chemosensors

Article Microwave-Assisted Synthesis of Amikacin Modified N,S co-Doped Carbon Dots for Escherichia coli Detection

Fajar Amelia Rachmawati Putri 1, Mudasir Mudasir 1 , Kinichi Morita 2 and Suherman Suherman 1,* 1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara Kotak Pos 21, Yogyakarta 55281, Indonesia; [email protected] (F.A.R.P.); [email protected] (M.M.) 2 New Business Development Office, Corporate R&D Division, Ushio INC. 1-6-5 Marunouchi, Chiyoda-ku, 100-8150, ; [email protected] * Correspondence: [email protected]; Tel.: +62-274-545188

 Received: 1 October 2019; Accepted: 25 November 2019; Published: 28 November 2019 

Abstract: Fluorescent amikacin modified nitrogen, sulfur co-doped carbon dots (amikacin modified N,S-CDs) were synthesized by a facile and low-cost one-step microwave-assisted specifically for selective detection of Gram-negative bacteria Escherichia coli (E. coli). Amikacin is a semi-synthetic amino glycoside antibiotic and it was employed in this study to increase the fluorescence response of N,S-CDs by providing binding ligand towards E. coli. The effect of thiourea content as the source of nitrogen and sulfur dopants was investigated prior to the preparation of amikacin modified N,S-CDs. The formation of amikacin modified N,S-CDs were characterized by using Fourier transform infrared (FTIR), X-ray diffraction (XRD), Transmission electron microscope (TEM), UV-Vis spectrophotometer, and spectrofluorometer. Amikacin modified N,S-CDs was identified to be successfully synthesized from the wavenumber shift of the C=O stretching mode. Amikacin modified N,S-CDs were amorphous with an average size of 7 nm. Fluorescence spectra showed that the highest intensity was obtained at thiourea content of 50% and amikacin mass of 25 mg. By comparing fluorescence responses of all the investigated amikacin modified N,S-CDs, the limit of detection (LOD) was attained by 25 mg 1 amikacin modified N,S-CDs at 1.526 cfu mL− .

Keywords: Carbon dots; amikacin; Escherichia coli; fluorescence; thiourea; citric acid

1. Introduction The contamination of Escherichia coli (E. coli) in water resources remains a serious environmental problem. 844 million people still lacked a basic drinking water service either utilize developed sources with a period of water collection more than 30 min. (limited services), employ exposed wells and springs (unimproved sources), or by using water straight from surface water sources, based on WHO and UNICEF data in 2015 [1]. Recently, the fluorescence-based method is used for bacterial detection. This method has drawn considerable concern, since it offers good selectivity, high sensitivity, and affordable means [2]. The fluorescence-based technique has been developed to overcome the constraints of conventional methods, namely sensitivity and time. Carbon dots (CDs) is one of the materials used based on fluorescence method. The CDs particularly size less than 10 nm and possess recognizable benefits, such as excellent water solubility and convenient functionalization, with diverse organic, polymeric, as well as inorganic compounds. Furthermore, CDs possess low toxicity and favorable compatibility, and therefore can be used as an eco-friendly alternative for biological purposes [3,4]. In addition, CDs offer cost-effective and

Chemosensors 2019, 7, 61; doi:10.3390/chemosensors7040061 www.mdpi.com/journal/chemosensors Chemosensors 2019, 7, 61 2 of 11 rapid synthetic routes for the preparation and application [3]. Many techniques have been conducted to synthesize CDs, such as on hydrothermal, solvothermal, and microwave synthesis [4]. In terms of the simple fabrication method, a novel one-pot hydrothermal was introduced to synthesize graphene quantum dots (GQDs) with two functional groups (-OH and -NH2) while using a polycyclic aromatic hydrocarbon as the precursor [5]. Applying the pyrolysis method is another way of preparing CDs in a facile synthetic route. The nitrogen-doped CDs (N-CDs) with excellent stability were directly synthesized within seven min. by pyrolyzing ethanolamine under air environment [6]. One application of CDs as a bacterial detector is utilizing amphiphilic CDs to perform detection and imaging of bacteria based on the attachment of synthesized material to bacterial cell surfaces. The study showed that amphiphilic CDs could bind to bacterial cells after incubation for 3 h and allowed for bacterial detection through both fluorescence spectroscopy and microscopy [7]. The modification of CDs can be done by using mannose and folic acids, which further to label E. coli bacteria within 1-h incubation [2]. The short period of incubation was also used for pathogenic bacteria detection utilizing magnetic CDs that were synthesized from magnetic nanoparticles and chitosan [8]. When considering these previous studies, fluorescent CDs are capable of greatly reducing the incubation period as a means to attain short to real-time detection. Despite having various outstanding performances, CDs still show relatively low intensity of fluorescence emission and offer a challenge for improving it. Modification of functional groups on the CDs surface is necessary for enhancing the intensity, and many researchers have performed doping with different atoms. The increment of intensity by heteroatom doping into CDs is caused by the changing of their electronic and chemical properties from the atoms [9]. Although the short duration of incubation has been discovered, selectivity as another main feature of bacterial detection is still a major flaw that constricts their practical application. Therefore, in this research, amikacin was introduced for improving the selectivity of synthesized CDs. Amikacin belongs to the aminoglycoside group of antibiotics and is well-known to be active against key Gram-negative bacteria. Amikacin is used to medicate urinary tract infections caused by E. coli and Klebsiella pneumoniae. Treatment with amikacin is also considered to be a safe alternative treatment option with high efficiency [10]. Previous research has reported a strategy to detect E. coli by modifying CDs while using colistin and found a linear relationship between concentrations of E. coli and fluorescent intensity of CDs colistin [11]. In this work, CDs were synthesized while using citric acid as the carbon source. Citric acid was chosen, since it is environmentally benign and contains hydroxyl groups that give CDs high water solubility. For the purpose of intensifying fluorescence emission, doping with different atoms was conducted by employing thiourea that served as N and S-containing precursors. Heteroatom doping into CDs has become an effective strategy for elevating the intensity of fluorescence emission. Amikacin was introduced through the microwave-assisted solid-phase synthesis in order to enhance the affinity and selectivity of N,S-CDs towards E. coli.

