Bare Eye Detection of Bacterial Enzymes of Pseudomonas aeruginosa with Polymer Modified Nanoporous Silicon Rugate Filters

Abstract

The fabrication, characterization and application of a nanoporous Silicon Rugate Filter (pSiRF) loaded with an enzymatically degradable polymer is reported as a bare eye detection optical sensor for enzymes of pathogenic bacteria, which is devoid of any dyes. The nanopores of pSiRF were filled with poly(lactic acid) (PLA), which, upon enzymatic degradation, resulted in a change in the effective refractive index of the pSiRF film, leading to a readily discernible color change of the sensor. The shifts in the characteristic fringe patterns before and after the enzymatic reaction were analyzed quantitatively by Reflectometric Interference Spectroscopy (RIfS) to estimate the apparent kinetics and its dependence on enzyme concentration. A clear color change from green to blue was observed by the bare eye after PLA degradation by proteinase K. Moreover, the color change was further confirmed in measurements in bacterial suspensions of the pathogen Pseudomonas aeruginosa (PAO1) as well as in situ in the corresponding bacterial supernatants. This study highlights the potential of the approach in point of care bacteria detection.

Keywords: Pseudomonas aeruginosa; bacteria detection; bacterial enzyme; biodegradable polymer; nanoporous silicon rugate filter; poly(lactic acid).

Scheme 1

Schematic of the pSiRF-based sensor for bacterial enzymes: (a) neat nanoporous pSiRF; (b) pSiRF after deposition of PLA inside the pores; (c) enzymatic degradation of PLA inside the pores; (d) pSiRF after the reaction. The color change of the pSiRF from blue (bare) to green (pores modified with PLA) is due to the increase in the effective refractive index of the sensor caused by the compositional change inside the pores. The original color is recovered after enzymatic degradation of PLA.

Figure 1

FESEM images of pSiRF sensor: (a) Top view; (b,c) cross-sectional views with low and high magnification, respectively, showing the modulated pore diameters. The schematic temporal current density profile corresponds to the data in panel (c).

Figure 2

RIfS spectra of pSiRF in air at 23 °C before (green) and after (blue) thermal oxidation in air at 600 °C for 1 h.

Figure 3

RIfS spectra and photographs of dried pSiRF: (a,b) air-filled pSiRF, (c,d) PLA-filled pSiRF and (e,f) pSiRF after enzymatic degradation of PLA with proteinase K (scale bars = 0.5 cm).

Figure 4

RIfS spectra of PLA coated pSiRF after incubation with 1.00 mg mL⁻¹ proteinase K for 0, 30, 60, 120, 240 and 360 min. The RIfS measurement was performed after the washing and drying steps of pSiRF.

Figure 5

Plot of ln(1 − x) (x: extent of reaction) and the pore filling as a function of incubation time for the degradation of PLA inside pSiRF in 0.10, 0.25, 0.50 and 1.00 mg mL⁻¹ of proteinase K solution at 25 °C for 0, 30, 60, 120, 240 and 360 min. The pore filling was estimated based on Figure S6. The data were presented as mean ± standard deviation. The solid lines correspond to linear least-squares fit of RIfS data. All measurements were conducted in the dried state.

Figure 6

Plot of apparent rate constant (k) versus proteinase K concentrations (0.10, 0.25, 0.50 and 1.00 mg mL⁻¹). The values for k were obtained from linear fits in Figure 5; the error bars denote the standard error from these fits. The solid line corresponds to the linear least-squares fit of the first three data points used to estimate the corresponding slope (0.022 mg mL⁻¹ min⁻¹) and serves, similar to the dotted line, as a guide to the eyes only.

Figure 7

Plot of in situ RIfS measurement of enzymatic reaction in PLA modified pSiRF with proteinase K solution (1.00 mg mL⁻¹) and Tris-HCl buffer at 25 °C.

Figure 8

Photographs of the pre-dried PLA-coated pSiRF before and after the immersion in bacteria-free LB medium and in P. aeruginosa (PAO1) suspension (bacteria starting concentration: 1.9 × 10⁹ CFU.mL⁻¹) in LB medium at 37 °C for 24 h (end concentration: 3.7 × 10⁹ CFU.mL⁻¹), scale bar = 1 cm.

Figure 9

Plot of in situ RIfS measurement during incubation of a PLA-modified pSiRF sensor in sterile P. aeruginosa (PAO1) supernatants obtained from a 1.9 × 10⁹ CFU mL⁻¹ suspension by filtration (black line) and in pure LB medium (red line). The PLA-modified pSiRF sensor was stable in LB for more than 22 h, whereas incubation in P. aeruginosa supernatant caused a quasi-linear blue shift in the wavelength. The spectral positions of the characteristic peaks were corrected by subtracting the wavelength obtained before incubation in the bacterial supernatant or in LB medium.