Three-dimensional imaging of the intracellular localization of growth hormone and prolactin and their mRNA using nanocrystal (Quantum dot) and confocal laser scanning microscopy techniques.

Semiconductor nanocrystals (Quantum dots, Qdots) have recently been used in biological research, because they do not fade on exposure to light, and they enable us to obtain multicolor imaging because of a narrow emission peak that can be excited via a single wavelength of light. There have been no reports of simultaneous localization of mRNA and protein using Qdots. We successfully applied these advantages of Qdot and confocal laser scanning microscopy (CLSM) to three-dimensional images of the intracellular localization of growth hormone and prolactin and to their mRNA. In situ hybridization and immunohistochemistry using Qdots combined with CLSM can optimally illustrate the relationship between protein and mRNA simultaneously in three dimensions. Such an approach enables us to visualize functional images of proteins in relation with mRNA synthesis and localization.     

Single-bead-based immunofluorescence assay for snake venom detection

In this communication, we reported a rapid and sensitive immunofluorescence method for the detection of snake venom by using microscale polystyrene beads as platform combined with semiconductor quantum dots (Qdots) as fluorescence label. Briefly, control rabbit IgG or capture antibody for venom was covalently immobilized onto the microspheres (surface activated with carboxyl group, dyed with different color) to form the control or capture beads. When incubated with the testing samples, the venom binds to the specific capture beads to form the complex through antibody-antigen interaction. Then, the second antibody conjugated Qdot was added, which targeted the Qdot to bind to the capture bead/antigen complex. The complex can be directly observed under a UV microscope. The system was applied to the testing of Naja kaouthia venom. Fluorescent microscopic images of QD-labeled capture beads demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of capture beads, indicating that the conjugated antibody molecules remained active and were able to recognize their specific target in solution. The detection limit of this method was 5-10 ng/mL. The detection could be completed within 3 h.     

Qdot nanocrystal conjugates conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in living cells

Quantum dots (Qdot Nanocrystal Conjugates; Quantum Dot, Hayward, CA) exhibit high fluorescence and low photobleaching compared with organic dyes, properties that should enhance their detection at low densities. In view of the properties of Qdots and the biological and pharmaceutical importance of G protein-coupled receptors (GPCRs), we attempted to use Qdots to label GPCRs in a variety of live cell types. An agonist consisting of biotinylated bombesin or ANG II was conjugated to Qdot Nanocrystal Conjugates coated with streptavidin through a biotin-streptavidin linkage (Qdot agonist). Herein we demonstrate that Qdot-bombesin conjugate can label the bombesin-preferring GPCR in living mouse Swiss 3T3 cells and in Rat-1 cells. Similarly, we used the Qdot-ANG II conjugate to label GPCR in intact rat intestinal epithelial cells (IEC)-18 and in a human pancreatic adenocarcinoma cell line of ductal origin, HPAF-II cells. We demonstrate that Qdot-ANG II is brighter and more photostable than agonist labeled with the organic dye Cy3. Our results demonstrate that Qdot technology can be adapted to monitor ligand binding to GPCRs. Combined with the narrow and symmetric emission profile of Qdot Nanocrystal Conjugates, this information suggests the potential for a new multiplex strategy to determine the effect of agonists and/or antagonists on agonist binding to several GPCRs simultaneously in living cells. 3T3 cells; intestinal epithelial cells; human pancreatic adenocarcinoma cells of ductal origin     

Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.     

A new capture test using conjugated peptides for the detection of HIV antibodies

Abstract A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591–611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314–329) IRIQRGPGRAFVTIGK and the p27 sequence (182–198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N -hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer''s patches ^1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes ⁴.Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.     

A new capture test using conjugated peptides for the detection of HIV antibodies.

A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

Random Walk of Processive, Quantum Dot-Labeled Myosin Va Molecules within the Actin Cortex of COS-7 Cells

Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (≤0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s⁻¹, similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network.     

Presence of IgG Anti-gp160/120 Antibodies Confers Higher HIV Capture Capacity to Erythrocytes from HIV-Positive Individuals

Background

HIV binding has been demonstrated in erythrocytes from HIV-positive and HIV-negative individuals. However, the presence of immunoglobulins G anti-HIV (IgG anti-HIV) in erythrocytes from HIV-positive individuals is still to be elucidated. Moreover, the capacity of erythrocytes from HIV-positive individuals to capture an additional amount of HIV has not been studied. Indeed, it is unknown if HIV binding to erythrocytes in HIV-positive persons could have consequences on the cell-free infectious virus available.

