Biolabeling Applications Research Articles

Solid-state synthesis of self-functional carbon quantum dots for detection of bacteria and tumor cells

We have developed a simple solid-state synthesis procedure to functionalize fluorescent carbon quantum dots (CQDs), using ammonium citrate as a carbon source and appropriate molecules as recognition ligands. Mannose and folic acid were used to modify CQDs to selectively label Escherichia coli (E. coli) and tumor cells, respectively. First, fluorescent-core CQDs (approximate size: 3nm) were synthesized through carbonization of ammonium citrate via dry heating. In the second step, CQDs were heated with mannose and folic acid to prepare mannose-functionalized CQDs (Man–CQDs) and folic acid-functionalized CQDs (FA–CQDs), respectively, through a dehydration reaction in the solid state. Solid-state synthesis of the self-functional CQDs is achievable without a coupling agent. We optimized the labeling efficiencies of self-functional Man–CQD and FA–CQD to cells by controlling the ratio of mannose or folic acid to CQDs, as well as the reaction temperature during synthesis. The solid-state synthesized Man–CQDs and FA–CQDs exhibited excitation-dependent fluorescence with excitation and emission maxima of 365 and 450nm, respectively, and a fluorescence quantum yield of approximately 9%. Man–CQDs can be used for selective labeling of E. coli and detection at concentrations as low as 100 colony forming units mL−1 in real samples (e.g., drinking water, apple juice, urine). Furthermore FA–CQDs are highly selective for labeling of folate receptor-overexpressing tumor cells. The synthesis of self-functional CQDs is simple, cost effective, and easily scaled up, and can be extended to the synthesis of various functional carbon nanomaterials, such as graphene oxide nanosheets, carbon nanotubes, fullerene nanoparticles and carbon nanodiamonds, with different ligands for other biolabeling applications and targeted therapies.

Microwave-Assisted Synthesis of Nitrogen-Doped Multi-Layer Graphene Quantum Dots with Oxygen-Rich Functional Groups

Strong green-luminescent nitrogen-doped multi-layer graphene quantum dots (N-GQDs) have been prepared via a microwave-assisted hydrothermal method using glucose and urea as the starting materials. The fabricated N-GQDs show a highly crystalline structure and consist of ~3–10 graphene layers with an N/C atomic ratio 5.7 %. The wavelength-dependent luminescence emission behaviour is observed with a photoluminescence quantum yield of 5.2 %. The combination of the unique optical properties introduced by nitrogen doping with the high solubility in aqueous medium offered by the surface oxygen-rich functional groups in N-GQDs provides additional advantages for their potential applications in biolabelling and bioimaging.

Biocompatible ZnS:Mn(2+) quantum dots/SiO2 nanocomposites as fluorescent probe for imaging HeLa cell.

ZnS:Mn(2+) quantum dots (QDs) were successfully embedded in SiO2 spheres by a reverse microemulsion method. The results showed that the monodispersed core/shell nanocomposites were uniform in size, with the majority of the SiO2 nanoparticles containing one QD in the center of the sphere. The shell thickness of SiO2 increased from 7 to 18nm as the hydrolysis time of TEOS increased from 20 to 40h. The quantum yield (QY) of the yellow-orange emission (coming from the Mn(2+) ions (4)T1-(6)A1 transition) for the ZnS:Mn(2+)(3%) QDs and ZnS:Mn(2+)(3%) QDs@SiO2 (when t=40h) nanocomposites was measured to be 34.5 and 22.4%, respectively. All samples showed no significant cytotoxicity against the HeLa cells even at a high concentration of 500μg/ml after incubation for 24h. The red fluorescence can be observed in the cytoplasm of the HeLa cell, further proving its biolabeling applications.

Optical properties of Er3+ doped KYbxF3x+1 (x=2, 3) upconverting nanoparticles

Well dispersed orthorhombic structure KYb2F7:2 mol.%Er3+ and KYb3F10:2 mol.%Er3+ nanoparticles were synthesized through solvothermal method. Measurements of X-ray diffraction (XRD), transmission electron microscopy (TEM) and upconversion spectra showed excellent morphology and optical properties of the as-prepared samples. The intensity ratio of the red emission (650 nm) to the green emission (545 nm) in these two materials was much stronger than that in other Er3+ doped materials. The transition mechanism of the processes was investigated and discussed. Due to their strong red emission, the samples are promising for applications in display, color tuning, and biolabeling.