2. Materials and Methods

2.1. Materials Citric acid monohydrate (C H O H O) and thiourea (CH N S) were purchased from Wako Pure 6 8 7· 2 4 2 Chemical Corporation-Japan, while amikacin (C22H43N5O13) was obtained from Sigma-Aldrich (Massachussets, USA). NaCl, CH3COOH, CH3COONa, H3PO4, NaH2PO4, Na2HPO4, NaOH, phosphate buffer saline (PBS) pH 7.4, and 0.1 µm filter membrane were provided by Merck (Darmstadt, Germany). All of the chemicals were used as received without further purification. The E. coli and Staphylococcus aureus (S. aureus) in concentration of 1 105 cfu mL 1 in PBS (pH 7.4) were purchased × − from Microbiology of Veterinary Medicine, Universitas Gadjah Mada (UGM). The E. coli bacteria were cultured in sterile Luria Bertani media that consisted of bacto-yeast extract, tryptone, and Chemosensors 2019, 7, 61 3 of 11

NaCl in deionized water. Commercially bottled mineral water was used for the detection of E. coli bacteria in real sample.

2.2. Characterization All of the fluorescence measurements were carried out while using RF-6000 (Kyoto, Japan) with xenon lamp as the source of excitation. UV-vis spectrophotometer JASCO V-670 (Tokyo, Japan) was employed to record the UV-vis absorption spectra. Fourier transform infrared (FTIR) spectra were measured on a Shimadzu IR Prestige-21, Fourier transform infrared spectrometer (Kyoto, Japan) 1 with potassium bromide pellet technique that ranged from 400 to 4000 cm− . The transmission electron microscope (TEM) image was recorded while using JEOL-JEM 1400 (Tokyo, Japan). The sample for TEM characterization was prepared by placing a drop of amikacin modified N,S-CDs solution on carbon-coated copper grid, followed by drying in air. X-ray diffraction (XRD) patterns were collected on a Miniflex Rigaku (Tokyo, Japan) that was equipped with Cu Kα radiation (λ = 0.154 nm). Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a JNM-ECZ500R (Tokyo, Japan), 500 Mhz Super Conductive Magnets.

2.3. Synthesis of N,S-CDs The N,S-CDs were synthesized by microwave-assisted treatment of citric acid and thiourea as the source of C and N-S, respectively. The effect of thiourea concentration was investigated by mixing 0.3 g of citric acid with various ratios of thiourea to citric acid mass, which were 0, 10, 25, 50, 75, and 100%. The solution was then heated in a microwave reactor of 180 ◦C for 30 min. The obtained products were dissolved in 25 mL. The purification products were conducted by centrifugation at 10000 rpm for 30 min. to remove the large solids, followed by filtration via a 0.1 µm filter membrane (Table S1 in Supplementary Material illustrates detailed variations).

2.4. Synthesis of Amikacin Modified N,S-CDs Amikacin modified N,S-CDs were synthesized by the same method while using the optimized percentage of thiourea that was obtained from previous means. The optimized ratio of thiourea to citric acid was separately mixed with various masses of amikacin, which were 12.5, 25, 50, and 75 mg (the information about the parameters used are designated in Table S1 in Supplementary Material).

2.5. Detection E. coli Bacteria E. coli concentration of 1 105 cfu mL 1 was dissolved for the range of 3.75 102 to × − × 1.2 104 cfu mL 1 in PBS solution pH 7.4 with a total volume of 10 mL. Each concentration of × − bacteria in 3 mL, 0.1 mL of all varied amikacin modified N,S-CDs were added separately and continued by vortexing for 5 min. Gentle shaking with orbital shaker was then performed at 200 rpm for 60 min. at room temperature. The same steps of the procedure were also carried out to compare the fluorescence response of amikacin modified N,S-CDs and N,S-CDs for sensing E. coli in a concentration of 1.2 104 cfu mL 1. The fluorescence spectra were eventually collected at 360 nm with the dilution of × − the samples up to 50 times. The selectivity of amikacin modified N,S-CDs was also examined towards E. coli and S.aureus in a concentration of 1.2 104 cfu mL 1, respectively. × − 2.6. Detection of E. coli Bacteria in Real Samples The E. coli concentration of 1 105 cfu mL 1 was diluted for the range of 3.75 102 × − × to 1.2 104 cfu mL 1 in commercially bottled mineral water with a total volume of 10 mL. × − Amikacin modified N,S-CDs with highest labeling efficiency was then added to 3 mL of each concentration of bacteria. The fluorescence spectra were recorded at optimum excitation wavelength by spectrofluorometer by applying the same procedure of detection of E. coli in PBS. Chemosensors 2019, 7, x FOR PEER REVIEW 4 of 12

