A New Method For Rapid Detection Of Point Mutation Based On Surface Plasmon Resonance Biosensor

In recent years, single nucleotide polymorphisms (SNPs)， as thepromising biomarkers stably inherited in genome，were widely applied invarious areas of biomedical research. Recent researches indicate that singlebase mutation existing in coding sequences or gene regulatory regions maycause genetic disorders， which result in increasing susceptibility to aparticular disease and different responses to drugs. Thus，the identificationof single-base mutations plays a central role in risk assessment, clinicaldiagnosis and treatment，especially in early diagnosis and personalizeddrug therapies of many genetic disease. Up to now, numerous newapproaches have been developed for SNPs detection.Among them，surfaceplasmon resonance (SPR) sensors are well developed and have become tobe the standard method for biomolecular interaction study. In addition,surface plasmon resonance sensors have shown great potential applicationsfor nucleic acids and proteins detection due to their label-free and real timeanalysis, easy of automation and rapid detection. In this work, we present anew method for point mutation detection that combines DNA chip technique， molecular biotechnology and SPR sensing technology andutilizes the property of DNA polymerase for discriminate mismatchedconfigurations. The research consists of two parts:1. Preparation of surface plasmon resonance biosensorIn this work, the Biacore XTMsystem and a bare gold sensor chip(Sensor chip Au) were used to sample detection.3’-thiolated DNA probeswere immobilized onto the sensor surface via molecular self-assembly, themethod employed herein is based on the chemical bonding of Au-S, thenMCH (6-mercapto-1-hexanol) solution was dropped onto the chip surfaceto minimize non-specific adsorption. To investigate the influence of flowrate on the signal, different flow rate (2μl/min、5μl/min、7μl/min、10μl/min) were studied with a sample volume of70μl. Analysis process canbe completed within15min. The sensor chip was regenerated withdifferent denaturing solution （NaOH， Gly-HCl）, with appropriateregeneration solution, the sensor chip can be reused more than30times.2. SPR biosensor for point mutation detection based on polymerizationextension reactionThe SPR biosensor was developed by integrating polymerizationextension reaction for sensitive and specific detection of single basemutation. Polymerization extension reactions were carried out under theoptimized experimental conditions with the mutation recognition of DNApolymerase. Then, the extension products were injected into the flow cell over the sensor chip surface to hybridize with the capture probes modifiedon the gold film surface to induce an SPR signal. Extension reactionconditions such as Mg2+concentration, reaction temperature and reactiontime were optimized. Furthermore, sensitivity and specificity of the assaywere also tested. The response signal exhibited a good linear relationshipwith the logarithm of target DNA concentration in the range form100pMto100nM, with the correlation coeffcient of0.9963, the detection limit of100pM can be achieved. The established method was proved to beconvenient，sensitive and specific．It might have the potential to become anefficient candidate technique for point mutation detection in biomedicalresearch and clinical diagnosis in the future.