Fourier spotting: a novel setup for single-color reflectometry

Johannes Siegel,Peter Fechner,Juergen H Werner,Markus Schubert,Marcel Berner,Guenther Proll

doi:10.1007/s00216-021-03802-w

Abstract

Single-color reflectrometry is a sensitive and robust detection method in optical biosensor applications, for example for bioanalysis. It is based on the interference of reflected monochromatic radiation and is label free. We present a novel setup for single-color reflectometry based on the patented technology of Berner et al. from 2016. Tilting areas of micro-mirrors allow us to encode the optical reflection signal of an analyte and reference channel into a particular carrier frequency with the amplitude being proportional to the local reflection. Therefore, a single photodiode is sufficient to collect the signals from both channels simultaneously. A 180∘ phase shift in the tilt frequency of two calibrated micro-mirror areas leads to a superposition of the analyte and reference signal which enables an efficient reduction of the baseline offset and potential baseline offset drift. A performance test reveals that we are able to detect changes of the refractive index n down to Δn < 0.01 of saline solutions as regents. A further test validates the detection of heterogeneous binding interaction. This test compromises immobilized testosterone-bovine serum albumin on a three-dimensional layer of biopolymer as ligand and monoclonal anti-testosterone antibodies as analyte. Antibody/antigen binding induces a local growth of the biolayer and change in the refractive index, which is measured via the local change of the reflection. Reproducible measurements enable for the analysis of the binding kinetics by determining the affinity constant KA = 1.59 × 10− 7 M− 1. In summary, this work shows that the concept of differential Fourier spotting as novel setup for single-color reflectometry is suitable for reliable bioanalysis.Graphical

Highlights

Almost all biosensors are based on a two-component system to measure the binding affinity between the ligate and the ligand [1]
Reproducible measurements of time-resolved biological binding interaction demonstrate the ability of differential Fourier spotting (DFS)-single-color reflectometry (SCORE) to be suitable as a biosensor to investigate molecular binding interactions
Saline solutions with varying concentrations are well suited for simulating changes in n2 which might originate from the change of refractive index of biopolymer in case of attaching analyte molecules to the immobilized target

Summary

Introduction

Almost all biosensors are based on a two-component system to measure the binding affinity between the ligate (target molecule, e.g., antibody) and the ligand (inhibitor, e.g., antigen) [1]. In case of RIfS, the use of the whole multi-wavelength spectrum in the reflection measurements includes signals from wavelengths which barely change in reflection during the binding interaction of the antigen/antibody layers. The combination of SCORE with optical modulation methods from the field of singlepixel imaging seems to be a pathway to overcome the required high computational power due to conventional camera systems by reducing the recorded data to a single time signal. Single-pixel imaging describes the detection of an object with various modulation schemes so that a one-element detector is sufficient to recover the image [9] These modulation schemes allow encoding the optical signals of parallelly measured analytic SCORE spots to a single time signal. Reproducible measurements of time-resolved biological binding interaction demonstrate the ability of DFS-SCORE to be suitable as a biosensor to investigate molecular binding interactions

Experimental setup

Measuring procedure

Results

Regeneration phase