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fluorescent poly(phenylenethynylene) composite for optical biosensors
Vacuum 84 (2010) 1244–1249
Contents lists available at ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Optical and morphological characterisation of a silver nanoparticle/fluorescent poly(phenylenethynylene) composite for optical biosensors Juan Carlos Ramos a, b, Antonio Ledezma a, Eduardo Arias a, Ivana Moggio a, *, Carlos Alberto Martı´nez b, Felipe Castillon c a b c
´n en Quı´mica Aplicada, Boulevard Enrique Reyna 140, 25253 Saltillo, Mexico Centro de Investigacio ´ rez, Henry Dunant 4016, C.P. 32310, Ciudad Jua ´ rez, Mexico ´noma de Ciudad Jua Universidad Auto Centro de Ciencia de la Materia Condensada de la UNAM, Apdo. Postal 2681, CP 22800 Ensenada, BC, Mexico
a b s t r a c t Keywords: Poly(phenylenethynylene) Silver nanoparticles Nanocomposite Thin films Fungi
Thiol silver nanoparticles prepared by the phase transfer method have been mixed with a fluorescent poly(phenylenethynylene) sequenced with dithioester-diethylsulfide moieties in order to develop a nanocomposite for its possible application in optical biosensors for the detection and attack of fungi such as Paecilomyces variotii. Films have been prepared by dipping technique and characterized by AFM, XPS, UV-Visible and fluorescence spectroscopy. Optical Absorption properties of the nanocomposite are similar to those of the polymer with an absorption tail in the visible which supports the presence of silver nanoparticles. Despite the lack of fluorescence of the nanoparticles, the composite emits in the yellow green region and the intensity of the fluorescence of the nanocomposite film decreases after the immersion in the culture thus permitting the detection of the fungus by this technique. The fungus can be deposited on films of both the polymer and nanocomposite, nevertheless only in the latter case, an attack on mycelium is observed revealing the fungicidal effect of silver nanoparticles in the nanocomposite. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Bacteria’s are considered as the main pathogenic microorganisms and studies about novel detection methods are usually related to them. However, fungi are a major health problem especially for patients with immunodeficiency [1]. Fungal infections are often very progressive, moreover several saprophytic or plant fungi can become opportunistic pathogens for humans, especially for patients who are exposed to antibiotics or immunosuppressive drugs [2]. Contrary to mycoses due to Candida or Aspergillus niger, infections due to opportunistic fungi are more difficult to be detected as the pathogen microorganism is not expected to be found in humans. Impact of these infections can be relevant because the fungus can easily become a host in any organs of the human body such as intestine, appendix, urinary system, etc. For this reason, a rapid diagnostic method could decrease the propagation of the microorganism in the body of already affected patients as well as increasing the health security of hospitals avoiding a large diffusion of infections. Biosensors have appeared in the last years as a faster, cheaper and easier diagnostic technology
* Corresponding author. Tel.: þ52 844 4389830; fax: þ52 844 4389839. E-mail address: [email protected] (I. Moggio). 0042-207X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vacuum.2009.10.034
with respect to classical analytical assays which require time for the culture growth and experience in handling and characterization of the microorganisms [3]. Among the different types of biosensors, optical biosensors present several advantages such as short response time, easy mode of detection, lack of electrical interference [4]. Conjugated polymers are optimal candidates as transducer elements for optical biosensors as they present specific and interesting optical properties due to the high p-electron delocalization through their backbone. Phenylenethynylenes are a class of semiconducting materials with interesting photo- and electroluminescent properties and their application in optical biosensors has been previously reported for the detection of Escherichia coli [5]. On the other hand, due to health impact of certain microorganisms, it could be useful to have a diagnostic method which permits not only to detect but also to attack and eliminate them. In this context, copper compounds are usually used in antifungal coating and recently this application was also proposed for polymer nanocomposites of copper nanoparticles [6]. The capacity of bulk silver compounds for attacking bacteria and fungi is well known [7–10]. In recent papers, the bactericide effect has also been demonstrated for silver nanoparticles for pathogen bacteria such as E. coli [11] and the HIV-1 virus [12]. As far as fungi are concerned, to the best of our knowledge there are no reports on the application of silver nanoparticles. In this respect, in present
J.C. Ramos et al. / Vacuum 84 (2010) 1244–1249
work we report the elaboration of a nanocomposite of silver nanoparticles with a fluorescent poly(phenylethynylene) sequenced with dithioester-diethylsulfide moieties, hereafter named pPET3OC12-sqS to develop a fungicidal system; where the fungi can be easily detected by fluorescence and simultaneously attacked by silver nanoparticles. We studied P. variotii which, despite being usually considered a saprophytic fungus, may become opportunistic causing a disease in humans and animals called paecilomycosis [13].
