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Fluorimetric detection of pathogenic bacteria using magnetic carbon dots
Accepted Manuscript Fluorimetric detection of pathogenic bacteria using Magnetic carbon dots Mukesh Lavkush Bhaisare, Gangaraju Gedda, M. Shahnawaz Khan, Hui-Fen Wu PII:
S0003-2670(16)30226-4
DOI:
10.1016/j.aca.2016.02.025
Reference:
ACA 234428
To appear in:
Analytica Chimica Acta
Received Date: 16 September 2015 Revised Date:
25 January 2016
Accepted Date: 18 February 2016
Please cite this article as: M.L. Bhaisare, G. Gedda, M.S. Khan, H.-F. Wu, Fluorimetric detection of pathogenic bacteria using Magnetic carbon dots, Analytica Chimica Acta (2016), doi: 10.1016/ j.aca.2016.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fluorimetric detection of pathogenic bacteria using Magnetic carbon dots Mukesh Lavkush Bhaisare1, Gangaraju Gedda4, M Shahnawaz Khan4, Hui-Fen Wu*, 1, 2, 3, 4
Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-
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1
Sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan 2
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan
Institute of Medical Science and Technology, National Sun Yat-Sen University, 80424, Taiwan 4
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3
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and
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Academia Sinica, Kaohsiung, 80424, Taiwan
*Corresponding author, Phone: +886-7-5252000 ext. 3955; Fax: +886-7-5253908 E-mail: [email protected] (Prof. H F Wu)
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Abstract
A novel and facile approach of pathogenic bacteria detection, which utilizes fluorescent sensing and bacteria capture with Magnetic carbon dots (Mag-CDs), was proposed in this work. Magnetic nanoparticles were synthesized and then decorated with C-dots, and further
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functionalized with amine groups (chitosan). In this way, bacteria were strongly anchored on the hybrid material Mag-CDs for highly sensitive fluorescent detection. The Mag-CDs were characterized by UV-vis, FT-IR spectra, TEM images, XRD, and EDX. The characterizations
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validate the fabrication of amine-Mag-CDs and the promising applications of this material. Fluorescence spectroscope and MALDI-MS were used for the detection and identification of bacterial strains, respectively. The limit of detection for S. aureus and E. coli was found to be 3×102 and 3.5×102 cfu mL-1, respectively. With these encouraging results, it is expected that it would open revenues for promising applications of Mag-CDs nanomaterial. Keywords: Magnetic carbon dots, Staphylococcus aureus, Escherichia coli, fluorescence detection, MALDI-MS. 1
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1. Introduction Pathogenic bacterial detection and quantification have been pursued in the long term and are extremely important in biological, food related items, and environmental samples [1]. Especially
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E. coli and S. aureus are opportunistic bacterial species leading to human infections; they are majorly found in urinary tract infections. The unique nano-based magnetic nanoparticles applied on bio-sensing methodology [2, 3] prompts us to apply them extendedly. In recent years, carbon nanomaterials have received considerable attention primarily because of their unique properties.
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Among various carbon nanomaterials like graphene sheet, graphene oxide, and carbon nanotube, carbon dots have attracted attention nowadays[4]. Carbon dots were accidentally discovered, which have unique properties such as, strong photoluminescence, wavelength-dependent
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emission, relative stability, attractive prospects for bioimaging, high photostability, tunable excitation and emission wavelength, exceptional biocompatibility and eco-friendliness[5-7], etc. Even presented in the combined form C-dots also have variety of potential applications with CaO, ZnO, TiO2, and Al2O3 [8], and so forth. Recently, much research interest has been extended to hybrid nanomaterials in biology, physics, engineering and chemistry fields. Carbon-coated nanomaterials have been extensively used for magnetic resonance imaging study, and carbon
[9, 10].
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dots can also be passivated with amine terminated compounds through amine functionalization
The hybrid nanomaterials of carbon dots coated magnetic nanoparticles combine both
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fluorescent and magnetic properties in one material. The particular functionalization of amine group is originated or sourced from precursor chitosan[11]. One of the advantages of carbon dots
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lie behind its feature of providing better dispersibility, stability to iron oxide nanoparticles compare to bare iron oxide nanoparticles. Additionally, C-dots coated iron oxide nanoparticles are prone to prevent the oxidation and corrosion of iron oxide nanoparticles [8]. Fe3O4/carbon synthesis was first proposed by Zhifei research group [12], the important application of iron oxide is iron oxide/gold nanoparticles, which is particularly of interest to the controlled magnetic field [13]. In general, the synthesis of carbon-coated materials have been proposed including electric arc discharge, catalytic pyrolysis of organic compounds, and the hydrothermal method [14]. Carbon/Fe3O4 nanoparticles have been specifically applied as sorbents in solid-phase extraction (SPE) from environmental water samples, because the carbon dots have been proved 2
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for high capacity adsorption and extraction efficiency due to large surface area to small volume ratios [14]. To date, carbon dots decorated on magnetic nanoparticles has not yet been studied. Furthermore, the fluorescent property of carbon dots and the amine-functionalized nature make
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them applicable for trapping or as probes for pathogenic bacteria by using Mag-CDs. Since last decade, research groups [15] such as; Arruda et al., 2009[16]; Gao et al., 2009[17]; Lu et al., 2010[18]; Sanvicens et al., 2009[19], have been engaged in development of efficient and cost-effective techniques based on magnetic nanoparticles for pathogen detection. Label-free
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magnetic nanoparticle-antibody conjugates were prepared for E. coli [20, 21]. Detection of bacteria using carbohydrate-based fluorescent polymer [22-25], nanoparticle mediated [26-30], fluorescent detection [31, 32], colorimetric bacterial sensing [33] have also been proposed.
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Specifically, marine pathogen detected by vancomycin-coated magnetic nanoparticles was proposed by Wan et al, 2014 [34]. Further development in detection and identification of bacteria by quaternized magnetic nanoparticle-fluorescent polymer system [34] is also known. Usually, magnetic nanoparticles were engineered with conjugation, tagging, chemical agent, and antibody applications [35]. Chitosan coated iron nanoparticles were synthesized and applied as environmental trace pollutants extraction [36-38] and drug sensors [39]. In our currently
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synthesized method, no tagging or use of polymer is needed and it provides many advantages such as fluorescent and magnetic properties by the straightforward and cost-effective approach for highly preconcentration capability for capture and sensitive detection for pathogenic bacteria. Instead of applying amine functionalization on iron oxide nanoparticles for pathogenic bacterial
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removal [40], this easy and feasible method uses the readily accessible precursor chitosan [41]
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and synthesizes in situ.
Previous reports have described the combination of carbon coated iron oxide nanoparticles used in magnetic resonance imaging [8]. We tried to synthesize the Mag-CDs and applied for detection of pathogenic bacteria using advanced fluorescence and MALDI-MS techniques. We demonstrated the synthesis and use of amine functionalized Mag-CDs as highly sensitive affinity probes for bacterial detection for the first time, utilizing the magnetic property of Mag-CDs for collecting and amine functionalized carbon dots for extracting pathogenic bacteria. 2. Experimental Section 3
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2.1 Materials and method FeCl2·4H2O was obtained from Alfa Aesar, Johnson Matthey Company (USA). FeCl3·6H2O was from Showa Chemical Co. LTD (Japan). Ammonia solution (28%) was purchased from Fluka
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(Steinheim, Germany). Acetic acid purchased from Sigma-Aldrich (USA). Chitosan (low molecular weight; 50,000–190,000 g mol−1, 75–85% deacetylation) and Trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Germany). Sinapic acid (SA) was obtained from Alfa Aesar (Ward Hill, MA 01835, USA). Ethanol and acetonitrile (ACN) were obtained from J.
