Femtomolar Detection Of Nucleic Acid Based On Functionalized Gold Nanoparticles

BackgroundAs the building block of biological inheritance and the carrier of genetic information,DNA plays an important role in life activities.High-sensitivity DNA detection can be used as an effective means for early detection of human genetic and cancer diseases,etc.From the 1950s,the discovery of DNA double helix opened the door for the studies of DNA detection.Traditional DNA detection technologies mainly include nucleic acid liquid phase hybridization,amplification and nucleic acid sequencing methods.These cl assical approaches for nucleic acid detection have been widely used in laboratory diagnoses.With the development of nanotechnology,DNA detection based on nanomaterial is also increasingly emerging.Among them,the gold nanoparticle plasmon sensing method is very suitable for Point-of-care Test（POCT）detection because of its simple operation.However,limited by the sensitivity of existing gold nanoparticle plasmon sensing methods,the need for trace detection is not met in nucleic acid detection.MethodsBased on the principle of gold nanoparticle plasmon sensing,this study examined surface functionalization on gold nanoparticles,using surface-modified polyethylene glycol（PEG）and single-strand DNA（ssDNA）to form steric hindrance and electrostatic mutual exclusion between gold nanoparticles,respectively.In addition to maintaining stability,the specific ssDNA sequence served as a bioreceptor for the nucleic acid to be tested,as chemically inert PEG can promote the modification of ssDNA on the surface of gold nanoparticles and improve the hybridization efficiency of ssDNA to be tested.Different from the classical sensing strategy of induced aggregation of analytes,our studies used the gold nanoparticles coupling effect to make the functionalized gold nanoparticles form a certain aggregation without precipitation.After the nucleic acid to be tested is added,it specifically bind to the ssDNA on the surface of the gold nanoparticles,increasing the distance between the gold nanoparticles to cause a disaggregation effect.The aforementioned process was monitored by a UV-Vis spectrometer.The aggregated gold nanoparticles showed the enhancement of the shoulder peak on the LSPR spectrum.After the addition of the nucleic acid to be tested,the shoulder peak intensity of the LSPR spectrum was decreased due to the disaggregation of the gold nanoparticles.We found that the intensity change of the shoulder peak is related to the concentration of the measured nucleic acid.Furthermore,we designed two different ssDNA probes.The first one is a ccpDNA probe that is fully complementary to the nucleic acid to be tested.As a CCP method,the nucleic acid to be tested bind to ccpDNA and blocked the interaction between ssDNA and PEG within the gold nanoparticles.The aggregated gold nanoparticles became disaggregated.The second is MIX method whereby an equal ratio mixture of two hcpDNA1probes and hcpDNA2 probes,which are half complementary to the nucleic acid to be tested.After the addition of the nucleic acid to be tested,it combines with the hcpDNA1 probe and the hcpDNA2 probe respectively to form a sandwich structure.At the same time,the interaction between the molecules on the surface of the gold nanoparticles was blocked,and the originally aggregated gold nanoparticles became a disaggregated form with a certain distance.Since the distance of the gold nanoparticles was increased,the disaggregation effect was also exhibited.Main resultsAfter the evaluation of the two methods of nucleic acid detection,we found that the sensitivity of the two methods were very high.The detection limit of CCP method is124*10^-18M（attoMol,aM）,and the detection limit of MIX method is 2.54*10^-15M（femtoMol,fM）,which can meet the requirement of trace DNA detection without the need to combine other amplification strategies.In terms of specificity,the MIX method is more specific than the CCP method and can recognize four base mismatched sequences.Finally,we also used the bimodal simulation to theoretically verify the effectiveness of the quantitative detection of this sensing method.ConclusionsIn summary,based on the LSPR effect of functionalized gold nanoparticles and the unconventional sensing strategy of gold nanoparticles aggregation to depolymerization,this study used two models of nucleic acid probes to achieve trace nucleic acid detection.To this end,we established,for the first time,a rapid,simple,and highly sensitive method for nucleic acid detection,which might form a basis for clinical trace nucleic a cid detection in the future.