An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum

Akanda, Md. Rajibul,Joung, Hyou-Arm,Tamilavan, Vellaiappillai,Park, Seonhwa,Kim, Sinyoung,Hyun, Myung Ho,Kim, Min-Gon,Yang, Haesik The Royal Society of Chemistry 2014 The Analyst Vol.139 No.6

<P>Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical–chemical–chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH<SUB>3</SUB>)<SUB>6</SUB><SUP>3+</SUP>, enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH<SUB>3</SUB>)<SUB>6</SUB><SUP>3+</SUP> is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [β-galactosidase, 4-amino-1-naphthyl β-<SMALL>D</SMALL>-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V <I>vs.</I> Ag/AgCl) minimizes the interference effect of electroactive species (<SMALL>L</SMALL>-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL<SUP>−1</SUP> for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples.</P> <P>Graphic Abstract</P><P>We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c3an02328a'> </P>

Anti-Inflammatory and Gastroprotective Roles of <i>Rabdosia inflexa</i> through Downregulation of Pro-Inflammatory Cytokines and MAPK/NF-κB Signaling Pathways

Akanda, Md Rashedunnabi,Kim, In-Shik,Ahn, Dongchoon,Tae, Hyun-Jin,Nam, Hyeon-Hwa,Choo, Byung-Kil,Kim, Kyunghwa,Park, Byung-Yong MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.2

<P>Globally, gastric ulcer is a vital health hazard for a human. <I>Rabdosia inflexa</I> (RI) has been used in traditional medicine for inflammatory diseases. The present study aimed to investigate the protective effect and related molecular mechanism of RI using lipopolysaccharide (LPS)-induced inflammation in RAW 246.7 cells and HCl/EtOH-induced gastric ulcer in mice. We applied 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitric oxide (NO), reactive oxygen species (ROS), histopathology, malondialdehyde (MDA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blot analyses to evaluate the protective role of RI. Study revealed that RI effectively attenuated LPS-promoted NO and ROS production in RAW 246.7 cells. In addition, RI mitigated gastric oxidative stress by inhibiting lipid peroxidation, elevating NO, and decreasing gastric inflammation. RI significantly halted elevated gene expression of pro-inflammatory cytokines such as tumor necrosis factor-α (<I>TNF-α</I>), interleukin-1β (<I>IL-1β</I>), interleukin-6 (<I>IL-6</I>), inducible nitric oxide synthetase (<I>iNOS</I>), and cyclooxygenase-2 (<I>COX-2</I>) in gastric tissue. Likewise, RI markedly attenuated the mitogen-activated protein kinases (MAPKs) phosphorylation, COX-2 expression, phosphorylation and degradation of inhibitor kappa B (IκBα) and activation of nuclear factor kappa B (NF-κB). Thus, experimental findings suggested that the anti-inflammatory and gastroprotective activities of RI might contribute to regulating pro-inflammatory cytokines and MAPK/NF-κB signaling pathways.</P>

Hair growth promoting activity of discarded biocomposite keratin extract

Akanda, Md Rashedunnabi,Kim, Hak-Yong,Park, Mira,Kim, In-Shik,Ahn, Dongchoon,Tae, Hyun-Jin,Park, Byung-Yong SAGE Publications 2017 Journal of biomaterials applications Vol.32 No.2

Optimization of Phosphatase- and Redox Cycling-Based Immunosensors and Its Application to Ultrasensitive Detection of Troponin I

Akanda, Md. Rajibul,Aziz, Md. Abdul,Jo, Kyungmin,Tamilavan, Vellaiappillai,Hyun, Myung Ho,Kim, Sinyoung,Yang, Haesik American Chemical Society 2011 ANALYTICAL CHEMISTRY - Vol.83 No.10

<P>The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium–tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl<SUB>2</SUB>, and the optimum conditions for antigen–antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen–antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (<SMALL>l</SMALL>-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (<SMALL>l</SMALL>-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2011/ancham.2011.83.issue-10/ac200447b/production/images/medium/ac-2011-00447b_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac200447b'>ACS Electronic Supporting Info</A></P>

Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical–Chemical–Chemical Redox Cycling for the Detection of <i>E. coli</i> O157:H7

Akanda, Md. Rajibul,Tamilavan, Vellaiappillai,Park, Seonhwa,Jo, Kyungmin,Hyun, Myung Ho,Yang, Haesik American Chemical Society 2013 ANALYTICAL CHEMISTRY - Vol.85 No.3

<P>Signal amplification by enzyme labels in enzyme-linked immunosorbent assays (ELISAs) is not sufficient for detecting a low number of bacterial pathogens. It is useful to employ approaches that involve multiple signal amplification such as enzymatic amplification plus redox cycling. An advantageous combination of an enzyme product [for fast electrochemical–chemical–chemical (ECC) redox cycling that involves the product] and an enzyme substrate (for slow side reactions and ECC redox cycling that involve the substrate) has been developed to obtain a low detection limit for <I>E. coli</I> O157:H7 in an electrochemical ELISA that employs redox cycling. In our search for an alkaline phosphatase substrate/product couple that is better than the most common couple of 4-aminophenyl phosphate (APP)/4-aminophenol (AP), we compared five couples: APP/AP, hydroquinone diphosphate (HQDP)/hydroquinone (HQ), <SMALL>l</SMALL>-ascorbic acid 2-phosphate/<SMALL>l</SMALL>-ascorbic acid, 4-amino-1-naphthyl phosphate/4-amino-1-naphthol, and 1-naphthyl phosphate/1-naphthol. In particular, we examined signal-to-background ratios in ECC redox cycling using Ru(NH<SUB>3</SUB>)<SUB>6</SUB><SUP>3+</SUP> and tris(2-carboxyethyl)phosphine as an oxidant and a reductant, respectively. The ECC redox cycling that involves HQ is faster than the cycling that involves AP, whereas the side reactions and ECC redox cycling that involve HQDP are negligible compared to the APP case. These results seem to be due to the fact that the formal potential of HQ is lower than that of AP and that the formal potential of HQDP is higher than that of APP. Enzymatic amplification plus ECC redox cycling based on a HQDP/HQ couple allows us to detect <I>E. coli</I> O157:H7 in a wide range of concentrations from 10<SUP>3</SUP> to 10<SUP>8</SUP> colony-forming units/mL.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2013/ancham.2013.85.issue-3/ac3028855/production/images/medium/ac-2012-028855_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac3028855'>ACS Electronic Supporting Info</A></P>

Involvement of MAPK/NF-κB signal transduction pathways: <i>Camellia japonica</i> mitigates inflammation and gastric ulcer

Akanda, Md Rashedunnabi,Park, Byung-Yong Elsevier 2017 BIOMEDICINE AND PHARMACOTHERAPY Vol.95 No.-

<P><B>Abstract</B></P> <P>Gastric ulcer is an important risk factor for human health globally. <I>Camellia japonica</I> (CJ) is a plant of which the fruits are used as traditional phytomedicine for inflammatory and immunomodulatory diseases; however, the underlying molecular mechanism has not been clarified. The present study aimed to investigate the immunopharmacological activities of <I>Camellia japonica</I> and validate its pharmacological targets. To evaluate the protective roles of <I>Camellia japonica</I> on LPS-induced inflammation in RAW 264.7 cells and HCl/EtOH-induced gastric ulcer in mice; we applied 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitric oxide (NO), reactive oxygen species (ROS), histopathology, malondialdehyde (MDA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and western blot analyses. We also determined the total phenolic and flavonoid content of <I>Camellia japonica</I> which might possess antioxidant and anti-inflammatory properties. We found the production of NO and ROS in RAW 246.7 cells were both suppressed by <I>Camellia japonica</I> <B>.</B> Moreover, <I>Camellia japonica</I> mitigated the HCl/EtOH-induced oxidative stress in gastric mucosa via the reduction of lipid peroxidation and elevation of NO production. Gastric mucosal damages were prominently improved by <I>Camellia japonica</I>, as confirmed by the histopathological evaluation. The gene expression of inflammatory cytokines and enzymes TNF-α, IL-6, IL-1β, iNOS, and COX-2 was notably downregulated by <I>Camellia japonica</I>. In addition, <I>Camellia japonica</I> markedly attenuated the MAPKs (ERK1/2, JNK, and p38) phosphorylation, COX-2 expression, and activation of transcription factor NF-κB and as well as phosphorylation and degradation of IκBα in gastric mucosa. Taken together, the intimated anti-inflammatory and gastroprotective mechanism of <I>Camellia japonica</I> is mediated by modulation of oxidative stress, inflammatory cytokines, and enzymes via suppression of MAPK/NF-κB signaling pathways.</P>

