Fluorescence resonance energy transfer in CdSe/ZnS-DNA conjugates: probing hybridization and DNA cleavage

Nucleic-acid-functionalized CdSe/ZnS quantum dots (QDs) were hybridized with the complementary Texas-Red-functionalized nucleic acid. The hybridization was monitored by following the fluorescence resonance energy transfer from the QDs to the dye units. Treatment of the QD/dye DNA duplex structure with DNase I resulted in the cleavage of the DNA and the recovery of the fluorescence properties of the CdSe/ZnS QDs. The luminescence properties of the QDs were, however, only partially recovered due to the nonspecific adsorption of the dye onto the QDs. Similarly, nucleic-acid-functionalized Au nanoparticles (Au NPs) were hybridized with the complementary Texas-Red-labeled nucleic acid. The hybridization was followed by the fluorescence quenching of the dye by the Au NPs. Treatment of the Au NP/dye DNA duplex with DNase I resulted in the cleavage of the DNA and the partial recovery of the dye fluorescence. The incomplete recovery of the dye fluorescence originated from the nonspecific binding of the dye units to the Au NPs. The nonspecific binding of the dye to the CdSe/ZnS QDs and the Au NPs is attributed to nonprotected surface vacancies in the two systems.     

Study the damage of DNA molecules induced by three kinds of aqueous nanoparticles

In this paper, the interaction of DNA molecules with aqueous CdTe quantum dots (CdTe QDs), CdTe/SiO₂ composite nanoparticles (CdTe/SiO₂ NPs), and Mn-doped ZnSe quantum dots (Mn:ZnSe d-dots) was studied with ethidium bromide as a probe. The purpose of this work was to study the damage of DNA molecules induced by these three kinds of water-soluble nanoparticles. It was found that ionic strength, pH value and UV irradiation influenced the PL emission properties of CdTe QDs, CdTe/SiO₂ NPs and Mn:ZnSe d-dots, and also influenced the interaction of DNA molecules with them. Among the three kinds of nanoparticles, DNA molecules were most easily damaged by CdTe QDs whether in the dark or under UV irradiation. The CdTe/SiO₂ NPs led to much less DNA damage when compared with CdTe QDs, as a silica overcoating layer could isolate the QDs from the external environment. Mn:ZnSe d-dots as a new class of non-cadmium doped QDs demonstrated almost no damage for DNA molecules, which have great potentials as fluorescent labels in the applications of biomedical assays, imaging of cells and tissues, even in vivo investigations.     

Controlling chemical reactivity at solid-solution interfaces by means of hydrophobic magnetic nanoparticles

Hydrophobic magnetic nanoparticles are employed to reversibly regulate the hydrophobic/hydrophilic properties of surfaces and to control the electrochemistry and bioelectochemistry at chemically modified electrodes. Selective bioelectrocatalytic transformations at relay-functionalized electrodes are accomplished by the magnetic attractions of the hydrophobic magnetic nanoparticles with coadsorbed hydrophobic redox relays to the electrode. The selective activation of one of two biocatalysts solubilized in the aqueous electrolyte solution in the absence or presence of hydrophobic magnetic nanoparticles results in the specific activation of bioelectrocatalytic processes. The magnetic attraction and retraction of hydrophobic magnetic nanoparticles to and from semiconductor nanoparticle (CdS)-functionalized electrodes enable the control of the photocurrent directions at the electrode from cathodic to anodic directions, respectively. The magnetic attraction of the hydrophobic magnetic nanoparticles to the surfaces is also employed to control biorecognition and biocatalytic transformations at solid supports. The magnetic attraction and retraction of the hydrophobic magnetic nanoparticles to and from the surfaces allow the blockage and activation of DNA hybridization, polymerization, and enzymatic digestion, respectively.     

Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors,DNA‐Sensors,and Enzyme Biosensors

Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial‐functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field‐effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen‐antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single‐base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field‐effect transistors (FET) upon the formation of antigen‐antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme‐based biosensors. The reconstitution of apo‐enzymes on cofactor‐functionalized electrodes and the formation of cofactor‐enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.     

Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.     

Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology

Biomolecule-metal or semiconductor nanoparticle (NP) hybrid systems combine the recognition and catalytic properties of biomolecules with the unique electronic and optical properties of NPs. This enables the application of the hybrid systems in developing new electronic and optical biosensors, to synthesize nanowires and nanocircuits, and to fabricate new devices. Metal NPs are employed as nano-connectors that activate redox enzymes, and they act as electrical or optical labels for biorecognition events. Similarly, semiconductor NPs act as optical probes for biorecognition processes. Double-stranded DNA or protein chains that are modified with metallic nanoclusters act as templates for the synthesis of metallic nanowires. The nanowires are used as building blocks to assemble nano-devices such as a transistor or a nanotransporter.     

