Polarization modulated spectroscopic ellipsometry-based surface plasmon resonance biosensor for E. coli K12 detection

Abstract

In this work, we report on the application of the polarization modulated spectroscopic ellipsometry-based surface plasmon resonance method for sensitive detection of microorganisms in Kretschmann configuration. So far, rotating analyzer and single wavelength polarization modulation methods have widely been investigated for phase sensitive surface plasmon resonance measurement. In this study, a much simpler optical setup relying on fast electro-optic phase modulator crystals is introduced for bacteria detection. A beta barium borate crystal connected to a function generator is adapted for generating phase shifts in the millisecond regime to extract the ellipsometric angles ( $\Psi$ and $\Delta$ ) under the surface plasmon resonance condition. For detection, the gold surface was functionalized with anti-Escherichia coli antibodies, and E. coli K12 was attached to them. We show that polarization modulated spectroscopic ellipsometry achieves a refractive index resolution in the order of ${10}^{-5}$ RIU, and a limit of detection of ${10}^2$ CFU/mL for E. coli K12 which is compatible with other surface plasmon resonance based phase sensitive methods with more complex detection concepts. As a follow-up step, an optical model can be developed to enhance this biosensor's performance, and applications for sorting and detecting other biological targets will be investigated.

Keywords: E. coli bacteria; Electro-optic polarization modulation; Spectroscopic ellipsometry; Surface plasmon resonance.

Fig. 1

Schematic configuration of the optical setup based on polarization modulated spectroscopic ellipsometry using an electro-optic phase modulator.

Fig. 2

Schematic of the ligand immobilization process on the gold surface: (a) Self-assembled monolayer formation of carboxymethylated dextran chains on the gold surface, (b) Covalently bonded thiol group to the gold surface, (c) Sensor surface activation using EDC/NHS mixture and (d) Covalent bonding between anti-E. coli antibody and NHS-esters.

Fig. 3

Ellipsometric angles ($\mathrm{\Psi }$ and $\mathrm{\Delta }$) for water as a reference medium for different gold film thicknesses and angles of incidence: (a-1) $\mathrm{\Delta }$ for 45 nm, (a-2) $\mathrm{\Psi }$ for 45 nm, (b-1) $\mathrm{\Delta }$ for 55 nm, (b-2) $\mathrm{\Psi }$ for 55 nm, (c-1) $\mathrm{\Delta }$ for 60 nm, (c-2) $\mathrm{\Psi }$ for 60 nm.

Fig. 4

Calculated [area under the curve/error] for (a) 45 nm, (b) 55 nm and (c) 60 nm gold layer thicknesses versus the RI for different water-glycerol concentrations. Red and black curves present the data for ${75}^{\circ }$ and ${80}^{\circ }$, respectively. Solid and dashed lines represent the data for $\mathrm{\Psi }$ and $\mathrm{\Delta }$, respectively.

Fig. 5

(a) In-situ measurement of the position of the resonance wavelength for different water-glycerol concentrations. Dark green and bright green curves represent the actual and smoothed data, respectively. (b) Intensity spectra near the plasmonic resonance wavelength for different water-glycerol concentrations. (c) Position of the resonance wavelength versus refractive index of water-glycerol solutions and fitted curve. (d) Calculated standard deviation for each water-glycerol concentration. (e) In-situ tracking of the position of the resonance wavelength for water as a reference medium for approximately 1 hour.

Fig. 6

(a) Sensogram of ligand immobilization steps on sensor surface using the amine coupling method. (b) Sensogram of bacteria injections through the surface from lowest to highest concentrations. Between each injection, PBS was used to wash the surface. (c) Optical image of attached single E. coli K12 cell on gold surface after the final washing step with PBS. (d) The position of the resonance wavelength for each concentration of bacteria and calculated quadratic fit with high correlation of ${\text{R}}^2$ = 0.98.

Fig. 7

(a-1) and (b-1) Two-dimensional in-situ sensogram measurements including ligand immobilization and bacteria detection steps for $\mathrm{\Psi }$ and $\mathrm{\Delta }$, respectively. (a-2) and (b-2) Cross sections of $\mathrm{\Psi }$ and $\mathrm{\Delta }$ spectra, respectively. (a-3) and (b-3) Three-dimensional in-situ sensogram measurements for $\mathrm{\Psi }$ and $\mathrm{\Delta }$, respectively.