Methodology/Principal Findings

IgGs anti-HIV associated to erythrocytes were found in 77.3% (58/75) of the HIV-positive individuals studied and the IgGs anti-gp160 and anti-p24 were the most frequently found. We found a positive association between detectable plasma viral load (pVL) and presence of IgGs anti-HIV associated to erythrocyte (p<0.005), though the anti-p24/160 were present with or without detectable pVL. The HIV capture capacity was higher in erythrocytes from HIV-positive than HIV-negative individuals (p<0.0001). Furthermore, among the HIV-positive individuals the higher viral capture capacity was associated with the presence of anti-gp160/gp120 on erythrocytes. Moreover, the viral capture by erythrocytes was independent of pVL (rho = 0.022, p = 0.8817). Additionally, reduction of cell-free infectious virus and available viral load was observed in the presence of erythrocytes from HIV-positive individuals.

Conclusions/Significance

Results suggest that in HIV-positive individuals, erythrocytes are capable of capturing high amounts of HIV by the presence of IgGs anti-gp160/120 on their membranes and this may produce a reduction in the available free virus. Finally, the current measurement of pVL would underestimate the real viral quantity due to the HIV binding through specific antibodies to erythrocytes.     

Near infrared sensing based on fluorescence resonance energy transfer between Mn:CdTe quantum dots and Au nanorods

A novel sensing system based on the near infrared (NIR) fluorescence resonance energy transfer (FRET) between Mn:CdTe quantum dots (Qdots) and Au nanorods (AuNRs) was established for the detection of human IgG. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) and AuNRs linked with rabbit anti-human IgG (AuNRs-Ab2) acted as fluorescence donors and acceptors, respectively. FRET occurred by human IgG with the specific antigen–antibody interaction. And human IgG was detected based on the modulation in FRET efficiency. The calibration graph was linear over the range of 0.05–2.5 μM of human IgG under optimal conditions. The proposed sensing system can decrease the interference of biomolecules in NIR region and increase FRET efficiency in optimizing the spectral overlap of AuNRs with Mn:CdTe Qdots. This method has great potential for multiplex assay with different donor–acceptor pairs.     

Direct observation to chemokine receptor 5 on T-lymphocyte cell surface using fluorescent metal nanoprobes

Chemokine receptor 5 (CCR5) is a cell surface protein required for HIV-1 infection. It is important to detect the amount and observe the spatial distribution of the CCR5 receptors on the cell surfaces. In this report, we describes the metal nanoparticles which were specially designed as molecular fluorescent probes for imaging of CCR5 receptors on the T-lymphocytic PM1 cell surfaces. These CCR5 monoclonal antibodies (mAbs) metal complexes were prepared by labeling mAbs with Alexa Fluor 680 followed by covalent binding the labeled mAbs on the 20 nm silver nanoparticles. Compared with the labeled mAbs without metal, the mAb-metal complexes were found to display enhanced emission intensity and shortened lifetime due to interactions between fluorophores and metal. The mAb-metal complexes were incubated with the PM1 cell lines. The confocal fluorescent intensity and lifetime cell images were recorded on single cells. It was observed that the mAb-metal complexes could be clearly distinguished from the cellular autofluorescence. By analyzing a pool of cell images, we observed that most CCR5 receptors appeared as clusters on the cell surfaces. The fluorophore-metal complexes developed in this report are generally useful for detection of cell surface receptors and provide a new class of probe to study the interaction between the CCR5 receptors with viral gp120 during HIV infections.     

Volatile fatty acids productivity by anaerobic co-digesting waste activated sludge and corn straw: Effect of feedstock proportion

Volatile fatty acids (VFAs) are the most suitable and biodegradable carbon substrates for many bioprocesses. This study explored a new approach to improve the VFAs production from anaerobic co-digesting waste activated sludge (WAS) with corn straw (CS). The effect of feedstock proportion on the acidification efficiency was investigated. The maximum VFAs yield (corresponding fermentation time) was substantially increased 69% (96 h), 45% (72 h), 13% (120 h) and 12% (120 h) with 50%, 35%, 25% and 20% CS proportion of feedstock, respectively. HAc (acetic acid) was consistently the most abundant, followed by HPr (propionic acid) and n-HBu (butyric acid) in the co-digesting tests. The increase of CS in feedstock led to more production of HAc and HPr. Moreover, the consumption of protein and carbohydrate were also improved remarkably from 2955 and 249 mg COD/L (individual WAS fermentation) to 6575 and 815 mg COD/L (50%_WAS:50%_CS co-digestion) from 120 onward, respectively. The highest contribution of CS to additional VFAs production was1113 mg VFAs (as COD)/g CS/L in the 65%_WAS:35%_CS co-digesting test. Our study indicated a valuable method to improve VFAs production from anaerobic co-digesting WAS and CS.     