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T. Baker (Phillipsburg, NJ, USA). Ultrapure water from a Milli-Q Plus water purification system (18.2 MΩ·cm, Millipore, and Bedford, MA, USA) was used for all experiments. All reagents used in the preparation process were of analytical reagent grade and were used without further
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purification.
Bacterial Source and cultivation A Biosafety, level-1 cabinet (Nuaire, Plymouth, MN, USA) was used for all bacteria experiments. The standard bacterial strains Staphylococcus aureus (BCRC 10451); Escherichia coli (BCRC 12570) were purchased from the culture collection at Bio resource Collection and Research Center (BCRC), Hsin-Chu, Taiwan. Both bacteria were cultured on Luria-Bertani (LB.) agar plates (Bio Basic Inc. SD 7002 amended with 15 g L−1 agar)
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for 24 h incubation at 37°C. The bacteria cells were transferred from Luria-Bertani agar plate to 1 mL PBS buffer solution (pH = 7.2) and used for further analysis. Bacterial concentration in the suspension was estimated by the traditional plate counting method. All the glassware and media
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used for the studies were autoclaved at 15 lbs. (pressure) for 45 min. 2.2 Instrumental Utility
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The structure and morphology of as-synthesized amine functionalized Mag-CDs were characterized by X-ray diffraction (XRD; Phillip, The Netherlands) and transmission electron microscopy (TEM, Phillip CM200, Switzerland). The Fourier transform infrared (FT-IR) spectra of Mag-CDs and iron oxide nanoparticles were obtained on a FT-IR spectrometer (Spectrum 100, Perkin Elmer, USA). The Energy-dispersive X-ray spectrometer (EDX) (JOEL 6700F, Japan) was used for the elemental detection of Mag-CDs. The excitations as well as the emission spectra were recorded using the fluorescence spectroscope (Hitachi F2700, Japan). A UV–vis. spectroscope (Thermo, Evolution 201, USA) 4
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was used to study the spectral properties of Mag-CDs. MALDI-TOF-MS analyses were performed employing delayed extraction in positive ion mode on a time-of-flight mass spectrometer (Microflex, Bruker Daltonics, Bremen, Germany) with a 1.25 m flight tube. Desorption and ionization was obtained by using a 337 nm nitrogen laser with a 3 ns pulse width.
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Available accelerating potential is in range of +20/-20 kV. In our experiment the applied voltage was set at 20 mV. Laser power was adjusted to slightly above the threshold to obtain good resolution and signal-to-noise ratios.
All mass spectra were obtained using an average of 200 laser shots. All experiments were
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performed in the linear positive mode with high laser energy. The dried droplet method was used for all experiments using sinapic acid (50 mM, acetonitrile: water 50:50) as the matrix.
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2.3 Synthesis of Magnetic Nanoparticles (MNPs)
Iron oxide nanoparticles were synthesized by co-precipitation of ferrous and ferric ions. FeCl2·4H2O (0.63 g) and FeCl3·6H2O (1.73 g) were dispersed in 30 mL of deionized water, then 35 mL of ammonia solution (28%) was added dropwisely prior to precipitation. The reaction mixture was kept in a round bottle flask and maintained in the nitrogen reflux for 5 h at 80°C. The
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synthesized nanoparticles were collected by applying an external magnet and washed three times thoroughly by deionized water. The nanoparticles were filtered and dried under vacuum at 55°C for 12 h. The iron oxide nanoparticles were ready for further modified on the C-dots.
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2.4 Magnetic nanoparticles decorated with Carbon dots (Mag-CD NPs) The synthesis procedure for the Mag-CD nanoparticles is described as below: 20 mL of 4%
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acetic acid was mixed with 2 g of chitosan. 0.1 g of Fe3O4 NPs was added. The reaction mixture was put directly in the (stainless steel) autoclave and kept in a reaction vessel in lab oven for 12 h at 180°C. After that, the oven was switched off and the oven temperature was decreased gradually. The reaction vessel was taken out from the oven and waited for some time to room temperature for the vessel. The reaction mixture was collected using centrifugation and washed thoroughly using DI water. The final product was separated by an external magnet. This material was kept for drying in the hot air oven and waiting for further characterization and application. 2.5 Fluorescence detection of E. coli and S. aureus 5
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A fluorescence spectrometer was applied for the detection of two pathogenic bacteria (S. aureus and E. coli) which were used in this study. Different counts of bacterial suspension have been prepared in PBS and urine for sensitivity and linearity detection. The stock suspension was prepared by dissolving 2 mg of Mag-CDs in 1000 µL solution and was dispersed in it. Afterwards,
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10 µL was pippetted out and added in each aforementioned suspension separately in 1 mL of bacterial suspension. After 2 min of enrichment, all the samples were analyzed using the fluorescence spectroscopy. The fluorescence emission spectra were recorded, and the linearity
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curves were plotted and calculated for LODs and R2.
2.6 Bacterial detection using Mag-CDs and measured by MALDI-MS
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The above-demonstrated method was applied additionally for mass analysis to confirm the robustness of detection. We have tried the detection of E. coli and S. aureus by applying MagCDs in urine spiked bacteria samples. The different counts of both bacterial samples were prepared in PBS solution. In each bacterial suspension, nanoparticles were added and vortexed for 2 min. The precipitate formation of bacteria connected on nanoparticles was separated by applying an external magnet for 2 min and the remaining suspension became transparently clear.
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These bacterial samples were washed thoroughly and mixed with SA matrix and spotted on MALDI plate. The observed results were recorded and the identity of bacteria was confirmed with reference to control.
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3. Results and Discussion
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3.1 The fabrication of Mag-CDs
The amine functionalized Mag-CDs were developed for detection of pathogenic bacteria E. coli and S. aureus. The schematic illustration of entire experiment was depicted in Fig. 1. Carbon dots anchored on the surface of iron oxide nanoparticles were synthesized via hydrothermal method at high temperature and the fluorescent Mag-CDs were characterized by UV-Vis, FT-IR, TEM, and EDX. The as-synthesized Mag-CDs were mixed with pathogenic bacteria samples and an external magnet was used for the separation of the aggregates from the suspension. Fluorescence detection was carried out and MALDI-MS analysis was performed further to identify the characteristic peaks of bacteria. 6
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3.2 The characterization of Mag-CDs Fig. 2 (a) and (b) showed the TEM images of iron oxide nanoparticles and Mag-CDs. The TEM images demonstrated that the shape of both nanomaterials is spherical with increased size of diameter in Mag-CDs compared with iron oxide nanoparticles. The average hydrodynamic
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diameter of the Mag-CDs was around 25 nm. The uniformly covering of C-dots on magnetic nanoparticles can be clearly observed in inset of Fig. 2 (b), the C-dots were aggregated homogeneously on the surface of iron oxide nanoparticles. Since the shapes of Mag-CDs remain the same, it indicates that the layer of C-dots was successfully and uniformly coated onto the
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magnetic particles. The interaction between C-dots and magnetic nanoparticles can be attributed to the collective van der Waals force and electrostatic attractions. Due to the difference of
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functional groups and the causing bonding, magnetic nanoparticles and Mag-CDs are distinctive in features.
From the FT-IR spectra in Fig. 2 (c), various characteristic peaks originated from Fe3O4 nanoparticles were shown. In this particular spectrum, absorption at 3414, 1626, 1039 and 585 cm-1 corresponds to O-H stretching, N-H stretching and bending, C-H stretching and Fe-O bond vibration, respectively.
