Carbon dot-MnO2 FRET system for fabrication of molecular logic gates

Accepted Manuscript Title: Carbon dot-MnO2 FRET System for Fabrication of Molecular Logic Gates Authors: Jayasmita Jana, Teresa Aditya, Mainak Ganguly, Tarasankar Pal PII: DOI: Reference:

S0925-4005(17)30356-8 http://dx.doi.org/doi:10.1016/j.snb.2017.02.129 SNB 21860

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

9-12-2016 19-2-2017 21-2-2017

Please cite this article as: Jayasmita Jana, Teresa Aditya, Mainak Ganguly, Tarasankar Pal, Carbon dot-MnO2 FRET System for Fabrication of Molecular Logic Gates, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.02.129 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.

Carbon dot-MnO2 FRET System for Fabrication of Molecular Logic Gates Jayasmita Jana,a Teresa Aditya,a Mainak Ganguly,b Tarasankar Pala* a

Department of Chemistry, Indian Institute of Technology, Kharagpur-721302, India

b

Department of Chemistry, Furman University, Greenville, South Carolina 29613, United States

*

Corresponding author. Phone: +91-03222 283320. E-mail: [email protected]

Graphical abstract

In situ generated carbondot-MnO2adduct works as a FRET system and paves the way to fabrication of molecular logic gates.

1

Highlights   

Carbon dot-MnO2 adduct has been prepared in situ that works as good FRET system. NAC sensing by ‘Turn On’ phenomenon. Molecular logic operations on the FRET system using NAC and H+ as inputs.

Abstract A stable deliverable solid, carbon dot-MnO2 adduct, has been exclusively synthesized from a solution phase redox reaction between KMnO4 and moderately fluorescing carbon dot. Here, fluorescence resonance energy transfer (FRET) phenomenon happens within the adduct where energy is transferred from carbon dot to MnO2 making the solid adduct nonfluorescent. The as-obtained non-fluorescing adduct can be used for N-acetyl-L-cysteine (NAC) detection (linear detection range is 67 nM - 0.11 mM and limit of detection is 1.27 nM) through fluorescence “Turn On” phenomenon. Also in acidic medium the FRET system is disturbed and the fluorescence of carbon dots is regenerated. Inspired by these results, the carbon dot-MnO2 adduct was engaged to perform preliminary logic operations, namely YES and AND using H+ and NAC as inputs. Due to its fast response and ease of operation, the in situ generated carbon dot-MnO2 adduct could be useful to design a molecular device for biomedical research. Also the as-synthesized carbon dot system was utilized to design NOT and IMPLICATION logic gates using KMnO4 and NAC as inputs. Keywords: FRET; Turn On; molecular logic gate; NAC 1. Introduction In recent years, the molecular logic gates, that are capable of performing Boolean logic operations in response to physical, chemical and biological inputs, have become veryinteresting topic for research in information technologyfor miniaturization and function density [1]. There are some reports of using proteins [2, 3], nucleic acid [4], carbon dots [5] to construct fundamental 16 logic gates and higher functions.Logic gates with one or more than one inputs have been developed to operate at the nano dimension [6]. N-acetyl-L-cysteine (NAC), the acetyl derivative of L-cysteine is biologically important compound. It works as an antioxidant due to presence of a sulfhydryl group and eventually becomes the reduced glutathione. NAC can scavenge free radicals and reactive 2

oxygen species which consume directly superoxide anion or hypochlorous acid and can act as a prospective radiation protector [7]. NAC is vastly used in pharmaceutical preparations for the treatment of influenza, smoking cessation, Sjogren’s syndrome, myoclonus epilepsy, hepatitis C, and hepatotoxicity due to acetaminophen overdose [8]. Also it is used as mucolytic agent to reduce the viscosity of pulmonary secretions in chronic respiratory illness. Several chronic and fatal diseases like cancer [9], cardiovascular and respiratory diseases [10], human immunodeficiency virus (HIV) infection [11], acetaminophen toxicity [12], neurodegenerative disorder [13] and the other diseases caused by free radicals production and oxidative damage can be treated using NAC. Also gastric, kidney and heart related problems can be treated using NAC. Such an important compound is determined by potential and electrochemical techniques [14-17]. However, NAC has a large oxidation overpotential which produces weak voltametric signals on unmodified electrode surface. To avoid this problem several physically and chemically modified electrodes are prepared [18,19]. Besides this certain fluorimetric [20], chromatographic [21], UV-Vis spectrophotometric [22], UPLCMS [23] techniques are employed for NAC sensing. Fluorescent carbon dots (CDs) have become another wonderful discovery of nanoscience [24]. Although they are generally considered as carbonaceous counterpart of the quantum dots, CDs are more advantageous for being cost effective, cytocompatible and high photostability [25]. CDs are used in catalysis, sensing, bioimaging, drug delivery and optoelectronic devices and they are also non-toxic [26]. The fluorescence of CDs originated from radiative recombination of electron-hole pair, emissive energy traps on the surface, quantum confinement, surface functional groups etc. [27-30]. CDs are good energy donors and they can be easily engaged into designing of certain energy donor-acceptor type assay in the presence of suitable acceptors. Fluorescence resonance energy transfer (FRET) is a kind of phenomenon that involves non-radiative energy transfer from a fluorescent donor to an energy acceptor while close proximity of 1–10 nm is encountered. As FRET technique possesses high sensitivity, it has been widely utilized in the fields of immunoassays, nucleic acid hybridization and interaction of bio macromolecules [31]. MoS2 [32], WS2 [33], MnO2 [34] etc. can be such energy acceptors due to their intriguing and unravellingenergy absorption capability and fast electron transfer rate. There are certain reports where such FRET assays are used for sensing of biomolecules like glutathione (GSH) [34], glucose [35] etc.

