Soni, Gautam V., Singer Alon, Yu Zhiliang, Sun Yingjie, McNally Ben and Mellera Amit. Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores. Review of Scientific Instruments 81; 2010: 014301-7 doi:10.1063/1.3277116
The article “synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores” is a 2010 publication in the Review of Scientific Instruments journal volume 81 from page 014301-7. The article is authored by Soni Gautam V., Singer Alon, Yu Zhiliang, Sun Yingjie, McNally Ben and Mellera Amit. Soni and colleagues discuss a new way of integrating “total internal reflection microscopy and electrical detection of biomolecules using nanopores” (p. 1) with both techniques being recognized as modalities of measuring single-molecules. This integration enables visualization of DNA and DNA-protein complexes while they undergo translocation via a nanopore. A good temporal resolution of about 1000 frames is produced while an excellent signal to background is enabled.
Using total internal reflection fluorescence (TIR) method, it is possible to do probing on SiN membrane which is of nanoscale diameter and put in-between to liquids (CsCl which acts as a buffer in the trans chamber and KCl in the cis chamber of the flow cell). This eventually gives way for low-fluorescence probing and detection of electrical signals and single fluorophores happens when the biomolecule is moving through the nanopore. SiN membrane images are collected and upon illumination with a laser beam and the images indicate a conjugate of biomolecules and fluorophores. The ionic current that passes through the 4 nm pore is measured by immersing Ag/AgCl electrodes in the chambers, in a cis and trans chambers for Ag and AgCl respectively.
The authors argue that this method is preferable since it can be used in current sequencing techniques which are dependent on nanopore technology. With that in mind, Soni et al (p 1) illustrate this possibility by probing double-stranded DNA as well as DNA-protein complexes using optic means while passing through a pore of approximately 4 nanometers. It is suggestive that this experiment showed that when electric force accts on protein-DNA complex, it at the nanoscale pore results into the duplex DNA unzipping. It is for this reason that the method is possibly applicable in the DNA sequencing methods that are composed of single molecules and these are viewed as belonging to the “next-generation”.
Soni et al (p. 7) describe their new method of customizing TIR illumination as one that acts as a complement to the well known ion-current measurements applied in nanopore-based sensing. The ability to having optical detection working simultaneously with ion-current measurement is shown to enable cause molecules to be excited and a nanoscale pore of approximately 4 nm which is put between two fluids helps in threading image molecules. Using necessary hardware (for both electrical functions and optical capabilities), changes that occur in the ionic current are detected and this happens simultaneously with detection of fluorescence signals from labeled biomolecules.
As such, this technique finds potential use in detecting fluorescently labeled double stranded DNA molecules as well as in detecting labeled DNA-protein complexes. The methods is noted to be highly sensitive with only few fluorophores being required for labeling he DNA or DNA-protein complexes, yet a temporal resolution (approximately 1 ms) is achieved. The additional component that is unique in this experiment is the optical detection which gives a new measurement coordinate in TIR illumination. Due to the useful characteristics of this duo detection method makes it find use in proving more details for biomolecular processes such as FRET detection or DNA molecules that may be having more than one species since this is done at a nanopore scale. Genome sequencing that uses nanopore scale can also benefit fro, multicolor measurements enabled by this method. In that case, it becomes possible to have multiple nanopores allowing signal probing simultaneously.
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