Min-Ah Woo

A label-free method for detecting biological thiols based on blocking of Hg2+-quenching of fluorescent gold nanoclusters

A novel, label-free, fluorescent, turn-on sensor for biological thiol detection that uses highly fluorescent gold nanoclusters (AuNCs), prepared by a bovine serum albumin (BSA)-templated green synthetic route, has been developed. The assay relies on blocking Hg(2+)-induced quenching of the fluorescence of AuNCs, caused by metallophilic Hg(2+)-Au(+) interactions, through selective coordination of biological thiols with Hg(2+) ions. Biological thiols entrap added Hg(2+) ions via a robust Hg-S interaction. This phenomenon prevents Hg(2+)-induced quenching and results in fluorescence from AuNCs. By employing this turn-on sensor, biological thiols, such as cysteine (Cys), glutathione (GSH) and homocysteine (Hcy), are successfully detected at concentrations as low as 8.3 nM for Cys, 9.4 nM for GSH, and 14.9 nM for Hcy. The diagnostic capability and potential in practical applications of this method have been demonstrated by detecting biological thiols in human blood serum.

HER2/neu Antibody Conjugated Poly(amino acid)-Coated Iron Oxide Nanoparticles for Breast Cancer MR Imaging

Superparamagnetic iron oxide nanoparticles are widely used as nanoprobes for magnetic resonance imaging (MRI). Water-soluble iron oxide nanoparticles were synthesized by coating iron oxide nanoparticles with a hydrophilic, biocompatible, biodegradable poly(amino acid) derivative, poly(2-hydroxyethyl aspartamide) graft copolymer for negative contrast enhancement on T2 weighted MRI. HER2/neu antibodies were conjugated on the surface of poly(amino acid) coated iron oxide nanoparticles for the detection of breast cancer. The antibody-grafted iron oxide nanoparticles (PAION-Ab) were about 31.1 nm in diameter. The T2 relaxivity of PAION-Ab was 246 L·mmol(-1)·sec(-1) greater than that of the commercial product such as Feridex. PAION-Ab showed low cytotoxicity even at relatively high concentrations. Furthermore, Prussian blue staining and in vitro MRI study with SKBR-3, breast cancer cells overexpressing HER2/neu receptors indicated that PAION-Ab exhibited excellent cancer cell detection ability and enhanced signal intensities in the T2-weighted image.

Colorimetric quantification of galactose using a nanostructured multi-catalyst system entrapping galactose oxidase and magnetic nanoparticles as peroxidase mimetics

A colorimetric method for quantification of galactose, which utilizes a nanostructured multi-catalyst system consisting of Fe(3)O(4) magnetic nanoparticles (MNPs) and galactose oxidase (Gal Ox) simultaneously entrapped in large pore sized mesocellular silica, is described. Gal Ox, immobilized in a silica matrix, promotes reaction of galactose to generate H(2)O(2) that subsequently activates MNPs in silica mesopores to convert a colorimetric substrate into a colored product. By using this colorimetric method, galactose can be specifically detected. Along with excellent reusability via application of simple magnetic capturing, enhanced operational stability was achieved by employing a cross-linked enzyme aggregate (CLEA) method for Gal Ox immobilization. This protocol leads to effective prevention of enzyme leaching from the pores of mesocellular silica. The analytical utility of the new colorimetric biosensor was demonstrated by its use in diagnosing galactosemia, a genetic metabolic disorder characterized by the inability to utilize galactose, through analysis of clinical dried blood spot specimens. A microscale well-plate format was employed that possesses a multiplexing capability. The multi-catalyst system entrapping Gal Ox and MNPs represents a new approach for rapid, convenient, and cost-effective quantification of galactose in human blood and it holds promise as an alternative method for galactosemia diagnosis, replacing the laborious procedures that are currently in use.

A Highly Efficient Colorimetric Immunoassay Using a Nanocomposite Entrapping Magnetic and Platinum Nanoparticles in Ordered Mesoporous Carbon

Nanocomposite to achieve ultrafast immunoassay: a new synergistically integrated nanocomposite consisting of magnetic and platinum nanoparticles, simultaneously entrapped in mesoporous carbon, is developed as a promising enzyme mimetic candidate to achieve ultrafast colorimetric immunoassays. Using new assay system, clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, can be detected in only 3 min at room temperature with high specificity and sensitivity.