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In our study we have used a FRET assay based on undoped CD and in situ generated MnO2adductin aqueous medium to detect NAC. MnO2nanostructures have been widely studied due to their potential application in catalysis, supercapacitor, battery, electrocatalysis, and gas sensor research [36]. CDs are synthesized from aqueous solution of ascorbic acid under modified hydrothermal treatment. The as-synthesized undoped CDs readily reduce KMnO4 to MnO2 and a CD-MnO2 adduct is generated in situ. Subsequently the inherent fluorescence of CDs is quenched. This quenched fluorescence is regenerated in the presence of NAC paving a solid platform for NAC sensing down to nanomolar (nM) level. To the best of our knowledge this is the first report of fluorometric NAC sensing using carbon dot-MnO2 adduct, a stable deliverable solid, exploiting the fluorescence generation out of FRET way of assessment. Also when the medium is made acidic the FRET system is ruptured fluorescence enhancement happens. So, using H+ and NAC as inputs we have constructed two kinds of logic gates. When individually they are used as single input we get YES gate and when they are subsequently used as inputs i.e. two input system, AND gate is constructed. Also the assynthesized CDs can also be used to design logic gates, namely NOT (H+ is single input) and IMPLICATION (H+ and NAC are two inputs) gates. 2. Experimental section 2.1. Chemicals and materials All the reagents used throughout the experiment were of AR grade. Triple distilled water was employed during the experiment. Ascorbic acid (AA), glutathione (GSH), Nacetyl-L-Cysteine (NAC), sodium chloride (NaCl), potassium chloride (KCl), iron(III) chloride hexahydrate (FeCl3.6H2O), magnesium sulfate (MgSO4),zinc chloride (ZnCl2)and all the amino acids were purchased from Sigma-Aldrich. All the reagents were used in the experiment without further purification. All glassware were cleaned with freshly prepared aqua regia, rinsed with sufficient amount of distilled water, and dried well before use. Urine sample was collected from a healthy donor. NAC containing oral pill ‘Keraglo Eva’ (Ipca Labs) was bought from the local market. 2.2. Instrumentation All UV−vis absorption spectra were recorded in an Evolution 201 spectrophotometer (Thermo Scientific). The fluorescence measurement was done at room temperature using a LS55 fluorescence spectrometer (Perkin-Elmer, Waltham, MA). X-ray photoelectron spectroscopy (XPS) was done with a VG Scientific ESCALAB MKII spectrometer (U.K.) 4

