Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Summary

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (R_ct) value and an increase of active surface area (0.41 cm²). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

Introduction

A major topic of public and scientific debate in recent years, food poisoning is mainly associated with 3 agents: microorganisms¹, chemicals², and parasites³. Contaminated food can cause serious health consequences in humans, especially in the higher risk group of those with weak immune systems, the elderly, pregnant women, babies, and young children⁴. With more than a million cases of acute diarrhea occurring annually in children under 5 years old in Africa, Asia, and Latin America, food poisoning is a major global disease^5,

Protocol

NOTE: All the chemical and biochemical reagents to be used should be of analytical grade and used without further purification. Prepare all solutions using sterile deionized water. Autoclave all glassware prior to sterilization.

Caution: Please use all appropriate safety practices when performing laboratory activities including the use of engineering controls (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).

1. Fabrication and Characterization of Modified Electrode using PLA-AuNPs

1. Preparation and characterization of....

Results

Formation of AuNPs was revealed through the change in color of the aqueous solution with sodium citrate present. This caused the color to change from light yellow to a deep ruby red. The generation of PLA-AuNPs was confirmed from the UV-vis spectra (Figure 1) where the growth of the surface plasmon resonance (SPR) peak was found at around 540 nm. The formation and existence of PLA-AuNPs was indicated at 500-600 nm wavelength ranges, depending on particle size.......

Discussion

The critical steps in a framework for successful development of this type of electrochemical biosensor are selection of appropriate biological recognition elements for the transducer (nucleic acid or DNA here); chemical approach for constructing the sensing layer of the transducer; transduction material; optimization of DNA immobilization and hybridization; and validation of the developed biosensor using real samples.

Core to the successful development of a sensitive and selective electrochemi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Merck	100056
Chloroform	Merck	102445
Diaminoethane tetraacetic acid	Promega	E5134
Dibasic sodium phosphate	Sigma-Aldrich	S9763
Disodium hydrogen phosphate	Sigma-Aldrich	255793
Ethanol	Sigma-Aldrich	16368
Gold (III) chloride trihydrate	Sigma-Aldrich	520918
Hydrochloric acid	Merck	100317
Methylene blue	Sigma-Aldrich	M44907
Monobasic sodium phosphate, monohydrate	Sigma-Aldrich	S3522
Phosphate-buffered saline	Sigma-Aldrich	P5119
Poly(lactic acid) resin, commercial grade 4042D	NatureWorks	4042D
Potassium chloride	R&M Chemicals	59435
Potassium dihydrogen phosphate	Sigma-Aldrich	P9791
Potassium hexacyanoferrate III	R&M Chemicals	208019
Sodium acetate anhydrous salt	Sigma-Aldrich	S2889
Sodium chloride	Sigma-Aldrich	S9888
Trisodium citrate	Sigma-Aldrich	S1804
Tris(hydroxymethyl) aminomethane	Fisher Scientific	T395-100
Tris-Base	Fisher Scientific	BP152-500
2X PCR Master Mix with Dual-Dye	Norgen Biotek	28240
Agarose gel	Merck	101236
Bolton Agar	Merck	100079
Bolton Broth	Merck	100079
CHROMagar Vibrio	CHROMagar	VB910
dNTPs	Promega	U1511
Nuclease-free water	Thermo Scientific	R0581
Eosin methylene blue agar	Merck	101347
GelRed	Biotium	41001
Glycerol	Merck	104092
Go Taq Buffer	Promega	M7911
Loading dye 100 bp DNA ladder	Promega	G2101
Loading dye 1kb DNA ladder	Promega	G5711
Magnesium chloride	Promega	91176
Mannitol egg yolk polymyxin agar	Merck	105267
McConkey Agar	Merck	105465
Nutrient Broth	Merck	105443
Taq polymerase	Merck	71003
Trypticase Soy Broth	Merck	105459
Trypticase Soy Agar	Merck	105458