Conjugated Quantum Dots Research Articles

Point-of-need quantitation of 2,4-dichlorophenoxyacetic acid using a ratiometric fluorescent nanoprobe and a smartphone-based sensing system

This study reports a new ratiometric fluorescence probe (referred to as rQD@PS@bCDs) and a smartphone-based sensing system for on-site quantitation of Fe3+, ascorbic acid (AA), alkaline phosphatase (ALP) and 2,4-dichlorophenoxyacetic acid (2,4-D). rQD@PS@bCDs with well-resolved dual emissions is facilely synthesized by covalent conjugation of red-emissive quantum dots (rQDs), the internal standard, with blue-emissive carbon dots (bCDs), the Fe3+-specific fluorescent indicator. 2,4-D can specifically inhibit the enzymatic activity of ALP, leading to suppress the production of AA which can reduce Fe3+ to Fe2+ and thereafter trigger the fluorescence response of rQD@PS@bCDs. Taking advantages of these highly specific cascade catalytic reactions, ratiometric fluorescence schemes are established for sensing Fe3+, AA, ALP and 2,4-D both in aqueous solution and on paper-based microarray. The smartphone-based fluorescence detector integrated with the rQD@PS@bCDs-based microarray demonstrated good sensing performance with a wide linear range and low detection limit, and is successfully applied to quantitate 2,4-D in cabbage and tap water samples. The reported method which enables point-of-need quantitation in a sample-in-result-out manner and highlights its features of cost-effectiveness and ease-of-use, is ideally for food safety inspection, environment surveillance, health monitoring at point-of-need, especially in low resource settings.

Development of a Quantum Dot-Based Fluorescence-Linked Immunosorbent Assay for Puerarin.

In this study, a rapid and highly sensitive fluorescence-linked immunosorbent assay for puerarin determination was developed by the conjugation of quantum dots with an antibody against puerarin. The linear range and detection limit of the fluorescence-linked immunosorbent assay were validated. The detection curve (y = -1041ln(x)+5366, R² = 0.999) was linear in the range of 7.8-125 ng/mL. The 50% inhibitory concentration determined by fluorescence-linked immunosorbent assay was 33.8 ng/mL puerarin in water. The limit of detection for PUE was 6.1 ng/mL. To our knowledge, this is the first report on the quantitative detection of a natural product using quantum dots as fluorescent markers. Furthermore, the newly developed fluorescence-linked immunosorbent assay was successfully applied to determine puerarin in several commercial Gegen Qinlian tablets, with a higher sensitivity than that of conventional enzyme-linked immunosorbent assays.

In Vitro and In Vivo Fluorescence Imaging of Antibody-Drug Conjugate-Induced Tumor Apoptosis Using Annexin V-EGFP Conjugated Quantum Dots.

Antibody–drug conjugates (ADCs) are conjugates of a monoclonal antibody and a cytotoxic drug that induce tumor apoptosis. The evaluation of ADC-induced tumor apoptosis is crucial for the development of ADCs for cancer therapy. To evaluate the efficacy of ADCs, we present in vitro and in vivo fluorescence imaging techniques for ADC-induced tumor apoptosis using annexin V–EGFP (EGFP: enhanced green fluorescent protein) conjugated quantum dots (annexin V–EGFP–QDs). This probe emits visible (VIS) and near-infrared (NIR) dual fluorescence at 515 nm (EGFP emission) and 850 nm (QD emission), which can be used for the detection of tumor apoptosis at the cellular and whole-body levels. By using annexin V–EGFP–QDs, we achieved VIS and NIR fluorescence imaging of human epidermal growth factor receptor 2-positive breast tumor apoptosis induced by an ADC, Kadcyla (trastuzumab emtansine). The results show that the in vitro and in vivo fluorescence imaging of ADC-induced tumor apoptosis using annexin V–EGFP–QDs is a useful tool to evaluate the efficacy of ADCs for cancer therapy.

Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures.

DNA nanostructures self-assemble into almost any arbitrary architecture, and when combined with their capability to precisely position and orient dyes, nanoparticles, and biological moieties, the technology reaches its potential. We present a simple yet multifaceted conjugation strategy based on metal coordination by a multi-histidine peptide tag (Histag). The versatility of the Histag as a means to conjugate to DNA nanostructures is shown by using Histags to capture semiconductor quantum dots (QDs) with numerical and positional precision onto a DNA origami breadboard. Additionally, Histag-expressing enzymes, such as the bioluminescent luciferase, can also be captured to the DNA origami breadboard with similar precision. DNA nanostructure conjugation of the QDs or luciferase is confirmed through imaging and/or energy transfer to organic dyes integrated into the DNA nanostructure.

