Malaysian Journal of Analytical Sciences Vol 20 No 5 (2016): 1020 - 1032

DOI: http://dx.doi.org/10.17576/mjas-2016-2005-06

 

 

 

FABRIKASI BIOSENSOR DNA PORSIN BERASASKAN KOMPLEKS RUTENIUM BIPIRIDINA

 

(Fabrication of Porcine DNA Biosensor Based on Ruthenium Bipyridine Complex)

 

Nurul Izni Abdullah Halid¹, Emma Izzati Zakariah¹, Lee Yook Heng¹, Nurul Huda Abd Karim¹, Haslina Ahmad², Siti Aishah Hasbullah¹*

 

¹Pusat Pengajian Sains Kimia dan Teknologi Makanan, Fakulti Sains dan Teknologi,

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

²Jabatan Kimia, Fakulti Sains,

Univesiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

 

*Pengarang utama: aishah80@ukm.edu.my

 

 

Received: 17 February 2016; Accepted: 9 June 2016

 

 

Abstrak

Biosensor DNA elektrokimia bagi pengesanan jujukan DNA porsin berasaskan kompleks rutenium (II) sebagai label aktif redoks telah dibangunkan. Sistem biosensor difabrikasi berdasarkan elektrod bercetak skrin (SPE) terubahsuai dengan zarah nano emas (AuNPs) sebagai transduser yang dipegunkan bersama mikrosfera poli (n-butilakrilat-N-akriloksisuksnimida) dan jujukan DNA prob terikat melalui ikatan kovalen. Kompleks interkalator rutenium (II), [Ru(bpy)₂PIP]²⁺ dan (bpy = bipiridina, PIP = 2-fenillimidazo[4,5-f[1,10-fenantrolina] digunakan dalam pengesanan DNA porsin. Pengukuran dan pengoptimuman biosensor telah dilakukan dengan menggunakan voltammetrik siklik (CV) dan voltammetrik denyutan pembezaan (DPV). Interaksi antara [Ru(bpy)₂PIP]²⁺ dengan beberapa jenis jujukan DNA telah dikaji iaitu, i) bebenang tunggal DNA prob, ii) selepas penghibridan DNA sasaran dan iii) selepas penghibridan dengan DNA bukan sasaran. Keputusan menunjukkan bahawa interaksi antara [Ru(bpy)₂PIP]²⁺ dengan DNA sasaran yang telah terhibrid memberikan rangsangan yang paling tinggi. Prestasi biosensor adalah dipengaruhi oleh beberapa faktor antaranya ialah kepekatan [Ru(bpy)₂PIP]²⁺ dan DNA prob, masa pemegunan dan penghibridan DNA prob, pH, kekuatan ion, kepekatan larutan penimbal dan suhu juga dikaji untuk menilai prestasi biosensor. Hasil kajian menunjukkan kepekatan kompleks [Ru(bpy)₂PIP]²⁺ dan DNA prob, masing-masing adalah 50 µM dan 2 µM. Masa yang optimum bagi pemegunan DNA prob dan penghibridan DNA sasaran adalah 7 jam dan 60 minit. Keadaan optimum bagi pH, kekuatan ion, kepekatan larutan penimbal dan suhu, masing-masing adalah pH 7.0, 1.0 M NaCl, 0.05 M potassium fosfat pada suhu 25°C. Julat linear bagi kepekatan DNA sasaran yang berbeza adalah antara 1.0 x 10^-13 M to 1.0 x 10^-8 M. Kajian ini adalah yang pertama melaporkan tentang penggunaan [Ru(bpy)₂PIP]²⁺ bagi pengesanan DNA porsin

 

Kata kunci:  biosensor, DNA porsin, kompleks rutenium bipiridina

 

Abstract

Electrochemical DNA biosensor for detection of porcine oligonucleotides based on ruthenium (II) as label redox complex has been developed. The system of biosensor is based on the modified screen printed carbon electrode (SPE) with goldnanoparticles (AuNPs) as transducer immobilized with poly(n-butylacrylate-N-acryloxysuccinimide) microsphere and porcine DNA probe sequences was attached onto it via the covalent bond. The ruthenium(II) complex, [Ru(bpy)₂PIP]²⁺ PIP = 2-phenylimidazo[4,5-f[1,10-phenanthroline] intercalator has been been used to determine prcine DNA. The biosensor was measured and optimized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The interaction of [Ru(bpy)₂PIP]²⁺ with various DNA with different sequences, i) single stranded probe DNA, ii) after hybridization with its complementary DNA and iii) after hybridization with mismatch complementary DNA were studied. The results indicated that the interaction of [Ru(bpy)₂PIP]²⁺ with hybridized complementary DNA gave the highest response. Thus, development of porcine DNA biosensor and influences of many factors such as [Ru(bpy)₂PIP]²⁺ complex and DNA probe concentration, DNA probe immobilization and hybridization time, pH, ionic strength, buffer concentration and temperatures were also studied to evaluate the performance of biosensor. The concentration of [Ru(bpy)₂PIP]²⁺ complex and DNA probe were found to be optimal at 50 µM and 2 µM, respectively. The optimal time for DNA probe immobilization and hybridization were 7 hours and 60 minutes, accordingly. The optimal condition of pH, ionic strength, buffer concentration and temperatures were at pH 7.0, 1.0 M NaCl, 0.05 M of Na-phosphate buffer and 25 °C, respectively. The linear range of different concentration complementary DNA was within the range between 1.0 x 10^-13 M to 1.0 x 10^-8 M. This study was first reported the used of [Ru(bpy)₂PIP]²⁺ for detection of porcine oligonucleotides

 

Keywords:  biosensor, porcine DNA, ruthenium bypyridine complex

 

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