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Pyrimidine based Functional Fluorescent Organic Nanoparticles Probe for Detection of Pseudomonas Aeruginosa Gaganpreet Kaur,a, Tilak Raj,b, Navneet Kaur a,* and Narinder Singh b,*
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Pyrimidine based sensor (1) has been synthesized through the facile one pot reaction of pyrrole -2carboxaldehyde, 2-aminobenzimidazole and 1, 3-dimethylbarbituric acid in methanol using few drops of HCl as a catalyst. Further, compound 1 was fabricated to its fluorescent organic nanoparticles (FONPs) and characterized using DLS and SEM. FONPs were further evaluated for the photo physical studies against bacteria and fungi. It was observed that FONPs selectively enhance the fluorescence intensity in the presence of gram ( ve) bacteria Pseudomonas Aeruginosa with the detection limit of 46 CFU. To the best of our knowledge, this study is the first report on the use of FONPs of pyrimidine derivative 1 for the detection of bacteria Pseudomonas Aeruginosa in various samples
Introduction Pseudomonas Aeruginosa is a ubiquitous gram negative bacterium, which colonizes in diverse natural and manmade environments.1 Due to its ability to survive on a wide range of nutritional sources and its tolerance to various physical conditions; it has been recognized as an emerging opportunistic human pathogen. It can be found on moist surfaces; medical equipments like breathing machines, catheters, etc. and are one of the leading causes of cross infections especially in patients who have been hospitalized for more than a week.2-4 It is often associated with lung infections in cystic fibrosis patients 5-8 and affect AIDS patients with immunocompromised host defense mechanisms9 and can be considered as an inevitable cause of patient morbidity and mortality. 10 Recent studies depicts Pseudomonas Aeruginosa as one of the leading causes of nosocomial pneumonia and healthcare associated pneumonia.1112 These infections are very hard to eradicate and present a therapeutic challenge due to the increasing resistance against different antibiotics and poses a serious concern in the medical field.13-14 In this context, ability to rapidly diagnose pathogens would be extremely beneficial to the medical community for steadfast treatment of various contagious diseases before the infection becomes chronic. Current detection techniques involve time consuming plate counting culture method. 15 Polymerase chain reaction (PCR) has also emerged as a rapid and accurate diagnostic technique, 16-18 In a recent study, a highly specific and sensitive Genome Exponential Amplification Reaction (GEAR) assay for the detection of E. coli has been designed.19 Recently, electrochemical sensing
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strategies have also provided a method to measure a chemical marker 2-aminoacetophenone, which is indicative of Pseudomonas Aeruginosa infection and proposes its implementation in breath sensing devices.20 Electrochemical identification of Pseudomonas Aeruginosa via addition of amino acids to up-regulate pyocyanin production has also been reported.21 Consequently, bacterial identification has been performed with the use of many techniques, viz., colorimetry, fluorimetry, electrochemistry, flow cytometry and mass spectrometry (mainly MALDI-MS).22-24 Although, the detection of bacteria has been widely explored with the use of various sensing materials and sophisticated instrumentation techniques, it is still a challenge to accomplish a sensitive, speedy, and inexpensive method. Therefore, a cost effective alternative diagnostic technique for rapid pathogen identification is the need of the hour to prevent the occurrence of extensive pathogenic outbreaks. Fluorimetry is a well established technique that offers highly accurate and reliable results. Its application in the field of bacterial detection has been widely explored. 25-28 The classical method of detecting bacteria by fluorescence microscopy or fluorimetry required labelling of the bacteria with fluorochromes during growth in broth. 29 In the last decade, the use of semiconductor quantum dots (QDs) instead of conventional organic dyes for biological labelling in fluorimetric detection has been studied. 30 In the current times nanoparticles and nanoclusters have opened new avenues for diagnostic applications.31-32 The use of CdSe/ZnS@SiO2, RuBpy-doped silica nanoparticles and multifunctional graphene magnetic nanosheets decorated with chitosan as fluorescent
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probes for bacterial detection has already been reported. 33-36 Recently, application of nanotechnology in the medical field has gained much attention and ensures the assessment of antimicrobial behaviour of metal nanoparticles.37-38 Taking the cognizance of the reported investigations, we proposed to synthesize pyrimidine derivative based receptor through three components, one pot cyclic condensation reaction. Pyrimidine derivative 1 was further engineered to fluorescent organic nanoparticles, FONPs in aqueous medium.39-40 The marvelous physiological properties and unswerving synthesis of pyrimidine derivative persuaded us to select this scaffold for proposed studies.41-42 Further, these FONPs were employed as an effective sensor for Pseudomonas Aeruginosa. This approach for detection of bacteria with the use of FONPs is IRXQG WR IROORZ ³IOXRUHVFHQFH WXUQ RQ´ PHFKDQLVP Current FONPs were further successfully evaluated for their antibacterial activity. The application of FONPs for the sensing of bacteria along with their antibacterial activity has not yet been exploited considerably. Hence, current study will offer new pathways for development of organic nanoparticles based highly sensitive and selective biosensors and may perhaps provide new dimensions to medical diagnosis.
