In‐capillary self‐assembly study of quantum dots and protein using fluorescence coupled capillary electrophoresis

As a vast number of novel materials in particular inorganic nanoparticles have been invented and introduced to all aspects of life, public concerns about how they might affect our ecosystem and human life continue to arise. Such incertitude roots at a fundamental question of how inorganic nanoparticles self‐assemble with biomolecules in solution. Various techniques have been developed to probe the interaction between particles and biomolecules, but very few if any can provide advantages of both rapid and convenient. Herein, we report a systematic investigation on quantum dots (QDs) and protein self‐assembly inside a capillary. QDs and protein were injected to a capillary one after another. They were mixed inside the capillary when a high voltage was applied. Online separation and detection were then achieved. This new method can also be used to study the self‐assembly kinetics of QDs and protein using the Hill equation, the K_D value for the self‐assembly of QDs and protein was calculated to be 8.8 μM. The obtained results were compared with the previous out of‐capillary method and confirmed the effectiveness of the present method.     

Capillary electrophoretic studies on displacement and proteolytic cleavage of surface bound oligohistidine peptide on quantum dots

Subtle changes in the chemical structure or the composition of surface bound ligands on quantum dots (QDs) remain difficult to detect. Here we describe a facile setup for fluorescence detection coupled capillary electrophoresis (CE-FL) and its application in monitoring ligand displacement on QDs through metal-affinity driven assembly. We also describe the use of CE-FL to monitor amide bond cleavage by a specific protease, based on Förster resonance energy transfer (FRET) between Cy5 and QDs spaced by a hexahistidine peptide (H6–Cy5). CE-FL allowed separation of unbound QDs and ligand bound QDs and also revealed an ordered assembly of H6–Cy5 on QDs. In a ligand displacement experiment, unlabeled hexahistidine peptide gradually displaced surface bound H6–Cy5 until finally reaching equilibrium. The displacement intermediates were clearly separated on CE-FL. Proteolytic cleavage of surface bound H6–Cy5 by thrombin was monitored by CE-FL through mobility shift, peak broadening, and FRET changes. Enzymatic parameters thus obtained were comparable with those measured by fluorescence spectroscopy.     

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.     

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.     

Characterization of quantum dots using capillary zone electrophoresis

Commercially available quantum dots (QDs) were characterized using CE. The CE instruments were laboratory-built, each being capable of both electrokinetic and hydrodynamic injection. Modes of detection include UV absorption and LIF. The CE-LIF system was further modified to handle microliter sample volumes during injection. Sodium phosphate (5-25 mM, pH 7.5-11) was found to be a good buffer electrolyte. Sodium mercaptoproprionate CdTe/CdS (ADS620) QDs and carboxylic acid CdSe/ZnS (T2-Evitag) QDs yielded high separation efficiencies of N = 1.5x10(6) plates at t(M) = 10 min and N = 1.0x10(5) plates at t(M) = 3.8 min, respectively. Apparently the EDC/sulfo-NHS bioconjugation chemistry worked well with the neutral T2-Evitag QDs, but not so well with the negatively charged ADS620 QDs. This preliminary knowledge will serve as a basis for new CE immunoassay studies of QD-biomolecule conjugates and their immunocomplexes with target analytes.     

Small molecule self‐assembly in polymer matrices

Using small molecules in polymer matrices is common in applications such as (i) plasticizing polymers to modify the glass transition and mechanical properties and (ii) dispersion of photoactive or electroactive small molecules in polymer matrices in organic‐electronic devices Aggregation of these small molecules and phase separation leading to crystallization often cannot be morphologically controlled. If these are designed with self‐assembling codes such as hydrogen bonding or aromatic interactions, their phase separation behavior would be distinctly different. This review summarizes the studies on morphologies in such situations, such as (i) sub‐surface assembly in polymer matrices, (ii) controlled polymerization‐induced phase separation to create polymer blends, (iii) using the polymer to direct the assembly of small molecules in liquid crystalline devices, (iv) functionalizing a polymer with self‐assembling small molecules to cause organo‐gelation which the polymer itself would not by itself, and (v) using such systems as templates to create porous polymer structures. Organic–inorganic hybrids using polymers as templates for nanostructures and imprinted porous membranes is an emerging area. Since self‐assembly is one of the dominating area of research with respect to both small molecules, polymers as well as the combination of the two, this review summarizes the studies on the aforementioned topics. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 451–478     

Determination of ζ‐potential,charge, and number of organic ligands on the surface of water soluble quantum dots by capillary electrophoresis