Semiconductor quantum dots and metal nanoparticles: syntheses,optical properties,and biological applications

We review the syntheses, optical properties, and biological applications of cadmium selenide (CdSe) and cadmium selenide–zinc sulfide (CdSe–ZnS) quantum dots (QDs) and gold (Au) and silver (Ag) nanoparticles (NPs). Specifically, we selected the syntheses of QDs and Au and Ag NPs in aqueous and organic phases, size- and shape-dependent photoluminescence (PL) of QDs and plasmon of metal NPs, and their bioimaging applications. The PL properties of QDs are discussed with reference to their band gap structure and various electronic transitions, relations of PL and photoactivated PL with surface defects, and blinking of single QDs. Optical properties of Ag and Au NPs are discussed with reference to their size- and shape-dependent surface plasmon bands, electron dynamics and relaxation, and surface-enhanced Raman scattering (SERS). The bioimaging applications are discussed with reference to in vitro and in vivo imaging of live cells, and in vivo imaging of cancers, tumor vasculature, and lymph nodes. Other aspects of the review are in vivo deep tissue imaging, multiphoton excitation, NIR fluorescence and SERS imaging, and toxic effects of NPs and their clearance from the body. Figure Semiconductor quantum dots and metal nanoparticles have extensive applications, e.g., in vitro and in vivo bioimaging Tamitake Itoh and Abdulaziz Anas contributed equally to this article.     

CdS量子点与金电极之间的光生电子交换

对光照下CdS量子点与金电极之间的光生电子交换过程进行了系统研究.首先,对基于电子隧穿的多个光生电子转移过程进行了理论分析,并进行了数学模型推导.其次,利用推导的数学模型在不同参数的条件下进行了仿真研究.最后,测量了CdS量子点修饰的金电极在不同偏置电压、不同光强和不同氧化物及还原物浓度的条件下产生的光电流,并将实验、理论及仿真结果三者相结合进行了分析讨论.实验结果与仿真结果都证明了理论模型的正确性.     

Biomimetic Synthesis of Ag2Se Quantum Dots with Enhanced Photothermal Properties and as “Gatekeepers” to Cap Mesoporous Silica Nanoparticles for Chemo–Photothermal Therapy

Ag₂Se quantum dots (QDs) with near‐infrared (NIR) fluorescence have been widely utilized in NIR fluorescence imaging in vivo because of their narrow bulk band gap and excellent biocompatibility. However, most of synthesis methods for Ag₂Se QDs are expensive and the reactants are toxic. Herein, a new protein‐templated biomimetic synthesis approach is proposed for the preparation of Ag₂Se QDs by employing bovine serum albumin (BSA) as a template and dispersant. The BSA‐templated Ag₂Se QDs (Ag₂Se@BSA QDs) showed NIR fluorescence with high fluorescence quantum yield (≈21.2 %), excellent biocompatibility and good dispersibility in different media. Moreover, the obtained Ag₂Se@BSA QDs exhibited remarkable photothermal conversion (≈27.8 %), which could be used in photothermal therapy. As a model application in biomedicine, the Ag₂Se@BSA QDs were used as “gatekeepers” to cap mesoporous silica nanoparticles (MSNs) by means of electrostatic interaction. By taking the advantages of NIR fluorescence and photothermal property of Ag₂Se@BSA QDs, the obtained MSN‐DOX‐Ag₂Se nanoparticles (MDA NPs) were employed as a nanoplatform for combined chemo‐photothermal therapy. Compared with free DOX and MDA NPs without NIR laser, the laser‐treated MDA NPs exhibited lower cell viability in vitro, implying that Ag₂Se@BSA QDs are highly promising photothermal agents and the MDA NPs are potential carriers for chemo–photothermal therapy.     

Bioinspired Artificial Photosynthetic Systems

Mimicking photosynthesis using artificial systems, as a means for solar energy conversion and green fuel generation, is one of the holy grails of modern science. This perspective presents recent advances towards developing artificial photosynthetic systems. In one approach, native photosystems are interfaced with electrodes to yield photobioelectrochemical cells that transform light energy into electrical power. This is exemplified by interfacing photosystem I (PSI) and photosystem II (PSII) as an electrically contacted assembly mimicking the native Z-scheme, and by the assembly of an electrically wired PSI/glucose oxidase biocatalytic conjugate on an electrode support. Illumination of the functionalized electrodes led to light-induced generation of electrical power, or to the generation of photocurrents using glucose as the fuel. The second approach introduces supramolecular photosensitizer nucleic acid/electron acceptor complexes as functional modules for effective photoinduced electron transfer stimulating the subsequent biocatalyzed generation of NADPH or the Pt-nanoparticle-catalyzed evolution of molecular hydrogen. Application of the DNA machineries for scaling-up the photosystems is demonstrated. A third approach presents the integration of artificial photosynthetic modules into dynamic nucleic acid networks undergoing reversible reconfiguration or dissipative transient operation in the presence of auxiliary triggers. Control over photoinduced electron transfer reactions and photosynthetic transformations by means of the dynamic networks is demonstrated.     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

