Studies on voltammetric determination of cadmium in samples containing native and digested proteins

This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples.     

Silanized quantum dots as labels in lateral flow test strips for C-reactive protein

The paper describes the first use of silanized semiconductor core-shell quantum dots as fluorescent labels for macromolecule, C-reactive protein determination in blood plasma. The controlled synthesis of CdSe cores, with successive shells of CdS, CdZnS, ZnS and coating with transparent, stable, and inert silica shell, provides quantum dots with a narrow emission band, high quantum yield, and prolonged signal stability. Finally, the quantum dots were conjugated with specific antibodies via carboxylic groups on the silica surface. The method was further used for the immunochromatographic assay of C-reactive protein, a diagnostically important inflammatory biomarker. Assays with both the fluorescent QDs and a widely used colloidal gold label were developed in parallel and compared. The silanized quantum dots provide a more sensitive assay with a detection limit of 1?ng/mL for C-reactive protein in standard solutions, whereas the common assay has a detection limit of 10?ng/mL. The possibility of quantitative evaluation of analyte content by a portable device was demonstrated; the accuracy of the measurements was in the range of 5%–10%. The tests were used to determine C-reactive proteins in human plasma samples. The selected optimized protocol for these samples is based on a 4-fold dilution. The final working range of the assay, 4–1,200?ng/mL, covers practically all important interval of C-reactive protein values for the characterization of acute, chronic, and local inflammatory processes. Due to their high physical stability and inertness as well as intense, stable, and reproducible fluorescence, silanized quantum dots may be applied for high-sensitive assays for different analytes.     

脂质体包覆CdSe/ZnSe核－壳量子点

本文提出了一种利用脂质体包覆量子点的方法。这种脂质体包覆的方法可以使量子点溶于水。被脂质体包覆的CdSe/ZnSe量子点仍具有很强的荧光，其荧光强度与未包覆的CdSe/ZnSe量子点处于同一数量级且具有很好的荧光稳定性。这种脂质体包覆的量子点有很好的生物相容性，利用它为荧光标记物，制备了鼠单克隆抗体CD95的免疫检测传感器。     

Calixarene-coated water-soluble CdSe-ZnS semiconductor quantum dots that are highly fluorescent and stable in aqueous solution

A simple method for the preparation of highly fluorescent and stable, water-soluble CdSe-ZnS quantum dots is reported using calix[4]arene carboxylic acids as surface coating agents; the coating of the surface with the calixarene and the conjugation of antibodies to the quantum dots are confirmed by fluorescence correlation spectroscopy.     

Immunoassay by capillary electrophoresis with quantum dots

The application of quantum dots in capillary electrophoresis immunoassay was studied for the first time. Quantum dots were conjugated with antibody and subsequently tested by electrophoretic separation of free antibody and antibody-antigen complex. Antibody was fluorescently labeled by quantum dots via conjugation procedures and its electrophoretic characteristics were effectively modified due to the attachment of quantum dots. The determination of human IgM by direct CE based immunoassay could be easily achieved by simply changing the pH value of separation buffer. Polymer additive influenced the separation too but the effect was not as significant as buffer pH adjustment. Satisfactory separation of complex from free antibody could be achieved with 20mM sodium tetraborate as separation buffer, at pH 9.8. The immunoassay application of quantum dots in CE offers considerable advantages and can be readily applied to other large bio-molecules.     

量子点标记的斑点免疫渗滤分析定量检测cTnI

利用量子点良好的光谱特征和光化学稳定性, 结合免疫分析技术, 对心肌肌钙蛋白I(cTnI)特异性进行定量检测. 用量子点标记cTnI的单克隆抗体(2F11), 通过SDS-PAGE电泳证明标记成功. 斑点免疫膜渗滤法证明标记后的2F11仍具有良好的生物学活性, 再将标记并纯化后的2F11与NC膜上不同浓度的cTnI进行免疫反应, 使用ImageMaster图像分析软件对膜上荧光斑点图像进行定量分析. 应用此方法测得cTnI的浓度和斑点处相对荧光值有良好的线性关系(R2=0.9966), 最低检出值为120 ng.     

Quantum Dot-mediated Photoproduction of Reactive Oxygen Species for Cancer Cell Annihilation

While semiconductor quantum dots produce little singlet oxygen, they may undergo Type I photoreactions to produce other reactive oxygen species (ROS) to kill cells. CdTe quantum dots coated with thioglycolic acid were used to test that possibility. Some thiol ligands were purposely removed to regenerate the surface electron traps that were passivated by the ligand. This allowed photoinduced electrons to dwell on the surface long enough to be gathered by nearby oxygen molecules to produce ROS. The photocytotoxicity of these quantum dots was tested on nasopharyngeal carcinoma cells. Photokilling was shown to be drug and light dose dependent. Using 0.6 μm quantum dots for incubation and 4.8 J cm⁻² for irradiation, about 80% of the cells were annihilated. These quantum dots promised to be potent sensitizers for photoannihilation of cancer cells.     