3. Results and Discussion

3.1. Optimization of Thiourea Content in N,S-CDs The effect of N and S atoms incorporation into CDs was carried out by varying contents of thiourea added to citric acid. Figure 1a depicts the fluorescence intensities at excitation wavelength of 360 nm was steadily enhanced along with higher thiourea content and it reached a maximum at 50%. The elevation of N,S-CDs fluorescence intensities could be attributed to the doping of N and S Chemosensors 2019, 7, 61 4 of 11 atoms, as they give extra electron for n-type doping. This type of doping favors the radiative relaxation pathway which affects the fluorescence intensity [12]. The abundant functional groups 3.containing Results andheteroatom Discussion were able to give new excitation energy traps, leading to the enhancement of fluorescence intensity [13]. The introduction of N and S atoms into CDs provided a new kind of 3.1.surface Optimization states (labeled of Thiourea as the Content N- and in S- N,S-CDsstate), where the excited electrons were trapped. This process broughtThe the eff ectconsequence of N and of S atomsthe high incorporation intensity of radiative into CDs recombination was carried out [14 by]. varying contents of thioureaThe added decrement to citric of fluorescenceacid. Figure1 intensitiesa depicts the appear fluorescences to be due intensities to the at higher excitation ratios wavelength of thiourea oflead 360ing nm to wasa further steadily carbonization enhanced alongstage that with is higher indicated thiourea by UV content-Vis absorption and it reached spectra a, maximum as shown atin 50%.Figure The 1b. elevation The higher of N,S-CDsabsorbance fluorescence at ca. 260 nm intensities represents could 휋-휋 be* transitions attributed toof thethe dopingaromatic of C=C N and [15 S] atoms,and signifies as they givean abundant extra electron sp2 for domain. n-type doping. This carbonic This type core of doping had no favors contribution the radiative to relaxation intensify pathwayfluorescence which intensity, affects which the fluorescence suggests that intensity the generation [12]. The of abundant fluorescence functional was not groups from containing electronic heteroatomconjugate structures were able [14 to]. give This new phenomenon excitation energywas emphasized traps, leading by the to thelower enhancement absorbance of peak fluorescence that was intensitycentred at [13 340]. The nm introductionattributed to of n-휋 N* and transitions S atoms originated into CDs fromprovided excited a new state kind energy of surface traps ofstates the (labeledsurface states as the [16 N-].and It asserts S-state), the where origin theof fluorescence excited electrons emission, were stating trapped. that This the processhigher fluorescence brought the consequenceintensity has ofresulted the high from intensity the introduction of radiative of recombination N and S atoms. [14 ].

135000 4 (a) 120000 (b)

105000 3

90000

75000 2 Intensity 60000 N,S-CDs 0 % 0% N,S-CDs 10 % Absorbance 10% 45000 N,S-CDs 25 % 25% N,S-CDs 50 % 50% 30000 1 N,S-CDs 75 % 75% N,S-CDs 100 % 100% 15000

0 0 375 450 525 600 675 200 300 400 500  (nm) EM  (nm) Figure 1.1. (a) FluorescenceFluorescence (diluted samples up to 50x) and (b) UV-visUV-vis absorptionabsorption spectraspectra of variedvaried N,S-CDsN,S-CDs synthesized.synthesized. Inset in (a) shows the fluorescence fluorescence intensityintensity atat 360 nm of excitation wavelength.

3.2. TheSynthesis decrement of Amikacin of fluorescence Modified N,S intensities-CDs appears to be due to the higher ratios of thiourea leading to a further carbonization stage that is indicated by UV-Vis absorption spectra, as shown in Figure1b. The higherBased absorbanceon the spectra at ca. depicted 260 nm representsin Figure 2π, -N,Sπ* transitions-CDs and ofamikacin the aromatic modified C=C[ N,S15]-CDs and signifiesshowed ansimilar abundant bands sp almost2 domain. at entire This carbonic wavenumbers core had, however no contribution two distinctive to intensify band fluorescences were observed intensity,. The whichsimilar suggests wavenumbers that the meant generation that the of surface fluorescence of amikacin was not modified from electronic N,S-CDs conjugate had the same structures functional [14]. Thisgroups phenomenon as N,S-CDs. was However emphasized, the byfirst the discrepancy lower absorbance that can peak be thatobserved was centred is the atvibrational 340 nm attributed band at -1 to2060 n-π cm* transitions of amikacin originated modified from N,S excited-CDs that state indicates energy trapsthat the of the addition surface of states amikacin [16]. Itprevent assertss thethe -1 originformation of fluorescence of –SCN bond. emission, Secondly, stating the thatshifting the higherof C=O fluorescence stretching vibration intensity at has around resulted 1713 from cm the of -1 introductionN,S-CDs to 1705 of N cm and of S atoms.amikacin modified N,S-CDs exhibited amide bond formation at the surface of N,S-CDs. It signifies that the interaction between N,S-CDs and amikacin was a covalent bond that 3.2.was Synthesisinduced ofby Amikacin the heating Modified process N,S-CDs in the microwave and followed by the formation of the amide bond. The functional groups taking part to set up the amide bonds were –OH groups of N,S-CDs and Based on the spectra depicted in Figure2, N,S-CDs and amikacin modified N,S-CDs showed –NH2 groups of amikacin by releasing some water molecules. The attachment of the amikacin to the similar bands almost at entire wavenumbers, however two distinctive bands were observed. The similar wavenumbers meant that the surface of amikacin modified N,S-CDs had the same functional groups 1 as N,S-CDs. However, the first discrepancy that can be observed is the vibrational band at 2060 cm− of amikacin modified N,S-CDs that indicates that the addition of amikacin prevents the formation 1 of –SCN bond. Secondly, the shifting of C=O stretching vibration at around 1713 cm− of N,S-CDs to 1 1705 cm− of amikacin modified N,S-CDs exhibited amide bond formation at the surface of N,S-CDs. It signifies that the interaction between N,S-CDs and amikacin was a covalent bond that was induced by the heating process in the microwave and followed by the formation of the amide bond. The functional groups taking part to set up the amide bonds were –OH groups of N,S-CDs and –NH2 groups of Chemosensors 2019, 7, x FOR PEER REVIEW 5 of 12

