研究目的
Investigating the development of a triplex DNA nanotweezer for the specific capture of melamine (MEL) and its application in highly selective MEL assays.
研究成果
The triplex DNA nanotweezer demonstrates high selectivity for MEL detection, facilitated by the TFO arm's role in enabling MEL binding at the AP site and subsequent fluorophore translocation. This method offers a promising approach for MEL assay in milk samples without prior separation, with potential applications in detecting other analytes using specific DNA modifications.
研究不足
The detection limit is influenced by the presence of CHE, which competes with MEL for binding at the AP site, potentially limiting sensitivity. The method's applicability to other analytes requires further exploration with different DNA modifications.
1:Experimental Design and Method Selection:
The study employs a triplex DNA nanotweezer design to capture MEL specifically, utilizing the triplex-forming oligonucleotide (TFO) arm's switching mechanism.
2:Sample Selection and Data Sources:
MEL and its hydrolysis products, along with milk samples, were used to test the nanotweezer's specificity and application.
3:List of Experimental Equipment and Materials:
Includes DNA strands synthesized by TaKaRa Biotechnology Co., Ltd., CHE from AAT Bioquest, Inc., and other analytical grade reagents.
4:Experimental Procedures and Operational Workflow:
Involves the preparation of AP-site triplex solutions, fluorescence measurements, DNA melting temperature measurements, and isothermal titration calorimetry (ITC) measurements.
5:Data Analysis Methods:
Fluorescence spectra and ITC data were analyzed to determine MEL binding and the mechanism of fluorophore translocation.
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FLSP920 spectrofluorometer
FLSP920
Edinburgh Instruments Ltd.
Used for acquiring fluorescence spectra.
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F2700 spectrofluorometer
F2700
Hitachi Ltd.
Used for acquiring fluorescence spectra.
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UV2550 spectrophotometer
UV2550
Shimadzu Corp.
Used for DNA melting temperature (Tm) measurements.
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DNA strands
TaKaRa Biotechnology Co.,Ltd.
Used as the primary material for constructing the triplex DNA nanotweezer.
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CHE
AAT Bioquest, Inc.
Employed as the fluorescent probe for detecting MEL.
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ITC200 microcalorimeter
ITC200
MicroCal, LLC
Used for isothermal titration calorimetry (ITC) measurements.
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