Characterization of the coupling of quantum dots and immunoglobulin antibodies

Water-soluble quantum dots (QDs) were used to label goat anti-human immunoglobulin antibodies (Abs), and the labeling process was characterized by column purification. The QDs obtained in organic solvent were modified with mercaptoacetic acid (MAA) and became water-soluble. These water-soluble QDs were linked to the antibodies using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The linking process was shown to be effective by ultra-filter centrifugation and column purification. After comparing the quantities of Abs and water-soluble QDs involved in the linking reaction via column purification, it was found that a molar Abs:QD ratio of >1.2 resulted in most of the water-soluble QDs becoming covalently linked to the Abs. The circular dichroism (CD) spectra of Abs and QD–Ab conjugates were very similar to each other, indicating that the secondary structure of Abs remained largely intact after the conjugation. Finally, antigen (Ag)–antibody (Ab) recognition reactions perfomed on the surface of a glass slide showed that the conjugate retained the activity of Abs. This work lends support to the idea of linking biomolecules to QDs, and thus should aid the application of QDs to the life sciences. Figure Firstly in this work, the conjugates of QDs-Ab were separated from EDC&NHS in the column of Sephadex G-100(left up). Then the bioactivity of QDs-Ab was analyzed in the immunoassay (right) and the immunofluorescent signals were detected (left bottom) finally     

Immunofluorescent Labeling of Human HepG2 Cells with CdTe Quantum Dot Probe Conjugated with Anti-pan CK MAb

A relatively sensitive, specific, and photostable method for the detection of cytokeratin of cancer cells via conjugation with cadmium telluride quantum dots(CdTe QDs) was described. Water soluble CdTe QDs were conjugated to anti-pan-cytokeratin(CK) monoclonal antibody(MAb) through coupling reagent [1-ethyl-3-(3-dimethyla- mino propyl)carbodiimide, EDC] and the conjugates were purified by dialysis. The expression of pan CK protein in HepG2 cells was observed by immunocytochemistry and direct immunofluoresce...     

Enhancement of sensitivity and specificity of the fluoroimmunoassay of Hepatitis B virus surface antigen through “flexible” coupling between quantum dots and antibody

Quantum dots (QDs) are widely used in the immune detection. Yet, the sensitivity and specificity of the immune detection are not satisfactory because the binding sites of QDs onto antibody (Ab) are often arbitrary and the influence of the large surface electronic potential energy of QDs on the directly conjugated Ab is nonnegligible. In this work, we provide a “flexible” coupling method, in which protein G (PG) is selected as the flexible bridge between the QDs and the Hepatitis B virus surface antibody (HBsAb), to improve the sensitivity and specificity of the fluoroimmunoassay compared to the directly covalent conjugation. Successful coupling of the HBsAb to our highly luminescent CdTe/CdS core/shell QDs is proven with Gel electrophoresis and atomic force microscopy (AFM). The assay results, based on the microelisa well plate as matrix to immobilize the sandwich structure, show that both sensitivity and specificity can be improved greatly through the flexible coupled QDs-PG-Ab conjugates.     

Characterization of CdTe/CdSe quantum dots-transferrin fluorescent probes for cellular labeling

In this paper, we prepared three types of transferrin-quantum dots conjugates (QDs-Tf) using three different methods (electrostatic interaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) coupling, denatured transferrin (dTf) coating). Fluorescence emission spectra, surface characteristics, zeta potentials of quantum dots (QDs) and QDs-Tf fluorescent probes were characterized by spectrophotometer, capillary electrophoresis, and dynamic light scattering. Fluorescent imaging of HeLa cells was also performed by QDs and QDs-Tf fluorescent probes. It was found that the fluorescence imaging performances of QDs-Tf probes prepared by electrostatic interaction and EDC coupling were better compared with the one prepared by dTf coating. Then a real-time single cell detection system was established to quantitatively evaluate cell labeling effects of QDs-Tf fluorescent probes. It was found that for cell labeling efficiency, the proportion of cells labeled by quantum dot probes to a group of cells, QDs-Tf probe prepared by EDC coupling showed the highest labeling efficiency (85.55 ± 3.88%), followed by electrostatic interaction (78.86 ± 9.57%), and dTf coating showed the lowest (40.09 ± 10.2%). This efficiency order was confirmed by flow cytometry results. This study demonstrated the relationship between conjugation methods and the resultant QDs-Tf probes and provided a foundation for choosing appropriate QDs-Tf probes in cell labeling.     

