研究目的
To develop a sensitive colorimetric method for detecting the Japanese cedar pollen allergen Cry j 2, addressing the need for environmental monitoring and medical diagnostics due to increasing pollen allergies.
研究成果
The developed colorimetric method for Cry j 2 detection achieves high sensitivity (detection limit of 0.2 ng/mL, 130-fold improvement over previous methods) and selectivity, with no false responses to non-target proteins when using ssDNA-AuNPs. It is effective in environmental samples like soil solutions, showing promise for practical use in environmental monitoring and biomedical diagnostics. The methodology can be generalized to other proteins with appropriate aptamers.
研究不足
The assay may have limitations in sensitivity and specificity when using unmodified AuNPs due to potential interference from non-target proteins like Der p 2, which can adsorb to AuNPs. Optimization of DNA grafting density and salt concentration is required to minimize false positives. The method's applicability to other proteins depends on the availability of specific aptamers.
1：Experimental Design and Method Selection:
The method involves catalytic DNA hairpin self-assembly for signal amplification and salt-induced non-cross-linking aggregation of DNA-modified gold nanoparticles for signal transduction.
2：Sample Selection and Data Sources:
Samples include Cry j 2 protein, other allergen proteins (Cry j 1, Phl p 5, Can f 2, Bla g 4, Der p 2), and soil solutions spiked with Cry j
3：List of Experimental Equipment and Materials:
Equipment includes UV/vis spectrophotometer (Cary 50, Varian), microplate reader (PerkinElmer), smartphone (Redmi Note 5, Xiaomi), Milli-Q water purification system (Millipore). Materials include citrate-capped AuNPs (15 nm, BBI), DNA strands (synthesized by Eurofins Genomics and Tsukuba Oligo Service), various allergen proteins (from Funakoshi and CosmoBio), and reagents (from Wako Pure Chemicals).
4：Experimental Procedures and Operational Workflow:
Procedures include preparation of ssDNA-AuNPs, fluorescent detection using molecular beacons, colorimetric detection using unmodified AuNPs and ssDNA-AuNPs, optimization of parameters (temperature, concentrations, incubation times), and application to soil samples.
5：Data Analysis Methods:
Data analysis involves measuring absorbance ratios (A610/A520 or A530/A610) using UV/vis spectroscopy, calculating signal-to-noise ratios, and determining detection limits based on standard deviations.
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