Urea based organic nanoparticles for selective determination of NADH

Abstract

Dipodal receptor 1 was synthesized using a single step procedure. The prepared receptor was subjected to organic nanoparticles (N1) and its sensor activities were tested with various biomolecules on the basis of changes in its photo-physical properties. Receptor responded selectively for reduced nicotineamide adenine dinucleotide; with a linear detection range upto 340 nM, having a detection limit of 96 nM, selective determination of NADH using N1 was not affected by the presence of any other potential interfering biomolecule or even in the presence of a higher concentration of salt.

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Introduction

In recent times the emphasis of modern research has been concentrated on human well-being and betterment. Therefore, researchers have tried to develop selective and sensitive methods to determine numerous moieties of human interest like environmental hazardous materials,¹ disease causing agents,² biomolecules of medicinal importance^3,4 etc. The pursuit of selectivity and sensitivity⁵ leads to another challenge, i.e. to make moieties which can selectively fit various sizes and shapes of analytes of interest.⁶ Supra-molecular chemistry addresses the challenge in a sufficient manner, as cavity size of the host can be maneuvered according to the size required.^7–10 It has been extensively used in determination of various analytes ranging from cations,¹¹ anions¹² to biomolecules.¹³ Determination of biomolecules itself presents a new challenge being very large in size and having very complex shapes like ATP, AMP, NADH, NADP, NAD⁺ etc.¹⁴ Hence, architecting host molecules for such moieties is very perplexing, encouraging and yet to be explored extensively.

Various sensors have been prepared for the determination of innumerable biomolecules like NADP,^15,16 NADH,^17,18 tryptophan,¹⁹ guanine,^20,21 adenine^20–24 etc. Out of these, biomolecules like dihydronicotinamide adenine dinucleotide (NADH) and its oxidized form i.e. nicotinamide adenine dinucleotide (NAD⁺) are of great biological importance, as they are used as cofactors in more than 300 dehydrogenases,²⁵ oxidoreductase enzymes²⁶ and play various vital roles in cellular metabolism of living beings.^27,28 NADH stimulates the formation of energy currency²⁹ (ATP) in the living beings by transferring the energy generated in oxidation of glucose, fructose like reduced species to energy bank of all eukaryotes³⁰ i.e. adenosine triphosphate (ATP) (Fig. 1).

Fig. 1 Diagrammatic representation NADH being an active intermediate for ATP formation.

Beside these, NADH is also of great medicinal importance as it improves the functioning of people suffering from diseases like Alzheimer, Parkinson's disease and depression etc.³¹ Due to such vital usage of NADH; various sensors and biosensors have been reported in recent time for NADH measurement, even for indirect determination of various analytes like glucose, ethanol and lactic acid owing to its oxido-reductive properties.^32,33 Various voltammetric and chronoamperometric methods have been employed spending various modifications like CNT³⁴ and other organic and inorganic moieties like cyanoferrate compounds, quinones,³⁵ phenazines³⁶ etc. as mediators on the electrode surface for determination of NADH. However, there are few reports which make use of fluorescence properties of the receptor on interaction with NADH. A well designed supra-molecule having a fluorophore and binding site can be used for such determination.

In the present work, we have chosen receptor 1 on the basis of structure requirement of NADH. NADH is having both electron acceptor and electron donating sites, so the identified compound should have multiple sites which can accommodate both types of sites provided by NADH. In NADH, two different electronic sites are provided by phosphates and NH₂ groups, while our selected molecules also provide the same with urea moiety and nitro terminal groups. The identified compound was prepared with slight modification in the reported method³⁷ using simple single step reaction and prepared their organic nano particles for improving the performance of the compound for NADH determination as the aggregates will have many binding sites on the surface of the globule and its usability can be extended to aqueous media. The organic nanoparticles of receptor 1 (N1) showed considerable changes in fluorimetric responses of the compound, which is further used for quantitative determination of NADH in aqueous samples.

Result and discussion

Synthesis

2-Aminothiophenol was coupled to form a disulfide linkage in presence of sodium hydroxide. Disulfide thus formed was then reacted with p-nitrophenyl isocyanate, dissolved in chloroform with continuous reflux for ten minutes (Scheme 1). The product thus formed was fully characterized using ¹H-NMR, ¹³C-NMR (Fig. S1 and S2†), CHN analysis.

Scheme 1 Synthesis of receptor 1.

Geometry optimization

Geometry of the receptor 1 was optimized (Fig. 2) using DMol 3 package with DFT (Density functional theory) calculations run through GGA with basis set DNP (Double numeric plus polarization) using water as a solvent. It was perceived that the highest occupied molecular orbitals (HOMO) are spread over both the podants of the dipodal receptor 1 (Fig. 3a), while lowest unoccupied molecular orbitals are concentrated on disulphide moiety (Fig. 3b). It showed that the incoming guest will come in the cavity formed between the two podants.

