Sains Malaysiana 53(6)(2024): 1295-1307

http://doi.org/10.17576/jsm-2024-5306-06

An Ultrasensitive Electrochemical Enzymatic Urea Biosensor Based on Aniline/N-Butyl Acrylate Conducting Polymer-Modified Screen-Printed Electrode

(Biosensor Urea Elektrokimia Enzimatik Ultrasensitif Berasaskan Elektrod Bercetak Skrin Terubah Suai Polimer Berkonduksi Anilina/N-Butil Akrilat)

ALIZAR ULIANAS^1,*, SHARINA ABU HANIFAH², LING LING TAN³, NORMAZIDA ROZI^2, LEE YOOK HENG², INDANG DEWATA¹, MAWARDI MAWARDI¹, YULKIFLI¹, RAHADIAN ZAINUL¹, ELSA YUNIARTI¹, OKTA SURYANI¹ & TRISNA KUMALA SARI¹

¹Biosensor & Chemical Sensor Research Centre, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Padang, Jl. Prof. Hamka, Padang 25131, Indonesia

²Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

³Southeast Asia Disaster Prevention Research Initiative (SEADPRI), Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

Received: 8 September 2023/Accepted: 17 May 2024

Abstract

An enzymatic electrochemical biosensor for urea detection was developed using the succinimide-modified aniline/n-butyl acrylate (nBA) conducting polymer. This aniline/nBA conducting polymer was synthesized by photopolymerization with the succinimide moiety incorporated during the photocuring procedure. The urease enzyme originating from Jack beans was chemically grafted on the succinimide-functionalized aniline/nBA conducting polymer, which was attached to a screen-printed carbon paste electrode (SPE). The enzymatic hydrolysis of urea by the urease electrode diminished the voltammetric biosensor response as a result of the cascaded chemical reaction between the enzymatically hydrolyzed hydroxide (OH^-) ion and the K₃Fe(CN)₆ redox species that generating the side products (Fe(OH)₃, CN^- ion, and KCN), which impeded the electron transfer of the redox mediator at electrode-electrolyte interface. In view of the amount of side products produced was proportional to the urea concentration associated in the enzymatic reaction with the immobilized urease enzymes, this has allowed the proposed enzymatic biosensor to demonstrate an inverse sensitivity concept of detecting urea concentration in an ultrasensitive manner. The electron transfer rate constant (k) of the urease electrode based on aniline/nBA hybrid material at the electrode-electrolyte interface was determined at 5.374×10^-5 cm s^-1. The linear response range of the enzymatic urea biosensor was obtained from 1×10^-10 mM to 1×10^-1 mM (R²=0.9834) by differential pulse voltammetry (DPV) with a limit of detection of 4.72×10^-11 mM at pH 5.0 and enzymatic hydrolysis time of 30 min. The voltammetric urea biosensor response showed good reproducibility with a promising relative standard deviation (RSD) acquired at 5.0% (n=9). The ultra-high sensitivity performance of the developed enzymatic biosensor based on aniline/nBA conducting polymer towards determination of urea concentrations at low levels has demonstrated superior performance across previously reported electrochemical urea biosensors based on various nanostructured conducting materials.

Keywords: Conducting polymer; electrochemical transducer; succinimide-modified polymer; urea biosensor

Abstrak

Biosensor elektrokimia enzimatik untuk pengesanan urea telah dibangunkan menggunakan suksinimida terubah suai polimer berkonduksi anilina/n-butil akrilat (nBA). Polimer berkonduksi anilina/nBA ini adalah disintesis melalui pempolimeran foto dengan bahagian suksinimida digabungkan semasa prosedur pempolimeran foto. Enzim urease yang berasal daripada kacang Jack telah diikat secara kimia dengan kumpulan berfungsi suksinimida pada polimer berkonduksi anilina/nBA yang dipegun pada elektrod karbon bercetak skrin (SPE). Hidrolisis enzimatik urea oleh elektrod urease mengurangkan rangsangan biosensor voltammetrik daripada tindak balas kimia melata antara ion hidroksida (OH^-) terhidrolisis secara enzimatik dan spesies redoks K₃Fe(CN)₆ yang menghasilkan produk sampingan, (Fe(OH)₃, ion CN^- dan KCN), yang menghalang pemindahan elektron pengantara redoks pada antara muka elektrod-elektrolit. Memandangkan jumlah produk sampingan yang dihasilkan adalah berkadaran dengan kepekatan urea yang terlibat dalam tindak balas enzimatik dengan enzim urease pegun, ini telah membenarkan biosensor enzim yang dicadangkan untuk menunjukkan konsep kepekaan songsang untuk mengesan kepekatan urea secara ultrasensitif. Pemalar kadar pemindahan elektron (k) elektrod urease berasaskan bahan hibrid anilina/nBA pada antara muka elektrod-elektrolit diperoleh pada 5.374×10^-5 cm s^-1. Julat rangsangan linear biosensor urea enzimatik diperoleh daripada 1×10^-10 mM hingga 1×10^-1 mM (R²=0.9834) dengan kaedah voltammetri denyutan pembeza (DPV) dengan had pengesanan 4.72×10^-11 mM pada pH 5.0 dan masa hidrolisis enzimatik selama 30 min. Rangsangan biosensor urea voltammetrik menunjukkan kebolehulangan yang baik dengan sisihan piawai relatif (RSD) yang menggalakkan diperoleh pada 5.0% (n=9). Prestasi kepekaan ultra tinggi biosensor enzimatik yang dibangunkan berasaskan polimer berkonduksi anilina/nBA terhadap penentuan kepekatan urea pada kepekatan rendah telah menunjukkan prestasi unggul merentas biosensor urea elektrokimia yang dilaporkan sebelum ini berasaskan pelbagai bahan berkonduksi berstruktur nano.

Kata kunci: Biosensor urea; polimer berkonduksi; polimer terubah suai suksinimida; transduser elektrokimia

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*Corresponding author; email: alizarulianas@fmipa.unp.ac.id