Neuroprotective Effects of Sigesbeckia pubescens Extract on Glutamate-Induced Oxidative Stress in HT22 Cells via Downregulation of MAPK/caspase-3 Pathways

Akanda, Md. Rashedunnabi,Kim, Myung-Jin,Kim, In-Shik,Ahn, Dongchoon,Tae, Hyun-Jin,Rahman, Md. Mahfujur,Park, Yang-Gyu,Seol, Jae-Won,Nam, Hyeon-Hwa,Choo, Byung-Kil,Park, Byung-Yong Springer-Verlag 2018 Cellular and molecular neurobiology Vol.38 No.2

<P>Sigesbeckia pubescens (SP) is a traditional Chinese medicine, possessing antioxidant and anti-inflammatory activities. In this study, we evaluate the neuroprotective activities of SP extract on glutamate-induced oxidative stress in HT22 cells and the molecular mechanism underlying neuroprotection. We applied 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), crystal violet, reactive oxygen species (ROS), lactate dehydrogenase (LDH), quantitative real-time polymerase chain reaction (qPCR), and western blot analyses for assessing the neuroprotective effects of SP extract. The experimental study revealed that SP considerably increased the cell viability, and reduced the oxidative stress promoted ROS and LDH generation in HT22 cells in a dose-dependent manner. Additionally, the morphology of HT22 cells was effectively improved by SP. Upregulated gene expressions of mitogen-activated protein kinase (MAPK) were markedly attenuated by SP. Similarly, SP notably suppressed the ROS-mediated phosphorylation of MAPK (pERK1/2, pJNK, and pp38) cascades and activation of apoptotic factor caspase-3 signaling pathway that overall contributed to the neuroprotection. Taken together, SP may exert neuroprotective effects via alteration of MAPK and caspase-3 pathways under oxidative stress condition. Therefore, SP is a potential agent for preventing oxidative stress-mediated neuronal cell death.</P>

Regulation of JAK2/STAT3 and NF-κB signal transduction pathways; <i>Veronica polita</i> alleviates dextran sulfate sodium-induced murine colitis

Akanda, Md Rashedunnabi,Nam, Hyeon-Hwa,Tian, Weishun,Islam, Anowarul,Choo, Byung-Kil,Park, Byung-Yong Elsevier 2018 BIOMEDICINE AND PHARMACOTHERAPY Vol.100 No.-

<P><B>Abstract</B></P> <P>Ulcerative colitis (UC) is a major inflammatory bowel disease (IBD) has become a worldwide emergent disease. <I>Veronica polita</I> (VP) is a medicinal herb that has strong antioxidant and anti-inflammatory properties. In the present study, we studied the protective effect of VP on dextran sulfate sodium (DSS)-induced experimental colitis in mice. Phytochemical screening of VP extract demonstrated the presence of high total phenolic and flavonoid contents. Compared with the DSS group, VP significantly reduced clinical symptoms with less weight loss, bloody stool, shortening of the colon, and the severity of colitis was considerably inhibited as evidenced by the reduced disease activity index (DAI) and degree of histological damage in the colon and spleen. Also, treatment with VP considerably decreased the nitric oxide (NO) and malondialdehyde (MDA) level. VP remarkably downregulated the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS) and cyclooxygenase-2 (COX-2) in the colon tissue. Likewise, activation of the signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-κB) was effectively blocked by VP. Taken together, these results demonstrate that VP has an ameliorative effect on colonic inflammation mediated by modulation of oxidative stress and inflammatory mediators by suppressing the JAK2/STAT3 and NF-κB signaling pathways.</P>

