Highly sensitive electrochemical detection of potential cytotoxicity of CdSe/ZnS quantum dots using neural cell chip

Cell chip was recently developed as a simple and highly sensitive tool for the toxicity assessment of various kinds of chemicals or nano-materials. Here, we report newly discovered potential cytotoxic effects of CdSe/ZnS quantum dots (QDs) on intracellular redox environment of neural cancer cells at very low concentrations which can be only detected by cell chip technology. Green (2.1 nm in diameter) and red (6.3 nm in diameter) QDs capped with cysteamine (CA) or thioglycolic acid (TA) were found to be toxic at 100 μg/mL when assessed by trypan blue and differential pulse voltammetry (DPV). However, in case of concentration-dependent cytotoxicity, toxic effects of TA-capped QDs on human neural cells were only measured by DPV method when conventional MTT assay did not show toxicity of TA-capped QDs at low concentrations (1-10 μg/mL). Red-TA QDs and Green-TA QDs were found to decrease electrochemical signals from cells at 10 μg/mL and 5 μg/mL, respectively, while cell viability decreased at 100 μg/mL and 50 μg/mL when assessed by MTT assay, respectively. The relative decreases of cell viability determined by MTT assay were 15% and 11.9% when cells were treated with 5-50 μg/mL of Red-TA QDs and 5-30 μg/mL of Green-TA QDs, respectively. However, DPV signals decreased 37.5% and 39.2% at the same concentration range, respectively. This means that redox environment of cells is more sensitive than other components and can be easily affected by CdSe/ZnS QDs even at low concentrations. Thus, our proposed neural cell chip can be applied to detect potential cytotoxicity of various kinds of molecular imaging agents simply and accurately.     

Formulation, characterization, and in vitro evaluation of quantum dots loaded in poly(lactide)-vitamin E TPGS nanoparticles for cellular and molecular imaging

We developed a novel system of poly(lactide acid)-d-alpha-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) nanoparticles (NPs) for quantum dots (QDs) formulation to improve imaging effects and reduce side effects as well as to promote a sustainable imaging. The QDs-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method, which were then characterized by laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and transmission electron microscope (TEM) for surface morphology. Surface chemistry of the QDs-loaded PLA-TPGS NPs was analyzed by X-ray photoelectron microscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR). Encapsulation efficiency of the QDs in the polymeric nanoparticles was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The photostability of the QDs formulated in the PLA-TPGS nanoparticles was investigated as changes in the florescence intensity versus the irradiation time. Confocal laser scanning microscopy (CLSM) was used to image the cellular uptake of the QDs-loaded NPs by MCF-7 cells. Methylthiazolyldiphenyl-tetrazolium (MTT) assay was employed to assess the viability of MCF-7 cells incubated with the QDs formulated by the PLA-TPGS NPs versus the mercaptoacetic acid (MAA)-coated QDs. It was found that the QDs formulated in the PLA-TPGS NPs can result in higher fluorescence intensity and higher photostability than the bare QDs as well as lower cytotoxicity than the MAA-coated QDs.     

Nanoscale time-lapse AFM imaging in solution for DNA aggregation

Revealing the behavior of biofunctional molecules (i.e., nucleic acids, nucleic acid binding reagents, enzymatic proteins, etc.) by monitoring them in solution is important for understanding the nanoscale dynamism of their interactions. Atomic force microscope (AFM) imaging with a dynamic force mode (DFM, i.e., tapping mode) in aqueous solution, has many advantages for the imaging of DNA morphological change at a single molecule scale. Hoechst 33258 (H33258) induces DNA condensation in the presence of its excess concentration. To have a better understanding of the condensation process of DNA with excess H33258, we tried to find the optimum conditions for carrying out time-lapse AFM imaging in aqueous solution. To immobilize DNA on the substrate surface, the mica was modified with the various concentrations of 3-aminopropyltriethoxysilane (APTES) solution. We observed that DNA was minimally immobilized on 0.002% APTES-modified mica surface. Then, we determined that the movement of DNA on the mica surface could be observed in the presence of 500 mM NaCl in 10 mM PBS (pH 7.0). Moreover, after the injection of 5 μM H33258, the partial condensation of DNA was observed.     

