A rhodamine embedded bio-compatible smart molecule mimicking a combinatorial logic circuit and ‘key-pad lock’ memory device for defending against information risk

Abstract

Organic molecules with the possibility of logic operations are highly useful building blocks for the development of molecule-based “intelligent” devices for information processing applications. We have designed herein a very simple bio-friendly chemosensor (L^C) equipped with a rhodamine fluorophore moiety. This probe showed a chromo-fluorescence response profile for Al³⁺ but a colorimetric response for Cu²⁺ metal. The absorption responses of L^C caused by these metal ions along with the “OFF–ON–OFF” fluorescence behavior of an L^C–Al³⁺ complex towards EDTA were employed for the development of a three-input and one output combinatorial molecular system. Interactions of the mentioned metal ions with L^C in controlled sequential experiments gave fluorescence responses, enabling us to fabricate a ‘key-pad-logic’ function. So, a single molecular system performing such multiple ‘Boolean’ operations not only simplifies the complexity of a chemical driven ‘Intelligence’ device but also enriches the security of such a device against information invasion due to the sequence controlled sensor–analyte interactions and may find potential applications in biocompatible molecular logic platforms.

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Introduction

In recent years the development of fluorescent molecular sensors triggered by several input signals and their applications in computation and analytical devices is gaining more and more research interest.¹ Their most striking feature is their ability to imitate the function of electronic logic gates and circuits² which makes them suitable for cellular imaging,³ clever chemosensing⁴ and digital molecular tags.⁵ Since the first pioneering proposal of a molecular AND gate by de Silva and co-workers,^2a a variety of molecule-based systems capable of acting as Boolean logic gates like AND, OR, NOT, INH, NOR, XNOR, XOR etc. have been realized with the prospect of creating more sophisticated and complex molecular circuits.^2c,6 It has been argued that the limitations of conventional silicon based devices in miniaturization down to the nanoscale level could be addressed successfully in a wireless manner with the appropriate design of molecular operators capable of executing various complicated and integrated logic operations.

In addition, recent interest has focused on a new strategy that enables the high-throughput, multi-analyte sensing shown by a molecule to be used to fabricate a combinatorial device.⁷ The combination of independent molecular gates as basic logic operators could in principle allow one to obtain any combinatorial logic circuit programmed into a single molecular switch. Such molecular switches can be used in complex circuits in the design and development of molecular electronic and photonic devices for information processing, sensing, and computation.⁷ So, it is necessary to design a chemical system that can respond to multiple inputs in order to improve complexity within the entire circuit of a single molecular switch.

But the possibility to develop such complex molecular logic systems along with the concept of constructing a sequence dependent memory device-like key-pad logic function within a single molecular system is more appealing and at the same time very challenging.^8–10 Among these various memory devices, molecular keypad systems have drawn more attention; as these systems are based on a sequence of a correct combination of chemical inputs that signifies a more secure and classified password, providing considerable advantages over many other simple molecular logic gate devices.⁹ As we know, the logic circuit behind the keypad of a bank machine is of the sequential type that remembers a “three key-strokes history” as well as reading the fourth number in the PIN-code before generating the output response to authorize the user. Given the correct input sequence, the output of the keypad lock is switched to the ‘on-state’, thereby authorizing the user to initiate further processing, and strengthening security to defend the hardware of computing devices as well as the confidential data stored within it against information invasion during its execution and communication processing. Such sequential integration of molecular logic gates brings about the realization of information storage processes.

Due to a poor coordination ability and a lack of spectroscopic characteristics, the detection of Al³⁺ has always been a problematic task.¹¹ The detection of Al³⁺ is of great interest due to its potential toxicity arising out of its widespread applications in automobiles, computers, packaging materials, electrical equipment, machinery, food additives and building construction.^12,13 It is well known that 40% of soil acidity is due to aluminium toxicity.¹⁴

