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Markers are regularly used to detect fraud and adulteration in fuel as well as to track products in a distribution chain. Herein, we tested the use of CdSe semiconductor fluorescent nanocrystals, quantum dots, as a new class of fuel markers. Quantum dots show bright fluorescence and they can be excited by light sources covering a broad spectral range, from ultraviolet up to the near infrared region. This class of markers presents great advantages over conventional ones, such as high detectability potential, fluorescence emission in multicolor wavelengths, high photostability, and easy preparation procedures. In this work, we prepared CdSe quantum dots by colloidal controlled growth in an organic medium to be used for evaluation of diesel oil. The detection method was either by electronic absorption or fluorescence spectroscopies. The nanomarkers could be detected down to 1 mg/kg level in diesel fuel. The overall results indicated that CdSe quantum dots have the potential for use as a new class of fuel nanomarkers.

Magnetic resonance imaging (MRI) is a powerful non-invasive diagnostic tool that enables distinguishing healthy from pathological tissues, with high anatomical detail. Nevertheless, MRI is quite limited in the investigation of molecular/cellular biochemical events, which can be reached by fluorescence-based techniques. Thus, we developed bimodal nanosystems consisting in hydrophilic quantum dots (QDs) directly conjugated to Gd(III)-DO3A monoamide chelates, a Gd(III)-DOTA derivative, allowing for the combination of the advantages of both MRI and fluorescence-based tools. These nanoparticulate systems can also improve MRI contrast, by increasing the local concentration of paramagnetic chelates. Transmetallation assays, optical characterization, and relaxometric analyses, showed that the developed bimodal nanoprobes have great chemical stability, bright fluorescence, and high relaxivities. Moreover, fluorescence correlation spectroscopy (FCS) analysis allowed us to distinguish nanosystems containing different amounts of chelates/QD. Also, inductively coupled plasma optical emission spectrometry (ICP – OES) indicated a conjugation yield higher than 75%. Our nanosystems showed effective longitudinal relaxivities per QD and per paramagnetic ion, at least 5 times [per Gd(III)] and 100 times (per QD) higher than the r1 for Gd(III)-DOTA chelates, suitable for T1-weighted imaging. Additionally, the bimodal nanoparticles presented negligible cytotoxicity, and efficiently labeled HeLa cells as shown by fluorescence. Thus, the developed nanosystems show potential as strategic probes for fluorescence analyses and MRI, being useful for investigating a variety of biological processes.

Background Site-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). Methods The Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. Results The FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. Conclusions The prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. General significance This study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.

The use of Quantum Dots (QDs) as fluorescent probes for understanding biological functions has emerged as an advantageous alternative over application of conventional fluorescent dyes. Intracellular delivery of QDs is currently a specific field of research. When QDs are tracking a specific target in live cells, they are mostly applied for extracellular membrane labeling. In order to study intracellular molecules and structures it is necessary to deliver free QDs into the cell cytosol. In this work, we adapted the freeze and thaw method to encapsulate water dispersed carboxyl-coated CdTe QDs into liposomes of different compositions, including cationic liposomes with fusogenic properties. We showed that labeled liposomes were able to fuse with live human stem cells and red blood cells in an endocytic-independent way. We followed the interactions of liposomes containing QDs with the cells. The results were minutely discussed and showed that QDs were delivered, but they were not freely diffused in the cytosol of those cells. We believe that this approach has the potential to be applied as a general route for encapsulation and delivery of any membrane-impermeant material into living cells.

Quantum Dots (QDs) are fluorescent semiconductor nanocrystals with unique optical properties, which are being highly applied in opto-electronic devices and biological sciences. Aiming at biological applications we developed highly blue fluorescent Cysteamine coated ZnSe QDs. They were prepared in aqueous medium at near physiological pH by a fast precipitation process followed by hydrothermal growth. The characteristic excitonic emission output was enhanced by decreasing their surface defects via an UV photoactivation procedure. We tested their in vitro cytotoxicity towards macrophage cells and probed their functionality by labeling two different cell types: Candida albicans and red blood cells by electrostatic interaction with the negative cell membranes. The overall results show that these positively charged QDs can be applied as new low toxic and promising platforms for biolabeling purposes.