surface of the N,S-CDs was further confirmed by 1HNMR spectroscopy (FigureS S1–S3 in Supplementary Materials). The 1HNMR spectrum of amikacin modified N,S-CDs in Figure S3 shows the attachment of H to sp3 C in the region of 1–3 ppm. The major peak that was observed was ascribed to the binding of H to cyclic 휋-system of N,S-CDs, which was also noticed in Figure S2 (supplementary data). The region of ca. 4 ppm in Figure S3 was attributed to the proton that was bonded to amide bond that formed between amikacin and N,S-CDs. To understand the further insight of amikacin modified N,S-CDs, the XRD pattern was investigated. Figure 3a displays single broad diffraction peak that is centred at around 26.64° with a Miller index of (002) in the absence of sharp peak, implying a highly disordered carbon structure. The obtained interlayer spacing is 3.34 Å and nearly suits with graphite. Analysis by the TEM technique was conducted to examine the size and shape of amikacin modified N,S-CDs. Figure 3b shows that amikacin modified N,S-CDs were spherical and well dispersed without noticeable aggregationm with an average size of 7 nm. Fluorescence emission spectra of amikacin modified N,S-CDs within different excitation wavelengths, as shown in Figure 4, could be divided into two sections. Firstly, excitation- independent emission lies at excitation wavelengths of 300–360 nm. In this range, the emission intensities steadily increased until reached a maximum at 360 nm. The peaks of emission showed no shift and they were constantly centered at 443 nm. This excitation-independent emission under 300– 360 nm represents a relatively uniform surface state for emissive traps [15]. On the contrary, the emission peaks were red-shifted from 447 to 469 nm at excitation wavelengths above 360 nm with gradually decreasing intensities. The excitation-dependent fluorescence emission phenomenon is ascribed to different surface states of amikacin modified N,S-CDs. The existence of multiple O-, N-, Chemosensors 2019, 7, 61 5 of 11 and C-containing functional groups on the surface of amikacin modified N,S-CDs generated a series of emissive traps due to diverse surface states with contrastive energy levels. The corresponding amikacinsurface state by releasing emissive some trap wateris controlling molecules. and Thedelivering attachment excitation of the-dependent amikacin toemission the surfaces at different of the N,S-CDsexcitation was wavelength further confirmeds [17]. by 1HNMR spectroscopy (Figures S1–S3 in Supplementary Materials).

curve translate: curve translate: curve translate: (a) 610 206 118 342 319 0 171 8 6 4 3 (b) 106 163 341 0 Transmittance (a.u.) Transmittance 6 7

610 (c) 118 342 319 8 6 4 1705 4000 3500 3000 2500 2000 1500 1000 500 -1 Wavenumber (cm ) FigureFigure 2. Fourier 2. Fourier transform transform infrared infrared (FTIR) (FTIR spectra) spectra of of (a )(a N,S-CDs,) N,S-CDs, (b ()b amikacin,) amikacin, and and ( c(c)) amikacin amikacin modified nitrogen, sulfurmodified co-doped nitrogen, carbon sulfur dots (N,S-CDs). co-doped carbon dots (N,S-CDs).

1 The HNMR spectrum of amikacin modified N,S-CDs in Figure S3 shows the attachment of H to sp3 C in the region of 1–3 ppm. The major peak that was observed was ascribed to the binding of H to cyclic π-system of N,S-CDs, which was also noticed in Figure S2 (supplementary data). The region of ca. 4 ppm in Figure S3 was attributed to the proton that was bonded to amide bond that formed between amikacin and N,S-CDs. To understand the further insight of amikacin modified N,S-CDs, the XRD pattern was investigated. Figure3a displays single broad di ffraction peak that is centred at around 26.64◦ with a Miller index of (002) in the absence of sharp peak, implying a highly disordered carbon structure. The obtained interlayer spacing is 3.34 Å and nearly suits with graphite. Analysis by the TEM technique was conducted to examine the size and shape of amikacin modified N,S-CDs. Figure3b shows that amikacin modified N,S-CDs were spherical and well dispersed without noticeable aggregationm with an average size of 7 nm. Fluorescence emission spectra of amikacin modified N,S-CDs within different excitation wavelengths, as shown in Figure4, could be divided into two sections. Firstly, excitation-independent emission lies at excitation wavelengths of 300–360 nm. In this range, the emission intensities steadily increased until reached a maximum at 360 nm. The peaks of emission showed no shift and they were constantly centered at 443 nm. This excitation-independent emission under 300–360 nm represents a relatively uniform surface state for emissive traps [15]. On the contrary, the emission peaks were red-shifted from 447 to 469 nm at excitation wavelengths above 360 nm with gradually decreasing intensities. The excitation-dependent fluorescence emission phenomenon is ascribed to different surface states of amikacin modified N,S-CDs. The existence of multiple O-, N-, and C-containing functional groups on the surface of amikacin modified N,S-CDs generated a series of emissive traps due to diverse surface states with contrastive energy levels. The corresponding surface state emissive trap is controlling and delivering excitation-dependent emissions at different excitation wavelengths [17]. ChemosensorsChemosensors2019 2019,,7 7,, 61 x FOR PEER REVIEW 66 of 1112 Chemosensors 2019, 7, x FOR PEER REVIEW B 6 of 12 1600 (002) B 1600 (002) 1400 (a) (b) 1400 (a) (b) 1200 1200 1000 1000

Intensity 800

Intensity 800 600 600 400 400 200 200 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 2 () 2 () Figure 3. Characterization results of amikacin modified N,S-CDs (a) X-ray diffraction (XRD) pattern Figure 3. Characterization results of amikacin modified N,S-CDs (a) X-ray diffraction (XRD) pattern Figureand (b 3.) t ransmissionCharacterization electron results microscope of amikacin (TEM modified) images N,S with-CDs magnification (a) X-ray diffraction of 40000x. ( XRD) pattern and (b) transmission electron microscope (TEM) images with magnification of 40,000 . and (b) transmission electron microscope (TEM) images with magnification of 40000x.×