Results and Discussion
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fabrication: (a) top down and (b) bottom up. 44 The nature of application for which the ONPs are required governs the appropriate selection of method. The meagre solubility of organic compounds in water remains a key challenge to study their properties in aqueous media. Since, our work is focused on the use of biological samples, which are often dispensed in water; we have developed the FONPs in pure water to counter the problem of reduced solubility; using a single step reprecipitation method through bottom up approach.45-46 A 1 mM solution of the compound 1 was prepared in DMSO. 1 mL of the functioning solution was gradually injected into 100 mL of water with a micro-syringe. The solution was then sonicated for a total of 10 minutes, making sure the temperature of the solution containing nanoparticles did not rise above 10 °C. As the organic molecules were exposed to water for a very short period of time, the water was expected to change their microenvironment, inducing nucleation and growth of the molecules to nanoparticles. Effect of water on photo-physical properties of compound 1 The UV-Vis absorption of compound 1 in DMSO and its nanoparticles FONPs dispersed in aqueous medium was recorded at a concentration of 10 µM each. Both the solutions displayed an absorbance maximum at a wavelength of 417 nm.
Synthesis Pyrimidine derivative (1) was synthesized in good yield via one pot three component condensation reaction of pyrrole-2carboxaldehyde, 2-aminobenzimidazole and 1,3dimethylbarbituric acid using HCl as a catalyst in methanol at 800C as shown in Scheme 1. After the completion of reaction, the solvent was evaporated under reduced pressure and crude product thus obtained was recrystallized from acetone/water solvent system. The synthesized product was characterized with different spectroscopic techniques i.e., 1H, 13C NMR, mass and elemental analysis.
(A)
(B)
Scheme 1: Synthesis of pyrimidine derivative 1
Development of FONPs Organic nanoparticles (ONPs) have gained tremendous attention due to their applications in material sciences and biosciences.38 Their minuscule size (ranging from 10 to 1000 nm) endows them with exceptional properties, which enable them to offer a multitude of applications in molecular recognition, drug delivery and particularly biosensing. 43 These applications lead to exploration of different methodologies for their synthesis. Literature reveals two approaches for ONP
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Fig. 1 (A) Absorption spectra of compound 1 in DMSO and its nanoparticles, FONPs in water; (B) Fluorescence emission spectra of compound 1 in DMSO and its nanoparticles, FONPs in water.
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On the change of solvent system from DMSO to water, a decrease in absorbance accompanied by the sharpening of peak was observed (Fig. 1A). Further, fluorescence spectra of both the solutions were also recorded to evaluate the effect of water content on the photo-physical properties of 1 by excitation at 417 nm. The emission spectra exhibited two significant effects on the change of solvent from DMSO to water; decrease in the fluorescence intensity and broadening of the peak (Fig. 1B). The decrease in fluorescence intensity due to increase in water content can be attributed to the formation of nano-aggregates. This effect of decrease in intensity can be referred to as ³DJJUHJDWLRQ FDXVHG TXHQFKLQJ´ (ACQ) which may be owed to stacking interactions.47 Effect of concentration of FONPs on the aggregation. The effect of changes of concentration of receptor 1 on the formation of FONPs was studied by taking the absorption spectra and emission spectra of FONPs by varying their concentrations (5, 10, 20, 30 and 50 µM, Fig. 2).
(A)
involvement of similar type of arrangements during formation of aggregates (Fig. 2A). In the same manner, enhanced fluorescence intensity was observed with increase in size (Fig. 2B). Although after a particular concentration a decrease in fluorescence intensity was observed (at 50 µM). The effect may EH GXH WR ³DXWR-TXHQFKLQJ´ RU ³VHOI-TXHQFKLQJ´ RI IOXRUHVFHQFH at very high concentrations due to severe aggregation. 48 Thus, the concentration of FONPs should be kept low and precisely adjusted to avoid the quenching effect in further studies However, it can be observed that there is no shift in the position of absorbance or fluorescence maximum with increase in size. A similar kind of arrangement of receptor molecules during formation of nano aggregates of different dimensions and uniformity in their dispersions in the aqueous media may be responsible for this effect. The change in size with the change in concentrations was also monitored by carrying out DLS studies which provides an estimation of the average particle size (Fig. 3A). It was observed that the particle size increased with the increase in concentrations of FONPs (Fig. 3B). The generation of larger particles with increase in concentration can be due to the controlled precipitation along with non polar interactions between the larger amounts of compound.
5 0µM
5 µM
(B) 50 µM
5 µM
Fig. 2 (A) Changes in the UV-Vis absorption spectra of FONPs at varying concentrations (5, 10, 20, 30 and 50 µM); (B) Changes in the emission spectra of FONPs at varying concentrations (5, 10, 20, 30 and 50 µM).
As the particle size increased (with increase in concentration), there was an increase in the absorbance which demonstrates the
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Fig. 3 (A) DLS histograms of FONPs ~ïî vu• š }v všŒ š]}v íì …DV ~ • ‰o}š showing the variation in size of FONPs as a function of concentration of compound in aqueous medium.
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Recognition studies of FONPs To assess the recognition behaviour of FONPs dispersed in water; changes in the emission profile (excited at 417 nm) were recorded on a fluorimeter with addition of a fixed number of colony forming units (CFU) of different bacteria Pseudomonas Aeruginosa, Staphylococcus Aureus, Escherichia Coli and Shigella Flexneri; and fungi such as Aspergillus Niger, Geotrichum Candidum, Candida Albicans and Candida Tropicalis to a fixed concentration (10 µM) of FONPs (Fig. 4A).
