Data Processing of SPR Curve Data to Maximize the Extraction of Changes in Electrochemical SPR Measurements

Abstract

We developed a novel measuring and data-processing method for performing electrochemical surface plasmon resonance (EC-SPR) on sensor surfaces for which detecting a specific SPR angle is difficult, such as a polymer having a non-uniform thickness with coloration. SPR measurements are used in medicine and basic research as an analytical method capable of molecular detection without labeling. However, SPR is not good for detecting small molecules with small refractive index changes. The proposed EC-SPR, which combines SPR measurements with an electrochemical reaction, makes it possible to measure small molecules without increasing the number of measurement steps. A drawback of EC-SPR is that it is difficult to detect a specific SPR angle on electron mediators, and it was found that it may not be possible to capture all the features produced. The novel method we describe here is different from the conventional one in which a specific SPR angle is obtained from an SPR curve; rather, it processes the SPR curve itself and can efficiently aggregate the feature displacements in the SPR curves that are dispersed through multiple angles. As an application, we used our method to detect small concentrations of H₂O₂ (LOD 0.7 μM) and glutamate (LOD 5 μM).

Keywords: electrochemical SPR (EC-SPR); glutamate; signal processing; surface plasmon resonance (SPR).

Figure 1

Mechanism of EC-SPR measurement. A- is a charge compensation molecule in a buffer consisting of chlorine ions and phosphate ions. The illustration enclosed in the purple dotted line is the molecular electronic transition reaction cascade from target capture to electron mediator oxidization. Reduced electron mediators attract two charge compensation molecules per molecule, but when oxidases capture the target molecule, the charge of the electron mediator changes, attracting three charge compensation molecules per molecule. Since SPR can detect the molecular concentration on the electrode surface, it can detect the difference in the charge compensation molecular weight attracted by the electron mediator.

Figure 2

EC-SPR measurement chip layout. (A) Chip layout of EC-SPR measurement. (B) Photograph of electrodes after the glass substrate and acrylic substrate were bonded. RE: Reference electrode, WE: Working electrode, CE: Counter electrode. (C) Photograph of reference electrode and working electrode on glass substrate. Each scale bars are 1 mm.

Figure 3

SPR measurements on electrodes subjected to potential sweep. (A) Time-course of SPR curve. Light intensity was standardized by zscore. Ox: oxidase state of osmium, 0 V: state with a charge of 0 V, RED: redox state of osmium. (B) Changes in SPR curve due to changes in the state of osmium. The inset is an enlarged view of the SPR curve between incident angles of 68 and 70 degrees. The triangles are the secondary dips in each SPR curve.

Figure 4

Photograph of electron mediator film applied on the electrode. Scale bar is 0.5 mm.

Figure 5

Feature quantity analysis obtained by KL conversion of image data of SPR curve. (A) SPR data deviation obtained from the completely oxidized and completely reduced states of the SPR curves. (B) Diagonal matrix obtained by KL conversion. (C) KL-converted SPR data.

Figure 6

Change in KL-converted SPR curve data when sweeping the potential. (A) KL-converted SPR curve data. KL-converted SPR data value was normalized using the 0.5 V value and 0.25 V value. (B) Current change detected by potentiostat. (C) Electronic sweep.

Figure 7

KL-converted SPR measurement data before normalization when repeated measurement is performed under the same voltage application condition.

Figure 8

Variation in KL-converted SPR data with H₂O₂ concentration. (A) Raw KL-converted SPR data showing transition with H₂O₂ concentration. (B) Calibration curve for H₂O₂ detection using the maximum slope of KL-converted SPR data.

Figure 9

Variation in KL-converted SPR data with glutamate concentration. (A) Raw KL-converted SPR data versus glutamate concentration. (B) Calibration curve for glutamate detection using the maximum slope of KL-converted SPR data.