Development of a Novel Quantum Dots and Graphene Oxide Based FRET Assay for Rapid Detection of invA Gene of Salmonella

Abstract

A novel, rapid and simple fluorescence resonance energy transfer (FRET) based Salmonella specific gene, invA, detection system was developed, in which quantum dots (QDs) and graphene oxide (GO) worked as fluorescent donor and quencher, respectively. By measuring the fluorescence intensity signal, the Salmonella specific invA gene could be sensitively and specifically detected with a limit of detection (LOD) of ∼4 nM of the invA gene in 20 min. The developed system has the potential to be used for Salmonella detection in food and environmental samples and further developed into a platform for detection of other bacterial pathogens.

Keywords: FRET assay; Salmonella; graphene oxide; invA; quantum dots; rapid detection.

FIGURE 1

Principle of GO-QDs FRET biosensor. In the system, carboxyl QDs (donor) and GO (quencher) were first conjugated with the capture probes B and A, respectively, with the aid of EDC/NHS. Upon the addition of the complementary invA oligo of Salmonella, the QD and GO conjugates could be brought into close proximity to make the FRET pair work, the energy emitted from excited QDs would be quenched by GO.

FIGURE 2

The emission spectra comparison between QDs and QD-capture B conjugate. The QD and QD-capture B conjugate were diluted in 1xPBS, pH = 7.4. The excitation wavelength was set at 320 nm.

FIGURE 3

The BSA passivation effect and quenching efficiency of the developed invA gene biosensor. (A) The BSA passivation effect in decreasing the non-specific adsorption between GO and QD conjugates. Briefly, in order to check the BSA passivation effect in preventing unspecific binding between QD and GO conjugates, the GO-capture A conjugate was further treated with or without 0.5 mg/mL BSA at RT for 30 min and then rinsed with DI-H₂O, then the fluorescence intensity was measured. The data were analyzed by OriginPro 8.5. (B) For the quenching efficiency assays, in 50 μl reaction volume, BSA passivated GO-capture A (60 μg/mL) was first incubated with serially diluted invA oligo at 55°C for 10 min, then 150 nM QD-capture B was added to the reaction mixture and incubated at 55°C for another 10 min. The fluorescence intensity was measured under 320 nm excitation wavelength and the values at 520 nm were extracted for the calculation of quenching efficiency. The only difference between the experimental assays and the control assays was that the BSA passivated GO-capture A which was included in the experimental assays was replaced by BSA passivated GO (without capture A) in the control assays. The SEM (Standard Error of the Mean) error bars were calculated from at least three replicates. The data were analyzed by GraphPad Prism.

FIGURE 4

The limit of detection (LOD) of the developed invA gene biosensor. The quenching efficiencies which were calculated as according to the listed formula were potted versus different concentrations of invA oligo. LOD = 3^∗S/k (S means standard deviation of negative control, k means slope).The SEM (Standard Error of the Mean) error bars were calculated from at least three replicates.

FIGURE 5

The specificity of the developed invA gene biosensor. The concentrations of invA oligo (fully complementary to probes), M1 (one-base mismatch), M2 (two-base mismatch) and control oligo (not complementary to neither probes) used were fixed at 400 nM. The SEM (Standard Error of the Mean) error bars were calculated from at least three replicates. The difference between invA oligo and control oligo groups was significant (P < 0.0001, two tail t-test), but the difference between invA oligo and M1 or M2 groups were not significant (P > 0.05, two tail t-test). The data was analyzed by GraphPad Prism. ^∗∗∗P < 0.0001.

FIGURE 6

Comparison between the ability of the invA gene PCR product and synthesized invA oligo in mediating changes in fluorescence intensity of the developed biosensor. In the control sample, only GO-capture A and QD-capture B were included, no invA gene PCR product or invA oligo was added to the reaction mixture.