Hybrid fluorescent nanoparticles from quantum dots coupled to cellulose nanocrystals

Carboxylated cellulose nanocrystals (CNCs) were decorated with CdSe/ZnS quantum dots (QDs) using a carbodiimide chemistry coupling approach. The one-step covalent modification was supported by nanoscale imaging, which showed QDs clustered on and around the CNCs after coupling. The QD–CNC hybrid nanoparticles remained colloidally stable in aqueous suspension and were fluorescent, exhibiting the broad excitation and narrow emission profile characteristic of the QDs. QD–CNCs in nanocomposite films imparted strong fluorescence within CNC-compatible matrices at relatively low loadings (0.15 nmol QDs/g of dry film), without altering the overall physical properties or self-assembly of the CNCs. The hybrid QD–CNCs may find applications in nanoparticle tracking, bio-imaging, optical/sensing devices, and anti-counterfeit technologies.     

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.     

Compatibility of quantum dots with immunobuffers, and its effect on signal/background of quantum dot-based immunoassay

In this work, the compatibility of quantum dots (QDs) with immunobuffers was studied by investigating the fluorescence stability of QDs in immunobuffers (in this research immunobuffers were defined as buffers for immunoaffinity binding or separation). Experimentally, the fluorescence signals of QDs with different surface chemistries (amine-terminated, streptavidin-coated, or antibody-conjugated) in commonly used immunobuffers were monitored versus time. The effect of some buffer composition on the compatibility of QDs with these buffers was also explored. Based on experimental data, the QD compatibility with these buffers is summarized, and it is found that a trace amount of bovine serum albumin added to most of these buffers helps QDs to achieve compatibility with them. Moreover, with QD as fluorescence label and C-reactive protein as a model analyte, a magnetic bead-based assay was performed using compatible and incompatible QD–immunobuffer systems. It is shown that compatible QD–immunobuffer systems can be used to achieve a higher assay signal/background ratio.      

NHS-mediated QDs-peptide/protein conjugation and its application for cell labeling

3-Mercaptopropyl acid-stabilized CdTe nanoparticles synthesized in aqueous solution are found to be able to conjugate with peptides or proteins mediated by N-hydroxysulfo-succinimide (NHS) but 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC). The reaction time and pH have been optimized. Gel-permeation HPLC was applied following the conjugation, which could quickly and simultaneously detect and purify the quantum dots (QDs) conjugates. The biological activities of QDs conjugates are maintained and give superior results in cell labeling. These results are encouraging regarding the application of QDs molecules for use in living cells, diagnostics and drug delivery.     

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.     

On the pH-dependent quenching of quantum dot photoluminescence by redox active dopamine

We investigated the charge transfer interactions between luminescent quantum dots (QDs) and redox active dopamine. For this, we used pH-insensitive ZnS-overcoated CdSe QDs rendered water-compatible using poly (ethylene glycol)-appended dihydrolipoic acid (DHLA-PEG), where a fraction of the ligands was amine-terminated to allow for controlled coupling of dopamine-isothiocyanate onto the nanocrystal. Using this sample configuration, we probed the effects of changing the density of dopamine and the buffer pH on the fluorescence properties of these conjugates. Using steady-state and time-resolved fluorescence, we measured a pronounced pH-dependent photoluminescence (PL) quenching for all QD-dopamine assemblies. Several parameters affect the PL loss. First, the quenching efficiency strongly depends on the number of dopamines per QD-conjugate. Second, the quenching efficiency is substantially increased in alkaline buffers. Third, this pH-dependent PL loss can be completely eliminated when oxygen-depleted buffers are used, indicating that oxygen plays a crucial role in the redox activity of dopamine. We attribute these findings to charge transfer interactions between QDs and mainly two forms of dopamine: the reduced catechol and oxidized quinone. As the pH of the dispersions is changed from acidic to basic, oxygen-catalyzed transformation progressively reduces the dopamine potential for oxidation and shifts the equilibrium toward increased concentration of quinones. Thus, in a conjugate, a QD can simultaneously interact with quinones (electron acceptors) and catechols (electron donors), producing pH-dependent PL quenching combined with shortening of the exciton lifetime. This also alters the recombination kinetics of the electron and hole of photoexcited QDs. Transient absorption measurements that probed intraband transitions supported those findings where a simultaneous pronounced change in the electron and hole relaxation rates was measured when the pH was changed from acidic to alkaline.     

Characterization of quantum dot bioconjugates by capillary electrophoresis with laser-induced fluorescent detection

In this paper, we present a universal, highly efficient and sensitive method for the characterization of quantum dot (QD) bioconjugates based on capillary electrophoresis with laser-induced fluorescent (LIF) detection. We first prepared CdTe QDs in aqueous phase by a chemical route with mercaptopropionic acid as a ligand, and then were coupled to certain proteins using bifunctional linkage reagent or electrostatic attraction. The QD bioconjugates were characterized by capillary electrophoresis with LIF detection. We found that QD bioconjugates were efficiently separated with free QDs by the optimization of buffer pH. Furthermore, we found that ultrafiltration was an effective and simple approach to purify QD conjugates with bovine serum albumin (BSA). Due to their broad absorption spectra and size dependent emission wavelength tunability, QDs can be excited to emit different colour fluorescence using a single wavelength laser source, and therefore, we believe that CE with LIF detection will become a universal and efficient tool for the characterization of QD bioconjugates.     