A number of analytical methods like chromatography,¹⁵ accelerator mass spectroscopy (AMS),¹⁶ graphite furnace atomic absorption spectrometry (GFAAS),¹⁷ neutron activation analysis (NAA),¹⁸ inductively coupled plasma-atomic emission spectrometry (ICP-AES),¹⁹ inductively coupled plasma-mass spectrometry (ICP-MS),²⁰ laser ablation microprobe mass analysis (LAMMA),²¹ electrothermal atomic absorption spectrometry (ETAAS),²² fluorescence chemosensing etc. are available for the detection of Al³⁺; out of these the last one is the best choice as it is the simplest, it is a sensitive, fast and inexpensive technique²³ offering significant advantages over other methods.²⁴ Hence, recently the design and synthesis of Al³⁺ selective fluorescent probes has received intense attention from chemists.^25–27 Compared to other metal ions, only a few fluorescent chemosensors have been reported for the detection of Al³⁺,^28–33 and most of the sensors are insoluble in polar solvents and require complicated synthetic procedures. For practical applications, it is necessary to develop Al³⁺ sensors that are easily prepared and possess selective and sensitive signalling mechanisms.

We demonstrate herein a new simple bio-compatible chemosensor equipped with rhodamine to mimic the advanced arithmetic logic operation within a combinatorial logic circuit as well as the memory logical function by investigating absorbance as well as fluorescence “ON and OFF” states as outputs with controlled experiments based on sensor–analyte interactions at physiological conditions. Such a molecular ‘Boolean’ logic operation improves not only the complexity of logic gate circuitry but also can be used in potential biocompatible molecular logic platforms. More importantly, it may be applied to ensure information security against the illegal invasion of data with respect to its sequence dependent key-pad logic function.

Experimental section

Materials and methods

All solvents used for the synthesis of ligands were of reagent-grade (Merck) unless otherwise mentioned. For spectroscopic (UV/Vis and fluorescence) studies HPLC-grade MeOH and double-distilled water were used. Rhodamine 6G hydrochloride and metal salts such as the perchlorates of Na⁺, K⁺, Ca²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Cu(NO₃)₂·2H₂O were purchased from Sigma-Aldrich and used as received. All other compounds were purchased from commercial sources and used as received.

Physical measurements

Elemental analyses were carried out using a Perkin-Elmer 240 elemental analyzer. Infrared spectra (400–4000 cm⁻¹) were recorded in the liquid state on a Nicolet Magna IR 750 series-II FTIR spectrometer. ¹NMR spectra were recorded in CD₃OD or DMSO-d₆ on a Bruker 300 MHz NMR spectrometer using tetramethylsilane (δ = 0) as an internal standard. ESI-MS⁺ (m/z) of the ligand and Al³⁺-complex were recorded on a Waters' HRMS spectrometer (Model: XEVO G2QTof). UV-Vis spectra were recorded on an Agilent diode-array spectrophotometer (Model, Agilent 8453). Steady-state fluorescence measurements were performed with a PTI QM-40 spectrofluorimeter.

Syntheses and characterization

Preparation of N-(rhodamine-6G)lactam-ethylenediamine (L^A). L^A was prepared according to a literature method.³⁴

Preparation of 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one³⁵ (L^B). 390 mmol of freshly vacuum distilled ethylacetoacetate and 0.3 g of sodium bicarbonate were heated until the liquid reached 200–210 °C. The time for heating was usually 7–8 h, during which period a bulk of distillate (mostly ethanol) was collected and the color of the reaction mixture became dark brown (Scheme 1). The resulting reaction mixture was distilled under reduced pressure (the desired crude product was collected at 140 °C – 12 mm). The crude product was re-crystallized using ethanol (yield 48%) for the further steps of the synthesis.³⁵¹H-NMR in CDCl₃: 2.26 (s, 3H), 2.66 (s, 3H), 5.93 (s, 1H) (Fig. S1, ESI†); ¹³C: 20.66, 30.02, 99.81, 101.39, 161.17, 169.08, 181.03, 205.20 (Fig. S2, ESI†); molecular formula (C₈H₈O₄): C, 57.14; H, 4.80; found C, 57.12; H 4.79; ESI-MS⁺ (m/z): 169.13 (C₈H₈O₄ + H⁺) (Fig. S3, ESI†).

Scheme 1 Methodology adopted for the synthesis of the target ligand (L^C).

