Objective Guanine is one of the four bases of deoxyribonucleic acid (DNA). It pairs with cytosine in the double helix structure of DNA to maintain the stability of life activities. However, guanine methylation can affect the normal operation of DNA. When guanine is methylated, it immediately depurinates and forms apurinic sites, causing DNA alkylation damage and increasing cytotoxicity. One of the byproducts of guanine methylation, 7-methylguanine (7-MG), is commonly used as a biomarker to assess alkylation damage. However, traditional medical methods, such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) used to detect 7-MG, are time-consuming, cumbersome, and costly. Therefore, medical research needs a new accurate and swift method to detect guanine methylation. Furthermore, THz fingerprint spectral characteristics enable it to effectively identify biomolecules. However, the detection limit of the traditional tablet pressing method is at milligram level, which cannot meet the application requirements of low concentration detection (microgram and less) in the biomedical field. Some researchers have proposed combining terahertz spectroscopy and metamaterial biosensors; however, these metamaterial biosensors are limited to the detection of a pure substance and cannot realize qualitative identification of substances and mixed quantitative analysis. The chip designed in this study was tested on binary and multicomponent mixtures to check if it could predict the concentration of 7-MG in mixture samples. Finally, the 7-MG content of the mixture was determined using the standard internal method and the variation function of a pure 7-MG product. The minimum detection limit is 6.30 μg, which is 500 times lower than 2.95 mg by the traditional tablet pressing method. Furthermore, when 7-methylguanine and other substances are mixed together, they exhibit different frequency shift changes on the chip, allowing high sensitivity qualitative differentiation and quantitative detection from the mixture. This study provides important reference value for the subsequent rapid detection of 7-MG content in human cell DNA, and the detection and treatment of diseases.
Methods In this paper, 7-methylguanine is considered as an example to design a metamaterial chip based on the capacitance and inductance effect to enhance THz detection sensitivity. First, the frequency shift response of 7-methylguanine and guanine was measured through terahertz time-domain spectroscopy. The chip used in this study was then tested on binary mixtures and multicomponent mixtures to check if it could predict the concentration of 7-MG in mixture samples. Finally, the 7-MG content of the mixture was determined using the standard internal method and the variation function of a pure 7-MG product.
Results and Discussions In this paper, the relation between chip frequency shift and the amount of 7-methylguanine (Fig. 4) is obtained by conducting experiments. When the amount of sample increases, i.e. When the concentration increases, the characteristic peak starts moving to a lower frequency (red shift). The nonlinear equation f(x)=aexp(bx)+cexp(dx) is obtained through function fitting, where f(x) is the frequency shift and x is the effective mass of the sample. The corresponding coefficients are: a=0.04336, b=0.002242, c=-0.04559, d=-0.05696, and the goodness of fit of determination coefficient R2=0.9915 can be obtained. The same test is performed on guanine (Fig. 5), the corresponding coefficient of guanine was a=0.06861, b=0.002499, c=-0.06831, d=-0.02969, and the goodness of fit of determination coefficient R2=0.9895 was obtained. These results show that for known samples, the content can be detected using the corresponding frequency shift relation; for unknown samples, the curve of unknown samples can be deduced by testing samples of different concentrations and fitting the frequency shift curve and comparing with the existing curve parameters to achieve qualitative analysis of the unknown sample. From the test of the binary mixture, the frequency shift of the mixture is found to be the superposition of the frequency shifts of individual substances in each group (Fig. 6). Then, the same conclusion was made by the testing the multicomponent mixture (Fig. 7). This shows that the frequency shift effect of each component in the mixture can be separately calculated, and the frequency shift amount follows the frequency shift rule of pure product. Simultaneously, by comparing the actual value and the calculated result of the mixture frequency shift, clearly, the total frequency shift of each substance is almost the superposition of the single-frequency shift of each substance, with an accuracy >85%.
Conclusions This paper provides a new method for nondestructive, rapid, and accurate detection of molecular methylation. Considering 7-MG and G as examples, a terahertz metamaterial chip is designed based on capacitive and inductive effect. The detection limit of the chip can reach 6.30 μg, which is about 500 times smaller than that of 2.95 mg measured using the traditional pressing method. Here, the metamaterial chip is covered with different concentrations of 7-MG and G. The specific change in absorption peak frequency shift allows for qualitative and quantitative analysis of 7-MG and G. Furthermore, the mixture test confirms that the frequency shift of the mixture is a superposition of the frequency shift of a single substance. Then, using the standard internal method and the variation function of a pure 7-MG product, the content of 7-MG in the mixture can be calculated. This method can also be used to identify other molecular methylation products, such as 6-methylguanine, which is formed through guanine methylation, and 5-methylcytosine, which is formed through cytosine methylation. Hence, the findings of this study can be used in the future to accurately detect human DNA methylation.