During carcinogenesis, changes in the glycosylation can modulate many biological processes. Thus, the interest in exploring and understanding the roles of carbohydrates as cancer biomarkers has been increasing. Lectins have been applied as useful tools in glycobiology, especially when associated with fluorescent reporters. Therefore, to take advantage of the physicochemical properties of quantum dots (QDs), herein, we conjugated Cramoll, a lectin that recognizes glucose/mannose residues, with those nanoparticles. We applied the conjugates to investigate the glycocode of normal, fibroadenoma (FB), and invasive ductal carcinoma (IDC) human breast tissues. Additionally, we proposed a method to quantitatively evaluate the tissue labeling intensity by a fluorescence microplate assay (FMA). Conjugates showed intense fluorescence and specificity. The lectin activity and secondary structure were also preserved after the conjugation with QDs. Moreover, fluorescence images showed that ductal cells of normal and FB tissues were preferentially labeled by conjugates, whereas both cells and stroma were strongly labeled in IDC. FMA showed in a quantitative, practical, and sensitive way that the level of exposed glucose/mannose residues increased accordingly to the sample malignancy degree. In conclusion, QDs-Cramoll conjugates can be considered effective, specific, and versatile probes to evaluate glycan profiles in normal and transformed tissues, by fluorescence microscopy as well as FMA quantification. Furthermore, FMA showed to be a potential method that can be applied with other fluorescent conjugates.

Quantum dots (QDs) are semiconductor nanocrystals, which present unique photophysical properties, enabling their application as new fluorescent platforms for biomedical sciences. Colloidal QDs are end-capped with organic or inorganic compounds, not only to prevent their agglomeration but also to provide reaction sites for the attachment of targeting (bio)molecules, nanoparticles or other interfaces, for specific biological purposes. The (bio)conjugation can involve non-covalent or covalent interactions, which can be accomplished through different strategies. The final assembly needs to maintain its chemical and optical stability and biochemical functionality. Although a relative good comprehension of the experimental procedures has been established, the bioconjugation process is still a challenge. The present manuscript aims to review the main (bio)conjugation strategies successfully applied to QDs, describing the steps necessary to prepare stable targeting fluorescent nanoplatforms, as well as some usual methods used to evaluate and optimize this process.

In this study we evaluated the absolute fluorescence quantum yield ( Φ ) of hydrophilic CdTe QDs in function of different pHs, modified from the alkaline to acid, by using two different chemicals compounds, the mercaptosuccinic acid (MSA-the stabilizing agent of the QDs synthesis) or hydrochloric acid (HCl). The pH control of QDs suspensions is essential for the use of fluorescent nanoparticles in biological systems. We used the eclipsing thermal lens spectroscopy technique to determine the absolute fluorescence quantum yield values. The results showed variations on the Φ values as a function of the pH, which allowed a better understanding of QDs emission characteristics, establishing parameters for their use in biomedical applications such as optical images of biological systems, immunoassays, flow cytometry, biosensors and others.

In this study, we present the optimization of the optical properties of Mn2+ doped ZnSe QDs (also referred to as d-dots) coated with thioglycolic acid in aqueous medium. The nanoparticles were characterized by ionic coupled plasma, electron paramagnetic resonance, transmission electron microscopy and X-ray diffractometry. By applying a controlled dose of UV irradiation, we obtained efficient orange emitting d-dots (4T1 6A1 transition centered at 580 nm). The results point out to a doping fraction in the nanoparticles smaller than 1% (0.89%), and that the Mn2+ ions are preferentially located close to the particle's surface. The UV photoactivation procedure has a definite influence on the emission intensity and on the colloidal stability of the particles. Photoactivated Mn2+ d-dots were conjugated to Concanavalin A and labeled specifically Candida albicans yeast cells, by targeting carbohydrate residues depicting the successful use of these QDs as fluorescent probes. (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 μm) was smaller when compared to C. albicans (4.0 μm) and C. parapsilosis sensu stricto (3.8 μm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