equipped with a Mg Kα excitation source (1253.6 eV) and a five-channeltron detection system. Fourier transform infrared (FTIR) studies were carried out with a Thermo-Nicolet continuum FTIR microscope. Transmission electron microscopy (TEM) analysis was performed with a H-9000 NAR instrument, Hitachi, using an accelerating voltage of 300 kV. Field emission scanningelectron microscopy (FESEM) was done with a supra 40, CarlZeiss Pvt. Ltd. instrument. Fluorescence lifetimes were measured with Easy lifeV (Optical Building Blocks Corporation) equipped with a 380 nm LED excitation source. A nonlinear least squares (χ2) fit was tested to determine the fit of the decay rate to a sum of exponentials and a visual inspection of the residuals and the autocorrelation function were used to determine the quality of the fit. The samples were taken in a quartz cuvette of 1 cm path length for fluorescence measurement. 2.3. Synthesis of carbon dots Ascorbic acid (AA) mediated CDs were synthesized by treating 0.01 M ascorbic acid in N2 environment under our lab designed modified hydrothermal treatment (MHT). In the typical synthesis 10 mL freshly prepared AA was poured into a 15 mL borosil screw capped test tube and N2 gas was injected for 20 minutes. Then the screw cap test tube underN2 was kept in front of 200 W bulb at a distance of 3 cm for 6 hours. The whole system was fitted in a 1 ft × 1ft × 1 ft closed wooden box. This method of heating a reaction mixture is termed as modified hydrothermal (MHT) reaction at ~180ºC [37, 38]. A pale yellow coloured solution was obtained after 6 hours that showed fluorescence emission at 443 nm when excited at 360 nm. The pH of the solution was found to be 6. This solution was taken for further experiment. 2.4. Synthesis of CD-MnO2 An aliquot of 0.4 mL of CD solution was taken in 3 mL vial. Then freshly prepared aqueous KMnO4 was added slowly. After some time (~20 min) a solid blackish-brown precipitation was observed. The precipitation was then washed thoroughly with distilled water and kept for further use. 2.5. Quantum yield Measurement The quantum yield (φ) of CDs was measured according to a reference point method. Quinine sulfate in 0.1m H2SO4 (literature quantum yield 0.54) [39] was used as the standard reference. The quantum yield of a sample (sl) was measured with respect to the known quantum yield of the standard reference (st) using the following formula, φsl=φst(Fsl/Fst)( η2sl/ η2st)(Ast/Asl) Where Fis the fluorescence intensity, η is the refractive index of the solvent, and Ais the optical density. The subscript "st" refers to standard with known QY and "sl" for the sample 5

3. Results and discussions 3.1. Synthesis and characterization of carbon dot Most of the carbon dot synthesis ends up with heteroatom doping. The chosen heteroatoms are generally nitrogen/sulfur/boron/phosphorous/copper etc. [40-43] and upon doping they become fluorescent. However, there are only a few reports of employment of undoped fluorescent carbon dots varying applications. Here in this work we have reported ascorbic acid (AA) mediated undoped carbon dots synthesis from anaerobic environment under our lab designed modified hydrothermal reaction condition in aqueous mediumusing a simple screw captest tube of 15 mL volume. The generated pale yellow coloured solution with undoped carbon dots (ACD) is fluorescent. The particles, ACD emit at 443 nm when the solution is excited at 360 nm (Fig. 1). Absorption spectrum of the ACD containing yellow solution shows a strong peak at 246 nm and a feeble peak appears at 282 nm which corresponds to n-σ* and n-π* transitions respectively. The measured quantum yield is 4.8% and the lifetime is 5.43 ns (Fig. S1). TEM image shows that the average particle size of ACDs is ~7 nm (Fig. 2). FTIR spectra show sharp peaks for C-O, C=C, C=O and C-H/O-H bonds (Fig S2, Supporting Information). XRD pattern shows a broad peak at 23.2o indicating the presence of (220) plane (Fig. S2). From XPS spectra, we find peaks at 284.05 eV and 532.09 eV for C1s and O1s respectively. Further deconvolution study shows the presence of peaks for C=C, C-O and C=O bonds (Fig. 2). Under MHT, AA undergoes carbonization to form tiny fluorescent particles. However, in this work the CDs are not surface passivated. So the quantum yield as well as lifetime values are quite low [44]. ACD is quite stable in terms of emission profile; even after 3 months of aging at room temperature, fluorescence remains unchanged (Fig. S3). 3.2. Synthesis and characterization of carbon dot-MnO2 adduct As expected the fluorescence of ACD containing yellow solution is drastically quenched right after the addition of sufficient amount of KMnO4 and a brown solid precipitation is obtained. Kinetic studies (relative intensity vs. time plot) show that the fluorescence of ACD containing solution is decreased gradually and completely quenched within 20 min (Fig. 3A).The system becomes nonfluorescent. When ACD containing solution is added to KMnO4, the characteristic absorption peak for MnO4- disappears and a new broad band over 300 nm to 600 nm for Mn(IV) is observed (Fig. 3B). After the introduction of sufficient amount of KMnO4 no further change of the broad band occurred. This indicates the 6

