Determination of minimal sequence for zearalenone aptamer by computational docking and application on an indirect competitive electrochemical aptasensor.

Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (Kd = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor.

Electrochemical determination of zearalenone using a label-free competitive aptasensor.

An electrochemical aptasensor is described for determination of the phytohormone of zearalenone (ZEA). The gold electrode was modified with ZEA via covalent attachment using cysteamine-hydrochloride and 1,4-phenylene diisocyanate linker. A truncated ZEA aptamer with a dissociation constant of 13.4 ± 2.1 nM was used in an aptasensor. The electrochemical property was investigated using square wave voltammetry for monitoring the change in the electron transfer using the ferro/ferricyanide system as redox probe. Under optimal experimental conditions, the response was best measured at a potential of 0.20 V (vs. Ag/AgCl). The signals depended on the competitive mechanism between the immobilised ZEA and free ZEA for the aptamer binding site. The aptasensor works in the range 0.01 to 1000 ng·mL-1 ZEA concentration, with a detection limit of 0.017 ng·mL-1. High degree of cross-reactivity with the other analogues of ZEA was observed, whereas none towards other mycotoxins. The aptasensor was further applied for the determination of ZEA in the extract of maize grain and showed good recovery percentages between 87 and 110%. Graphical abstract Schematic representation of the electrochemical determination of zearalenone based on indirect competitive assay. Step a Immobilisation of ZEA on the surface of gold electrode via covalent attachment, b competition for the ZEA aptamer binding site between immobilised and free ZEA, and c current signal of the binding event based on SWV technique.

Aspergillus section Flavi and Aflatoxins: Occurrence, Detection, and Identification in Raw Peanuts and Peanut-Based Products Along the Supply Chain.

Aflatoxin contamination in foods is a global concern as they are carcinogenic, teratogenic and mutagenic compounds. The aflatoxin-producing fungi, mainly from the Aspergillus section Flavi, are ubiquitous in nature and readily contaminate various food commodities, thereby affecting human's health. The incidence of aflatoxigenic Aspergillus spp. and aflatoxins in various types of food, especially raw peanuts and peanut-based products along the supply chain has been a concern particularly in countries having tropical and sub-tropical climate, including Malaysia. These climatic conditions naturally support the growth of Aspergillus section Flavi, especially A. flavus, particularly when raw peanuts and peanut-based products are stored under inappropriate conditions. Peanut supply chain generally consists of several major stakeholders which include the producers, collectors, exporters, importers, manufacturers, retailers and finally, the consumers. A thorough examination of the processes along the supply chain reveals that Aspergillus section Flavi and aflatoxins could occur at any step along the chain, from farm to table. Thus, this review aims to give an overview on the prevalence of Aspergillus section Flavi and the occurrence of aflatoxins in raw peanuts and peanut-based products, the impact of aflatoxins on global trade, and aflatoxin management in peanuts with a special focus on peanut supply chain in Malaysia. Furthermore, aflatoxin detection and quantification methods as well as the identification of Aspergillus section Flavi are also reviewed herein. This review could help to shed light to the researchers, peanut stakeholders and consumers on the risk of aflatoxin contamination in peanuts along the supply chain.

Etlingera elatior-Mediated Synthesis of Gold Nanoparticles and Their Application as Electrochemical Current Enhancer.

This work presents a simple green synthesis of gold nanoparticles (AuNPs) by using an aqueous extract of Etlingera elatior (torch ginger). The metabolites present in E. elatior, including sugars, proteins, polyphenols, and flavonoids, were known to play important roles in reducing metal ions and supporting the subsequent stability of nanoparticles. The present work aimed to investigate the ability of the E. elatior extract to synthesise AuNPs via the reduction of gold (III) chloride hydrate and characterise the properties of the nanoparticles produced. The antioxidant properties of the E. elatior extract were evaluated by analysing the total phenolic and total flavonoid contents. To ascertain the formation of AuNPs, the synthesised particles were characterised using the ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) microscopy, and dynamic light scattering (DLS) measurement. The properties of the green synthesised AuNPs were shown to be comparable to the AuNPs produced using a conventional reducing agent, sodium citrate. The UV-Vis measured the surface plasmon resonance of the AuNPs, and a band centered at 529 nm was obtained. The FTIR results proved that the extract contained the O-H functional group that is responsible for capping the nanoparticles. The HRTEM images showed that the green synthesized AuNPs were of various shapes and the average of the nanoparticles' hydrodynamic diameter was 31.5 ± 0.5 nm. Meanwhile, the zeta potential of -32.0 ± 0.4 mV indicates the high stability and negative charge of the AuNPs. We further successfully demonstrated that using the green synthesised AuNPs as the nanocomposite to modify the working surface of screen-printed carbon electrode (SPCE/Cs/AuNPs) enhanced the rate of electron transfer and provided a sensitive platform for the detection of Cu(II) ions.

Electrochemical Immunosensor for Detection of Aflatoxin B1 Based on Indirect Competitive ELISA.

Mycotoxins are the secondary toxic metabolites produced naturally by fungi. Analysis of mycotoxins is essential to minimize the consumption of contaminated food and feed. In this present work, an ultrasensitive electrochemical immunosensor for the detection of aflatoxin B1 (AFB1) was successfully developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Various parameters of ELISA, including antigen⁻antibody concentration, blocking agents, incubation time, temperature and pH of reagents, were first optimized in a 96-well microtiter plate to study the antigen⁻antibody interaction and optimize the optimum parameters of the assay. The optimized assay was transferred onto the multi-walled carbon nanotubes/chitosan/screen-printed carbon electrode (MWCNTs/CS/SPCE) by covalent attachment with the aid of 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Competition occurred between aflatoxin B1-bovine serum albumin (AFB1⁻BSA) and free AFB1 (in peanut sample and standard) for the binding site of a fixed amount of anti-AFB1 antibody. Differential pulse voltammetry (DPV) analysis was used for the detection based on the reduction peak of TMB(ox). The developed immunosensor showed a linear range of 0.0001 to 10 ng/mL with detection limit of 0.3 pg/mL. AFB1 analysis in spiked peanut samples resulted in recoveries between 80% and 127%. The precision of the developed immunosensor was evaluated by RSD values (n = 5) as 4.78% and 2.71% for reproducibility and repeatability, respectively.

Electrochemical Immunosensor for the Detection of Aflatoxin B1 in Palm Kernel Cake and Feed Samples.

Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B1 (AFB1) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB1 was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement. N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB1-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB1-BSA and free AFB1 for the binding site of a fixed amount of anti-AFB1 antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox) was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB1 in real samples.