Preparation of the target molecule (L^C). 0.5 mmol (0.228 g) of N-(rhodamine-6G)lactam-ethylenediamine in MeOH (10 mL) was added dropwise over a period of 30 min to a 10 mL methanolic solution of 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one (L^B) (0.55 mmol, 0.428 g) containing 2 drops of acetic acid under hot (ca. 50–60 °C) conditions. Then the reaction mixture was stirred under reflux for 2 h. An off-white precipitate was formed, which was collected by filtration after cooling the solution The residue was washed thoroughly with cold methanol/ether (1 : 1 v/v) to isolate L^C in a pure form (78% yield). M.p. 214.4 °C. ¹H NMR (CD₃OD): δ = 1.28 (t, 6H), 1.82 (s, 6H), 2.12 (s, 3H), 2.29 (s, 3H), 3.26 (q, 4H), 3.29 (2H), 3.4 (t, 2H), 5.67 (s, 1H), 6.11 (s, 2H), 6.34 (s, 2H), 7.05 (m, 1H), 7.56 (m, 2H), 7.89 (m, 1H); (Fig. S4, ESI†). ¹³C-NMR: (DMSO-d₆) δ_ppm: 13.23, 15.55, 16.88, 18.14, 37.68, 38.35, 41.55, 65.65, 95.90, 96.15, 104.54, 106.96, 118.74, 122.39, 123.75, 127.65, 128.20, 130.51, 132.14, 148.17, 151.83, 153.54, 162.67, 134.62, 169.23, 176.59, 183.97 (Fig. S5, ESI†). CHN analysis for C₃₆H₃₈N₄O₅: calculated (%): C, 71.27; H, 6.31; N, 9.23. Found (%): C, 71.26; H, 6.32; N 9.24. ESI-MS⁺ (m/z): 607.328 (C₃₆H₃₈N₄O₅ + H⁺) (Fig. S6, ESI†). IR: 1688 cm⁻¹ (spirolactam ring) (Fig. S7, ESI†).

Cell culture. HepG2 (human hepatocellular liver carcinoma) and HCT116 (human colon cancer) cell lines from NCCS, Pune, India, were grown in DMEM supplemented with 10% FBS and antibiotics (penicillin – 100 μg mL⁻¹; streptomycin – 50 μg mL⁻¹) at 37 °C in 95% air, 5% CO₂ incubator cells.

Cell cytotoxicity assay. To determine % cell viability of L^C, an MTT assay was performed. HepG2 and HCT116 cells (1 × 10⁴ cells per well) were cultured in a 96-well plate at 37 °C, and exposed to varying concentrations of L^C, 1, 10, 20, 50 and 100 μM, for 12 h. 20 μl of MTT solution [5 mg mL⁻¹ in 1× phosphate-buffered saline (PBS)] was added to each well of a 96-well culture plate and incubated continuously at 37 °C for a period of 4 h. All media were removed from the wells and 100 μl of DMSO was added to each well and absorbance was measured at 550 nm (EMax Precision MicroPlate Reader, Molecular Devices, USA). All experiments were performed in triplicate and the relative cell viability (%) was expressed as a percentage relative to the untreated control cells.

Cell imaging study. HepG2 and HCT116 cells were cultured and incubated in a 35 × 10 mm culture dish with a 10 μM solution of L^C prepared by dissolving L^C in mixed solvent (DMSO : water = 1 : 9 (v/v)) in the culture medium, which had been allowed to incubate for 30 min at 37 °C. After incubation, cells were washed twice with 1× PBS. Bright field and fluorescence images of HepG2 and HCT116 cells were taken using a fluorescence microscope (Leica DM3000, Germany) with an objective lens of 20× magnification. Fluorescence images of HepG2 and HCT116 cells were taken separately from another set of experiments where cells were incubated with 10 μM L^C + 10 μM Al³⁺ for 30 min at 37 °C. Similarly, in another set of experiments, cells were incubated with 10 μM L^C + 10 μM Al³⁺ for 30 min at 37 °C and fluorescence images were taken. HepG2 and HCT116 cells showed almost equal fluorescence intensities with L^C forming a complex with Al³⁺.