Aptamer Conjugated Indium Phosphide Quantum Dots with a Zinc Sulphide Shell as Photoluminescent Labels for Acinetobacter baumannii.

Acinetobacter baumannii is a remarkable microorganism known for its diversity of habitat and its multi-drug resistance, resulting in hard-to-treat infections. Thus, a sensitive method for the identification and detection of Acinetobacter baumannii is vital. However, current methods used for the detection of pathogens have not improved in the past decades and suffer from long process times and low detection limits. A cheap, quick, and easy detection mechanism is needed. In this work, we successfully prepared indium phosphide quantum dots with a zinc sulphide shell, conjugated to a targeting aptamer ligand, to specifically label Acinetobacter baumannii. The system retained both the photophysical properties of the quantum dots and the folded structure and molecular recognition function of the aptamer, therefore successfully targeting Acinetobacter baumannii. Confocal microscopy and transmission electron microscopy showed the fluorescent quantum dots surrounding the Acinetobacter baumannii cells confirming the specificity of the aptamer conjugated to indium phosphide quantum dots with a zinc sulphide shell. Controls were undertaken with a different bacteria species, showing no binding of the aptamer conjugated quantum dots. Our strategy offers a novel method to detect bacteria and engineer a scalable platform for fluorescence detection, therefore improving current methods and allowing for better treatment.

Graphene Oxide Synergy with the Conjugation of DNA and Quantum Dots for the Sensitive Detection of Ochratoxin A

Graphene Oxide Synergy with the Conjugation of DNA and Quantum Dots for the Sensitive Detection of Ochratoxin A

Manganese phthalocyanine and its graphene quantum dot conjugate: Synthesis, characterization electrochemistry, spectroelectrochemistry, electropolymerization, and electrochromism

Manganese phthalocyanine (MnPc) bearing peripherally substituted octacarbazole moieties was synthesized and characterized by matrix-assisted laser desorption/ionization spectrometry (MALDI), FT-IR, and UV–Vis spectroscopy. The complex was then conjugated to graphene quantum dots (GQDs) via π-π stacking and characterized by energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), energy dispersive spectroscopy, and X-ray diffraction. Electrochemical performances of MnPc and its nanoconjugate were investigated using cyclic voltammetry (CV), square wave voltammetry (SWV) and spectroelectrochemistry (SEC) to determine the influence of the carbazole substituents and graphene quantum dots on the redox mechanism of MnPc. Although the carbazole substituents did not significantly influence the reduction processes of MnPc, they induced MnPc coating onto the electrode surface due to cationic electropolymerization of the carbazole substituents. The composite comprising graphene quantum dots and MnPc exhibited redox responses that were comparable to those of MnPc.

Antibody Self-Assembly Maximizes Cytoplasmic Immunostaining Accuracy of Compact Quantum Dots

Antibody conjugates of quantum dots (QDs) are expected to transform immunofluorescence staining by expanding multiplexed analysis and improving target quantification. Recently, a new generation of small QDs coated with multidentate polymers has improved QD labeling density in diverse biospecimens, but new challenges prevent their routine use. In particular, these QDs exhibit nonspecific binding to fixed cell nuclei and their antibody conjugates have random attachment orientations. This report describes four high-efficiency chemical approaches to conjugate antibodies to compact QDs. Methods include click chemistry and self-assembly through polyhistidine coordination, both with and without adaptor proteins that directionally orient antibodies. Specific and nonspecific labeling are independently analyzed after application of diverse blocking agent classes, and a new assay is developed to quantitatively measure intracellular labeling density based on microtubule stain connectivity. Results show that protein conjugation to the QD surface is required to simultaneously eliminate nonspecific binding and maintain antigen specificity. Of the four conjugation schemes, polyhistidine-based coordination of adaptor proteins with antibody self-assembly yields the highest intracellular staining density and the simplest conjugation procedure. Therefore, antibody and adaptor protein orientation, in addition to blocking optimization, are important determinants of labeling outcomes, insights that can inform translational development of these more compact nanomaterials.

Direct and Efficient Conjugation of Quantum Dots to DNA Nanostructures with Peptide-PNA.

DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.

Efficient Energy Funneling in Spatially Tailored Segmented Conjugated Block Copolymer Nanofiber-Quantum Dot or Rod Conjugates.