Ethylenediamine-assisted hydrothermal synthesis of nitrogen-doped carbon quantum dots as fluorescent probes for sensitive biosensing and bioimaging

In this work, nitrogen-doped carbon quantum dots (N-CQDs) with high fluorescence quantum yield (46.2%) were successfully synthesized through one-pot hydrothermal carbonization approach assisted by ethylenediamine as surface passivation agent, wherein alanine acted as a single carbon source, ethylenediamine as the surface passivation agent. The obtained biocompatible N-CQDs could effectively label both the MCF-7 cell membranes and the nucleus, suggesting the potential application in biolabeling and bioimaging. Furthermore, the addition of dihydronicotinamide adenine dinucleotide (NADH) could dramatically quench the fluorescence of N-CQDs. Therefore, a novel protocol for NADH determination was established and its mechanism was discussed. This proposed fluorescence sensor displayed an enhanced performance for NADH determination with the linear range up to 80μM and the detection limit down to 25.1nM.

Silica-modified monodisperse hexagonal lanthanide nanocrystals: synthesis and biological properties

Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.

Alloyed CuInS2–ZnS nanorods: synthesis, structure and optical properties

Alloyed CuInS2–ZnS nanocrystals are promising candidates for application in biolabeling, photocatalysis, solar energy conversion, and light emitting diodes. When charge transport is of importance, elongated nanoparticles are advantageous, because of their higher electrical conductivity compared to the quasi-spherical ones. However, still little is known about the growth mechanism of such nanostructures composed of quaternary materials. Here, CuInS2–ZnS nanorods were synthesized by a heating-up method, and their Zn content was controlled by changing the composition of the reaction solution. A mixture of oleylamine and oleic acid is used as solvent. Copper, indium, and zinc acetate are the sources of the cations, while sulfur monomers stem from the thermal decomposition of tert-dodecanethiol. The growth of CuInS2–ZnS nanorods starts with the formation of copper sulfide particles. They are gradually converted to CuInS2–ZnS by incorporation of indium and zinc ions. Alloyed CuInS2–ZnS nanorods are the only product, independent of the amount of zinc applied; Raman spectroscopy measurements show no separate ZnS phase. At longer reaction time, the nanorods aggregate to form dimers. The onset of the absorption and the position of the maximum of the emission as well as the fluorescence lifetime depend on the composition of the nanorods.

Evaluation of novel fluorescence probes for conjugation purposes using the traceless Staudinger Ligation

A robust and versatile method for the synthesis of selected azide, alkyne and phosphane functionalized coumarin derivatives from the corresponding 7-amino and 7-hydroxy analogs for an application in bioorthogonal conjugation and labeling reactions like the Staudinger Ligation or the Huisgen-click reaction is presented. Further, the convenient preparation of modified coumarins was performed and subsequent regio- and chemo-selective labeling reactions delivered the desired biologically active derivatives in high yields. These new compounds may thus constitute attractive scaffolds for designing novel fluorescence building blocks for various challenging biolabeling applications.

Synthesis mechanism and optical properties of well nanoflower-shaped ZnO fabricated by a facile method

Photoluminescent ZnO nanoparticles have wide applications in biolabeling. A dual phase hydrothermal method has been developed in this paper to synthesize nanoflower-shaped ZnO nanoparticles. Hydrogen peroxide was identified as a unique oxygenic source to promote the formation of ZnO nanoflowers from the organic zinc precursor. The reaction mechanism for the formation of ZnO nanoflowers was proposed and studied by Fourier transform infrared (FTIR). The as-prepared hydrophobic colloidal ZnO nanoparticles could be subsequently modified to water-soluble ZnO nanoflowers via a ligand exchange process with aminethanethiol HCl. The structure and optical properties of the ZnO nanoparticles were studied by transmission electron microscopy, X-ray diffraction, and photoluminescence measurement (PL). Both types of ZnO nanoflowers demonstrated good photoluminescent properties which could have wide applications.