白术多糖WAM-1结构的色谱分析和原子力显微镜观察

通过热水浸提法从草本植物白术根茎提取的水溶性粗多糖,经DEAE-52纤维素柱层析分离和Sephadex G-200凝胶过滤柱层析纯化,得到组分WAM-1.采用高效液相色谱(HPLC)检测WAM-1的纯度,气相色谱(GC)对其单糖组分进行分析,原子力显微镜(AFM)对其分子外貌进行观测.结果显示:WAM-1为均一多糖,由葡萄糖和半乳糖以3.01:1摩尔比构成;在不同浓度溶液条件下,WAM-1分子以不同形态存在,多糖溶液的浓度对WAM-1的分子链构象及链间相互作用形式产生影响,推测可能与WAM-1分子内、分子间的氢键缔合作用有关.多糖浓度为10μg/mL时,可清晰的观察到WAM-1是以刚性链状形态存在,且具有多分支结构.     

In vitro and in vivo anticandidal efficacy of green synthesized gold nanoparticles using Spirulina maxima polysaccharide

This study, for the first time, demonstrated an unprecedented approach for the green synthesis of gold (Au) nanoparticles (NPs) using the polysaccharide of Spirulina maxima as a reducing agent. Time-kill kinetic analysis was used to evaluate the antifungal activity of the green synthesized Au NPs against the pathogenic Candida albicans (C. albicans). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were found to be 32 μg/mL and 64 μg/mL, respectively. Ultra-structural analysis indicated prominent damage on cell wall of the C. albicans after Au NPs treatment, and suggested that the treatment could increase the membrane permeability and disintegration of cells leading to cellular death. The results of propidium iodide (PI) uptake assay showed the higher level of cell death in Au NPs treated C. albicans cells, further confirming the loss of plasma membrane integrity. Cytotoxicity analysis of Au NPs on HEK293T and A549 cells showed no cytotoxic effect up to 64 μg/mL of Au NPs concentration, indicating the potential use in in vivo studies. Also, the recovery of C. albicans infected zebrafish after Au NPs therapy suggest green synthesized Au NPs from S. maxima polysaccharide as a prospective anticandidal agent.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

The binding mode of the DNA bisintercalator luzopeptin investigated using atomic force microscopy

The luzopeptins are DNA bisintercalating antibiotics that contain a decadepsipeptide to which are attached two quinoline chromophores. We have used atomic force microscopy (AFM) to investigate the interaction between luzopeptin B and DNA in an attempt to shed light on the binding mode of this antibiotic. AFM images provided contour lengths which were used as a direct measure of bisintercalation. Binding of luzopeptin B was investigated using two different DNA sequences, one having a GC content of 42% and the other 59%, which revealed a higher degree of bisintercalation into the DNA sequences having the lower GC content. The measured increment in contour length was found to plateau at values corresponding to binding of one drug molecule every 40 and 72 bp to the 42 and 59% GC sequences, respectively. In addition to the length increase, a higher proportion of DNA molecules displaying complex morphology was observed as the concentration of luzopeptin was increased. Such molecules were not included in the measurements of contour length. We propose that the various manifestations of complex morphology arise from both inter- and intramolecular cross-linking of the DNA caused by binding of luzopeptin, providing direct evidence of cross-linked species by AFM imaging.     

量子点细胞毒性及其作用机制

纳米粒子(NPS)在工业和研究中的使用急剧增加,因而这种材料面临一个其潜在毒性的问题。不幸的是,对纳米颗粒与纳米/生物界面可能发生的相互作用没有足够的了解。广大科技工作者正在积极寻求日益关注的纳米技术对人类的影响答案。我们将从NPS在生物媒体中的浓度,尺寸大小,电荷,和配位体的稳定性方面来了解纳米粒子的性质和他们在生物环境中对细胞毒所起的作用;并初步探讨已知的机制,量子点可以破坏细胞,包括氧化应激引起的活性氧(ROS)。微小浓度量子点足以造成长期持久的,甚至是跨代的影响。本文讨论了从纳摩尔到皮摩尔浓度的诱导细胞损伤的量子点(QDS)的浓度,这意味着含镉量子点可以发挥表观遗传毒性,纳米基因毒性,重金属基因的毒性。在此为评估包括量子点的在内的纳米毒性的的纳米材料,我们采用量子点作为一个例证,来阐述以科学为基础的发展到纳米毒理学的相关的问题。     