completion of redox reaction between ACD and KMnO4. From TEM image we see an aggregated structure (Fig. 3C) for the brown precipitate. FTIR studies show the presence of Mn-O, C-O-C and C-O bands at 510 cm-1, 1067 cm-1, and 1640 cm-1 respectively [45] (Fig. S4). XPS study of the obtained solid brown product shows peaks at 84.5 e and 89.9 eV for Mn3s and 641.1 eV, 652.7eVfor Mn2p3/2 and Mn2p1/2. XPS peak at 284.3 eV and 530 eVshows the presence of C1s and O1s. Further deconvolution of elemental C shows the presence of C=C, C-O, C=O and C-C=O bonds (Fig. 4 inset). Also the deconvolution of O1s spectra shows the presence of Mn-O, Mn-O-C and C-O bonds (Fig. 4). XRD data shows peaks at 2θ = 20.1, 25.9, 37.5, 39.9, 41.2, 51.7, 65.1 and 70.6 for (200), (220), (211), (330), (420), (440), (002) and (541) for MnO2 [JCPDS file no. 44-0141]. TEM and FESEM images show an aggregated structure (Fig. S5). Thus we conclude that the product is anACD-MnO2 adduct. As ACD is engaged in such interaction, the fluorescence is quenched. It is found that the supernatant is nonfluorescent. Further addition of KMnO4 to the supernatant shows up KMnO4 colour only (confirmed from visible band of KMnO4) and no precipitate is generated. This indicates complete and quantitative attachment of ACD with MnO2. Washing and repetitive sonication caused no change of the ACD-MnO2 adduct indicating the stability of the adduct. This is due to the ACD surface mediated redox reaction. Only conc. HCl washing disintegrates the ACD-MnO2 association leaving ACDs apart after the dissolution of MnO2. However the fluorescence of the conc. HCl added ACD-MnO2 system is lower compared to the inherent fluorescence of the as-synthesized ACD. As in HCl added ACD-MnO2 system, the free ACD is the fluorescing species, the system will behave as acidified ACD system only. It is presumably due to the protonation induced inhibition of the emissive sites of ACD resulting in the decrease in fluorescence intensity [46]. It is found that the emissive behaviour of the as-synthesized ACD in acidic medium (pH=2.1) is quite similar to the ACD-MnO2 solution washed with conc. HCl (pH=2.1). Fig. S6A shows the fluorescence spectral profile of as-synthesized ACD, ACD at pH=2.1 and ACD-MnO2 with conc. HCl (pH=2.1). It should be mentioned that in with the increase in OH- ion concentration in medium, the fluorescence of ACD improves but the fluorescence of ACD-MnO2 does not change. Again we have performed the same experiment employing commercially available active carbon (AC) suspension (0.1 gm/3 mL) in water. It was revealed that AC does not reduce KMnO4 solution which was authenticated from absorbance measurement (Fig. S7).Thus the importance of trapped electrons in ACD for the observed redox reaction is proved. 3.3. Energy transfer phenomenon within the adduct 7

The mechanism of the observed redox reaction as well as in situ Fluorescence Resonance Energy Transfer (FRET) proposition is quite interesting. It was indicated by Lin et al. that the N doped CDs reduce KMnO4 to water soluble Mn(II) [47]. This results in chemiluminescence from CD. They have assumed that oxidant KMnO4 injects hole into CDs and they in turn get reduced. In the present case, as-synthesized ACD reduces KMnO4 to MnO2 and then the presence of MnO2 ceases the emission of ACD as MnO2 compels the ACD to enter into a donor-acceptor interaction as evident from the experimental results. It is interesting to see that doped and undoped CDs show different reduction ability towards KMnO4. The reduction capability depends on the redox potential of the moiety. The formal potential of ACD is -0.19 V. We have synthesized N doped CDs (NCD) using NH4Br and ascorbic acid to compare the redox potential values of doped and undoped CDs. Our synthesized NCD possess a formal potential -0.209 V. From the potential values it is clear that NCD has higher reducing ability than ACD. However we have worked with ACD to see the activity of undoped carbon dots. In the present case, ACD reduces KMnO4 to MnO2 and finally the ACD-MnO2 adduct is formed. The absorbance spectrum of MnO2 is quite broad (300 nm to 600 nm) which overlaps with the fluorescence excitation and emission of ACD causing quenching of emission of ACD (Fig. S8). The in situ generated MnO2 acts as energy acceptor while ACD works as energy donor and efficient FRET scenario sets in. Subsequently, concentrations of the donor ACD and the acceptor MnO2 are optimized. The experiment is shown in Fig.5 where fluorescence of the ACD (0.40 mL) is significantly quenched with the gradual addition of KMnO4 from 0 to 2 mM, and then reaches a plateau even with higher concentrations. Also a point should be noted that if there remains excess KMnO4 in solution, it is just expelled out by repetitive washing with water. The maximum % fluorescence quenching efficiency of this system is observed to be 98.6% [quenching efficiency = I/I0, Fig. 5]. The fluorescence lifetime measurement shows the lifetime values for ACD and ACD–MnO2 adduct are 5.43 ns and 0.49 ns respectively (Fig. S9). The energy transfer efficiency (E) of the process can be calculated from the lifetime values of donor (τD) and donor-acceptor (τD-A) species using the following equation [48] E = 1 – (τD-A / τD) In the present study the E value is found to be 0.9097. Thus the FRET efficiency 90.97 % is in good agreement with the calculated quenching efficiency (98.6 %). This is a kind of 8