Facile and Green Synthesis of CdSe Quantum Dots in Protein Matrix: Tuning of Morphology and Optical Properties

Herein, we have demonstrated a facile and green approach for the synthesis of Cadmium selenide (CdSe) quantum dots (QDs). The process was mediated by bovine serum albumin (BSA) and it was found that BSA plays the dual role of reducing agent as well as a stabilizing agent. The QDs exhibited sharp excitonic absorption features at ~500 nm and subsequently showed reasonably good photoluminescence (PL) at room temperature. The PL is seen to be strongly dependent on the concentration of the precursors and hence, the luminescence of these QDs could be conveniently tuned across the visible spectrum simply by varying molar ratio of the precursors. It can be envisaged from the fact that a red-shift of about 100 nm in the PL peak position was observed when the molar ratio of the precursors ([Cd2+]:[Se2-], in mM) was varied from 10:5 to 10:40. Subsequently, the charge carrier relaxation dynamics associated with the different molar ratio of precursors has been investigated and very interesting information regarding the energy level structures of these QDs were revealed. Most importantly, in conjunction with the optical tuning, the nanomorphology of these nanoparticles was found to vary with the change in molar ratios of Se and Cd precursors. This aspect can provide a new direction of controlling the shape of CdSe nanoparticles. The possible mechanism of the formation as well as for the shape variation of these nanoparticles with the molar ratios of precursors has been proposed, taking into account the role of amino acid residues (present in BSA). Moreover, the QDs were water soluble and possessed fairly good colloidal stability therefore, can have potential applications in catalysis and bio-labeling. On the whole, the present methodology of protein assisted synthesis is relatively new especially for semiconducting nanomaterials and may provide some unique and interesting aspects to control and fine tune the morphology vis-à-vis, their optical properties.

Aqueous self-assembly and surface-functionalized nanodots for live cell imaging and labeling

Nanoparticles have enormous potential for bioimaging and biolabeling applications, in which conventional organically based fluorescent labels degrade and fail to provide long-term tracking. Thus, the development of approaches to make fluorescent probes water soluble and label cells efficient is desirable for most biological applications. Here, we report on the fabrication and characterization of self-assembled nanodots (SANDs) from 3-aminopropyltriethoxysilane (APTES) as a probe for protein labeling. We show that fluorescent SAND probes exhibit both bright photoluminescence and biocompatibility in an aqueous environment. Selective in vitro imaging using protein and carbohydrate labeling of hepatoma cell lines are demonstrated using biocompatible SANDs conjugated with avidin and galactose, respectively. Cytotoxicity tests show that conjugated SAND particles have negligible effects on cell proliferation. Unlike other synthetic systems that require multistep treatments to achieve robust surface functionalization and to develop flexible bioconjugation strategies, our results demonstrate the versatility of this one-step SAND fabrication method for creating multicolor fluorescent probes with the tailored functionalities, efficient emission, as well as excellent biocompatibility, required for broad biological use. Open image in new window

Hydrothermal Preparation of Photoluminescent Graphene Quantum Dots Characterized Excitation‐Independent Emission and its Application as a Bioimaging Reagent

Zero‐dimensional photoluminescent (PL) graphene quantum dots (GQDs) that can be used as the cell‐imaging reagent are prepared by a hydrothermal route using the graphene oxide (GO) as the carbon source. Under the optimized hydrothermal conditions, an initial hydrogen peroxide concentration of 0.5 mg mL−1 at 180 °C for 120 min, the GO sheets can be cut into nanocrystals with lateral dimensions in the range of 1.5–5.5 nm and an average thickness of around 1.1 nm. The as‐prepared GQDs exhibit an abundance of hydrophilic hydroxy and carboxyl groups and emit bright blue luminescence with up‐conversion properties in a water solution at neutral pH. Most interestingly, they indicate excitation‐independent emission characteristics, and the surface state is demonstrated to have a key role in the PL properties. The fluorescence quantum yield of the GQDs is tested to be around 6.99% using quinine sulfate as a standard. In addition, the as‐prepared GQDs can enter into HeLa cells easily as a fluorescent imaging reagent without any further functionalization, indicating they are aqueous stability, biocompatibility, and promising for potential applications in biolabeling and solution state optoelectronics.

