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A) Digital photograph of the streptavidin coated 96-well plate used in the DNA-hybridization assay for P. falciparum CSP gene, B) schematic of a pipettor used to transfer incubated PAPP from the 96-well plate into the microcavity in the VEG 4X1™ chip attached to the connector that allows access to all the electrodes in the 4 cavities, and C) a scanning electron micrograph of the 50-mm diameter cavity showing the built-in microelectrodes.
Source publication
An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The...
Contexts in source publication
Context 1
... solid-phase enzyme-linked DNA-hybridization assay starts with the immobilization of 50 µL biotinylated 1 0 P on streptavidin coated 96-well plates ( Figure 1A) using 50 ng/mL solution (unless otherwise indicated) in HB. The streptavidin-coated microwells in a 96-well plate were pre-incubated with 50 uL HB for 30 min at room temperature (RT) in a vortex mixer at 0 setting (This mixing condition was implemented throughout the procedure). ...
Context 2
... 1 0 P+T+ 2 0 P ~AP immobilized microwells were washed, blocked, and rinsed thoroughly before exposure to the enzyme substrate PAPP. , 3 (40) 1-11 (2007) ) unless CC License in place (see abstract The electroactive PAP R was generated by adding 20 uL of 4 mM PAPP in 0.1 M Tris, pH 9.0 in each of the modified microwells, covered, and incubated with shaking for 30 min at RT after which a 1 uL aliquot was placed on top of a 50-µm-diameter microcavity. Control experiments verified that conversion of PAPP to PAP R over this 30 min period under these conditions does not occur when alkaline phosphatase is absent. ...
Context 3
... rapid, sensitive, low-cost, and reliable microelectrochemical detection of signals from a 96-well plate ( Figure 1A) solid-phase enzyme-linked DNA-hybridization assay for Plasmodium falciparum has been demonstrated. Electrochemical detection of the signals was collected using the embedded individually addressable microelectrodes along the wall of 50-um diameter cavities on VEG 4X1™ containing an array of 4 x 1 cavities shown in Figure 1B). ...
Context 4
... rapid, sensitive, low-cost, and reliable microelectrochemical detection of signals from a 96-well plate ( Figure 1A) solid-phase enzyme-linked DNA-hybridization assay for Plasmodium falciparum has been demonstrated. Electrochemical detection of the signals was collected using the embedded individually addressable microelectrodes along the wall of 50-um diameter cavities on VEG 4X1™ containing an array of 4 x 1 cavities shown in Figure 1B). Figure 1A shows a digital photograph of the streptavidin coated 96-well plate that was used in the DNA-hybridization detection of the circumsporozoite protein gene of Plasmodium falciparum. ...
Context 5
... detection of the signals was collected using the embedded individually addressable microelectrodes along the wall of 50-um diameter cavities on VEG 4X1™ containing an array of 4 x 1 cavities shown in Figure 1B). Figure 1A shows a digital photograph of the streptavidin coated 96-well plate that was used in the DNA-hybridization detection of the circumsporozoite protein gene of Plasmodium falciparum. ...
Citations
... By applying highly sensitive and specific immunocytochemical and molecular methods [7,8] it is now possible to detect disseminated tumor cells (DTC) in bone marrow and circulating tumor cells (CTC) in peripheral blood at the single cell level in the background of millions of normal cells. In the peripheral blood, CTC are detectable months to years after complete removal of the primary tumors, indicating that these cells might circulate between different metastatic sites [9]. ...
... Immunoassay detection as well as immunohistochemical methods are needed for rapid and accurate detection at the onset of the disease. Quantum dots as labels of antibodies for fluorescent signal generation have become a viable alternative to enzyme and fluorescent dyes [7,8,[14][15][16][17][18][19][20][21][22][23][24]. In this project, we used two kinds of antibodies. ...
We report the development of early stage detection of breast cancer cells using quantum dots (QDs) as fluorescent signal generator for detection. The QDs based imaging of breast cancer cells involved two kinds of antibodies: the first was for labeling the cells and the second was for imaging the breast cancer cells. Anti-Her2/neu (10Ab) was used for labeling the captured SK-BR3 cells. The second antibody against anti-Her2/neu (20Ab) that was conjugated to QDs (20Ab~QDs) forms the complete assay, SK-BR3 + 10Ab + 20Ab~QDs, to generate the fluorescent cells for imaging. Fluorescent images of the complete assay for SK-BR3 cells were evaluated under a microscope with a UV light source. The preliminary results showed that the breast cancer cell SK-BR3 in the complete assay were successfully observed as fluorescent cells that had brighter signals compared with those labeled with organic dye using similar parameters and the same number of cells.