2. Materials and methods The synthesis and physicochemical characterization of pPEOC4 and pPET3OC12-sqS (Fig. 1) have been reported in ref [14,15]. Silver nanoparticles were synthesized using the phase transfer method reported first by Brust et al. for gold particles [16]. In a typical synthesis, 0.55 g of tetraoctylammonium bromide was dissolved in 20 ml of toluene at room temperature under vigorous stirring. Then, 2 ml of a 0.1 M silver nitrate solution in distilled water was added to the reaction flask. One hour later, 0.3 ml of 1-dodecanethiol was added. Finally, 6.3 ml of 0.25 M sodium boronhydride solution in distilled water was slowly added dropwise. After three hours, the reaction was stopped and the product was recovered from organic phase by precipitation using cold ethanol. Nanoparticles were re-dissolved in toluene and precipitated again in cold ethanol and, finally, metal nanoparticles were dissolved and stored in toluene. The final concentration evaluated by drying a known volume of the solution, was 2.2 mg/ml. The nanocomposite is obtained by mixing the toluene solution of nanoparticles with a 1 mg/ml CHCl3 solution of pPET3OC12-sqS in a relation 1:1 v/v for three hours under strong magnetic stirring. The yellow solution remained stable without aggregation or precipitation after one year from its preparation. For the deposition of the films, Corning glass or quartz slides by SPI. Inc have been used as substrates and treated with sulfochromic mixture as described in published literature [17]. Then each lime has been immersed in the nanocomposite solution by dipping using a KSV Langmuir-Blodgett equipment at a speed of 68.55 mm/min for 10 min followed by drying in air. A direct measurement of the thickness by profilometry was not possible. However, on the basis of the absorption peak due to the polymer and previously found absorption coefficient for pPET3OC12-sqS thin films [15], a thickness of around 10 nm can be estimated. As a sample control for the microbiological assay, films of pPET3OC12-sqS and silver nanoparticles have been prepared under the same conditions as those of the nanocomposite. For the microbiological tests, cultures on potato dextrose agar of the fungus P. variotii have been incubated for 7 days to prepare a microorganism suspension in distilled water containing both spores and mycelium. Films were immersed in the
Fig. 1. Chemical structure of pPET3OC12-sqS and pPEOC4.
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microorganism suspension for 20 min with the same procedure and equipment used for their elaboration. Then, these were drawn out and washed up to 6 times with distilled water. Each washing was realized by dipping in distilled water for 2 min and water was renewed every time. Attack to the fungus was studied with a Leica ATC 2000 optical microscope and by laser scanning confocal microscopy (Zeiss Pascal 5). Also the microorganism suspension has been analyzed before and after the film immersion by optical microscopy by casting few drops between two microscope slides. UV-Visible and emission spectra were recorded on a Shimadzu 2401 spectrophotometer and a Perkin Elmer LS50B spectrofluorimeter, respectively. The excitation wavelength was chosen 10 nm under the absorption lower energy peak. Quantum yields (q.y.) were obtained according to the procedure reported earlier [18] and quinine sulfate (q.y. ¼ 0.54) was used as standard. The AFM analysis was carried out on a Digital Dimension 3100 microscope in tapping mode at a rate of 0.3–0.5 Hz. TEM analysis was performed in a JEOL 2010F microscope operating at 200 kV equipped with a Schottkytype field emission gun, an ultra-high resolution pole piece (Cs ¼ 0.5 mm) and an energy dispersive X-ray spectrometer (EDS) unit. Samples for TEM were prepared by depositing a drop of the original suspension on a lacey carbon coated Cu grid and allowed to evaporate. The SEM analysis was carried out on a SM510 TOPCON microscope. The X-Ray photoelectron spectroscopy (XPS) analysis was carried out on a modified laser ablation system, Riber LDM-32, using a Cameca Mac3 analyzer. The base pressure in the analysis chamber was approximately 1010 mbar. The X-ray Al Ka line at 1486.6 eV was used for excitation. The binding energies were calibrated with reference to Cu 2 p3/2 at 932.67 eV and Ag 3d5/2 at 368.26 eV, respectively. The resolution attained with this set-up is 1.1 eV measured on the C 1 s signal of a graphite target. Spectra were collected by acquiring data at every 0.2 eV and the energy resolution was 0.8 eV. The wide-scan and core-level spectra for C 1 s were obtained. A short-time scan of C 1 s was first acquired to check that no degradation occurs during the spectra acquisition due to X-ray exposure. Background subtraction was done using the Tougaard method [19]. In addition, wide-scan spectra were gathered acquiring data at every 1.0 eV with an energy resolution of 3 eV. Charging effect was corrected by shifting the binding energies considering the C 1 s signal at 284.6 eV. A non-linear fit, using Gaussian curves, was performed maintaining the Full-Width at Half-Maximum (FWHM) constant for all components in a particular spectrum. 3. Results and discussion The poly(phenylethynylene) pPET3OC12-sqS, in its chemical structure presents the main conjugated chain sequenced with flexible dithioester-diethylsulfide segments. The conjugated backbone imparts high fluorescence to the polymer, while the sulfur atoms of the flexible sequence are intended to promote the affinity with silver. In Fig. 2a, the UV-Visible spectrum of the pPET3OC12sqS/Ag nanocomposite in toluene is reported. Two bands, observed at 327 and 394 nm as found for the polymer solution [15], correspond to the electronic transitions of the dodecanoxy substituted benzene and to the p p* electronic transitions of the conjugated chains, respectively. Suspension of silver nanoparticles in toluene exhibits a broad absorption band from 300 to 800 nm, with a maximum at 454 nm (as shown in inserted figure) due to the plasmon band. This absorption cannot be observed in the composite suspension probably because it is masked by the stronger absorbing polymer band. The absorption coefficient of pPET3OC12-sqS is in fact, 50.7 gl1 cm1 for pPET3OC12-sqS [15] contra 0.33 gl1 cm1 for the suspension of silver nanoparticles. Nevertheless, the
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at 327 and 403 nm can be distinguished, in agreement with the spectrum of the pPET3OC12-sqS sample [15]. No evident features due to silver are observed, as expected on the basis of the results discussed for the solutions and considering the low intensity of the spectrum. It is interesting to notice that after immersing the films in the microorganism suspension (dashed lines), the spectrum for the pPET3OC12-sqS/Ag nanoparticles film presents the same absorbance as the pristine sample. A slight broadening of the baseline observed results from a certain loss of homogeneity. On the contrary, for the spectrum of pPET3OC12-sqS film, a strong decrease in the absorbance occurs. This behaviour is due to a partial dissolution of the polymer in water, as previously reported [15]. This result indicates that the presence of the nanoparticles in the nanocomposite decreases the polymer solubility in water confirming the formation of a composite through the interactions of polar groups of pPET3OC12-sqS with silver. As shown in Fig. 3b, the pPET3OC12-sqS/Ag nanoparticles film presents a broad fluorescence band centered at around 481 nm, as the polymer film. The fluorescence intensity decreases strongly as a consequence of the detection of the fungus. This behaviour cannot be ascribed to a partial dissolution of the film according to what was previously discussed and to the fact that no emission was found in the microorganism suspension after the immersion of the film. More likely, the fungus which is deposited on the film produces
Fig. 2. a) UV-Visible spectra of pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 (solid line), pPET3OC12-sqS in CHCl3 (dashed line) and Ag nanoparticles in toluene (dotted line). Inserted figure: magnification of the same spectra in the 450–600 nm region. (b) Emission spectra of pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 (solid line) and pPET3OC12-sqS in CHCl3 (dashed line). Inserted figure: photo taken under UV (lexc ¼ 365 nm) irradiation. From left to right, pPET3OC12-sqS in CHCl3, pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 solution, silver nanoparticles in toluene and toluene. Notice that the bluish colour of the silver nanoparticles suspension is due to toluene as evidenced by the comparison with the solvent emission.
