In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications.     

A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL⁻¹ for CEA, with a detection limit of 0.3 ng mL⁻¹. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.     

Capillary electrophoresis of quantum dots: Minireview

It has been already three decades, since the fluorescent nanocrystals called quantum dots (QDs) appeared and attracted attention of a broad scientific community. Their excellent not only optical but also electronic properties predetermined QDs for utilization in a variety of areas. Besides lasers, solar cells, and/or computers, QDs have established themselves in the field of (bio)chemical labeling as well as medical imaging. However, due to the numerous application possibilities of QDs, there are high demands on their properties that need to be precisely controlled and characterized. CE with its versatile modes and possibilities of detection was found to be an effective tool not only for characterization of QDs size and/or surface properties but also for monitoring of their interactions with other molecules of interest. In this minireview, we are giving short insight in analysis of QDs by CE, and summarizing the advantages of this method for QDs characterization.     

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.     

Study of the photophysical behavior of tetrasulfonated metallophthalocyanines in the presence of CdTe quantum dots

Förster resonance energy transfer (FRET) studies were carried out with CdTe quantum dots (QDs) synthesized in aqueous phase and various tetrasulfonated metallophthalocyanines (MPcS₄, M = aluminum ((OH)AlPcS₄), zinc (ZnPcS₄), silicon ((OH)₂SiPcS₄) and germanium ((OH)₂GePcS₄) in a H₂O:MeOH (1:1) solvent mixture. The QDs studied were capped with thioglycolic acid (TGA) or mercaptopropionic acid (MPA) with sizes ranging from 2.3 to 3.7 nm. Non-radiative energy transfer from QDs emission to MPcS₄ complexes was observed. Study of the photophysics of the MPcs in the presence of the QDs revealed high triplet state quantum yields (Φ_T, ranging from 0.41 to 0.85 in the presence of QDs), with corresponding long triplet state lifetimes (τ_T, which ranged from 140 to 610 μs in the presence of QDs) to allow for photosensitized reactions to occur. The efficiency of energy transfer and the donor–acceptor distance between the MPcs and the QDs were also evaluated.     

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors

The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD₆₀₅) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.     

Investigation of the weak binding of a tetrahistidine‐tagged peptide to quantum dots by using capillary electrophoresis with fluorescence detection

Capillary electrophoresis with fluorescence detection was utilized to probe the self‐assembly between cyanine group dye labeled tetrahistidine containing peptide and CdSe/ZnS quantum dots, inside the capillary. Quantum dots and cyanine group dye labeled tetrahistidine containing peptide were injected into the capillary one after the other and allowed to self‐assemble. Their self‐assembly resulted into a measurable Förster resonance energy transfer signal between quantum dots and cyanine group dye labeled tetrahistidine containing peptide. The Förster resonance energy transfer signal increased upon increasing the cyanine group dye labeled tetrahistidine containing peptide/quantum dot molar ratio and reached a plateau at the 32/1 molar ratio. Additionally, the Förster resonance energy transfer signal was also affected by the increment of the interval time of injection and the sampling time. Online ligand exchange experiments were used to assess, the potential of a monovalent ligand of imidazole and a hexavalent ligand peptide, to displace surface bound cyanine group dye labeled peptide ligands from the quantum dots surface. Under optimal conditions, a linear relationship between the integrated peak areas and hexavalent ligand peptide was obtained at a hexavalent ligand concentration range of 0−0.5 mM. Therefore, the present assay has the potential to be applied in the online ligands detection.     

Capillary electrophoresis,a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single‐stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.     

Resolving quantum dots and peptide assembly and disassembly using bending capillary electrophoresis

Despite the numerous techniques developed for the studying nanoparticle and peptide interaction nowadays, sensitive and convenient assay in the process of flow, especially to simulate the self‐assembly of quantum dots (QDs) and peptide inflow in blood vessels, still remains big challenges. Here, we report a novel assay for studying the self‐assembly of QDs and peptide, based on CE using a bending capillary. We demonstrate that the semicircles numbers of the bending capillary affect the self‐assembly kinetics of CdSe/ZnS QDs and ATTO‐D₃LVPRGSGP₉G₂H₆ peptide. Moreover, benefitting from this novel assay, the effect of the position on the self‐assembly has also been realized. More importantly, we also demonstrate that this novel assay can be used for studying the stability of the QDs–peptide complex inflow. We believe that our novel assay proposed in this work could be further used as a general strategy for the studying nanoparticle–biomolecule interaction or biomolecule–biomolecule interaction.     

Capillary electrophoretic studies on quantum dots and histidine appended peptides self‐assembly

Herein, we designed four peptides appended with different numbers of histidine (His_n‐peptide). We launched a systematic investigation on quantum dots (QDs) and His_n‐peptide self‐assembly in solution using fluorescence coupled CE (CE‐FL). The results indicated that CE‐FL was a powerful method to probe how ligands interaction on the surface of nanoparticles. The self‐assembly of QDs and peptide was determined by the numbers of histidine. We also observed that longer polyhistidine tags (n ≤ 6) could improve the self‐assembly efficiency. Furthermore, the formation and separation of QD‐peptide assembly were also studied by CE‐FL inside a capillary. The total time for the mixing, self‐assembly, separation, and detection was less than 10 min. Our method greatly expands the application of CE‐FL in QDs‐based biolabeling and bioanalysis.     