Fig. 2 DFT optimized structure of receptor 1.

Fig. 3 Structure showing (a) highest occupied molecular orbitals (HOMO) (b) lowest unoccupied molecular orbitals (LUMO).

To confirm the results the structure of receptor 1 with NADH was also optimized using same parameters. It was perceived that NADH fits well into the cavity formed by the podants of receptor 1 and is held their by strong inter and intra molecular hydrogen bonding (Fig. 4).

Fig. 4 DFT optimized structure of complex of receptor 1 with NADH, showing inter and intramolecular hydrogen bonding of the receptor with NADH.

Organic nanoparticles formation

Wide ranges of domains like electronics, photonics, biotechnology etc. are benefited with the introduction of nanoparticle science in the fields of organic and inorganic chemistry.³⁸ Researchers have shown their interest in development of methods for formation of organic and inorganic nanoparticles. Nanoparticles have direct application in various fields and can be molded according to the requirement, which can directly impact the society.³⁹ Organic nanoparticles are the solid particles of organic compounds suspended in aqueous media having size 10 nm to 1 μm.⁴⁰ Single step re-precipitation method was employed over various methods available for ONP formation because it is easy to use, economical and results obtained are quite reproducible. Organic compound dissolved in organic solvent is slowly injected into aqueous media forming lipophilic solution by interfacial deposition of organic molecules in organic solvent suspended in water. Various organic solvents were used for determination of best suited solvent for organic nanoparticle formation. Solution of receptor was formed by dissolving receptor 1 in 1 mL of different organic solvents and was injected slowly in 100 mL of water. It was found that tetrahydrofuran (THF)–water system provides the desired suspension formation by displacing water molecules. Effect of addition of water on formation of ONP was also studied and it was observed that 1 : 99 THF to water ratio gave the optimum sized ONP. While, higher concentration of THF causes non-stable and irregular formation of ONP, which in turn leads to agglomeration and settling of the compound.

Effect of solvent was also studied using fluorescence spectroscopy as solvent was shifted from THF to water. It was perceived that receptor 1 is having a very weak emission profile at 480 nm, which got split into two separate peaks at 420 and 460 nm on formation of ONP. The peaks showed considerable enhancement and hypsochromic shift. The enhancement in the fluorescence intensity can be attributed to aggregation induced emission i.e. organic molecules lose their excited energy by various translational and rotational processes like bending, twisting etc. in normal solvent mode, but in ONP they are confined to rigid solid particles causing them to retain their energy. The retained energy is further expressed as enhancement in fluorescence intensity. Shifting of solvent from THF to water can be the cause of solvatochromic shift in the fluorescence profile of receptor 1 (Fig. 5).

Fig. 5 Emission profile of N1 in THF : water (1 : 99; v/v) and receptor 1 in THF.

The particle size of the organic nanoparticles was monitored using DLS (Dynamic light scattering) and found that, N1 had a size distribution around 45 nm (Fig. 6).

Fig. 6 DLS histogram of organic nanoparticles of receptor 1 (N1).

Recognition studies

N1 was subjected to various biomolecules (NADP, NADH, NAD, ATP, ADP, guanine, cytosine, cysteine, uracil, adenine etc.) to test its binding ability in aqueous media qualitatively and quantitatively using fluorescence spectroscopic measurements. Upon addition of 350 nM of biomolecules to the fixed concentration of N1 (200 nM), no or negligible change is observed in all the molecules except NADH, which exhibited sharp enhancement in fluorescence profile of N1 (Fig. 7).

Fig. 7 Changes in emission profile of N1 (200 nM) in aqueous medium upon addition of 350 nM of particular biomolecule.

The enhancement in the fluorescence intensity on addition of NADH to N1 has been further explored by performing the titration of N1 (Fig. 8a and b) with NADH by adding small aliquots of it in solution of ONPs for projecting it as sensors for determination of NADH in various samples of analytical and biological importance. It was perceived that N1 respond linearly (r² = 0.9836) up to 340 nM concentration of NADH with a detection limit of 96 nM, having a calibration slope of 0.41169.

Fig. 8 (a) Change in fluorescence emission spectra of N1 (200 nM) upon successive addition of NADH (0–340 nM); (b) linear regression graph for successive additions of NADH (0–340 nM) to ONP (200 nM).

