Malaysian Journal of Analytical Sciences, Vol 26 No 6 (2022): 1191 - 1204

 

DEVELOPMENT OF AN ELECTROCHEMICAL IMMUNOSENSOR STRIP FOR EARLY DETECTION OF RICE BACTERIAL LEAF BLIGHT (BLB) DISEASE AND ITS APPLICATION ON A PORTABLE DEVICE

 

(Pembangunan Strip Imunosensor Elektrokimia Untuk Pengesanan Awal Penyakit Hawar Daun Bakteria Padi dan Aplikasinya Pada Peranti Mudah Alih)

 

Hazana Razali1, Norhafniza Awaludin1, Nurul Hidayah Husin1, Sahira Akmar Zulkepli1, Rafidah Abd Rahman1, Mohammad Rejab Ismail1, Kogeethavani Ramachandran2, Faridah Salam1,  Nur Azura Mohd Said1*

 

1Biotechnology & Nanotechnology Research Centre,

MARDI Headquarter, Persiaran MARDI-UPM, 43400 Serdang, Selangor, Malaysia

2Paddy& Rice Research Centre,

MARDI Seberang Perai, 13200 Seberang Perai, Pulau Pinang

 

*Corresponding author : nazurams@mardi.gov.my

 

 

Received: 8 February 2022; Accepted: 27 April 2022 ; Published:  27 December 2022

 

 

Abstract

We described here an electrochemical immunosensor strip based on a screen-printed carbon electrode (SPCE) for early detection of rice bacterial leaf blight (BLB) disease. The causal agent for this destructive disease has been identified as Xanthomonas oryzae pv. oryzae (Xoo). In order to circumvent the disease outbreak, an early detection system is required. Polyclonal antibody against Xoo was employed and immobilized on the SPCE strips modified with polypyrrole (PPy) and functionalized multi-walled carbon nanotube (fMWCNT) network. The anti-Xoo antibody is conjugated with horseradish peroxidase (HRP) as an enzyme label and used as the detection agent in the sensor development. Electrochemical detection was carried out via the chronoamperometry technique at a set potential of -200 mV. A fixed anti-Xoo antibody concentration at 0.03 mg/mL on the working electrode of the strip surface produced a standard linear curve for Xoo detection (R2 = 0.9746). Two extraction methods for rice leaves (scissors-cutting and grinding) were compared for real samples application analysis. The scissors-cutting method had less matrix interference effect and gave a higher recovery rate than the grinding method. The optimal immunosensor configuration was then compared with the PCR technique for Xoo detection in inoculated leaves in a controlled environment. A good correlation of 92.7% was achieved between the two methods. The immunosensor strips were then tested on an Android-based portable biosensor device for on-site detection of BLB in hotspot areas at Bagan Terap, Selangor Northwest and Sg. Burong, Tanjung Karang. On-field detection has indicated that the immunosensor strips can detect BLB disease as early as 15 days after transplant (DAT) before symptoms appear.

 

Keywords: bacterial leaf blight, early detection, immunosensor, rice disease,  Xanthomonas oryzae pv.oryzae

 

 

Abstrak

Satu strip imunosensor elektrokimia berasaskan elektrod karbon bercetak skrin (SPCE) untuk pengesanan awal penyakit hawar daun bakteria (BLB) pada pokok padi dilaporkan dalam kajian ini. Agen penyakit padi ini telah dikenalpasti disebabkan oleh bakteria Xanthomonas oryzae pv. oryzea (Xoo). Bagi mengekang penyebaran penyakit padi ini, satu sistem pengesanan awal penyakit padi adalah diperlukan. Antibodi poliklonal terhadap Xoo digunakan dalam kajian ini dan dipegunkan ke atas permukaan strip yang telah diubah suai dengan jaringan polipirol (PPy) dan nanotiub karbon berbilang dinding berfungsi (fMWCNT). Antibodi anti-Xoo dikonjugasikan dengan horseradish peroxidase (HRP) sebagai label enzim; dan digunakan sebagai agen pengesanan dalam pembangunan sensor. Pengesanan elektrokimia dilakukan dengan menggunakan teknik kronoamperometri pada set potensi -200 mV. Pada kepekatan antibodi 0.03 mg/mLyang dipegunkan pada permukaan elektrod bekerja strip, satu graf piawai linear untuk pengesanan Xoo berjaya dibangunkan (R2 = 0.9746). Bagi aplikasi sampel sebenar, dua kaedah pengekstrakan daun padi (menggunting daun dan pengisaran daun) telah dibandingkan. Kaedah menggunting daun didapati memberikan kesan matriks yang lebih rendah dan kadar pulangan yang lebih tinggi berbanding kaedah pengisaran daun. Kaedah sensor ini kemudian dibandingkan dengan teknik PCR bagi analisis sampel daun teraruh dengan bakteria Xoo dalam persekitaran yang terkawal. Korelasi yang baik dengan nilai 92.7% telah dicapai bagi kedua-dua kaedah tersebut. Strip imunosensor ini kemudiannya diuji pada satu peranti biosensor mudah alih berasaskan Android untuk pengesanan BLB di lapangan di kawasan titik panas Bagan Terap, Barat Laut Selangor dan Sg. Burong, Tanjung Karang. Kajian lapangan menunjukkan bahawa strip pengesanan imunosensor ini berupaya mengesan penyakit BLB seawal hari ke-15 selepas transplan (HLT) sebelum kemunculan simptom penyakit.

 

Kata kunci: hawar daun bakteria, pengesanan awal, imunosensor, penyakit padi, Xanthomonas oryzae pv. oryzae (Xoo)

 

Graphical Abstract

 

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