(A)
emission profile of FONPs did not show any significant change with the addition of other bacteria and fungi under study. This enhancement in the fluorescence intensity may be a result of interaction of Pseudomonas Aeruginosa with the nanoparticles of receptor molecules, FONPs. To further investigate the binding behaviour of FONPs and Pseudomonas Aeruginosa, a titration was carried out (Fig. 4B) in which varying CFUs of Pseudomonas Aeruginosa (4.0 × 103 - 1.8 × 104 CFU) were successively added to a fixed concentration of FONPs (10 µM). It was observed that the increase in the CFUs of Pseudomonas Aeruginosa added to host solution (FONPs) led to a continuous enhancement in the fluorescence intensity of the band. The linear regression graph was plotted to study the linearity of the titrations (Fig. 5).
y = 0.00643 x + 199.69 R2 = 0.9897
Fig. 5 Linear regression graph between the colonies of Pseudomonas Aeruginosa added and increase in the fluorescence intensity
(B) CFU
Further, various experiments were carried out to determine the effect of pH, salt effect and use of buffer (pH-7.4) and different metal ions on recognition profile of FONPS. However, no noticeable changes were observed (Figure S1-S6). Therefore, the proposed FONPS effortlessly meet the demands for sensing of bacteria in diverse biological samples Scanning electron microscopy of Bacteria
Fig. 4 (A) Changes in the emission profile of FONPs (10 µM) dispersed in water upon addition of different bacteria and fungi; (B) Changes in the emission profile of FONPs (10 µM) dispersed in water upon successive addition of Pseudomonas Aeruginosa.
The addition of Pseudomonas Aeruginosa produced a distinct enhancement in the fluorescence intensity, although the
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The morphology of nanoparticles was also studied by recording their SEM images (Fig. 6A) which displayed the formation of needle shaped nanomaterials. To get an in depth knowledge of the processes going on at microbial scale, SEM images of Pseudomonas Aeruginosa were also recorded to visualize the effect of addition of FONPs (Fig. 6 B and C). The imaging of cells was accomplished by treating the samples with glutaraldehyde for a night and centrifuged and washed using deionised water. The suspension was put on an analysis plate and dried in air. All the images were examined in a scanning mode, with Jeol JSM-6610LV scanning electron microscope, which operated at 15KeV. On comparing the SEM images of detected bacteria, Pseudomonas Aeruginosa before and after treatment with the FONPs, we found that the FONPs promote changes in the morphology of the bacterial cells which can be a
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Fig. 6. (A) SEM images of FONPs; (B) SEM image of Pseudomonas Aeruginosa before treatment with FONPs. (C) SEM image of Pseudomonas Aeruginosa after treatment with FONPs.
Antibacterial activity of FONPs
The outcomes of SEM analysis persuaded us to assess the antibacterial characteristics of FONPs on the detected bacteria, Pseudomonas Aeruginosa. For this purpose, different concentrations of FONPs were prepared and added to Pseudomonas Aeruginosa. The effect of change in concentration of FONPs on the bacterial growth was monitored by recording the UV-Vis optical density, OD at 600 nm as a function of time (an indirect method of measuring bacterial cells). Fig. 7 shows a comparison of bacterial growth in the presence of different concentrations of FONPs over 8 hours.
Real Sample analysis Pseudomonas Aeruginosa is often found in lakes and rivers and is a leading cause of skin and ear infections from exposure to infected water during recreational activities in swimming pools and spas, specifically in whirlpools and hot tubs. 49 The high temperatures of whirlpools and hot tubs are appropriate for the growth of Pseudomonas Aeruginosa. To evaluate the practical application of fluorescent organic nanoparticles, FONPs for the detection of Pseudomonas Aeruginosa, different water samples were collected from river, lake and spa. The bacteria and fungi were incubated in these water samples. The results obtained from fluorimetric sensing with FONPs were also compared with conventional plate counting method and were found to have good agreement. The results in Table 1 reveal that the proposed sensor can be successfully employed as a bacterial detection probe for Pseudomonas Aeruginosa in environmental samples. Table 1. Real time analysis of FONPs S. no.
Sample
Plate count method (CFU/100mL)
Proposed sensor FONPs (CFU/100mL)
1
River water
2.70 X 103
2.69 X 103
2
Lake water
3.15 X 103
3.17 X 103
3
Spa water
4.6 X 10
1
4.64 X 101
Conclusions In conclusions, compound 1 is synthesized through the one pot reaction, which was further processed to their fluorescent organic nanoparticles using reprecipitation method. Further, FONPs of compound were evaluated for biosensor potential against different strains of gram +ve and gram ±ve bacteria and different fungi. Results of investigation revealed that FONPs acts as potential sensor for gram (±ve) bacteria Pseudomonas Aeruginosa with a detection limit of 46 CFU. Additionally, the proposed sensor displays good antibacterial properties over a period of time.
Experimental Fig. 7. OD v/s Time plot depicting antibacterial activity of FONPs at different concentrations.
Apparently, the increase in concentration of FONPs has a negative impact on the growth of bacteria. It is evident, that there is only a minute increase in the OD for the sample corresponding to 100 µg/ml FONPs concentration, as compared to that of 10 µg/ml. These outcomes particularly emphasize on the role of FONPs in suppressing bacterial growth. Consequently, the developed FONPs can also be employed as antibacterial after they have been exercised as a sensor for bacterial detection.
