Solid-state Nanopore Based Detection Of Single Base And Noise Characteristics

The wide-spread applications of nanopore devices in the field of DNA sequencing technology mainly owe to its simple and practical technical characteristics.Compared with other DNA sequencing methods,neither fluorescence label nor PCR amplification is required in nanopore based DNA sequencing approaches.By applying a bias voltage at both entrances of the nanopore,the electric field distributes concentratedly in the channel.When a DNA strand reaches the transition area at the entrance of the nanopore via Brownian motion,it can be accelerated immediately and enter the sensing region of the nanopore gradually.The temporary blocking caused by the translocated DNA in the nanopore induces a change of open current,the degree of which depends on the relationship between the channel diameter and the DNA structure.Therefore,the ionic current signal generated by the modulation effect of the blocking reflects the structural characteristics of the analyte.Graphene,as the most representative two-dimensional thin film material,has gradually shown its potential in nanopore DNA sequencing in recent years.In this work,the single-layer graphene nanopore is taken as the research object,and the design routine,manufacture procedure of graphene nanopore based detection devices are studied.The influence of the free-standing area of single-layer graphene on the noise characteristics of nanopore is analyzed and discussed in great detail.In addition,using the hydrophobic association effect between room temperature ionic liquids and DNA,a series of graphene nanopore based DNA detection experiments have been carried out.Through statistics and analyses of the modulation signals,we have successfully realized the recognition of deoxynucleotide monomers.In the fabrication process of single-layer graphene nanopores,silicon nitride(Si N_x)thin film windows for supporting single-layer graphene membranes were fabricated through photolithography and wet etching.Focused ion beam was used to sputter sub-micrometer holes which were equivalent to the free-standing area of graphene on Si N_x films.Through traditional wet transfer method,the two-dimensional monolayer graphene film was covered on top of the through-hole of Si N_x to form free-standing monolayer graphene membrane.A direct writing method of helium ion beam milling then was utilized to puncture the graphene to produce the nanopore.Under the point exposure mode,the diameter of the fabricated nanopore was precisely controlled by adjusting the exposure dose while the preset ion beam current and helium gas pressure were kept unchanged.The limitation of exposure time that was capable to drill the single-layer graphene film was studied.Moreover,the relationship between the exposure time and the nanopore diameter was analyzed and discussed.In the study of the noise characteristics of graphene nanopores,eight graphene nanopores with similar geometry but different free-standing area were designed and fabricated.The noise power spectral density(PSD)of graphene nanopores with different free-standing area was derived from the Fourier transform of recorded open current of these graphene nanopores.PSDs demonstrate that in the graphene nanopore regime,the diameter of its free-standing region has an impact on both the low-frequency and high-frequency noise characteristics.The results of previous reports proposed that the pore resistance of graphene nanopore was dominated by the diameter of graphene nanopore.However,the experimental results in this work reveal a discrepancy since the free-standing diameter of the nanometer-scaled graphene nanopore contributes to the pore resistance simultaneously.Considering that the resistance dominates noise level at low-frequency,the size of the free-standing region should be considered as an influence factor when designing and fabricating nanopore devices and studying the noise characteristics of nanopores.However,the equivalent channel length of the device is contributed by Si N_xopening at the same time when its size is comparable to the diameter graphene nanopore resulting in reduced spatial resolution.Therefore,the Si N_x opening is supposed to be much greater than graphene nanopore although it demonstrates lower noise level when the graphene membrane is supported by a small Si N_x opening.Taking the noise characteristics,spatial resolution of graphene nanopore and the positioning of Si N_x opening in helium ions beam milling,the Si N_x opening should be at least 20 times bigger than graphene nanopore.The single-atom thickness of graphene brings about a vital character to the high spatial resolution in the nanopore field.Combined with room temperature ionic liquids(RTILs)to slow down the DNA migration rate,the results of DNA recognition demonstrate a successful application of graphene nanopores to achieve single-base resolution for the first time.The results indicate that nanopores in monolayer graphene can be applied to effectively distinguish four kinds of deoxynucleotide homopolymers including poly-d N20s,poly-d N5s and poly-d N3s.At the same time,monolayer graphene nanopores functioned well in classification of the conformations of translocated DNA.In a series of detection of d NTPs,it can be clearly found that due to the limitation of spatial hindrance and channel diameter,graphene nanopores in various apertures exhibit different capability to discriminate d NTPs-BMIM~+composites,which results in diverse distributions of modulation signals,i.e.single peak and double peak Gaussian distribution.These results provide theoretical basis and reference standard for the design,fabrication,and application of graphene nanopore devices for DNA sequencing.