食品由来病原体を検出する電気化学的 DNA のバイオ センサーの開発

Foodborne pathogens contribute to a large proportion of public health problems globally, which significantly affect the rate of human mortality and morbidity. Conventional methods for the detection of foodborne pathogens require complicated sample handling and are time consuming. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection and practicality.

Development of biosensors is based on modified electrode with polylactic acid stabilized gold nanoparticles. Screen printed carbon electrode was modified to increase the working electrode active surface area. Methylene blue used as the redox complex has a strong bond with single stranded DNA of immobilized probe DNA.

This relationship of high afinity represented by a high peak current. Hybridization of single stranded DNA with its complementary sequence replaces the bonded methylene blue molecules, which reduces the current of the differential pulse voltammetry. This may have been due to a smaller amount of methylene blue gathered on the surface of modified electrode with complementary double stranded DNA, which was caused by unreachable interaction between guanine bases.

The color of prepared solutions change beginning from yellow to blackish and finally to dark ruby red. This indicated that the gold salt has been reduced by the citrate ions. The dissolved polylactic acid was mixed with the previously prepared gold nanoparticle solution and then homogeneously stirred at room temperature, which then denoted as polylactic acid stabilized gold nanoparticles.

Ultraviolet visible spectroscopy, x-ray diffraction, transmission electron microscopy, and field emission scanning electron microscopy with energy dispersive x-ray spectrometry were used to characterize the polylactic acid stabilized gold nanoparticles. About 25 microliters of the homogeneous solution of polylactic acid stabilized gold nanoparticles was quickly pipetted onto the screen printed carbon electrode and air dried for 24 hours prior to use. The modified electrodes were then electrochemically characterized in potassium ferrocyanide to measure active surface area, electrochemical impedance spectroscopy, repeatability, reproducibility, and stability.

Optimization of the immobilization condition was determined using three factors, the concentration of single stranded DNA ranging from 0.2 to 1.4 micromolar, time ranging from 30 to 220 minutes, and temperature ranging from 25 to 75 degrees Celsius. On the other hand, optimization of the hybridization condition was determined via two factors, which are time ranging from five to 35 minutes and temperature ranging from 25 to 75 degrees Celsius. The treated modified electrodes were subsequently immersed in 20 micromolar methylene blue for 30 minutes.

The non-specifically absorbed DNA and excess methylene blue were removed by washing with saline acetate buffer pH 4.5 and then rinsed with deionized water. The peak current of methylene blue reduction was measured using the differential pulse voltammetry technique. The differential pulse voltammetry measurement was executed using phosphate buffer saline pH 7.0 that contained no indicator.

A similar procedure was conducted for all interactions. Vibrio parahaemolyticus as reference strains and eight other bacterial strains, namely Campylobacter jejuni, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritis, Klebsiella pneumoniae, Escherichia coli O157:H7, Bacillus cereus, and Vibrio alginolyticus of the common foodborne pathogen were employed for the electrochemical DNA biosensor validation in this study. A routine subculture of bacterial strains on their respective agar was conducted every two weeks to maintain their viability.

The quantity of Vibrio parahaemolyticus cells was determined using a spread plate technique. One millimeter of the bacteria cell culture was transferred into nine millimeter of trypticase soy broth containing 3%sodium chloride to obtain a dilution factor of 10. Next, 0.1 milliliter of each dilution factor starting from to one time dilution factor of 10 to 10 times dilution factor of 10 was spread on a plate of CHROMagar Vibrio for colony counting, respectively.

The cockles were obtained from the wet market and then quickly brought to the laboratory in an ice cooler box for analysis. The cockles were divided into two groups, namely the treated and untreated group with the assumption that the cockles were harvested uniformly from the start of the harvest until placing them in cold storage at the market. Cockles in the treated group were pretreated by storing them at minus 20 degrees Celsius for 24 hours, followed by exposure to ultraviolet light at 20 degrees Celsius for four hours prior to DNA extraction.

