Nanopore sensors can detect changes in ion current through nanopores, achieving single-molecule sensitivity. They have been successfully used to detect biomarkers, including nucleic acids, proteins, and small molecules. However, when quantitatively analyzing ultra-low concentration (sub picomolar) molecules, there is a problem of long response time, as the time constant for the analyte to diffuse into the nanopore is square to the distance that the molecule must diffuse. Meanwhile, in complex samples such as biological fluids, other substances translocated through nanopore sensors in the sample may also exhibit resistance pulses in ion currents, which means that the selectivity of clinical samples may be a challenge.
To overcome these challenges, researchers from the University of New South Wales in Australia have proposed an optical nanopore blocking sensor method that can quickly detect substances at ultra-low concentrations.
The model used by the researchers to analyze the drug is vascular endothelial growth factor (VEGF). Fluorescent polystyrene nanoparticles (PSNPs) are modified with anti VEGF antibodies to form Ab PSNPs, while the surface of the nanopore array is modified with anti VEGF aptamers to form aptamer nanopores. By applying an electric field on the nanopores, Ab PSNPs are brought into the aptamer nanopores. Quantify the amount of VEGF at sub picomolar concentrations by calculating the ratio of blocking and unblocking events in a nanopore array (676 nanopores).
Researchers did not determine blocking events through changes in ion current, but instead used fluorescent nanoparticles and wide field microscopy as readout mechanisms to achieve simultaneous monitoring of hundreds of nanopores, significantly increasing the number of detectable blocking events and enabling nanopore blocking sensors to achieve quantification.
Firstly, researchers measured the closure event of exposed PSNP using bare nanopores without any surface modification. When no voltage is applied, there is no fluorescence signal; When a voltage of 1.5 V was applied to the trans chamber, many fluorescent signals were observed, proving that nanoparticles can be driven into the nanopores through an electric field.
In order to enhance the anti pollution performance of nanopore surfaces and reduce non-specific binding, researchers studied the chemical functionalization of nanopore surfaces and the removal efficiency of non-specific particles from nanopores of different diameters. The experiment successfully demonstrated that specific and non-specific binding events can be distinguished through electric field control, and by selecting appropriate nanopore diameters and electric field conditions, non-specific bound nanoparticles can be effectively removed, improving the selectivity and accuracy of the sensor.
Next, the researchers demonstrated the ability to detect the target protein VEGF based on the number of particles that can be removed from the pores (non-specific events) and the number of particles left in the pores when a negative voltage is applied (specific events). The experimental results indicate that the use of optical nanopore blocking sensors can successfully distinguish between specific and non-specific events. By calculating the percentage of particles remaining in the pores after applying negative voltage, the concentration of VEGF can be quantitatively analyzed.
Finally, the researchers conducted quantitative detection experiments using different concentrations of VEGF and fixed concentrations of Ab PSNP, with VEGF concentrations ranging from 0 to 7.895 pM. The binding ratios of Ab PSNP to VEGF at different concentrations are shown in the following figure. Meanwhile, the lowest detectable concentration of VEGF in the experiment was 78.75 fM (3 pg/mL).
In summary, the researchers demonstrated a technique for independently monitoring fluorescent nanoparticle blocking/unblocking events in a high-density nanopore array (676 nanopores), which can be used for quantitative analysis of ultra-low concentrations of analytes using single-molecule counting, and demonstrated its ability to quantify protein VEGF in the sub picomolar range.
In the future, this technology may be extended to the detection of other analytes present at ultra-low concentrations in biological samples. By increasing the number of nanopores detected, the sensitivity of the detection can be improved.
Source: Sensor Expert Network