Background Quantum dots (QDs) are outstanding nanomaterials of great interest to life sciences. Their conjugation versatility added to unique optical properties, highlight these nanocrystals as very promising fluorescent probes. Among uncountable new nanosystems, in the last years, QDs conjugated to glycans or lectins have aroused a growing attention and their application as a tool to study biological and functional properties has increased. Scope of review This review describes the strategies, reported in the literature, to conjugate QDs to lectins or carbohydrates, providing valuable information for the elaboration, improvement, and application of these nanoconjugates. It also presents the main applications of these nanosystems in glycobiology, such as their potential to study microorganisms, the development of diseases such as cancer, as well as to develop biosensors. Major conclusions The development of glyconanoparticles based on QDs emerged in the last decade. Many works reporting the conjugation of QDs with carbohydrates and lectins have been published, using different strategies and reagents. These bioconjugates enabled studies that are very sensitive and specific, with potential to detect and elucidate the glycocode expressed in various normal or pathologic conditions. General significance Produce a quick reference source over the main advances reached in the glyconanotechnology using QDs as fluorescent probes.

Quantum dots (QDs) are fluorescent nanocrystals that present unique optical properties, especially a high photostability. However, their use for intracellular studies is still limited since their passage through the living cell membranes does not occur passively. In this work, we adapted the ethanol injection method to encapsulate cationic hydrophilic QDs into fusogenic liposomes, to deliver them in living cells. Liposomes were characterized using zeta potential, dynamic light scattering (DLS), fluorescence microscopy and transmission electron microscopy (TEM). Red blood cells (RBCs) were applied as models in this study to probe the liposome fusion with the cell membrane since RBCs do not present endocytic activity. Therefore, HeLa cells were also applied to test the QDs delivery by the liposomes. The TEM and the fluorescence microscopy confirmed the QDs encapsulation, with an efficiency of 43%, determined by UV-vis spectroscopy. Zeta potential showed that the QDs-loaded fusogenic liposomes were positively charged and presented an average size of 343nm, determined by DLS. Furthermore, fluorescence microscopy analyses of RBCs and HeLa cells confirmed the liposomes fusion with the cell membrane and suggested the release of QDs into cells. Thus, we expect that this work will contribute to improve the use of QDs as fluorescent probes to intracellular studies.

Metallic nanoparticles (NPs) have been used to improve the sensibility of biosensors and bioassays either by enhancing radiative emission or inducing quenching process on fluorescent probes. The aim of this research was to study the interaction of silver and silver-pectin NPs with water-dispersed carboxyl-coated cadmium telluride (CdTe) quantum dots (QDs). Metallic NPs were observed to change the emission of these fluorophores through local field effects. In a solution-base platform, an increase of 82 % was observed for the CdTe emission due to the interaction of QDs and silver-pectin NPs. QDs interaction with silver NPs without pectin was also investigated and a smaller emission enhancement of 20 % was detected. We observed that the NPs’ nature and QDs’ surface charge and concentration are important parameters for NPs-QDs interaction. Moreover, the presence of the pectin polymer shows to be a key component to the observed fluorescence enhancement.

Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well-known features which make them very useful tools for biological labeling purposes. Low photobleaching rates, high chemical stability and active surface allowing conjugation to living cells explain the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with highly fluorescent core–shell CdTe–CdS (diameter=3–7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules. The conjugated nanocrystals were incubated with living yeast cells, in order to investigate their glucose up-take activity in real time, by confocal microscopy analysis.

Paulo E Cabral Filho,1 Maria IA Pereira,1 Heloise P Fernandes,2 Andre A de Thomaz,3 Carlos L Cesar,3 Beate S Santos,4 Maria L Barjas-Castro,2 Adriana Fontes1 1Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Pernambuco, 2Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, 3Departamento de Eletrônica Quântica, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, Campinas, São Paulo, 4Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Abstract: New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs]) as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC) antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I) lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion, this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes. Keywords: erythrocytes, nanoparticles, ABO antigens, carbohydrates, covalent binding