The freezing temperature and ultraviolet exposure used for the control group was expected to kill it or at least limit the naturally accumulating foodborne pathogens in the cockles. A higher pasteurization regime of 70 degrees Celsius was not applied, as the aim of the controlled condition in this study was to mimic the actual situation of the fresh cockles. Meanwhile, samples from the untreated group were directly analyzed without any pretreatment as soon as they arrived in the laboratory.

Cockles were washed in distilled water and scrubbed free of dirt before removal of the tissues from the shells using sterile forceps and a laminar flow cabinet. About 10 grams of cockle tissue samples were homogenized with the homogenizer in 19 milliliters of sterile trypticase soy broth containing 3%sodium chloride for 60 seconds. A known amount of foodborne pathogens was then added to nine millimeters of the homogenized sample broth for the spiked samples.

The unspiked samples were used as a negative control. About one milliliter of samples were pipetted into an Eppendorf tube for DNA sample extraction which would be used for the biosensor and polymerase chain reaction assay. The genomic DNA of the foodborne pathogen was then extracted from the spiked and unspiked samples.

Genomic DNA was extracted following a modified boiled lysis procedure. The genomic DNA was centrifuged at 12, 000 rotations per minute for three minutes at four degrees Celsius to obtain a clear suspension and the supernatant kept at minus 20 degrees Celsius for further use. A biophotometer was used to determine the concentration and purity of the extracted genomic DNA.

The identity of all strains was then determined using standard biochemical assays and verified by 16 SR RNA gene sequencing. Finally, the genomic DNA was denatured at 92 degrees Celsius for two minutes and rapidly cooled in ice water prior to application of the biosensor. Further confirmation of foodborne pathogens was done using polymerase chain reaction targeting the respective gene.

The amplified product and their sizes were determined via electrophoresis on 1.5%agarose gel. The gel images were captured using a gel documentation system. The generation of polylactic acid stabilized gold nanoparticles was confirmed from the ultraviolet visible spectra, which the growth of the surface plasma and resonance peak was founded around 540 nanometers.

The formation and existence of polylactic acid stabilized gold nanoparticles was indicated at 500 to 600 nanometer wave length ranges, depending on particle size. All crystallite peaks of polylactic acid stabilized gold nanoparticles were observed at the two theater at the 31.7, 38.2, 44.4, 64.7, and 77.7 degrees. The crystallite peak intensities also increased as broader diffraction peaks entered at 31.7 degrees, implying that the cold nanoparticles were embedded in the polylactic acid and suggesting the formation of polyphasic gold nanostructures.

From the transmission electron microscopy particle distribution curve and images of polylactic acid stabilized gold nanoparticles, it could be seen that gold nanoparticles are almost spherical in shape. The size of the nanoparticles was in range of about 37 nanometers, which is slightly bigger than that obtained from Scherrer's formula, suggesting a poly-crystalline nature of the as synthesized nanoparticles. The main size of polylactic acid stabilized gold nanoparticles was calculated using the Sherrer's equation by determining the width of the 100 Bragg's reflection.

From the table, the crystallite size of polylactic acid stabilized gold nanoparticles was calculated as 27 nanometers. From the field emission scanning electron microscopy images and with energy dispersive x-ray spectra, the modified screen printed carbon electrode had the highest weight percentage of gold, greatest density, and largest structure of nanoparticle coverage. The energy dispersive x-ray spectrum confirmed that the as produced polylactic acid stabilized gold nanoparticles were composed of gold only and displayed no other element except the peak corresponding to carbon and oxygen.

The anodic and cathodic peak potentials were consistent at different scan rates suggesting that the electrochemical reaction was reversible based on peak separation. The active surface area of the modified electrodes was calculated as 0.26 centimeters squared and 0.41 centimeters squared for the bare electrode and modified electrode, respectively. This result confirmed that the improvement afforded by polylactic acid stabilized gold nanoparticles on the active surface area was relatively higher compared with the bare electrode.