absorption tail found between 450 and 600 nm (see magnification of the 400–600 nm region in the inserted Fig. 2) indicates the presence of the silver nanoparticles. As fluorescence (Fig. 2b) is concerned, the pPET3OC12-sqS/Ag nanoparticles suspension emits in the bluegreen region (photo in the inserted Fig. 2) with an emission maximum at 454 nm coinciding with the emission of the polymer [15]. Despite of the lack of fluorescence of silver nanoparticles, the nanocomposite exhibits a quantum yield of 0.53, practically identical to that found for the polymer alone [15] indicating that no energy transfer occurs. This result together with the fact that no energy shift is observed for the absorption or emission spectra passing form the polymer to the nanocomposite samples, is consistent with the hypothesis that the conjugated moiety of pPET3OC12-sqS is not involved in the assembly with silver. The optical properties of the composite in thin films (Fig. 3) are similar to those already discussed for the solution. In the absorption spectrum of pPET3OC12-sqS/Ag nanoparticles (Fig. 3a), two peaks
Fig. 3. a) UV-Visible spectra of films of pPET3OC12-sqS/Ag nanoparticles and pPET3OC12-sqS (inserted figure) before (solid lines) and after (dash lines) the immersion in P. variotii suspension. b) Emission spectra of film of pPET3OC12-sqS/Ag nanoparticles before (solid lines) and after (dash lines) the immersion in P. variotii suspension.
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a quenching of the polymer emission. This result clearly indicates the possibility to use nanocomposite films for the detection of this microorganism. Preparation of the composite has indeed a technological relevance because this biosensor can be applied not only for the detection but also for attacking the fungus as discussed below. In Fig. 4, the optical micrographs relative to the microbiological tests with P. variotii are shown. The pristine culture presents spherical spores which are typical of this fungus and mycelium. Both parts are transferred to the pPET3OC12-sqS film (top) as well as on the pPET3OC12-sqS/Ag nanoparticles film (bottom). The fact that the fungus can be transferred onto both samples indicates that pPET3OC12-sqS favours the affinity with the microorganism, probably because of the interactions of the flexible sequences with proteins present in the cellular membrane. In order to support this hypothesis, we performed the same test with polymer pPEOC4 which does not contain polar groups in its chemical structure. In this case, no transfer was detected. The interesting point is that only for the nanocomposite film, the mycelium presents morphological differences along its structure (dark and light segments as indicated by the arrows in Fig. 4b) which could suggest cellular damage. In order to evaluate this damage in better detail, we analyzed the samples by laser confocal microscopy at higher magnifications. Fig. 5 shows images for the pPET3OC12-sqS/Ag nanoparticles film after the immersion in P. variotii. As observed by optical microscopy,
Fig. 4. Optical micrographs of the microorgamism tests with P. variotii. top) film of pPET3OC12-sqS after immersion. bottom) film of pPET3OC12-sqS/Ag nanoparticles after immersion. Arrows indicate segments with morphological differences.
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the deposition of spores and mycelium is evident (Fig. 5a). In some mycelium segments, even pores can be found (Fig. 5b) from which cellular material could be eventually go out producing empty cells and fragmentation due to a complete collapse (Figs. 5a and 5b, see indications in the Figures). Similar results are found for the film of the silver nanoparticles alone, where a segment with empty cell can be observed (Fig. 6, depicted by an arrow). On the contrary, it is to be mentioned that no damage has been found for the fungus deposited on pPET3OC12-sqS films. All these results are consistent with the fact that the combination of fluorescent polymer with the nanoparticles in the composite film allows the construction of an optical biosensor for both the detection and attack of fungi. In order to understand
Fig. 5. Laser confocal micrographs of pPET3OC12-sqS/Ag nanoparticles film after immersion in the P. variotii suspension. a) spores and mycelium deposition- in mycelium, fragmentation due to collapse is observed, b) a mycelium fragment in which pores can be observed.