Results and discussion

General features

Owing to the interesting photophysical properties of the rhodamine moiety, it is an attractive choice for the design of a receptor platform for various metal ions.³⁴ Also, the various chemical output (absorbance or fluorescence) signals on such a platform may provide a good opportunity to fabricate interesting ‘Boolean’ logic operations at physiological conditions. The recent development of a multiple logic gate function on a single rhodamine based platform confirms this opinion.^7a,b But this field is not well explored at an advanced level for the rhodamine system. All of the logical arithmetic developed from rhodamine derivatives is devoid of any concept related to memory devices with a combinatorial logic circuit. However, we have synthesized the target probe by a simple Schiff's condensation reaction between N-(rhodamine-6G)lactam-ethylenediamine and 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one in methanolic conditions and thoroughly characterized it by NMR and ESI⁺-mass spectrometry analysis.

Spectroscopic interaction of metal ions with L^C

The electronic absorption and emission properties of L^C interacting with several metal ions [Mⁿ⁺ = Na⁺, K⁺, Ca²⁺, Cr³⁺, Ag⁺, Pb²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺, Cd²⁺, Hg²⁺ and Al³⁺] were investigated in MeOH : buffer (7 : 3, v/v HEPES buffer at pH 7) at 25 °C and the corresponding spectral changes of L^C upon the addition of 5 equiv. of each metal ion are illustrated in Fig. 1. It is revealed that on addition of Al³⁺ and Cu²⁺ ions to a solution of the ligand there occurs a significant change in absorption spectra along with the development of an absorption band at ∼525 nm, clearly demonstrating the formation of the ring-opened amide form of L^C upon binding to Al³⁺ or Cu²⁺ ions (Fig. 1A and Fig. S8, ESI†).¹³ No such change is observable by the addition of other metal ions. The steady-state fluorescence studies, under identical reaction conditions, reveal that upon excitation at 505 nm the free ligand itself is very weakly or non-fluorescent, while in the presence of Al³⁺ there is a significant fluorescence enhancement (FE) at 553 nm due to an Al³⁺ induced opening of the spirolactam ring of L^C.³⁶ No such prominent FE takes place for other metal ions including Cu²⁺ (Fig. 1B and Fig. S9, ESI†). In the case of Cu²⁺ the opening of the spirolactam ring of L^C does occur and is expected to be fluorescent too; but the paramagnetic (d⁹) Cu²⁺ fully quenches the fluorescence making the system silent toward emission.³⁷ Hence the above observations clearly demonstrate that the present probe, L^C has the ability to detect oxophilic Al³⁺ both chromo/fluorometrically and Cu²⁺ ions only colorimetrically.

Fig. 1 (A) UV-Vis spectral studies and (B) fluorescence studies of the selective binding of L^C (20 μM) toward Al³⁺ over other metal ions in HEPES buffer at pH 7.0 in H₂O–MeOH = 3 : 7 (v/v) at 25 °C.

Spectroscopic determination of the stoichiometry and binding constants of L^C–Al³⁺ and L^C–Cu²⁺

Looking at the strong response towards the change in absorption properties of L^C through Al³⁺ and Cu²⁺ chelation induced opening of the spirolactam ring, we were interested to find out their binding stoichiometries and corresponding binding constants. The composition of each of the metal complexes was determined by Job's method (Fig. 2A(b) and B(b)) and it was found to be 1 : 1 for both the complexes which was further confirmed by mass spectrometric analysis. ESI-MS⁺ (m/z): 632.59 [Al(C₃₆H₃₇N₄O₅)]⁺ and 650.27 [Al(C₃₆H₃₇N₄O₅)(H₂O)]⁺ for Al³⁺ and 669.27 [Cu(C₃₆H₃₇N₄O₅) + H]⁺ for Cu²⁺ (Fig. S10 and S11, ESI†).

Fig. 2 (A) (a) Change in the absorption spectra of L^C (20 μM) upon the addition of [Al³⁺] = 0–100 μM, (b) the 1 : 1 binding stoichiometry shown by Job's plot, (c) the B–H plot of absorbance (at 525 nm) vs. [Al³⁺] for the corresponding UV-Visible titration. (B) (a) Change in the absorption spectra of L^C (20 μM) upon the addition of [Cu²⁺] = 0–100 μM, (b) the 1 : 1 binding stoichiometry shown by Job's plot, (c) the B–H plot of absorbance (at 525 nm) vs. [Cu²⁺] for the corresponding UV-Vis titration. Conditions: HEPES buffer at pH 7.0 in H₂O–MeOH = 3 : 7 (v/v) at 25 °C.