inherent FRET property which is reported here. Previously Achilleos et al. [49] have reported inherent FRET for core-shell particles. However in our case the FRET distance (R) has not been measured. The measurement of R depends on the value of R0 (R0 is the distance at which 50% of the energy is transferred) with some precision. R = R0[(1-E)/E]1/6 The value of R0 requires the knowledge of proper orientation of donor [50]. In an aggregated adduct measurement of such orientation is not possible. 3.4. Detection of N-acetyl-L-cysteine This MnO2 induced quenching can be reversed by using N-acetyl-L-Cysteine (NAC). This may be due to reduction of MnO2 to Mn2+ by NAC. In this process NAC is converted to its disulfide form [19]. The plausible reaction is as follows

2

+ MnO2+ 2H+

+ Mn2+ + 2H2O

Due to disappearance of MnO2, ACD is now free and the fluorescence of ACD is restored. From the TEM image (Fig. 6A) we confirm the degradation of adduct and the presence of tiny ACD particles in the system. The presence of stretching frequency at 493 cm-1 corresponding to S-S bond in the FTIR spectra [51] of ACD-MnO2-NAC system indicates that the interaction follows the above mentioned path (Fig. S10). With gradual increase in NAC concentration, the fluorescence of the system is gradually increased. Thus NAC can be sensed by a ‘Turn On’ phenomenon. The kinetics of this experiment is measured by monitoring change in fluorescence with respect to time. It is seen that fluorescence increases initially and a stable value is obtained after five minutes of waiting. Thus, an equilibrium time of five minutes is considered for the determination of NAC (Fig. 7A). We can detect NAC down to 6.7 × 10-9 M. The fluorescence enhancement is found to be linearly dependent on the concentrations of NAC ranging from 67 nM to 0.11 mM with a perfect correlation coefficient of 0.98 (Fig. 7C and D) indicating the potential of this system for quantitative analysis of NAC. The limit of detection has been calculated to be 1.27 nM according to 3σ (signal to noise) criteria [52]. Table S1 represents a comparative account of the NAC sensing in this context which shows that our prescribed process works well than some conventional methods. The interference of different amino acids (4-hydroxy proline, alanine, arginine, 9

aspargine, aspartic acid, Cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, serine, threonine, tyrosine, valine) GSH, Ca(II), Fe(III), K(I), Mg(II), Na(I) and Zn(II)ions have been investigated (Fig. 8A). Simultaneous addition of these compounds with NAC does not hamper fluorescence enhancement. However, it is found that GSH causes interference by exhibiting similar enhancement in fluorescence at 443 nm. This interference is removed by swiping excitation wavelength. By sweeping the excitation wavelength, we have found that 320 nm is an appropriate wavelength at which selectively ACD-MnO2-NAC system shows emission at 420 nm while ACDMnO2-GSH system shows emission at 430 nm proving that 320 nm is the best-suited excitationwavelength to overcome the interferences due to GSH (Fig. 8B). Thus NAC sensing through the prescribed method is made interference free. This newly prescribed sensing technique of NAC is utilized to detect NAC in pharmaceutical samples. In this respect an oral pill named ‘Keraglo Eva’ was examined. This medicine is used to treat hair fall problems. The medicine contains 0.05 g NAC/0.26984 g pill (Fig. S10). The recovered NAC concentration goes good with the claimed concentration of NAC in the pill with a percentage relative standard deviation (% RSD) of 0.46% (Table S2). In another piece of experiment, NAC sensing was done in the urine sample collected from a healthy donor. NAC can be used to cure urinary tract infections caused by bacteria [53]. We have determined the amount of NAC in the urine sample using standard addition method. The collected urine sample was diluted 100 times for the detection of spiked NAC in the sample. Then by using ACD-MnO2 system, NAC was recovered to a satisfactory extent from different sets of experiment (Table S3). This experiment was repeated for five times to ensure the reproducibility of the prescribed method. The as-synthesized ACD-MnO2 adduct can be used for clinical purpose. Thus the prescribed method of NAC determination is competently applied for pharmaceutical drug samples and real samples. 3.5. Fabrication of molecular logic gates Molecular logic gate with biological and chemical compounds as inputs have emerged as an alternative for silicon based logic gates. The molecular logic gates are generally used in biocatalysis, biomolecular recognition, biosensing, etc. processes using simple Boolean operations. Different molecular logic gates are used in practical life. Here are few instances. Wang et al. [54] constructed cellular AND gate for the detection of arsenic (As3+) and mercury (Hg2+). Chen et al. had used two-analyte logic gates OR and AND to devise a strip logical system for proteins and small molecules based on target-induced assembly of split 10