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Fig. 6. a) SEM image of a film of pure Ag nanoparticles after immersion in the P. variotii suspension.
the mechanism of the attack, AFM, TEM and XPS studies on the nanocomposite samples were carried out. Silver nanoparticles show spherical morphology and an average diameter of 6.8 nm as found by TEM (Fig. 7). Unfortunately, the TEM characterization of the nanocomposite was not possible. The polymer forms a thin layer on the copper grid trapping the particles. When the electron beam hits the film, the polymer burns and the nanoparticles agglomerate. Morphological analysis by AFM (Fig. 8a) reveals that the pPET3OC12-sqS/Ag nanoparticles film presents a granular morphology analogously to that found for pPET3OC12-sqS [15]. Isolated nanoparticles cannot be distinguished even in nanometer size scans (Fig. 8b). A reasonable explanation could be that the nanoparticles are capped by a polymer layer. However, due to their very low dimension and loss of resolution of tapping images of nanometer size, this hypothesis cannot be completely demonstrated by this technique. Fig. 8. AFM bi-dimensional images of the nanocomposite films in a) 50 50 mm2 and b) 500 500 nm2.
Fig. 7. TEM image of silver nanoparticles studied in this work. Inserted figure: particle size distribution.
Fig. 9 shows the XPS spectra for the silver nanoparticles and the nanocomposite sample. The bonding nature and chemical states on the surface of the films with silver nanoparticle sample (top) shows a major contribution in the Ag3d high resolution spectra of a peak centred at 368.3 eV which is assigned to Ag 3d5/2 in Agþ form [20]. The XPS Ag3d high resolution spectrum for the nanocomposite (bottom) shows no evidence of silver contribution. Taking into account the limit in penetration depth of the X-rays, this result could be ascribed to the fact that the nanoparticles are covered by the polymer, supporting the morphological characterization by AFM. Optical and morphological characterizations seem to indicate that pPET3OC12-sqS is assembled over nanoparticles. This hypothesis is reliable, considering the much greater size of the polymer with respect to the nanoparticles and the fact that it is expected to interact through the flexible groups partially with silver (to make the nanocomposite) and to the fungus (to permit its deposition). However, microbiological tests indicate the attack of the fungus. A possible mechanism for the attack could involve a local direct contact of silver nanoparticles with the fungus i.e. certain facets of the silver nanoparticles are exposed on the surface. The evidence by laser scanning microscopy suggests that this contact occurs at specific points which could be related to the part
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with dithioester-diethylsulfide moieties and microbiological tests with P. variotii. Optical and morphological characterizations of films of the nanocomposite suggest that the silver nanoparticles are covered by the polymer. Nevertheless, the microbiological tests indicate an attack of the mycelium that could be due to the contact of some facets of nanoparticles with the fungus. Despite the fact that attack is thus limited, the importance of the work is that with this nanocomposite, it is possible to construct optical biosensors for the simultaneous detection and attack of fungus. Future work will address quantifying the response by studying spore counting and spore viability. On the basis of those assays, changes in the nanocomposite composition or in the silver nanoparticles characteristics (size and capping agent) will be considered, in order to increase the fungicidal properties of this biosensor. Acknowledgements This work was financially supported by CONACyT through the project 51504-R and CIQA (project FD0003). Special acknowledgements to Antonio Diaz, for his technical assistance in XPS measurements, Esmeralda Saucedo for SEM analysis, Selene Sepulveda for TEM. References
Fig. 9. XPS spectra of the Ag3d region for the silver nanoparticles (top) and the nanocomposite (bottom).
of nanocomposite film where the polymer layer does not completely cap all the particles. Nevertheless, the delivery of silver to the cultures and consequently its attack of the fungus cannot be discarded as the amount of silver present in the nanocomposite films is below the detection limit of the atomic absorption measurements. As a final remark, the importance of this work is that we have demonstrated the viability of obtaining an optical biosensor for the detection and attack of P. variotii. Nevertheless as the mycelium, where the attack was evident, is a growing part of the fungus which cannot be quantified, a quantitative study of the device response was not possible for the moment. 4. Conclusions In this work we report the development of a nanocomposite of silver nanoparticles with a fluorescent poly(phenylethynylene) sequenced
[1] Meunier F. Fungal infections in the compromised host. In: Rubin RH, editor. Clinical approach to infections in the compromised host. New York: Plenum Publishing; 1989. p. 193–220. [2] Perfect JR, Schell WA. The new fungal opportunists are coming. Clin Infect Dis 1996;22:S112–118. [3] Liron Z, Bromberg A, Fisher M, editors. Novel approaches in biosensors and rapid diagnostic assays. New York: Kluwer Academic/Plenum Publishers; 1999. [4] Ligler FS, Taitt CAR, editors. Optical biosensors: present and future. Amsterdam: Elsevier Science BV; 2002. [5] Disney MD, Zheng J, Swager TM, Seeberger PH. Detection of bacteria with carbohydrate-functionalized fluorescent polymers. J Am Chem Soc 2004;126:13343–6. [6] Cioffi N, Torsi L, Ditaranto N, Tantillo G, Ghibelli L, Sabbatini L, et al. Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties. Chem Mater 2005;17:5255–62. [7] Clement JL, Jarrett PS. Antibacterial silver. Met Based Drugs 1994;1:467–82. [8] George N, Faoagali J, Muller M. Silvazine (silver sulfadiazine and chlorhexidine) activity against 200 clinical isolates. Bur 1997;23:493–5. [9] Fox CL, Modak SM. Mechanism of silver sulfadiazine action on burn wound infections. Antimicrobial Agents Chemother 1974;5:582–8. [10] Russell AD, Hugo WB. Antimicrobial activity and action of silver. Progr Med Chem 1994;31:351–70. [11] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramı´rez JT, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:1–8. [12] Elechiguerra JL, Buro´ JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, et al. Interactions of silver nanoparticles with HIV-1. J Nanobiotechnology 2005;3:6. [13] Brown AH, Smith G. The genus paecylomyces bainier and its perfect stage byssochlamys westing. Trans Brit Mycol Soc 1957;40:17–89. [14] Wautelet P, Moroni M, Oswald L, Le Moigne J, Pham TA, Bigot JY, et al. Rigid rod conjugated polymers for nonlinear optics. 