Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F62156489%3A43210%2F20%3A43917098" target="_blank" >RIV/62156489:43210/20:43917098 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216305:26620/20:PU135056

Výsledek na webu

<a href="https://doi.org/10.1016/j.aca.2019.11.031" target="_blank" >https://doi.org/10.1016/j.aca.2019.11.031</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.aca.2019.11.031" target="_blank" >10.1016/j.aca.2019.11.031</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement

Popis výsledku v původním jazyce

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 μM with good precision (RSDs &lt; 4.1%). Furthermore, the assay of CYP3A4 activity on the developed paper-based device provided comparable results with those obtained from conventional well-plates (p &gt; 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 +- 0.35 μM and 0.43 +- 0.05 μM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 oC can increase storage stability, offering the activity of 86.3 +- 0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.

Název v anglickém jazyce

Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement

Popis výsledku anglicky

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 μM with good precision (RSDs &lt; 4.1%). Furthermore, the assay of CYP3A4 activity on the developed paper-based device provided comparable results with those obtained from conventional well-plates (p &gt; 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 +- 0.35 μM and 0.43 +- 0.05 μM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 oC can increase storage stability, offering the activity of 86.3 +- 0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytica Chimica Acta

ISSN

0003-2670

e-ISSN

—

Svazek periodika

1098

Číslo periodika v rámci svazku

15 February

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

8

Strana od-do

86-93

Kód UT WoS článku

000506887100008

EID výsledku v databázi Scopus

2-s2.0-85076021300