Ultrasensitive Colorimetric Method To Detect The Dioxin-like Chemicals By Gold Nanoparticles With Silver Enhancement

Gold nanoparticles are these particles with diameters between 1 and 100 nanometers. Gold nanoparticles have been used in biotechnology over the last 4 decades as immunocytochemical probes as well as biological tags. By functionalizing particles with oligonucleotides and tailoring their surface properties, there has developed a series of new and practical biodetection systems. Combined with the surface-based readouts and the property of the nanoparticles, the scanometric assay can detect the trace level of the nucleotide target without the enzyme amplification. The"bio-barcode"assay, which quantified the target molecule by amplifying the barcode DNA labeled on the surface of the nanoparticles, has paved its way to the trace level of the protein and nucleotide molecule detection field.Dioxin-like chemicals (DLCs), including polychlorinated dibenzo-p-dioxins (PCDDs), biphenyls (PCBs) and dibenzofurans (PCDFs), represent a large group of compounds. They are ubiquitous environmental pollutants which resistance to biological and chemical degradation. They are the chemicals with high bioaccumulation and biomagnifying in the food chain. It is necessary to strengthen the monitoring of DLCs and the High-resolution gas chromatography- High-resolution mass spectrometry (HRGC/HRMS) is considered as the golden standard for the identification and quantitation of DLCs. For the procedures are very costly and time-consuming, a variety of rapid and inexpensive screening bioassays capable of detecting and estimating the relative potency of complex mixtures of DLCs have been developed. Here we developed a new method for trace level of nucleotide target molecule detecting and quantifying based on the gold nanoparticle at first. This method can detect the nucleotide molecule at trace level in the sample. Then we bring the double-strand nucleotide probe including the core sequence of the Dioxin-Response-Element. We obtained the complex of TCDD-AhR-Arnt-DRE after the TCDD induction mechanism in vitro. After the complex was especially immobilized to the solid phase carrier by the anti- Arnt body, we obtained the amplified signal of the gold nanoparticle through the Silver Enhancement Solution and developed a new bio-analytic technology for DLCs detecting.Part one: Development of a novel nucleotide quantification method by the gold nanoparticleObjection: To develop a novel trace level of nucleotide target molecular quantification method based on the gold nanoparticle technology. Method: Gold nanopaticle probe, biotin-labeled probe and the target will form hybridization product with gold nanoparticle at the end and biotin at the other end of the double-strand product after incubation in the especial ion and temperature conditions for a time. Then the hybridization will be immobilized to the microplate through the avidin-biotin system. The Silver Enhancement Solution will be introduced after the microplate was washed by 1×PBS and 2×PBN to remove the free gold nanoparticle probes. The silver ions will be deoxidized to silver metal around the gold nanoparticles and the signal will be amplified. The signal was record by the Microplate Reader at 630nm as the OD value of the solution. Result: (Ⅰ) During the procedure, the color change of the solution was obviously according to the concentration of the target, while not observed for target-free groups. At a point time, the concentration of the target is higher the OD value is higher. (Ⅱ) In the range of 10-16 mol/l to 10-12 mol/l, the OD value of the solution induce by the target has concentration dependence at the same reaction time. The dose-response relationship curve about the target nuceotide concentration against the OD Value was set up and the R2=0.9713. We can detect the lowest concentration of 100aM for the single-strand target by this method. (Ⅲ) For the double-strand DNA include the target sequence, the same dose-response relationship curve can be obtain as the single-strand nucleotide. But we can only get the lowest concentration of 10fM for double-strand for the property of the double-strand. Conclusion: The gold nanoparticle technology using in the nucleotide quantification is a sensitive, rapid, simple and cost-effective method, and it can detect the even trace level of the target nucleotide specifically in the sample.Part two: Ultrasensitive colorimetric method to identify and quantify 2,3,7,8- TCDD by the gold nanoparticleObjection: To develop a ultrasensitive colorimetric method to identify and quantify 2,3,7,8- TCDD by the gold nanoparticles. Method: SD rat hepatic cytosol containing AhR and relative protein was prepared by centrifugation and was incubated with TCDD to transform the AhR. The mixture was incubated with the DRE-gold nanoparticle probe containing one DRE site to form the TCDD-AhR-Arnt-DRE complex. The complex was then immoblized on the surface of the Micro plate by the Arnt-antibody embedded previous. The unbinding probes in the mixture were then washed using 1×PBS and then 2×PBN solution. At last, it was treated with Silver Enhancement solution and read by Microplate Reader at 630nm. Results: (Ⅰ) The color change of the solution was obviously according to the concentration of TCDD, while not observed for DMSO-treated groups. At a point time, the concentration is higher the OD value is higher. (Ⅱ) In the range of 10-14 mol/l to 10-8 mol/l, the OD value of the solution induced by TCDD has dose dependence at the same reaction time. The dose-response relationship curve about TCDD concentration against the OD Value was set up and the R2=0.9731. We can detect the lowest concentration of 10fM for TCDD by this method. (Ⅲ) Just as the other bio-analytical assay, the method developed here evaluated the TEQs value of the TCDD. The method reported here was 100 times more sensitive than the other bio-analytical method. Conclusion: the gold nanoparticle technology is a sensitive, rapid, simple and cost-effective method to screen the TCDD.Here we employed the gold nanoparticle technology to the TCDD identification and quantitation. We expect to develop a useful fast screening method for dioxin-like chemicals with the sensitive and simple properties of the gold nanoparticle technology. It can be used to prescreen DLCs in environmental samples due to its consistency with the other methods, with extremely low cost. It can either evaluate the biological and toxicological effects of dioxin or be carried out easily in the field.