A Novel Colorimetric Immunoassay Utilizing the Peroxidase Mimicking Activity of Magnetic Nanoparticles

A simple colorimetric immunoassay system, based on the peroxidase mimicking activity of Fe3O4 magnetic nanoparticles (MNPs), has been developed to detect clinically important antigenic molecules. MNPs with ca. 10 nm in diameter were synthesized and conjugated with specific antibodies against target molecules, such as rotaviruses and breast cancer cells. Conjugation of the MNPs with antibodies (MNP-Abs) enabled specific recognition of the corresponding target antigenic molecules through the generation of color signals arising from the colorimetric reaction between the selected peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2. Based on the MNP-promoted colorimetric reaction, the target molecules were detected and quantified by measuring absorbance intensities corresponding to the oxidized form of TMB. Owing to the higher stabilities and economic feasibilities of MNPs as compared to horseradish peroxidase (HRP), the new colorimetric system employing MNP-Abs has the potential of serving as a potent immunoassay that should substitute for conventional HRP-based immunoassays. The strategy employed to develop the new methodology has the potential of being extended to the construction of simple diagnostic systems for a variety of biomolecules related to human cancers and infectious diseases, particularly in the realm of point-of-care applications.

Aptamer-based cell imaging reagents capable of fluorescence switching

We describe an aptamer-conjugated polydiacetylene imaging probe (ACP) that shows highly specific fluorescence switching upon binding to epithelial cancer cells that overexpress the tumor biomarker protein EpCAM (epithelial cell adhesion molecule) on their surface.

Cell-Based Quantification of Homocysteine Utilizing Bioluminescent Escherichia coli Auxotrophs

A cell-based quantitative assay system for Hcy has been developed by utilizing two Escherichia coli auxotrophs that grow in the presence of methionine (Met) and either homocysteine (Hcy) or Met, respectively. A bioluminescent reporter gene, which produces luminescence as cells grow, was inserted into the auxotrophs, so that cell growth can be readily determined. When the relative luminescence unit (RLU) values from the two auxotrophs immobilized within agarose gels arrayed on a well plate were measured, the amount of Hcy was quantitatively determined on the basis of differences between two RLU values corresponding to cell growth of two auxotrophs with excellent levels of precision and reproducibility. Finally, the diagnostic utility of this assay system was verified by its employment in reliably determining different stages of hyperhomocysteinemia in human plasma samples providing CVs of within and between assays that are less than 2.9% and 7.1%, respectively, and recovery rates of within and between assays that are in the range of 99.1-103.5% and 97.5-105.5%, respectively. In contrast to existing conventional methods, the new system developed in this effort is simple, rapid, and cost-effective. As a result, it has great potential to serve as a viable alternative for Hcy quantification in the diagnosis of hyperhomocysteinemia.

Cell-based galactosemia diagnosis system based on a galactose assay using a bioluminescent Escherichia coli array.

A new cell-based galactose assay system, which is comprised of two bioluminescent Escherichia coli strains immobilized within an agarose gel arrayed on a well plate, has been developed. For this purpose, a galT knockout strain [galT(-) cell] of E. coli was genetically constructed so that cell growth is not promoted by galactose but rather by glucose present in a sample. Another E. coli W strain (normal cell), which grows normally in the presence of either glucose or galactose, was employed. A luminescent reporter gene, which produces luminescence as cells grow, was inserted into both of the E. coli strains, so that cell growth could be monitored in a facile manner. The two strains were separately grown for 4 h on gel arrays to which test samples were individually supplied. The relative luminescence unit (RLU) values caused by cell growth were determined for each array, one of which is resulted by glucose only and the other of which is resulted by both glucose and galactose present in the sample. By employing this protocol, galactose concentrations present in the test sample are reflected in the differences between the RLU values for each array. The practical utility of the new assay system was demonstrated by its use in determining galactose levels in clinical blood spot specimens coming from newborn babies. Because it can be employed to diagnosis of galactosemia in newborn babies in a more rapid, convenient, and cost-effective manner, this cell-based solid-phase galactose assay system should become a powerful alternative to conventional methods, which require labor-intensive and time-consuming procedures and/or complicated and expensive equipment.

Cell-based method utilizing fluorescent Escherichia coli auxotrophs for quantification of multiple amino acids.

A cell-based assay system for simultaneous quantification of the three amino acids, phenylalanine (Phe), methionine (Met), and leucine (Leu) in a single biological sample, was developed and applied in the multiplex diagnosis of three key metabolic diseases of newborn babies. The assay utilizes three Escherichia coli auxotrophs, which grow only in the presence of the corresponding target amino acids and which contain three different fluorescent reporter plasmids that produce distinguishable fluorescence signals (red, green, and cyan) in concert with cell growth. To mixtures of the three auxotrophs, immobilized on agarose gels arrayed on a well plate, is added a test sample. Following incubation, the concentrations of the three amino acids in the sample are simultaneously determined by measuring the intensities of three fluorescence signals that correspond to the reporter plasmids. The clinical utility of this assay system was demonstrated by employing it to identify metabolic diseases of newborn babies through the quantification of Phe, Met, and Leu in clinically derived dried blood spot specimens. The general strategy developed in this effort should be applicable to the design of new assay systems for the quantification of multiple amino acids derived from complex biological samples and, as such, to expand the utilization of cell-based analytical systems that replace conventional, yet laborious methods currently in use.