135000 135000 120000 120000 105000 105000 90000 90000 75000 300 nm 310 nm 75000 300 nm

Intensity 320 nm 60000 310 nm 330 nm

Intensity 320 nm 60000 340 nm 330 nm 45000 350 nm 340 nm 360 nm 45000 350 nm 370 nm 30000 360 nm 380 nm 370 nm 30000 390 nm 380 nm 15000 400 nm 390 nm 15000 400 nm 0 0 375 450 525 600 675 375 450  525 (nm) 600 675 EM  (nm) EM FigureFigure 4. 4.Fluorescence Fluorescence spectra spectra of 25 of mg 25 amikacin mg amikacin modified modified N,S-CDs at N,S diff-CDserent atexcitation different wavelengths. excitation FigureInsetwavelengths. presents 4. Fluorescence theInset fluorescence presents spectra the intensity fluorescence of 25 at mg each intensity amikacin excitation at modified each wavelength. excitation N,S-CDs wavelength. at different excitation wavelengths. Inset presents the fluorescence intensity at each excitation wavelength. AsAs illustratedillustrated inin FigureFigure5 5aa,, there there was was an an increment increment of of fluorescence fluorescence intensities intensities until until the the maximum maximum oneone wasAswas illustrated obtainedobtained atinat 25Figure25 mgmg of of5a amikacin,amikacin there was andand an thenthenincrement followedfollowed of fluorescence byby aa sharpsharp decrement.decrement. intensities until ItIt could the bebemaximum possiblypossibly onehappenhappen was dueobtaineddue toto thethe at introducedintroduced 25 mg of amikacin NN atoms and still then oofferedffered followed aa positivepositive by a eeffect ffsharpect byby decrement. providingproviding Itexcitedexcited could energy enerbe possiblygy trapstraps happenthroughthrough due N-N- toandand the S-states.S introduced-states. ThisThis N tendencytendency atoms still waswas offered confirmedconfirmed a positive by UV-VisUV effect-Vis by spectraspectra providing pointedpointed excited lowerlower ener absorbanceabsorbancegy traps throughpeakpeak thanthan N- threethreeand S other-otherstates. amikacinamikacin This tendency modifiedmodified was N,S-CDsN,S confirmed-CDs atat ca.byca. UV 234234-V nm,nmis spectra, whichwhich pointed couldcould bebe lower ascribedascribed absorbance toto π휋--π휋* * peaktransitionstransitions than three of of the the other aromatic aromatic amikacin C= C[ C=C 15modified ].[15 The]. The contrary N,S contrary-CDs was at noticed,ca. was 234 noticed wherenm, which, higherwhere could absorbancehigher be ascribed absorbance was achieved to 휋 was-휋* transitionsatachieved ca. 337 nmat of ca. (Figure the 337 aromatic nm5b). (Figure C=C 5b ).[15 ]. The contrary was noticed, where higher absorbance was achievedAA dramaticdramatic at ca. 337 decrementdecrement nm (Figure occurred,occurred, 5b). sincesince thethe higherhigher massmass ofof amikacinamikacin inducedinduced aa furtherfurther processprocess ofof carbonizationcarbonizationA dramatic andand decrement denoteddenoted occurred, byby elevatedelevated since absorbanceabsorbance the higher atat mass ca.ca. 234 of nm,nmamikacin, as displayed induced in a UV-VisUVfurther-Vis absorptionprocessabsorption of carbonizationspectraspectra (Figure(Figure and5 5bb). denoted).It Itimplied implied by that elevatedthat electronicelectronic absorbanceconjugate conjugate at ca.structures structures 234 nm, brought asbrought displayed no no notable notable in UV-e Veffectffisect absorption toto deliverdeliver spectrathethe highhigh (Fig intensityure 5b). ofIt of impliedfluorescence fluorescence that electronic [14 [14]].. Lower Lower conjugate absorbance absorbance structures peak peak centredbrought centred atno atca. notable ca.337 337nm effect nmfavoured to favoured deliver this thethisprocess high process tointensity occur to occur by of preventing fluorescence by preventing the [14 arrang the]. Lower arrangementement absorbance of N- ofand N- peakS- andsurface centred S-surface states at ca.for states the337 for trappingnm the favoured trapping of excited this of processexcitedelectrons. to electrons. occur by preventing the arrangement of N- and S-surface states for the trapping of excited electrons.

Chemosensors 2019, 7, 61 7 of 11 Chemosensors 2019, 7, x FOR PEER REVIEW 7 of 12

135000 4 120000 (a) (b) 105000 3 90000

75000

Intensity 60000 2

12.5 mg amikacin Absorbance 45000 12.5 mg amikacin 25 mg amikacin 25 mg amikacin 50 mg amikacin 50 mg amikacin 30000 75 mg amikacin 1 75 mg amikacin 15000

0 0 375 450 525 600 675 200 250 300 350 400 450 500  (nm) EM  (nm)

Figure 5. (a) Fluorescence and (b) UV-vis absorption spectra of all amikacin modified N,S-CDs at 360 nm

of emission wavelength. Inset in (a) shows the fluorescence intensity at 360 nm of excitation wavelength.