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To confirm the chemical composition of synthesized nanoparticles, FTIR spectra were obtained. The presence of Fe3O4 core could be identified by the strong stretching absorption band at 585 cm-1, which corresponded to the Fe–O bond (Fig. 2c). The peak present in the 585 cm-1 region is found in bare and chitosan-coated nanoparticle’s spectra, confirming that the products
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contained magnetite. The peaks around 1630 ± 10 cm-1, which can be assigned to the NH2 group bend scissoring and stretching, are present in both Fe3O4 nanoparticle and Mag-CDs nanoparticle’s spectra, proving that magnetite nanoparticles were successfully coated by carbon
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dots. One of the most significant peaks in 1108 cm-1 for Mag-CDs is the vibration of C-O-C, which confirms the attachment of carbon dots. All characteristic peaks of Iron oxide and carbon dots precursor from chitosan were present in the spectrum of Mag-CDs. Results indicated that the MNPs were successfully coated with carbon dots and these results are compatible with the articles in the literature (Li et al. 2008; Zhang et al. 2010a; Ma et al. 2007)[42-44]. The EDX spectrum of Mag-CDs was shown in Fig. 2 (d). The surface molar ratio of carbon and iron is 56.15% to 11.27%, respectively, which indicates that carbon materials are uniformly and 7
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successfully coated on magnetic nanoparticles. The peaks of carbon and iron in the spectra confirmed that the Mag-CDs were made up of both elements. Iron oxide nanoparticles and Mag-CDs showed obvious optical absorbance in the UV-Vis range (Fig. 3 (a)). The strong absorption of Mag-CDs which was observed at 290 nm as bell-
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shaped is absent in that of iron oxide nanoparticles. The characteristic absorption of Mag-CDs ranges from 200-400 nm and decreases to level off in visible range represents the presence of functional groups grafted on the nanomaterial. Thus, the Mag-CDs were used for further detection of bacteria using fluorescence spectroscopy excited at UV range for preferable results.
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The fluorescence spectra of Mag-CDs were recorded in Fig. 3 (b). The λex-dependent λem of Cdots which is also applicable to Mag-CDs has been proved confirmed previously [45]. The Mag-
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CDs were excited from 280 nm to 400 nm with increment of 20 nm. It was observed that the nanomaterial showed significantly high intensity of fluorescence at the end of UV and starting on visible wavelength range. The Mag-CDs exhibited red shift along with decrease in fluorescence intensity. The highest fluorescence intensity was observed when excited at 320 nm. Therefore, this wavelength was selected to perform further experiment and the corresponding emission patterns were observed. Another peculiar observation is that the fluorescence displayed in the
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range of 250 nm to 600 nm.
The crystal phase of iron oxide and Mag-CDs were further investigated by the XRD. As shown in Fig. 3 (c), both peak patterns are similar at respective degrees, which indicates that amorphous C-dots coating was uniformly performed without any alterations in crystal phase of iron oxide
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nanoparticles. This confirmed the synthesis of Mag-CDs was successfully conducted without changing the properties of iron oxide. The optical pictures demonstrate the fluorescent and
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magnetic properties of Mag-CDs. In Fig. 3 (d), the upper pictures demonstrate the Mag-CDs in normal light and the fluorescent feature under UV irradiation. The lower pictures demonstrate the magnetic property of this material with an external magnet. 3.3 The efficacy evaluation of Mag-CDs using Fluorescence spectrometric analysis The fluorescence study for detection of gram-positive and gram-negative bacteria, S. aureus and E. coli was described in Fig. 4. The fluorescence curves for S. aureus in PBS (Fig. 4 (a)) and in urine (4 (c)) were demonstrated as well as the corresponding linearity graphs were plotted respectively (Fig. 4 (b)) and (Fig. 4 (d)). The linear regression curves for S. aureus in PBS and in urine samples are y = −387.997 + 1.432x with R2 = 0.995 and y = 447.755 + 0.395x with R2 = 8
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0.992, respectively. All measurements were conducted with different counts of S. aureus with constant quantity of Mag-CDs. It has been observed in Fig. 4 (a) that as bacterial count increases the fluorescence intensity increases accordingly and gradually. This peculiar correlation between the numbers of bacteria with nanomaterial properties is particularly useful for detection. The
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optimal minimum amount of Mag-CDs was used to attach and collecting bacteria due to collective van der Waal force and electrostatic interaction, which provides a sensitive detection protocol. Based on the bacterial counts, the intensity of fluorescence will exhibit accordingly. The same phenomenon has been discovered by Nandi’s group for pathogenic bacteria deyection based
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carbon dots only [32]. The limit of detection of our approach for S. aureus is 3×102 cfu mL-1. Even at such low colony of bacteria, it can also be entrapped by Mag-CDs due to the electrostatic
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attraction. The bacterial count of 4.6×103 cfu mL-1 showed highest fluorescence intensity compared to other counts. And the significant fluorescence intensity increased with bacterial count showed a trend of parallel response. Based on the fluorescence spectra, the linearity graph was plotted (Fig. 4 (b)) to show the phenomenon of fluorescence results. This spectrum was plotted as fluorescence intensity against bacterial count, herein the linear line showed a perfect linear response with R2 = 0.99. Furthermore, we have tried our Mag-CDs for the detection of S.
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aureus in urine matrix (Fig. 4 (c)). The different count of bacteria spiked in equal amount of urine. The fluorescence measurement of urine has been performed to subtract its own background fluorescence. As the increase in count of bacteria, fluorescence intensity increased and was observed in the wavelength range of 350-560 nm. It is obvious that S. aureus has been
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successfully detected by Mag-CDs. The efficiency of nanomaterial in urine is almost the same as in PBS. The minimum count of S. aureus detected were 3×102 cfu mL-1 with significant
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fluorescence intensity. Similarly, the dependency study was carried out by plotting of linearity graph for the fluorescence detection of S. aureus (Fig. 4 (d)). The results showed the linear graph of fluorescence intensity against bacterial count with R2 = 0.99 over the large range from 102-103 cfu mL-1. In case of urine as the matrix for pathogenic bacteria detection, the nanomaterial usually causes interference during measurement. Therefore, the blank urine sample was detected. From this, we observed that it has fluorescent moieties which affect detection. LOD and LOQ were calculated as 425 and 1287 cfu mL-1 respectively, and those for the urine samples were estimated as 171 and 518 cfu mL-1 respectively. It is worth to note that a recovery study showed a matrix effect value of 110, an enhancement of matrix to the emission spectra. 9
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Likewise, Mag-CDs were applied for detection of E. coli in PBS (Fig. 4 (e), (f)) and urine (Fig. 4(g), (h)) as well. Firstly, fluorescence intensity was measured with different counts of E. coli at excitation wavelength of 320 nm. We have found that increasing the count of E. coli the corresponding fluorescence intensity increased gradually as shown in Fig. 4 (e). The minimum
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bacterial count has been measured as low as 4×102 cfu mL-1. The corresponding linearity graph was provided in Fig. 4 (f), where the bacterial count exhibits excellent linearity with fluorescence intensity where y = −651.85 + 1.996x and it is confirmed by R2 = 0.98. Almost all the counts of E. coli showed relatively linear pattern with fluorescence. Furthermore, we have spiked E. coli in
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urine and detected by Mag-CDs. The amine groups on the surface of carbon dots electrostatically attract toward the overall negatively surface charged of E. coli. In Fig. 4 (g), it showed
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fluorescence response toward different count of E. coli. The increase in fluorescence intensity with bacteria count was observed in the case of urine as well. For the same graph we have plotted linearity curve, which implies that the linear response between fluorescence intensity with bacterial count by y = 809.114 + 0.262x with the coefficient of determination R2 = 0.96. In the real sample analysis, generally the deviation resulted from matrix effect in biological fluids. LOD and LOQ were calculated as 352 and 1067 cfu mL-1, respectively, and those for the urine samples
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were estimated at 586 and 1776 cfu mL-1 respectively.