The number of charges and/or organic ligands covalently attached to the surface of CdTe quantum dot nanoparticles has been determined from their electrophoretic mobilities measured in capillaries filled with free electrolyte buffers. Three sizes of water soluble CdTe quantum dots with 3‐mercaptopropionic and thioglycolic acids as surface ligands were prepared. Their electrophoretic mobilities in different pH and ionic strength values of separation buffers were measured by capillary electrophoresis with laser induced fluorescence detection. The ζ‐potentials determined from electrophoretic mobilities using analytical solution of Henry function proposed by Ohshima were in the range from ?30 to ?100 mV. Charges of QDs were calculated from ζ‐potentials. As a result, numbers of organic ligands bonded to QDs surface were determined to be 13, 14, and 15 for the sizes of 3.1, 3.5, and 3.9 nm, respectively. The dissociation constants of organic ligands bonded on QDs surfaces estimated from the dependence of QDs charge on pH of the separation buffer were 7.8 and 7.9 for 3‐mercaptopropionic acid and 6.9 for thioglycolic acid.     

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size‐dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD–protein, QD–DNA, QD–small molecule), are also described. Finally, future perspectives are discussed.     

Temperature‐induced self‐assembly and metal‐ion stabilization of histidine functional block copolymers

Histidine functional block copolymers are thermally self‐assembled into polymer micelles with poly‐N‐isopropylacrylamide in the core and the histidine functionality in the corona. The thermally induced self‐assemblies are reversible until treated with Cu²⁺ ions at 50 °C. Upon treatment with 0.5 equivalents of Cu²⁺ relative to the histidine moieties, metal‐ion coordination locks the self‐assemblies. The self‐assembly behavior of histidine functional block copolymers is explored at different values of pH using DLS and ¹H NMR. Metal‐ion coordination locking of the histidine functional micelles is also explored at different pH values, with stable micelles forming at pH 9, observed by DLS and imaged by atomic force microscopy. The thermal self‐assembly of glycine functional block copolymers at pH 5, 7, and 9 is similar to the histidine functional materials; however, the self‐assemblies do not become stable after the addition of Cu²⁺, indicating that the imidazole plays a crucial role in metal‐ion coordination that locks the micelles. The reversibility of the histidine‐copper complex locking mechanism is demonstrated by the addition of acid to protonate the imidazole and destabilize the polymer self‐assemblies. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1964–1973     

Advances in square arrays through self‐assembly and directed self‐assembly of block copolymers

This review covers recent advances in developing square arrays in thin films using block copolymers. Theoretical and experimental results from self‐assembly of block copolymers in bulk and thin films, directed self‐assembly of block copolymers confined in small wells, on substrates with arrays of posts, and on chemically nanopatterned substrates, as well as applications as nanolithography are reviewed. Some future work and hypothesis are discussed. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

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.     

β‐cyclodextrin‐based polymeric nano‐receptor: the self‐assembly of cyclodextrin‐appended comb‐copolymer

Well‐defined β‐cyclodextrin (β‐CD)‐appended biocompatible comb‐copolymer ethyl cellulose‐graft‐poly (ε‐caprolactone) (EC‐g‐PCL) was synthesized via the combination of ring‐opening polymerization (ROP) and click chemistry. The resulting products were characterized by ¹H NMR, FT‐IR spectroscopy, and GPC. The synthesized comb‐copolymer could assemble to micelles, with the surface covered by β‐CD. The inclusion with ferrocene derivation was investigated by cyclic voltammetric (CV) experiments, which indicated the potential application of the micelles as nano‐receptors for molecule recognization and controlled drug release. Copyright © 2011 John Wiley & Sons, Ltd.     

High‐resolution quantification by charge‐dominant electrophoretic mobility shift of quantum dots

Conjugation of biomolecules to colloidal nanoparticles, such as quantum dots (QDs), often leads to change in mobility. We discover that linking DNA molecules to quantum dots alters their surface charge density without significantly increasing the hydrodynamic radius, causing a prominent shift in electrophoretic mobility. In this study, a high‐resolution molecular quantification method named quantification by QDs electrophoretic mobility shift (qQEMS) is developed based on the charge‐dominant transformation that closely associates DNA quantity to QDs electrophoretic mobility. The versatility of qQEMS is demonstrated by a number of quantification assays in which DNA molecules functioned as enzyme substrates, target‐specific probes, and competitive charge carriers. qQEMS shows a great potential as a generic and versatile quantification platform for a wide range of applications.     

