Fluorometric gene assay for determination of Escherichia coli 0157:H7 using graphene quantum dots and carbon dots with gold and silver nanoparticles
Escherichia coli (E. coli) O157:H7 is considered as harmful bacteria which can result in severe infections to human. Current detection methods are prolonged and inefficient due to extensive sample preparation and lengthy test procedures. Thus, there is a requirement for simplicity of technique...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/97858/1/ITMA%202020%2016%20UPMIR.pdf |
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| Summary: | Escherichia coli (E. coli) O157:H7 is considered as harmful bacteria which can result in
severe infections to human. Current detection methods are prolonged and inefficient due
to extensive sample preparation and lengthy test procedures. Thus, there is a requirement
for simplicity of techniques which are capable of sensing E. coli O157:H7 at high
sensitivity, specific, less toxicity and agile. A fluorescence quenching assay involving
graphene quantum dots (GQDs) and carbon dots (CDs) with gold (AuNPs) and silver
(AgNPs) nanoparticles for the determination of E. coli O157:H7 have been explored.
GQDs and CDs act as the fluorophore, while AuNPs and AgNPs as the quencher. Short
target oligos (20 bp) have been utilized to establish distance between fluorophore and
quencher in close proximity. Then, the fluorophore and quencher were complexing
adjacently to trigger the fluorescence quenching mechanism following the target oligos
co-hybridization. The complex immediately returns to the ground state by absorption of
light without the release of photons. Several essential parameters such as reaction time
and wavelength maximum of emission have been optimized to enhance the efficiency of
fluorescence quenching. An excitation/emission wavelength of 400 nm/530 nm and 340
nm/450 nm were used for GQDs and CDs, respectively. The net intensity fluorescence
quenching of GQDs and CDs was enhanced proportionally with the increment
concentrations of target oligos. A linear correlation between the fluorescence quenching
of GQDs/CDs and the logarithm concentration of target oligos in the series of 0.1 nM to
150 nM (GQDs-AuNPs), 0.01 nM to 200 nM (CDs-AuNPs) and 0.001 nM to 200 nM
(CDs-AgNPs) (slope = 42.74, R2
= 0.991; slope = 675.6, R2 = 0.992; slope = 217.6, R2
= 0.977) and the detection limit (LOD) of 1.10 ± 0.58 nM, 1.00 ± 0.71 nM and 1.01 ±
0.71 nM, respectively. The proposed method was utilized for verification of selectivity
and specificity towards different oligonucleotide sequence and bacteria strain with
satisfactory results. The practicability of the assay was also verified by evaluating the
amplicon (fliC gene, 381 bp) of genomic DNA isolated from food samples spiked with
E. coli O157:H7. It is noteworthy that the determined t-value is less than the critical t�value (tcalc. < 2.78) indicating that the developed method and real time PCR method are
comparable and in good agreement. |
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