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Kang, Cheolho,Kang, Juyeon,Lee, Nam-Sihk,Yoon, Young Ho,Yang, Haesik American Chemical Society 2017 ANALYTICAL CHEMISTRY - Vol.89 No.15
<P>The most common enzyme labels in enzyme-linked immunosorbent assays are alkaline phosphatase and horseradish peroxidase, which, however, have some limitations for use in electrochemical immunosensors. This Article reports that the small and thermostable DT-diaphorase (DT-D) and electrochemically inactive 4-nitroso-1-naphthol (4-NO-1-N) can be used as a bifunctional enzyme label and a rapidly reacting substrate, respectively, for electrochemical immunosensors. This enzyme–substrate combination allows high signal amplification via rapid enzymatic amplification and electrochemical redox cycling. DT-D can convert an electrochemically inactive nitroso or nitro compound into an electrochemically active amine compound, which can then be involved in electrochemical–chemical (EC) and electrochemical–enzymatic (EN) redox cycling. Six nitroso and nitro compounds are tested in terms of signal-to-background ratio. Among them, 4-NO-1-N exhibits the highest signal-to-background ratio. The electrochemical immunosensor using DT-D and 4-NO-1-N detects parathyroid hormone (PTH) in phosphate-buffered saline containing bovine serum albumin over a wide range of concentrations with a low detection limit of 2 pg/mL. When the PTH concentration in clinical serum samples is measured using the developed immunosensor, the calculated concentrations are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of DT-D as an enzyme label is highly promising for sensitive electrochemical detection and point-of-care testing.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2017/ancham.2017.89.issue-15/acs.analchem.7b01223/production/images/medium/ac-2017-012233_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac7b01223'>ACS Electronic Supporting Info</A></P>
Nitrosoreductase-Like Nanocatalyst for Ultrasensitive and Stable Biosensing
Nandhakumar, Ponnusamy,Kim, Byeongyoon,Lee, Nam-Sihk,Yoon, Young Ho,Lee, Kwangyeol,Yang, Haesik American Chemical Society 2018 ANALYTICAL CHEMISTRY - Vol.90 No.1
<P>Enzyme-like nanocatalytic reactions developed for high signal amplification in biosensors are of limited use because of their low reaction rates and/or unwanted side reactions in aqueous electrolyte solutions containing dissolved O<SUB>2</SUB>. Herein, we report a nitrosoreductase-like catalytic reaction, employing 4-nitroso-1-naphthol, Pd nanoparticles, and H<SUB>3</SUB>N–BH<SUB>3</SUB>, which affords a high reaction rate and minimal side reactions, enabling its use in ultrasensitive electrochemical biosensors. 4-Nitroso-1-naphthol was chosen after five hydroxy-nitro(so)arene compounds were compared in terms of high signal and low background levels. Importantly, the nanocatalytic reaction occurs without the self-hydrolysis and induction period observed in the nanocatalytic reduction of nitroarenes by NaBH<SUB>4</SUB>. The high signal level results from (i) fast nanocatalytic 4-nitroso-1-naphthol reduction, (ii) fast electrochemical redox cycling, and (iii) the low influence of dissolved O<SUB>2</SUB>. The low background level results from (i) slow direct reaction between 4-nitroso-1-naphthol and H<SUB>3</SUB>N–BH<SUB>3</SUB>, (ii) slow electrode-mediated reaction between 4-nitroso-1-naphthol and H<SUB>3</SUB>N–BH<SUB>3</SUB>, and (iii) slow electrooxidation of H<SUB>3</SUB>N–BH<SUB>3</SUB> at electrode. When applied to the detection of parathyroid hormone, the detection limit of the newly developed biosensor was ∼0.3 pg/mL. The nitrosoreductase-like nanocatalytic reaction is highly promising for ultrasensitive and stable biosensing.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2018/ancham.2018.90.issue-1/acs.analchem.7b03364/production/images/medium/ac-2017-03364x_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac7b03364'>ACS Electronic Supporting Info</A></P>
Identification of Structural Domains Affecting Transactivation Potential of Nm23
Cho, Seong-Jun,Lee, Nam-Sihk,Jung, Yong-Sam,Lee, Hansoo,Lee, Kong-Joo,Kim, Eunhee,Chae, Suhn-Kee 梨花女子大學校 藥學硏究所 2001 藥學硏究論文集 Vol.- No.10
The strong transactivation activity of the C-terminal half(amino acids 76-152) of Nm23 was reported previously. Here we examined a structural domain preventing or necessary to its transactivation activity. The C-terminal 1/4 (amino acids 109-052) was sufficient form transactivation, but the C-terminal half with a longer N-terinal extension (amino acids 58-152) caused the loss of the transactivation ability. Furthermore, co-expression of the N-termanal half with the C-terminus of Nm23-H1 blocked the transactivation activity of the C-terminal half, where direct interaction of both truncated proteins was demonstrated in vitro. Transactivation activities in the C-terminal halves of the known mutants (P96S, H118F, S120G, and S120A) exhibiting differential antimetastasis effects were also tested. Significant reduction of transactivation activity was observed only in H118F, indicating that NPD kinase active-site histidine is required. This suggests that transactivation potential of Nm23 is related to NDP kinase activity but not to metastasis suppressor activity.
Nandhakumar, Ponnusamy,Haque, Al-Monsur Jiaul,Lee, Nam-Sihk,Yoon, Young Ho,Yang, Haesik American Chemical Society 2018 ANALYTICAL CHEMISTRY - Vol.90 No.18
<P>Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)<SUB>2</SUB>Cl<SUB>2</SUB> between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN)<SUB>6</SUB><SUP>4-</SUP>, and Ru(NH<SUB>3</SUB>)<SUB>6</SUB><SUP>3+</SUP>, the electrochemical and redox cycling behaviors of Os(bpy)<SUB>2</SUB>Cl<SUB>2</SUB> are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.</P> [FIG OMISSION]</BR>
Dong IL Kim,Sul Ki Lim,Nam Sihk Lee,Ho Jae Han,Seong Soo Kang,Soo Hyun Park 한국실험동물학회 2006 Laboratory Animal Research Vol.22 No.3
Hyperglycemia is known to be a major factor to induce the development of diabetes mellitus. Liver is a very important organ to xenotransplantation and the development of diabetes mellitus. Pig is known to be a most suitable species in transplantation of human organs. However, the effect of high glucose on cell proliferation in pig hepatocytes was not elucidated. Thus, we examined the effect of high glucose on cell proliferation and its related signal pathways in primary cultured pig hepatocytes. In the present study, high glucose stimulates [³H]-thymidine incorporation. However, mannitol and ʟ-glucose didn't affect [³H]-thymidine incorporation. In addition, high glucose-induced stimulation of [³H]-thymidine incorporation was blocked by PD98059 (a p44/42 mitogen activated protein kinase [MAPK] inhibitor) and 58203580 (a p38 MAPK inhibitor) but not by SP600125 (a SAPK/JNK inhibitor). Indeed, high glucose stimulated p38 MAPK and p44/42 MAPK activation in a time dependent manner. High glucose-induced stimulation of [³H]-thymidine incorporation was blocked by staurosporine, H-7, and bisindolylmaleimide I (protein kinase C [PKC] inhibitors). Indeed, high glucose translocated PKC from cytosolic to membrane fraction. In conclusion, high glucose stimulates cell proliferation via PKC and MAPK activation in cultured pig hepatocytes.