3.3. StabilityFigure of 5. Amikacin (a) Fluorescence Modified and N,S-CDs (b) UV-vis absorption spectra of all amikacin modified N,S-CDs at 360 nm of emission wavelength. Inset in (a) shows the fluorescence intensity at 360 nm of excitation Based on Figure6a, the fluorescence intensities of the amikacin modified N,S-CDs were susceptive wavelength. . to pH due to the presence of some functional groups, such as –COOH, –OH, and –NH2, on their surfaces.3.3. Stability Fluorescence of Amikacin intensities Modified were N,S- greatlyCDs weaken under highly acidic condition, but gradually decreasedChemosensors in2019 a strong, 7, x FOR alkaline PEER REVIEW environment. The optimum fluorescence intensity was achieved at8pH of 12 7. Based on Figure 6a, the fluorescence intensities of the amikacin modified N,S-CDs were

2 susceptive135000 to pH due to the presence of some functional135000 groups, such as –COOH, –OH, and –NH , on their surfaces. Fluorescence intensities were greatly weak(b) en under highly acidic condition, but 120000 (a) 120000 gradually decreased in a strong alkaline environment. The optimum fluorescence intensity was 105000 105000 achieved at pH 7. 90000 90000The steep decrease in the fluorescence intensities at highly acidic condition due to the formation 0 mol L-1 2 75000 75000 0.2 mol L-1 of hydrogen bonding between –COOH, –OH, 3 and –NH2 moieties on the surface of 25 mg amikacin 0.4 mol L-1

Intensity 4 -1 60000 Intensity 60000 0.6 mol L modified N,S-CDs, leading particle aggregation, 5 and resulting in the quenching of fluorescence-1 0.8 mol L 6 -1 1.0 mol L emission.45000 In strong alkaline condition, decreased 7 fluorescence45000 intensities have occurred as the -1result 1.2 mol L 8 -1 of the deprotonation of carboxylic acid moieties, which led to the build up of negative charge 1.4 mol Lon the 30000 9 30000 1.6 mol L-1 surface of amikacin modified N,S-CDs. The presence 10 of a large quantity of negatively charge 1.8d mol surface L-1 15000 11 15000 2.0 mol L-1 functional groups would decrease the fluorescence 12 emissions due to its strong electron-withdrawing 0 0 ability375 reducing450 the radiativ525 e recombination600 675 between electrons375 450 and holes525, thus quenching600 some675

EM (nm)  (nm) emission signals [18]. EM However, as the pH value lower than 6, the emission spectra were blue-shifted along with the Figure 6. Fluorescence spectra of amikacin modified N, S-CDs at (a) various pH values and (b) different decreaseconcentration in pH of values. NaCl. InsetsObvious display blue the-shifted fluorescence wavelength intensity occurred of N,S-CDs-amikacin due to sp2 (ato) at sp varied3 hybridization pH changevaluesFigure within and 6. Fluorescence (b ) the under π- eachconjugated spectra concentration of system amikacin of NaCl. of modified carbon generated N, S-CDs at loss (a) ofvarious the C pH-ring values aromaticity and (b) , thus alterdifferenting the concentration optical properties. of NaCl. This Insets defective display conjugationthe fluorescence system intensity resulted of N,S in-CDs an -incrementamikacin (a of) at HOMO The steep decrease in the fluorescence intensities at highly acidic condition due to the formation andvaried LUMO pH values band gapand ([19b) under]. Consequently each concentration, the fluorescence of NaCl. emission of the protonated N,S-CDs- ofamikacin hydrogen should bonding fall between at a higher –COOH, energy. –OH, and –NH2 moieties on the surface of 25 mg amikacin modified3.4. FluorescenceThe N,S-CDs, influence Detection leadingof ionic of E.strength particle Coli aggregation,was also determined and resulting in various in theconcentration quenchings of NaCl. fluorescence Based on emission.Figure 6b, In strongthe increment alkaline condition, in ionic strengthdecreased of fluorescence NaCl medium intensities resulte haved in occurred a slight as reduction the result in Interaction between amikacin modified N,S-CDs and E. coli was proposed based on the offluorescence the deprotonation intensity, of carboxylic which suggested acid moieties, the good which potential led to of the 25 build mg amikacin up of negative modified charge N,S- onCDs the for electrostatic interaction between the positive charge of amikacin and negative charge of LPS. surfacesensing of amikacinapplications modified under N,S-CDs.physiological The presencecondition of [20 a]. large quantity of negatively charged surface Amikacin modified N,S-CDs contain positive charge as a consequence of amino groups existence, functional groups would decrease the fluorescence emissions due to its strong electron-withdrawing abilitywhereas reducing LPS, as the the target radiative, is negatively recombination charged between because electrons of the phosphate and holes, groups thus quenchingin lipid A. When some emissioncomparing signals to amikacin [18]. modified N,S-CDs, an insignificant quenching effect due to the existence of E. coliHowever, was portrayed as the pHby N,S value-CDs lower in Figure than 6, 7a. the It emissioncould be spectraascribed were to the blue-shifted lack of their along capability with the to decreaseeffectively in attach pH values. the outer Obvious membrane blue-shifted E. coli due wavelength to the disso occurredciation of due S-containing to sp2 to spfunctional3 hybridization groups changemaking within the surface the πs-conjugated of N,S-CDs system negatively of carbon charge generatedd. loss of the C-ring aromaticity, thus altering the opticalThe specificity properties. of amikacin This defective modified conjugation N,S-CDs for system Gram resulted-negative in bacteria an increment (E. coli of) detection HOMO andwas examined by performing a similar procedure to Gram-positive bacteria cell (S. aureus). As revealed by Figure 7, the fluorescence intensity of S. aureus after treatment with amikacin modified N,S-CDs was still high when compared to E. coli. It signified that amikacin modified N,S-CDs was only selective towards Gram-negative bacteria. This selectivity could be related to the compositional discrepancy in the cell walls of the two bacteria, thus creating different ways to interact with amikacin modified N,S-CDs. The detection efficiency of each amikacin modified N,S-CDs was determined by employing a series of various concentrated E. coli with a constant quantity of the synthesized materials. As described in Figure 7, fluorescence intensity gradually decreased as bacterial concentration increased. This linear relationship implies that amikacin modified N,S-CDs creates more intense interaction with a higher amount of E. coli. It also signifies that the strong quenching effect was developed due to the abundance of E. coli. The highest detection efficiency was exhibited by 25 mg amikacin modified N,S-CDs, with a coefficient of determination (R2) of 0.9906. The limit of detection (LOD) for E. coli after treatment with all amikacin modified N,S-CDs was examined from the linear regression curve of quenching percentage vs log of E. coli concentration. The percentage of quenching was calculated from [(S0 - S)/S0] × 100%, where S0 and S are the intensities of the before and after the bacteria addition, respectively. The 25 mg amikacin modified N,S-CDs exhibited the lowest LOD of 1.526 cfu mL-1, which indicated that the synthesized material had the highest sensitivity towards such low concentration of E. coli bacteria. The detection of E. coli by using 12.5, 50, and 75 mg amikacin modified N,S-CDs resulted LODs of 1.836, 2.020, and 2.838 cfu mL-1 (Table 1). It signified that 75 mg amikacin modified with N,S-CDs had the least sensitivity in application to detect E. coli bacteria.