Liposomes have been used to deliver DNA, drugs and, more recently, nanoparticles such as quantum dots, into living cells. Their electrostatic interaction with cell’s surface (negatively charged) can lead to membrane destabilization and/or fusion, facilitating intracellular release of those compounds. Nevertheless, cationic lipids can modify living cells homeostasis, depending on their concentration. In this study, we observed that the DOTAP cationic lipid concentrations influence the red blood cells (RBCs) homeostasis. We used fluorescent fusogenic liposomes composed by three lipids: DOPE, DOTAP and DPPE-Rhodamine (1:0.1/0.3/0.5/0.8/1:0.1 mM respectively), varying DOTAP from 0.1 to 1 mM. To probe liposomes ability to fuse with cells, RBCs (1% in saline) were utilized. Liposomes were characterized by zeta potential, dynamic light scattering (DLS), fluorescence and transmission electron microscopy. Their interaction with RBCs was evaluated by fluorescence microscopy and flow cytometry. Zeta potential results showed that, from 0.1 to 1 mM concentration, the charge increases, due to the addition of DOTAP. Liposomes’ diameter does not vary significantly when more DOTAP was added, except for the one containing 0.1 mM of DOTAP, according to DLS results. Flow cytometry and microscopy analysis showed that for all DOTAP’ concentration applied, the liposomes were capable to label RBCs. However, as higher the amount of DOTAP in liposomes, the more harmful they were to cells. Thus, the results showed that it is possible to use lower concentrations of DOTAP keeping the fusogenic liposomes’s ability and cell homeostasis. This is important to guarantee a greater efficiency in the delivery of nanoparticles or other active samples into cells.

The application of fluorescent II-VI semiconductor quantum dots (QDs) as active photosensitizers in photodymanic inactivation (PDI) is still being evaluated. In the present study, we prepared 3 nm size CdTe QDs coated with mercaptosuccinic acid and conjugated them electrostatically with Zn(II) meso-tetrakis (N-ethyl-2-pyridinium-2-yl) porphyrin (ZnTE-2-PyP or ZnP), thus producing QDs-ZnP conjugates. We evaluated the capability of the systems, bare QDs and conjugates, to produce reactive oxygen species (ROS) and applied them in photodynamic inactivation in cultures of Candida albicans by irradiating the QDs and testing the hypothesis of a possible combined contribution of the PDI action. Tests of in vitro cytotoxicity and phototoxicity in fibroblasts were also performed in the presence and absence of light irradiation. The overall results showed an efficient ROS production for all tested systems and a low cytotoxicity (cell viability &gt, 90%) in the absence of radiation. Fibroblasts incubated with the QDs-ZnP and subjected to irradiation showed a higher cytotoxicity (cell viability &lt, 90%) depending on QD concentration compared to the bare groups. The PDI effects of bare CdTe QD on Candida albicans demonstrated a lower reduction of the cell viability (~1 log10) compared to bare ZnP which showed a high microbicidal activity (~3 log10) when photoactivated. The QD-ZnP conjugates also showed reduced photodynamic activity against C. albicans compared to bare ZnP and we suggest that the conjugation with QDs prevents the transmembrane cellular uptake of the ZnP molecules, reducing their photoactivity.

This work reports on highly fluorescent and superparamagnetic bimodal nanoparticles (BNPs) obtained by a simple and efficient method as probes for fluorescence analysis and/or contrast agents for MRI. These promising BNPs with small dimensions (ca. 17 nm) consist of superparamagnetic iron oxide nanoparticles (SPIONs) covalently bound with CdTe quantum dots (ca. 3 nm). The chemical structure of the magnetic part of BNPs is predominantly magnetite, with minor goethite and maghemite contributions, as shown by Mossbauer spectroscopy, which is compatible with the x-ray diffraction data. Their size evaluation by different techniques showed that the SPION derivatization process, in order to produce the BNPs, does not lead to a large size increase. The BNPs saturation magnetization, when corrected for the organic content of the sample, is ca. 68 emu g-1, which is only slightly reduced relative to the bare nanoparticles. This indicates that the SPION surface functionalization does not change considerably the magnetic properties. The BNP aqueous suspensions presented stability, high fluorescence, high relaxivity ratio (r 2/r 1 equal to 25) and labeled efficiently HeLa cells as can be seen by fluorescence analysis. These BNP properties point to their applications as fluorescent probes as well as negative T 2-weighted MRI contrast agents. Moreover, their potential magnetic response could also be used for fast bioseparation applications.

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

We present here a new and alternative method that uses a Fluorescence Plate Reader in a different approach, not to study protein-protein interactions, but to evaluate the efficiency of the protein bioconjugation to quantum dots (QDs). The method is based on the QDs' native fluorescence and was successfully tested by employing two different QDs-proteins conjugation methodologies, one by promoting covalent binding and other by inducing adsorption processes. For testing, we used bioconjugates between carboxyl coated CdTe QDs and bovine serum albumin, concanavalin A lectin and anti-A antibody. Flow cytometry and fluorescence spectroscopy studies corroborated the results found by the Fluorescence Plate Reader assay. This kind of analysis is important because poor bioconjugation efficiency leads to unsuccessful applications of the fluorescent bioconjugates. We believe that our method presents the possibility of performing semi-quantitative and simultaneous analysis of different samples with accuracy taking the advantage of the high sensitivity of optical based measurements.

