Mechanistic Studies on Magnesium Ion Influencing Biosensor Technologies

Abstract

The ultimate goal of this study is to apply biosensor technologies to environmental samples. In order to apply biosensor technologies to actual environmental samples, it is important to understand the interactions with various substances existing in the environmental samples and to eliminate the inhibition phenomena caused by these substances. Therefore, the inhibitory factors were analyzed in environmental samples for the gene quantification method (i.e., NanoGene assay) and examined the mechanisms. As a result, Mg2+ showed a unique and interesting tendency depending on its concentration. At higher concentration of Mg2+, the gene quantification was over-estimated. At lower and environmental relevant concentration of Mg2+, the gene quantification was inhibited (under-estimated). Over-estimation of gene quantity with Mg2+ at the concentrations of 10-3 M or higher was observed and this is because Mg2+ may serve as a bridge between MB-QD565-probe DNA and QD655-signaling DNA complexes during the NanoGene assay. It was confirmed by Fourier transform infrared spectroscopy (FT-IR), zeta potential, and scanning electron microscope (SEM). On the other hand, the inhibition phenomenon (under-estimation of gene quantification) was observed at a concentration of 10-3 M or less and it might be due to a non-specific reaction caused by the electrical binding between Mg2+ and DNA. Circular dichroism (CD) and zeta potential analyses indicated Mg2+ can cause the conformational change and the subsequent aggregation of DNA. A pretreatment apparatus employing the electrical discharge with submerged liquid anode has been developed and used for Mg2+ inhibited samples prior to DNA hybridization. Electrical discharge apparatus creates a cold plasma around the sample, resulting in the generation of massive electron clouds and ionized species. It was hypothesized that those rapidly available electrons and ionized species can alter the nonspecific binding between Mg2+ and DNA, which hinders DNA hybridization and the subsequent gene quantification by the NanoGene assay. The inhibition by Mg2+ to the DNA hybridization during the NanoGene assay was overcome by the electrical discharge treatment (2 – 4 min) prior to the DNA hybridization. % fluorescence has changed back to almost 100 % fluorescence without Mg2+ inhibition, which shows the compatible or better recovery compared to EDTA chelation. CD and zeta potential analysis also indicated electrical discharge treatment may possibly cause the dispersion of DNA-Mg2+ aggregation, resulting in the suitable proximity for DNA hybridization. In summary, the main inhibition mechanisms between magnesium ions and the elements constituting the NanoGene assay were identified, and the following mitigation was suggested. It is expected that not only the analysis results of the NanoGene assay can be obtained more accurately, but also development and application of environmental samples in the field of biosensors can be promoted. ;본 연구의 최종 목표는 바이오센서 기술의 환경 시료 적용이다. 바이오센서 기술을 실제 환경 시료에 적용시키기 위해서는 환경 시료 내에 존재하는 다양한 물질들과의 상호작용을 이해하고 이러한 물질들로 인해 발생되는 저해 현상을 없애는 것이 중요하다. 따라서 본 연구에서는 유전자 정량 바이오센서 (나노진 기법) 기술에서의 환경시료 속 저해 인자들을 분석하고 그 기작을 연구하였다. 본 연구에서는 마그네슘 이온이 나노진 기법에서 농도에 따라 특이적 경향성을 보이는 것을 발견하였으며, 10-3 M 농도를 기준으로 상반된 두 가지 경향성에 대한 기작을 살펴보았다. 10-3 M이상의 농도에서 발견되는 이상 현상인 과상향 측정은 마그네슘 이온이 유전자 혼성화 과정에서 유전자 복합체끼리의 결합을 유도하는 기작을 일으키기 때문으로 밝혔으며, 이를 푸리에 변환 적외선 분광도계 (FT-IR), 제타포텐셜, 전자현미경 등을 통해 결과를 뒷받침하였다. 10-3 M 이하의 농도에서 발견되는 저해 현상은 마그네슘 이온과 DNA와의 전기적 결합으로 인한 비특이적 반응이 주요 저해 기작이라는 것을 발견하였으며, 이러한 저해 현상을 미세 전류 방출 기기를 개발하여 해결하였다. 또한 이를 원평광 이색성 (Circualr Dichroism)과 제타포텐셜 등을 이용해 그 결과를 뒷받침하였다. 결론적으로 유전자 혼성화 과정에서의 저해 현상에 대한 기작 연구를 통해 마그네슘 이온과 DNA와의 주요 반응 기작을 파악함으로써 나노진 기법의 분석 결과를 보다 정확하게 얻을 수 있을 뿐만 아니라, 유전자 혼성화 과정을 거치는 바이오센서 분야에서의 발전 및 환경시료 적용을 도모할 수 있을 것으로 기대한다.