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All the chemicals were purchased from commercial suppliers and were used without additional purification. 1H and 13C NMR spectra were recorded on JEOL II 400 spectrometer (400 MHz with TMS as internal standard; chemical shifts are expressed in ppm). Mass analysis was performed on Shimadzu 79037 GCMS GC-2010 plus. The CHN analysis was performed on Perkin Elmer 2400 CHN Elemental Analyser. Scanning electron microscopic studies were conducted with drying the aqueous solutions of materials (10 µM). SEM images were taken with Jeol JSM-6610LV scanning electron microscope which operated at 15KeV. The particle size of nanoparticles was determined with Dynamic Light Scattering (DLS) using external probe feature of Microtrac Ultra Nanotrac Particle Size
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probable cause of cell death. This observation indicates that besides the detection of bacteria, the treatment of developed nanoparticles has an added advantage of acting as an antibacterial over a period of time.
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Analyzer instrument. For analysing, 10 µM concentration of solution was used and presented results are the average of 20 scans× 240 pixels recorded with camtasia recorder at 12 frames/s. Shimadzu RF-5301 spectrofluorophotometer was used for fluorimetric measurements. UV-Vis studies were done on SpectroScan 30 spectrophotometer. SYNTHESIS OF COMPOUND 1 The compound (1) was synthesized with good yield via one pot three component condensation reaction of pyrrole-2carboxaldehyde (95.10 mg, 1 mmol), 2-aminobenzimidazole (199.7 mg, 1.5 mmol) and 1,3-dimethylbarbituric acid (156.1 mg, 1 mmol) using HCl (2 mol %) as a catalyst in 10 ml methanol at 80 0C as shown in Scheme 1. After the completion of reaction (TLC), the solvent was evaporated under reduced pressure and crude product thus obtained was recrystallized from acetone/water solvent system. The synthesized product was characterized with different spectroscopic techniques i.e., 1 H, 13C NMR, mass and elemental analysis. A brown solid was obtained in 78.9%, yield. mp. >185 °C; 1H NMR (400 MHz, CDCl3 / SSP 11.04(brs, 1H, NH), 9.11(brs, 1H, NH), 8.34(d, 1H, ArH),7.91(d, 1H, ArH), 7.41(s, 1H, ArH), 7.14(d, 1H, ArH), 6.54(m, 2H, ArHs), 6.23(s, 1H, ArH), 6.59(s, 1H, CH), 3.42(s, 3H, CH3), 3.40(s, 3H, CH3); 13C NMR (100MHz, CDCl3 / (ppm) 163.80(C=O), 163.46(C=O), 151.63, 143.11, 140.06, 138.02, 135.35, 131.52, 130.25, 129.65, 123.11, 121.91, 120.01, 114.75, 110.06, 105.66, 29.00, 28.51. MS (ESI) : m/z 371 (M + Na+) ; Anal. calcd. for C, 62.06; H, 4.63; N, 24.12; Found C, 61.96; H, 4.53; N, 24.02. FABRICATION OF FONPS The FONPS were prepared by using re-precipitation method. A solution of compound 1 (1 mM) was prepared in DMSO. 1 mL of this solution was gradually injected into 100 mL of water with a micro-syringe. The nanoparticles were obtained by sonication for about 15 minutes, ensuring that the temperature of the solution did not rise above 10°C during sonication. MICROBIAL CULTURE Bacterial cultures such as Gram negative bacteria Escherichia Coli (MTCC-119), Pseudomonas Aeruginosa (MTCC 741), Shigella Flexneri (MTCC-1457); gram positive bacteria Staphylococcus Aureus (MTCC-740) and fungal cultures, fungi Aspergillus niger (MTCC-281), Geotrichum candidum (MTCC-3993), Candida albicans (MTCC-227) and Candida tropicalis (MTCC-230) were originally obtained from Microbial Type Culture Collection, IMTECH, Chandigarh, India and kept frozen. For the experiment, an aliquot of concerned bacteria or fungi was thawed and diluted in the nutrient broth culture and incubated at 37 0C until it turned turbid (7-8 hours). The cultured bacteria or fungi was centrifuged at 3000 rpm for 10 minutes, washed with phosphate buffered saline and resuspended in sterile saline. Cell numbers were determined by serial dilutions and plating onto standard agar plates (incubated at 370C till the formation of colonies) and by counting the number of colony forming units (CFU).
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RECOGNITION STUDIES The recognition studies were performed at 25 ± 1 °C, and the solutions were shaken for a sufficient time before recording the spectra. The binding ability of FONPs (10 0 LQ DTXHRXV medium was determined by addition of different bacteria to 5 mL solution of FONPs taken in volumetric flasks. The volumetric flasks were allowed to stand for 10 minutes before the spectra were recorded. For Pseudomonas Aeruginosa titrations, the bacterium was added to volumetric flasks containing solutions of FONPs in aqueous medium. pH titrations and salt effect were performed to understand the effect of pH and salts on the recognition profile of FONPs.
Acknowledgements GK is thankful to CSIR, New Delhi for the junior research fellowship. TR acknowledges UGC India for his Post Doctoral project fellowship (Project no. PDFSS-SC-PUN-34).
Notes and references a
Centre for Nanoscience & Nanotechnology (UIEAST), Panjab University Chandigarh 160014, India. E-mail: [email protected], Tel: +911722534464 b Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab 140001, India. E-mail: [email protected], Tel: +911881242176, Fax: +911881223395 *Corresponding authors. ‚ %RWK DXWKRUV KDYH FRQWULEXWHG HTXDOO\ Electronic Supplementary Information (ESI) available: Characterization data including NMR, IR, Mass spectra; photophysical properties including fluorescence spectra; supporting data for theoretical calculations. See DOI: 10.1039/b000000x/ 1.
