Animals ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Fluorescence Resonance Energy Transfer ; Fluorometry ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry

OBJECTIVESTo summarize neonatal screening for phenylketonuria (PKU) and congenital hypothyroidism (CH) in China, to further clarify incidence of the two kinds of diseases in newly-born babies, and to explore issues in neonatal screening and their solutions.

METHODSNeonatal screening for PKU and CH was conducted by 39 neonatal screening centers all over the country, sponsored by the Group of Neonatal Screening, Chinese Society of Child Health Care, Chinese Preventive Medical Association and the Center for Neonatal Screening Quality Control Laboratory, National Center for Clinical Laboratories (NCCL). In each infant a heel prick blood sample was collected at 72 hours postnatal onto standard filter paper. PKU was screened by bacterial inhibition assay and fluorometric method, and CH was screened by TSH measurement by time-resolved fluorescence immunoassay (TRFIA), fluorescence enzyme immunoassay (FEIA) and enzyme immunoassay (EIA).

RESULTSFrom 1985 to 2001 in China, totally of 5 817 280 newborns were screened for PKU, 522 cases of PKU detected with an incidence of 1:11 144, and 5 524 019 newborns were screened for CH, 1 836 cases of CH detected with an incidence of 1:3 009. Annual average number of newborns screened for congenital genetic diseases was increased by 45.5% in recent six years.

CONCLUSIONSNeonatal screening was developed quickly in China in recent years, especially in some developed cities, such as Shanghai with a coverage of 98.3% in 2001. But, its coverage was about only 10% in China as a whole. In development of neonatal screening, it is necessary to attach more importance to quality of screening and increasing coverage of screening, as well as gradual development of new screening techniques for other neonatal preventable diseases, in addition to PKU and CH, and their application, and improvement of level of child health care in China.

China ; epidemiology ; Congenital Hypothyroidism ; Fluorescence Polarization Immunoassay ; Fluorometry ; Humans ; Hypothyroidism ; epidemiology ; Immunoenzyme Techniques ; Infant, Newborn ; Neonatal Screening ; Phenylketonurias ; epidemiology ; Thyrotropin ; blood

No abstract available.
Cyclosporine* ; Fluorescence Polarization Immunoassay* ; Fluorescence Polarization* ; Fluorescence*

OBJECTIVETo establish a highly sensitive fluorometric nanobiosensor for determination of aqueous mercury ions (Hg(2+)) using optimized mercury-specific oligonucleotide (MSO) probes and graphene oxide (GO).

METHODSThe nanobiosensor was assembled by attaching the self-designed MSO(1) (5' end labeled with fluorophore carboxyfluorescein (FAM), denoted as FAM-MSO(1)) and MSO(2) to the surface of GO through strong non-covalent bonding forces. Upon the addition of Hg(2+), the formation of the T-Hg(2+)-T configuration desorbed the FAM-MSO(1) and MSO(2) from the surface of GO, resulting in a restoration of the fluorescence of FAM-MSO(1). Using the specific mispairing of T-Hg(2+)-T and the changes in fluorescent signals in solutions, quantitative analysis of Hg(2+) could be performed.

RESULTSThe average thickness of the prepared GO sheets was only 1.4 nm. For the Hg(2+) nanobiosensor, the optimum concentrations of FAM-MSO(1) and MSO(2) were both 1 µmol/L, the optimum volume of 0.5 g/L GO was 5 µL, and the limit of detection was 10 pmol/L; it had low cross-reactivity with 10 other kinds of non-specific metal ions; the fluorescence recovery efficiency was up to 65% in the re-determination of Hg(2+) after addition of Na(2)S(2)O(3).

CONCLUSIONThe MSO/GO-based nanobiosensor is convenient to operate, highly sensitive, highly specific, highly accurate, and reusable. It can be applied to determine trace amount of Hg(2+) in aqueous solutions.

Biosensing Techniques ; Fluorometry ; Graphite ; Mercury ; analysis ; Nanotechnology ; Oligonucleotide Probes ; Water

PURPOSE: This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. MATERIALS AND METHODS: Biopanning of a phage-displayed peptide library was performed on KIM-1–coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1–overexpressing and KIM-1–low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1–overexpressing and KIM1–low expressing tumors in mice was examined by whole-body fluorescence imaging. RESULTS: A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1–overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1–overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1–low expressing HEK293 normal cells. Co-localization and competition assays using an anti–KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1–overexpressing A498 renal tumor compared to KIM1–low expressing HepG2 liver tumor in mice. CONCLUSION: The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.
Animals ; Bacteriophages ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Fluorometry ; Kidney Neoplasms ; Kidney ; Liver ; Lung ; Mass Screening ; Mice ; Optical Imaging ; Peptide Library ; Peptides

A fluorescence polarization immunoassay (FPIA) was developed for the analysis ofaflatoxins (AFs) using an anti-aflatoxin B1 (AFB1) monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 min. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.
Aflatoxins ; analysis ; Fluorescence Polarization Immunoassay