2. Synthesis and characterization of phenylene-ethynylene oligomers. Macromolecules 1996;29:446–55. [15] Va´zquez E, Esquivel Aguilar A, Moggio I, Arias E, Romero J, Barrientos H, et al. Immobilization of the enzyme b-lactamase by self-assembly on thin films of a poly(phenyleneethynylene) sequenced with flexible segments containing sulfur atoms. Mat Sc Eng C 2007;27:787–93. [16] Brust M, Walter M, Berthell D, Schiffrin DJ, Whyman R. Synthesis of thiolderivatised gold nanoparticles in a two-phase liquid-liquid system. J Chem Soc Chem Commun 1994:801–2. [17] Espinosa C, Moggio I, Arias-Marı´n E, Romero-Garcı´a J, Cruz-Silva R, Le Moigne J, et al. Layer-by-layer assembled films of a rigid poly(phenyl-ethynylene) and alternate poly(phenyl-ethynylene)/poly(aniline). Synth Met 2003;139:155–61. [18] Morisaki Y, Ishida T, Chujo Y. Synthesis and properties of novel through-space p-conjugated polymers based on poly(p-phenylenevinylene)s having a [2,2] paracyclophane skeleton in the main chain. Macromolecules 2002;35:7872–7. [19] Tougaard S. Practical algorithm for background subtraction. Surf Sci 1989;216:343–60. [20] Li Xianfeng, Hao Xiufeng, Hui Na. Preparation of nanosilver particles into sulfonated poly(ether ether ketone) (S-PEEK) nanostructures by electrospinning. Mater Lett 2006;61:421–6.
Contents lists available at ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Optical and morphological characterisation of a silver nanoparticle/fluorescent poly(phenylenethynylene) composite for optical biosensors Juan Carlos Ramos a, b, Antonio Ledezma a, Eduardo Arias a, Ivana Moggio a, *, Carlos Alberto Martı´nez b, Felipe Castillon c a b c
´n en Quı´mica Aplicada, Boulevard Enrique Reyna 140, 25253 Saltillo, Mexico Centro de Investigacio ´ rez, Henry Dunant 4016, C.P. 32310, Ciudad Jua ´ rez, Mexico ´noma de Ciudad Jua Universidad Auto Centro de Ciencia de la Materia Condensada de la UNAM, Apdo. Postal 2681, CP 22800 Ensenada, BC, Mexico
a b s t r a c t Keywords: Poly(phenylenethynylene) Silver nanoparticles Nanocomposite Thin films Fungi
Thiol silver nanoparticles prepared by the phase transfer method have been mixed with a fluorescent poly(phenylenethynylene) sequenced with dithioester-diethylsulfide moieties in order to develop a nanocomposite for its possible application in optical biosensors for the detection and attack of fungi such as Paecilomyces variotii. Films have been prepared by dipping technique and characterized by AFM, XPS, UV-Visible and fluorescence spectroscopy. Optical Absorption properties of the nanocomposite are similar to those of the polymer with an absorption tail in the visible which supports the presence of silver nanoparticles. Despite the lack of fluorescence of the nanoparticles, the composite emits in the yellow green region and the intensity of the fluorescence of the nanocomposite film decreases after the immersion in the culture thus permitting the detection of the fungus by this technique. The fungus can be deposited on films of both the polymer and nanocomposite, nevertheless only in the latter case, an attack on mycelium is observed revealing the fungicidal effect of silver nanoparticles in the nanocomposite. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Bacteria’s are considered as the main pathogenic microorganisms and studies about novel detection methods are usually related to them. However, fungi are a major health problem especially for patients with immunodeficiency [1]. Fungal infections are often very progressive, moreover several saprophytic or plant fungi can become opportunistic pathogens for humans, especially for patients who are exposed to antibiotics or immunosuppressive drugs [2]. Contrary to mycoses due to Candida or Aspergillus niger, infections due to opportunistic fungi are more difficult to be detected as the pathogen microorganism is not expected to be found in humans. Impact of these infections can be relevant because the fungus can easily become a host in any organs of the human body such as intestine, appendix, urinary system, etc. For this reason, a rapid diagnostic method could decrease the propagation of the microorganism in the body of already affected patients as well as increasing the health security of hospitals avoiding a large diffusion of infections. Biosensors have appeared in the last years as a faster, cheaper and easier diagnostic technology
* Corresponding author. Tel.: þ52 844 4389830; fax: þ52 844 4389839. E-mail address: [email protected] (I. Moggio). 0042-207X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vacuum.2009.10.034
with respect to classical analytical assays which require time for the culture growth and experience in handling and characterization of the microorganisms [3]. Among the different types of biosensors, optical biosensors present several advantages such as short response time, easy mode of detection, lack of electrical interference [4]. Conjugated polymers are optimal candidates as transducer elements for optical biosensors as they present specific and interesting optical properties due to the high p-electron delocalization through their backbone. Phenylenethynylenes are a class of semiconducting materials with interesting photo- and electroluminescent properties and their application in optical biosensors has been previously reported for the detection of Escherichia coli [5]. On the other hand, due to health impact of certain microorganisms, it could be useful to have a diagnostic method which permits not only to detect but also to attack and eliminate them. In this context, copper compounds are usually used in antifungal coating and recently this application was also proposed for polymer nanocomposites of copper nanoparticles [6]. The capacity of bulk silver compounds for attacking bacteria and fungi is well known [7–10]. In recent papers, the bactericide effect has also been demonstrated for silver nanoparticles for pathogen bacteria such as E. coli [11] and the HIV-1 virus [12]. As far as fungi are concerned, to the best of our knowledge there are no reports on the application of silver nanoparticles. In this respect, in present
J.C. Ramos et al. / Vacuum 84 (2010) 1244–1249
work we report the elaboration of a nanocomposite of silver nanoparticles with a fluorescent poly(phenylethynylene) sequenced with dithioester-diethylsulfide moieties, hereafter named pPET3OC12-sqS to develop a fungicidal system; where the fungi can be easily detected by fluorescence and simultaneously attacked by silver nanoparticles. We studied P. variotii which, despite being usually considered a saprophytic fungus, may become opportunistic causing a disease in humans and animals called paecilomycosis [13].