In Vivo and In Vitro Hepatoprotective Effects of <i> Geranium koreanum</i> Methanolic Extract via Downregulation of MAPK/Caspase-3 Pathway

Akanda, Md Rashedunnabi,Kim, In-Shik,Ahn, Dongchoon,Tae, Hyun-Jin,Tian, Weishun,Nam, Hyeon-Hwa,Choo, Byung-Kil,Park, Byung-Yong Hindawi 2017 Evidence-based Complementary and Alternative Medic Vol.2017 No.-

<P><I>Geranium koreanum</I> (GK) is an indigenous Chinese herbal medicine widely used for the treatment of various inflammation and liver disorders. However, the exact mechanism of action of GK remains unknown. This study aimed to investigate the protective effect and related molecular mechanism of GK on NaAsO<SUB>2</SUB>-induced cytotoxicity in HepG2 cells and liver damage in mice. The cytoprotective role of GK was assessed on HepG2 cells using MTT assay. Oxidative stress and lactate dehydrogenase levels were measured with ROS and LDH assay. Histopathology and serum enzymes levels were estimated. The molecular mechanism was evaluated by qPCR and immunoblotting to ensure the hepatoprotective role of GK against NaAsO<SUB>2</SUB> intoxication in mice. We found cotreatment with GK significantly attenuated NaAsO<SUB>2</SUB>-induced cell viability loss, intracellular ROS, and LDH release. Hepatic histopathology and serum biochemical parameters, ALT, and AST were notably improved by cotreatment with GK. Beside, GK markedly altered both mRNA and protein expression level of MAPK. The proapoptotic and antiapoptotic protein Bax/Bcl-2 ratio was significantly regulated by GK. Moreover, GK remarkably suppressed the postapoptotic transcription protein cleaved caspase-3 expression. The present study reveals that GK possesses hepatoprotective activity which is probably involved in the modulation of the MAPK/caspase-3 pathway.</P>

Hepatoprotective Role of <i>Hydrangea macrophylla</i> against Sodium Arsenite-Induced Mitochondrial-Dependent Oxidative Stress via the Inhibition of MAPK/Caspase-3 Pathways

Akanda, Md Rashedunnabi,Tae, Hyun-Jin,Kim, In-Shik,Ahn, Dongchoon,Tian, Weishun,Islam, Anowarul,Nam, Hyeon-Hwa,Choo, Byung-Kil,Park, Byung-Yong MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.7

<P>Sodium arsenite (NaAsO<SUB>2</SUB>) has been recognized as a worldwide health concern. <I>Hydrangea macrophylla</I> (HM) is used as traditional Chinese medicine possessing antioxidant activities. The study was performed to investigate the therapeutic role and underlying molecular mechanism of HM on NaAsO<SUB>2</SUB>-induced toxicity in human liver cancer (HepG2) cells and liver in mice. The hepatoprotective role of HM in HepG2 cells was assessed by using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), reactive oxygen species (ROS), and lactate dehydrogenase (LDH) assays. Histopathology, lipid peroxidation, serum biochemistry, quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were performed to determine the protective role of HM against NaAsO<SUB>2</SUB> intoxication in liver tissue. In this study, we found that co-treatment with HM significantly attenuated the NaAsO<SUB>2</SUB>-induced cell viability loss, intracellular ROS, and LDH release in HepG2 cells in a dose-dependent manner. Hepatic histopathology, lipid peroxidation, and the serum biochemical parameters alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were notably improved by HM. HM effectively downregulated the both gene and protein expression level of the mitogen-activated protein kinase (MAPK) cascade. Moreover, HM well-regulated the Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) ratio, remarkably suppressed the release of cytochrome <I>c</I>, and blocked the expression of the post-apoptotic transcription factor caspase-3. Therefore, our study provides new insights into the hepatoprotective role of HM through its reduction in apoptosis, which likely involves in the modulation of MAPK/caspase-3 signaling pathways.</P>