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Cite this: DOI: 10.1039/x0xx00000x
Pyrimidine based Functional Fluorescent Organic Nanoparticles Probe for Detection of Pseudomonas Aeruginosa Gaganpreet Kaur,a, Tilak Raj,b, Navneet Kaur a,* and Narinder Singh b,*
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Pyrimidine based sensor (1) has been synthesized through the facile one pot reaction of pyrrole -2carboxaldehyde, 2-aminobenzimidazole and 1, 3-dimethylbarbituric acid in methanol using few drops of HCl as a catalyst. Further, compound 1 was fabricated to its fluorescent organic nanoparticles (FONPs) and characterized using DLS and SEM. FONPs were further evaluated for the photo physical studies against bacteria and fungi. It was observed that FONPs selectively enhance the fluorescence intensity in the presence of gram ( ve) bacteria Pseudomonas Aeruginosa with the detection limit of 46 CFU. To the best of our knowledge, this study is the first report on the use of FONPs of pyrimidine derivative 1 for the detection of bacteria Pseudomonas Aeruginosa in various samples
Introduction Pseudomonas Aeruginosa is a ubiquitous gram negative bacterium, which colonizes in diverse natural and manmade environments.1 Due to its ability to survive on a wide range of nutritional sources and its tolerance to various physical conditions; it has been recognized as an emerging opportunistic human pathogen. It can be found on moist surfaces; medical equipments like breathing machines, catheters, etc. and are one of the leading causes of cross infections especially in patients who have been hospitalized for more than a week.2-4 It is often associated with lung infections in cystic fibrosis patients 5-8 and affect AIDS patients with immunocompromised host defense mechanisms9 and can be considered as an inevitable cause of patient morbidity and mortality. 10 Recent studies depicts Pseudomonas Aeruginosa as one of the leading causes of nosocomial pneumonia and healthcare associated pneumonia.1112 These infections are very hard to eradicate and present a therapeutic challenge due to the increasing resistance against different antibiotics and poses a serious concern in the medical field.13-14 In this context, ability to rapidly diagnose pathogens would be extremely beneficial to the medical community for steadfast treatment of various contagious diseases before the infection becomes chronic. Current detection techniques involve time consuming plate counting culture method. 15 Polymerase chain reaction (PCR) has also emerged as a rapid and accurate diagnostic technique, 16-18 In a recent study, a highly specific and sensitive Genome Exponential Amplification Reaction (GEAR) assay for the detection of E. coli has been designed.19 Recently, electrochemical sensing
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strategies have also provided a method to measure a chemical marker 2-aminoacetophenone, which is indicative of Pseudomonas Aeruginosa infection and proposes its implementation in breath sensing devices.20 Electrochemical identification of Pseudomonas Aeruginosa via addition of amino acids to up-regulate pyocyanin production has also been reported.21 Consequently, bacterial identification has been performed with the use of many techniques, viz., colorimetry, fluorimetry, electrochemistry, flow cytometry and mass spectrometry (mainly MALDI-MS).22-24 Although, the detection of bacteria has been widely explored with the use of various sensing materials and sophisticated instrumentation techniques, it is still a challenge to accomplish a sensitive, speedy, and inexpensive method. Therefore, a cost effective alternative diagnostic technique for rapid pathogen identification is the need of the hour to prevent the occurrence of extensive pathogenic outbreaks. Fluorimetry is a well established technique that offers highly accurate and reliable results. Its application in the field of bacterial detection has been widely explored. 25-28 The classical method of detecting bacteria by fluorescence microscopy or fluorimetry required labelling of the bacteria with fluorochromes during growth in broth. 29 In the last decade, the use of semiconductor quantum dots (QDs) instead of conventional organic dyes for biological labelling in fluorimetric detection has been studied. 30 In the current times nanoparticles and nanoclusters have opened new avenues for diagnostic applications.31-32 The use of CdSe/ZnS@SiO2, RuBpy-doped silica nanoparticles and multifunctional graphene magnetic nanosheets decorated with chitosan as fluorescent
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probes for bacterial detection has already been reported. 33-36 Recently, application of nanotechnology in the medical field has gained much attention and ensures the assessment of antimicrobial behaviour of metal nanoparticles.37-38 Taking the cognizance of the reported investigations, we proposed to synthesize pyrimidine derivative based receptor through three components, one pot cyclic condensation reaction. Pyrimidine derivative 1 was further engineered to fluorescent organic nanoparticles, FONPs in aqueous medium.39-40 The marvelous physiological properties and unswerving synthesis of pyrimidine derivative persuaded us to select this scaffold for proposed studies.41-42 Further, these FONPs were employed as an effective sensor for Pseudomonas Aeruginosa. This approach for detection of bacteria with the use of FONPs is IRXQG WR IROORZ ³IOXRUHVFHQFH WXUQ RQ´ PHFKDQLVP Current FONPs were further successfully evaluated for their antibacterial activity. The application of FONPs for the sensing of bacteria along with their antibacterial activity has not yet been exploited considerably. Hence, current study will offer new pathways for development of organic nanoparticles based highly sensitive and selective biosensors and may perhaps provide new dimensions to medical diagnosis.