The value of charge transfer resistance for the bare electrode was 1, 932 ohm, while the modified electrode decreased to 1, 444 ohm. The decrease of charge transfer resistance value in modified electrode may have resulted from a decrease in local dielectric constant and/or an increase in the thickness of the electric double layer, suggesting that potassium ferrocyanide functioned by absorption at the metal or solution interface. The peak currents increased progressively with the modification of the bare electrode using polylactic acid stabilized gold nanoparticles which showed that a higher sensitivity was the inverse of the lower charge transfer resistance.

The relative standard deviation of modified electrode was derived as 4.26%The electrode with polylactic acid stabilized gold nanoparticles modification gave better relative standard deviation after several measurements. Relative standard deviation values of 4.05%for modified electrode indicating better reproducibility of the electrode fabrication for polylactic acid stabilized gold nanoparticles. The optimized concentration of the DNA was 1.2 micromolar single stranded DNA.

The optimum immobilization time of the single stranded DNA was selected as 180 minutes. The optimum immobilization temperature of the single stranded DNA was selected as 55 degrees Celsius. The optimum hybridization time of the double stranded DNA was selected as 10 minutes.

The optimum hybridization temperature of the double stranded DNA was selected as 35 degrees Celsius. Complementary DNA exhibited the lowest peak current of 0.73 microampere among the probed DNA, non-complementary DNA, three bases mismatched DNA, and one base mismatched DNA. It is obvious that the high current from the DNA that was targeted is smaller than the current of the other sequences of oligonucleotide.

This result indicated that the biosensor could be used to discriminate with high sensitivity and specificity. The constructed DNA biosensor had a selectivity of hybridization detection via immobilized probe DNA on the surface of the modified electrode. Continuous decrease in differential pulse voltammetry peak current of methylene blue on the modified electrode was triggered as a result of an increase in concentration of complementary DNA.

A linear correlation between the log of various target DNA concentrations 0.2 picomolar to 0.02 millimolar and the log of the methylene blue reduction peak current is depicted with a linear regression coefficient of 0.96. The percentage recovery for the fabricated DNA biosensor slowly decreased to not less than 80%of its initial signal after six months of storage, indicating that the probe of single stranded DNA on the surface of modified electrode is highly stable. Good long term stability at a temperature below 45 degrees Celsius might be the reason for the strong reaction between polylactic acid stabilized gold nanoparticles and single stranded DNA.

The polymerase chain reaction amplification products for nine different species of bacteria, which were Bacillus cereus, Listeria monocytogenes, Campylobacter jejuni, Escherichia coli, Vibrio alginolyticus, Salmonella typhimurium, Salmonella enteritis, Klebsiella pneumoniae, and Vibrio parahaemolyticus were screened. The differential pulse voltammetry peak current obtained after a cross reactivity assessment shows a very clear detection of Vibrio parahaemolyticus in comparison with other bacteria species. It can be seen by decreasing the number of base pairs, the observed differential pulse voltammetry signal increases.

This is related to the larger quantity of methylene blue molecules down to the probes. From the polymerase chain reaction analysis, Vibrio parahaemolyticus was found in both the treated and untreated spiked samples, as shown by lane seven, eight, nine, 10, 11, and 12. Whereas the treated and untreated unspiked samples at lane one, two, three, four, five, and six were devoid of Vibrio parahaemolyticus.

The detection results using the developed DNA biosensor which successfully determined the presence of Vibrio parahaemolyticus in the treated group consisting of spiked and unspiked samples. These results positively correlate with the previously discussed polymerase chain reaction results. The application of polylactic acid stabilized gold nanoparticles has a great potential for application as a modify for further sense of fabrication.

A high selectivity could be achieved when the redox complex binds more strongly with the un-hybridized biorecognition molecule, which may be an innovation worth following up on.