Localization imaging using blinking quantum dots

The blinking phenomena of the quantum dots have been utilized in the super-resolution localization microscopy to map out the locations of individual quantum dots on a total internal reflection microscope. Our result indicated that the reconstructed image of quantum dots agreed with the topographic image measured by atomic force microscopy. Because of the superior optical properties of the quantum dots, the high localization resolution can be achieved in the shorter acquisition time with larger detected photon numbers. When the cells were labeled with quantum dots, the sub-cellular structures could be clearly seen in the reconstructed images taken by a commercial microscope without using complicated optical systems, special photo-switchable dye pairs or photo-activated fluorescence proteins.     

γ-Fe2O3 Nanoparticles Covered with Glutathione-Modified Quantum Dots as a Fluorescent Nanotransporter

The present paper describes the synthesis, characterization, and utilization of multi-functional magnetic conjugates that integrate optical and magnetic properties in a single structure for use in many biomedical applications. Spontaneous interaction with eukaryotic cell membrane (HEK-239 cell culture) was determined using fluorescence microscopy, and fluorescence analyses. Both, differences in excitation, and emission wavelength were observed, caused by glutathione intake by cells, resulting in disintegration of core–shell structure of quantum dots, as well as adhesion of conjugate onto cell surface. When compared with quantum dots fluorescent properties, HEK-239 cells with incorporated nanoconjugate exhibited two excitation maxima (λ _ex = 430 and 390 nm). Simultaneously, application of ideal λ _ex for quantum dots (λ _ex = 430 nm), resulted in two emission maxima (λ = 740 and 750 nm). This nanoconjugate fulfills the requirements of term theranostics, because it can be further functionalized with biomolecules as DNA, proteins, peptides or antibodies, and thus serves as a tool for therapy in combination with simultaneous treatment.     

γ-Fe2O3 Nanoparticles Covered with Glutathione-Modified Quantum Dots as a Fluorescent Nanotransporter

The present paper describes the synthesis, characterization, and utilization of multi-functional magnetic conjugates that integrate optical and magnetic properties in a single structure for use in many biomedical applications. Spontaneous interaction with eukaryotic cell membrane (HEK-239 cell culture) was determined using fluorescence microscopy, and fluorescence analyses. Both, differences in excitation, and emission wavelength were observed, caused by glutathione intake by cells, resulting in disintegration of core–shell structure of quantum dots, as well as adhesion of conjugate onto cell surface. When compared with quantum dots fluorescent properties, HEK-239 cells with incorporated nanoconjugate exhibited two excitation maxima (λ _ex = 430 and 390 nm). Simultaneously, application of ideal λ _ex for quantum dots (λ _ex = 430 nm), resulted in two emission maxima (λ = 740 and 750 nm). This nanoconjugate fulfills the requirements of term theranostics, because it can be further functionalized with biomolecules as DNA, proteins, peptides or antibodies, and thus serves as a tool for therapy in combination with simultaneous treatment.

     

Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays

Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of their use as a versatile label. CNPs can be loaded with a range of biomolecules, such as DNA, antibodies, and proteins (e.g., neutravidin or a fusion protein of neutravidin with an enzyme), and the resulting conjugates can be used to detect analytes of high or low molecular mass.     

In‐capillary probing of quantum dots and fluorescent protein self‐assembly and displacement using Förster resonance energy transfer

Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real‐time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His₆‐mCherry/quantum dot molar ratio. The kinetics of the self‐assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of‐ and in‐capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in‐capillary probing system.     

Live cell surface labeling with fluorescent Ag nanocluster conjugates

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.     

Specific Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay Based on Quantum Dots

In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43 %. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC?·?HCl, 150 μL of 4 mg/mL NHS, buffer system of Na₂HPO₄-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764 %. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.     

