Digital Detection Of Biomolecules Encoded With Scattering Spectrum Of Plasmonic Nanoparticles

Plasmonic nanoparticles exhibit special properties that are not available for bulk materials such as size effect,surface effect and quantum tunnel effect.Owing to the unique localized surface plasmon resonance(LSPR)effect,plasmonic nanoparticles absorb and scatter light very strongly at specific range of wavelength depend on their size,shape and their composition.In addition,plasmonic nanoparticles have some advantages such as ease of synthesis and surface modification,good optical stability,non-toxicity to cells and good biological compatibility.Due to these excellent physical and chemical properties,plasmonic nanoparticles have been widely applied in high single-to-noise optical imaging,photothermal therapy and chemical sensing.This paper focuses on the utilization of the ability of signle-particle-detection of plasmonic nanoparticles in the ultrasensitive biomolecule deteciton and presents several aspects of work as follows:First,discrete dipole approximation was used to simulate the optical cross-sections of several types of typical plasmonic nanoparticles(gold nanoparticles,gold nanorods,gold/silver core-shell plasmonic nanoparticles)with different shapes,structures and materials.The simulation results showed that the localized surface plasmon resonance peak wavelengths of these plasmonic nanoparticles can cover the whole visible region by adjusting the sizes and fine structures.Practicaly,gold nanospheres,gold nanorod,gold@silver core-shell nanoparticles were synthesized and characterized by transmission electron microscopy and UV-Vis extinction spectrum.A reliable and stable method was explored to conjugate DNA probes onto the surface of the synthesized particles for the further application in biochemical analysis.Second,a computing program based on the chromaticity analysis of individual particle for automatically identification and counting of the plasmonic nanoparticles with various colors(corresponding to the various LSPR peak wavelength)in dark field images is developed.In addition,an easy-to-attainable method to confine and enrich the nanoparticles in a limited area was explored,which can greatly reduce the distribution area of plasmonic nanoparticles on glass slide without the need to sacrifice the image quality.The combination of the single particle counting and the enrichment method to greatly improve the detection efficiency enable the application of single particle identification to be directly used in the low-abundance detection.Third,a novel ultrasensitive,rapid and low-cost digital DNA assay method was developed.This method is principly based on magnetic separation and the specific binding between complementary DNA sequences.Target DNA in sample solution can thus be easily replaced by gold nanorod in 1:1 ratio in liquid reaction environment.As a result,the problem of quantification of the target DNA is transformed to quantify the gold nanorod which can be easily achieved by the method we have developed.In the proof-of-concept experiments,we selected a DNA sequence related with human papillomavirus as a model system.The experimental results showed that the detection limit of the proposed method was about 6.5 aM and the linear dynamic range was 3-4 logs,which could rival with the digital PCR,a commercialized technology with the highest detection sensitivity.Fourth,considering that the scattering spectrum of plasmonic nanoparticles can be continuously controlled in the process of synthesis,we developed a highly sensitive,multiplexed DNA assay method.This method makes use of magnetic bead which is modified with several kinds of DNA probes to capture the target DNA molecules in sample solution and color-encoded plasmonic nanoparticles to encode and signal the target through DNA hybridization.The plasmonic nanoparticles are imaged by the dark field microscopy and classified by their-colors.Particles with the same color indicate the existence of the same target DNA.The simultaneous detection of multiple target DNA can be achieved by counting the particles with the matched colors.This method takes advantage of two main features of the conventional suspension array: the highly efficient liquid phase target binding kinetics and the color-encoding system.Remarkably,the ability of single molecule detection with high sampling rate gives a good performance in detection sensitivity.As a proof of concept,three kinds of representative plasmonic nanoparticles(gold nanorod,gold nanoparticle and gold@silver nanoparticle)were employed to signal three dissimilar virus gene segments,Ebola virus(EV),Variola virus(VV)and Bacillus anthracis(BA),respectively.Experiment results showed that the proposed method was good at selectivity and specificity and detection limit at low fM level were obtained at the current stage without the further optimization of the experiments.As compared with the conventional suspension array,the proposed method exhibits an at least two orders of magnitude improvement in detection sensitivity,showing its great potential in practical applications.