Supporting Information for the Article:

RAPID IDENTIFICATION OF PSEUDOMONAS SPP. VIA RAMAN SPECTROSCOPY USING PYOVERDINE

AS CAPTURE PROBE

Susanne Pahlow, Stephan Stöckel, Sibyll Pollok, Dana Cialla-May, Petra Rösch, Karina Weber

and Jürgen Popp

CONTENTS

1) Isolation of Pseudomonas spp. with a Pyoverdine Modified Chip

Figure S-1. P. aeruginosa DSM 22644

Figure S-2. P. aeruginosa (environmental isolate)

Figure S-3. P. fluorescens DSM 50090

Figure S-4. P. fluorescens DSM 50106

Figure S-5. P. chlororaphis DSM 50083

2) Characterization of the Pyoverdine Modified Chip Surface via Raman Spectroscopy

Figure S-6. Background spectra of chip substrates and reference spectrum of pyoverdine.

1) Isolation of Pseudomonas spp. with a Pyoverdine Modified Chip

In order to prove that the binding of the bacterial cells is actually due to the pyoverdine modification, be-

low several microscopic images, acquired after staining with crystal violet, are shown. Next to

P. psychrophila we could successfully isolate P. aeruginosa (DSM 22644), P. aeruginosa (environmental

isolate), P. fluorescens (DSM 50090), P. fluorescens (DSM 50106) and P. chlororaphis (DSM 50083) as

can be seen in Figures S1 – S5. It becomes apparent, that the different Pseudomonas species specifically

bind to the pyoverdine modified areas. Each figure shows an aluminum field modified with pyoverdine

and one blank field of the very same chip. On the pyoverdine fields the areas where the pyoverdine was

immobilized can be nicely recognized as circular spots. Some unspecific binding does occur however

since the experiments were carried out with very high cell concentrations of 109 cells/ml.

Figure S-1. a) Schematic display of the isolation protocol for demonstrating the specific capture of Pseudomonas spp. due to the pyover-

dine modification. b) Microscopic images of a pyoverdine modified aluminum field and an unmodified field after incubation with P. aeru-

ginosa shown in different magnifications.

Figure S-2. a) Schematic display of the isolation protocol for demonstrating the specific capture of Pseudomonas spp. due to the pyover-

dine modification. b) Microscopic images of a pyoverdine modified aluminum field and an unmodified field after incubation with P. aeru-

ginosa shown in different magnifications.

Figure S-3. a) Schematic display of the isolation protocol for demonstrating the specific capture of Pseudomonas spp. due to the pyover-

dine modification. b) Microscopic images of a pyoverdine modified aluminum field and an unmodified field after incubation with P. fluo-

rescens shown in different magnifications.

Figure S-4. a) Schematic display of the isolation protocol for demonstrating the specific capture of Pseudomonas spp. due to the pyover-

dine modification. b) Microscopic images of a pyoverdine modified aluminum field and an unmodified field after incubation with P. fluo-

rescens shown in different magnifications.

Figure S-5. a) Schematic display of the isolation protocol for demonstrating the specific capture of Pseudomonas spp. due to the pyover-

dine modification. b) Microscopic images of a pyoverdine modified aluminum field and an unmodified field after incubation with P. chlo-

roraphis shown in different magnifications.

2) Characterization of the Pyoverdine Modified Chip Surface via Raman Spectroscopy

The concentration of pyoverdine used for the isolation experiments was too low to yield a distinct spec-

trum with characteristic marker bands. In order to obtain a Raman spectrum of this substance, we spotted

the ferric complex of pyoverdine in a concentration of 1 mg/ml and subsequently measured the dried sub-

stance. The spectrum is depicted in Figure S6a. The band positions are in excellent agreement with a

study of Wu et al., who used pyoverdine as a biomarker for Raman based detection of P. aeruginosa.1 In

Figure S6b the background spectra of the silanized aluminum and the pyoverdine modified aluminum are

shown on a smaller scale. No distinct bands neither from the silane nor the siderophore can be recog-

nized. The peak at 2328 cm-1 arises from ambient N2.

Figure S-6. a) Background spectra of the GOPS silanized and the pyoverdine modified Al substrates including a reference spectrum of

pyoverdine. b) Background spectra of the GOPS silanized and the pyoverdine modified Al substrates shown on a smaller scale.

1. X. Wu, J. Chen, Y. Zhao, S.M. Zughaier, Rapid detection of Pseudomonas aeruginosa biomarkers in biological fluids using surface-

enhanced Raman scattering, in: SPIE Sensing Technology+ Applications, International Society for Optics and Photonics, 2014, pp.

91070A-91070A-91010.