Quantum dots (QDs) have been used as fluorescent probes in biological and medical fields such as bioimaging, bioanalytical, and immunofluorescence assays. For these applications, it is important to characterize the QD-protein bioconjugates. This chapter provides details on a versatile method to confirm quantum dot-protein conjugation including the required materials and instrumentation in order to perform the step-by-step semiquantitative analysis of the bioconjugation efficiency by using fluorescence plate readings. Although the protocols to confirm the QD-protein attachment shown here were developed for CdTe QDs coated with specific ligands and proteins, the principles are the same for other QDs-protein bioconjugates.

We present and discuss the application of colloidal semiconductor quantum dots for diagnostic purposes, with special emphasis for cancer. We prepared and applied core–shell cadmium sulfide–cadmium hydroxide (CdS/Cd(OH)2) semiconductor quantum dots in aqueous medium. Tissue and cells labeling was evaluated by laser scanning confocal microscopy as well as by conventional fluorescence microscopy. The procedure presented in this work, shown to be a promising tool for fast, low-cost and precise cancer diagnostic protocols.

Semiconductor quantum dots are a promising class of materials in the labeling of biological systems. In the present study we show the marking pattern of Trypanosoma cruzi (T. cruzi) live parasites using PEGylated CdS/Cd(OH)2 fluorescent nanocrystals. The analysis obtained by confocal fluorescence microscopy and transmission electron microscopy indicates that only the endocytic paths of parasites were labeled. The parasites were alive after the incubation with the CdS/Cd(OH)2–PEG suspension. Labeling the T. cruzi with quantum dots can help to better understand the endocytosis process and also the cellular differentiation.

Semiconductor nanoparticles in the quantum confinement regime used as biolabels present many advantages over the other chemical species used as fluorophores. They are composed of 2000–6000 atoms rendering a far greater photostability and allowing for long time bioimaging experiments. In this work we present a synthetic route for the obtention of large quantities of highly fluorescent CdSe and CdTe/CdS core–shell nanocrystals based on aqueous colloidal chemistry. The methodologies were optimized and the systems were characterized by optical spectroscopy, transmission electronic microscopy and X-Ray diffractometry. The fluorescent biolabels were tested in live macrophages.

Semiconductor quantum dots based on II–VI materials may be prepared to develop good biolabeling properties. In this study we present some well-succeeded results related to the preparation, functionalization and bioconjugation of CdY (Y = S, Se and Te) to biological systems (live cells and fixed tissues). These nanostructured materials were prepared using colloidal synthesis in aqueous media resulting nanoparticles with very good optical properties and an excellent resistance to photodegradation.

Fluorescence provides a unique method for understanding how biomolecules interact with each other in many levels, from single cell to whole organisms. Researchers commonly use fluorescence-based techniques mainly due to its high specificity and sensitivity (capable of detecting even a single molecule) (Michalet et al., 2006; Giepmans et al., 2006). The evolution of tools based on fluorescence including new techniques (like multiphoton microscopy), new lasers and new fluorescent probes (presenting new features when compared to the traditional organic fluorophores), allowing us to take advantages of the full potential of fluorescence. This is the case of Quantum dots (QDs) that since the end the 90 s have been increasingly used in biological and biomedical field.

Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological conditions.

Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell antibodies. For this reason, simple, cheap and reproducible routes of QDs´s syntheses are the main goal of many researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of equipments.

We present and discuss results and features related to the synthesis of water-soluble semiconductor quantum dots and their application as fluorescent biomarkers in cancer diagnostics. We have prepared and applied different core-shell quantum dots, such as cadmium telluride-cadmium sulfide, CdTe-CdS, and cadmium sulfide-cadmium hydroxide, CdS/Cd(OH)(2), in living healthy and neoplastic cells and tissues samples. The CdS/Cd(OH)(2) quantum dots presented the best results, maintaining high levels of luminescence as well as high photostability in cells and tissues. Labeled tissues and cells were analyzed by their resulting fluorescence, via conventional fluorescence microscopy or via laser scanning confocal microscopy. The procedure presented in this work was shown to be efficient as a potential tool for fast and precise cancer diagnostics.

