Final Report Summary - NANOBACTERPHAGESERS (Design of Novel Portable-Sensors Based on Suspension Arrays Composed of Monoclonal Antibody andBacteriophage Carrying Magnetically Loaded Nanoparticles and Surface Enhanced Raman Spectroscopy)
As stated in the original proposal (Annex 1) project aims to develop nanotechnology-based systems carrying three different bioprobes, i.e. antibodies, aptamers and bacteriophages for detection of pathogenic bacteria in several aqueous media mainly in environmental water based on suspension arrays together with a portable custom-designed detection system. The six objectives (six work packages-WPs) outlined in the original project proposal were as follows: (i) Design and fabrication of nanostructured sensors platforms for simultaneous Raman and Fluorescence enhancement; (ii) production of specific nanosorbents and their mixtures as suspension arrays against the target bacteria for capturing units; (iii) selection, and propagation of target bacteria and bacteriophages specifically infecting those bacteria as bioprobes; (iv) selection modification and production of aptamers against target bacteria as alternative bioprobes; (v) design and construction of a portable prototypes of “Raman Spectrometer/Fluorescence Detector” systems and finally (vi) validation of the materials and systems developed.
However, one of the partners - Aptares (a SME from Germany) was not able to continue due to economical problems – therefore WP4 aiming aptamer development was removed from the project and the New (Modified) Annex 1 was prepared which was approved officially by the EU office in September 2014. The second period was started at November 1, 2013, therefore there was a significant time between this date and the approval of the New Annex, however we have continued our activities in this period also. The Final Publishable Summary in the five WPs are given below.
WP1: Several geometries have been simulated and optimized during the whole Project period - which exhibited broadband absorption - by applying several computational methods and simulations Note that surface enhancement by using nanostructures optical signals both in “Raman Spectrometer/Fluorescence Detectors” could greatly improved Raman signal intensity, which in turn allows use of lower cost detectors having high sensitivity means detection of target at even lower target concentrations. Plasmonic metals were used for nanostructuring, after our several attempts, silver was chosen as the main/most successful plasmonic material. Initially electron beam lithography has been applied successfully. This technique produced very uniform enhancement, however it was difficult/expensive to apply on large areas due to the use of electron beam lithography. Then we have switched to a Metal-Insulator-Metal type design, that allowed control of the optical resonances not only by island size but also by the thickness of the insulator layer. Our efforts have resulted in wide area disposable substrates that were demonstrated to show single molecule level enhancement that can be detected by a low cost camera system, such as a smart phone (see Supplement – WP1). We also designed a low cost attachment for Raman spectroscopy, using a diode laser source, excitation and Raman filters and dichroic mirrors. We were able to observe both Raman and fluorescence enhancement using a portable miniature instrument. We have then investigated detection of biological molecules using our substrates. We have collaborated with ELV-BP and HU/BMT for biological samples as well as UNAM groups.
WP2: In the first period, superparamagnetic nanoparticles (“magnetite”) nano-particles (<40 nm) were synthesized by co-precipitation of Fe+2 and Fe+3 ions with NaOH and stabilized with TMAOH. Then a functional shell was created by different techniques. In the first approach MMA and AA monomers were polymerized and a coat with COOH functional groups was obtained. This layer was labelled with fluorescence dyes (Rhodamin B and FITC) and streptavidin layer was then adsorbed. Biotinilyted antibodies against different serotypes of Escherichia coli were then immobilized. Nanoparticles labelled with two different fluorescence dyes were mixed and suspension arrays were formed and tested against target bacteria (together with some other non-target bacteria mixtures) using Fluorescence microscopy. High selectivity and specificity were achieved. Alternatively, magnetite nanoparticles were coated with a gold layer (shell). Both chemical and biological approaches were used. In chemical approach the gold layer was deposited onto the nanoparticles from HAuCl4 solution using different reducing agents (citrates, etc.). In the biological approach gold coating was achieved using a L.vulgare plant extract as the reducing agent. Both approaches were successfully created gold layers which were confirmed by different techniques. The gold layers were modified with bifunctional self-assembling molecules then antibodies were immobilized using activation agents.
In this second period, we have focused on detection of bacteria with bacteriophages using nanoparticles having plasmonic properties. After getting very good results in the preliminary studies we have decided to focus on both silver and gold nanoparticles. Different Ag and Au nanoparticles with different properties were synthesized and used in the SERS experiments for detection of the target bacteria using also bacteriophages which was one of the main targets of this Project (see also the sections WP3 and WP4). The secondments of ELV-BP and GT also joined these studies at the facilities of HU and BMT in Ankara. Especially related SERS experiments were also done in Urla-İzmir (at İYTE - Prof. H. Zareie’s labs) (see Supplement - WP2).