UV titrations for the interaction between the receptor L^C and Al³⁺ or Cu²⁺ in MeOH–aqueous buffer solution (7 : 3 v/v, HEPES at pH 7) at 25 °C for the complexation process were done in order to find out the stability constants. These were evaluated by the linear least-square fitting of the absorption data at 525 nm to the well known Benesi–Hildebrand equation (1).

where, A₀ and A_max are the absorbances of the pure ligand and the ligand in the presence of excess metal ions, respectively. The evaluated parameters are K_d = (5.8 ± 0.21) × 10⁻⁶ M and n = 1 for Al³⁺ and K_d = (5.53 ± 0.13) × 10⁻⁶ M and n = 1 for Cu²⁺.

Similarly, the fluorescence titration (Fig. 3a) of L^C (10 μM) against variable Al³⁺ concentrations (0–50 μM) in MeOH–aqueous buffer solution (7 : 3 v/v, HEPES at pH 7) at 25 °C gives K_d = (6.07 ± 0.12) × 10⁻⁶ M for Al³⁺ by fitting the corresponding fluorescent intensity at 553 nm to the well known Benesi–Hildebrand equation (n = 1) (Fig. 3b). There is an excellent agreement between the K_d values for the L^C–Al³⁺ system obtained from absorption and fluorescence titration data and this clearly manifests itself in the self-consistency of our results. It looked quite interesting that the association constants of the L^C–Al³⁺ system were almost similar to that of the L^C–Cu²⁺ system determined by absorption titrations.

Fig. 3 (a) Change in the emission spectra of L^C (10 μM) upon the addition of Al³⁺ in HEPES buffer at pH 7.0 in H₂O–MeOH = 3 : 7 (v/v) at 25 °C, [Al³⁺] = 0–50 μM; (b) B–H plot of fluorescence (at 553 nm) vs. [Al³⁺] for the corresponding emission titration. (c) Fluorescence intensity vs. pH plot at 553 nm L^C (10 μM, denoted by black mark) and L^C–Al³⁺ complex (denoted by red mark).

Effect of pH and reversibility study

To study the practical applicability of the probe, the effect of pH on the fluorescence response of L^C towards Al³⁺ was investigated. Experimental results show that the free L^C is highly fluorescent at pH ≤ 2 which arises due to the opening of the spirolactam ring by strong protonation on the secondary amine-N atom (Fig. 3c). On a gradual increase in pH of the medium the fluorescence intensity decreases gradually, reaches a minimum at pH ∼ 6.0 and then remains almost constant up to pH 12.0. However, in the presence of Al³⁺, there was an obvious fluorescence switch ON of L^C at pH 5.0 to 9.0 indicating the fact that the probe L^C can work well at physiological pH conditions. Above pH ≥ 9.0 the fluorescence intensity falls rapidly and becomes constant at pH ≥ 11.0 and this arises mainly due to the formation of [Al(OH)₃], which comes out of the L^C–Al³⁺ complex and re-establishes the non-fluorescent spirolactam ring.

We subsequently studied the chemical reversibility behaviour of the binding of L^C and Al³⁺ in methanol–water solution. With the addition of EDTA to the L^C–Al³⁺ complex, release of Al³⁺ from this complex took place resulting in the bleaching of the absorption band at 525 nm and the emission band at around 553 nm. The corresponding L^C–Cu²⁺ complex also showed reversibility in the presence of EDTA (Fig. S12 and S13, ESI†). This reversibility assay indicates the reusability of the chemo-sensor L^C.³⁸

