Sum-frequency generation spectroscopy applied to model biosensors systems

doi:10.1016/j.tsf.2004.06.108

Thin Solid Films

Volumes 464–465, October 2004, Pages 373-378

https://doi.org/10.1016/j.tsf.2004.06.108 Get rights and content

Abstract

Vibrational information recorded by infrared-visible sum frequency generation spectroscopy was used to study the adsorption of a derivated vitamin (biocytin) on different substrates and its subsequent reaction with a protein (avidin). No reaction is observed on metallic substrates. When the experiments are carried out with a CaF₂ substrate in the total internal reflection configuration, significant changes of the CH and NH vibrations can be related to the specific bonding of avidin to biocytin.

Introduction

In a general way, a biosensor is a device capable of detecting a biological phenomenon by a physical signal. DNA microarrays also known as “gene chips” are one example of such biosensors where complementary DNA sequences are detected through fluorescence of added chromophores. The physical techniques that have been applied so far to the biosensors include atomic force microscopy (AFM) [1], [2], visible–ultraviolet absorption spectroscopy [3], [4], surface plasmon resonance (SPR) [5], [6], Raman spectroscopy [7] and, recently, Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) [8], [9]. However, in that latter case, like for the chromophores needed for the fluorescence, it was necessary to use markers in order to distinguish between reacted and non-reacted species.

Like infrared and Raman spectroscopies, infrared-visible sum-frequency generation (SFG) [10] spectroscopy aims at identifying adsorbed species through their vibrational fingerprints. However, unlike the other two spectroscopies, SFG has the unrivaled advantage of being intrinsically sensitive to surface (or interface) vibrations of centrosymmetric media and consequently seems better appropriate to biosensor detection where only surface or interface phenomena matter. In the classical SFG configuration schematized in Fig. 1, two pulsed laser beams impinge on the sample surface, the first one being visible at fixed frequency (ω_vis), and the second one being infrared (ω_ir) and tunable over the vibrational fingerprint of the target molecules. The high intensity of the lasers is sufficient to reveal the second order components of the sample susceptibility that will mix the frequencies of the two lasers and give rise to non linear effects like optical rectification, second harmonic (SHG), sum (SFG) and difference (DFG) frequency generations. Within the electric dipole approximation, all these processes should vanish inside centrosymmetric media, but not at their surfaces or interfaces, which is the reason why they can be considered as surface/interface sensitive. In a sum-frequency generation (SFG) spectroscopy experiment, the photons generated at the sum of the two laser frequencies (ω_vis+ω_ir) are recorded as a function of the variable infrared frequency. The resonant part of the spectrum is related to the vibrational fingerprint of the sample surface, shifted in the visible by the nonlinear process. As an example of SFG application to biological samples, let us mention the observation of conformational changes of a biopolymer (PEG) in water [11], the investigation of protein adsorption at aqueous interfaces [12], the study of molecular packing of proteins [13] and of polymer surfaces [14], [15], [16].

The system “biocytin–avidin” has been selected as an appropriate template for testing the possible application of SFG spectroscopy to real biosensors. An almost identical system “biotin–avidin”, because of the strong affinity of the two biomolecules, has been considered as the paradigm of ligand-receptor complex and has therefore been subjected to numerous investigations [17], [18], [19], [20], [21], [22]. We proceed first by immobilizing a biocytin monolayer on various metallic and insulating substrates and characterize it by SFG and FT-IRRAS. The recognition of avidin by biocytin is then analyzed by observing the differences in the frequency spectra induced after the adsorbed biocytin is immersed in an avidin solution. The specificity of the recognition process and of the SFG analysis is then confirmed by measurements carried out with two other protein solutions: avidin previously saturated with biotin and bovine serum albumin (BSA).

Section snippets

Experimental description

Biotin [C₅H₇N₂OS–(CH₂)₄–COOH] is commonly referred to as vitamin H. It has the structure of a bicycle ring attached to an alkane chain terminated by a carboxylic group. Biocytin is the reaction product of biotin with lysine (C₆H₁₄N₂O₂). Its chemical formula is C₁₆H₂₈N₄O₄S and its structure is represented in Fig. 2. Ag(111) and Pt(111) single crystals, polycrystalline Au films (Arrandee) and a CaF2 prism (λ/10 flatness, Foctek) were used as substrates. All substrates were sonicated in acetone,

Results and discussion

On Pt(111) and Au substrates, biocytinilated thiol had to be used instead of biocytin which showed poor adhesion properties. In that case, like for well-known alkanethiols, the bonding to the metal proceeds via the thiol group and self-assembly is expected on the surface. This is confirmed by the absence of the ν(S–H) vibration, expected between 2400 and 2600 cm⁻¹, in the PM-IRRAS measurements shown in Fig. 3 (upper curve). On the other hand, from results published in the literature on the

Conclusion

Infrared-visible SFG was first used to study the adsorption of biocytin on various kinds of substrates. We found that biocytin molecules build a fairly defective self-assembled monolayer on all investigated surfaces. Metallic substrates are not suitable for investigating avidin because the protein layer is damaged by the laser beams. Only the TIR configuration with CaF₂ as transparent substrate allowed studying the sample after interaction with avidin. Modifications in the CH and NH spectral

Acknowledgements

C.H. and A.P. are scientific research worker and research associate of the Belgian National Fund for Scientific Research (F.N.R.S), respectively. This work is supported by the Ministry of the Walloon Region (Belgium) and by the Interuniversity Research Program 05/01 on “Quantum size effects in nanostructured materials”, initiated by the Belgian Office for Scientific, Technical and Cultural Affairs.

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Sum-frequency generation spectroscopy applied to model biosensors systems

Abstract

Introduction

Section snippets

Experimental description

Results and discussion

Conclusion

Acknowledgements

Colloids Surf., A Physicochem. Eng. Asp.

Nature

Langmuir

Annu. Rev. Phys. Chem.

Biochem. J.

Langmuir

Single Mol.

Langmuir

Langmuir

Langmuir

J. Raman Spectrosc.

J. Raman Spectrosc.