... Biological sensors such as immunoassay or bioassays for clinical diagnostics have been increasing in number and are used in many areas for immediate, sensitive and accurate detection [13][14][15][16][17][18][19][20][21][22][23]. Different transducers that can be used as a basis for quantitative detection are available. ...
Quantum dots (QDs) have received considerable attention due to their advantages that are suitable for biological studies, especially for multiplexed staining assays and immunological assays. Here we report an application of QDs for the detection of breast cancer cells. Breast cancer is the second most common type of cancer and the fifth most common cause of cancer deaths in the US and the world. The development of QD based sensing for breast cancer cells involved three kinds of antibodies, the first was for capture, the second was for labeling and the third was for imaging the breast cancer cells. The capture antibody, anti-Her2/neu, was covalently immobilized on silanized glass slide which was used to capture the SK-BR3 breast cancer cells. A second antibody, anti-EpCAM was then used for labeling the captured SK-BR3 cells. The third antibody against anti-EpCAM that was conjugated to QDs was used for imaging the captured cells. Images of SK-BR3 cells captured on the glass slide were evaluated under a UV source microscope. The preliminary studies showed that the imaging with QDs-antibody conjugates was successful. The QD breast cancer cell sensing had a brighter signal compared to the organic dye using similar parameters and the same number of cells. Future directions will involve elimination of non-specific signal as well as quantification to establish the limit of detection for the QD-based cell sensing.
... Nanomedicine aspires to develop valuable set of research tools and devices (2,3) as well as possible commercial applications in the pharmaceutical industry that includes targeted drug delivery systems, new therapies, and in vivo imaging (4). Nanoparticle-based diagnostics, neuro-electronic interfaces, and nanoelectronicbased sensors are among the on-going and active areas of research involving nanotechnology (5)(6)(7). In the molecular level, it is speculated that cell repair can be carried out by molecular nanorobots that could revolutionize the medical field. ...
... Furthermore, the small size of nanoparticles (10 to 100 nanometers), allows them to preferentially diffuse and accumulate at tumor sites that lack an effective lymphatic drainage system (14). Highly sensitive and specific immunocytochemical and molecular methods [5,6] makes it possible to detect disseminated tumor cells (DTC) in bone marrow and circulating tumor cells (CTC) in peripheral blood at the single cell level in the presence of millions of normal cells. In the peripheral blood, CTC are detectable months to years after complete removal of the primary tumors, indicating that these cells might circulate between different metastatic sites [7].Nanotechnology is anticipated to lead to advancements in imaging for understanding biological processes at the molecular level (1,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). ...
... The initial studies on the application on quantum dots for breast cancer cell detection showed very promising results. Quantum dots as labels of antibodies for fluorescent signal generation have become a viable alternative to enzyme and fluorescent dyes [5,6,[19][20][21][22][23][24][25]. In this project, we used two kinds of antibody, anti-EpCAM antibody to target the over expressed surface proteins on SK-BR3. ...
Nanotechnology is currently undergoing unprecedented development in various fields. There has been a widespread interest in the application of nanomaterials in medicine with its promise of improving imaging, diagnostics, and therapy. The recent advances in engineering and technology have led to the development of new nanoscale platforms such as quantum dots, gold nanocrystals, superparamagnetic nanocrystals, and other semiconductor nanoparticles. Literature on the applications of quantum dots in life sciences has recently increased in number. This may have led to predictions that nanotechnology in life sciences research will contribute $3.4 billion by 2010 while institutions have predicted that the market for nanotechnology and corresponding products will reach $1 trillion in 2012 (1).
Ocean NanoTech is at the height of developmental stages of nanoparticle production for biological applications. Ocean’s high quantum-yield quantum dots (QDs) is currently being tested and used for cell imaging, as wells as for the detection of proteins, DNA, whole cells, and whole organisms. Imaging of cells involves conjugation of QDs to highly sensitive and specific antibody to form QD˜Ab conjugates that attach to specific protein target on the cell surface. Attachment of the QD˜Ab on the cell surface allows imaging of the cell under a fluorescence microscope. QD based imaging can be used in a multiplex immunoassay detection of several types of cells (or microorganisms) in a single sample when several size tunable quantum dots are used as reporter probes.
We report the QD imaging of breast cancer cells. Using the breast cancer cell line SK-BR3, which expresses high levels of her2 antigens on the cell surface, anti-her2 were conjugated to Ocean’s quantum dots, QSH620. To eliminate non-specific binding of the QD˜20Ab Ocean’s super blocking buffer BBB and BBG were used. Preliminary results of in vitro studies indicated that QD based systems can be used to image cells. We anticipate that this system can be transferred to in vivo detection.