Synthesis and self‐assembly of multiple‐responsive magnetic nanogels

In this paper, temperature and pH‐sensitive interpenetrating polymer network (IPN) nanogels (NGs) were firstly prepared, and magnetic hybrid NGs were made through in‐situ precipitation of Fe²⁺ and Fe³⁺ into the IPN NGs. Under the optimized condition, the resulting hybrid NG dispersion with up to 17.3 wt% magnetite was stable, while the size distribution of the NGs is broad due to the formation of Fe₃O₄ nanoparticles outside the NGs. In order to synthesize relatively uniform magnetic NGs, magnetite content was reduced to 8.1 wt% magnetite. The NGs with 8.1 wt% magnetite can quickly self‐assemble into colloidal crystals induced by magnet, while such NGs slowly self‐assembled into colloidal crystals without external magnetic field. Furthermore, the reflection wavelength of the self‐assembled magnetic NGs showed red‐shift with increasing pH and temperature.     

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.     

Hybrid organic–inorganic block copolymer nano‐objects from RAFT polymerization‐induced self‐assembly

A simple route to organic–inorganic (O/I) nano‐objects with different morphologies through polymerization‐induced block copolymer self‐assembly is described. The synthetic strategy relies on the chain‐extension of polyhedral oligomeric silsesquioxanes (POSS)‐containing macro‐CTA (PMAiBuPOSS₁₃ and PMAiBuPOSS₁₉) with styrene at 120 °C in octane, a selective solvent of the POSS‐containing block. The polymerization system was proven to afford a plethora of O/I nano‐objects, such as spherical micelles, cylindrical micelles, and vesicles depending on the respective molar masses of the PMAiBuPOSS and polystyrene (PS) blocks. The cooling procedure was also proven to be a crucial step to generate particles with a unique morphology. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4558–4564     

Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser‐induced fluorescence detection

Quantum dots (QDs) are one of the most promising nanomaterials, due to their size‐dependent characteristics as well as easily controllable size during the synthesis process. They are promising label material and their interaction with biomolecules is of great interest for science. In this study, CdTe QDs were synthesized under optimal conditions for 2 nm size. Characterization and verification of QDs synthesis procedure were done by fluorimetric method and with CE. Afterwards, QDs interaction with chicken genomic DNA and 500 bpDNA fragment was observed employing CE‐LIF and gel electrophoresis. Performed interaction relies on possible matching between size of QDs and major groove of the DNA, which is approximately 2.1 nm.     

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.     

Ratiometric fluorescence detection of mercuric ion based on the nanohybrid of fluorescence carbon dots and quantum dots

A novel nanohybrid ratiometric fluorescence probe comprised of carbon dots (C-dots) and hydrophilic CdSe@ZnS quantum dots (QDs) has been developed by simply mixing the blue-emission C-dots with red-emission carboxylmethyldithiocarbamate modified CdSe@ZnS QDs (GDTC-QDs). The nanohybrid ratiometric fluorescence probe exhibits dual emissions at 436 nm and 629 nm under a single excitation wavelength. Due to the strong chelating ability of GDTC on the surface of QDs to mercuric ion (Hg²⁺), the fluorescence of the GDTC-QDs in the nanohybrid system could be selectively quenched in the presence of Hg²⁺ while the fluorescence of the C-dots remained constant, resulting in an obviously distinguishable fluorescence color evolution (from red to blue) of the nanohybrid system. The detection limit of this method was found to be as low as 0.1 μM. Furthermore, the recovery result for Hg²⁺ in real samples including tap water and lake water by this method was satisfying, suggesting its potential application for Hg²⁺ sensing.     

Temperature‐induced self‐assembly of degalactosylated xyloglucan at low concentration

Xyloglucan is a natural polysaccharide having a cellulose‐like backbone and hydroxyl groups‐rich side‐chains. In its native form the polymer is water‐soluble and forms gel only in presence of selected co‐solutes. When a given fraction of galactosyl residues are removed by enzymatic reaction, the polymer acquires the ability to form a gel in aqueous solution at physiological temperatures, a property of great interest for biomedical/pharmaceutical applications. This work presents data on the effect of a temperature increase on degalactosylated xyloglucan dispersed in water at concentration low enough not to run into macroscopic gelation. Results obtained over a wide interval of length scales show that, on increasing temperature, individual polymer chains and pre‐existing clusters self‐assemble into larger structures. The process implies a structural rearrangement over a few nanometers scale and an increase of dynamics homogeneity. The relation of these findings to coil‐globule transition and phase separation is discussed. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 1727–1735