An optical biosensor was developed for the detection of pathogenic E. coli
O157:H7, by using FTIR spectroscopy to provide mid-infrared fingerprints of pathogens present in buffer. The spectroscopic fingerprint of pathogens originates from the various functional groups related to proteins, lipids, and carbohydrates, and their mid-infrared (MIR) spectra can be used for the identification and structural characterization of different pathogens and subspecies [27
]. MIR spectra are additive and sensitive, and allow the fingerprinting and quantification of the pathogen of interest, transforming the traditional devices into biosensing systems with high sensitivity.
In particular, mesoporous titania thin films synthesized with the sol–gel method, were used to immobilize biomolecules (antibodies and pathogens) thanks to the high surface area due to their nano-organization, visible in a AFM image (). This was possible due to a high control of the gelation process on the films and subsequent thermal treatments that avoided the denaturation of biomolecules in environments that have a high alcohol concentration and extreme pH values, hence obtaining ordered and reproducible substrates. With this objective, special attention was paid to the thermal treatments of films to completely remove the inorganic template, EtOH and HCl.
Microscopically ordered structure of a mesoporous titania film observed by AFM analysis.
Mesoporous titania was used as a substrate for the features described above, but also because it has excellent biocompatibility, stability (12 months at RT), and reproducibility, and can interact with biological molecules due to the formation of coordinated linkages between titania films, organic crosslinkers and amino or carboxyl groups of the antibodies or bacteria. In the present work the immobilization of bacteria was studied directly on titania films, on titania films functionalized with APTES and on titania films functionalized with APTES, GA and antibodies (Ab).
Detection of E. coli with TiO2–APTES–GA–anti E. coli O157:H7-Ab
The first method used illustrates the detection of E. coli O157:H7 through the immobilization of antibodies on titania films functionalized with APTES and GA. In the first step () titania thin films were functionalized with APTES; in particular the spectra of the films before and after the functionalization process were reported. The peaks due to APTES (inset spectrum) have been attributed to N–H stretching at 3300 cm−1, N–CH2 stretching around 2800 cm−1, NH2 scissoring and N–H bending at 1615 cm−1, aliphatic C–N stretching at 1020–1220 cm−1, NH2 wagging and twisting at 850–750 cm−1 and N–H wagging at 715 cm−1.
Figure 2 (a) FTIR spectrum of mesoporous titania thin films (solid line) and films functionalized with APTES (dashed line). The reference spectrum of APTES is reported in the inset. (b) FTIR spectrum of titania films functionalized with APTES (solid line) and (more ...)
The second step was based on the reaction between APTES and GA, which was used to crosslink the APTES with antibodies due to the formation of an imine. Here, the terminal amino groups of APTES were changed to aldehydic groups that, in the following step, were covalently coupled with the amino groups of the antibody. The APTES–GA linking is shown in , in which the bands due to the formation of imines in the area between 1900 and 1600 cm−1, and the bands related to the stretching of C–N, C–O, C–C groups in the range 1500–1200 cm−1 are visible. The GA spectrum is included as a reference in the inset.
The functionalization process was also visible on the film surface, as reported in , due to the change in the colour of films. To complete the sensor fabrication, antibodies against E. coli O157:H7 were linked to the substrate as reported in and . For the final detection of E. coli O157:H7 this chip was immersed in a PBS buffer with E. coli O157:H7 (108 CFU/mL) for 30 min, washed and analysed.
Titania films before functionalization (yellow), after APTES treatment (pink) and after the linking with GA (blue).
Structure of the chemical linking, TiO2–APTES–GA–antibody.
The reported spectrum () shows similar peaks for the film with the pathogen (dotted line) in comparison to films without the pathogen (solid line); however, new peaks appeared in particular in the regions 1300–2000 cm−1 (protein peaks of the bacterium), 3700–4000 cm−1 and 1200–800 cm−1 (signals of nucleic acids of the bacterium), which unfortunately in this region overlapped with the spectrum of the Ab and of the functionalized titania. The peaks in the 1630–1697 cm−1 region are due to amide I bands of the proteins in the cell and to their secondary structure. In the region 1402–1457 cm−1, bands due to carbohydrates, glycoproteins, lipids and their characteristic C–O–H in-plane bending peaks and C(CH3)2 symmetric stretching were present. Finally, in the range 900–1100 cm−1, peaks due to the DNA/RNA backbone and phosphate groups of nucleic acids due to the symmetric and asymmetric stretching of P=O and P–O–C groups were visible. The spectrum of E. coli deposited on Si is provided as a reference in the inset.
Detection of E. coli with other functionalization methods
To understand the best method for pathogen capture, the direct absorption of the pathogens (E. coli O157:H7 and K12) on a titania thin film (), on films with only the specific antibody for E. coli O157:H7, and on films with APTES and Ab () were tested. shows the FTIR spectra of different strains of E. coli (O157:H7 and K12) adsorbed onto the film surface with electrostatic interactions, illustrating that the substrate cannot discriminate between different strains and the different spectra of the pathogens are clearly visible. In contrast, in films with specific antibodies, only the binding of E. coli O157:H7 was visible and is reported in and 5c, although in films with APTES the presence of pathogens is poor.
Figure 5 (a) FTIR spectrum of mesoporous titania films before (solid line) and after the immobilization of pathogens E. coli O157:H7 (dotted line) and E. coli K12 (dashed line). (b) FTIR spectrum of mesoporous titania films functionalized with anti-E. coli O157:H7-antibody (more ...)
Comparing the different immobilization techniques of detection ( and ), the best result was obtained with the method that provided the covalent binding of the Ab on the film surface with APTES and GA (, dotted line), as expected. The other methods allowed the immobilization of bacteria, although not selectively, or with a low sensitivity of the device. In fact only the device shown in is selective and does not allow the linking of other subspecies of E. coli (E. coli K12), while on titania films () it was possible to entrap pathogens with electrostatic interactions not differentiating between pathogens.
Determination of the detection limits of E. coli O157:H7
Tests to establish the detection limit of the device were carried out using serial dilutions of E. coli ranging in concentration from 1 × 108 CFU/mL to 10 CFU/mL; the dilutions were validated with the standard colony counting method () and DNA analysis was achieved by RT–PCR (). These experiments were evaluated on mesoporous titania films functionalized with APTES–GA–anti-E. coli O157:H7-antibody, and a limit of detection of 1 × 102 CFU/mL () was achieved for E. coli O157:H7. A test with E. coli K12 was also carried out, but the spectrum of the functionalized chip did not show any peak due to this strain, because of the selectivity of the antibody, illustrating the specificity of the binding. Finally, colony micrographs of functionalized films after the immobilization of E. coli O157:H7 at different concentrations were collected with an optical microscope ().
Colony counting method on a Petri plate with PCA and E. coli O157:H7 at a dilution of 10−6.
RT–PCR of DNA extracted from the nutrient broth. The blue line is the blank, the light green curve is the reference sample and the green curve is the DNA analysed.
Figure 8 FTIR spectra of titania films alone (solid line) and functionalized with APTES–GA–anti-E. coli O157:H7-Ab after exposure to different concentrations of E. coli O157:H7 (108–102 CFU/mL) and E. coli K12 (108 CFU/mL) in order from (more ...)
Figure 9 Colony micrographs of E. coli O157:H7 immobilized on mesoporous titania films functionalized with APTES–GA–anti-E. coli O157:H7-Ab, after exposure to different concentrations of E. coli O157:H7. (a) 106 CFU/mL; (b) 104 CFU/mL; (c) 102 (more ...)