Bacteria growth conditions and characteristics of the strains
The bacterial strains used in this work are listed in Table 2.2. E. coli MG1655 (Katushka), E. coli UTI89 (pathogenic) and the three modified strains AAEC185 were kindly gifted by dr. Julie Bouckaert from from Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Villeneuve d’Ascq; the strain E. coli K12 JM101TR E. coli K12 JM101TR 93,94 as well as all solid and liquid growth medias, the antibiotics and the isopropyl β-D-1-thiogalactopyranoside (IPTG) were kindly provided by the Biochemistry laboratory in Ecole Polytechnique, Palaiseau. The pathogenic S. aureus bacteria was grown by Endre Jakab and tested by Monica Potara in Interdisciplinary Research Institute in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania.
The growth of all the bacteria strain except S. aureus was performed in the Biochemistry laboratory in Ecole Polytechnique Both E. coli Katushka and E. coli JM101 strains were grown in 2x YT medium, all at 37°C with shaking until the OD600 reached 0.5-1. Part of the culture (10 mL) were washed with PBS 1X or Milli-Q water (20 mL), re-suspended in 1mL of PBS 1X or Milli-Q water (1 mL) and diluted to required concentration. E. coli AAEC185 (pUT2002) and E.coli (AAEC185) pMMB66 were grown in LB medium with antibiotic selection (25 μg/ml Chloramphenicol for pUT2002 and 100 μg/ml ampicillin for pMMB66) with shaking, at 37°C overnight. Part of E.coli (AAEC185) pMMB66 was washed with PBS 1X or Milli-Q water and diluted to required concentration as described above. In case of E.coli (AAEC185) pUT2002 part of first overnight culture was inoculated in fresh LB media (varying volumes but keeping 100x dilution) in a flask with a broad liquid air interface, with 1µM of IPTG added to the medium and incubated without shaking for about 48h. The obtained culture was washed with PBS 1X or Milli-Q water and diluted to required concentration.
S. aureus strain (ATCC 25923) was cultured overnight in a shaking incubator (ES 20/60, Biosan, Riga, Latvia) in Mueller-Hinton broth at 37ºC and 200 rpm until the light absorbance at 600 nm reached 1.0 (corresponding to 109 CFU mL-1 – Spekol UV VIS 3.02, Analytic Jena, Jena, Germany). The bacterial suspension was pelleted at 5000 x g for 10 min at 20ºC then washed three times with ultrapure water (Purelab Ultra Genetic, ELGA LabWater, High Wycombe, UK). After that, a ten-fold dilution series was prepared using ultrapure water. S. aureus strain (ATCC 43300 used for SEM images) was grown in Brain Heart Infusion (BHI) broth at 37°C with shaking until the OD600 reached 0.5-1.
Caracteristics of the strains
E. coli Katushka was obtained from the transformation of E. coli K12 MG1655 with the pDONR221-nadB-cat recombinant plasmid as described before.95 E. coli AAEC185 (pUT2002) and E. coli AAEC185 (pMMB66) are K12 strains modified with plasmids as follows: pUT2002 plasmid which carries the fim operon with a deletion in the FimH gene encoding the FimH adhesion96 and pMMB66 plasmid which is a low copy number plasmid with the lacI repressor and tac promoter, controlling the expression of the cloned wild-type fimH gene.97 To resume, E. coli AAEC185 (pUT2002) is characterized by a depletion of FimH protein, presenting only FimA fimbriae protein when it is induced during growth using isopropyl β-D-1-thiogalactopyranoside IPTG (as described in Table 2.2 and in Growth protocols section), E. coli AAEC185 (pMMB66) is characterized by a depletion of both FimH and FimA proteins and the strain modified with both plasminds E. coli AAEC185 (pUT2002pMMB66) presents both fimbriae proteins when it is induced using IPTG (as described in Table 2.2 and in Growth protocols section). FimA protein is located along the type1 fimbriae (pili) and FimH protein at the end of the fimbriae (pili) and they are adhesion receptors binding domains. Only E. coli Katushka presents flagella. Characteristics of the E. coli K12 AAEC185 compared with E. coli Katushka are shown in Figure 2.1.
Probes: antibodies and mannoside
Two types of probes were used for the development of the surface of the biosensor: antibodies interacting specifically with FimA protein found along the type1 fimbriae on E. coli type bacteria and a mannoside derivative (mannose carrying an amino group) for specific interaction with FimH protein found on the edge of type1 fimbriae. 95 In case of antibody probes, both serum and purified antibodies were employed.
a) Serum Antibodies
Serum antibodies (rat polyclonal anti-FimA antibodies) were kindly offered by Dr. Julie Bouckaert (UGSF, Villeneuve d’Ascq). For the production of anti-FimA antibodies, FimA protein extracted from bacterial fimbriae (Figure 2.2) was purified and sent for an immunization program to Eurogentec.
Polyclonal serum antibodies tested using ELISA method (protocol and results-Eurogentec) were used as received. The stock used was SYR474GP giving the highest immune response in ELISA test (black curve).
b) Purified antibodies
One part of serum antibodies stock (V= 3,5 mL) was purified by affinity chromatography using HiFliQ Protein G FPLC Column from Generon. Prior to injection on protein G column delipidation and filtration steps were performed.
For delipidation the serum was treated with a solution of 10% dextran sulphate and 1 M calcium chloride (for 1 mL of serum 1 mL of calcium chloride and 0.14 mL of dextran sulphate were added). After 15 minutes interaction the obtained precipitate was discarded and the supernatant was washed three times with the binding buffer and concentrated (binding buffer 20 mM sodium phosphate/0,8 M ammonium sulphate pH 7.4). The as-obtained stock was injected on protein G column for purification (flow rate 1mL/min). Two elution buffers were used: 0.1 M sodium citrate pH 5.46 and 0.2 M glycin pH 2.5. The corresponding chromatogram is displayed in Figure 2.4.
