Black Phosphorus Mimics The Phospholipid Plasma Membrane To Enhance Polymerase Chain Reaction Efficiency

Phosphor.us.is an.essential element for many life forms and multiple biological systems,such as the phospholipids of the plasma membrane.The plasma membrane provides the reaction substrate for many important biological reactions.For example,it can supply binding sites for the bacterial chromosomal replication origin to initial DNA replication;many enzyme-catalyzed biological reactions occur on the plasma membrane.Among all of the allotropes of phosphorus,black phosphorus is the most stable form and can be exfoliated to a few layers black phosphorus(BP),a two-dimensional nanomaterial with large surface area,excellent mechanical flexibility,biocompatibility and biodegradability.Polymerase chain reaction(PCR)is a main biological technique to amplify DNA fragments in vitro,but its efficiency and yield are limited sometimes.In this work,the bionic phospholipid bilayer model can be used to bind DNA templates,primers and DNA polymerases by the characteristics of the bionic structure of BP like phospholipid membranes to improve the efficiency of the DNA synthesis process in polymerase chain reaction(PCR).In this work,two-dimensional black phosphorus was prepared by liquid exfoliation with a thickness of 4 nm and a size of 200-300 nm.The two-dimensional black phosphorus has a large surface area,good biocompatibility and degradability,the surface is negatively charged,hydrophilic,the structure and composition are similar to the phospholipid membrane,which is favorable for the DNA polymerase and the adsorption of PCR reaction components.In order to study the effects of black phosphorus on the PCR,we used BP as a PCR additive.The study found that BP can significantly improve the efficiency of PCR in the following aspects:(1)BP increases the yield and specificity of PCR without affecting the fidelity of DNA polymerase.(2)Reduce the number of PCR cycles from 30 laps to 20 laps and reduce the extension time from 1 minute to 0.5 minute,thereby the PCR reaction time was shortened by 40 min.(3)Reduce the concentration of the DNA amplification template from 0.4 ?g ?L-1 by 2 pg ?L-1.The required primer concentration was reduced from 0.4 mM to 0.08 mM.(4)Long target sequences(>5 kb)can be amplified.(5)Annealing temperature decreased from 65 ? to 25 ?,extending the annealing temperature range.(6)BP can enhance the effect of conventional Taq polymerase and high-fidelity DNA polymerase and is suitable for a variety of different polymerase reaction systems.(7)Compared with other nanomaterials,including graphene oxide,few-layer boron nitride nanosheets and gold nanoparticles,the improvement of BP on PCR is more significant.Finally,the mechanism was studied by Zeta potential,microbalance,isothermal titration calorimetry,fluorescence spectroscopy,RT-PCR and fluorescence spots.The results confirmed that BP can effectively adsorb the components of the PCR reaction such as DNA,polymerase,primers and dNTP.During PCR,BP has stronger adsorption effect on single-stranded DNA than double-stranded DNA.In the early stage of the PCR reaction,BP serves as a DNA amplification substrate,which enhances DNA replication efficiency and yield.