Ultra-sensitive Digital Detection Of Biomolecules Based On Microscopic Imaging Of Coding Nanoparticles

It is very important to achieve qualitative identification and quantitative detection of biomolecules with very low concentrations in many fields,such as the early diagnosis of diseases,bacterial and virus detection,food safety and environmental monitoring.This work proposes a highly sensitive and reliable method for biomolecular detection,based on the encoding of individual target biomolecules by a single noble metal nanoparticles.Using a dark field microscopic imaging system,the number of coding nanoparticles witch is proportional to that of the target molecules is counted and ultra-sensitively detection of target molecules was thus achieved.Due to the local surface Plasmon Resonance(LSPR)phenomenon of noble metal nanoparticles,when irradiated with white light,the particles scatter light strongly at a specific wavelength.The wavelength of the scattered light depend on the type,size and shape of the particles.Under dark field microscopy,each particle will show up as a colored bright spot in the dark field image.Different nanoparticles are differentiated from color they appears.In this work,spherical gold nanoparticles with an average particle size of 49 nm and rod-shaped gold nanoparticles with an average aspect ratio of 2.12 were prepared by a two-step liquid phase reduction method,which corresponded to green bright spots and red bright spots in dark field images respectively.In order to overcome the adverse effect of the Brownian motion,the slide was modified with positively charged molecules to adsorb the nanoparticles.To obtain reliable counting of the nanoparticles,it is necessary to image and enumerate all the nanoparticles on the slides,due to the nonuniformly distributed feature of the coding nanoparticles,which is practically tedious and labor costly.To overcome this weakness and improve the detection speed,spherical nanoparticles with known concentration was used as the internal standard,and the ratio of red and green dots in the image was used to calculate the concentration of rod-shaped particles.Two different DNA probes were modified on the surface of rod-shaped particles and magnetic beads,respectively which were each complementary to one-half the target DNA.When the target DNA is present,a sandwich structure "reporting particle-target DNA-magnetic beads" is formed through hybridization of DNA,and digital encoding of every target DNA molecule with a microscopically countable nanoparticle is completed.Upon magnetic separation,free rod-shaped particles were thoroughly removed.Following de-hybridization,the coding particles that dissociated from the surface of the MBs were re-suspended in a buffer containing 11-mercaptoundecanoicacid(MUA)functionalized spherical gold nanoparticles with certain concentration as an internal standard.Based on the internal standard counting method,the concentration of the coding rod-shaped particles is obtained,which represents the concentration of target DNA.The limit of detection using this method was ?3 f M,which was affected by the background signal,and the detection efficiency is about 70%.The rod-shaped nanoparticles have two plasma modes,and the light scattered by them may have different polarization properties with that of the isotropic particles.In this work,the Monte Carlo method is used to simulate the polarization characteristics of light scattered by rod-shaped particles,and the linear polarization parameters of the scattered light are extracted which is mainly affected by the angle between the particle and the incident direction of light.Then,based on the dark field imaging system after polarization modification,the linear polarization of the light scattered by the rod-shaped particles is measured,which is consistent with the simulation results.Achieving the linear polarization distribution of a large number of particles and based on this setting the linear polarization threshold to identify and count the rod-shaped particles.Then digital encoding individual target DNA by a single rod-shaped particles and counting the number based on the linear polarization parameter,thereby obtaining the concentration of the target DNA molecule.In this method,the limit of detection is about 2.5f M.The innovation points of this work: The concentration of nanoparticles was obtained reliably by directly counting the number by internal standards,and the direct conversion between target molecule concentration and reported particle concentration was achieved through clever structural design.The linear polarization parameter of the scattered light of nanoparticles was measured by modifying the conventional dark field microscope.Based on the optical microscope,coding method and digital detection method were combined into the same detection system,whitch had a series of advantage such as high sensitivity,high reliability,and good expandability.