aptamers [55]. Kahan-Hanum et al. [56] had used cellular miRNAs and mRNAs to construct a programmable DNAzyme library composed of various Boolean logic gates, including YES, AND, NOT, OR, NAND, ANDNOT, XOR, NOR and 3-input-AND gate. Biomolecular logic based biosensors are used for in vivo detection. In this area the biofuel powered sensor may be helpful as shown by Zhou et al. [57]. Different field will be significantly benefitted using the digital system with proper analytical data. In this work we have constructed YES, NOT, AND, IMPLICATION logic gates using KMnO4, NAC and H+ as inputs for ACD as well as ACD-MnO2 system. In this experiment two types of signal (0 and 1) for single input and four types of signals (0,0; 0,1; 1,0; and 1,1) from binary input are used to obtain output. ACD is fluorescent and for this case when the fluorescence is switched on, output =1. H+ (input = 1) lowers fluorescence of ACD so output = 0. Thus a NOT gate has been constructed. Again on the basis of the fact that, consecutive addition of H+ (Input 1) and NAC (Input 2) cannot improve the fluorescence (output = 0), IMPLICATION logic operation has been implemented employing ACD as gate (Fig. 9). ACD-MnO2 itself is nonfluorescent so in absence of any of the inputs the output = 0. When pH is decreased (presence of excess H+) the fluorescence is recovered, output = 1 and in the presence of NAC the fluorescence ‘Turn On’ happens, output = 1. Thus with H+ and NAC as single inputs YES logic operation can be done with ACD-MnO2 adduct as gate. Again, simultaneousaddition of both the inputs (H+ and NAC) causes enhancement in fluorescence intensity, then output =1. Thus the AND logic operation has been accomplished using ACD-MnO2 adduct as gate (Fig. 10). Hence, by utilizing the FRET system, different logic operations have been performed. 4. Conclusion In a nutshell, a new platform for free electron mediated KMnO4 reduction in solution phase has been reported. The chosen carbon dot vs. KMnO4 redox system leaves an adduct enabling FRET to appear in situ. Sensing of NAC through fluorescence ‘Turn On’ phenomenon has been key information using carbon dot-MnO2 adduct where donor-acceptor relationship is established beyond doubt.Production, separation and purification of the material is simpleand user friendly which becomes a suitable material for successful fabrication of molecular logic gates. Acknowledgements

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The authors are thankful to the UGC, DST, NST, and CSIR, New Delhi, India, and the IIT Kharagpur for financial assistance.

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[50]

Jayasmita Jana is a Ph.D student at Department of Chemistry, IIT Kharagpur, India. Her research interest is focussed on the synthesis and characterization of carbon dots from biocompatible materials. Teresa Aditya is a Ph.D student at Department of Chemistry, IIT Kharagpur, India. Her research interest is focussed on the study of the morphologically different metal oxide nanostructures and their applications. Dr. Mainak Ganguly did his postdoctoral research at Department of Chemistry, Furman University, United States. His research interest is focussed on the synthesis of fluorescent clusters and sensing. Dr. Tarasankar Pal is a Professor at Department of Chemistry, IIT Kharagpur, India. His research interest is focussed on Inorganic Chemistry, Synthesis of Metal & Oxide Nanoparticles, Homo- & Heterogeneous Catalysis, Surface Enhanced Raman Scattering (SERS) Study, Luminescence Spectroscopy, Analytical and Environmental Chemistry, Micellar Chemistry, Supercapacitor, Electrochemical Catalysis.