La mitochondrie est restée longtemps cantonnée au rôle de centrale énergétique cellulaire. On sait désormais qu'elle est aussi la principale source d'espèces réactives oxygénées, impliquées dans le stress oxydant et la signalisation inter- cellulaire. Le dérèglement de l'activité mitochondriale est ainsi susceptible d'être la cause de l'apparition et de la progression de maladies associées au vieillissement, comme le cancer et les maladies neurodégénératives. Dans le cadre de la leucémie aiguë myéloïde, des études menées par l'équipe dirigée par Jean-Emmanuel Sarry du Centre de Recherche en Cancérologie de Toulouse, ont montré qu'il est possible de sensibiliser les cellules jusqu'alors résistantes à la chimiothérapie, en ciblant préalablement la fonction mitochondriale. C'est dans ce contexte que s'inscrit le sujet de cette de thèse, portant sur la conception et la réalisation de micro-capteurs électrochimiques dédiées à l'analyse du métabolisme mitochondrial, à l'échelle de la mitochondrie isolée. La fabrication des microsystèmes s'est déroulée dans la salle blanche du Laboratoire d'Analyse et d'Architecture des Systèmes de Toulouse, sous l'encadrement des chercheurs Jérôme Launay et Pierre Temple-Boyer, spécialisés dans la conception de capteurs pour la détection d'espèces en phase liquide. Finalement, un système complet assurant le couplage à la microscopie et à la gestion des fluides a été fabriqué, validé, et breveté. Les résultats obtenus nous permettent d'envisager l'analyse à l'échelle de la mitochondrie unique par une approche parallélisée, ce qui n'a encore jamais été réalisé.
The breast cancer is the most common cause of cancer death in women. To establish an early stage in situ imaging of breast cancer cells, green quantum dots (QDs) are used as a fluorescent signal generator. The QDs based imaging of breast cancer cells involves anti-HER2/neu antibody for labeling the over expressed HER2 on the surface of breast cancer cells. The complete assay involves breast cancer cells, biotin labeled antibody and streptavidin conjugated QDs. The breast cancer cells are grown in culture plates and exposed to the biotin labeled antibodies, and then exposed to streptavidin labeled QDs to utilize the strong and stable biotin-streptavidin interaction. Fluorescent images of the complete assay for breast cancer cells are evaluated on a microscope with a UV light source. Results show that the breast cancer cells in the complete assay are used as fluorescent cells with brighter signals compared with those labeled by the organic dye using similar parameters and the same number of cells.
A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection.
An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin-magnetic nanoparticles and used for targeting the HBsAg. By addition of horseradish peroxidase conjugated with secondary antibody (HRP-HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed in to 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred in to an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear towards HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at signal/noise ratio of 3.
This title covers recent advances in a variety of biomedical applications of nanostructured materials, as the field evolves from prototype device to real-world application. It presents the main types of nanomaterial used in medical application today: semiconductor nanomaterials, Magnetic nanomaterials, metal nanoparticles, Carbon nanomaterials, Hydrogel nanocomposites, Liposomes, Dendrimers, Polymer nanocomposites, and Biodegradable polymers. Structurally the work is demarcated into the six most popular areas of research: (1) biocompatibility of nanomaterials with living organisms in their various manifestations (2) nanobiosensors for clinical diagnostics, detecting biomolecules which are useful in the clinical diagnosis of genetic, metabolically acquired, induced or infectious disease (3) targeted drug delivery for nanomaterials in their various modifications (4) nanomedical devices and structures which are used in the development of implantable medical devices and structures such as nanorobots (5) nanopharmacology, as novel nanoparticles are increasingly engineered to diagnose conditions and recognize pathogens, identify ideal pharmaceutical agents to treat the condition or pathogens,fuel high-yield production of matched pharmaceuticals (potentially in vivo), locate, attach or enter target tissue, structures or pathogens; and dispense the ideal mass of matched biological compound to the target regions (6) nanotoxicology and remediation, which focuses on finished and on-going various toxicity evaluations on various nanomaterials that are used and currently being developed for medical applications
Discusses the most important biomedical applications and devices of nanomaterials: drug delivery, medical imaging, gene therapy, nanorobots, biosensors and diagnostics
Focuses on current commercialized techniques and applications, ensuring the work is entirely relevant to a rapidly evolving field
Reviews the most recent studies on nanomaterial toxicity, thereby responding to the widescale private, policy and public interest in nanoscience