3.4 The MALDI-MS analysis of S. aureus and E. coli by Mag-CDs Even for such sensitive instrument as fluorescence spectroscopy, the identification of peaks were conducted by MALDI-MS for additional support to the fluorescence results. The bacterial
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counts for E. coli and S. aureus were in the range of 104 to 105 cfu mL-1. The MADLI-MS spectra were depicted in Fig. 5 (a) and Fig. 5 (b) for S. aureus and E. coli, respectively. The spectra were
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composed of bacterial control which is for the bacterial identification, urine, and different colony numbers of bacterial cells extracted from urine samples. The different macromolecules present in cell walls of gram-positive and gram-negative were distinctly characterized. In Fig. 5 (a), the masses observed at m/z 3893, 5068.9, 5568.2, 6059.9, 6954.8, 8237.7, 8989.0 were characteristic signals for S. aureus. The background spectra of urine had also been collected, no specific signal was found in the particular range of masses. Afterward, the spectrum for the bacterial count (3× 102 cfu mL-1) has been detected; in this spectrum, we have observed the characteristic peaks at m/z 5727.1, 6062.4, 6679.2, 8240.2, and 8994.9 which are similar to the control. The presence of these peaks implies that even with the small amount the Mag-CDs can easily detect bacteria by 10
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using MALDI-MS. Subsequently, we have collected mass spectra for 3×102, 1.1×103, 1.7×103 and 2.3×103 cfu mL-1. All these mass spectra showed the similar peaks as in the control with high intensity and unambiguousness. In addition, it demonstrated the increase intensity with increase in colony count of S. aureus. Furthermore, we have accomplished the separation and detection of E.
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coli in urine using Mag-CDs (Fig. 5 (b)). Herein, E. coli control, urine control, and increasing count of E. coli in urine were detected. The characteristic peaks viz. m/z 5494.2, 6911.4, 7335.5, 7777.6, 8935.6, 9212.7, 9553.4, and 10576.0 were observed in the spectrum of the E. coli control. These peaks assigned also 3.5×102 cfu mL-1, 1.1×103 cfu mL-1, 1.55×103 cfu mL-1, 2.9×103 cfu
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mL-1 different count of E. coli spiked in urine. These results confirmed that even at 102 cfu mL-1 the observed peaks increases in intensity with increase in count of bacteria. The separation of
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bacteria from urine matrix has been successfully demonstrated by Mag-CDs. The peaks were distinctly clear, separated and high intensity similar to those of bacterial control in mass spectrum. These results confirmed that Mag-CDs are effective of bacteria enrichment because of the functionalization of amine group over carbon dots decorated on magnetic nanoparticles. The electrostatic attraction of amine group and overall surface negative charge on pathogenic bacteria attracts toward each other and hence being sufficiently enriched. The poly-cationic functional
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groups from chitosan precursor C-dots were significantly interacted with the overall negative charge on the surface of bacteria due to electrostatic interactions, hydrophobic interaction, and hydrogen bonding. Although, the zeta potential of chitosan or C-dots were in positive values, which easily attracts toward bacteria surface negative charge. During MALDI-MS analysis the
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entrapment of bacteria showed in high amount and the signal was enhanced by using SA matrix.
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These results completely agree with the fluorescence spectroscopic results.
4. Conclusions
A new nanomaterial with fluorescent and magnetic properties for the detection of pathogenic bacteria was proposed in this work. The amine functionalized Mag-CDs have demonstrated outstanding features of sensing and enrichment. The Mag-CDs were characterized by UV-vis, FT-IR spectra, TEM images, XRD and EDX. UV-vis, EDX and FT-IR measurements confirmed the grafting of amine group on the nanomaterial. Fluorescence spectroscopy and MALDI-MS were used for the detection and identification of bacterial strains S. aureus and E. coli in PBS 11
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and urine matrix, respectively. Parallel response between fluorescence and bacterial count was found with linear relationship. The limit of detection for S. aureus and E. coli was found to be 3×102 and 3.5×102 cfu mL-1, respectively. The current approach is a powerful tool for future detection of pathogenic bacteria by fluorescence method in the clinical microbiology and
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biomedicine field.
Acknowledgements
The authors acknowledge the financial support from the ministry of science and technology of
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Taiwan.
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Harruff, X. Wang, H. Wang, Journal of the American Chemical Society, 128 (2006) 7756.
Figures and captions
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Fig. 1 Schematic representation of experimental concepts. Fig. 2 Characterization I: TEM images of (a) Iron oxide nanoparticles and (b) Mag-CDs. (c) FTIR spectra of Iron oxide and Mag-CDs, and (d) EDX of Mag-CDs. Fig. 3 Characterization II: (a) UV-vis spectra of Mag-CDs and Iron oxide nanoparticles. (b) Emission spectra of the Mag-CDs at different excitation wavelength as indicated. (c) The XRD patterns of the Iron oxide nanoparticles and Mag-CDs. (d) Optical pictures demonstrate the fluorescent and magnetic properties of Mag-CDs. Concentration of Mag-CDs was 20 µg 14
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Fig. 4 (a) Photoluminescence spectra of S. aureus with different concentrations and (b) the calibration curve of the pathogenic bacteria. (c) Photoluminescence spectra of S. aureus with different concentrations in urine sample and (d) the calibration curve of the pathogenic bacteria. (e) Photoluminescence spectra of E. coli with different concentrations and (f) the calibration
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curve of the pathogenic bacteria. (g) Photoluminescence spectra of E. coli with different concentrations in urine sample and (h) the calibration curve of the pathogenic bacteria Concentration of Mag-CDs was 20 µg.
Fig. 5 MALDI-MS spectra of (a) S. aureus, including bacteria control, urine control, 3×102 cfu
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mL-1, 1.1×103 cfu mL-1, 1.7×103 cfu mL-1, and 2.3×103 cfu mL-1 (b ) E. coli, including bacteria control, urine control, 3.5×103 cfu mL-1, 1.1×103 cfu mL-1, 1.5×103 cfu mL-1, and 2.9×103 cfu
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mL-1.
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Figure captions
Fig. 1 Schematic representation of experimental concepts.
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Fig. 2 Characterization I: TEM images of (a) Iron oxide nanoparticles and (b) Mag-CDs. (c) FTIR spectra of Iron oxide and Magnetic Cdots, and (d) EDX of Mag-CDs.
Fig. 3 Characterization II: (a) UV-vis spectra of Mag-CDs and Iron oxide nanoparticles. (b) Emission spectra of the Mag-CDs at different
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excitation wavelength as indicated. (c) The XRD patterns of the Iron oxide nanoparticles and Mag-CDs. (d) Optical pictures demonstrate the fluorescent and magnetic properties of Mag-CDs. The concentration of Mag-CDs was 20 µg.
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Fig. 4 (a) Photoluminescence spectra of S. aureus with different concentrations and (b) the calibration curve of the pathogenic bacteria. (c) Photoluminescence spectra of S. aureus with different concentrations in urine sample and (d) the calibration curve of the pathogenic bacteria. (e) Photoluminescence spectra of E. coli with different concentrations and (f) the calibration curve of the pathogenic bacteria. (g)
The concentration of Mag-CDs was 20 µg.
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Photoluminescence spectra of E. coli with different concentrations in urine sample and (h) the calibration curve of the pathogenic bacteria.
Fig. 5 MALDI-MS spectra of (a) S. aureus, including bacteria control, urine control, 3×102 cfu mL-1, 1.1×103 cfu mL-1, 1.7×103 cfu mL-1,
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2.9×103 cfu mL-1.
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and 2.3×103 cfu mL-1 (b ) E. coli, including bacteria control, urine control, 3.5×103 cfu mL-1 , 1.1×103 cfu mL-1, 1.5×103 cfu mL-1, and
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Element
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(a) S. aureus
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(b) E. coli
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3. Achieved very low limit of detection of pathogenic bacteria using this technique.