15

Figure Legends

Fig. 1: Spectral profile of as-synthesized ACD in solution. (a) Absorption profile; (b) excitation profile and (c) emission profile with the respective λmax values. Fig. 2: (A) TEM image of ACD obtained from aqueous solution of ascorbic acid after MHT. (B) Broad range XPS spectra of as-synthesized ACD. Narrow range XPS for (C) C1s and (D) O1s of the ACD. Fig. 3: (A)Relative fluorescence intensity vs. time plot for kineticstudy of ACD-MnO2 formation. Condition: λex = 360 nm, room temperature, [ACD] = 0.40 mL in 3 mL water, [KMnO 4] = 3.3 × 10-4 M. (B) Absorption spectra of KMnO4 and ACD-MnO2. (C) TEM image of ACD-MnO2. (D) Digital image showing steps for the formation of ACD-MnO2 from ACD at different time. Fig. 4: XPS spectrua of ACD-MnO2adduct (A) broad range spectrum (Inset: narrow range XPS for C1s of ACD), narrow range XPS of (B) O1s, (C) Mn2p and (D) Mn3s of ACD-MnO2 adduct. Fig. 5: (A) Fluorescence spectral profile for ACD and ACD-MnO2adduct, (B) Experimentally recorded relative fluorescence intensity change of ACD upon the addition of different concentrations of KMnO4, indication of ACD-MnO2 adduct formation through successive fluorescence intensity quenching. I= fluorescence intensity of ACD-MnO2 and I0= fluorescence intensity of ACD.Condition: λex = 360 nm, room temperature, [ACD] = 0.40 mL in3 mL water. Fig. 6: (A) TEM image of ACD-MnO2-NAC system. Fig. 7: (A) Relative fluorescence intensity vs. time plot for kinetic study of the interaction between as-synthesized ACD-MnO2 and NAC. (B) Bar diagram showing the effect of different bio-compounds on the fluorescence of ACD. Control is only ACD-MnO2 adduct. I= fluorescence intensity of ACD-MnO2-NAC/others and I0= fluorescence intensity of ACD-MnO2.Condition: λex = 360 nm, λem = 443 nm, room temperature, [ACD-MnO2] = 0.60 mL in 3 mL water, [NAC/others] = 6.7 × 10-4 M. (C) Fluorescence spectral profile of ACD-MnO2 in the presence of different concentrations of NAC. (D) Relative fluorescence intensity of ACD-MnO2 as a function of NAC concentration.(Inset: linear detection range). Condition: λex = 360 nm, λem = 443 nm, [ACD-MnO2] = 0.60 mL in 3 mL water, room temperature. Fig. 8: (A) Bar diagram showing the effect of simultaneous addition of bio-compounds and NAC. Blank is only ACD-MnO2 adduct. Condition: λex = 360 nm, room temperature, [ACDMnO2] = 0.60 mL in 3 mL water, [NAC/others] = 6.7 × 10-4 M. (B) Effect of NAC and GSH on the emission maxima of 3 mL of as-synthesized ACD-MnO2 at different excitation wavelengths, 280 nm, 320 nm and 360 nm. (C) Fluorescence spectral profile of ACD-MnO2 in the presence of NAC, GSH and both NAC-GSH at different excitation wavelengths, 320 nm and 360 nm. Condition:[ACD-MnO2] = 0.60 mL in 3 mL water, [NAC/GSH] = 6.7 × 10−4 M. Fig. 9: Fluorescence spectra of the (A) NOT, with KMnO4 as single input and (B) IMPLICATION gate with KMnO4 and NAC as binary inputs. (Inset in each case shows the corresponding column diagram of the relative fluorescence intensities: the dashed line shows the threshold). (C) Truth table and electronic equivalent circuitry for NOT and IMPLICATION logic operations on ACD. Fig. 10: Fluorescence spectra of the (A) YES, with H+ as single input, (B) YES, with NAC as single input and (C) AND gate with H+ and NAC as binary inputs. (Inset in each case shows the corresponding column diagram of the relative fluorescence intensities: the dashed line shows the threshold). (D) Truth table and electronic equivalent circuitry for YES and AND logic operations on ACD-MnO2.

16

1.2 443 nm

363 nm

1.0

1.0

Normalized absorbance (a.u.)

Normalized fluorescence intensity (a.u.)

1.2

246 nm absorbance emission excitation

0.8

a

0.6

282 nm

0.6

c

b

0.4

0.8

0.4

0.2

0.2

0.0 200

300

0.0 600

400 500 Wavelength (nm)

Fig. 1

A

O1s

Intensity (a.u.)

B

200

C1s

300

400

500

600

Binding energy (eV)

C1s

O1s

C

D

C-O

C=O

282

285

288

Binding energy (eV)

Intensity (a.u.)

Intensity (a.u.)

C=C

528

532

536

Binding energy (eV)

Fig. 2

17

1.2

Normalized absorbance (a.u.)

A

C

B

1.0 0.8

KMnO4 ACD-MnO2

0.6 0.4 0.2 0.0 200

300

400

500

600

700

Wavelength (nm)

D

Vacuum drying

KMnO4

ACD

RT

in aqueous medium

After 0 min of KMnO4 addition

Vacuum dried ACD-MnO2

ACD-MnO2 After 20 min of KMnO4 addition

Fig. 3

A

Mn2p3/2 Mn2p 1/2

B

O1s

O1s C1s C=C

C-O 285

Intensity (a.u.)

