Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites

Abstract

There is a general necessity to improve the specificity and efficiency of the polymerase chain reaction (PCR), and exploring the PCR-enhancing mechanism still remains a great challenge. In this paper we report the use of branched polyethyleneimine (PEI)-based derivatives and hybrid nanocomposites as a novel class of enhancers to improve the specificity and efficiency of a nonspecific PCR system. We show that the surface-charge polarity of PEI and PEI derivatives plays a major role in their effectiveness to enhance the PCR. Positively charged amine-terminated pristine PEI, partially (50%) acetylated PEI (PEI-Ac₅₀), and completely acetylated PEI (PEI-Ac) are able to improve PCR efficiency and specificity with an optimum concentration order of PEI < PEI-Ac₅₀ < PEI-Ac, whereas negatively charged carboxyl-terminated PEI (PEI-SAH; SAH denotes succinamic acid groups) and neutralized PEI modified with both polyethylene glycol (PEG) and acetyl (Ac) groups (PEI-PEG-Ac) are unable to improve PCR specificity and efficiency even at concentrations three orders of magnitude higher than that of PEI. Our data clearly suggests that the PCR-enhancing effect is primarily based on the interaction between the PCR components and the PEI derivatives, where electrostatic interaction plays a major role in concentrating the PCR components locally on the backbones of the branched PEI. In addition, multiwalled carbon nanotubes modified with PEI and PEI-stabilized gold nanoparticles are also able to improve the PCR specificity and efficiency with an optimum PEI concentration less than that of the PEI alone, indicating that the inorganic component of the nanocomposites may help improve the interaction between PEI and the PCR components. The developed PEI-based derivatives or nanocomposites may be used as efficient additives to enhance other PCR systems for different biomedical applications.

Keywords:

• polyethyleneimine
• gold nanoparticles
• multiwalled carbon nanotubes
• nanocomposites
• polymerase chain reaction
• specificity
• efficiency

Acknowledgments

This research is financially supported by the Program for New Century Excellent Talents in University, the National Natural Science Foundation of China (20974019, 81101150, and 81071747), the Fund of the Science and Technology Commission of Shanghai Municipality (11 nm0506400), the Fundamental Research Funds for the Central Universities (for XC, RG, MS, and XS), and the Key Laboratory of Textile Science and Technology, Ministry of Education, “111 Project”, B07024. JW thanks the Shanghai Education Committee Key Discipline and Specialties Foundation (J50208) and Shanghai Pujiang Program (10PJ1406400). XS gratefully acknowledges the Fundação para a Ciência e a Tecnologia (FCT) and Santander Bank for the Invited Chair in Nanotechnology.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figure

Figure S1 The effects of commercial colloid gold nanoparticles (AuNPs) on the nonspecific polymerase chain reaction system. (A) The effect of AuNPs with a diameter of 5 nm. Lane M is the DL 2000 marker, and lane 6 is the negative control amplified without template and additives. AuNPs (5 nm) were added into the polymerase chain reaction mixture. From lane 1 to 5, its final Au concentration is 0, 116, 232, 348, and 464 μg/L, respectively. (B) The effect of AuNPs with a diameter of 20 nm. Lane M is the DL 2000 marker, and lane 8 is the negative control amplified without template and additives. AuNPs (20 nm) were added into the polymerase chain reaction mixture. From lane 1 to 7, its final Au concentration is 0, 2.32, 3.48, 6.96, 11.6, 16.24, and 23.2 mg/L, respectively.