We present and discuss results and features related to the synthesis of water-soluble semiconductor quantum dots and their application as fluorescent biomarkers in cancer diagnostics. We have prepared and applied different core-shell quantum dots, such as cadmium telluride-cadmium sulfide, CdTe-CdS, and cadmium sulfide-cadmium hydroxide, CdS/Cd(OH)(2), in living healthy and neoplastic cells and tissues samples. The CdS/Cd(OH)(2) quantum dots presented the best results, maintaining high levels of luminescence as well as high photostability in cells and tissues. Labeled tissues and cells were analyzed by their resulting fluorescence, via conventional fluorescence microscopy or via laser scanning confocal microscopy. The procedure presented in this work was shown to be efficient as a potential tool for fast and precise cancer diagnostics.

In this work we used a setup consisting of an optical tweezers combined with a nonlinear microspectroscopy system to perform scanning microscopy and obtain emission spectra using two photon excited (TPE) luminescence of captured single living cells labeled with core-shell fluorescent semiconductor quantum dots (QDs). The QDs were obtained via colloidal synthesis in aqueous medium with an adequate physiological resulting pH. Sodium polyphosphate was used as the stabilizing agent. The results obtained show the potential presented by this system as well as by these II-VI fluorescent semiconductor quantum dots to perform spectroscopy in living trapped cells in any neighborhood and dynamically observe the cell chemical reactions in real time.

Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well known features which make them very useful tools for biological labeling purposes. Low photo-bleaching rates, high chemical stability, active surface allowing conjugation to living cells, explains the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with high fluorescent core-shell CdTe-CdS (diameter = 3-7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules, incubated with living yeast cells, in order to investigate their glucose up-take activity.

In this work we present the preparation, characterization and conjugation of colloidal core shell CdS-Cd(OH) 2 quantum dots to health and cancer glial rats living cells in culture media. The particles were obtained via colloidal synthesis in aqueous medium, with final pH=7.3-7.4. Laser Scan Confocal Microscopy (LSCM) and Fluorescence Microscopy were used to evaluate fluorescence intensities and patterns of health and cancer (glioblastoma) glial cells labeled with the quantum dots in different time intervals. Health and cancer glial cells clearly differ in their fluorescence intensities and patterns. These different fluorescence intensities and patterns may be associated to differences concerning cellular membrane and metabolic features of health and cancer cells. The results obtained indicate the potential of the methodology for fast and precise cancer diagnostics.

In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λ exc =795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.

We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.

Semiconductor nanocrystals composed by few hundred to a few thousand atoms also known as quantum dots have received substantial attention due to their size tunable narrow-emission spectra and several other advantages over organic molecules as fluorescent labels for biological applications, including resistence to photodegradation, improved brightness and only one laser excitation that enable the monitoring of several processes simultaneously. In this work we have synthesized and characterized thiol-capped CdTe and bioconjugated them to macrophages. We have mapped the fluoroscence intensity along the macrophage's body in our set up consisting of an optical tweezer plus a non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited luminescence.

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

Semiconductor quantum dots (QDs) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QDs are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching, and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10 to 100 fold. Recent research has shown that QDs can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids and small molecules for use as fluorescent probes. The aim of the present study was to evaluate the application of a Cadmium Sulfide QDs method for the analysis of the expression and distribution of A antigen on living red blood cell surface. Highly luminescent and stable cadmium sulfide colloidal particles were obtained in the nanometer size range. Their surface was functionalized with commercially available monoclonal anti-A antibody via an one-pot glutaraldehyde cross linking procedure, followed by their conjugation to red cells. Samples were obtained from blood bank red cell units, of groups A (n=3), A2 (n=2) and O (n=3). Confocal microscopy (LSM 510 - Carl Zeiss, Jena, Germany) was used and for each cell type, the images were reproduced at least three times. The acquisition confocal image parameters, such as pinhole, filters, beam splitters and photomultiplier gain, were maintained constant to permit a comparative analysis of the luminescence intensity maps. Analyzing the intensity map of group A erythrocytes, a homogeneous emission profile was observed throughout the cell surface, suggesting a homogeneous distribution of A antigen. In contrast, the intensity map analysis of the confocal images showed a lower intensity pattern of the A2 group erythrocytes studied, suggesting different antigen expression on cell surface. The O group cells showed no emission. This result suggests that this new efficient, fast and simple labeling procedure may be employed as a quantitative tool to investigate the expression and distribution of molecules in red blood cells.

