| The varieties and plant areas of transgenic soybeans were increasing since the first transgenic soybean was successfully developed. Focuses on these considerations of security and environmental impact have been heightened in recent years. Therefore, it is essential to develop the fast,sensitive, high throughput and automatic detection methods to protect consumers' rights and interests. This has become the research highlights at home and abroad. It is significant to improve the transgenic detection technology, control the security of transgenic soybean and maintain social stability. In this study, these photoelectrochemical (PEC) and fluorescent biosensors were developed to detect the promoter of cauliflower mosaic virus 35s (P35S) or / and terminator nopaline synthase (TNOS) based on the fluorescent material of quantum dots. By comparing these approved methods, the optimum method was selected to combine with machine vision to fabricate the fast, sensitive, high throughput and automatic detection device for the transgenic soybean. The detailed researches were as follows:1. The gold nanoparticles-reduce grapheme oxide (AuNPs-rGO) was used to be substrate which was modified onto the surface of electrode to fix abundant thiol-functional probes to form the fixed probes. Silica nanospheres (SiO2) with good monodispersity and uniform structure were employed as the carrier for CdTe QDs to achieve the core-shell SiO2@CdTe nanoparticles. The amino-functional probes were connected with the SiO2@CdTe to form the capture probes. The fixed probes and capture probes were hybridized with the two terminals of target DNA of P35S through the high specific DNA hybridizationto fabricate the sandwich structure PEC biosensor for transgenic soybean detection. The study found that the photocurrent of SiO2@CdTe was 2.5-fold of CdTe,and the AuNPs-rGO possessed the high specific surface area. These two merits were employed to be dual signal amplification strategy and the DNA probes acted the recognition elements for the PEC biosensor with the advantages of good selectivity, sensitivity, reproducibility and stability. By optimizing the conditions, the PEC biosensor showed a wide linear range of 0.1 pM?500 pM and low limit of detection (LOD) of 0.05 pM (S/N=3), and it was used to detect real transgenic soybean samples.2. A novel homogeneous fluorescence resonance energy transfer(FRET) assay with nitrogen-doped graphene quantum dots (NGQDs) and silver nanoparticles (AgNPs) as the fluorescence resonance energy donor-acceptor pairs was developed for P35S of transgenic soybeans gene detection. The amino-functional probes were connected with the NGQDs to form the capture probes by amidation reaction. The thiol-functional probes were fixed onto the surface of AgNPs to act the fixed probes. Upon the recognition of target DNA of P35S,the distance between NGQDs and AgNPs was narrowed by specific DNA hybridization between probes and P35S to trigger FRET to occur the generation of fluorescence quenching. Under the optimized conditions,the fabricated homogeneous FRET assay displayed a wide linear range of 0.1?500.0 nM and a low LOD 0.03 nM (S/N = 3). This proposed assay revealed high specificity to detect P35S with acceptable precision and excellent stability. This method was successfully applied to identify the real transgenic soybean.3. A quantum dots (QDs) multifunctional method for P35S and TNOS detection simultaneously has been developed using Fe3O4@Au magnetic bead (MBs) as the recognition and concentration element with a heavy CdTe QDs as the label. Multiple CdTe QDs were loaded both in and on a versatile SiO2 nanocarrier (QDs@SiO2@QDs) to produce a large amplification factor. The capture probes of TNOS (CT) and the capture probes of P35S (CP) were labeled onto the surface of different colors of QDs@SiO2@QDs through amidation reaction to form gQDs@SiO2@gQDs-CT and rQDs@SiO2@rQDs-CP, respectively. The thiolated fixed probes of TNOS (FT) and the thiolated fixed probes of P35S (FP) were conjugated on the MBs through Au-S covalent binding to form MBs-FT and MBs-FP. Upon the recognition of target DNA of TNOS and P35S, the specific DNA hybridizations were occurred between probes and P35S to connect the QDs@SiO2@QDs and MBs. After a simple magnetic separation, these QDs@SiO2@QDs which linked with the target DNA were separated from liquid to trigger the fluorescence signal decrease in the solution. Under the optimized conditions, the fabricated magnetic controlled assay exhibited a good dynamic range of 0.2 nM?800 nM for TNOS with the low LOD of 0.07 nM and 0.1nM?800 nM for P35S with LOD of 0.04 nM (S/N=3). By employing this trategy, an efficient magnetic controlled assay has been developed for real transgenic soybean detection.4.A simple, sensitive and selective assay was constructed based on homogeneous FRET between CdTe QDs and multiwalled carbon nanotubes@graphene oxide nanoribbons (MWCNTs@GONRs) to form the fluorescent "on-off-on" switching for simultaneous monitoring dual target DNAs of P35S and TNOS from transgenic soybean. The capture DNAs were immobilized with corresponding QDs to obtain strong fluorescent signals (turning on). The strong ?-? stacking interaction between single-stranded DNA (ssDNA) probes and MWCNTs@GONRs led to minimal background fluorescence due to the FRET process(turning off). The targets of P35s and TNOS were recognized by dual fluorescent probes to form double-stranded DNA (dsDNA) through the specific hybridization between target DNAs and ssDNA probes. And the dsDNA were released from the surface of MWCNTs@GONRs, which leaded the dual fluorescent probes to generate the strong fluorescent emissions (turning on). Therefore, this proposed homogeneous assay can be achieved to detect P35s and TNOS simultaneously by monitoring the relevant fluorescent emissions. Under the optimized conditions, the fabricated assay exhibited a good dynamic range of 1.5 nM?1000 nM for TNOS with the low LOD of 0.5 nM and 1.2 nM?900 nM for P35S with LOD of 0.35 nM (S/N=3). Moreover, this approved assay can distinguish complementary and mismatched nucleic acid sequences with excellent sensitivity, selectivity, stability and reproducibility. It has potential to be a tool for daily detection of genetically modified organism with the merits of feasibility and reliability.5. Through comparing these approved methods, a detection device was fabricated based on the CdTe QDs and machine vision for transgenic foreign DNA. The fluorescent "on-off-on" switching strategy was employed to trigger chromogenic reaction for target DNA of TNOS and P35S. The experimental result was analyzed by images collection,segmentation and processing of the data characteristics. This fabricated device could be employed to detect TNOS and P35 with the merits of simplicity, automation and intelligence. |
PDF Full Text Request