Electrochemical sensing of silver tags labelled DNA immobilized onto disposable graphite electrodes

An electrochemical approach for the improved electrochemical sensing of DNA was developed in this study based on the oxidation signals of silver and DNA base, guanine by using disposable pencil graphite electrode (PGE) electrodes. The easy surface modification of disposable electrodes PGEs with nucleic acids was performed by passive adsorption using amino linked DNA oligonucleotide attached onto the surface of silver nanoparticles (Ag-NPs). Firstly, the microscopic characterization of silver nanoparticles was investigated by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and the electrochemical behaviour of these NPs was studied by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. Then, the overall performance of this novel electrochemical DNA sensing method based on these nanoparticles is studied and discussed in terms of optimum analytical conditions, such as; the effect of DNA concentration, NPs concentration and different buffer solutions, etc. in order to obtain silver and guanine oxidation signals in higher sensitivity and selectivity. The main features related with this electrochemical assay based on silver nanoparticles are discussed and compared with other assays reported in the literatures.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Detection of influenza A virus based on fluorescence resonance energy transfer from quantum dots to carbon nanotubes

In this paper, a simple and sensitive approach for H5N1 DNA detection was described based on the fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to carbon nanotubes (CNTs) in a QDs-ssDNA/oxCNTs system, in which the QDs (CdTe) modified with ssDNA were used as donors. In the initial stage, with the strong interaction between ssDNA and oxCNTs, QDs fluorescence was effectively quenched. Upon the recognition of the target, the effective competitive bindings of it to QDs-ssDNA occurred, which decreased the interactions between the QDs-ssDNA and oxCNTs, leading to the recovery of the QDs fluorescence. The recovered fluorescence of QDs was linearly proportional to the concentration of the target in the range of 0.01–20 μM with a detection limit of 9.39 nM. Moreover, even a single-base mismatched target with the same concentration of target DNA can only recover a limited low fluorescence of QDs, illustrating the good anti-interference performance of this QDs-ssDNA/oxCNTs system. This FRET platform in the QDs-ssDNA/oxCNTs system was facilitated to the simple, sensitive and quantitative detection of virus nucleic acids and could have a wide range of applications in molecular diagnosis.     

Turn‐On Luminescent Probes for the Real‐Time Monitoring of Endogenous Hydroxyl Radicals in Living Cells

The utilization of semiconductor quantum dots (QDs) as optical labels for biosensing and biorecognition has made substantial progress. However, the development of a suitable QD‐based luminescent probe that is capable of detecting individual reactive oxygen species (ROS) represents a great challenge, mainly because the fluorescence of QDs is quenched by a wide variety of ROS. To overcome this limitation, a novel QD‐based turn‐on luminescent probe for the specific detection of ^.OH has been designed, and its application in monitoring the endogenous release of ^.OH species in living cells is demonstrated. Metal citrate complexes on the surfaces of the QDs can act as electron donors, injecting electrons into the LUMO of the QDs, while ^.OH can inject holes into the HOMO of the QDs. Accordingly, electron–hole pairs are produced, which could emit strong luminescence by electron–hole recombination. Importantly, this luminescent probe does not respond to other ROS.     

Quantum dots on electrodes—new tools for bioelectroanalysis

The review covers recent developments in which quantum dots (QDs) are combined with electrodes for detection of analytes. Special focus will be on the generation of photocurrents and the possibility of spatially resolved, light-directed analysis. Different modes for combining biochemical reactions with QDs will be discussed. Other applications involve the use of QDs as labels in binding analysis. Different methods have been developed for read-out. In addition to photocurrent analysis, voltammetric detection of metals and electrochemiluminescence (ECL) can be used. In the latter, light is the sensor signal. ECL-based systems combine the advantage of very sensitive analytical detection with rather simple instrumentation.

碲化镉量子点光催化降解溴化乙锭的研究

以巯基乙酸为稳定剂, 水相合成出不同粒径的碲化镉量子点, 对其进行适当的光照处理, 以提升其光学性质和稳定性. 将其用作光催化剂, 以紫外灯为光源, 降解具有强诱变作用的常用核酸荧光染料——溴化乙锭. 考察了光照时间和溶液 pH 对降解率的影响. 以溴化乙锭在620 nm处的特征荧光发射峰为参数表征降解率, 在反应4 h后, 对15 mg/L溴化乙锭水溶液的降解率近90%, 同时, 对琼脂糖凝胶中的溴化乙锭也有降解作用.