Kinetics study on recombinant alkaline phosphatase and correlation with the generated fluorescent signal

Susini, Vanessa; Rossi, Veronica Lucia; Sanesi, Antonio; Drazek, Laurent

doi:10.1080/15321819.2017.1408022

Cited by 2 publications

(3 citation statements)

References 26 publications

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“…The turnover number of FPK–AP would be lower than that of FPK–NA. The turnover numbers of the two proteases under the experimental conditions of this study would be quite smaller than those of HRP and ALP (10–1000). − Thus, in the scheme of Figure S-1b, a low detection limit could not be obtained although the proteolytic reaction using a thrombin label was combined with the EC redox cycling reaction. Instead, a low detection limit could be obtained in the scheme of Figure a using a propagating cascade reaction of two proteases, although each turnover number of ecarin and thrombin is low.…”

Section: Resultsmentioning

confidence: 81%

“…In the second type, the second enzyme [a protease zymogen (or proenzyme)] is activated by the first enzyme (the same or different type of protease). ,− For example, inactive trypsinogen is converted into active trypsin by enterokinase . In general, the turnover numbers ( k cat ) of proteases are smaller than those of common enzymes used for signal amplification in biosensors [horseradish oxidase (HRP) and ALP] (10–1000 s –1 ). − Thus, proteases are not used as enzyme labels in conventional affinity biosensors. Instead, proteases have been employed in biosensors using the second type of propagating cascade reactions. ,− ,− …”

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confidence: 99%

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Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Park

Kim

et al. 2019

Anal. Chem.

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Propagating cascade reactions based on two proteases are promising for obtaining high signal amplification. However, in many cases, biosensors that use cascade reactions do not have low detection limits because of the inherent slowness of proteolytic reactions. Here, we report a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical–chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the other two. The measured concentrations of TSH in clinical serum are found to agree well with those determined using a commercial instrument.

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Section: Resultsmentioning

confidence: 81%

mentioning

confidence: 99%

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Park

Kim

et al. 2019

Anal. Chem.

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“…Hydrolase enzymes include carboxyl esterases, phosphatases, and proteases/peptidases that break a carboxyl ester bond, a phosphoester bond, and an amide bond, respectively. , Hydrolysis of a carboxylic ester bond among the three chemical bonds is the easiest in terms of bond stability. However, carboxyl esterases (including lipases) show limited use as catalytic labels. − Probably, the first reason is that the turnover numbers ( k cat ) of carboxyl esterases are lower than those of HRP and ALP (10–1000 s –1 ). − The second reason is that many signaling substrates of carboxyl esterases are slowly hydrolyzed in the absence of esterases because acid- and base-catalyzed hydrolysis occurs in aqueous electrolyte solutions. , The third reason is that enzyme kinetics of carboxyl esterases are highly dependent on the type of ester substrate . If any enzyme shows a high carboxyl esterase activity and if any substrate is stable but enzymatically rapidly hydrolyzed, the enzyme could be used for carboxyl ester hydrolysis instead of common carboxyl esterases.…”

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confidence: 99%

Carboxyl Esterase-Like Activity of DT-Diaphorase and Its Use for Signal Amplification

Nandhakumar

Ichzan

Lee³

et al. 2019

ACS Sens.

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Carboxyl esterases show limited use as catalytic labels in bioassays because of slow enzymatic reaction. We report that DT-diaphorase from Bacillus stearothermophilus (DT-D, EC 1.6.99.-) shows high carboxyl esterase-like activity in the presence of reduced β-nicotinamide adenine dinucleotide (NADH) and may be used as a better catalytic label than carboxyl esterases. DT-D is a redox enzyme and can participate in signal-amplifying redox cycling. Thus, an electrochemical immunosensor using a DT-D label allows for triple signal amplification based on (i) hydrolysis of a carboxyl ester, (ii) electrochemical–chemical (EC) redox cycling involving an electrode, a hydrolysis product, and NADH, and (iii) electrochemical–enzymatic (EN) redox cycling involving an electrode, a hydrolysis product, DT-D, and NADH. Ester hydrolysis by DT-D is confirmed via spectrophotometric measurement of a chromogenic substrate (4-nitrophenyl acetate) and 1H NMR spectra. Among two phenyl acetates and four naphthyl acetates considered, 4-aminonaphthalene-1-yl acetate (4-NH2-NAc) is chosen as the best acetyl ester substrate because 4-NH2-NAc is stable, its hydrolysis is slow in the absence of DT-D, its hydrolysis is very fast in the presence of DT-D, and EC and EN redox cycling involving the hydrolysis product (4-amino-1-naphthol) is rapid. However, hydrolysis of 4-NH2-NAc by esterase from porcine liver (EC 3.1.1.1.) is very slow. When DT-D is applied to sandwich-type detection of thyroid-stimulating hormone in artificial serum, the detection limit is ∼2 pg/mL, indicating that the developed immunosensor is highly sensitive because of triple signal amplification. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common alkaline phosphatase and horseradish peroxidase.

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Kinetics study on recombinant alkaline phosphatase and correlation with the generated fluorescent signal

Abstract: 4-MUP, 4-Methylumbelliferyl phosphate; 4-MU, 4-Methylumbelliferone; RFV, Relative Fluorescent Values; RFU, Relative Fluorescent Units; QDs, Quantum Dots; LoD, Limit of Detection.

Cited by 2 publications

References 26 publications

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Carboxyl Esterase-Like Activity of DT-Diaphorase and Its Use for Signal Amplification

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