Novel Au+-doped ZnxCd1−xS/ZnS core/shell quantum dots

Au+ ions have been successfully incorporated into the lattice of ZnxCd1−xS quantum dots as luminescent centers via an aqueous method. The dopant emission can be tuned in the range of 445 to 570 nm, and the highest quantum yield can reach as high as 30%. Our results showed that Au+ ions can be used as an excellent luminescent dopant in doped quantum dots. Finally, these water-soluble quantum dots can be used directly in PL imaging of HepG2 cells without conjugating any biological targets, indicating a high potential application in bio-labeling.

Nanostructural transformations of silver nanoclusters occurring during their synthesis and after interaction with UV-light

Metal nanoclusters (NCs), composed of several to tens of atoms, are a new class of fluorescent nanoprobes. NCs present distinct and favourable optical, electrical, and chemical properties as compared to other large-scale nanomaterials or bulk materials with the same chemical composition, being attractive for bio-imaging and bio-labelling applications. As this is a new nanomaterial class, fundamental questions regarding cluster growth mechanisms, stability, and functionality remain. Thus, new analytical tools for the analysis and characterization of such NCs are urgently needed to help in their synthesis optimization and final quality control. Information obtained from the use of a gentle separation technique, asymmetric flow field-flow fractionation, coupled on-line to molecular and elemental spectroscopic detection (spectrophotometry, fluorimetry and inductively coupled plasma mass spectrometry) along with transmission electron microscopy provide new insights into the structural transformations occurring during the growth of highly fluorescent silver nanoclusters (AgNCs) and also after their continuous irradiation with UV-light. These studies reveal that the mechanism of AgNCs growth goes through the previous formation of the corresponding unstable plasmonic metal nanoparticle. Moreover, photo-stability of the synthesized fluorescent NCs was investigated, in order to establish its manipulation and possible applications. Obtained results prove that the continuous exposure of AgNCs to UV-light provokes the loss of their optical properties driven by a photo-oxidation reaction.

Optical spectroscopic studies on GdF3:Tb3+,Yb3+ nanocrystals

Rare earth ions (Ln3+) doped optical materials manifest sharp and tunable emission, high photostability, long luminescent lifetime and low cytotoxicity, and find diverse applications in optics, optoelectronic devices and bio-labelling. Herein, we employed a conventional hydrothermal approach to control the size and tune the phase as well as the morphology of GdF3:Tb3+,Yb3+ nanocrystals, wherein the effects of the synthetic conditions, such as pH, time, the ratio of the Gd to F and the surfactant, have been detailed investigated, producing sphere, platelet, rice, flower-shaped GdF3 nanoparticles. Diffuse reflectance spectra, photoluminescence emission and excitation spectra, and photoluminescence lifetime measurements were employed to characterize the optical spectroscopic properties of GdF3:Tb3+,Yb3+ nanocrystals. No efficient energy transfer from Tb3+ to Yb3+ is observed in GdF3:Tb3+,Yb3+ nanocrystals and the Yb3+ shows a concentration quenching effect at high doping levels.

Meso‐Ester and Carboxylic Acid Substituted BODIPYs with Far‐Red and Near‐Infrared Emission for Bioimaging Applications

A series of meso-ester-substituted BODIPY derivatives 1-6 are synthesized and characterized. In particular, dyes functionalized with oligo(ethylene glycol) ether styryl or naphthalene vinylene groups at the α positions of the BODIPY core (3-6) become partially soluble in water, and their absorptions and emissions are located in the far-red or near-infrared region. Three synthetic approaches are attempted to access the meso-carboxylic acid (COOH)-substituted BODIPYs 7 and 8 from the meso-ester-substituted BODIPYs. Two feasible synthetic routes are developed successfully, including one short route with only three steps. The meso-COOH-substituted BODIPY 7 is completely soluble in pure water, and its fluorescence maximum reaches around 650 nm with a fluorescence quantum yield of up to 15 %. Time-dependent density functional theory calculations are conducted to understand the structure-optical properties relationship, and it is revealed that the Stokes shift is dependent mainly on the geometric change from the ground state to the first excited singlet state. Furthermore, cell staining tests demonstrate that the meso-ester-substituted BODIPYs (1 and 3-6) and one of the meso-COOH-substituted BODIPYs (8) are very membrane-permeable. These features make these meso-ester- and meso-COOH-substituted BODIPY dyes attractive for bioimaging and biolabeling applications in living cells.