One‐pot synthesis and characterization CdTe:Zn2+ quantum dots and its molecular interaction with calf thymus DNA

Tremendous research efforts have been dedicated to fabricating high‐quality Zn‐doped CdTe quantum dots (QDs) for any potential biomedical applications. In particular, the correlation of issues regarding how QDs interact with DNA is of greatest importance. Herein, a pH‐responsive study of the interactions between CdTe:Zn²⁺ quantum dots with 4 different sizes and calf thymus DNA (ctDNA) was conducted using multispectroscopic techniques and electrochemical investigation. Fluorescence studies revealed that this interaction process is predominantly a static process and groove binding was the main binding mode for CdTe:Zn²⁺ QDs to ctDNA. The calculated negative values of enthalpy (?45.06 kJ mol^?1) and entropy (?133.62 J mol^?1 K^?1) with temperature changes indicated that the hydrogen bonds and van der Waals interactions played major roles in the reaction. Furthermore, circular dichroism spectroscopy and Fourier transform infrared spectrometry analyses indicate that the normal conformation of ctDNA is discombobulated by CdTe:Zn²⁺ QDs. In addition, the electrochemical behavior of the affinity of CdTe:Zn²⁺ QDs for ctDNA agreed well with the results obtained from fluorescence experiments. This study might be meaningful for understanding the molecular binding mechanism of QDs for DNA and provides a basis for QD‐labeled systems.     

A simple and effective sample preparation method for atomic force microscopy visualization of individual DNA molecules in situ

A simple, controllable and effective sample preparation method was established for atomic force microscopy (AFM) imaging of individual DNA molecules in aqueous solution. Firstly, magnesium ion (Mg²⁺) at a concentration of 5.0–10.0 mM as a positively charged bridge was transferred onto mica to immobilize DNA molecules. Then Mg²⁺-modified mica was used to investigate DNA molecules in any buffer without magnesium ion by AFM. AFM images demonstrated that DNA molecules can be successfully observed in solution with good resolution, reproducibility, and stability. Further, this DNA sample preparation method makes AFM successful to investigate DNA molecular interaction in situ and DNA/chitosan complex in gene delivery.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Specific detection of DNA using quantum dots and magnetic beads for large volume samples

Here we present a sensitive DNA detection protocol using quantum dots (QDs) and magnetic beads (MBs) for large volume samples. In this study, QDs, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were used to isolate and concentrate the signals. The presence of target DNAs leads to the sandwich hybridization between the functionalized QDs, the target DNAs and the MBs. In fact, the QDs-MBs complex, which is bound using the target DNA, can be isolated and then concentrated. The binding of the QDs to the surface of the MBs was confirmed by confocal microscopy and Cd elemental analysis. It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of non-complementary DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5 pM in large volume samples up to 40 mL was successfully detected by using a magnet-assisted concentration protocol which consequently results in the enhancement of the sensitivity more than 100-fold.     

Nucleation temperature‐controlled synthesis and in vitro toxicity evaluation of l‐cysteine‐capped Mn:ZnS quantum dots for intracellular imaging

Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long‐term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of l ‐cysteine as a capping agent for Mn‐doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non‐toxic, water‐dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with l ‐cysteine as a capping agent were found to be non‐toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

Molecular spectroscopic studies on the interactions of rhein and emodin with thioglycolic acid‐capped core/shell CdTe/CdS quantum dots and their analytical applications