Hybrid systems composed of conjugated polymers and inorganic semiconductor nanocrystals such as quantum dots (QDs) and nanorods (QRs) represent highly desirable multifunctional materials for applications from energy harvesting to light emission and sensing. Herein, we describe energy transfer studies between low-dispersity segmented conjugated polymer micellar nanofibers integrated with quantum dots that are spatially confined to discrete regions in the hybrid assembly via noncovalent interactions. The nanofibers were prepared from diblock copolymers with a crystallizable poly(di-n-hexylfluorene) (PDHF) core-forming block and different corona-forming blocks using the seeded-growth "living" crystallization-driven self-assembly method. The highly ordered crystalline PDHF core in the fibers functions as a donor and permits long-range exciton transport (>200 nm). Energy can therefore be funneled through the fiber core to QDs and QRs that function as acceptor materials and which are noncovalently bound to spatially defined coronal regions of poly(2-vinylpyridine) (P2VP) or quaternized polyfluorene (QPF). Using steady-state and time-resolved spectroscopy, we demonstrate that efficient energy transfer (over 70%) occurs from the crystalline PDHF donor core to the acceptor CdSe QRs attached at the fiber termini. The emission of the PDHF donor in the hybrid conjugate was extensively quenched (by 84%), and a subsequent 4-fold enhancement of the QR emission in solution was observed. These results indicate that the conjugates prepared in this work show promise for potential applications in fields such as light-emitting diodes, photovoltaics, chemical sensors, and photocatalysis.

Direct conjugation of fluorescent quantum dots with E. coli via surface-displayed histidine-containing peptides

Biocompatible approaches to labeling bacteria with fluorescent nanoparticles are essential in order to create living bacterial bioconjugates for imaging, biosensors, medicine, and other applications. Herein we report the direct conjugation of carboxyl quantum dots (QDs) with E. coli outer membrane via surface-displayed binding peptides. The histidine-containing peptide H6G9 was displayed at the N-terminus of membrane-embedded enhanced circularly permuted outer membrane protein X (eCPX) scaffold, which was expressed upon chemical induction. The presence of the binding peptide creates an environment distinct from the negatively charged E. coli surface and provides strong binding affinity to carboxyl quantum dots (QDs). Transmission electron microscopy (TEM) analysis of E. coli-QD bioconjugates revealed high loading densities of these QDs immobilized on the cell surface, even when adding a very low concentration (10 μg/mL) of QDs in order to reduce the cell exposure. These hybrid cells strongly fluoresce with each of the distinct colors of loaded QDs with different emission wavelengths, which can be easily visualized by fluorescence microscopy or differentiated using flow cytometry. Importantly, the E. coli–QD bioconjugates were highly viable and maintained the ability to grow and divide. This study demonstrates a simple, direct, and highly efficient method for labelling bacteria with QDs, without significantly compromising the vitality of the cells.

Role of PEGylated CdSe-ZnS quantum dots on structural and functional properties of electrospun polycaprolactone scaffolds for blood vessel tissue engineering

Conjugation of quantum dots (Q-dots) into tissue engineering scaffolds is extremely beneficial for designing composite scaffolds imparting multifunctional properties. In the present work, we report the efficacy of incorporating the PEGylated CdSe-ZnS (Q-dots) into electrospun poly(caprolactone)(PCL) scaffolds whose properties were evaluated for small diameter blood vessel applications. Incorporation of Q-dots was found to result in significant reduction in fiber diameter of electrospun scaffolds. Corresponding pore size ranges varied from 12 to 36 µm for composite scaffold compared with 12–60 µm in bare scaffold. Both PCL and composite scaffolds were found to possess better mechanical strength and elasticity than that of native arteries. Contact angle measurements revealed hydrophobic nature of scaffolds which significantly improved upon modification with fibrin. PCL/Q-dot composite scaffolds were found to possess optimum micro-structure for supporting uniform endothelial cell growth after 5 days of culture compared to the pristine PCL scaffold.

Electrochemical detection of dopamine using phthalocyanine-nitrogen-doped graphene quantum dot conjugates

Two cobalt phthalocyanine molecules were synthesized, a symmetrically substituted CoPc (1) and an unsymmetrically substituted CoPc (2). These CoPcs were π-π stacked onto nitrogen-doped graphene quantum dots (NGQDs). The CoPcs, NGQDs and their respective conjugates were used as electro-catalytic probes in the electrochemical oxidation of dopamine. The molecules were adsorbed onto a glassy carbon electrode (GCE) using the drop dry method, the GCE was used to support those catalysts. The electrooxidation of dopamine was studied using cyclic voltammetry and chronoamperometry. An improvement in the sensitivity and limit of detection was observed upon conjugation.