Results and Discussion
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fabrication: (a) top down and (b) bottom up. 44 The nature of application for which the ONPs are required governs the appropriate selection of method. The meagre solubility of organic compounds in water remains a key challenge to study their properties in aqueous media. Since, our work is focused on the use of biological samples, which are often dispensed in water; we have developed the FONPs in pure water to counter the problem of reduced solubility; using a single step reprecipitation method through bottom up approach.45-46 A 1 mM solution of the compound 1 was prepared in DMSO. 1 mL of the functioning solution was gradually injected into 100 mL of water with a micro-syringe. The solution was then sonicated for a total of 10 minutes, making sure the temperature of the solution containing nanoparticles did not rise above 10 °C. As the organic molecules were exposed to water for a very short period of time, the water was expected to change their microenvironment, inducing nucleation and growth of the molecules to nanoparticles. Effect of water on photo-physical properties of compound 1 The UV-Vis absorption of compound 1 in DMSO and its nanoparticles FONPs dispersed in aqueous medium was recorded at a concentration of 10 µM each. Both the solutions displayed an absorbance maximum at a wavelength of 417 nm.
Synthesis Pyrimidine derivative (1) was synthesized in good yield via one pot three component condensation reaction of pyrrole-2carboxaldehyde, 2-aminobenzimidazole and 1,3dimethylbarbituric acid using HCl as a catalyst in methanol at 800C as shown in Scheme 1. After the completion of reaction, the solvent was evaporated under reduced pressure and crude product thus obtained was recrystallized from acetone/water solvent system. The synthesized product was characterized with different spectroscopic techniques i.e., 1H, 13C NMR, mass and elemental analysis.
(A)
(B)
Scheme 1: Synthesis of pyrimidine derivative 1
Development of FONPs Organic nanoparticles (ONPs) have gained tremendous attention due to their applications in material sciences and biosciences.38 Their minuscule size (ranging from 10 to 1000 nm) endows them with exceptional properties, which enable them to offer a multitude of applications in molecular recognition, drug delivery and particularly biosensing. 43 These applications lead to exploration of different methodologies for their synthesis. Literature reveals two approaches for ONP
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Fig. 1 (A) Absorption spectra of compound 1 in DMSO and its nanoparticles, FONPs in water; (B) Fluorescence emission spectra of compound 1 in DMSO and its nanoparticles, FONPs in water.
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On the change of solvent system from DMSO to water, a decrease in absorbance accompanied by the sharpening of peak was observed (Fig. 1A). Further, fluorescence spectra of both the solutions were also recorded to evaluate the effect of water content on the photo-physical properties of 1 by excitation at 417 nm. The emission spectra exhibited two significant effects on the change of solvent from DMSO to water; decrease in the fluorescence intensity and broadening of the peak (Fig. 1B). The decrease in fluorescence intensity due to increase in water content can be attributed to the formation of nano-aggregates. This effect of decrease in intensity can be referred to as ³DJJUHJDWLRQ FDXVHG TXHQFKLQJ´ (ACQ) which may be owed to stacking interactions.47 Effect of concentration of FONPs on the aggregation. The effect of changes of concentration of receptor 1 on the formation of FONPs was studied by taking the absorption spectra and emission spectra of FONPs by varying their concentrations (5, 10, 20, 30 and 50 µM, Fig. 2).
(A)
involvement of similar type of arrangements during formation of aggregates (Fig. 2A). In the same manner, enhanced fluorescence intensity was observed with increase in size (Fig. 2B). Although after a particular concentration a decrease in fluorescence intensity was observed (at 50 µM). The effect may EH GXH WR ³DXWR-TXHQFKLQJ´ RU ³VHOI-TXHQFKLQJ´ RI IOXRUHVFHQFH at very high concentrations due to severe aggregation. 48 Thus, the concentration of FONPs should be kept low and precisely adjusted to avoid the quenching effect in further studies However, it can be observed that there is no shift in the position of absorbance or fluorescence maximum with increase in size. A similar kind of arrangement of receptor molecules during formation of nano aggregates of different dimensions and uniformity in their dispersions in the aqueous media may be responsible for this effect. The change in size with the change in concentrations was also monitored by carrying out DLS studies which provides an estimation of the average particle size (Fig. 3A). It was observed that the particle size increased with the increase in concentrations of FONPs (Fig. 3B). The generation of larger particles with increase in concentration can be due to the controlled precipitation along with non polar interactions between the larger amounts of compound.
5 0µM
5 µM
(B) 50 µM
5 µM
Fig. 2 (A) Changes in the UV-Vis absorption spectra of FONPs at varying concentrations (5, 10, 20, 30 and 50 µM); (B) Changes in the emission spectra of FONPs at varying concentrations (5, 10, 20, 30 and 50 µM).
As the particle size increased (with increase in concentration), there was an increase in the absorbance which demonstrates the
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Fig. 3 (A) DLS histograms of FONPs ~ïî vu• š }v všŒ š]}v íì …DV ~ • ‰o}š showing the variation in size of FONPs as a function of concentration of compound in aqueous medium.
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Recognition studies of FONPs To assess the recognition behaviour of FONPs dispersed in water; changes in the emission profile (excited at 417 nm) were recorded on a fluorimeter with addition of a fixed number of colony forming units (CFU) of different bacteria Pseudomonas Aeruginosa, Staphylococcus Aureus, Escherichia Coli and Shigella Flexneri; and fungi such as Aspergillus Niger, Geotrichum Candidum, Candida Albicans and Candida Tropicalis to a fixed concentration (10 µM) of FONPs (Fig. 4A).