2. Materials and methods The synthesis and physicochemical characterization of pPEOC4 and pPET3OC12-sqS (Fig. 1) have been reported in ref [14,15]. Silver nanoparticles were synthesized using the phase transfer method reported first by Brust et al. for gold particles [16]. In a typical synthesis, 0.55 g of tetraoctylammonium bromide was dissolved in 20 ml of toluene at room temperature under vigorous stirring. Then, 2 ml of a 0.1 M silver nitrate solution in distilled water was added to the reaction flask. One hour later, 0.3 ml of 1-dodecanethiol was added. Finally, 6.3 ml of 0.25 M sodium boronhydride solution in distilled water was slowly added dropwise. After three hours, the reaction was stopped and the product was recovered from organic phase by precipitation using cold ethanol. Nanoparticles were re-dissolved in toluene and precipitated again in cold ethanol and, finally, metal nanoparticles were dissolved and stored in toluene. The final concentration evaluated by drying a known volume of the solution, was 2.2 mg/ml. The nanocomposite is obtained by mixing the toluene solution of nanoparticles with a 1 mg/ml CHCl3 solution of pPET3OC12-sqS in a relation 1:1 v/v for three hours under strong magnetic stirring. The yellow solution remained stable without aggregation or precipitation after one year from its preparation. For the deposition of the films, Corning glass or quartz slides by SPI. Inc have been used as substrates and treated with sulfochromic mixture as described in published literature [17]. Then each lime has been immersed in the nanocomposite solution by dipping using a KSV Langmuir-Blodgett equipment at a speed of 68.55 mm/min for 10 min followed by drying in air. A direct measurement of the thickness by profilometry was not possible. However, on the basis of the absorption peak due to the polymer and previously found absorption coefficient for pPET3OC12-sqS thin films [15], a thickness of around 10 nm can be estimated. As a sample control for the microbiological assay, films of pPET3OC12-sqS and silver nanoparticles have been prepared under the same conditions as those of the nanocomposite. For the microbiological tests, cultures on potato dextrose agar of the fungus P. variotii have been incubated for 7 days to prepare a microorganism suspension in distilled water containing both spores and mycelium. Films were immersed in the
Fig. 1. Chemical structure of pPET3OC12-sqS and pPEOC4.
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microorganism suspension for 20 min with the same procedure and equipment used for their elaboration. Then, these were drawn out and washed up to 6 times with distilled water. Each washing was realized by dipping in distilled water for 2 min and water was renewed every time. Attack to the fungus was studied with a Leica ATC 2000 optical microscope and by laser scanning confocal microscopy (Zeiss Pascal 5). Also the microorganism suspension has been analyzed before and after the film immersion by optical microscopy by casting few drops between two microscope slides. UV-Visible and emission spectra were recorded on a Shimadzu 2401 spectrophotometer and a Perkin Elmer LS50B spectrofluorimeter, respectively. The excitation wavelength was chosen 10 nm under the absorption lower energy peak. Quantum yields (q.y.) were obtained according to the procedure reported earlier [18] and quinine sulfate (q.y. ¼ 0.54) was used as standard. The AFM analysis was carried out on a Digital Dimension 3100 microscope in tapping mode at a rate of 0.3–0.5 Hz. TEM analysis was performed in a JEOL 2010F microscope operating at 200 kV equipped with a Schottkytype field emission gun, an ultra-high resolution pole piece (Cs ¼ 0.5 mm) and an energy dispersive X-ray spectrometer (EDS) unit. Samples for TEM were prepared by depositing a drop of the original suspension on a lacey carbon coated Cu grid and allowed to evaporate. The SEM analysis was carried out on a SM510 TOPCON microscope. The X-Ray photoelectron spectroscopy (XPS) analysis was carried out on a modified laser ablation system, Riber LDM-32, using a Cameca Mac3 analyzer. The base pressure in the analysis chamber was approximately 1010 mbar. The X-ray Al Ka line at 1486.6 eV was used for excitation. The binding energies were calibrated with reference to Cu 2 p3/2 at 932.67 eV and Ag 3d5/2 at 368.26 eV, respectively. The resolution attained with this set-up is 1.1 eV measured on the C 1 s signal of a graphite target. Spectra were collected by acquiring data at every 0.2 eV and the energy resolution was 0.8 eV. The wide-scan and core-level spectra for C 1 s were obtained. A short-time scan of C 1 s was first acquired to check that no degradation occurs during the spectra acquisition due to X-ray exposure. Background subtraction was done using the Tougaard method [19]. In addition, wide-scan spectra were gathered acquiring data at every 1.0 eV with an energy resolution of 3 eV. Charging effect was corrected by shifting the binding energies considering the C 1 s signal at 284.6 eV. A non-linear fit, using Gaussian curves, was performed maintaining the Full-Width at Half-Maximum (FWHM) constant for all components in a particular spectrum. 3. Results and discussion The poly(phenylethynylene) pPET3OC12-sqS, in its chemical structure presents the main conjugated chain sequenced with flexible dithioester-diethylsulfide segments. The conjugated backbone imparts high fluorescence to the polymer, while the sulfur atoms of the flexible sequence are intended to promote the affinity with silver. In Fig. 2a, the UV-Visible spectrum of the pPET3OC12sqS/Ag nanocomposite in toluene is reported. Two bands, observed at 327 and 394 nm as found for the polymer solution [15], correspond to the electronic transitions of the dodecanoxy substituted benzene and to the p p* electronic transitions of the conjugated chains, respectively. Suspension of silver nanoparticles in toluene exhibits a broad absorption band from 300 to 800 nm, with a maximum at 454 nm (as shown in inserted figure) due to the plasmon band. This absorption cannot be observed in the composite suspension probably because it is masked by the stronger absorbing polymer band. The absorption coefficient of pPET3OC12-sqS is in fact, 50.7 gl1 cm1 for pPET3OC12-sqS [15] contra 0.33 gl1 cm1 for the suspension of silver nanoparticles. Nevertheless, the