Two fractions of purified antibodies were obtained after elution, 1st at pH = 5.46 and 2nd one at pH = 2.51. Only the fraction at pH = 2.51 (elution 2 in Figure 2.4 ) was employed for the modification of the surface of the biosensor (Chapter 5) after neutralization (addition of 1M Tris pH = 9) and buffer exchange (the buffer was replaced by PBS 1X by multiple filtrations using Amicon filtering membranes). The concentration of the final stock of anti-FimA antibody (determined by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer) was 2.861 mg/mL in PBS 1X, stock used in all experiments.
For FimH-mannoside interactions 4-aminophenyl α-D-mannopyranoside hydrochloride was used kindly gifted by Dr. Julie Bouckaert. The stock at 8 mM dissolved in DMSO/MilliQ water was diluted into required spotting buffers as it will be described in Slides preparation section.
Cleaning procedure of the slides
Microscope glass slides were copiously rinsed with water then with TFD4 type detergent (Franklab), before being immersed in absolute ethanol for 15 min. After vigorously rinsing with deionized water, the slides were further immerged into piranha solution (1/3 H2O2/H2SO4; caution: very corrosive) for 15 min. After a final rinse with ultrapure Milli-Q water, the clean slides were dried under nitrogen flow.
Thermal deposition and annealing- preparation of SERS-active substrates Silver thin films were deposited on clean microscope glass slides using a home-made thermal evaporator as follows: a 10 cm long silver wire (99.99% purity, 0.25 mm diameter, supplied by Goodfellow) was deposited on a platinum crucible which was heated up by applying a 4.8 A current (Joule effect). The deposition was made under
a pressure of 25-35 10-6 Torr. Post-deposition annealing of Ag covered slides was carried out at 500 °C for 1 min under argon atmosphere using a rapid thermal annealer (Jipelec Jet First 100). Images of the home-made thermal deposition machine and of the annealing oven are displayed in Apparatus section (Figures 2.9 and 2.10)
Deposition of a-Si1-xCx:H thin films by plasma-enhanced chemical vapor deposition (PECVD) a-Si1-xCx:H thin films (3-5 nm) were deposited onto the glass slides (or on top of metallic films deposited on glass slides) using a homemade plasma-enhanced chemical vapor deposition (PECVD) reactor in low power regime (0.1 W/cm2) and at low temperature (150 or 250°C ) as described by Solomon et al.99 Methane (CH4) and silane (SiH4) were mixed inside the deposition chamber in specific ratios, depending on the desired x, in a flow rate of 2 L/h and keeping the pressure of gases at ~40 mTorr. For example, a-Si0.8C0.2:H was produced by mixing a methane and silane in the ratio 6.2/69.7, a-Si0.9C0.1:H, methane/silane in the ratio 13.3/51.1 and a-Si:H using only silane. The layer thickness was controlled by the deposition time (1 nm in 3 s): for 3 nm of thickness the deposition time is 9 s; for 5 nm 15 s. The reaction of the gases inside the reactor which leads to the formation of thin films on the substrates (glass slides) was initiated by radiofrequency-created plasma (13.56 MHz). Image of the reactor and the sample holder are displayed in Figure 2.11 in Apparatus section.
Substrates a-Si1-xCx:H deposited on glass were exposed to HF vapor for 15 s and placed into Schlenk tubes containing deoxygenated neat undecylenic acid at room temperature (the acid was heated at 100°C during 30 minutes and allowed to cool at room temperature before adding the slides).91 The tubes were subsequently introduced into a UV chamber and exposed to 312 nm irradiation for 3h. The interfaces were then rinsed for 30 min with hot acetic acid (75°C, two times), and finally with PBS 1X/0.1% SDS for 15 min, followed by 5 min PBS 0.2X, 5 min PBS 0.1X and 2 min Milli-Q water. The samples dried under nitrogen flow were stored under vacuum at room temperature until the next use.
NHS ester-functionalized surfaces
The acid-functionalized surface was subsequently immersed in 10 mL of a mixture of 10 mM EDC and 10 mM NHS for 90 min at 15°C. 100 The as-obtained samples were copiously rinsed with Milli-Q water and dried under nitrogen flow.
Table of contents :
Chapter 1 State of the art
1.2 Detection of bacteria
1.3 Detection of bacteria using SERS
1.4 Objective of the thesis
Chapter 2 Experimental methods and setup
2.2 Slides preparation/fabrication
2.3. Bacteria trapping and bacteria/NPs interactions
2.4. Samples characterization
Chapter 3 SERS substrates based on silver thin films
3.2 Silver SERS substrates: production, characterization and tests using model molecules
3.3 Study of bacteria deposited on SERS substrates
3.4 PCA for discrimination between strains
Chapter 4 New architecture proposed for the biosensor and new SERS strategy
4.2. Influence of the amorphous silicon layer on SERS exaltation
4.3 New strategy: SERS detection using NPs colloids
Chapter 5 Development and optimization of the biosensor
5.2 Optimization of spotting and blocking steps
5.3 Specificity, reusability and stability of the biosensor
5.4 Grafting of new probes on the biosensor’s surfaces
Chapter 6 SERS detection of bacteria using the biosensor
6.2 Tests with the entire biosensor architecture and optimization of SERS parameters
6.3 Detection of bacteria using fluidic system
6.4 Detection of bacteria in different complex media (serum, milk, water)
General conclusion and perspectives