Elsevier

Analytica Chimica Acta

Volume 1098, 15 February 2020, Pages 86-93
Analytica Chimica Acta

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

https://doi.org/10.1016/j.aca.2019.11.031Get rights and content

Highlights

  • Sol-gel thin film immobilized CYP450 increases the storage stability of enzyme.

  • The sol-gel formulation can be deposited on paper-based devices using a pipette.

  • Handheld microscope and ImageJ allow fluorescence imaging and product measurement.

  • The devices are simple, low-cost and efficient for enzyme activity measurement.

Abstract

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 < 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 > 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 °C 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.

Introduction

Human cytochrome P450 (CYP450) is involved mainly in Phase I metabolism in the liver and is responsible for the biotransformation of a variety of xenobiotics including therapeutic drugs, medicinal plants and some endogenous substances. Functional human hepatic CYP450 isozymes, i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, contribute to the biotransformation of most exogenous substances including 90% of all drugs in clinical uses [1,2]. Among these isozymes, CYP3A4 is the most abundantly expressed CYP450 isoenzyme. It has been involved in the metabolism of almost 50% of all drugs available on the market as well as in high number of clinically significant drug-drug and medicinal plant-drug interactions leading to unexpected adverse effects, reduction of drug responses or increase of the actions of a particular drug [3]. Hence, in vitro measurement of effects of new drug candidates or medicinal plants on CYP450 and in particular CYP3A4 activities is essential for guiding in vivo and clinical drug interaction studies in order to avoid enzyme interactions.

Several analytical techniques for the determination of CYP450 activities have been reported (e.g. HPLC-UV and fluorescence detections [4], LC-MS [5], LC-MS/MS [6,7], microplate assay with fluorescence detection [8,9]). Advanced miniaturized microfluidic biosensors have been integrated in poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) microfluidic devices equipped with electrochemical biosensors. The enzymes were immobilized on electrodes to facilitate electron transfer. The microfluidic devices were utilized for the determination of enzyme substrates, which showed significances in environmental monitoring, pharmaceutical industry, drug metabolism profiling and clinical practice [[10], [11], [12], [13], [14], [15], [16]]. In addition, fluorescence detection-based microfluidic devices have been reported for the measurement of CYP450 enzymatic activity of hepatocyte [17,18]. The microfluidic CYP450 reactor followed by fluorescence detection [19] and online coupling with LC-MS [20] for determination of metabolites have been demonstrated in a study of CYP450-mediated drug metabolism.

Paper-based devices have been on the rise as the trend towards miniaturized analytical systems offering low-cost, portability, disposability, low sample and solvent consumption and rapid analysis. This technique has been demonstrated in a variety of applications including health diagnostics, environmental monitoring and food quality testing [[21], [22], [23]]. A microfluidic lateral flow assay of CYP2A6 isozyme activity using conventional fluorescence microscope has been presented [24]. A functionalized calcium carbonate coated paper was utilized to increase liquid uptake capacity and enhance the liquid wicking ability, which resulted in an improvement of sensitivity. Although a lateral flow paper assay of human metabolism mediated by CYP450 has been presented, fresh preparation of enzyme solution for each experiment was required since the enzyme is generally unstable and has to be kept at −80 °C. Moreover, the free enzyme solution could not be impregnated on the paper-based device as a dry reagent without immobilization.

The enzyme immobilization is an entrapment of the enzyme within the polymer matrix, which is the polymerization of monomer/low molecular weight polymer around the enzyme. In order to avoid enzyme denaturation, an aqueous polymerization or gelation reaction has to be achieved in mild conditions. Furthermore, the pore size and interconnectivity of the pores in the polymer could facilitate the migration of substrate and product and prevent enzyme leaching. Enzymes have been successfully immobilized in sol-gels and hydrogels without a significant loss of enzyme activity [25]. In addition, a sol-gel immobilization technique offers advantages such as optical transparency, compatibility with various organic moieties, and stability in harsh environments [26]. The immobilization of CYP450 isozymes in alginate fabricated on glass slide has been used for assessment of CYP450 activity using the wide-field optical imaging system. CYP3A4 exhibited excellent storage stability in alginate, with the activity of ≈90% storage for 3 weeks at −80 °C [27]. CYP450 isozymes has also been immobilized in the silica sol-gel and coated on plastic well-plate. The activity remained at 98% after 3-weeks storage at 4 °C [8].

In our studies, we immobilized CYP3A4 in silica sol–gel and coated as thin film on the paper-based devices for assessing the effects of drugs or medicinal herbs on CYP3A4 activities. The silica film is biologically inert and optically transparent, thus, the fluorescence product (resorufin) can be accurately quantified using a handheld fluorescence microscope and image analysis software (Fig. 1). The detection system is portable, easy to use and requires only a simple equipment. The entrapped CYP3A4 in the sol-gel matrices could retain its bioactivity and remain accessible to external reagents by diffusion through the porous silica. Finally, we evaluated the kinetic parameters of the immobilized enzyme and present utilization of the paper-based sol-gel thin films immobilized CYP3A4 for the study of known drug inhibitors/inducers and medicinal plant extracts.

Section snippets

Chemicals and reagents

Dithiothreitol, cytochrome P450 3A4 (CYP3A4) human recombinant (expressed in Saccharomyces cerevisiae), resorufin, resorufin benzyl ether, carbamazepine, sodium silicate solution (eq. to 2.5 M) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyoxyethylene-polyoxypropylene-block copolymer or poloxamer 188 (Pluronic® F-68), 50%w/w colloidal silica (LUDOX® TM-50, eq. to 8.3 M) and β-nicotinamide adenine dinucleotide-2′-phosphate reduced tetrasodium salt

Sol-gel immobilized CYP3A4

Sodium silicate as a starting material was used in order to avoid alcohol by product (i.e. methanol), which could inhibit the CYP450 activity [8]. Silicates condense at the colloidal silica particle and produce SiO2 network following acidification. Silicate-silica mixed system could offer a variety of sol-gel characteristics, which can be categorized into 4 groups (Fig. 2): 1) no detectable gel formed 2) flowing gel 3) non-flowing gel and 4) rigid gel [33]. The desired gel formulation is the

Conclusions

The immobilization of CYP3A4 in silica-based sol-gel provides the improvement of the CYP450 storage stability, which would be of crucial importance for easily degradable biomolecules. The method versatility also includes low-cost, high speed, simplicity and optical transparency of the sol-gel. Therefore, it could be compatible with the optical detection. The sol-gel immobilized CYP3A4 can be conveniently deposited on the cellulose fiber and the gel can be rapidly formed at the room temperature.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the Thailand Research Fund, the Office of the Higher Education Commission (Thailand) and Mahidol University through Research Grant for New Scholar (Grant No. MRG5980063). Authors would like to thanks the support from Faculty of Pharmacy, Mahidol University.

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