Atomic force microscopy fishing and mass spectrometry identification of gp120 on immobilized aptamers

Abstract

Atomic force microscopy (AFM) was applied to carry out direct and label-free detection of gp120 human immunodeficiency virus type 1 envelope glycoprotein as a target protein. This approach was based on the AFM fishing of gp120 from the analyte solution using anti-gp120 aptamers immobilized on the AFM chip to count gp120/aptamer complexes that were formed on the chip surface. The comparison of image contrasts of fished gp120 against the background of immobilized aptamers and anti-gp120 antibodies on the AFM images was conducted. It was shown that an image contrast of the protein/aptamer complexes was two-fold higher than the contrast of the protein/antibody complexes. Mass spectrometry identification provided an additional confirmation of the target protein presence on the AFM chips after biospecific fishing to avoid any artifacts.

Keywords: aptamer; atomic force microscopy; gp120 HIV-1 envelope glycoprotein; mass spectrometry.

Figure 1

Atomic force microscopy (AFM) images of aptamer chip surface with immobilized aptamers (A) and image of the control area without aptamers before biospecific fishing of gp120. (B) Density of visualized object distribution with heights ρ(h) over the area with immobilized anti-gp120 aptamers. (C) Density of visualized object distribution. Notes: Experimental conditions were standard cantilever, tapping mode in air, relative humidity 60%, temperature of 22°C, scan size 2.0×2.0 μm²; NTEGRA Prima AFM. Arrows point to several objects with height ~1 nm.

Figure 2

Atomic force microscopy (AFM) topography image (A) and recognition image (B) of the chip surface with immobilized anti-gp120 aptamers before biospecific fishing of gp120. Notes: Experimental conditions were gp120-functionalized cantilever, PicoTREC mode in buffer, 10 mM Modified Dulbecco’s phosphate buffer, pH 7.4, temperature of 22°C; scan size 3.0×3.0 μm², Agilent 5500 AFM. Dark spots on the recognition image are recognition events on binding of aptamer on the surface and tip-tethered gp120. Some coincidences of objects and recognition events are indicated by circles as an example.

Figure 3

Atomic force microscopy (AFM) images of aptamer chip surface after biospecific fishing of gp120. Notes: (A, B) AFM images of the area with immobilized aptamers, (C) AFM image of the control area without aptamers, and (D) density of visualized object distribution with heights ρ(h) obtained over the area with immobilized aptamers after fishing of gp120 at different concentrations. Experimental conditions were concentrations of the analyte solutions (A, C) C_gp120=0.8×10⁻⁹ M and (B) C_gp120=0.8×10⁻¹¹ M; standard cantilever, tapping mode in air, relative humidity 60%, temperature of 22°C, scan size 2.0×2.0 μm²; NTEGRA Prima AFM. Arrows point to gp120/aptamer complexes. (D) ρ(h) for C_gp120=0.8×10⁻¹¹ M (solid line), for C_gp120=0.8×10⁻⁹ M (dashed line) and for C_gp120=0 (dotted line).

Figure 4

Atomic force microscopy (AFM) images of antibody chip surface after biospecific fishing of gp120. Notes: (A) AFM image of the area with immobilized antibodies before biospecific fishing of gp120, (B) AFM image of the area with immobilized antibodies after biospecific fishing of gp120, (C) AFM image of the control area without antibodies, and (D) density of visualized objects distribution with heights ρ(h) obtained over the area with immobilized antibodies. Experimental conditions were concentration of the analyte solution C =10^–8 gp120 M; standard cantilever, tapping mode in air, relative humidity 60%, temperature of 22°C, scan size 2.0×2.0 μm²; NTEGRA Prima AFM. (D) ρ(h) after fishing of gp120 (solid line) and before fishing of gp120 (dashed line).

Figure 5

Mass spectra of objects on the atomic force microscopy (AFM) chips with molecular probes before and after biospecific fishing of gp120: before fishing on the AFM chip with immobilized aptamers (A) and antibodies (B); after fishing on the AFM chip with immobilized aptamers (C) and antibodies (D). Notes: Experimental conditions were concentrations of the analyte solutions (C) C_gp120=0.8×10⁻¹¹ M and (D) C_gp120=10⁻⁸ M. Trypsin autolysis peaks are pointed by circles (•), peaks of antibodies are pointed by triangles (▲), gp120 peptides are marked as S/N with the corresponding values of the signal/noise ratio. Abbreviation: au, arbitrary units.