WP3: aims “selection/production/characterization of target bacteria and bacterio- phages” - was led by ELV-BP and contributed by UB, HU, BMT and UNAM. Several bacteria and their selected bacteriophages were propagated, characterized and activities/performances of phages were successfully done. Firstly the target bacteria related to the species Escherichia coli (E.coli) Enterococcus faecalis (E.faecalis) and Enterococcus faecim (E.faecim) have been collected from environmental (water and/or soil), human sources (feces) and food samples (meat), in total over 140 samples have been used. The total number of these bacterial strains attains 100, among which 50 strains are related to E.coli and 50 to Enterococcus sp. Isolation and selection of specific bacteriophages effective against different bacterial species of E.coli E.faecalis and E.faecim have been started. At this stage in total 20 bacteriophage clones related to E.coli and Enterococcus sp. have been isolated from 35 sewage water samples and screened against a collection of appropriate bacterial strains (100 cultures). Initial screening showed that an average activity of E.coli related phages varies between 50-60%, whole and average activity of Enterococcus related phage clones is in the range of 50-70%. The isolated phages have been studied for their plaque and virion morphology (electron microscopic imaging), host ranges, one step growth curve (allowing estimation of the phage adsorption period on the host cell wall, latent period, phage burst size, etc.), genetic and serology relatedness to other phages.
In the second reporting period mainly two group of studies related to bacteria and bacteriophages - selection/propagation/use - were performed: (i) The investigations which were realized together with UB, ELV-BP and UNAM; and (ii) the studies which were performed by the HU and BMT partners together with the secondees from ELV-BP and GT in the facilities of HU and BMT in Ankara and in Urla-İzmir (at İYTE - Prof. H. Zareie’s labs).
In the first group of studies, ELV-BP and UB have worked closely with to isolate, identify and characterise strains of E.coli and Enterococcus species susceptible to a selection of E. coli and Enterococcus-specific bacteriophages. Three researchers from ELV-BP performed secondments at UB and one from UB at ELV-BP. These visits were very successful and enabled exchange of knowledge, techniques and experience to be shared between the parties. A post-doctoral ER from UB is expected to visit ELV-BP in 2015 to conclude the isolation and characterisation studies. One ESR and an ER visited UNAM. The phages belonging to three morphological groups: Myoviridae, Siphoviridae and Podoviridae have been chosen for testing by use of Raman spectroscopy. In contrast to infrared (IR) spectroscopy, Raman spectroscopy offers other advantages for water monitoring because water itself does not significantly affect Raman spectral data. Although Raman sensitivity is low in comparison to IR spectroscopy, this can be increased by applying signal-enhancement methods (SERS), in which metal particles are placed in direct contact with the sample. When the incident laser wavelength matches the resonant frequency of the metal, Raman scattering is enhanced. Since the applied test-phages were not specifically purified the obtained Raman spectra reflected the background picks, which covered the bacteriophages picks.
In the second group of studies three bacteria has been selected as targets namely Escherichia coli (E.coli) Staphylococcus aureus (S.aureus) and Salmonella infantis (S.infantis) both are among the most abundant bacteria – some species are highly pathogenic. Similar protocols were applied for the propagation of their three specific bacteriophages. The methods and characterization protocols/results are briefly given in the Supplement – WP3 which are mainly taken from the PhD thesis of Dr. Farzaneh Moghtader (Supervisor - Prof.Dr. Erhan Bişkin) who was one of the secondees of NanobacterphageSERS project.
WP4: In the second project period, we have focused mainly on SERS systems. HU, BMT and GT groups worked together and designed and constructed some prototypes and tested them. These optical devices that were quite close to production were briefly described in the Supplement - W4. However, due to economical problems in GT, it was not possible to complete prototype production of the SERS/fluorescence readers for validation tests. Therefore, the performance/validation experiments were done by using the Raman systems (both at BMT facilities and other partners labs - at İYTE - Prof. H. Zareie’s labs, Urla, İzmir) - in which the researchers and secondees mainly from HU, BMT, ELV-BP and GT were also participated.
