Detection Of Nucleic Acid Using Terahertz Spectroscopy

Background:Terahertz(THz)wave is an electromagnetic wave which the frequency is in the range of 0.1-10 THz(1THz = 1012Hz).Due to the intermolecular and intramolecular weak interactions,skeletal vibration and dipole rotation of biological macromolecular are located in the THz spectrum,Laying a good foundation of Terahertz spectroscopy in nucleic acids,proteins,sugars and other biological macromolecules.However,the current appliction of THz spectroscopy is mainly concentrated in the sample which is a solid state or a dry state of a single type of molecular,It can not be applied to the detection of clinical biological samples which is in liquid phase and has a variety of biological macromolecules,The reason is that the strong absorption of THz in water molecules in the solution obscures the characteristic peaks of biological macromolecules.Purpose:Based on the results of pre-project nucleic acid amplification,we detected the nucleic acids under liquid conditions to initially explore water sensitivity.we use a metamaterial structure made by split ring resonator to detect the nucleic acid of isothermal exponential amplification reaction(EXPAR)in the dry state and to explore the advantages and disadvantages of terahertz metamaterial applications.Methods:1.We used NCBI website to find the target gene sequence and designed four pair of primers for polymerase chain reaction;We searched article to design primers and templates for isothermal exponential amplification reaction,optimized the reaction conditions such as primer,template and isothermal exponential amplification reaction temperature for increasing the yield of targeted DNA fragment.We use phosphorothioate to modificate template sequence base for reducing the earlier non-specific amplificaton of no template control in EXPAR.2.We used THz-TDS to detect nucleic acid in aqueous solution which was purified after PCR amplification,and then we obtained the time-domain and frequency-domain spectra of four fragments of DNA in aqueous solution and deionized water;A test pool with the sample 120 μm thickness was constructed to mitigate the water sensitivity.3.The metamaterials of split ring resonator were designed and fabricated to improve the THz detection sensitivity,THz-TDS system was used to detect the reaction difference before and after nucleic acid amplification in a nitrogen-enriched environment.Results:1.The results of conventional PCR showed that the reaction conditions were not need optimized.The optimum reaction temperature of EXPAR was 57.6℃,and the amplification of NTC was obviously weakened by phosphorothioate modification of template sequence.but also led to its primer requirements increased by 104 times.2.Four pairs of primers were designed to amplify successfully 145bp,202bp,281bp and 357bp DNA different length fragments.THz-TDS system can not detect their characteristic absorption peaks under liquid phase,but their absorption intensities are different at the same molar concentration and the difference of DNA absorption coefficient is monotonous,and the absorption coefficient of 357bp DNA fragment is the smallest.3.At the metamaterial which was SRRs structural,The sensitivity of a 145 bp DNA fragment was 118.3ng/GHz;the frequency offset of before amplification was △f1=(54±3)GHz and the post-amplification frequency offset was △f2=(60 ± 5)GHz.Conclusions:1.The design of the detection pool in the THz-TDS can detect and distinguish the length of different fragments of DNA.THz-TDS technology is expected to become a new clinical detection of quantitative means.2.Through the modification of the thiolated oligonucleotide of EXPAR template sequence,the phenomenon of NTC amplification was effectively solved,EXPAR which is a high efficiency and temperature uniformity nucleic acid amplification technology will get more applications3.Metamaterials have very high sensitivity to trace biomacromolecules,but lack specificity for biological macromolecules.