The photophysical studies of a mixture of CdTe quantum dots and negatively charged zinc phthalocyanines

Fluorescence resonance energy transfer (FRET) studies were carried out with quantum dots capped with thioglycolic acid (TGA) and 2-mercaptoethanol (2-ME) and negatively charged phthalocyanines {Zn tetracarboxy (ZnTCPc), Zn octacarboxy (ZnOCPc) and Zn tetrasulfo (ZnTSPc) phthalocyinines} in a 0.1 NaOH:EtOH (50:50) solvent mixture. The best overlap between emission spectra of the donor (QDs) and the absorption spectra of the acceptor (ZnPc derivatives) was observed for TGA capped QDs, very little overlap was obtained for 2-ME QDs. ZnTSPc gave the highest FRET efficiency (0.3), with ZnOCPc and ZnTCPc giving a FRET efficiency of 0.2. The Φ_T values of the MPcs generally decreased in the presence of the QD whereas the triplet lifetimes (τ_T) of the ZnPc derivatives were higher in the presence of QDs.     

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.     

A comparative physicochemical study of unsymmetrical indium phthalocyanines in the presence of magnetic nanoparticles or quantum dots

Asymmetric indium phthalocyanine (3, containing an NH₂ group) was conjugated (via an amide bond) to magnetic nanoparticle (MNP) functionalized with carboxylic acid or glutathione-capped CdTe/ZnSe/ZnO quantum dots to form 3-MNPs or 3-QDs. Techniques such as time-resolved fluorescence measurements, transmission electron microscopy, XPS, elemental analysis, FTIR, NMR (¹H, ¹³C, and cozy), electronic spectroscopy, as well as mass spectroscopy were employed to characterize 3 and its nanoconjugates. The phthalocyanine conjugated to quantum dot (3-QDs) possesses the lowest Ф_pd higher Ф_? and Ф_T as well as longer triplet lifetimes compares to 3-MNPs and free phthalocyanine.     

Ultrasensitive determination of 1,4-dihydroxybenzene based on fluorescence resonance energy quenching of luminescent quantum dots modified on surface of silica nanoparticles

An ultrasensitive solid-phase fluorescence resonance energy quenching (FREQ) method for determination of 1,4-dihydroxybenzene (DHB) using mercaptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) immobilized on silica nanoparticles (NPs) as donors was developed. In the method, silica NPs were first modified with 3-aminopropyltriethoxysilane (APTS). Then, MSA-capped CdTe QDs were immobilized on the surface of the APTS-modified silica NPs. Finally, DHB in the solution was attached to the empty sites on the surface of silica NPs with QDs through electrostatic interaction. The fluorescence emission of the QDs was quenched by the proximal DHB molecules on the silica NPs. The quenching efficiency of the solid-phase FREQ method was 200-times higher than that of the solution-phase FREQ method. Using the ultrasensitive solid-phase FREQ method, DHB as low as 2.4 × 10⁻¹² mol/L could be detected. The method was applied to quantify trace DHB in water samples.     

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.     

Multienzyme detection and in-situ monitoring of enzyme activity by bending CE using quantum dots-based polypeptide substrate

Multienzyme detection and monitoring enzyme activity in situ are significant for the disease to diagnose. This study aims to develop a quantum dots (QDs)-based nanoprobe Cyanine5-DDDLEVLFQFPGLVPRGSGGHHHHHH-QDs (Cy5-LEVLVP-QD), which is able to detect two enzymes inside a bent capillary using CE. Cy5-LEVLVP and QDs were allowed to bind with each other through metal affinity interaction and then injected the Cy5-LEVLVP-QD complex into a capillary with different bends, followed by related enzyme that can cleave the Cy5-LEVLVP peptide. The fluorescence of Cy5 was excited by QDs due to Förster resonance energy transfer. By monitoring the peaks produced by the original Cy5-LEVLVP-QD complex and a significant fluorescence change, sensitive analysis of two different enzymes was conducted. Therefore, the novel approach of using capillaries with semicircular bends could prove particularly useful for enzyme investigating in disease.     

Simultaneous monitoring of quantum dots and their assembly and disassembly with PreScission protease using capillary electrophoresis with fluorescence detection

A novel assay was developed for the simultaneous monitoring of quantum dots and their assembly and disassembly with PreScission protease using capillary electrophoresis with fluorescence detection. Quantum dots and PreScission protease were injected into a capillary sequentially, then mixed and assembled via a thioether bond upon coupling to glutathione S‐transferase tag inside the capillary. The in‐capillary assembly was influenced by the molar ratio and the time interval of injection. Furthermore, the simultaneous monitoring of quantum dots and their assembly with PreScission protease and glutathione induced disassembly was achieved by adjusting the sampling sequence and the time interval of injection. More importantly, the in‐capillary assay could be also applied to the online detection of glutathione.     

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.     

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.     

Investigation of multivalent interactions between conjugate of quantum dots with c‐Myc peptide tag and the anti‐c‐Myc antibody by capillary electrophoresis with fluorescence detection

Herein, we report an assay for detecting the binding of a multivalent peptide and antibody within a capillary with the use of fluorescence coupled capillary electrophoresis. Quantum dots and a c‐Myc tag containing peptide EQKLISEEDLG₄H₆ were injected sequentially and formed a multivalent quantum dot‐EQKLISEEDLG₄H₆ assembly within the capillary. The efficiency of the quantum dot‐peptide self‐assembly was affected by the peptide/quantum dot molar ratio, sampling time, and interval time. Finally, the binding of the monoclonal anti‐c‐Myc antibody and the multivalent quantum dot‐EQKLISEEDLG₄H₆ ligand was studied using an in‐capillary assay. The microscopic dissociation constant for the self‐assembly of monoclonal anti‐c‐Myc antibody and quantum dot‐EQKLISEEDLG₄H₆ was determined to be 14.1 μM with a stoichiometry of the peptide‐antibody complex of 1.7 determined after fitting this to the Hill equation. This method can be further extended to detect a wide range of biomolecule–biomolecule binding interactions.