研究目的
To prepare non-fluorescent graphene oxide quantum dots (GOQDs) as fluorescence quenchers in quantitative real-time PCR (qRT-PCR) to improve the specificity and sensitivity of DNA detection.
研究成果
The study successfully prepared non-fluorescent GOQDs via an improved Fenton reaction and demonstrated their effectiveness as fluorescence quenchers in qRT-PCR. The GOQDs reduced the background fluorescence intensity and improved the specificity of the qRT-PCR reaction. The method could detect DNA sequences with high specificity in a wide range of concentrations, indicating its potential for sensitive and specific DNA detection in biological and medical fields.
研究不足
The study did not explore the long-term stability of GOQDs in qRT-PCR systems or their potential toxicity in biological applications. The detection limit of 104 copies per ml may not be sufficient for some ultra-sensitive applications.
1:Experimental Design and Method Selection:
The study employed an improved Fenton reaction to prepare GOQDs with a large content of hydroxyl groups and low fluorescence intensity. The GOQDs were then used as fluorescence quenchers in qRT-PCR.
2:Sample Selection and Data Sources:
The study used graphite as the starting material for GOQDs synthesis and various fluorescent dyes (6-FAM, JOE, Cy3) to test the quenching ability of GOQDs.
3:List of Experimental Equipment and Materials:
Equipment included AFM, XPS, FT-IR spectrometer, UV-vis spectrophotometer, and fluorescence spectrophotometer. Materials included graphite, potassium nitrate, potassium permanganate, sulphuric acid, hydrogen peroxide, and various DNA sequences.
4:Experimental Procedures and Operational Workflow:
The improved Fenton reaction was used to synthesize GOQDs. The GOQDs were characterized and then tested for their fluorescence quenching ability. The GOQDs were then applied in qRT-PCR to detect DNA sequences.
5:Data Analysis Methods:
The fluorescence intensity was measured using a fluorescence spectrophotometer. The specificity and sensitivity of the qRT-PCR method were evaluated based on the amplification curves and agarose gel electrophoresis results.
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Atomic force microscopy
Multimode Nanoscope V
Bruker
Characterization of GOQDs
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X-ray photoelectron spectroscopy
Thermo ESCALAB 250XI
Thermo
Characterization of GOQDs
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Fourier transform infrared spectroscopy
NICOLET 5700
Waltham
Characterization of GOQDs
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Fluorescence Quantitative Gradient Polymerase Chain Reactor
qTOWER 2.2
analytikJena
qRT-PCR experiments
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Graphite
crystalline powder, 100 mesh
Qingdao Laixi Colloidal Graphite Factory
Starting material for GOQDs synthesis
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Potassium nitrate
Chengdu Kelong Chemical Reagent Factory
Chemical reagent for GOQDs synthesis
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Potassium permanganate
Chengdu Kelong Chemical Reagent Factory
Chemical reagent for GOQDs synthesis
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Sulphuric acid
Chengdu Kelong Chemical Reagent Factory
Chemical reagent for GOQDs synthesis
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Hydrogen peroxide
30%
Chengdu Kelong Chemical Reagent Factory
Chemical reagent for GOQDs synthesis
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Dialysis bag
1000 Da
Shanghai Yuanye Biological Science & Technology Company
Purification of GOQDs solution
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Water purification system
ELGA
ELGA
Preparation of ultrapure water
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Ultraviolet-visible spectrophotometer
MAPADA UV-6300
Shanghai
Characterization of GOQDs
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Fluorescence spectrophotometer
Lengguang F97PRO
Shanghai
Measurement of fluorescence intensity
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