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LUMO band gap [19]. Consequently, the fluorescence emission of the protonated N,S-CDs-amikacin should fall at a higher energy. The influence of ionic strength was also determined in various concentrations of NaCl. Based on Figure6b, the increment in ionic strength of NaCl medium resulted in a slight reduction in fluorescence intensity, which suggested the good potential of 25 mg amikacin modified N,S-CDs for sensing applications under physiological condition [20].

3.4. Fluorescence Detection of E. coli Interaction between amikacin modified N,S-CDs and E. coli was proposed based on the electrostatic interaction between the positive charge of amikacin and negative charge of LPS. Amikacin modified N,S-CDs contain positive charge as a consequence of amino groups existence, whereas LPS, as the target, is negatively charged because of the phosphate groups in lipid A. When comparing to amikacin modified N,S-CDs, an insignificant quenching effect due to the existence of E. coli was portrayed by N,S-CDs in Figure7a. It could be ascribed to the lack of their capability to e ffectively attach the outer membrane E. coli due to the dissociation of S-containing functional groups making the surfaces of ChemosensorsN,S-CDs negatively 2019, 7, x FOR charged. PEER REVIEW 9 of 12

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120000 (a) 120000 (b) Amikacin modified N,S-CDs N,S-CDs 105000 105000 N,S-CDs + E. coli Amikacin modified N,S-CDs + E. coli Amikacin modified N,S-CDs + S. aureus 90000 90000

75000 75000 Intensity

60000 Intensity 60000

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0 0 375 450 525 600 675 375 450 525 600 675  (nm) EM  (nm) EM 4 Figure 7. Fluorescence emission spectra of (a) E. coli labeled N,S-CDs, (b) E. coli and S. aureus of 1.2x10 1 cfu mL− each labelled with 25 mg amikacin modified N,S-CDs 360 nm excitation wavelength. Figure 7. Fluorescence emission spectra of (a) E. coli labeled N,S-CDs, (b) E. coli and S. aureus of 1.2x104 The specificity of amikacin modified N,S-CDs for Gram-negative bacteria (E. coli) detection was cfu mL-1 each labelled with 25 mg amikacin modified N,S-CDs 360 nm excitation wavelength. examined by performing a similar procedure to Gram-positive bacteria cell (S. aureus). As revealed by FigureTable7, the 1. fluorescenceLimit of detection intensity (LOD) of valuesS. aureus at 360after nm excitation treatment labelled with amikacin with various modified amikacin N,S-CDs modified was still highN,S when-CDs. compared to E. coli. It signified that amikacin modified N,S-CDs was only selective towards Gram-negative bacteria. This selectivity could be related to the compositional discrepancy in the cell Materials R2 LOD (cfu mL-1) walls of the two bacteria, thus creating different ways to interact with amikacin modified N,S-CDs. 12.5 mg amikacin modified N,S-CDs 0.9808 1.836 The detection efficiency of each amikacin modified N,S-CDs was determined by employing a 25.0 mg amikacin modified N,S-CDs 0.9906 1.526 series of various50.0 concentrated mg amikacin E.modified coli with N,S a constant-CDs quantity of the0.9745 synthesized materials.2.020 As described in Figure7, fluorescence75.0 mg amikacin intensity modified gradually N,S-CDs decreased as bacterial0.9666 concentration increased.2.838 This linear relationship implies that amikacin modified N,S-CDs creates more intense interaction with a higher 3.5.amount Detection of E. coliof E.. It C alsooli in signifies Real Sample that the strong quenching effect was developed due to the abundance of E. coli. The highest detection efficiency was exhibited by 25 mg amikacin modified N,S-CDs, with a The applicability of amikacin modified N,S-CDs was analyzed in commercially bottled mineral coefficient of determination (R2) of 0.9906. The limit of detection (LOD) for E. coli after treatment with water. Based on Figure 8, it was also found that increasing E. coli concentrations were followed by a all amikacin modified N,S-CDs was examined from the linear regression curve of quenching percentage decrement of fluorescence intensity. The detection limit for E. coli that was attained from the mineral vs log of E. coli concentration. The percentage of quenching was calculated from [(S0 S)/S0] 100%, water was 3.04 cfu mL-1. The result implied that amikacin modified N,S-CDs have potential− practise× where S0 and S are the intensities of the before and after the bacteria addition, respectively. The 25 mg in detecting target bacteria in real samples. Table 2 presents the comparison1 of LOD with some other amikacin modified N,S-CDs exhibited the lowest LOD of 1.526 cfu mL− , which indicated that the previously reported papers based on optical bacterial sensors. synthesized material had the highest sensitivity towards such low concentration of E. coli bacteria.

