Quantum dots versus organic dyes as fluorescent labels

Suitable labels are at the core of Luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)--inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry--as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.     

量子点荧光标记技术的研究热点及面临的挑战

半导体量子点作为新型荧光标记物,在生物医学领域具有重要应用．与传统的有机染料及荧光蛋白等荧光标记物相比,半导体量子点具有发光颜色可调、激发范围宽、发射光谱窄、化学及光稳定性好、表面化学丰富以及生物偶联技术成熟等诸多优势,为生命体系的靶向示踪,高灵敏、原位、实时、动态荧光成像,DNA及蛋白质检测,靶向药物,临床医学,生物芯片和传感器等研究提供了新的发展契机．基于作者在半导体量子点生物荧光成像和安全性评价研究的基础,综述了半导体量子点荧光标记物在生命科学与医学领域应用的研究热点,并对半导体量子点荧光标记技术走向实用面临的挑战进行了评述．     

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.     

Quantum dots and prion proteins

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.     

Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates' surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05-40 ng mL(-1) and 0.05-25 ng mL(-1), respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.     

Quantum dots in nanobiotechnology

Research on semiconductor nanocristals (also known as quantum dots of QD) in the field of nanobiotechnology is rapidly evolving thanks to progresses in their synthesis and their surface chemistry. Two types of materials, water soluble and biocompatible single QD and beads containing QDs, are becoming available and exciting applications based on these new materials are developed. We will present the recent progress in the synthesis of these materials and their applications. We will discuss the problems that remain to be solved and the perspectives.     

Quantum dots as nano plug-in's for efficient NADH resonance energy routing

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD(+)/NADH with QDs was used to monitor the reaction of NAD(+)-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000-0.01 ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD(+) and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028-0.4 U). Hence, it was possible to detect formaldehyde in the range 1000-0.01 ng/mL with a limit of detection (LOD) at 0.01 ng/mL and regression coefficient R(2)=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD(+)-dependent enzymes and detection of dehydrogenases in general.     

Quantum dots brighten biological imaging

Quantum dots (QDs) are novel photostable semiconductor nanocrystals possessing wide excitation spectra and narrow, symmetrical emission spectra and can be conjugated to a wide range of biological targets, including proteins, antibodies and nucleic acid probes. These characteristics have provoked considerable interest in their use for bioimaging. Much investigation has been performed into their use for multiplex immunohistochemistry and in situ hybridisation which, when combined with multispectral imaging, has enabled quantitation and colocalisation of gene expression in clinical tissue. Many advances have recently been made using QDs for live cell and in vivo imaging, in which QD-labelled molecules can be tracked and visualised in 3-D. This review aims to outline the beneficial properties presented by QDs along with important advances in their biological application.     

Quantum dots go with the flow

RNA interference (RNAi) and automated high-throughput screening is a promising combination. But the first systematic large-scale mapping of genetic interactions in an animal shows that manual methods still have advantages over sophisticated automated screens.     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

Quantum dots as a novel immunofluorescent detection system for Cryptosporidium parvum and Giardia lamblia

Semiconductor quantum dot-conjugated antibodies were successfully developed to label Cryptosporidium parvum and Giardia lamblia. This novel fluorescence system exhibited superior photostability, gave 1.5- to 9-fold-higher signal-to-noise ratios than traditional organic dyes in detecting C. parvum, and allowed dual-color detection for C. parvum and G. lamblia.     

Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging

Delivery of imaging agents to the brain is highly important for the diagnosis and treatment of central nervous system (CNS) diseases, as well as the elucidation of their pathophysiology. Quantum dots (QDs) provide a novel probe with unique physical, chemical, and optical properties, and become a promising tool for in vivo molecular and cellular imaging. However, their poor stability and low blood-brain barrier permeability severely limit their ability to enter into and act on their target sites in the CNS following parenteral administration. Here, we developed a QDs-based imaging platform for brain imaging by incorporating QDs into the core of poly(ethylene glycol)-poly(lactic acid) nanoparticles, which was then functionalized with wheat germ agglutinin and delivered into the brain via nasal application. The resulting nanoparticles, with high payload capacity, are water-soluble, stable, and showed excellent and safe brain targeting and imaging properties. With PEG functional terminal groups available on the nanoparticles surface, this nanoprobe allows for conjugation of various biological ligands, holding considerable potential for the development of specific imaging agents for various CNS diseases.     

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.     

Quantum dots and peptides: a bright future together

Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include, broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QDs-labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions.     

Quantum dots encapsulated with amphiphilic alginate as bioprobe for fast screening anti-dengue virus agents

The increasing threats of viral diseases have gained worldwide attention in recent years. Quite a few infectious diseases are still lacking effective prevention or treatment. The pace of developing antiviral agents could be expedited by the availability of quick and efficient drug screening platforms. In this study, quantum dot (QD), an emerging probe for biological imaging and medical diagnostics, was employed to form complexes with virus and used as fluorescent imaging probes for exploring potential antiviral therapeutics. Inorganic CdSe/ZnS QDs synthesized in organic phase were encapsulated by amphiphilic alginate to attain biocompatible water-soluble QDs via phase transfer. Virus employed for this study was dengue virus which is a notorious one in tropical and subtropical regions of the world. To construct a QD-virus imaging modality capable of providing meaningful information, preservation of viral infectivity after tagging virus with QDs is of utmost importance. In order to form colloidal complexes of QD-virus, electrostatic repulsion force generated from both negatively charged virus and QDs was neutralized by various concentrations of cationic polybrene. Results showed that BHK-21 cells infected with dengue viruses incorporated with QDs exhibited bright fluorescence intracellularly within 30 min. To demonstrate the potency of QD-virus complexes as bioprobes for screening antiviral agents, BHK-21 cells were incubated for one hour with allophycocyanin purified from blue-green algae and then infected with QD-virus complexes. Based on the developed cell-based imaging assay, allophycocyanin with concentration of 125 microg/mL led to extremely weak intracellular fluorescence post-infection of QD-virus complexes for 30 min. That is, the efficacy of anti-dengue viral activity of the algae extract was clearly illustrated by the inorganic-organic hybrid platform constructed in current study.     

Quantum dots to monitor RNAi delivery and improve gene silencing

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell–cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.     

Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles

Successful drug delivery by functionalized nanocarriers largely depends on their efficient intracellular transport which has not yet been fully understood. We developed a new tracking technique by encapsulating quantum dots into the core of wheat germ agglutinin-conjugated nanoparticles (WGA-NP) to track cellular transport of functionalized nanocarriers. The resulting nanoparticles showed no changes in particle size, zeta potential or biobinding activity, and the loaded probe presented excellent photostability and tracking ability. Taking advantage of these properties, cellular transport profiles of WGA-NP in Caco-2 cells was demonstrated. The cellular uptake begins with binding of WGA to its receptor at the cell surface. The subsequent endocytosis happened in a cytoskeleton-dependent manner and by means of clathrin and caveolae-mediated mechanisms. After endosome creating, transport occurs to both trans-Golgi and lysosome. Our study provides new evidences for quantum dots as a cellular tracking probe of nanocarriers and helps understand intracellular transport profile of lectin-functionalized nanoparticles.