CdSe Quantum Dots in Human Models Derived from ALS Patients: Characterization, Nuclear Penetration Studies and Multiplexing.

CdSe quantum dots (QDs) are valuable tools for deciphering molecular mechanisms in cells. Their conjugation with antibodies offers a unique staining source with optimal characteristics, including increased photostability and narrow emission spectra, allowing for improved multiplexing capabilities using a single excitation source. In combination with pathology models derived from patients, they have great potential to contribute to quantitative molecular profiling and promote personalized medicine. However, the commercial availability of diverse CdSe QDs is still limited and characterization techniques must be performed to these materials or the conjugates developed in the lab to assure a proper function and reproducibility. Furthermore, while there is significant data of QDs experiments in cell lines, the literature with primary human cells is scarce, and QD behavior in these systems may be different. Rigorous characterization data of commercially available QDs and their conjugates with biomolecules of interest is needed in order to establish their potential for target labelling and expand their use among research labs. Here we compare the characterization and labelling performance of different QD conjugates in SH-SY5Y cell line, fibroblasts and immortalized lymphocytes derived from amyotrophic lateral sclerosis patients.

Multiphoton Deep-Tissue Imaging of Micrometastases and Disseminated Cancer Cells Using Conjugates of Quantum Dots and Single-Domain Antibodies.

Early detection of malignant tumors, micrometastases, and disseminated tumor cells is one of the effective way of fighting cancer. Among the many existing imaging methods like computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), optical imaging with fluorescent probes is one of the most promising alternatives because it is fast, inexpensive, safe, sensitive, and specific. However, traditional fluorescent probes, based on organic fluorescent dyes, suffer from the low signal-to-noise ratio. Furthermore, conventional organic fluorescent dyes are unsuitable for deep tissue imaging because of the strong visible light absorption by biological tissues. The use of fluorescent semiconductor nanocrystals, or quantum dots (QDs), may overcome this limitation due to their large multiphoton cross section, which ensures efficient imaging of thick tissue sections inaccessible with conventional fluorescent probes. Moreover, the lower photobleaching and higher brightness of fluorescence signals from QDs ensures a much better discrimination of positive signals from the background. The use of fluorescent nanoprobes based on QDs conjugated to uniformly oriented high-affinity single-domain antibodies (sdAbs) may significantly increase the sensitivity and specificity due to better recognition of analytes and deeper penetration into tissues due to small size of such nanoprobes.Here, we describe a protocol for the fabrication of nanoprobes based on sdAbs and QDs, preparation of experimental xenograft mouse models for quality control, and multiphoton imaging of deep-tissue solid tumors, micrometastases, and disseminated tumor cells.

Monitoring of Cell‐Dependent Reduced States Using Aptamer‐Functionalized Reduction‐Sensitive Quantum Dots

Conjugation of quantum dot (QD) with oxidized nile blue (NB) is known to be able to strongly decrease fluorescence intensity of QD. In the present study, NB‐conjugated QD (QD‐NB) was functionalized with anti‐human epidermal growth factor receptor 2 aptamer to form aptamer‐functionalized QD‐NB (Apt‐QD‐NB), as a new biosensing platform to monitor aptamer‐dependent internalizations and reduction‐dependent transformations. Our study shows that more QD fluorescence signals in cells can be induced by the specific internalization of higher amounts of Apt‐QD‐NB and that the higher ratio of reduced NB over oxidized NB resulting from the different reducing environment of the cells can lead to brighter QD signals. Apt‐QD‐NBs are expected to be used as optical labels that can monitor intracellular metabolic pathways at the single‐cell level, because the use of Apt‐QD‐NBs to investigate drug effectiveness and cellular redox conditions offers exciting opportunities for drug screening.

Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection.

We have developed a novel detection system that couples clustered regularly interspaced short palindromic repeat-Cas recognition of target sequences, Cas-mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for simple detection of viral nucleic acid targets. After target recognition and Cas-mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic beads. A complementary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to uncleaved probes on the magnetic beads. After separating hybridized quantum dots, the collected supernatant is illuminated by a portable ultraviolet flashlight, and it provides a simple “Yes-or-No” nucleic acid detection answer. By using a DNA target matching part of the African swine fever virus, detection limits of ∼0.5 and ∼1.25 nM are achieved in buffer and porcine plasma, respectively. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple assay for nucleic acid screening in both hospitals and point-of-care settings.