S0003-2670(16)30226-4
DOI:
10.1016/j.aca.2016.02.025
Reference:
ACA 234428
To appear in:
Analytica Chimica Acta
Received Date: 16 September 2015 Revised Date:
25 January 2016
Accepted Date: 18 February 2016
Please cite this article as: M.L. Bhaisare, G. Gedda, M.S. Khan, H.-F. Wu, Fluorimetric detection of pathogenic bacteria using Magnetic carbon dots, Analytica Chimica Acta (2016), doi: 10.1016/ j.aca.2016.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fluorimetric detection of pathogenic bacteria using Magnetic carbon dots Mukesh Lavkush Bhaisare1, Gangaraju Gedda4, M Shahnawaz Khan4, Hui-Fen Wu*, 1, 2, 3, 4
Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-
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1
Sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan 2
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan
Institute of Medical Science and Technology, National Sun Yat-Sen University, 80424, Taiwan 4
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Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and
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Academia Sinica, Kaohsiung, 80424, Taiwan
*Corresponding author, Phone: +886-7-5252000 ext. 3955; Fax: +886-7-5253908 E-mail: [email protected] (Prof. H F Wu)
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Abstract
A novel and facile approach of pathogenic bacteria detection, which utilizes fluorescent sensing and bacteria capture with Magnetic carbon dots (Mag-CDs), was proposed in this work. Magnetic nanoparticles were synthesized and then decorated with C-dots, and further
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functionalized with amine groups (chitosan). In this way, bacteria were strongly anchored on the hybrid material Mag-CDs for highly sensitive fluorescent detection. The Mag-CDs were characterized by UV-vis, FT-IR spectra, TEM images, XRD, and EDX. The characterizations
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validate the fabrication of amine-Mag-CDs and the promising applications of this material. Fluorescence spectroscope and MALDI-MS were used for the detection and identification of bacterial strains, respectively. The limit of detection for S. aureus and E. coli was found to be 3×102 and 3.5×102 cfu mL-1, respectively. With these encouraging results, it is expected that it would open revenues for promising applications of Mag-CDs nanomaterial. Keywords: Magnetic carbon dots, Staphylococcus aureus, Escherichia coli, fluorescence detection, MALDI-MS. 1
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1. Introduction Pathogenic bacterial detection and quantification have been pursued in the long term and are extremely important in biological, food related items, and environmental samples [1]. Especially
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E. coli and S. aureus are opportunistic bacterial species leading to human infections; they are majorly found in urinary tract infections. The unique nano-based magnetic nanoparticles applied on bio-sensing methodology [2, 3] prompts us to apply them extendedly. In recent years, carbon nanomaterials have received considerable attention primarily because of their unique properties.
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Among various carbon nanomaterials like graphene sheet, graphene oxide, and carbon nanotube, carbon dots have attracted attention nowadays[4]. Carbon dots were accidentally discovered, which have unique properties such as, strong photoluminescence, wavelength-dependent
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emission, relative stability, attractive prospects for bioimaging, high photostability, tunable excitation and emission wavelength, exceptional biocompatibility and eco-friendliness[5-7], etc. Even presented in the combined form C-dots also have variety of potential applications with CaO, ZnO, TiO2, and Al2O3 [8], and so forth. Recently, much research interest has been extended to hybrid nanomaterials in biology, physics, engineering and chemistry fields. Carbon-coated nanomaterials have been extensively used for magnetic resonance imaging study, and carbon
[9, 10].
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dots can also be passivated with amine terminated compounds through amine functionalization
The hybrid nanomaterials of carbon dots coated magnetic nanoparticles combine both
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fluorescent and magnetic properties in one material. The particular functionalization of amine group is originated or sourced from precursor chitosan[11]. One of the advantages of carbon dots
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lie behind its feature of providing better dispersibility, stability to iron oxide nanoparticles compare to bare iron oxide nanoparticles. Additionally, C-dots coated iron oxide nanoparticles are prone to prevent the oxidation and corrosion of iron oxide nanoparticles [8]. Fe3O4/carbon synthesis was first proposed by Zhifei research group [12], the important application of iron oxide is iron oxide/gold nanoparticles, which is particularly of interest to the controlled magnetic field [13]. In general, the synthesis of carbon-coated materials have been proposed including electric arc discharge, catalytic pyrolysis of organic compounds, and the hydrothermal method [14]. Carbon/Fe3O4 nanoparticles have been specifically applied as sorbents in solid-phase extraction (SPE) from environmental water samples, because the carbon dots have been proved 2
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for high capacity adsorption and extraction efficiency due to large surface area to small volume ratios [14]. To date, carbon dots decorated on magnetic nanoparticles has not yet been studied. Furthermore, the fluorescent property of carbon dots and the amine-functionalized nature make
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them applicable for trapping or as probes for pathogenic bacteria by using Mag-CDs. Since last decade, research groups [15] such as; Arruda et al., 2009[16]; Gao et al., 2009[17]; Lu et al., 2010[18]; Sanvicens et al., 2009[19], have been engaged in development of efficient and cost-effective techniques based on magnetic nanoparticles for pathogen detection. Label-free
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magnetic nanoparticle-antibody conjugates were prepared for E. coli [20, 21]. Detection of bacteria using carbohydrate-based fluorescent polymer [22-25], nanoparticle mediated [26-30], fluorescent detection [31, 32], colorimetric bacterial sensing [33] have also been proposed.
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Specifically, marine pathogen detected by vancomycin-coated magnetic nanoparticles was proposed by Wan et al, 2014 [34]. Further development in detection and identification of bacteria by quaternized magnetic nanoparticle-fluorescent polymer system [34] is also known. Usually, magnetic nanoparticles were engineered with conjugation, tagging, chemical agent, and antibody applications [35]. Chitosan coated iron nanoparticles were synthesized and applied as environmental trace pollutants extraction [36-38] and drug sensors [39]. In our currently
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synthesized method, no tagging or use of polymer is needed and it provides many advantages such as fluorescent and magnetic properties by the straightforward and cost-effective approach for highly preconcentration capability for capture and sensitive detection for pathogenic bacteria. Instead of applying amine functionalization on iron oxide nanoparticles for pathogenic bacterial
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removal [40], this easy and feasible method uses the readily accessible precursor chitosan [41]
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and synthesizes in situ.
Previous reports have described the combination of carbon coated iron oxide nanoparticles used in magnetic resonance imaging [8]. We tried to synthesize the Mag-CDs and applied for detection of pathogenic bacteria using advanced fluorescence and MALDI-MS techniques. We demonstrated the synthesis and use of amine functionalized Mag-CDs as highly sensitive affinity probes for bacterial detection for the first time, utilizing the magnetic property of Mag-CDs for collecting and amine functionalized carbon dots for extracting pathogenic bacteria. 2. Experimental Section 3
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2.1 Materials and method FeCl2·4H2O was obtained from Alfa Aesar, Johnson Matthey Company (USA). FeCl3·6H2O was from Showa Chemical Co. LTD (Japan). Ammonia solution (28%) was purchased from Fluka
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(Steinheim, Germany). Acetic acid purchased from Sigma-Aldrich (USA). Chitosan (low molecular weight; 50,000–190,000 g mol−1, 75–85% deacetylation) and Trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Germany). Sinapic acid (SA) was obtained from Alfa Aesar (Ward Hill, MA 01835, USA). Ethanol and acetonitrile (ACN) were obtained from J.
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T. Baker (Phillipsburg, NJ, USA). Ultrapure water from a Milli-Q Plus water purification system (18.2 MΩ·cm, Millipore, and Bedford, MA, USA) was used for all experiments. All reagents used in the preparation process were of analytical reagent grade and were used without further
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purification.