BACKGOUND: Renal tubular epithelial cells are primary target for hypoxic injury. Hypoxia induced tubular cell apoptosis has been reported previously and thought to be important mechanism of renal dysfunction in ischemic ARF, but precise signaling mechanisms need to be defined. The aim of this study is to clarify intracellular signaling mechanism mediating apoptosis by hypoxic stimuli in cultured tubular cells. METHODS: HK-2 cells were placed in hypoxic chamber (O2<1%) for 24 hrs in minimal essential medium. DNA fragmentation was detected by Hoechst 33342 stain and FACS. The activation of caspase was measured by fluorometry and activations of p-38, ERK, and JNK were examined by western blot analysis. RESULTS: Hypoxia induced caspase 3 activation and apoptosis at 24 hrs and this was accompanied by increased phosphorylation of p-38, ERK1/2, and JNK. Pretreatment of p-38 inhibitor (SB 203280) and JNK inhibitor (SP600125) did not afftect the activation of caspase 3 and apoptosis but inhibition of ERK1/2 by PD98059 resulted in partial inhibition of caspase 3 and apoptosis induced by hypoxia. CONCLUSION: ERK 1/2 activation can be an upstream signal in hypoxia induced caspase 3 activation and apoptosis in tubular cells.
Anoxia* ; Apoptosis* ; Blotting, Western ; Caspase 3 ; DNA Fragmentation ; Epithelial Cells ; Fluorometry ; Negotiating ; Phosphorylation

BACKGROUND/AIMS: Glucosamine is widely taken as a functional food, and some studies reported its anti-inflammatory effects. K+ channels and intracellular signal play important roles in the activation of immune cells such as T lymphocytes. Therefore we aimed to examine the effects of glucosamine on the cell physiological parameters. METHODS: In Jurkat-T lymphocytes, intracellular [Ca2+] ([Ca2+]i) was measured using fura-2 fluorimetry, and voltage-gated K+ current (I(Kv)) was measured using whole-cell clamp technique. Ca2+-activated K+ current (I(Kca)) was measured in HEK293 cells over expressing SK4 using inside-out patch clamp technique. RESULTS: An acute application of glucosamine (0.5 mM) affected neither the increase in [Ca2+]i induced by CD3 stimulation (anti-CD3 Ab, 5 microgram/mL) nor the I(Kv) in Jurkat-T cells. A chronic stimulation of with anti-CD3 Ab (5 microgram/mL, 24~36 hr) largely increased the amplitude of IKv. However, the combined treatment with glucosamine (0.1 mM) did not block the increase of I(Kv). The I(KCa) in SK4-overexpressing cells was slightly decreased by glucosamine (0.5 mM). CONCLUSIONS: While glucosamine had a minor inhibitory effect on SK4 K+ channels, the anti-inflammatory effects of glucosamine could not be explained by the effects on the Ca2+ signaling in T lymphocytes.
Calcium ; Fluorometry ; Functional Food ; Fura-2 ; Glucosamine ; HEK293 Cells ; Ion Channels ; Lymphocytes ; T-Lymphocytes

Enzymes play such a pivotal role in cellular metabolism that enzyme assays are important for bio-engineering, disease diagnoses and drug discovery. Among the reported methods, fluoremetry has attracted more and more attention due to its high sensitivity and possibility of continuous dynamic monitoring. The recent progresses and applications in enzyme assays using fluorescent probes were reviewed. Different methods were classified into direct fluorescence detection and indirect fluorescence detection according to their labeled substrates and detection mechanisms. Our writing purpose is to provide the readers with a flavor of the kinds of tools and strategies available in enzyme assays with fluorescent probes. Also, the research situation and prospects were disucssed
Animals ; Enzyme Assays ; methods ; trends ; Fluorescent Dyes ; Fluorometry ; methods ; Humans ; Microscopy, Fluorescence

Digoxin plays a part in healing of congestive heart failure in clinic. Its therapeutic dose is very approximate to toxic dose and even they overlap each other sometimes. There are many influencing factors on blood drug level of digoxin. Pharmacodynamics and pharmacokinetics varies with different individuality. It is indispensable to detecting blood drug level in order to treat disease and prevent intoxication. Integrating with the detecting-methods of blood drug level of digoxin home and broad, characteristic of many methods are summarized from sensitivity, linearity range, cross-reaction and precision. These methods include radio immunoassay, enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay and HPLC-MS-MS. These methods are popular for their specialized ascendancy. The cost of radio immunoassay is low. Enzyme immunoassay has good specificity. Sensitivity and stability of chemiluminescence immunoassay is very excellent. Fluorescence polarization immunoassay is sensitive and convenient. HPLC-MS-MS has high resolution and good specificity. One of the development tendencies is to combine two or more methods in detecting the blood drug level of digoxin which contribute to these methods integrated use.
Chemistry Techniques, Analytical ; methods ; Chromatography, High Pressure Liquid ; Digoxin ; blood ; Enzyme-Linked Immunosorbent Assay ; Fluorescence Polarization Immunoassay ; Fluoroimmunoassay ; Humans ; Radioimmunoassay ; Reproducibility of Results ; Tandem Mass Spectrometry