(A)
emission profile of FONPs did not show any significant change with the addition of other bacteria and fungi under study. This enhancement in the fluorescence intensity may be a result of interaction of Pseudomonas Aeruginosa with the nanoparticles of receptor molecules, FONPs. To further investigate the binding behaviour of FONPs and Pseudomonas Aeruginosa, a titration was carried out (Fig. 4B) in which varying CFUs of Pseudomonas Aeruginosa (4.0 × 103 - 1.8 × 104 CFU) were successively added to a fixed concentration of FONPs (10 µM). It was observed that the increase in the CFUs of Pseudomonas Aeruginosa added to host solution (FONPs) led to a continuous enhancement in the fluorescence intensity of the band. The linear regression graph was plotted to study the linearity of the titrations (Fig. 5).
y = 0.00643 x + 199.69 R2 = 0.9897
Fig. 5 Linear regression graph between the colonies of Pseudomonas Aeruginosa added and increase in the fluorescence intensity
(B) CFU
Further, various experiments were carried out to determine the effect of pH, salt effect and use of buffer (pH-7.4) and different metal ions on recognition profile of FONPS. However, no noticeable changes were observed (Figure S1-S6). Therefore, the proposed FONPS effortlessly meet the demands for sensing of bacteria in diverse biological samples Scanning electron microscopy of Bacteria
Fig. 4 (A) Changes in the emission profile of FONPs (10 µM) dispersed in water upon addition of different bacteria and fungi; (B) Changes in the emission profile of FONPs (10 µM) dispersed in water upon successive addition of Pseudomonas Aeruginosa.
The addition of Pseudomonas Aeruginosa produced a distinct enhancement in the fluorescence intensity, although the
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The morphology of nanoparticles was also studied by recording their SEM images (Fig. 6A) which displayed the formation of needle shaped nanomaterials. To get an in depth knowledge of the processes going on at microbial scale, SEM images of Pseudomonas Aeruginosa were also recorded to visualize the effect of addition of FONPs (Fig. 6 B and C). The imaging of cells was accomplished by treating the samples with glutaraldehyde for a night and centrifuged and washed using deionised water. The suspension was put on an analysis plate and dried in air. All the images were examined in a scanning mode, with Jeol JSM-6610LV scanning electron microscope, which operated at 15KeV. On comparing the SEM images of detected bacteria, Pseudomonas Aeruginosa before and after treatment with the FONPs, we found that the FONPs promote changes in the morphology of the bacterial cells which can be a
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Fig. 6. (A) SEM images of FONPs; (B) SEM image of Pseudomonas Aeruginosa before treatment with FONPs. (C) SEM image of Pseudomonas Aeruginosa after treatment with FONPs.
Antibacterial activity of FONPs
The outcomes of SEM analysis persuaded us to assess the antibacterial characteristics of FONPs on the detected bacteria, Pseudomonas Aeruginosa. For this purpose, different concentrations of FONPs were prepared and added to Pseudomonas Aeruginosa. The effect of change in concentration of FONPs on the bacterial growth was monitored by recording the UV-Vis optical density, OD at 600 nm as a function of time (an indirect method of measuring bacterial cells). Fig. 7 shows a comparison of bacterial growth in the presence of different concentrations of FONPs over 8 hours.
Real Sample analysis Pseudomonas Aeruginosa is often found in lakes and rivers and is a leading cause of skin and ear infections from exposure to infected water during recreational activities in swimming pools and spas, specifically in whirlpools and hot tubs. 49 The high temperatures of whirlpools and hot tubs are appropriate for the growth of Pseudomonas Aeruginosa. To evaluate the practical application of fluorescent organic nanoparticles, FONPs for the detection of Pseudomonas Aeruginosa, different water samples were collected from river, lake and spa. The bacteria and fungi were incubated in these water samples. The results obtained from fluorimetric sensing with FONPs were also compared with conventional plate counting method and were found to have good agreement. The results in Table 1 reveal that the proposed sensor can be successfully employed as a bacterial detection probe for Pseudomonas Aeruginosa in environmental samples. Table 1. Real time analysis of FONPs S. no.
Sample
Plate count method (CFU/100mL)
Proposed sensor FONPs (CFU/100mL)
1
River water
2.70 X 103
2.69 X 103
2
Lake water
3.15 X 103
3.17 X 103
3
Spa water
4.6 X 10
1
4.64 X 101
Conclusions In conclusions, compound 1 is synthesized through the one pot reaction, which was further processed to their fluorescent organic nanoparticles using reprecipitation method. Further, FONPs of compound were evaluated for biosensor potential against different strains of gram +ve and gram ±ve bacteria and different fungi. Results of investigation revealed that FONPs acts as potential sensor for gram (±ve) bacteria Pseudomonas Aeruginosa with a detection limit of 46 CFU. Additionally, the proposed sensor displays good antibacterial properties over a period of time.
Experimental Fig. 7. OD v/s Time plot depicting antibacterial activity of FONPs at different concentrations.
Apparently, the increase in concentration of FONPs has a negative impact on the growth of bacteria. It is evident, that there is only a minute increase in the OD for the sample corresponding to 100 µg/ml FONPs concentration, as compared to that of 10 µg/ml. These outcomes particularly emphasize on the role of FONPs in suppressing bacterial growth. Consequently, the developed FONPs can also be employed as antibacterial after they have been exercised as a sensor for bacterial detection.