Bacterial Source and cultivation A Biosafety, level-1 cabinet (Nuaire, Plymouth, MN, USA) was used for all bacteria experiments. The standard bacterial strains Staphylococcus aureus (BCRC 10451); Escherichia coli (BCRC 12570) were purchased from the culture collection at Bio resource Collection and Research Center (BCRC), Hsin-Chu, Taiwan. Both bacteria were cultured on Luria-Bertani (LB.) agar plates (Bio Basic Inc. SD 7002 amended with 15 g L−1 agar)
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for 24 h incubation at 37°C. The bacteria cells were transferred from Luria-Bertani agar plate to 1 mL PBS buffer solution (pH = 7.2) and used for further analysis. Bacterial concentration in the suspension was estimated by the traditional plate counting method. All the glassware and media
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used for the studies were autoclaved at 15 lbs. (pressure) for 45 min. 2.2 Instrumental Utility
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The structure and morphology of as-synthesized amine functionalized Mag-CDs were characterized by X-ray diffraction (XRD; Phillip, The Netherlands) and transmission electron microscopy (TEM, Phillip CM200, Switzerland). The Fourier transform infrared (FT-IR) spectra of Mag-CDs and iron oxide nanoparticles were obtained on a FT-IR spectrometer (Spectrum 100, Perkin Elmer, USA). The Energy-dispersive X-ray spectrometer (EDX) (JOEL 6700F, Japan) was used for the elemental detection of Mag-CDs. The excitations as well as the emission spectra were recorded using the fluorescence spectroscope (Hitachi F2700, Japan). A UV–vis. spectroscope (Thermo, Evolution 201, USA) 4
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was used to study the spectral properties of Mag-CDs. MALDI-TOF-MS analyses were performed employing delayed extraction in positive ion mode on a time-of-flight mass spectrometer (Microflex, Bruker Daltonics, Bremen, Germany) with a 1.25 m flight tube. Desorption and ionization was obtained by using a 337 nm nitrogen laser with a 3 ns pulse width.
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Available accelerating potential is in range of +20/-20 kV. In our experiment the applied voltage was set at 20 mV. Laser power was adjusted to slightly above the threshold to obtain good resolution and signal-to-noise ratios.
All mass spectra were obtained using an average of 200 laser shots. All experiments were
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performed in the linear positive mode with high laser energy. The dried droplet method was used for all experiments using sinapic acid (50 mM, acetonitrile: water 50:50) as the matrix.
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2.3 Synthesis of Magnetic Nanoparticles (MNPs)
Iron oxide nanoparticles were synthesized by co-precipitation of ferrous and ferric ions. FeCl2·4H2O (0.63 g) and FeCl3·6H2O (1.73 g) were dispersed in 30 mL of deionized water, then 35 mL of ammonia solution (28%) was added dropwisely prior to precipitation. The reaction mixture was kept in a round bottle flask and maintained in the nitrogen reflux for 5 h at 80°C. The
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synthesized nanoparticles were collected by applying an external magnet and washed three times thoroughly by deionized water. The nanoparticles were filtered and dried under vacuum at 55°C for 12 h. The iron oxide nanoparticles were ready for further modified on the C-dots.
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2.4 Magnetic nanoparticles decorated with Carbon dots (Mag-CD NPs) The synthesis procedure for the Mag-CD nanoparticles is described as below: 20 mL of 4%
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acetic acid was mixed with 2 g of chitosan. 0.1 g of Fe3O4 NPs was added. The reaction mixture was put directly in the (stainless steel) autoclave and kept in a reaction vessel in lab oven for 12 h at 180°C. After that, the oven was switched off and the oven temperature was decreased gradually. The reaction vessel was taken out from the oven and waited for some time to room temperature for the vessel. The reaction mixture was collected using centrifugation and washed thoroughly using DI water. The final product was separated by an external magnet. This material was kept for drying in the hot air oven and waiting for further characterization and application. 2.5 Fluorescence detection of E. coli and S. aureus 5
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A fluorescence spectrometer was applied for the detection of two pathogenic bacteria (S. aureus and E. coli) which were used in this study. Different counts of bacterial suspension have been prepared in PBS and urine for sensitivity and linearity detection. The stock suspension was prepared by dissolving 2 mg of Mag-CDs in 1000 µL solution and was dispersed in it. Afterwards,
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10 µL was pippetted out and added in each aforementioned suspension separately in 1 mL of bacterial suspension. After 2 min of enrichment, all the samples were analyzed using the fluorescence spectroscopy. The fluorescence emission spectra were recorded, and the linearity
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curves were plotted and calculated for LODs and R2.
2.6 Bacterial detection using Mag-CDs and measured by MALDI-MS
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The above-demonstrated method was applied additionally for mass analysis to confirm the robustness of detection. We have tried the detection of E. coli and S. aureus by applying MagCDs in urine spiked bacteria samples. The different counts of both bacterial samples were prepared in PBS solution. In each bacterial suspension, nanoparticles were added and vortexed for 2 min. The precipitate formation of bacteria connected on nanoparticles was separated by applying an external magnet for 2 min and the remaining suspension became transparently clear.
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These bacterial samples were washed thoroughly and mixed with SA matrix and spotted on MALDI plate. The observed results were recorded and the identity of bacteria was confirmed with reference to control.
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3. Results and Discussion
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3.1 The fabrication of Mag-CDs
The amine functionalized Mag-CDs were developed for detection of pathogenic bacteria E. coli and S. aureus. The schematic illustration of entire experiment was depicted in Fig. 1. Carbon dots anchored on the surface of iron oxide nanoparticles were synthesized via hydrothermal method at high temperature and the fluorescent Mag-CDs were characterized by UV-Vis, FT-IR, TEM, and EDX. The as-synthesized Mag-CDs were mixed with pathogenic bacteria samples and an external magnet was used for the separation of the aggregates from the suspension. Fluorescence detection was carried out and MALDI-MS analysis was performed further to identify the characteristic peaks of bacteria. 6
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3.2 The characterization of Mag-CDs Fig. 2 (a) and (b) showed the TEM images of iron oxide nanoparticles and Mag-CDs. The TEM images demonstrated that the shape of both nanomaterials is spherical with increased size of diameter in Mag-CDs compared with iron oxide nanoparticles. The average hydrodynamic
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diameter of the Mag-CDs was around 25 nm. The uniformly covering of C-dots on magnetic nanoparticles can be clearly observed in inset of Fig. 2 (b), the C-dots were aggregated homogeneously on the surface of iron oxide nanoparticles. Since the shapes of Mag-CDs remain the same, it indicates that the layer of C-dots was successfully and uniformly coated onto the
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magnetic particles. The interaction between C-dots and magnetic nanoparticles can be attributed to the collective van der Waals force and electrostatic attractions. Due to the difference of
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functional groups and the causing bonding, magnetic nanoparticles and Mag-CDs are distinctive in features.
From the FT-IR spectra in Fig. 2 (c), various characteristic peaks originated from Fe3O4 nanoparticles were shown. In this particular spectrum, absorption at 3414, 1626, 1039 and 585 cm-1 corresponds to O-H stretching, N-H stretching and bending, C-H stretching and Fe-O bond vibration, respectively.
