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Real Time RT-PCR02:57

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Real-time PCR for direct aptamer quantification on functionalized graphene surfaces.

Viviane C F Dos Santos1,2, Nathalie B F Almeida3,4, Thiago A S L de Sousa3

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This study introduces a real-time PCR method for quantifying DNA aptamers on graphene surfaces. This novel technique precisely measures aptamer immobilization, enhancing biosensor development.

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Area of Science:

  • Biotechnology
  • Materials Science
  • Analytical Chemistry

Background:

  • Graphene's unique properties make it a promising material for biosensor development.
  • Accurate quantification of immobilized aptamers is crucial for optimizing biosensor performance.
  • Existing methods for aptamer quantification on surfaces have limitations.

Purpose of the Study:

  • To develop and validate a real-time PCR strategy for direct detection and quantification of DNA aptamers on functionalized graphene.
  • To compare the efficiency of different graphene functionalization methods for aptamer immobilization.
  • To assess the selectivity of the functionalization methods for graphene surfaces.

Main Methods:

  • Development of a real-time PCR assay for DNA aptamer quantification.
  • Functionalization of graphene surfaces with a Staphylococcus aureus aptamer (SA20) using amino and pyrene modifications.
  • Surface modification of graphene with thionine prior to amino-SA20 functionalization.
  • Testing the selectivity of functionalization on graphene versus silicon dioxide surfaces.

Main Results:

  • Real-time PCR enabled aptamer quantification across a broad range (0.05 fg to 2.5 ng).
  • Graphene functionalization with amino-modified SA20 (following thionine modification) was significantly more efficient than pyrene modification.
  • The investigated functionalization methods demonstrated selectivity for graphene over bare silicon dioxide surfaces.

Conclusions:

  • A novel, precise, and quantitative real-time PCR methodology for aptamers on graphene surfaces has been established.
  • This technique offers a significant advancement for aptamer immobilization studies and biosensor development.
  • The findings provide insights into optimizing graphene-based aptasensor fabrication.