WP5: As mentioned before, GT had economical problems especially in the last part of this second period, therefore it was not possible to produce prototypes of the SERS system and deliver to UB and ELV-BP for validation of the whole system for detection of pathogenic target bacteria. Therefore, we have focused on detection of pathogenic bacteria by using bacteriophages and nanoparticles with photonic properties (silver and gold nanoparticles) using the Raman systems (both at BMT facilities and other partners - at İYTE - Prof. H. Zareie’s labs in Urla, İzmir) – in which the researchers and secondees mainly from HU and ELV-BP have actively participated. Three target bacteria and their specific bacteriophages were used in the SERS experiments, successfully (see Supplement - WP5).
However, one of the partners - Aptares (a SME from Germany) was not able to continue due to economical problems – therefore WP4 aiming aptamer development was removed from the project and the New (Modified) Annex 1 was prepared which was approved officially by the EU office in September 2014. The second period was started at November 1, 2013, therefore there was a significant time between this date and the approval of the New Annex, however we have continued our activities in this period also. The Final Publishable Summary in the five WPs are given below.
WP1: Several geometries have been simulated and optimized during the whole Project period - which exhibited broadband absorption - by applying several computational methods and simulations Note that surface enhancement by using nanostructures optical signals both in “Raman Spectrometer/Fluorescence Detectors” could greatly improved Raman signal intensity, which in turn allows use of lower cost detectors having high sensitivity means detection of target at even lower target concentrations. Plasmonic metals were used for nanostructuring, after our several attempts, silver was chosen as the main/most successful plasmonic material. Initially electron beam lithography has been applied successfully. This technique produced very uniform enhancement, however it was difficult/expensive to apply on large areas due to the use of electron beam lithography. Then we have switched to a Metal-Insulator-Metal type design, that allowed control of the optical resonances not only by island size but also by the thickness of the insulator layer. Our efforts have resulted in wide area disposable substrates that were demonstrated to show single molecule level enhancement that can be detected by a low cost camera system, such as a smart phone (see Supplement – WP1). We also designed a low cost attachment for Raman spectroscopy, using a diode laser source, excitation and Raman filters and dichroic mirrors. We were able to observe both Raman and fluorescence enhancement using a portable miniature instrument. We have then investigated detection of biological molecules using our substrates. We have collaborated with ELV-BP and HU/BMT for biological samples as well as UNAM groups.
WP2: In the first period, superparamagnetic nanoparticles (“magnetite”) nano-particles (<40 nm) were synthesized by co-precipitation of Fe+2 and Fe+3 ions with NaOH and stabilized with TMAOH. Then a functional shell was created by different techniques. In the first approach MMA and AA monomers were polymerized and a coat with COOH functional groups was obtained. This layer was labelled with fluorescence dyes (Rhodamin B and FITC) and streptavidin layer was then adsorbed. Biotinilyted antibodies against different serotypes of Escherichia coli were then immobilized. Nanoparticles labelled with two different fluorescence dyes were mixed and suspension arrays were formed and tested against target bacteria (together with some other non-target bacteria mixtures) using Fluorescence microscopy. High selectivity and specificity were achieved. Alternatively, magnetite nanoparticles were coated with a gold layer (shell). Both chemical and biological approaches were used. In chemical approach the gold layer was deposited onto the nanoparticles from HAuCl4 solution using different reducing agents (citrates, etc.). In the biological approach gold coating was achieved using a L.vulgare plant extract as the reducing agent. Both approaches were successfully created gold layers which were confirmed by different techniques. The gold layers were modified with bifunctional self-assembling molecules then antibodies were immobilized using activation agents.
In this second period, we have focused on detection of bacteria with bacteriophages using nanoparticles having plasmonic properties. After getting very good results in the preliminary studies we have decided to focus on both silver and gold nanoparticles. Different Ag and Au nanoparticles with different properties were synthesized and used in the SERS experiments for detection of the target bacteria using also bacteriophages which was one of the main targets of this Project (see also the sections WP3 and WP4). The secondments of ELV-BP and GT also joined these studies at the facilities of HU and BMT in Ankara. Especially related SERS experiments were also done in Urla-İzmir (at İYTE - Prof. H. Zareie’s labs) (see Supplement - WP2).
