We used infrared-visible sum-frequency generation (SFG) spectroscopy to study a model biosensor based on the well-known interaction between a protein (avidin) and a vitamin (biocytin). This experimental tool is ideal to study such system because it is sensitive to the interface and allows the measurement of its vibrational fingerprint. We worked in two steps: in a first step, we characterized the formation of the biocytin layer and secondly we studied its interaction with different target molecules. In our experiments, we used different types of substrates, metallic or not.
First, we studied adsorbed biocytin molecules on metallic substrates (Pt, Au, Ag) in the stretching C-H spectral range. For the biocytin/Pt spectrum, the observation of peaks attributed to the alkane chain and aromatic methylene stretching vibration modes attests the formation of an oriented biocytin self-assembled monolayer. We can observe the same resonances and draw the same conclusions on Au and Ag.
After immersion of the sensor in the avidin solution, the spectra do not change. This means that there is no significant recognition of the protein.
In a second time, we used a calcium fluoride (CaF2) prism as substrate. It is optically transparent between 0.2 and 7 µm and can therefore be used in a total internal reflection configuration (TIR). We investigated the stretching C-H and N-H spectral ranges. For the layer of biocytin, the resonances show a similar structure as on the metallic substrates. After interaction with the avidin solution, significant modifications of the SFG spectra attest the molecular recognition.
In a final step, we checked the sensor selectivity by substituting avidin with bovine serum albumina (BSA) and with avidin pre-saturated with biocytin. In those cases, no significant modification of the spectra is observed, demonstrating the specific binding between avidin and biocytin.