Evidence in support of the spirolactam ring opening mechanism

The mechanistic pathway for the reaction of L^C with Al³⁺via the opening of the spirolactam ring has been well established through ¹H, ¹³C-NMR and IR studies.³⁹ IR studies revealed that the characteristic stretching frequency for the ‘C=O’ in the amide group of the rhodamine moiety at 1688 cm⁻¹ was shifted to a lower wave number of 1612 cm⁻¹ in the presence of Al³⁺ (Fig. S14, ESI†). This larger shift (Δν = 66 cm⁻¹) towards a lower wave number signifies a higher polarization of the C=O bond upon efficient binding to the Al³⁺ ion, indicating the cleavage of the above mentioned bond. Also in ¹H NMR (CD₃OD), the ring protons (i and j; see the labelling in Fig. S15, ESI†) of the rhodamine part moved in the downfield direction (Δδ_i = 0.717 ppm, Δδ_j = 0.638 ppm) in the presence of Al³⁺ ions. Also in ¹³C-NMR (DMSO-d₆) the disappearance of the signal at 65.65 ppm for the tertiary carbon (sp³ hybridized) of the spirolactam ring of L^C (labelled with a star mark, see Fig. S6 and S16, ESI†) upon addition of Al³⁺ convincingly supports the opening of the spirolactam ring and coordination through the O atom of the –CONH group of the rhodamine moiety with an Al³⁺ ion.⁴⁰ Due to the paramagnetic nature of Cu²⁺, it was not included for NMR studies.^37d

Demonstration of various ‘Boolean’ logic operations

Based on various spectroscopic observations like the chromo-fluorogenic dual signalling response of Al³⁺ and the colorimetric response of the chemosensor L^C towards Cu²⁺ along with the very close K_d values of L^C–Al³⁺ and L^C–Cu²⁺ complexes it will be quite interesting to see what will happen if they co-exist in a system to coordinate with L^C. So, investigating the different absorbance and fluorescence “ON and OFF” states of L^C through controlled experiments some interesting chemistry related to mimicking advanced logic operations and various output information is noted, which could be interpreted within the framework of various molecular logic circuits.⁴¹ In order to correlate various arithmetic logic operations, we arbitrarily choose threshold values of 0.20 for absorbance and 2 × 10⁶ for fluorescence intensity. Any spectral values of outputs lower than the respective threshold limits are taken as 0 (OFF-state) while those greater than the threshold limit are taken as 1 (ON-state).

Using Cu²⁺ (In 1), Al³⁺ (In 2) and EDTA (In 3) as inputs, an integrated logic circuit is constructed in the L^C system (Fig. 4). In the presence of both inputs Cu²⁺ and Al³⁺, the output at 525 nm is 1 (OD > 0.20); while in the presence of EDTA the resultant output is always 0 (OD < 0.2). Hence, this combinational complex logic circuit incorporates an AND gate that integrates an OR and a NOT gate. Fig. 5 illustrates the corresponding truth table and logic circuit.

Fig. 4 (A) UV-Vis spectra of L^C (in CH₃OH : H₂O 7 : 3; pH 7.0) under different input conditions. (B) Corresponding bar diagram at 525 nm in the presence of different input sequences.

Fig. 5 A three-input, one-output molecular switch representation of the absorbance of L^C based on Fig. 4. (A) Truth table. (B) Combinatorial logic scheme.

As the binding constants of the L^C–Al³⁺ and L^C–Cu²⁺ systems are comparable, so the introduction of the first input will be dominant over the second one. Based on this idea a ‘Keypad lock’ for device security can be constructed in a sequential logical manner.^9a In the absence of any chemical input, the receptor L^C shows no prominent emission band at 553 nm, i.e. output is 0 (OFF-state). Now, for the first input sequence, Al³⁺ and Cu²⁺ were used as inputs A (In A) and B (In B), respectively. Operation by Al³⁺ as (In A) to receptor L^C, gives the output “1” (ON-state) and with the sole sequential addition of another input Cu²⁺ (In B), the output still remained ‘1’ (ON-state), i.e. for both cases fluorescence intensity crosses the threshold limit (fluorescence intensity > 2 × 10⁶, output 1). But, on reversal of the input sequence, i.e. for Cu²⁺ as the first input followed by Al³⁺, it causes the fluorescence intensity to be far below its threshold limit and the corresponding output is taken as zero. On the basis of the above observations (Fig. 6A–D) our system can be used as a confidential password entry for a keypad lock; inputs Al³⁺ and Cu²⁺ were designated as ‘N’ and ‘E’ respectively. Out of two possibilities of inputs: (a) first addition of ‘N’ followed by ‘E’ where the receptor (L^C) showed emission ‘ON’ at 553 nm and this “switch ON” is represented by ‘W’ creating a secret code ‘NEW’. While on the other hand, by reversing the order of inputs, i.e. the first input ‘E’ followed by ‘N’, the fluorescence at 553 nm remained switched “OFF” and is represented by ‘N’, which is the wrong entry (END), and failed to open the keypad lock; i.e. a three-digit identification number is obtained by the sequence controlled sensor–analyte interaction with fluorescence. Thus, a sequence dependence of inputs is mandatory for the construction of the molecular keypad lock, i.e. ‘NEW’ only (Fig. 7B). It is due to the fact that the use of numerical digits (0–9) and letters (A–Z) as ‘PIN’ numbers in a two-digit password allows a total of more than 700 different combinations,^9d this adds to the complexity of cracking the keypad lock and improves remarkably the security of the molecular devices. Therefore, only an authorized user who knows the exact password can open the lock, which is a new approach for protecting information at the molecular scale.