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All the chemicals were purchased from commercial suppliers and were used without additional purification. 1H and 13C NMR spectra were recorded on JEOL II 400 spectrometer (400 MHz with TMS as internal standard; chemical shifts are expressed in ppm). Mass analysis was performed on Shimadzu 79037 GCMS GC-2010 plus. The CHN analysis was performed on Perkin Elmer 2400 CHN Elemental Analyser. Scanning electron microscopic studies were conducted with drying the aqueous solutions of materials (10 µM). SEM images were taken with Jeol JSM-6610LV scanning electron microscope which operated at 15KeV. The particle size of nanoparticles was determined with Dynamic Light Scattering (DLS) using external probe feature of Microtrac Ultra Nanotrac Particle Size
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probable cause of cell death. This observation indicates that besides the detection of bacteria, the treatment of developed nanoparticles has an added advantage of acting as an antibacterial over a period of time.
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Analyzer instrument. For analysing, 10 µM concentration of solution was used and presented results are the average of 20 scans× 240 pixels recorded with camtasia recorder at 12 frames/s. Shimadzu RF-5301 spectrofluorophotometer was used for fluorimetric measurements. UV-Vis studies were done on SpectroScan 30 spectrophotometer. SYNTHESIS OF COMPOUND 1 The compound (1) was synthesized with good yield via one pot three component condensation reaction of pyrrole-2carboxaldehyde (95.10 mg, 1 mmol), 2-aminobenzimidazole (199.7 mg, 1.5 mmol) and 1,3-dimethylbarbituric acid (156.1 mg, 1 mmol) using HCl (2 mol %) as a catalyst in 10 ml methanol at 80 0C as shown in Scheme 1. After the completion of reaction (TLC), the solvent was evaporated under reduced pressure and crude product thus obtained was recrystallized from acetone/water solvent system. The synthesized product was characterized with different spectroscopic techniques i.e., 1 H, 13C NMR, mass and elemental analysis. A brown solid was obtained in 78.9%, yield. mp. >185 °C; 1H NMR (400 MHz, CDCl3 / SSP 11.04(brs, 1H, NH), 9.11(brs, 1H, NH), 8.34(d, 1H, ArH),7.91(d, 1H, ArH), 7.41(s, 1H, ArH), 7.14(d, 1H, ArH), 6.54(m, 2H, ArHs), 6.23(s, 1H, ArH), 6.59(s, 1H, CH), 3.42(s, 3H, CH3), 3.40(s, 3H, CH3); 13C NMR (100MHz, CDCl3 / (ppm) 163.80(C=O), 163.46(C=O), 151.63, 143.11, 140.06, 138.02, 135.35, 131.52, 130.25, 129.65, 123.11, 121.91, 120.01, 114.75, 110.06, 105.66, 29.00, 28.51. MS (ESI) : m/z 371 (M + Na+) ; Anal. calcd. for C, 62.06; H, 4.63; N, 24.12; Found C, 61.96; H, 4.53; N, 24.02. FABRICATION OF FONPS The FONPS were prepared by using re-precipitation method. A solution of compound 1 (1 mM) was prepared in DMSO. 1 mL of this solution was gradually injected into 100 mL of water with a micro-syringe. The nanoparticles were obtained by sonication for about 15 minutes, ensuring that the temperature of the solution did not rise above 10°C during sonication. MICROBIAL CULTURE Bacterial cultures such as Gram negative bacteria Escherichia Coli (MTCC-119), Pseudomonas Aeruginosa (MTCC 741), Shigella Flexneri (MTCC-1457); gram positive bacteria Staphylococcus Aureus (MTCC-740) and fungal cultures, fungi Aspergillus niger (MTCC-281), Geotrichum candidum (MTCC-3993), Candida albicans (MTCC-227) and Candida tropicalis (MTCC-230) were originally obtained from Microbial Type Culture Collection, IMTECH, Chandigarh, India and kept frozen. For the experiment, an aliquot of concerned bacteria or fungi was thawed and diluted in the nutrient broth culture and incubated at 37 0C until it turned turbid (7-8 hours). The cultured bacteria or fungi was centrifuged at 3000 rpm for 10 minutes, washed with phosphate buffered saline and resuspended in sterile saline. Cell numbers were determined by serial dilutions and plating onto standard agar plates (incubated at 370C till the formation of colonies) and by counting the number of colony forming units (CFU).
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RECOGNITION STUDIES The recognition studies were performed at 25 ± 1 °C, and the solutions were shaken for a sufficient time before recording the spectra. The binding ability of FONPs (10 0 LQ DTXHRXV medium was determined by addition of different bacteria to 5 mL solution of FONPs taken in volumetric flasks. The volumetric flasks were allowed to stand for 10 minutes before the spectra were recorded. For Pseudomonas Aeruginosa titrations, the bacterium was added to volumetric flasks containing solutions of FONPs in aqueous medium. pH titrations and salt effect were performed to understand the effect of pH and salts on the recognition profile of FONPs.
Acknowledgements GK is thankful to CSIR, New Delhi for the junior research fellowship. TR acknowledges UGC India for his Post Doctoral project fellowship (Project no. PDFSS-SC-PUN-34).
Notes and references a
Centre for Nanoscience & Nanotechnology (UIEAST), Panjab University Chandigarh 160014, India. E-mail: [email protected], Tel: +911722534464 b Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab 140001, India. E-mail: [email protected], Tel: +911881242176, Fax: +911881223395 *Corresponding authors. ‚ %RWK DXWKRUV KDYH FRQWULEXWHG HTXDOO\ Electronic Supplementary Information (ESI) available: Characterization data including NMR, IR, Mass spectra; photophysical properties including fluorescence spectra; supporting data for theoretical calculations. See DOI: 10.1039/b000000x/ 1.
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