WP3: aims “selection/production/characterization of target bacteria and bacterio- phages” - was led by ELV-BP and contributed by UB, HU, BMT and UNAM. Several bacteria and their selected bacteriophages were propagated, characterized and activities/performances of phages were successfully done. Firstly the target bacteria related to the species Escherichia coli (E.coli) Enterococcus faecalis (E.faecalis) and Enterococcus faecim (E.faecim) have been collected from environmental (water and/or soil), human sources (feces) and food samples (meat), in total over 140 samples have been used. The total number of these bacterial strains attains 100, among which 50 strains are related to E.coli and 50 to Enterococcus sp. Isolation and selection of specific bacteriophages effective against different bacterial species of E.coli E.faecalis and E.faecim have been started. At this stage in total 20 bacteriophage clones related to E.coli and Enterococcus sp. have been isolated from 35 sewage water samples and screened against a collection of appropriate bacterial strains (100 cultures). Initial screening showed that an average activity of E.coli related phages varies between 50-60%, whole and average activity of Enterococcus related phage clones is in the range of 50-70%. The isolated phages have been studied for their plaque and virion morphology (electron microscopic imaging), host ranges, one step growth curve (allowing estimation of the phage adsorption period on the host cell wall, latent period, phage burst size, etc.), genetic and serology relatedness to other phages.
In the second reporting period mainly two group of studies related to bacteria and bacteriophages - selection/propagation/use - were performed: (i) The investigations which were realized together with UB, ELV-BP and UNAM; and (ii) the studies which were performed by the HU and BMT partners together with the secondees from ELV-BP and GT in the facilities of HU and BMT in Ankara and in Urla-İzmir (at İYTE - Prof. H. Zareie’s labs).
In the first group of studies, ELV-BP and UB have worked closely with to isolate, identify and characterise strains of E.coli and Enterococcus species susceptible to a selection of E. coli and Enterococcus-specific bacteriophages. Three researchers from ELV-BP performed secondments at UB and one from UB at ELV-BP. These visits were very successful and enabled exchange of knowledge, techniques and experience to be shared between the parties. A post-doctoral ER from UB is expected to visit ELV-BP in 2015 to conclude the isolation and characterisation studies. One ESR and an ER visited UNAM. The phages belonging to three morphological groups: Myoviridae, Siphoviridae and Podoviridae have been chosen for testing by use of Raman spectroscopy. In contrast to infrared (IR) spectroscopy, Raman spectroscopy offers other advantages for water monitoring because water itself does not significantly affect Raman spectral data. Although Raman sensitivity is low in comparison to IR spectroscopy, this can be increased by applying signal-enhancement methods (SERS), in which metal particles are placed in direct contact with the sample. When the incident laser wavelength matches the resonant frequency of the metal, Raman scattering is enhanced. Since the applied test-phages were not specifically purified the obtained Raman spectra reflected the background picks, which covered the bacteriophages picks.
In the second group of studies three bacteria has been selected as targets namely Escherichia coli (E.coli) Staphylococcus aureus (S.aureus) and Salmonella infantis (S.infantis) both are among the most abundant bacteria – some species are highly pathogenic. Similar protocols were applied for the propagation of their three specific bacteriophages. The methods and characterization protocols/results are briefly given in the Supplement – WP3 which are mainly taken from the PhD thesis of Dr. Farzaneh Moghtader (Supervisor - Prof.Dr. Erhan Bişkin) who was one of the secondees of NanobacterphageSERS project.
WP4: In the second project period, we have focused mainly on SERS systems. HU, BMT and GT groups worked together and designed and constructed some prototypes and tested them. These optical devices that were quite close to production were briefly described in the Supplement - W4. However, due to economical problems in GT, it was not possible to complete prototype production of the SERS/fluorescence readers for validation tests. Therefore, the performance/validation experiments were done by using the Raman systems (both at BMT facilities and other partners labs - at İYTE - Prof. H. Zareie’s labs, Urla, İzmir) - in which the researchers and secondees mainly from HU, BMT, ELV-BP and GT were also participated.
WP5: As mentioned before, GT had economical problems especially in the last part of this second period, therefore it was not possible to produce prototypes of the SERS system and deliver to UB and ELV-BP for validation of the whole system for detection of pathogenic target bacteria. Therefore, we have focused on detection of pathogenic bacteria by using bacteriophages and nanoparticles with photonic properties (silver and gold nanoparticles) using the Raman systems (both at BMT facilities and other partners - at İYTE - Prof. H. Zareie’s labs in Urla, İzmir) – in which the researchers and secondees mainly from HU and ELV-BP have actively participated. Three target bacteria and their specific bacteriophages were used in the SERS experiments, successfully (see Supplement - WP5).