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at 327 and 403 nm can be distinguished, in agreement with the spectrum of the pPET3OC12-sqS sample [15]. No evident features due to silver are observed, as expected on the basis of the results discussed for the solutions and considering the low intensity of the spectrum. It is interesting to notice that after immersing the films in the microorganism suspension (dashed lines), the spectrum for the pPET3OC12-sqS/Ag nanoparticles film presents the same absorbance as the pristine sample. A slight broadening of the baseline observed results from a certain loss of homogeneity. On the contrary, for the spectrum of pPET3OC12-sqS film, a strong decrease in the absorbance occurs. This behaviour is due to a partial dissolution of the polymer in water, as previously reported [15]. This result indicates that the presence of the nanoparticles in the nanocomposite decreases the polymer solubility in water confirming the formation of a composite through the interactions of polar groups of pPET3OC12-sqS with silver. As shown in Fig. 3b, the pPET3OC12-sqS/Ag nanoparticles film presents a broad fluorescence band centered at around 481 nm, as the polymer film. The fluorescence intensity decreases strongly as a consequence of the detection of the fungus. This behaviour cannot be ascribed to a partial dissolution of the film according to what was previously discussed and to the fact that no emission was found in the microorganism suspension after the immersion of the film. More likely, the fungus which is deposited on the film produces
Fig. 2. a) UV-Visible spectra of pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 (solid line), pPET3OC12-sqS in CHCl3 (dashed line) and Ag nanoparticles in toluene (dotted line). Inserted figure: magnification of the same spectra in the 450–600 nm region. (b) Emission spectra of pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 (solid line) and pPET3OC12-sqS in CHCl3 (dashed line). Inserted figure: photo taken under UV (lexc ¼ 365 nm) irradiation. From left to right, pPET3OC12-sqS in CHCl3, pPET3OC12-sqS/Ag nanoparticles in CHCl3/toluene 1:1 solution, silver nanoparticles in toluene and toluene. Notice that the bluish colour of the silver nanoparticles suspension is due to toluene as evidenced by the comparison with the solvent emission.
absorption tail found between 450 and 600 nm (see magnification of the 400–600 nm region in the inserted Fig. 2) indicates the presence of the silver nanoparticles. As fluorescence (Fig. 2b) is concerned, the pPET3OC12-sqS/Ag nanoparticles suspension emits in the bluegreen region (photo in the inserted Fig. 2) with an emission maximum at 454 nm coinciding with the emission of the polymer [15]. Despite of the lack of fluorescence of silver nanoparticles, the nanocomposite exhibits a quantum yield of 0.53, practically identical to that found for the polymer alone [15] indicating that no energy transfer occurs. This result together with the fact that no energy shift is observed for the absorption or emission spectra passing form the polymer to the nanocomposite samples, is consistent with the hypothesis that the conjugated moiety of pPET3OC12-sqS is not involved in the assembly with silver. The optical properties of the composite in thin films (Fig. 3) are similar to those already discussed for the solution. In the absorption spectrum of pPET3OC12-sqS/Ag nanoparticles (Fig. 3a), two peaks
Fig. 3. a) UV-Visible spectra of films of pPET3OC12-sqS/Ag nanoparticles and pPET3OC12-sqS (inserted figure) before (solid lines) and after (dash lines) the immersion in P. variotii suspension. b) Emission spectra of film of pPET3OC12-sqS/Ag nanoparticles before (solid lines) and after (dash lines) the immersion in P. variotii suspension.
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a quenching of the polymer emission. This result clearly indicates the possibility to use nanocomposite films for the detection of this microorganism. Preparation of the composite has indeed a technological relevance because this biosensor can be applied not only for the detection but also for attacking the fungus as discussed below. In Fig. 4, the optical micrographs relative to the microbiological tests with P. variotii are shown. The pristine culture presents spherical spores which are typical of this fungus and mycelium. Both parts are transferred to the pPET3OC12-sqS film (top) as well as on the pPET3OC12-sqS/Ag nanoparticles film (bottom). The fact that the fungus can be transferred onto both samples indicates that pPET3OC12-sqS favours the affinity with the microorganism, probably because of the interactions of the flexible sequences with proteins present in the cellular membrane. In order to support this hypothesis, we performed the same test with polymer pPEOC4 which does not contain polar groups in its chemical structure. In this case, no transfer was detected. The interesting point is that only for the nanocomposite film, the mycelium presents morphological differences along its structure (dark and light segments as indicated by the arrows in Fig. 4b) which could suggest cellular damage. In order to evaluate this damage in better detail, we analyzed the samples by laser confocal microscopy at higher magnifications. Fig. 5 shows images for the pPET3OC12-sqS/Ag nanoparticles film after the immersion in P. variotii. As observed by optical microscopy,
Fig. 4. Optical micrographs of the microorgamism tests with P. variotii. top) film of pPET3OC12-sqS after immersion. bottom) film of pPET3OC12-sqS/Ag nanoparticles after immersion. Arrows indicate segments with morphological differences.