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To confirm the chemical composition of synthesized nanoparticles, FTIR spectra were obtained. The presence of Fe3O4 core could be identified by the strong stretching absorption band at 585 cm-1, which corresponded to the Fe–O bond (Fig. 2c). The peak present in the 585 cm-1 region is found in bare and chitosan-coated nanoparticle’s spectra, confirming that the products
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contained magnetite. The peaks around 1630 ± 10 cm-1, which can be assigned to the NH2 group bend scissoring and stretching, are present in both Fe3O4 nanoparticle and Mag-CDs nanoparticle’s spectra, proving that magnetite nanoparticles were successfully coated by carbon
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dots. One of the most significant peaks in 1108 cm-1 for Mag-CDs is the vibration of C-O-C, which confirms the attachment of carbon dots. All characteristic peaks of Iron oxide and carbon dots precursor from chitosan were present in the spectrum of Mag-CDs. Results indicated that the MNPs were successfully coated with carbon dots and these results are compatible with the articles in the literature (Li et al. 2008; Zhang et al. 2010a; Ma et al. 2007)[42-44]. The EDX spectrum of Mag-CDs was shown in Fig. 2 (d). The surface molar ratio of carbon and iron is 56.15% to 11.27%, respectively, which indicates that carbon materials are uniformly and 7
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successfully coated on magnetic nanoparticles. The peaks of carbon and iron in the spectra confirmed that the Mag-CDs were made up of both elements. Iron oxide nanoparticles and Mag-CDs showed obvious optical absorbance in the UV-Vis range (Fig. 3 (a)). The strong absorption of Mag-CDs which was observed at 290 nm as bell-
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shaped is absent in that of iron oxide nanoparticles. The characteristic absorption of Mag-CDs ranges from 200-400 nm and decreases to level off in visible range represents the presence of functional groups grafted on the nanomaterial. Thus, the Mag-CDs were used for further detection of bacteria using fluorescence spectroscopy excited at UV range for preferable results.
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The fluorescence spectra of Mag-CDs were recorded in Fig. 3 (b). The λex-dependent λem of Cdots which is also applicable to Mag-CDs has been proved confirmed previously [45]. The Mag-
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CDs were excited from 280 nm to 400 nm with increment of 20 nm. It was observed that the nanomaterial showed significantly high intensity of fluorescence at the end of UV and starting on visible wavelength range. The Mag-CDs exhibited red shift along with decrease in fluorescence intensity. The highest fluorescence intensity was observed when excited at 320 nm. Therefore, this wavelength was selected to perform further experiment and the corresponding emission patterns were observed. Another peculiar observation is that the fluorescence displayed in the
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range of 250 nm to 600 nm.
The crystal phase of iron oxide and Mag-CDs were further investigated by the XRD. As shown in Fig. 3 (c), both peak patterns are similar at respective degrees, which indicates that amorphous C-dots coating was uniformly performed without any alterations in crystal phase of iron oxide
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nanoparticles. This confirmed the synthesis of Mag-CDs was successfully conducted without changing the properties of iron oxide. The optical pictures demonstrate the fluorescent and
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magnetic properties of Mag-CDs. In Fig. 3 (d), the upper pictures demonstrate the Mag-CDs in normal light and the fluorescent feature under UV irradiation. The lower pictures demonstrate the magnetic property of this material with an external magnet. 3.3 The efficacy evaluation of Mag-CDs using Fluorescence spectrometric analysis The fluorescence study for detection of gram-positive and gram-negative bacteria, S. aureus and E. coli was described in Fig. 4. The fluorescence curves for S. aureus in PBS (Fig. 4 (a)) and in urine (4 (c)) were demonstrated as well as the corresponding linearity graphs were plotted respectively (Fig. 4 (b)) and (Fig. 4 (d)). The linear regression curves for S. aureus in PBS and in urine samples are y = −387.997 + 1.432x with R2 = 0.995 and y = 447.755 + 0.395x with R2 = 8
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0.992, respectively. All measurements were conducted with different counts of S. aureus with constant quantity of Mag-CDs. It has been observed in Fig. 4 (a) that as bacterial count increases the fluorescence intensity increases accordingly and gradually. This peculiar correlation between the numbers of bacteria with nanomaterial properties is particularly useful for detection. The
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optimal minimum amount of Mag-CDs was used to attach and collecting bacteria due to collective van der Waal force and electrostatic interaction, which provides a sensitive detection protocol. Based on the bacterial counts, the intensity of fluorescence will exhibit accordingly. The same phenomenon has been discovered by Nandi’s group for pathogenic bacteria deyection based
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carbon dots only [32]. The limit of detection of our approach for S. aureus is 3×102 cfu mL-1. Even at such low colony of bacteria, it can also be entrapped by Mag-CDs due to the electrostatic
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attraction. The bacterial count of 4.6×103 cfu mL-1 showed highest fluorescence intensity compared to other counts. And the significant fluorescence intensity increased with bacterial count showed a trend of parallel response. Based on the fluorescence spectra, the linearity graph was plotted (Fig. 4 (b)) to show the phenomenon of fluorescence results. This spectrum was plotted as fluorescence intensity against bacterial count, herein the linear line showed a perfect linear response with R2 = 0.99. Furthermore, we have tried our Mag-CDs for the detection of S.
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aureus in urine matrix (Fig. 4 (c)). The different count of bacteria spiked in equal amount of urine. The fluorescence measurement of urine has been performed to subtract its own background fluorescence. As the increase in count of bacteria, fluorescence intensity increased and was observed in the wavelength range of 350-560 nm. It is obvious that S. aureus has been
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successfully detected by Mag-CDs. The efficiency of nanomaterial in urine is almost the same as in PBS. The minimum count of S. aureus detected were 3×102 cfu mL-1 with significant
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fluorescence intensity. Similarly, the dependency study was carried out by plotting of linearity graph for the fluorescence detection of S. aureus (Fig. 4 (d)). The results showed the linear graph of fluorescence intensity against bacterial count with R2 = 0.99 over the large range from 102-103 cfu mL-1. In case of urine as the matrix for pathogenic bacteria detection, the nanomaterial usually causes interference during measurement. Therefore, the blank urine sample was detected. From this, we observed that it has fluorescent moieties which affect detection. LOD and LOQ were calculated as 425 and 1287 cfu mL-1 respectively, and those for the urine samples were estimated as 171 and 518 cfu mL-1 respectively. It is worth to note that a recovery study showed a matrix effect value of 110, an enhancement of matrix to the emission spectra. 9
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Likewise, Mag-CDs were applied for detection of E. coli in PBS (Fig. 4 (e), (f)) and urine (Fig. 4(g), (h)) as well. Firstly, fluorescence intensity was measured with different counts of E. coli at excitation wavelength of 320 nm. We have found that increasing the count of E. coli the corresponding fluorescence intensity increased gradually as shown in Fig. 4 (e). The minimum
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bacterial count has been measured as low as 4×102 cfu mL-1. The corresponding linearity graph was provided in Fig. 4 (f), where the bacterial count exhibits excellent linearity with fluorescence intensity where y = −651.85 + 1.996x and it is confirmed by R2 = 0.98. Almost all the counts of E. coli showed relatively linear pattern with fluorescence. Furthermore, we have spiked E. coli in
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urine and detected by Mag-CDs. The amine groups on the surface of carbon dots electrostatically attract toward the overall negatively surface charged of E. coli. In Fig. 4 (g), it showed
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fluorescence response toward different count of E. coli. The increase in fluorescence intensity with bacteria count was observed in the case of urine as well. For the same graph we have plotted linearity curve, which implies that the linear response between fluorescence intensity with bacterial count by y = 809.114 + 0.262x with the coefficient of determination R2 = 0.96. In the real sample analysis, generally the deviation resulted from matrix effect in biological fluids. LOD and LOQ were calculated as 352 and 1067 cfu mL-1, respectively, and those for the urine samples
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were estimated at 586 and 1776 cfu mL-1 respectively.