Water‐soluble thioglycolic acid (TGA)‐capped core/shell CdTe/CdS quantum dots (QDs) were synthesized. The interactions of rhein and emodin with TGA‐CdTe/CdS QDs were evaluated by fluorescence and ultraviolet‐visible absorption spectroscopy. Experimental results showed that the high fluorescence intensity of TGA‐CdTe/CdS QDs could be effectively quenched in the presence of rhein (or emodin) at 570 nm, which may have resulted from an electron transfer process from excited TGA‐CdTe/CdS QDs to rhein (or emodin). The quenching intensity was in proportion to the concentration of both rhein and emodin in a certain range. Under optimized conditions, the linear ranges of TGA‐CdTe/CdS QDs fluorescence intensity versus the concentration of rhein and emodin were 0.09650–60 µg/mL and 0.1175–70 µg/mL with a correlation coefficient of 0.9984 and 0.9965, respectively. The corresponding detection limits (3σ/S) of rhein and emodin were 28.9 and 35.2 ng/mL, respectively. This proposed method was applied to determine rhein and emodin in human urine samples successfully with remarkable advantages such as high sensitivity, short analysis time, low cost and easy operation. Based on this, a simple, rapid and highly sensitive method to determine rhein (or emodin) was proposed. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of synergistic effects of antibiotics and Zno NPs against isolated E. Coli and A. Baumannii bacterial strains from clinical samples

The mortality rates has been increased globally due to multidrug resistant (MDR) E.coli and A.baumanii bacterial strains and also there is an emerging resistance of the Enterobacteriaceae family of bacteria to Carbapenem antibiotics (CRE) in Saudi Arabia. The main aim of our research study is to isolate E.coli and A. baumannii bacterial species from various collected clinical samples and to evaluate the MIC and FICI of Colistin, Ciprofloxacin, Meropenem and ZnO NPs and in combination of Colistin, Ciprofloxacin, Meropenem with ZnO NPs.The clinical isolated strains of A. baumannii (MRO-17-13) and A. baumannii (MRO-17–25) was found to be sensitive towards colistin with 0.5 μg/mL concentration, whereas, all the isolated A. baumannii strains showed similar MIC value 2 mg/mL when tested with ZnO NPs, the MIC value for the ZnO NPs was found to be similar for all the E.coli strains 0.25 mg/mL. The effects of all Ciprofloxacin concentrations used in the study were bacteriostatic against E. coli (01UR19006568-01) strain but 1 mg/mL concentration of ZnO NPs alone is showed bactericidal activity, ZnO NPs effect was found to be concentration dependent, as highest concentration of ZnO NPs showed strongest antibacterial effect. In conclusion, more investigation is required to evaluate the acceptable concentration of Zno NPs and antibiotics selected to avoid toxicity and must be tested against more clinically isolated gram-negative bacterial strains.     

Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids.

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.     

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.     

Characterization of multivalent lactose quantum dots and its application in carbohydrate–protein interactions study and cell imaging

We have previously reported a facile and convenient method for the preparation of a new type of lactose-CdSeS/ZnS quantum dots conjugates (Lac-QDs) that exhibit biocompatibility, noncytotoxicity and specificity to leukocytes. In order to further study the carbohydrate–protein interactions, a series of Lac-QDs with different lactose densities and a PEGylated (n = 3) lactose-QDs conjugate (LacPEG-QDs) with more flexible sugar ligands were prepared. The amount of the sugar molecules on QDs can be determined by NMR, which was in agreement with the results from TGA determination. The formula of the conjugates was determined with ICP-OES. The interactions between the conjugated QDs and the PNA protein were measured using SPR, which revealed that higher lactose density favored binding affinity under the same concentration, and Lac-QDs exhibit higher affinity than LacPEG-QDs. We further used a solid phase assay to assess the anti-adhesion activity of Lac-QDs and LacPEG-QDs on the cell level. The results showed that Lac-QDs had stronger activity in preventing THP1 from adhering to HUVEC than LacPEG-QDs, which was consistent with the SPR results. We reasoned that decrease in the conformational entropy induced by appropriate restriction of sugar flexibility could enhance the binding affinity of glyco-QDs, which implies that entropy change may be the main contributor to the interaction between high valent glyco-QDs and protein. The fabrication of lactose on QDs provides a fluorescent multivalent carbohydrate probe that can be used as mimics of glycoprotein for the study of carbohydrate–protein interactions and cell imaging.