Liposomes are being studied as drug delivery systems, membrane models, as well as encapsulating systems of nanostructured materials for cellular imaging and diagnostics applications. Taking advantage of the use of Quantum Dots (QDs) as fluorescent probes over many conventional fluorescent dyes, several research groups are developing methods to encapsulate lypophilic and hydrophilic QDs into liposomes either in the aqueous compartment or in their bilayer in order to have a multifunctional system and provide therapy and diagnostics at the same time [1].In this work, Cadmium Telluride/Cadmium Sulfide (CdTe/CdS) quantum dots functionalized with mercaptopropionic acid (MPA) was synthesized in aqueous medium and characterized by optical spectroscopy and X-Ray diffractometry. Synthesis was carried out by adding Te−2 to a Cd+2/MPA refluxed for 7 h at ∼ 95°C. Cationic multifluorescent quantum dot liposomes (QD-Ls) formed by phosphatidylcholine (PC) and 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP) (80:20) were prepared by lipid film hydration with pure water. QDs were encapsulated by a new method using freeze-and-thaw cycle. Liposomes (100 or 800 nm in diameter) encapsulating QDs were characterized by zeta potential, size distribution, fluorescence and transmission electron microscopy and show great potential for diagnostic purposes.[1] G. D. Bothun, A. E. Rabideau, and M. A. Stoner, J. Phys. Chem B Lett 2009 113, 7725-7728.Support: FACEPE, CNPq, INCT de Fotonica.

In this work we show and discuss the results obtained by using highly luminescent colloidal hydrophilic semiconductor Quantum Dots, synthesized in aqueous medium, physiological pH and functionalized with organic compounds for precise diagnostic of breast (ductal filling carcinoma), brain (glioblastoma) and cervical cancer. No presente artigo são apresentados e discutidos, resultados obtidos pelo uso de quantum dots de semicondutores altamente luminescentes, obtidos por métodos de síntese coloidal. Os quantum dots apresentam superfície hidrofílica e pH fisiológico. Foram sintetizados em meio aquoso e funcionalizados com compostos orgânicos para utilização em diagnótico preciso de câncer de mama (carcinoma ductal infiltrante), câncer de cérebro (glioblastoma) e câncer cervical (colo de útero).

Fluorescent semiconductor nanocrystals called “quantum dots” (QDs) have been synthesized for applications in many areas, such as biomedical sciences as biosensors, disease diagnostics and cell labeling, as well as in electronics and optics. In this work, cadmium telluride (CdTe) QDs functionalized with mercaptosuccinic acid (MSA) was synthesized in aqueous medium by adding a solution of Te2- ions to a solution of cadmium perchlorate and MSA, followed by reflux under argon for 10 h at 95°C. These QDs were characterized by optical spectroscopy, X-ray diffractommetry and transmission electron microscopy (TEM). Cationic liposomes composed of egg phosphatidylcholine (PC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were prepared by hydrating a lipid film at a lipid ratio of 80:20 respectively. This method consists in drying the lipids and the formation of a film under N2 for 30 min and drying it for 3 h under vacuum to remove residual solvent from the system. The hydration step was performed by adding pure water followed by stirring. The QDs have been synthesized previously encapsulated within these vesicles by the method of freezing and thawing. These vesicles were characterized by Zeta potential, Fluorescence Microscopy and TEM. Primary astrocytes from newborn Wistar rats (Pn 0 - 3, Pn = day of postnatal) were isolated and cultured in Dulbecco's Modified Eagle's Medium supplemented with F −12 (DMEM/F-12) and 10% fetal bovine serum. Upon 80% confluence, the culture medium was removed and fresh serum-free medium was added. Liposomes with and without QDs were tested in these cells with a high unspecific labeling character. The results demonstrate that liposomes present an effective entrapment of these fluorescent probes and are potential candidates for the development of diagnostics of pathologies related to glial tumor cells such as gliomas and glioblastomas.