3.4 The MALDI-MS analysis of S. aureus and E. coli by Mag-CDs Even for such sensitive instrument as fluorescence spectroscopy, the identification of peaks were conducted by MALDI-MS for additional support to the fluorescence results. The bacterial
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counts for E. coli and S. aureus were in the range of 104 to 105 cfu mL-1. The MADLI-MS spectra were depicted in Fig. 5 (a) and Fig. 5 (b) for S. aureus and E. coli, respectively. The spectra were
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composed of bacterial control which is for the bacterial identification, urine, and different colony numbers of bacterial cells extracted from urine samples. The different macromolecules present in cell walls of gram-positive and gram-negative were distinctly characterized. In Fig. 5 (a), the masses observed at m/z 3893, 5068.9, 5568.2, 6059.9, 6954.8, 8237.7, 8989.0 were characteristic signals for S. aureus. The background spectra of urine had also been collected, no specific signal was found in the particular range of masses. Afterward, the spectrum for the bacterial count (3× 102 cfu mL-1) has been detected; in this spectrum, we have observed the characteristic peaks at m/z 5727.1, 6062.4, 6679.2, 8240.2, and 8994.9 which are similar to the control. The presence of these peaks implies that even with the small amount the Mag-CDs can easily detect bacteria by 10
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using MALDI-MS. Subsequently, we have collected mass spectra for 3×102, 1.1×103, 1.7×103 and 2.3×103 cfu mL-1. All these mass spectra showed the similar peaks as in the control with high intensity and unambiguousness. In addition, it demonstrated the increase intensity with increase in colony count of S. aureus. Furthermore, we have accomplished the separation and detection of E.
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coli in urine using Mag-CDs (Fig. 5 (b)). Herein, E. coli control, urine control, and increasing count of E. coli in urine were detected. The characteristic peaks viz. m/z 5494.2, 6911.4, 7335.5, 7777.6, 8935.6, 9212.7, 9553.4, and 10576.0 were observed in the spectrum of the E. coli control. These peaks assigned also 3.5×102 cfu mL-1, 1.1×103 cfu mL-1, 1.55×103 cfu mL-1, 2.9×103 cfu
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mL-1 different count of E. coli spiked in urine. These results confirmed that even at 102 cfu mL-1 the observed peaks increases in intensity with increase in count of bacteria. The separation of
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bacteria from urine matrix has been successfully demonstrated by Mag-CDs. The peaks were distinctly clear, separated and high intensity similar to those of bacterial control in mass spectrum. These results confirmed that Mag-CDs are effective of bacteria enrichment because of the functionalization of amine group over carbon dots decorated on magnetic nanoparticles. The electrostatic attraction of amine group and overall surface negative charge on pathogenic bacteria attracts toward each other and hence being sufficiently enriched. The poly-cationic functional
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groups from chitosan precursor C-dots were significantly interacted with the overall negative charge on the surface of bacteria due to electrostatic interactions, hydrophobic interaction, and hydrogen bonding. Although, the zeta potential of chitosan or C-dots were in positive values, which easily attracts toward bacteria surface negative charge. During MALDI-MS analysis the
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entrapment of bacteria showed in high amount and the signal was enhanced by using SA matrix.
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These results completely agree with the fluorescence spectroscopic results.
4. Conclusions
A new nanomaterial with fluorescent and magnetic properties for the detection of pathogenic bacteria was proposed in this work. The amine functionalized Mag-CDs have demonstrated outstanding features of sensing and enrichment. The Mag-CDs were characterized by UV-vis, FT-IR spectra, TEM images, XRD and EDX. UV-vis, EDX and FT-IR measurements confirmed the grafting of amine group on the nanomaterial. Fluorescence spectroscopy and MALDI-MS were used for the detection and identification of bacterial strains S. aureus and E. coli in PBS 11
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and urine matrix, respectively. Parallel response between fluorescence and bacterial count was found with linear relationship. The limit of detection for S. aureus and E. coli was found to be 3×102 and 3.5×102 cfu mL-1, respectively. The current approach is a powerful tool for future detection of pathogenic bacteria by fluorescence method in the clinical microbiology and
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biomedicine field.
Acknowledgements
The authors acknowledge the financial support from the ministry of science and technology of
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Taiwan.
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Figures and captions
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Fig. 1 Schematic representation of experimental concepts. Fig. 2 Characterization I: TEM images of (a) Iron oxide nanoparticles and (b) Mag-CDs. (c) FTIR spectra of Iron oxide and Mag-CDs, and (d) EDX of Mag-CDs. Fig. 3 Characterization II: (a) UV-vis spectra of Mag-CDs and Iron oxide nanoparticles. (b) Emission spectra of the Mag-CDs at different excitation wavelength as indicated. (c) The XRD patterns of the Iron oxide nanoparticles and Mag-CDs. (d) Optical pictures demonstrate the fluorescent and magnetic properties of Mag-CDs. Concentration of Mag-CDs was 20 µg 14
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Fig. 4 (a) Photoluminescence spectra of S. aureus with different concentrations and (b) the calibration curve of the pathogenic bacteria. (c) Photoluminescence spectra of S. aureus with different concentrations in urine sample and (d) the calibration curve of the pathogenic bacteria. (e) Photoluminescence spectra of E. coli with different concentrations and (f) the calibration
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curve of the pathogenic bacteria. (g) Photoluminescence spectra of E. coli with different concentrations in urine sample and (h) the calibration curve of the pathogenic bacteria Concentration of Mag-CDs was 20 µg.
Fig. 5 MALDI-MS spectra of (a) S. aureus, including bacteria control, urine control, 3×102 cfu
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mL-1, 1.1×103 cfu mL-1, 1.7×103 cfu mL-1, and 2.3×103 cfu mL-1 (b ) E. coli, including bacteria control, urine control, 3.5×103 cfu mL-1, 1.1×103 cfu mL-1, 1.5×103 cfu mL-1, and 2.9×103 cfu
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mL-1.
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Figure captions
Fig. 1 Schematic representation of experimental concepts.
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Fig. 2 Characterization I: TEM images of (a) Iron oxide nanoparticles and (b) Mag-CDs. (c) FTIR spectra of Iron oxide and Magnetic Cdots, and (d) EDX of Mag-CDs.
Fig. 3 Characterization II: (a) UV-vis spectra of Mag-CDs and Iron oxide nanoparticles. (b) Emission spectra of the Mag-CDs at different
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excitation wavelength as indicated. (c) The XRD patterns of the Iron oxide nanoparticles and Mag-CDs. (d) Optical pictures demonstrate the fluorescent and magnetic properties of Mag-CDs. The concentration of Mag-CDs was 20 µg.
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Fig. 4 (a) Photoluminescence spectra of S. aureus with different concentrations and (b) the calibration curve of the pathogenic bacteria. (c) Photoluminescence spectra of S. aureus with different concentrations in urine sample and (d) the calibration curve of the pathogenic bacteria. (e) Photoluminescence spectra of E. coli with different concentrations and (f) the calibration curve of the pathogenic bacteria. (g)
The concentration of Mag-CDs was 20 µg.
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Photoluminescence spectra of E. coli with different concentrations in urine sample and (h) the calibration curve of the pathogenic bacteria.
Fig. 5 MALDI-MS spectra of (a) S. aureus, including bacteria control, urine control, 3×102 cfu mL-1, 1.1×103 cfu mL-1, 1.7×103 cfu mL-1,
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2.9×103 cfu mL-1.
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and 2.3×103 cfu mL-1 (b ) E. coli, including bacteria control, urine control, 3.5×103 cfu mL-1 , 1.1×103 cfu mL-1, 1.5×103 cfu mL-1, and
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Fig. 1
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Fig. 2
Element
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32.58 11.27
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Fig. 4
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(a) S. aureus
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Control
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Fig. 5
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(b) E. coli
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Fig. 5
3.5×103 cfu mL-1
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3. Achieved very low limit of detection of pathogenic bacteria using this technique.