Cell-SELEX-based selection of aptamers that recognize distinct targets on metastatic colorectal cancer cells.

The development of diagnostic/therapeutic strategies against metastasis-related molecular targets is critical for improving the survival rate of cancer patients. Subtractive Cell-SELEX was performed using highly metastatic colorectal cancer (CRC) LoVo cells and non-metastatic HCT-8 cells as the target and negative cells, respectively, for the selection of metastatic-specific aptamers. This process generated seven aptamers that displayed highly specific binding to the target cells with Kds in the nanomolar range. Based on the distinct chemical/biological properties of their individual cell surface targets, the aptamers were separately functionalized: the receptor-targeting aptamer W14 was used as a carrier for doxorubicin, resulting in the specific delivery of the drug to the target cells and a significant reduction of its cytotoxicity to non-target cells, and the non-receptor-binding aptamer W3 was used as a molecular probe conjugated to quantum dots for the targeted imaging of metastatic cancer cell lines, spontaneous lung metastasis murine tissue, and metastatic CRC patient tissues. In addition, these aptamers can be used in combination due to their lack of detectable mutual-binding interference. The study demonstrates that a panel of aptamers that recognize distinct features of target molecules can be obtained through single Cell-SELEX selection, and the selected aptamers may be individually functionalized for specific applications and/or utilized in combination.

Mercury(II)-mediated formation of imide-Hg-imide complexes.

Three different kinds of cyclic imides with imido protons are chosen to interact with Hg(II): succinimide, the simplest cyclic imide (five-membered ring); phthalimide (conjugated five-membered ring); and 1,8-naphthalimide (conjugated six-membered ring). Based on the results of MS, (1)H-NMR, XPS, IR spectroscopy and fluorescence response analyses, it is suggested that N-unsubstituted cyclic imides react specifically with Hg(II) and form imide-Hg-imide complexes through an imido proton-metal exchange process. The reaction is reversible and occurs rapidly at moderate to high pH. This discovery expands the comprehension of the specific interaction of Hg(II) with the nucleobase thymine, and may open up new possibilities in designing novel ligands for sensing and removing Hg(II) based on cyclic imides.

[Level of glutamate on the auditory pathway of inferior colliculus after exposure to noise observed by microdialysis in vivo].

OBJECTIVE: To explore the quality and quantity of glutamate(Glu) on the auditory pathway of inferior colliculus when exposed to conditioning noise or traumatic sound to find the relationship between the change of neurotransmitters and the protection of hearing, and the mechanism in the phenomena. METHODS: Twenty Sprague-Dawley rats were divided into four groups: control group, conditioning group, intensive noise group and combining group. The hearing function was measured by ABR. Glu was detected by microdialysis and HPLC. RESULTS: The ABR levels after exposure were (47.0+/-2.7) dB SPL (intensive noise group ),(31.0+/-2.2) dB SPL (conditioning group ) and (36.0+/-2.2) dB SPL (combining group ).The levels of Glu were (310.5+/-78.5)x 10(-7)mol/L (intense noise group ),(162.9+/-64.1)x10(-7)mol/L (conditioning group ); (113.6+/-38.1)x10(-7)mol/L (combining group),(56.3+/-23.9)x10(-7)mol/L (control group). CONCLUSION: The level of Glu on the auditory pathway of inferior colliculus was higher when exposed to noise . Glu was exhausted when given conditioning sound, and the level of Glu was lower when given following intense noise. High level of Glu can damage hearing, so exhaustion of Glu in sound condition may contribute to the protection.

QCM-FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III.

In this work, we describe a method of constructing a film of linear poly(glycidyl methacrylate) (PGMA) polymer onto the surface of quartz crystal microbalance (QCM) electrode as a coating material that allows easy coupling of heparin molecules onto the electrode and facilitates the determination of the interaction between heparin and antithrombin III (AT III). The PGMA film was characterized with atomic force microscopy (AFM) and infra-red spectroscopy. The coupling of heparin was accomplished in one step solution reaction. A home-made quartz crystal microbalance-flow injection analysis (QCM-FIA) system with data analysis software developed in our laboratory was used to determine the interaction. The interactions between immobilized heparin and AT III were studied with various concentrations under various conditions. The obtained constants are kass=(1.49+/-0.12)x10(3)mol-1ls-1, kdiss=(3.94+/-0.63)x10(-2)s-1, KA=(3.82+/-0.33)x10(4)mol-1l.

Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents.

A quartz crystal microbalance (QCM) biosensor integrated into a flow injection analysis (FIA) system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Two sulfa-drugs, sulfamethazine (SMZ) and sulfamethoxazole (SMO), were, respectively, immobilized on the gold electrodes of the piezoelectric crystals using appropriate procedures based on self-assembly of the dithiothreitol (DTT). The binding interactions of the two immobilized drug ligands, with various proteins in solution, were followed as changes in the resonant frequency of the modified crystals. Results obtained from this rapid screen analysis clearly indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar. SMZ-immobilized sensor showed specific interaction only with IgG, while SMO-immobilized sensor showed negligible specific binding with IgG, but binding with trypsin and chymotrypsin. Further studies on the specific interaction between immobilized SMZ and three different species of IgG--human IgG, goat IgG and mouse IgG were carried out and the marked species-dependent difference was observed. The resultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive both the kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) for IgG-SMZ interactions. For the interactions, KA were 5.48 x 10(5), 2.75 x 10(5) and 1.86 x 10(5) M(-1) for human IgG, goat IgG and mouse IgG, respectively. The kinetic data provided further insight into the structural/functional relationships of different IgG on a molecular level.

Real time kinetic analysis of the interaction between immunoglobulin G and histidine using quartz crystal microbalance biosensor in solution.

Quartz crystal microbalance (QCM) biosensor integrated in a flow injection analysis (FIA) system was used for the investigation of the specific interaction between immunoglobin G (IgG) and histidine. The histidine was immobilized on the gold electrodes of the piezoelectric crystal using appropriate procedures based on self-assembling of the dithiothreitol (DTT). The specific interaction of the immobilized ligand with IgG in solution was followed as a change in the resonant frequency of the modified crystal and studied in real time without any additional labels. With the mass sensitive biosensor system, the differences in affinity of three different species of IgG: human IgG, goat IgG and mouse IgG were easily distinguished and their respective kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) were determined from the curves of frequency versus time. For the interactions, KA were 2.92 x 10(4), 3.23 x 10(4) and 4.08 x 10(4) M(-1) for human IgG, goat IgG and mouse IgG, respectively.