Antibody microarrays for label-free cell-based applications

The recent advances in microtechnologies have shown the interest of developing microarrays dedicated to cell analysis. In this way, miniaturized cell analyzing platforms use several detection techniques requiring specific solid supports for microarray read-out (colorimetric, fluorescent, electrochemical, acoustic, optical…). Real-time and label-free techniques, such as Surface Plasmon Resonance imaging (SPRi), arouse increasing interest for applications in miniaturized formats. Thus, we focused our study on chemical methods for antibody-based microarray fabrication dedicated to the SPRi analysis of cells or cellular activity. Three different approaches were designed and developed for specific applications. In the first case, a polypyrrole-based chemistry was used to array antibody-microarray for specific capture of whole living cells. In the second case, the polypyrrole-based chemistry was complexified in a three molecular level assembly using DNA and antibody conjugates to allow the specific release of cells after their capture. Finally, in the third case, a thiol-based chemistry was developed for long incubation times of biological samples of high complexity. This last approach was focused on the simultaneous study of both cell type characterization and secretory activity (detection of proteins secreted by cells). This paper describes three original methods allowing a rapid and efficient analysis of cellular sample on-chip using immunoaffinity-based assays.     

A sensitive electrochemiluminescent sensor chip based on the ssDNA-Ru(II) complex and aptamer for the determination of thrombin

In this work, an electrochemiluminescence (ECL) sensor chip for sensitive detection of thrombin (TB) was prepared using a screen-printed electrode (SPE) as a working electrode and an aptamer as a specific recognition moiety. To produce an ECL sensor chip, a layer of pL-Cys was immobilized on the surface of the SPE using the cyclic voltammetry scanning method. A layer of gold nanoparticles (AuNPs) was assembled through an Au–S bond and hairpin DNA was further immobilized on the electrode surface. Ru(bpy)₂(mcpbpy)²⁺, as a luminescent reagent, was covalently bound to single-stranded DNA (ssDNA) to prepare a luminescence probe ssDNA-Ru. The probe was hybridized with TB aptamer to form a capture probe. In the presence of TB, the TB aptamer in the capture probe bound to TB, causing the release of ssDNA-Ru that could bind to hairpin DNA on the electrode surface. The Ru(II) complex as a luminescent reagent was assembled onto the electrode, and pL-Cys was used as a co-reactant to enhance the ECL efficiency. The ECL signal of the sensor chip generated based on the above principles had a linear relationship with log TB concentration at the range 10 fM to1 nM, and the detection limit was 0.2 fM. Finally, TB detection using this method was verified using real blood samples. This work provides a new method using an aptamer as a foundation and SPE as a material for the detection of biological substances.     

Development of On-Chip Multi-Imaging Flow Cytometry for Identification of Imaging Biomarkers of Clustered Circulating Tumor Cells

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as “imaging biomarkers”, with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm² and (2) nucleus area larger than 90 µm² were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm² were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers—cluster area, nuclei area, nuclei number, and ratio of perimeter—can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.     

人类免疫缺陷病毒-1进入细胞的分子机制及相关药物的研究

艾滋病（AIDS）是由人类免疫缺陷病毒（HIV）侵染表达CD4表面抗原（CD4⁺）的T淋巴细胞而引起的.艾滋病病毒进入CD4⁺T淋巴细胞首先是通过病毒与细胞膜的融合来完成的.该融合过程涉及到病毒表面膜蛋白（gp120和gp41）与细胞表面受体蛋白（CD4和CCR5等）之间的相互作用.根据对这些蛋白质分子结构及作用机制的认识,从破坏病毒与细胞的融合入手,设计新型的抗艾滋药物及疫苗,已成为目前药物开发的新热点.     

Micropillar array chip for integrated white blood cell isolation and PCR

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.