Water-soluble, highly emissive, color-tunable, and stable Cu-doped ZnSeS/ZnS core/shell nanocrystals

Water-soluble, highly emissive, color-tunable, and stable Cu:ZnSeS/ZnS core/shell doped nanocrystals (d-dots) with pure dopant emission have been synthesized through a simple aqueous synthetic route. The influence of various experimental variables, including introduction of chloride ions, amount of mercaptopropanoic acid (MPA), pH value and Cu-doping concentration, on the optical properties of Cu dopant emission have been systematically investigated. Through epitaxial growth of a ZnS shell on Cu:ZnSeS cores, the fabricated Cu:ZnSeS/ZnS d-dots exhibit a high PL QY of up to 40%, which is the highest ever reported for any type of water-soluble Cu-doped selenide-based II–VI semiconductor nanocrystals (NCs). Moreover, the serious photostability problem of Cu-based d-dots in the presence of air and light is solved and the quenching of the excitonic emission is suppressed as well. The stable testing of these highly luminescent d-dots shows that the Cu:ZnSeS/ZnS d-dots have better photostability and preserve more than ~90% of the initial intensity in the presence of air and light 30 days later. This new class of green emissive material shows their potential applications in biolabeling, bioimaging, and light emitting devices.

Encapsulated Cd3P2 quantum dots emitting from the visible to the near infrared for bio-labelling applications

Quantum dot composites (PS@Cd3P2, SiO2@Cd3P2) were prepared and employed for the first time as fluorescent probes for biological imaging.

Characterization of the nonlinear optical properties of nanocrystals by Hyper Rayleigh Scattering.

BackgroundHarmonic Nanoparticles are a new family of exogenous markers for multiphoton imaging exerting optical contrast by second harmonic (SH) generation. In this tutorial, we present the application of Hyper-Rayleigh Scattering (HRS) for a quantitative assessment of the nonlinear optical properties of these particles and discuss the underlying theory and some crucial experimental aspects.MethodsThe second harmonic properties of BaTiO3, KNbO3, KiTiOPO4 (KTP), LiNbO3 and ZnO nanocrystals (NCs) are investigated by HRS measurements after careful preparation and characterization of colloidal suspensions.ResultsA detailed analysis of the experimental results is presented with emphasis on the theoretical background and on the influence of some experimental parameters including the accurate determination of the nanocrystal size and concentration. The SH generation efficiency and averaged nonlinear optical coefficients are then derived and compared for six different types of NCs.ConclusionsAfter preparation of colloidal NC suspensions and careful examination of their size, concentration and possible aggregation state, HRS appears as a valuable tool to quantitatively assess the SH efficiency of noncentrosymmetric NCs. All the investigated nanomaterials show high SH conversion efficiencies, demonstrating a good potential for bio-labelling applications.

Synthesis of highly fluorescent gold nanoclusters using egg white proteins

Gold nanoclusters (AuNCs) have gained interest during the recent years because of their low toxicity and finer size for the bioimaging and biolabeling applications in comparison to the semiconductor quantum dot analogues. Diverse materials such as sulfur compounds, peptides, dendrimers, proteins, etc., are exploited for the preparation of AuNCs. Henceforth, highly fluorescent, water-soluble, and few atom-containing gold nanoclusters are created using a rapid, straightforward, and green method. In this regard for the first time chicken egg white (CEW), one of the most unique materials, is utilized in an aqueous solution under basic conditions at physiological temperature for the preparation of AuNCs. Tyrosine and tryptophan amino acid residues are responsible for the conversion of Au ions to Au0 under alkaline condtions. CEW contains four major proteins of which the main constituent protein, ovalbumin also leads to the formation of the AuNCs with a higher fluorescence emission compared to the CEW. The ratios between the different reaction partners are very crucial, along with temperature and time for the preparation of AuNCs with high photoluminescence emission. The limited vibrational motion of the proteins under alkaline condition and the bulkiness of the proteins help in the formation of AuNCs.