Intensity (a.u.)

Intensity (a.u.)

O-C=O

C=O

288 291 Binding energy (a.u.)

C1s

Mn-O

Mn-O-C

Mn3s

C-O

200

400

600

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800

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536

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D

Mn2p3/2 Mn2p1/2

Intensity (a.u.)

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Mn3s

E = 4.6 eV

E = 11.6 eV

84

640

645

650

Binding energy (eV)

655

Fig. 4

87 Binding energy (eV)

90

Fig. 4 18

B

1.0

70 60

ACD ACD-MnO2

0.8

50

I0/I

A

Normalized Fluorescence Intensity (a.u.)

80 1.2

0.6

40 30 20

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10 0.2

0 0.0

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450 500 Wavelength (nm)

550

-4

-3

-3

-3

5.0x10 1.0x10 1.5x10 2.0x10 2.5x10 Concentration M

Fig. 5

Fig. 6

19

-3

20

35

B

A

16

25

12

20

I/I0

I/I0

30

15

8 10 5

4

0

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2

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4

5

6

C

Normalized fluorescence intensity (a.u.)

Time (min)

1.2

NAC concentration variation

0M -9 6.7 x 10 M -8 6.7 x 10 M -7 6.7 x 10 M -6 6.7 x 10 M -5 6.7 x 10 M -5 3.8 X 10 M -4 1.1 X 10 M -4 3.8 X 10 M -4 6.7 X 10 M

1.0 0.8 0.6 0.4

D

8

6

4

3

2

2

R = 0.98

I/I0

0

I/I0

0

Control Alanine Agrinine Asparagine Aspartic acid Cysteine Glycine Glutamic acid Histidine 4-Hydroxy proline Isoleucine Leucine Lysine Methionine Phenyl alanine Serine Threonine Tyrosin Valine GSH NAC Ca(II) Fe(III) K(I) Na(I) Mg(II) Zn(II)

ACD-MnO2-NAC

2

1

0.2 0.0

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450

500

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-5

8.0x10

-5

1.2x10

Concentration M

0 -4

2.0x10

-4

4.0x10

-4

6.0x10

Concentration (M)

Wavelength (nm)

Fig. 7

20

-4

Emission wavelength (nm) Emission wavelength (nm)

B 450 450

440 440

430 430

420 420

390 390 NAC NAC GSH GSH

410 410

400 400

280 280 300 320 340 360 300 320 340 360 Excitation Wavelength (nm) Excitation Wavelength (nm) Normalized fluorescence intensity (a.u.)

Blank NAC+Alanine NAC+Agrinine NAC+Asparagine NAC+Aspartic acid NAC+Cysteine NCA+Glycine NAC+Glutamic acid NAC+Histidine NAC+4-Hydroxy proline NAC+Isoleucine NAC+Leucine NAC+Lysine NAC+Methionine NAC+Phenyl alanine NAC+Serine NAC+Threonine NAC+Tyrosin NAC+Valine NAC+GSH NAC+Ca(II) NAC+Fe(III) NAC+K(I) NAC+Na(I) NAC+Mg(II) NAC+Zn(II)

I/I0 A 35

30

25

20

15

10

5

0

1.2

1.0

0.0 350

C exnm (1) ACD-MnO2

(2) ACD-MnO2-NAC

(3) ACD-MnO2-GSH

0.8 (4) ACD-MnO2-NAC+GSH exnm (5) ACD-MnO2

0.6 (6) ACD-MnO2-NAC

(7) ACD-MnO2-GSH

0.4 (8) ACD-MnO2-NAC+GSH

0.2

400 Wavelength (nm) 450 500 550

Fig. 8

21

0.0

0

1

0.4 ACD ACD+KMnO4

0.2 0.0 400

450

500

Input

550

0.0

0.6

Input

ACD ACD+KMnO4 ACD+NAC ACD+KMnO4+NAC

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(1,1 )

0.6

(0,1 )

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0.5

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(0,0 )

1.2

Normalized fluorescence intensity (a.u.)

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1.2

I/I0

Normalized fluorescence intensity (a.u.)

A

450

500

550

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C

Fig. 9

22

0

1

ACD-MnO2

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+

ACD-MnO2 + H

0.2 0.0 400

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Input

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Fig.10

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(1, 1)

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Normalized fluorescence intensity (a.u.)

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A

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I/I0

Normalized fluorescence intensity (a.u.)

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Input