Received Date:
22 November 2018 Available Online:
15 March 2019
Fund Project:
Project supported by the National Natural Science Foundation of China (Nos. 51675101, 51435003, 51375092) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX18_0019)
Abstract:
Immunotherapy for cancer is a method to treat cancer by using the body's own immune system. Programmed death receptor 1 (PD-1) is one of the checkpoints in the immunotherapy. The signal pathway PD-1 (programmed death receptor 1)/PD-L1 (ligand of PD-1) is closely related to the immune escape of the cancer cells. The inhibitor drugs for PD-1 checkpoint, essentially the monoclonal antibodies of PD-1 or PD-L1 which is essentially the immune checkpoints inhibitors could block the PD-1/PD-L1 pathway and reactivate T-cells to kill cancer cells, and as a result, the immunotherapy for cancer is realized. In order to study the binding process of PD-1 drugs and PD-1 antigen in vivo, in this work, solid-state nanopore as a single molecule method is used to detect the binding of PD-1 antibody and antigen. The PD-1 antibody as well as antigen is driven though the same nanopore under the same experimental condition by the external electric field. Since the antibody's block is about 0.01297 while the antigen's block is 0.00404, the PD-1 antibody is distinguished with the PD-1 antigen according to the theoretical formula. Driving the PD-1 antigen though the nanopore modified by PD-1 antibody (a series of experiments are conducted for characterization) under the same temperature and buffer concentration, the antibody-antigen complexes are detected and distinguished with PD-1 protein and its antibody through the relative current drop analysis and the current drop achieved before. The results suggest that the antibody and antigen have a specific binding (the smaller peak represents the free PD-1 antibody and antigen) and the binding process can be detected by nano-sensors. So the nanopore is able to distinguish the antibody, the antigen and the complexes without any labling. And in the future, the nanopore technology may be a rapid and label-free way for patients and doctors to evaluate the drugs' efficiency.
Figure 1.
(a) The raw current trace of the PD-1, (b) the raw current trace of the PD-1 antibody, (c) the relative current drop ΔI/I0 histogram of the PD-1 (the curve satisfies Gauss distribution and the Gauss peak is about 0.00404), (d) the relative current drop ΔI/I0 histogram of the PD-1 antibody (the Gauss peak is about 0.01297), (e) hydrodynamic measurements of PD-1 antigen without electric field using dynamic light scattering (DLS) (hydrodynamic diameter (Dh) of PD-1 antigen is about 5.10 nm) and (f) hydrodynamic measurements of PD-1 antibody without electric field using dynamic light scattering (DLS) (hydrodynamic diameter (Dh) of PD-1 antibody is about 10.08 nm)
Figure 2.
(a) Nanopore antibody modification and (b) characterization of the modified nanopore
The square line represents the I-V curve with equal concentration buffer solution. The dot line represents the I-V curve with concentration gradient buffer solution. The triangle line represents the I-V curve of the modified nanopore with concentration gradient buffer solution
Figure 3.
(a) The modified nanopore, (b) the scatter of the data and (c) the histogram of the complexes' relative current drop (two Gauss peaks are 0.00685 and 0.02128)
Figure 4.
(a) The structure of the fabricated nanopore, and the black circle area is the actual pore, (b) the enlarged image of the actual pore (the left one is the sectional view of the nanopore, and the right one is the top view of the pore) and (c) the I-V curve with an inserted SEM image of fabricated pore after zero calibration (the whole experiment is conducted in pH＝8, 0.5 mol/L KCl solution)
Figure 1
(a) The raw current trace of the PD-1, (b) the raw current trace of the PD-1 antibody, (c) the relative current drop ΔI/I0 histogram of the PD-1 (the curve satisfies Gauss distribution and the Gauss peak is about 0.00404), (d) the relative current drop ΔI/I0 histogram of the PD-1 antibody (the Gauss peak is about 0.01297), (e) hydrodynamic measurements of PD-1 antigen without electric field using dynamic light scattering (DLS) (hydrodynamic diameter (Dh) of PD-1 antigen is about 5.10 nm) and (f) hydrodynamic measurements of PD-1 antibody without electric field using dynamic light scattering (DLS) (hydrodynamic diameter (Dh) of PD-1 antibody is about 10.08 nm)
Figure 2
(a) Nanopore antibody modification and (b) characterization of the modified nanopore
The square line represents the I-V curve with equal concentration buffer solution. The dot line represents the I-V curve with concentration gradient buffer solution. The triangle line represents the I-V curve of the modified nanopore with concentration gradient buffer solution
Figure 3
(a) The modified nanopore, (b) the scatter of the data and (c) the histogram of the complexes' relative current drop (two Gauss peaks are 0.00685 and 0.02128)
Figure 4
(a) The structure of the fabricated nanopore, and the black circle area is the actual pore, (b) the enlarged image of the actual pore (the left one is the sectional view of the nanopore, and the right one is the top view of the pore) and (c) the I-V curve with an inserted SEM image of fabricated pore after zero calibration (the whole experiment is conducted in pH＝8, 0.5 mol/L KCl solution)
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