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Biomedical subjects

Weihong Tan

Publications and source records attributed to Weihong Tan.

At least 19 recordsLinked to original sources
PubMed2006

Atomic force microscopy study of different effects of natural and semisynthetic beta-lactam on the cell envelope of Escherichia coli.

The effects of native and semisynthetic beta-lactam on the surface of E. coli cells were investigated using tapping mode atomic force microscopy (AFM). These studies were done on both single cell and cell colonies. This allowed us to achieve a complete understanding of the effects, since previous related AFM analysis was only performed on single cells. Our results indicated that although amoxycillin and penicillin could both induce nanoporous damage to the envelope of E. coli, the distribution of the pores was different: those induced by penicillin were randomly distributed on the cell surface, while those induced by amoxycillin were far more numerous and mainly on the two ends of the cell. These findings could explain why the effect of amoxycillin is stronger than that of native penicillin. Ofloxacin was used as a control due to its inactive membrane, and no cell wall damages were observed. Our results demonstrate that AFM is a useful tool in discerning and verifying antibiotic mechanisms and can be helpful to explain the relationship between chemical structure and the function of antibiotics.

Anti-Bacterial Agents↗
PubMed2006

Quantitative intracellular molecular profiling using a one-dimensional flow system.

We report on the development of one-dimensional microfluidic bead arrays for rapid and quantitative molecular profiling of human cancer cells. This new bioanalytical platform integrates the rapid binding kinetics of suspension bead carriers, the multiplexing and encoding capabilities of gene/protein chips, and the liquid handling advantages of microfluidic devices. Using antibody-conjugated beads in a two-site "sandwich" format, we demonstrate that the proteomic contents of as few as 56 human lung epithelial cancer cells can be determined with high sensitivity and specificity. The results indicate that each cell contains approximately 6 x 10(5) copies of the tumor suppressor protein P53. We have further examined the expression changes of P53, c-Myc, and beta-Actin as a function of anticancer drug treatment and have validated these changes by using Western blotting. This ability to quantitatively analyze normal and diseased cells raises new possibilities in studying cancer heterogeneity and circulating tumor cells.

Animals↗
PubMed2006

Hybrid molecular probe for nucleic acid analysis in biological samples.

The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.

Animals↗
PubMed2006

Aptamers evolved from live cells as effective molecular probes for cancer study.

Using cell-based aptamer selection, we have developed a strategy to use the differences at the molecular level between any two types of cells for the identification of molecular signatures on the surface of targeted cells. A group of aptamers have been generated for the specific recognition of leukemia cells. The selected aptamers can bind to target cells with an equilibrium dissociation constant (K(d)) in the nanomolar-to-picomolar range. The cell-based selection process is simple, fast, straightforward, and reproducible, and, most importantly, can be done without prior knowledge of target molecules. The selected aptamers can specifically recognize target leukemia cells mixed with normal human bone marrow aspirates and can also identify cancer cells closely related to the target cell line in real clinical specimens. The cell-based aptamer selection holds a great promise in developing specific molecular probes for cancer diagnosis and cancer biomarker discovery.

Binding Sites↗
PubMed2006

Selection of DNA ligands for protein kinase C-delta.

Protein kinase Cs are a family of serine and threonine kinases that mediate a wide variety of cellular signalling processes such as cell growth, differentiation, apoptosis and tumor development. We have selected high-affinity DNA aptamers for PKCdelta by capillary electrophoresis based SELEX (systematic evolution of ligands by exponential enrichment, CE-SELEX). We have demonstrated that fluorescently tagged PB9 aptamer can specifically recognize PKCdelta under in vitro conditions. The Kd of the aptamer-protein binding is 122 nM. These aptamers will enable us to apply fluorescently labelled probes to study the spatiotemporal dynamics and activation of individual endogenous PKC isoforms during various cell signalling processes.

Aptamers, Nucleotide↗
PubMed2006

Electrical switching of DNA monolayers investigated by surface plasmon resonance.

The switching of DNA monolayers between a "lying" and a "standing" state initiated by applying electric field, and the subsequent DNA hybridization at different states were investigated in a contactless, label-free mode by surface plasmon resonance (SPR) technique. The results showed that the strength of the electric field and surface coverage could influence the switching of DNA monolayers. In addition, it was found that DNA hybridization efficiency could be enhanced or decreased when DNA probes stood straight up or lay flat on the gold surface, depending on the potential of the gold substrate. The enhancement of DNA hybridization efficiency reached the maximum when surface coverage reached 5.87 x 10(12) molecules/cm(2) and the potential of gold substrate was more negative than -0.7 V (versus ITO-coated glass). The research may be helpful for the construction of sensitive biosensors, biochips, and nanoscale electronic devices.

Base Sequence↗
PubMed2006

Enhanced surface plasmon resonance with the modified catalytic growth of Au nanoparticles.

The catalytic growth of Au nanoparticles (AuNPs) has been employed in several analytical methods for improving the detection sensitivity, or integrated with the enzyme reactions for the quantitative detection of the respective substrates. However, the catalytic growth of Au nanoparticles do not work in some situations, such as surface plasmon resonance (SPR), electrochemistry, where metal matrices were used, because metal matrices used in these techniques, e.g. Au, are susceptible to metal deposition, which increased the background seriously. In this work, a SiO(2) layer was vapor-deposited on the gold film. The inhibition of metal deposition by this SiO(2) layer was investigated by SPR sensor. The results showed that the SiO(2) layer could avoid the deposition of metal on Au film. With the low background achieved by SiO(2)-coated Au films, sensitive detection of DNA hybridization using the catalytic growth of Au nanoparticles enhanced SPR was demonstrated. The work described here maybe helpful for the development of sensitive bioanalytical methods.