135000 120000 (a) (b) 105000

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0 375 450 525 600 675

 (nm) EM Figure 8. (a) Fluorescence spectra and (b) linear plot of E. coli after labelling with 25 mg amikacin modified N,S-CDs at 360 nm excitation wavelength.

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120000 (a) 120000 (b) Amikacin modified N,S-CDs N,S-CDs 105000 105000 N,S-CDs + E. coli Amikacin modified N,S-CDs + E. coli Amikacin modified N,S-CDs + S. aureus 90000 90000

75000 75000 Intensity

60000 Intensity 60000

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0 0 375 450 525 600 675 375 450 525 600 675 Chemosensors 2019, 7, 61  (nm) 9 of 11 EM  (nm) EM

The detection of E. coli by using 12.5, 50, and 75 mg amikacin modified N,S-CDs resulted LODs of 1 1.836,Figure 2.020, 7. and Fluorescence 2.838 cfu emission mL− (Table spectra1). of It ( signifieda) E. coli labeled that 75 N,S mg-CDs, amikacin (b) E. coli modified and S. aureus with of N,S-CDs 1.2x104 had the leastcfu mL sensitivity-1 each labelled in application with 25 mg to amikacin detect E. modified coli bacteria. N,S-CDs 360 nm excitation wavelength.

Table 1. 1. LimitLimit of detection of detection (LOD (LOD)) values values at 360 at nm 360 excitation nm excitation labelled labelled with various with amikacin various modified amikacin N,Smodified-CDs. N,S-CDs.

2 2 1 -1 MaterialsMaterials R R LOD (cfuLOD mL− )(cfu mL ) 12.512.5 mg mg amikacin amikacin modified modified N,S N,S-CDs-CDs 0.98080.9808 1.836 1.836 25.025.0 mg mg amikacin amikacin modified modified N,S N,S-CDs-CDs 0.99060.9906 1.526 1.526 50.050.0 mg mg amikacin amikacin modified modified N,S N,S-CDs-CDs 0.97450.9745 2.020 2.020 75.075.0 mg mg amikacin amikacin modified modified N,S N,S-CDs-CDs 0.96660.9666 2.838 2.838

3.5. DetectionDetection ofof E.E. coliColi in in Real Real Sample Sample The a applicabilitypplicability of amikacin modified modified N,S N,S-CDs-CDs was analyzed in commercial commerciallyly bottled mineral water. Based on Figure 88,, itit waswas alsoalso foundfound thatthat increasingincreasing E. coli concentrations were followed by a decrement of fluorescence fluorescence intensity. The detection limit for E. coli thatthat was attained from the the mineral mineral water was 3.04 cfu mL-11. The result implied that amikacin modified N,S-CDs have potential practise water was 3.04 cfu mL− . The result implied that amikacin modified N,S-CDs have potential practise inin detecting detecting target bacteria in real samples. Table Table 22 presentspresents thethe comparisoncomparison ofof LODLOD withwith somesome otherother previously reported papers based on optical bacterial sensors.sensors.

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 (nm) EM Figure 8. ((aa)) Fluorescence Fluorescence spectra spectra and and ( (bb)) linear linear plot plot of of E.E. coli coli after labelling with 25 mg amikacin modifiedmodified N,S N,S-CDs-CDs at 360 nm excitation wavelength. Table 2. The comparative list of LOD and linear range of this work and previously reported work for detection of E. coli.

1 1 Material Linear Range (cfu mL− ) LOD (cfu mL− ) Method Reference Carbon dots@colistin 3.81 102–2.44 104 460 Fluorescent sensing [21] (CDs@colistin) × × Amine-functionalized 3.50 102–2.90 103 3.5 102 Fluorescent sensing [8] Magnetic-CDs × × × CDs@amikacin 7.625 102–3.904 105 552 Fluorescent sensing [22] × × CdTe/CdS quantum dots Immunochromatogra-phy 102–109 104 [23] (CdTe/CdS QDs) test strips Mannose-modified fluorescent carbon quantum 102–108 450 Fluorescent labelling [24] dots (Man-CQDs) Amikacin modified N,S-CDs 0–1.20 104 3.04 Fluorescent sensing This work × Chemosensors 2019, 7, 61 10 of 11

4. Conclusions In summary, fluorescent amikacin modified N,S-CDs have been successfully fabricated by applying the microwave-assisted solid phase method. It was concluded that the ratio of thiourea content plays an important role in altering the fluorescence intensity of N,S-CDs. The incorporation of amikacin into N,S-CDs exhibits higher affinity and selectivity toward E. coli. Moreover, the synthesized amikacin modified N,S-CDs are applicable in detecting E. coli in commercially bottled mineral water with a 1 limit of detection at 3.04 cfu mL− . The recent approach can be considered as a background for future detection of pathogenic bacteria by employing fluorescence-based material.

Supplementary Materials: Table S1: Synthesis of N,S-CDs; Table S2: Synthesis of amikacin modified N,S-CDs, Figure S1: 1HNMR of Amikacin; Figure S2: 1HNMR of N,S-CDs; Figure S3: 1HNMR of amikacin modified N,S-CDs. Author Contributions: F.A.R.P. synthesized and characterized CDs as well as data acquisition. M.M. contributed to the data analysis. K.M. reviewed and edited the manuscript. S.S. designed the experimental and wrote the initial manuscript. Funding: This research was funded by The Ministry of Research, Technology, and Higher Education of Republic Indonesia under the name of PTM Project grant number 2860/UN1.DITLIT/DIT-LIT/LT/2019. Acknowledgments: The authors are also thankful to Department of Chemistry, Universitas Gadjah Mada for providing facilities during the research and activities. Conflicts of Interest: The authors declare no conflict of interest.

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