Progress on optical measurements in single-molecule analysis with nanopores

Nanopore technology is widely used for single molecule/single particle detection due to its advantages of label-free sensing, low cost, portability and ultra-high sensitivity. The ionic current based electrical measurement is the common method for collecting nanopore signals. However, the electrical measurements suffer from several deficiencies such as the non-ideal noise components, low throughput and insufficient temporal or spatial resolution. Therefore, many strategies have been proposed to overcome these limitations. In this review, we summarize the progress of optical detection techniques with high bandwidth and high throughput that can be used to supplement or/and replace electrical measurements. It has been demonstrated that single-molecule optical detection using fluorescently labeled analytes (such as DNA) in nanopores is feasible. Moreover, methods to further improve the signal-to-noise ratio (SNR) of optical measurements in nanopores, including background noise suppression and the use of spectrally resolvable fluorescent groups, were also assessed. To avoid the fluorescence labeling of analytes, Ca2+ fluorescent probe-based ion flux imaging can achieve the optical measurement of unlabeled analytes directly. In addition to fluorescence imaging, plasmonic nanopores also provide solutions for optical nanopore sensing with the advantages of tunable optical/electrical sensitivity and scalable parallel detection. Plasmonic structures can enhance electromagnetic fields and focus them to a "hot spot" region located only at the nanopore depending on the structure of the pore, and the characteristic optical signals of the analyte are affected as it translocates through the pore. The SNR of optical signals can be improved by designing pores with reasonable size, shape, material and dielectric environment for enhancing the confinement of the electromagnetic field. The highly constrained electromagnetic field of plasmons have been used to help resolve single fluorescent molecules. Appropriate modifications of the surface of the plasmonic nanostructure can effectively suppress the fluorescence background and further enhance the fluorescence signal. In addition to enhancing labeled fluorescence emission, the unlabeled resonance shift and Raman scattering spectroscopy can also be performed directly for measuring single molecules. The principle of detecting the plasmonic resonance shift is that when the analytes enter the plasmonic nanopore, the local refractive index changes, which affects the intensity and frequency of the light scattered. Single DNA molecules have already been shown to transport through nanopores which can be efficiently detected by monitoring the intensity of scattered light. In addition, unlabeled surface-enhanced Raman scatting (SERS) can not only provide rich fingerprint information of molecular fine structure characteristics, but also be used to identify and measure a variety of complex samples, with chemical contrast. SERS with single molecule sensitivity is very important for the potential application of nanopores in chemical identification of different types of molecules. To achieve accurate and real-time measurement of the reaction of local targets, nanopipettes and SERS technology can be further combined. The highly sensitive plasmonic nanopipettes enable the measurement of single particles, single biomolecules and the environmental stimulus. However, controlling the molecular translocation through the pore is the main challenge for the enhanced SERS sensing at present. The typical sub-millisecond translocation time is too short to obtain the full spectral information with SERS. To address this limitation, several techniques based on the plasmonic trapping effect have been proposed to regulate the translocation speed. To conclude, nanopore technology based on single-molecule optical sensing has made great progress, but there are still several major challenges that needs to be solved. We believe that optical nanopore technology with high temporal and spatial resolution may open up a new horizon for nanopore analysis.