应用。为了高效发酵合成透明质酸寡糖和解决发酵过程的溶氧问题,文中通过在兽疫链球菌WSH-24中过表达透明质酸合酶HasA以及优化表达水蛭来源的透明质酸酶LHAase。重组菌株摇瓶发酵24h,透明质酸寡糖积累至0.97g/L,比野生菌提高了182.0%。在3L发酵罐中发酵24 h,透明质酸寡糖生产强度为294.2 mg/(L·h),HA积累至7.06 g/L,比野生菌的罐上水平提高了112.4%。文中所构建的发酵合成透明质酸寡糖的兽疫链球菌重组菌株具有重要的应用前景。     

透明质酸在肿瘤治疗中的应用

透明质酸(hyaluronic acid,HA)是脊椎动物细胞间基质的重要组成成分,它是一种线性生物多聚糖,具有良好的生物相容性、生物可降解、无毒、无免疫原性等特点,在生物医药领域有广泛的应用。本文简要介绍透明质酸的结构特点及其靶向作用机制,综述近年来透明质酸作为药物载体和靶向因子在肿瘤治疗中的研究现状。     

两种UDP-葡萄糖脱氢酶对透明质酸生物转化的影响

分别从大肠杆菌和化脓链球菌中扩增出编码UDP-葡萄糖脱氢酶基因ecohas B和spyhas B,并将其插入T7表达载体p RX2构建重组质粒p RXEB和p RXSB。在大肠杆菌BL21(DE3)中重组表达,并对经镍柱纯化后的UDP-葡萄糖脱氢酶的酶学性质进行分析。酶学性质研究表明:spy Has B的最适反应温度是30℃,最适p H 10,最适条件下的比活力是12.2 U/mg;eco Has B的最适反应温度是30℃,最适p H 9,最适条件下的比活力是5.55 U/mg。从多杀巴氏杆菌扩增出的透明质酸合成酶基因pmuhas A分别与ecohas B和spyhas B构建共表达载体p BPAEB和p BPASB。将其转化到大肠杆菌BW25113中,经生物转化生产透明质酸(HA),并对转化条件进行了优化。结果表明:重组菌株进行透明质酸转化时,UDP-葡萄糖脱氢酶酶活力越高,稳定性越好,HA产量越高;转化条件优化后,p BPAEB/BW25113和p BPASB/BW25113在摇瓶中的产量分别是1.52和1.70 g/L,比之前报道的提高了2-3倍。     

Conformational and functional effects of MPA‐CdTe quantum dots on SOD: Evaluating the mechanism of oxidative stress induced by quantum dots in the mouse nephrocytes

The application of quantum dots (QDs) is restricted by the biosafety issue. QDs contribute to the adverse effects of organisms probably because of the ability to induce oxidative stress via changing the activity of antioxidant enzyme, for example, superoxide dismutase (SOD). But the underlying molecular mechanisms still remain unclear. This study investigated the harmful effects of oxidative stress induced by mercaptopropionic acid capped CdTe QDs (MPA‐CdTe QDs) on the mouse primary nephrocytes as well as the structure and function of SOD molecule and explored the underlying molecular mechanism. After 24‐hour MPA‐CdTe QD exposure, the activation level of extracellular regulated protein kinase (ERK) signaling pathway and cysteinyl‐directed aspartate‐specific proteases (Caspases) significantly increased, which led to the increasing level of reactive oxygen species (ROS) and cell apoptosis; the group pretreated with ROS scavenger N‐acetyl‐L‐cysteine (NAC) significantly reduced the apoptotic cell percentage, indicating that ROS played a critical role in QD‐induced cytotoxicity. Further molecular experiments showed that the interacting processes between the MPA‐CdTe QDs and SOD were spontaneous which changed the conformation, secondary structure of SOD. The interaction significantly resulted in the tightening of polypeptide chains and the shrinkage of SOD, leading to the inhibition of molecular SOD activity. This study demonstrates the adverse effects of QDs, revealing their potential risk in biomedical applications.     

Anti-calcification of bovine pericardium for bioprosthetic heart valves after surface modification with hyaluronic acid derivatives

Surface modification of glutaraldehyde fixed bovine pericardium (GFBP) was successfully carried out with hyaluronic acid (HA) derivatives. At first, HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional group into the carboxyl group of HA backbone. Then, GFBP was surface modified by grafting HA-ADH to the free aldehyde groups on the tissue and the subsequent HA-ADH hydrogel coating. HA-ADH hydrogels could be prepared through selective crosslinking at low pH between hydrazide groups of HA-ADH and crosslinkers containing succinimmidyl moieties with minimized protein denaturation. When HA-ADH hydrogels were prepared at low pH of 4.8 in the presence of erythropoietin (EPO) as a model protein, EPO release was continued up to 85% of total amount of loaded EPO for 4 days. To the contrary, only 30% of EPO was released from HA-ADH hydrogels prepared at pH=7.4, which might be due to the denaturation of EPO during the crosslinking reaction. Because the carboxyl groups on the glucuronic acid residues are recognition sites for HA degradation by hyaluronidase, the HA-ADH hydrogels degraded more slowly than HA hydrogels prepared by the crosslinking reaction of divinyl sulfone with hydroxyl groups of HA. Following a two-week subcutaneous implantation in osteopontin-null mice, clinically significant levels of calcification were observed for the positive controls without any surface modification. However, the calcification of surface modified GFBP with HA-ADH and HA-ADH hydrogels was drastically reduced by more than 85% of the positive controls. The anti-calcification effect of HA surface modification was also confirmed by microscopic analysis of explan ted tissue after staining with Alizarin Red S for calcium, which followed the trend as observed with calcium quantification.     

采用计算流体力学技术研究搅拌对兽疫链球菌发酵生产透明质酸的影响

搅拌是影响透明质酸(HA)发酵的一个重要因素,然而有关搅拌对HA发酵影响的认识存在较大争议。本研究采用计算流体力学(CFD)技术深入研究了搅拌对菌体生长和HA合成的影响。结果表明,菌体量和HA产量受搅拌转速的影响很小,而HA分子量随着转速的增加呈现出先增加后降低的趋势。分阶段控制转速研究表明转速对HA分子量的影响主要体现在HA合成阶段。CFD计算结果表明随着搅拌转速的增加,混合时间降低的同时反应器内部的剪切速率明显增加。最终通过改变搅拌桨组合方式的手段有效地解决了上述矛盾,并使得HA分子量提高23.9%。     

In vitro sliding of actin filaments labelled with single quantum dots

We recently refined the in vitro motility assay for studies of actomyosin function to achieve rectified myosin induced sliding of actin filaments. This paves the way, both for detailed functional studies of actomyosin and for nanotechnological applications. In the latter applications it would be desirable to use actin filaments for transportation of cargoes (e.g., enzymes) between different predetermined locations on a chip. We here describe how single quantum dot labelling of isolated actin filaments simultaneously provides handles for cargo attachment and bright and photostable fluorescence labels facilitating cargo detection and filament tracking. Labelling was achieved with preserved actomyosin function using streptavidin-coated CdSe quantum dots (Qdots). These nanocrystals have several unique physical properties and the present work describes their first use for functional studies of isolated proteins outside the cell. The results, in addition to the nanotechnology developments, open for new types of in vitro assays of isolated biomolecules.