水溶性量子点标记狂犬病P蛋白单克隆抗体的研究

利用共价偶联的方式,在水溶性缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(Sulfo-NHS)促进作用下,将400 μL的2 g/L狂犬病P蛋白抗体与适量的聚丙烯酸修饰后的水溶性硫脲修饰ZnO掺Cd量子点进行共价偶联反应,经磷酸盐缓冲液(PBS,0.01 mol/L,pH 7.4)透析纯化得到目标偶联物,采用荧光发射光谱、生物质谱、酶联免疫法等对偶联物进行表征.结果表明:偶联后的量子点荧光最大发射波长红移了10 nm,荧光强度随着狂犬病P蛋白抗原浓度的增加而逐渐增强;量子点标记狂犬病P蛋白抗体后的分子离子峰在m/z 67580处,比狂犬病P蛋白抗体分子离子峰增大了1453.由此证实狂犬病P蛋白抗体成功偶联到水溶性量子点上,且结构未受破坏.     

Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

Development of an amperometric immunosensor based on flow injection analysis for the detection of red blood cells

An amperometric immunosensor, based on a non-competitive sandwich assay and flow injection analysis (FIA), was developed for the detection of human red blood cells (RBCs). A dual working electrode, on which specific IgM and nonspecific IgM were chemically immobilised to form sensing and blank electrodes, respectively, was employed to determine the binding of specific blood cells and non-specific adsorption in one determination. Horseradish peroxidase (HRP)-labelled antiblood group A IgM was used in the assay. Sensor preparation involved chemical immobilisation of the IgMs on glassy carbon electrodes using l-ethyl-3(3-dimethyl aminopropyl)carbodiimide (EDC) as a coupling reagent in the presence of N-hydroxysuccinimide (NHS). The interference contributions, such as the non-specific adsorption of the enzyme conjugate and the blood cells, were determined and removed. A quantitative relationship between the cell binding response and its concentration was obtained in the region 1 − 30 × 10⁸ cells ml⁻¹.     

Quantum dot-mediated detection of gamma-aminobutyric acid binding sites on the surface of living pollen protoplasts in tobacco

gamma-Aminobutyric acid (GABA) is an inhibitory transmitter in the central nervous system of mammals. Recent investigations showed that it also plays an important role in regulating pollen tube growth and orientation in plants. To determine whether GABA receptors are also present on the membrane of pollen protoplasts, a fluorescence probe of quantum dots (QDs) was constructed and applied. The water-soluble CdSe-ZnS (core-shell) QDs were first synthesized and verified to possess good optical properties. GABA was then bioconjugated to the QDs in the presence of 1-ethyl-3-(3)-dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make the fluorescence probe. Using the probe, GABA binding sites were detected on the protoplast membrane of both pollen and somatic cells. Both the fluorescent signals on the surface of the protoplasts and the Ca(2+) oscillation assayed via the Ca(2+) probe Fluo-3/AM inside the protoplasts provided evidence that the potential GABA(B) receptors are present on the plant protoplast membrane.     

亲水性修饰的量子点的结构对荧光性能的影响研究

亲水性量子点的荧光性能是其作为生物检测探针的一个重要质量指标. 不同结构的量子点在亲水性修饰过程中, 其抵抗荧光淬灭的能力差异较大. 设计与制备具有不同结构和成分的核、核壳量子点, 再通过双亲性高分子对其亲水性改性, 利用荧光光谱监测亲水性修饰过程中的荧光性能变化来度量所合成量子点的光化学稳定性. 实验结果表明,在表面亲水性修饰过程中, 未包覆壳层的裸核量子点其抵抗荧光淬灭的能力最弱; 包覆壳层的核壳量子点, 其抵抗荧光淬灭的能力增强, 且壳层越多, 抵抗能力越强. 壳层的结构和成分直接影响核壳量子点抵抗荧光淬灭的能力, 具有合理晶格匹配的核壳量子点, 其抵抗荧光淬灭的能力较强. 另外, 通过优化设计与制备的核壳量子点经表面亲水性修饰后, 再偶联叶酸, 构建出特异性生物荧光探针, 对乳腺癌细胞进行靶向性标记后, 利用流式细胞仪进行细胞检测分析. 实验结果表明, 通过优化制备的核壳量子点, 亲水性修饰后仍具有很好的荧光性能, 偶联叶酸后具有较好的细胞靶向性.     

Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.     

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.     

Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.     

Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene

We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a “turn-on” effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2–3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.