Washing-Free Displacement Immunosensor for Cortisol in Human Serum Containing Numerous Interfering Species

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>

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>

Au nanoparticle‐catalyzed electron transfer from ammonia‐borane to Ru( NH 3 ) 6 3+

박선화,Bhatia Aman,Nandhakumar Ponnusamy,김지현,양해식 대한화학회 2024 Bulletin of the Korean Chemical Society Vol.45 No.4

Metal nanoparticle (NP)‐catalyzed electron transfer (ET) from a reducing agent to a metal complex is useful for signal amplification in biosensors. For efficient ET, the metal complex must undergo rapid outer‐sphere reactions, be highly water‐soluble, and effectively penetrate bio/organic layers on metal NPs. Our study identifies Ru(NH 3 ) 6 3+ as well‐suited for this purpose. Among reducing agents, ammonia‐borane (AB) enables rapid metal NP‐catalyzed ET, with Au, Pt, and Pd NPs displaying similar catalytic activities. The pseudo second‐order rate constant for 20‐nm Au NP‐catalyzed ET from AB to Ru(NH 3 ) 6 3+ (1.4 × 10 8  M −1  s −1 ) approaches the diffusion‐controlled rate constant. Despite immunoglobulin G and bovine serum albumin passively adsorbed on Au NPs, catalytic activity remains largely unaffected. Applying Au NP‐catalyzed ET to prostate‐specific antigen detection in human serum achieves a low detection limit of 10 pg/mL. These findings highlight the potential of Ru(NH 3 ) 6 3+ and AB in designing biosensors based on rapid catalytic reaction. Metal nanoparticle (NP)-catalyzed electron transfer (ET) from a reducing agent to a metal complex is useful for signal amplification in biosensors. For efficient ET, the metal complex must undergo rapid outer-sphere reactions, be highly watersoluble, and effectively penetrate bio/organic layers on metal NPs. Our study identifies Ru(NH3)6 3+ as well-suited for this purpose. Among reducing agents, ammonia-borane (AB) enables rapid metal NP-catalyzed ET, with Au, Pt, and Pd NPs displaying similar catalytic activities. The pseudo second-order rate constant for 20-nm Au NP-catalyzed ET from AB to Ru(NH3)6 3+ (1.4 108 M1 s1) approaches the diffusion-controlled rate constant. Despite immunoglobulin G and bovine serum albumin passively adsorbed on Au NPs, catalytic activity remains largely unaffected. Applying Au NP-catalyzed ET to prostate-specific antigen detection in human serum achieves a low detection limit of 10 pg/mL. These findings highlight the potential of Ru(NH3)6 3+ and AB in designing biosensors based on rapid catalytic reaction.

Rapid and Sensitive Detection of <i>Aspergillus niger</i> Using a Single-Mediator System Combined with Redox Cycling

Kwon, Jungwook,Cho, Eun-Min,Nandhakumar, Ponnusamy,Yang, Sung Ik,Yang, Haesik American Chemical Society 2018 ANALYTICAL CHEMISTRY - Vol.90 No.22

<P>Rapid and sensitive mold detection is becoming increasingly important, especially in indoor environments. Common mold detection methods based on double-mediated electron transfer between an electrode and molds are not highly sensitive and reproducible, although they are rapid and simple. Here, we report a sensitive and reproducible detection method specific to <I>Aspergillus niger</I> (<I>A. niger</I>), based on a single-mediator system combined with electrochemical-chemical (EC) redox cycling. Intracellular NAD(P)H-oxidizing enzymes in molds can convert electro-inactive hydroxy-nitro(so)arenes into electro-active hydroxy-aminoarenes. Since the membrane and wall of <I>A. niger</I> is well permeable to both a substrate (4-nitro-1-naphthol) and a reduced product (4-amino-1-naphthol) in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of <I>A. niger</I> due to two reactions: (i) enzymatic reduction of the substrate to the reduced product and (ii) electrochemical oxidation of the reduced product to an oxidized product. When a reducing agent (NADH) is present in the solution, the oxidized product is reduced back to the reduced product and then electrochemically reoxidized. This EC redox cycling significantly amplifies the electrochemical signal. Moreover, the background level is low and highly reproducible because the substrate and the reducing agent are electro-inactive at an applied potential of 0.20 V. The calculated detection limit for <I>A. niger</I> in a common double-mediator system consisting of Fe(CN)<SUB>6</SUB><SUP>3-</SUP> and menadione is ∼2 × 10<SUP>4</SUP> colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is ∼2 × 10<SUP>3</SUP> CFU/mL, indicating that the newly developed single-mediator system is more sensitive. Importantly, the detection method requires only an incubation period of 10 min and does not require a washing step, an electrode modification step, or a specific probe.</P> [FIG OMISSION]</BR>

Use of a Phosphatase-Like DT-Diaphorase Label for the Detection of Outer Membrane Vesicles

Ichzan, Andi Muhammad,Lee, Sohee,San Fang, Chiew,Nandhakumar, Ponnusamy,Ha, Hyeri,Joo, Jung Min,Kim, Kwang-sun,Yang, Haesik American Chemical Society 2019 ANALYTICAL CHEMISTRY - Vol.91 No.7

<P>DT-diaphorase (DT-D) is known to mainly catalyze the two-electron reduction of quinones and nitro(so) compounds. Detection of Gram-negative bacterial outer membrane vesicles (OMVs) that contain pyrogenic lipopolysaccharides (LPSs, also called endotoxins) is required for evaluating the toxic effects of analytical samples. Here, we report that DT-D has a high dephosphorylation activity: DT-D catalyzes reductive dephosphorylation of a phosphate-containing substrate in the presence of NADH. We also report that sensitive and simple OMV detection is possible with a sandwich-type electrochemical immunosensor using DT-D and two identical LPS-binding antibodies as a catalytic label and two sandwich probes, respectively. The absorbance change in a solution containing 4-nitrophenyl phosphate indicates that dephosphorylation occurs in the presence of both DT-D and NADH. Among the three phosphate-containing substrates [4-aminophenyl phosphate, ascorbic acid phosphate, and 1-amino-2-naphthyl phosphate (ANP)] that can be converted into electrochemically active products after dephosphorylation, ANP shows the highest electrochemical signal-to-background ratio, because (i) the dephosphorylation of ANP by DT-D is fast, (ii) the electrochemical oxidation of the dephosphorylated product (1-amino-2-naphthol, AN) is rapid, even at a bare indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The two redox cycling processes include an electrochemical-enzymatic redox cycling and an electrochemical-chemical redox cycling. The electrochemical signal in a neutral buffer (tris buffer, pH 7.5) is comparable to that in a basic buffer (tris buffer, pH 9.5). When the immunosensor is applied to the detection of OMV from <I>Escherichia coli</I>, the detection limit is found to be 8 ng/mL. This detection strategy is highly promising for the detection of biomaterials, including other extracellular vesicles.</P> [FIG OMISSION]</BR>