High resolution detection of high mass proteins up to 80,000 Da via multifunctional CdS quantum dots in laser desorption/ionization mass spectrometry

CdS quantum dots (∼5 nm) are used as multifunctional nanoprobes as an effective matrix for large proteins, peptides and as affinity probes for the enrichment of tryptic digest proteins (lysozyme, myoglobin and cytochrome c) in laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS). The use of CdS quantum dots (CdS QDs) as the matrix allows acquisition of high resolution LDI mass spectra for large proteins (5000-80,000 Da). The enhancement of mass resolution is especially notable for large proteins such as BSA, HSA and transferrin (34-49 times) when compared with those obtained by using SA as the matrix. This technique demonstrates the potentiality of LDI-TOF-MS as an appropriate analytical tool for the analysis of high-molecular-weight biomolecules with high mass resolution. In addition, CdS QDs are also used as matrices for background-free detection of small biomolecules (peptides) and as affinity probes for the enrichment of tryptic digest proteins in LDI-TOF-MS.     

Quantum dots protect against MPP+-induced neurotoxicity in a cell model of Parkinson’s disease through autophagy induction

Autophagy is a basic cellular process that decomposes damaged organelles and aberrant proteins. Dysregulation of autophagy is implicated in pathogenesis of neurodegenerative disorders, including Parkinson's disease(PD). Pharmacological compounds that stimulate autophagy can provide neuroprotection in models of PD. Nanoparticles have emerged as regulators of autophagy and have been tested in adjuvant therapy for diseases. In this present study, we explore the effects of quantum dots(QDs) that can induce autophagy in a cellular model of Parkinson's disease. Cd Te/Cd S/Zn S QDs protect differentiated rat pheochromocytoma PC12 cells from MPP+-induced cell damage, including reduced viability, apoptosis and accumulation of α-Synuclein, a characteristic protein of PD. The protective function of QDs is autophagy-dependent. In addition, we investigate the interaction between quantum dots and autophagic pathways and identify beclin1 as an essential factor for QDs-induced autophagy. Our results reveal new promise of QDs in the theranostic of neurodegenerative diseases.     

Simple bio-conjugation of polymer-coated quantum dots with antibodies for fluorescence-based immunoassays

A simple procedure for the bio-conjugation of amphiphilic polymer-coated quantum dots with antibodies is described. The formed bio-conjugates are purified from an excess of free antibodies and of free nanoparticles by size-exclusion HPLC and then characterized by fluorescence emission and MALDI-TOFMS. The applicability of the approach is demonstrated for aflatoxin B1 detection.     

Studies on bioconjugation of quantum dots using capillary electrophoresis and fluorescence correlation spectroscopy

In this paper, we systematically investigated the conjugation of quantum dots (QDs) with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) methods. Commercial QDs and aqueous-synthesized QDs in our lab were used as labeling probes, certain bio-macromolecules, such as proteins, antibodies, and enzymes, were used as mode samples, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfo-succinimide (Sulfo-NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio-macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of bio-macromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo-NHS as linking agents. Furthermore, we on-line investigated the kinetic process of QDs-bioconjugation by FCS and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer. Our work described here will be very helpful for the improvement of the QDs conjugation efficiency in bioapplications.     

ZnS∶Co半导体量子点的制备及其光电化学性质

采用低温水热技术,分别以柠檬酸(CA)和巯基丙酸(MPA)为稳定剂,在70℃的水相中合成了单分散的,粒子尺寸约为4 nm的ZnS∶Co半导体量子点.研究了稳定剂、Co2+掺杂剂及其掺杂量对掺杂量子点发光性能和结构的影响.XRD结果表明,Co2+离子主要掺杂在量子点表面,对主体ZnS晶格没有影响.当采用MPA为稳定剂,掺杂量为5%(摩尔分数)时,掺杂量子点的荧光发射强度最高;而同样掺杂量下采用CA为稳定剂时,量子点的荧光发射强度有所下降.循环伏安研究显示,与空白ZnS量子点相比,Co2+离子的掺杂在ZnS的禁带中形成杂质能级,相应地,ZnS∶Co量子点的吸收边发生红移.与未掺杂ZnS量子点相比,掺杂量子点具有较少的表面非辐射复合中心,因而荧光发射强度显著提高.     

Supramolecular nanostructures based on bacterial reaction center proteins and quantum dots

Design of the nanostructures based on membrane proteins (the key functional elements of biomembranes) and colloid nanoparticles is a fascinating field at the interface of biochemistry and colloids, nanotechnology and biomedicine. The review discusses the main achievements in the field of ultrathin films prepared from bacterial reaction center proteins and light-harvesting complexes, as well as these complexes tagged with quantum dots. The principles of preparation of these thin films and their structure and properties at different interfaces are described; as well as their characteristics estimated using a combination of the modern interfacial techniques (absorption and fluorescence spectroscopy, atomic force and Brewster angle microscopy, etc.) are discussed. Further approaches to develop the nanostructures based on the membrane proteins and quantum dots are suggested. These supramolecular nanostructures are promising prototypes of the materials for photovoltaic, optoelectronic and biosensing applications.