Fig. 6 (A) Emission output of L^C following excitation at 505 nm with different input sequences: (A) Al³⁺ as the first input A followed by Cu²⁺ as the second input B; (B) Cu²⁺ as the first input A followed by Al³⁺ as the second input B. The corresponding emission output bar diagram at 553 nm is shown in an inset: (C) In A (Al³⁺ first) and In B (Cu²⁺ second); (D) In B (Cu²⁺ first) and In A (Al³⁺ second). The threshold limit (fluorescence intensity = 2 × 10⁶ nm) was taken for the concerned logic operation.

Fig. 7 (A) Combined truth table based on Fig. 6 for key-pad logic operation. (B) Sequence-dependent crossword puzzle with the right code.

Cell imaging experiments

To determine whether L^C has any cytotoxic effect, an MTT assay⁴² was performed to check its viability on HepG2 and HCT116 cells. No significant cell cytotoxicity was observed for both HepG2 and HCT116 cells with up to 100 μM of L^C for 24 hours (Fig. S17, ESI†). So, this probe is found to be suitable for bioimaging of intracellular Al³⁺ ions. The intracellular imaging behaviours of L^C on HepG2 and HCT116 cells displayed no fluorescence on treating with 10 μM of L^C only (Fig. 8). However, an excellent fluorescence was observed inside the cells, when HepG2 and HCT116 cells were incubated with 10 μM of L^C followed by 10 μM of Al³⁺. This suggests that L^C with a rhodamine fluorophore platform is useful for the intracellular detection of Al³⁺.

Fig. 8 The phase contrast and fluorescence images of HepG2 and HCT116 cells were captured after incubation with L^C or L^C + Al³⁺ for 30 min at 37 °C. HepG2 and HCT116 cells showed almost equal fluorescence intensities with L^C forming a complex with Al³⁺.

Conclusion

We have designed, and easily synthesized herein, a very simple bio-compatible chemosensor L^C bearing rhodamine as a signaling moiety. Based on the receptor–analyte interactions along with its chemosensing behaviour we have fabricated three inputs and one output, and a one output combinatorial molecular logic system with an absorption response. Also controlled sequential experiments with an emission response enabled us to fabricate a ‘key-pad-logic’ function. So, a single molecular system performing such interesting ‘Boolean’ operations not only improves the complexity of a chemical driven ‘Intelligence’ device but also enriches the security of such a device against information invasion through sequence controlled sensor–analyte-interactions. Its bio-compatible nature along with these logical functions may be used in constructing potential biocompatible molecular logic platforms too. Such kinds of ‘Lab-on-molecule’ for the development of a chemical driven smart molecular circuit is in progress in our laboratory.

Acknowledgements

Financial support from DST (Ref. SR/S1/IC-20/2012) and CSIR (Ref No. 01(2490)/11/EMR-II), New Delhi, India, are gratefully acknowledged. T. M. acknowledges the fellowship from UGC (UGC-NET).

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Footnote

† Electronic supplementary information (ESI) available: Additional detailed characterization of synthesized materials along with fluorescence and UV-Vis spectra. See DOI: 10.1039/c5nj02579f

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