This thesis deals mainly about developing simple and ultrasensitive electrochemical immunosensors by using new catalytic reactions of enzymes and nanoparticles.
Electrochemical immunoassays have been widely used to detect a diverse range of biomolecules because they are highly sensitive and spec...
This thesis deals mainly about developing simple and ultrasensitive electrochemical immunosensors by using new catalytic reactions of enzymes and nanoparticles.
Electrochemical immunoassays have been widely used to detect a diverse range of biomolecules because they are highly sensitive and specific. Among the different types of immunoassays, most commonly used ones are sandwich-type and, which directly quantifies the target species by using secondary antibody with some catalytic label. This catalytic label could either be a redox enzyme or a redox nanocatalyst (i.e. nanoparticles), which involves the label-mediated redox cycling and that allows rapid and high signal amplification, along with the stable catalytic activity. These types of redox cycling are electrochemical-enzymatic (EN) redox cycling and electrochemical-nanocatalytic (ENc) redox cycling, respectively.
NAD(P)H dependent DT-diaphorase (DI) enzyme label can hydrolyze the phosphate esters (phosphate-containing substrates) identical to alkaline phosphatase. We found that esterase-like catalytic reaction of DI, can hydrolyze the ester containing substrate 4-NH2-N-1-A rapidly in the presence of NADH. This new catalytic reaction was applied to the sensitive detection of TSH. The high signal-to-background ratio resulted from (i) rapid enzymatic hydrolysis of 4-NH2-N-1-A, (ii) fast EC redox cycling involving ITO electrode, hydrolyzed enzyme product (4-NH2-N) and NADH, (iii) fast EN redox cycling involving ITO electrode, 4-NH2-N, DI and NADH, (iv) slow direct reaction between 4-NH2-N-1-A, and NADH, and (v) slow electrode mediated reaction between 4-NH2-N-1-A and NADH. Finally, TSH was detected in artificial serum over a wide range of concentrations with a detection limit of ~2 pg/mL. Measured concentrations of TSH in clinical serum samples were agreed well with the concentrations obtained using a commercial instrument.
We have developed an ultrasensitive immunosensor using the catalytic reduction (nitrosoreductase-like catalytic reaction) of 4-NO-1-N by PdNP/H3N−BH3. Among five nitro and nitroso-arene substrates and four reductants, 4-NO-1-N and H3N−BH3 pair showed the highest signal-to-background ratio. A high electrochemical signal observed from (i) low interference of dissolved oxygen and (ii) rapid catalytic reduction of 4-NO-1-N, and (iii) rapid EC and ENc redox cycling. The slow reaction rate of the following reactions, (i) direct electrooxidation of H3N−BH3 on the sensing surface, (ii) direct reduction of 4-NO-1-N by H3N−BH3, and (iii) electrode mediated reduction of 4-NO-1-N by H3N−BH3 are responsible for the low background levels. The developed method was successfully applied to the PTH detection with a detection limit of ~0.3 pg/mL.
Although sandwich-type immunosensors are sensitive they are not suitable for small molecules (such as nonprotein hormones and some organic molecular species) with only a single binding site to antibodies. To overcome above mentioned limitation, one-site immunometric assays such as competitive and (competitive) displacement are used for the small molecule detection. These detection methods have a simple detection procedure over sandwich-type assays, but the washing steps hinder the development of the miniaturized detection tools. On the basis of miniaturization, proximity-dependent washing-free immunosensors are recently developed which involves the faster electron transfer between an electrode-bound enzyme label to electrode than an un-bound one with the help of redox mediator (EN redox cycling). In the washing-free immunosensor, to achieve high electrochemical signal background ratio, the concentration of the redox enzyme label is very essential. When the redox enzyme label concentration is too low, it results in the less difference between the target and nontarget signals. When the concentration is too high, the un-bound label causes a higher background level. Both cases limit the applicability of washing-free competitive immunoassays. Another one-site immunometric assay is (competitive) displacement-type assay and it shows high specificity and high sensitivity over competitive-type assay. In displacement assay, initially, the signal labeled antibody allowed to form a noncovalent complex on target immobilized surface, and finally target analyte is introduced into the complex to induce the displacement of the labeled antibody from the electrode surface. Consequently, the concentration of displaced antibody from the electrode surface is directly proportional to the concentration of the target analyte present in the sample solution. This one-site immunometric assay allows the high signal change in washing-free detection.
We have developed a new displacement-type washing-free immunosensor using the fast EN redox cycling of DI with Os(bpy)2Cl2 and NADH. Proximity-dependent electron mediation of Os(bpy)2Cl2 allowed us to distinguish the bound and displaced DI labeled in the solution. In terms of sensitivity and low detection limit, the washing-free displacement-type assay was performed better than other ‘signal-off’ one-site immunometric assays. Moreover, the different combination of cortisol-BSA and BSA (1:10) on the sensing surface enhanced the displacement of bound DI labeled antibody. This newly developed low-interference immunosensor enabled us to detect cortisol in artificial serum with a detection limit of ~30 pM.