Catalysis↗
PubMed2006

IL-17 receptor knockout mice have enhanced myelotoxicity and impaired hemopoietic recovery following gamma irradiation.

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline, the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is, with the exception of increased splenic progenitor numbers, indistinguishable from normal control mice. However, when challenged with gamma irradiation, hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals, with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells, gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad), the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL-17Ra in normal mice, resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression, in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte, CFU-high proliferative potential), as well as 2- and 3-fold increases of bone marrow precursors, respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury.

Animals↗
PubMed2006

Immunofluorescent labeling of cancer cells with quantum dots synthesized in aqueous solution.

Thioglycolic-acid-stabilized CdTe quantum dots, synthesized directly in aqueous solution, are successfully conjugated with biotin and polyethylene glycol. Using these conjugates, we report the development of this kind of water-soluble quantum dot for immunofluorescent labeling of cancer cells. The results show that these conjugates have very low nonspecific binding and good stability against photobleaching, enabling them to be applied in many biological fields, such as cellular labeling, intracellular tracking, and other imaging applications.

Cell Line, Tumor↗
PubMed2006

FloDots: luminescent nanoparticles.

Luminescent dye-doped silica nanoparticles (FloDots) have been developed for ultrasensitive bioanalysis and diagnosis in the past several years. Those novel nanoparticles are highly luminescent and extremely photostable. In this paper, we review the preparation, characterization, bioconjugation and bioapplication of FloDots. All the results clearly demonstrated that FloDots have many advantages over currently used luminescent probes, such as traditional fluorophores and quantum dots.

Humans↗
PubMed2006

Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells.

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.

Aptamers, Nucleotide↗
PubMed2006

Surface modification of silica nanoparticles to reduce aggregation and nonspecific binding.

In this article, a systematic study of the design and development of surface-modification schemes for silica nanoparticles is presented. The nanoparticle surface design involves an optimum balance of the use of inert and active surface functional groups to achieve minimal nanoparticle aggregation and reduce nanoparticle nonspecific binding. Silica nanoparticles were prepared in a water-in-oil microemulsion and subsequently surface modified via cohydrolysis with tetraethyl orthosilicate (TEOS) and various organosilane reagents. Nanoparticles with different functional groups, including carboxylate, amine, amine/phosphonate, poly(ethylene glycol), octadecyl, and carboxylate/octadecyl groups, were produced. Aggregation studies using SEM, dynamic light scattering, and zeta potential analysis indicate that severe aggregation among amine-modified silica nanoparticles can be reduced by adding inert functional groups, such as methyl phosphonate, to the surface. To determine the effect of various surface-modification schemes on nanoparticle nonspecific binding, the interaction between functionalized silica nanoparticles and a DNA chip was also studied using confocal imaging/fluorescence microscopy. Dye-doped silica nanoparticles functionalized with octadecyl and carboxylate groups showed minimal nonspecific binding. Using these surface-modification schemes, fluorescent dye-doped silica nanoparticles can be more readily conjugated with biomolecules and used as highly fluorescent, sensitive, and reproducible labels in bioanalytical applications.

Coated Materials, Biocompatible↗
PubMed2006

Nuclease resistance of telomere-like oligonucleotides monitored in live cells by fluorescence anisotropy imaging.

Telomeres carry important biological functions such as the protection of chromosomes. In this paper, we have developed a fluorescence anisotropy imaging system for monitoring DNA digestion inside live cells. The nuclease-resistant capability of telomere-like ssDNAs in nuclei of human breast cancer cells is studied. We found that those oligonucleotides were clearly more stable than regular DNA sequences during the time course of the experiments. We conclude that the G-quadruplex structure of the telomere-like ssDNA makes it inherently more stable in intracellular environments than non-G-quadruplex structures. This will help us understand why the G-quadruplex forming telomere sequences were adopted by almost all eukaryotic cells to protect the ends of chromosomes. This is the first time such a phenomenon was observed in live cells. Our fluorescence anisotropy imaging provides an efficient way to directly monitor DNA digestion in any region of live cells in real time, providing insights into many important and related intracellular processes.

Breast Neoplasms↗
PubMed2006

Multicolor FRET silica nanoparticles by single wavelength excitation.

Fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stöber synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size, and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift, which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems.

Journal Article↗
PubMed2006

Using molecular beacons for sensitive fluorescence assays of the enzymatic cleavage of nucleic acids.

A novel method for DNA enzymatic cleavage assays using molecular beacons (MBs) as the substrate for nuclease is described. An MB is a hairpin-shaped DNA probe that is labeled with a fluorescent dye at one end and a quencher at the other end. The loop sequence of the MB can be used as the substrate for single-stranded specific nucleases, whereas the stem of the MB can be designed as the substrate for restriction enzymes. The enzymatic cleavage breaks the MB into fragments and leads to the distance separation of the quencher and the fluorophore, resulting in an increase in the fluorescent signal. Up to an 80-fold signal-to-noise ratio was observed when these probes were cleaved by nucleases. Taking advantage of the MB's detection-without-separation property, this method allows for the real-time detection of DNA cleavage, which is useful for the characterization of DNA nuclease activity as well as the study of steady-state cleavage reaction kinetics. With its simplicity, convenience, high sensitivity, and excellent reproducibility, this method has the potential to be used in the study of both natural and artificial nucleic acid-cleaving enzymes.

DNA↗
PubMed2005

Light-switching excimer probes for rapid protein monitoring in complex biological fluids.

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.

Body Fluids↗
PubMed2005

Locked nucleic acid molecular beacons.

A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.

Fluorescence↗