Nano-Phase Separation and Analyte Binding in Aptasensors Investigated by Nano-IR Spectroscopy

Abstract

Biosensors based on DNA aptamer receptors are increasingly used in diagnostic applications. To improve the sensitivity and specificity of aptasensors, parameters affecting the stability and binding efficiency of the receptor layer need to be identified and studied. For example, the blocking step, i.e., the addition of inert molecules to the receptor layer, can improve sensor performance, but can also cause phase separation into nanodomains of unknown composition and structure. Here, nano-IR spectroscopy is used together with complementary macroscopic spectroscopic methods to study the nano-structural variations during the fabrication of a recently developed SARS-CoV-2 aptasensor. The blocking step by polyethylene glycol (PEG) causes a significant thickening of the receptor layer and a phase separation into nanodomains consisting of an aptamer-rich and a slightly thicker PEG-rich phase. The unambiguous chemical identification of the nanodomains is achieved by analysis of nano-IR images. Furthermore, bound analyte (spike protein of SARS-CoV-2) is detected at the single molecule level. Detailed analysis of the local nano-IR spectra revealed structural properties such as the amorphous state of the PEG molecules within the nanodomains and a strong change in the secondary structure of the analyte. This study significantly advances the understanding of nanoscale chemical processes in the receptor layer of aptasensors.

Keywords: aptasensor; nano‐IR spectroscopy; nano‐phase separation; self‐assembled‐monolayer; single molecule spectroscopy.

Figure 1

Modification steps during sensor fabrication and application. I: aptamer immobilization, II: PEG‐blocking, III: S protein binding.

Figure 2

Results of TM AFM measurements during biosensor fabrication. Typical topographies of Au(111)_sc (a), Au(111)_sc/C9t (b), and Au(111)_sc/PEG‐C9t (c) and the respective line scans (d). 50×50‐pixel squares in the topography images indicate selected areas for roughness determination.

Figure 3

Layer thicknesses of I: Au(111)/C9t, II: Au(111)/PEG‐C9t, and III: Au(111)/(PEG‐C9t)+ S protein determined by spectroscopic ellipsometry. The error bars represent the maximum errors arising from the standard deviations of substrate and sample measurements.

Figure 4

IRRA spectra of fabrication steps and application of aptasensor in the low (a) and high wavenumber range (b). Peak assignments are presented in Table  1 and in the text.

Figure 5

Results of TM AFM‐IR measurements on Au(111)_sc/C9t. Averaged nano‐IR point spectra, recorded at 5 positions (a,b) (The spectra before correction and the position of recording are given in Figure S4, Supporting Information). Nano‐IR mappings at maximum of ν _s (C═O) at 1720 cm⁻¹ (c) (Associated topography and phase images are given in Figure S4, Supporting Information). IR absorbance‐height scatter plot with point density represented by the color (d) created from the data in the square indicated in (c).

Figure 6

Results of TM AFM‐IR measurements on Au(111)_sc/PEG‐C9t. Averaged nano‐IR point spectra recorded on 3–4 different positions per spectrum (a, b). Nano‐IR mapping at maximum of aptamer ν(C = O) at 1680 cm⁻¹ (c) (Associated topography and phase image are given in Figure S7, Supporting Information). IR absorbance‐height scatter plot with point density represented by the color (d) created from the data in the square indicated in c.

Figure 7

Results of TM AFM of Au(111)_sc/(PEG‐C9t)+ S protein topography image (a) and line profiles (b). The line profiles are in corresponding colors to the lines in the topography image.

Figure 8

Results of TM AFM‐IR measurements on Au(111)_sc/(PEG‐C9t)+ S protein. Averaged nano‐IR point spectra recorded on proteins and receptor layer (a). Comparison of solution spectrum of S protein recorded by ATR‐IR with difference spectrum S proteins minus receptor layer recorded by AFM‐IR(b). Topography image of proteins complex on receptor layer (c). Averaged nano‐IR point spectra recorded on proteins and nano domains in the receptor layer (d).