Therefore, the developed immunosensors with new catalytic reactions of enzymes and nanoparticles are highly promising for the simple and sensitive point-of-care testing.
This thesis deals mainly about developing simple and ultrasensitive electrochemical immunosensors by using new catalytic reactions of enzymes and nanoparticles.
Electrochemical immunoassays have been widely used to detect a diverse range of biomolecules because they are highly sensitive and specific. Among the different types of immunoassays, most commonly used ones are sandwich-type and, which directly quantifies the target species by using secondary antibody with some catalytic label. This catalytic label could either be a redox enzyme or a redox nanocatalyst (i.e. nanoparticles), which involves the label-mediated redox cycling and that allows rapid and high signal amplification, along with the stable catalytic activity. These types of redox cycling are electrochemical-enzymatic (EN) redox cycling and electrochemical-nanocatalytic (ENc) redox cycling, respectively.
NAD(P)H dependent DT-diaphorase (DI) enzyme label can hydrolyze the phosphate esters (phosphate-containing substrates) identical to alkaline phosphatase. We found that esterase-like catalytic reaction of DI, can hydrolyze the ester containing substrate 4-NH2-N-1-A rapidly in the presence of NADH. This new catalytic reaction was applied to the sensitive detection of TSH. The high signal-to-background ratio resulted from (i) rapid enzymatic hydrolysis of 4-NH2-N-1-A, (ii) fast EC redox cycling involving ITO electrode, hydrolyzed enzyme product (4-NH2-N) and NADH, (iii) fast EN redox cycling involving ITO electrode, 4-NH2-N, DI and NADH, (iv) slow direct reaction between 4-NH2-N-1-A, and NADH, and (v) slow electrode mediated reaction between 4-NH2-N-1-A and NADH. Finally, TSH was detected in artificial serum over a wide range of concentrations with a detection limit of ~2 pg/mL. Measured concentrations of TSH in clinical serum samples were agreed well with the concentrations obtained using a commercial instrument.
We have developed an ultrasensitive immunosensor using the catalytic reduction (nitrosoreductase-like catalytic reaction) of 4-NO-1-N by PdNP/H3N−BH3. Among five nitro and nitroso-arene substrates and four reductants, 4-NO-1-N and H3N−BH3 pair showed the highest signal-to-background ratio. A high electrochemical signal observed from (i) low interference of dissolved oxygen and (ii) rapid catalytic reduction of 4-NO-1-N, and (iii) rapid EC and ENc redox cycling. The slow reaction rate of the following reactions, (i) direct electrooxidation of H3N−BH3 on the sensing surface, (ii) direct reduction of 4-NO-1-N by H3N−BH3, and (iii) electrode mediated reduction of 4-NO-1-N by H3N−BH3 are responsible for the low background levels. The developed method was successfully applied to the PTH detection with a detection limit of ~0.3 pg/mL.
Although sandwich-type immunosensors are sensitive they are not suitable for small molecules (such as nonprotein hormones and some organic molecular species) with only a single binding site to antibodies. To overcome above mentioned limitation, one-site immunometric assays such as competitive and (competitive) displacement are used for the small molecule detection. These detection methods have a simple detection procedure over sandwich-type assays, but the washing steps hinder the development of the miniaturized detection tools. On the basis of miniaturization, proximity-dependent washing-free immunosensors are recently developed which involves the faster electron transfer between an electrode-bound enzyme label to electrode than an un-bound one with the help of redox mediator (EN redox cycling). In the washing-free immunosensor, to achieve high electrochemical signal background ratio, the concentration of the redox enzyme label is very essential. When the redox enzyme label concentration is too low, it results in the less difference between the target and nontarget signals. When the concentration is too high, the un-bound label causes a higher background level. Both cases limit the applicability of washing-free competitive immunoassays. Another one-site immunometric assay is (competitive) displacement-type assay and it shows high specificity and high sensitivity over competitive-type assay. In displacement assay, initially, the signal labeled antibody allowed to form a noncovalent complex on target immobilized surface, and finally target analyte is introduced into the complex to induce the displacement of the labeled antibody from the electrode surface. Consequently, the concentration of displaced antibody from the electrode surface is directly proportional to the concentration of the target analyte present in the sample solution. This one-site immunometric assay allows the high signal change in washing-free detection.
We have developed a new displacement-type washing-free immunosensor using the fast EN redox cycling of DI with Os(bpy)2Cl2 and NADH. Proximity-dependent electron mediation of Os(bpy)2Cl2 allowed us to distinguish the bound and displaced DI labeled in the solution. In terms of sensitivity and low detection limit, the washing-free displacement-type assay was performed better than other ‘signal-off’ one-site immunometric assays. Moreover, the different combination of cortisol-BSA and BSA (1:10) on the sensing surface enhanced the displacement of bound DI labeled antibody. This newly developed low-interference immunosensor enabled us to detect cortisol in artificial serum with a detection limit of ~30 pM.
Therefore, the developed immunosensors with new catalytic reactions of enzymes and nanoparticles are highly promising for the simple and sensitive point-of-care testing.
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