Toward a nanopaper-based and solid phase immunoassay using FRET for the rapid detection of bacteria

In this study, we propose a novel sensitive solid-based immunosensor developed on a plasmonic nanopaper platform for the detection of Escherichia coli (E. coli) bacteria. This plasmonic nanopaper that comprises of carboxylated bacterial cellulose (CBC) impregnated with gold nanoparticles (AuNP-CBC), employed as a quencher and a sustainable functionalized platform to be conjugated with protein A. Thus, the conjugated protein A allows the aligned linkage of EAb-QD (anti-E. coli conjugated quantum dot) and EAb-AF (anti-E. coli conjugated Alexa Fluor 488). Interestingly, once E. coli was captured by the AuNP-CBC/EAb-QD or AuNP-CBC/EAb-AF, the energy transfer from the QD or Alexa Fluor fluorophores is triggered due to the conformational change in the antibody structure and this, in turn, causes a decrease in the distance between fluorophores and the quencher nanopaper and, therefore diminishing their photoluminescence. The immunosensors performed successfully to recognize E. coli at concentrations as low as 50 CFU mL−1 in the standard buffer. The examined functionality of the immunosensors in a real matrix such as chicken extract and lettuce juice demonstrated a highly efficient response while QD is the main fluorophore with a limit of detection around 100 CFU mL−1.

Aqueous Synthesis of DNA-Functionalized Near-Infrared AgInS2/ZnS Core/Shell Quantum Dots.

Biocompatibility, biofunctionality, and chemical stability are essential criteria to be fulfilled by quantum dot (QD) emitters for bio-imaging and -sensing applications. In addition to these criteria, achieving efficient near-infrared (NIR) emission with nontoxic QDs remains very challenging. In this perspective, we developed water-soluble NIR-emitting AgInS2/ZnS core/shell (AIS/ZnS) QDs functionalized with DNA. The newly established aqueous route relying on a two-step hot-injection synthesis led to highly luminescent chalcopyrite-type AIS/ZnS core/shell QDs with an unprecedented photoluminescence quantum yield (PLQY) of 55% at 700 nm and a long photoluminescence (PL) decay time of 900 ns. Fast and slow hot injection of the precursors were compared for the AIS core QD synthesis, yielding a completely different behavior in terms of size, size distribution, stoichiometry, and crystal structure. The PL peak positions of both types of core QDs were 710 (fast) and 760 nm (slow injection) with PLQYs of 36 and 8%, respectively. The slow and successive incorporation of the Zn and S precursors during the subsequent shell growth step on the stronger emitting cores promoted the formation of a three-monolayer thick ZnS shell, evidenced by the increase of the average QD size from 3.0 to 4.8 nm. Bioconjugation of the AIS/ZnS QDs with hexylthiol-modified DNA was achieved during the ZnS shell growth, resulting in a grafting level of 5-6 DNA single strands per QD. The successful chemical conjugation of DNA was attested by UV-vis spectroscopy and agarose gel electrophoresis. Importantly, surface plasmon resonance imaging experiments using complementary DNA strands further corroborated the successful coupling and the stability of the AIS/ZnS-DNA QD conjugates as well as the preservation of the biological activity of the anchored DNA. The strong NIR emission and biocompatibility of these AIS/ZnS-DNA QDs provide a high potential for their use in biomedical applications.

Wound therapy via a photo-responsively antibacterial nano-graphene quantum dots conjugate

Common bacterial pathogens have become resistant to traditional antibiotics, representing an indispensable public health crisis. Photodynamic therapy (PDT), especially when common visible light sources are used as photodynamic power, is a promising bactericidal method. Based on the special photodynamic properties triggered by commonly available light emitting diode (LED) lamps, a kind of graphene quantum dots (GQDs) based composite system (termed GQDs@hMSN(EM)) was prepared through loading both GQDs and erythromycin (EM) into the hollow mesoporous silica nanoparticle (hMSN), aiming to achieve joint antimicrobial effect. Bacterial density experiments confirmed that GQDs@hMSN(EM) had combined antimicrobial effects from photodynamic effect and drug release on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In animal models, the healing degree of wounds infected by bacteria also confirmed that GQDs@hMSN(EM) group had the best therapeutic effect, with the significantly reduced inflammatory factors in blood. Different from traditional GQDs synthesized by solvothermal method, the as-prepared GQDs@hMSN can produce singlet oxygen (1O2) under light exposure to destroy the structure of bacteria, thus achieving highly efficient antimicrobial effect. The GQDs@hMSN(EM) in this work possesses good antimicrobial activity, sufficient drug loading, and controllable drug release ability, which provides a new opportunity for GQDs-based nanoplatform to enhance antimicrobial effect and reduce their drug resistance.