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the deposition of spores and mycelium is evident (Fig. 5a). In some mycelium segments, even pores can be found (Fig. 5b) from which cellular material could be eventually go out producing empty cells and fragmentation due to a complete collapse (Figs. 5a and 5b, see indications in the Figures). Similar results are found for the film of the silver nanoparticles alone, where a segment with empty cell can be observed (Fig. 6, depicted by an arrow). On the contrary, it is to be mentioned that no damage has been found for the fungus deposited on pPET3OC12-sqS films. All these results are consistent with the fact that the combination of fluorescent polymer with the nanoparticles in the composite film allows the construction of an optical biosensor for both the detection and attack of fungi. In order to understand
Fig. 5. Laser confocal micrographs of pPET3OC12-sqS/Ag nanoparticles film after immersion in the P. variotii suspension. a) spores and mycelium deposition- in mycelium, fragmentation due to collapse is observed, b) a mycelium fragment in which pores can be observed.
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Fig. 6. a) SEM image of a film of pure Ag nanoparticles after immersion in the P. variotii suspension.
the mechanism of the attack, AFM, TEM and XPS studies on the nanocomposite samples were carried out. Silver nanoparticles show spherical morphology and an average diameter of 6.8 nm as found by TEM (Fig. 7). Unfortunately, the TEM characterization of the nanocomposite was not possible. The polymer forms a thin layer on the copper grid trapping the particles. When the electron beam hits the film, the polymer burns and the nanoparticles agglomerate. Morphological analysis by AFM (Fig. 8a) reveals that the pPET3OC12-sqS/Ag nanoparticles film presents a granular morphology analogously to that found for pPET3OC12-sqS [15]. Isolated nanoparticles cannot be distinguished even in nanometer size scans (Fig. 8b). A reasonable explanation could be that the nanoparticles are capped by a polymer layer. However, due to their very low dimension and loss of resolution of tapping images of nanometer size, this hypothesis cannot be completely demonstrated by this technique. Fig. 8. AFM bi-dimensional images of the nanocomposite films in a) 50 50 mm2 and b) 500 500 nm2.
Fig. 7. TEM image of silver nanoparticles studied in this work. Inserted figure: particle size distribution.
Fig. 9 shows the XPS spectra for the silver nanoparticles and the nanocomposite sample. The bonding nature and chemical states on the surface of the films with silver nanoparticle sample (top) shows a major contribution in the Ag3d high resolution spectra of a peak centred at 368.3 eV which is assigned to Ag 3d5/2 in Agþ form [20]. The XPS Ag3d high resolution spectrum for the nanocomposite (bottom) shows no evidence of silver contribution. Taking into account the limit in penetration depth of the X-rays, this result could be ascribed to the fact that the nanoparticles are covered by the polymer, supporting the morphological characterization by AFM. Optical and morphological characterizations seem to indicate that pPET3OC12-sqS is assembled over nanoparticles. This hypothesis is reliable, considering the much greater size of the polymer with respect to the nanoparticles and the fact that it is expected to interact through the flexible groups partially with silver (to make the nanocomposite) and to the fungus (to permit its deposition). However, microbiological tests indicate the attack of the fungus. A possible mechanism for the attack could involve a local direct contact of silver nanoparticles with the fungus i.e. certain facets of the silver nanoparticles are exposed on the surface. The evidence by laser scanning microscopy suggests that this contact occurs at specific points which could be related to the part
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with dithioester-diethylsulfide moieties and microbiological tests with P. variotii. Optical and morphological characterizations of films of the nanocomposite suggest that the silver nanoparticles are covered by the polymer. Nevertheless, the microbiological tests indicate an attack of the mycelium that could be due to the contact of some facets of nanoparticles with the fungus. Despite the fact that attack is thus limited, the importance of the work is that with this nanocomposite, it is possible to construct optical biosensors for the simultaneous detection and attack of fungus. Future work will address quantifying the response by studying spore counting and spore viability. On the basis of those assays, changes in the nanocomposite composition or in the silver nanoparticles characteristics (size and capping agent) will be considered, in order to increase the fungicidal properties of this biosensor. Acknowledgements This work was financially supported by CONACyT through the project 51504-R and CIQA (project FD0003). Special acknowledgements to Antonio Diaz, for his technical assistance in XPS measurements, Esmeralda Saucedo for SEM analysis, Selene Sepulveda for TEM. References
Fig. 9. XPS spectra of the Ag3d region for the silver nanoparticles (top) and the nanocomposite (bottom).
of nanocomposite film where the polymer layer does not completely cap all the particles. Nevertheless, the delivery of silver to the cultures and consequently its attack of the fungus cannot be discarded as the amount of silver present in the nanocomposite films is below the detection limit of the atomic absorption measurements. As a final remark, the importance of this work is that we have demonstrated the viability of obtaining an optical biosensor for the detection and attack of P. variotii. Nevertheless as the mycelium, where the attack was evident, is a growing part of the fungus which cannot be quantified, a quantitative study of the device response was not possible for the moment. 4. Conclusions In this work we report the development of a nanocomposite of silver nanoparticles with a fluorescent poly(phenylethynylene) sequenced
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