To further establish workability of N1 in competitive medium, interference studies have been carried out by adding 350 nM of other biomolecules (NADP, NAD, ATP, adenine, guanine, cytocine, cysteine, uracil, etc.) to the solution of N1 having 350 nM of NADH. No or very negligible change in fluorescence profile of N1 in presence of NADH have been observed (Fig. 9), which clearly authenticated that the prepared ONPs can selectively determine NADH in all real life samples i.e. in presence of other potential interfering biomolecules without any interference.

Fig. 9 Interference studies of N1 for determination of NADH in presence of other biomolecules in aqueous media.

To extend the study for the practical applicability of N1 for selective determination of NADH, the fluorescence profile of both N1 were recorded at different pH by varying the pH of solution using sodium hydroxide (0.1 M) and dilute hydrochloric acid (0.1 M). It was observed that with in pH 6–9, no change in fluorescence spectra of N1. However, as the pH of the solution decreased below 6 the emission intensity decreases considerably with subsequent decrease in pH. Similarly with increase in pH of the solution above 9, emission intensity of both ONPs decreased with each subsequent increase in pH (Fig. S3†). The decrease in emission intensity on increasing and decreasing the pH of the solution can be attributed to change in conditions to form hydrogen bonding between NADH and N1. The response of N1 was also determined as a function of time for determination of NADH. Different concentration of NADH (50, 100, 150, 200, 250 nM) was added to fixed concentration of N1 (Fig. 10) respectively and the fluorescence spectra was measured as a function of time. It is quite evident from the spectra that N1 showed increment in fluorescence intensity up to 90 s i.e. N1 interacted with NADH in initial 90 seconds, beyond which hardly any change in emission profiles of both was detected.

Fig. 10 Plot of time dependence change in the fluorescence intensity of N1 (200 nM) mixed with different concentrations of NADH (50, 100, 150, 200, 250 nM).

In addition to above, salt effect was also measured on the performance of N1 (200 nM) by adding high concentration of tetrabutyl ammonium perchlorate salt (0–100 equiv.) on their respective fluorescence profiles. It was perceived that no change in fluorescence emission spectra of N1 was observed, even in presence of high concentration of tetrabutyl ammonium perchlorate (Fig. S4†). The prepared receptor was subjected to comparison with recently reported literature for determination of NADH using various techniques (Table S1†). It was concluded that the present sensor has dynamic detection range with detection limit considerably better than its contemporary sensors.

Experimental

General information

All chemicals used were of analytical grade and were purchased from Sigma Aldrich Co. ¹H and ¹³C NMR spectra were recorded on JEOL instrument operated at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR (chemical shifts are expressed in ppm). The CHN analysis was performed using a Perkin Elmer 2400 CHN Elemental Analyser. The fluorescence measurements were performed on a Perkin Elmer LS55 Fluorescence spectrophotometer. The particle size of nano-aggregates was determined with Dynamic Light Scattering (DLS) using external probe feature of Metrohm Microtrac Ultra Nanotrac Particle Size Analyser.

Synthesis of receptors

Disulphide linkage was formed by coupling reaction of 2-aminothiophenol by adding NaOH (0.03 M, 1.2 g) to 2-aminothiophenol (0.01 M, 1.25 g) dissolved in 30 mL of methanol. Reaction mixture was stirred for 2 hours at room temperature. Yellow crystalline product was separated out and was further used for the synthesis of receptor 1. 1.42 g (0.05 M) of separated product was added to 1.64 g (0.1 M) of para-nitrophenyl isocyanate dissolved in 50 mL of chloroform taken in 250 mL round bottom flask. The reaction mixture was refluxed for 10 min and a yellow powder was precipitated out. The precipitates were filtered, air dried and characterized using ¹H NMR (400 MHz, DMSO) δ: 7.21 (t, 2H, ArH), 7.36 (t, 2H, ArH), 7.68 (d, 2H, ArH), 7.86 (d, 2H, ArH), 7.98 (d, 4H, ArH), 8.25 (d, 4H, ArH), 11.20 (s, 2H, NH); ¹³C NMR (100 MHz, DMSO) δ: 161.28, 152.18, 146.72, 141.30, 130.84, 126.62, 126.01, 123.80, 121.95, 120.27, 117.67 and elemental analysis calculated C 54.16%; H 3.5%; N 14.58%, found C 54.04%; H 3.33%; N 14.43%.

Synthesis of organic nanoparticles (N1)

Out of the various methods available for the formation of organic nano particles, single step re-precipitation method was used due to its ease of implementation and reproducibility. A typical procedure adopted is as follow: a solution of receptor 1 was prepared in various concentrations using THF as solvent and named it as working solution. 1 mL of the working solution was slowly injected into 100 mL of water with micro-syringe under sonication and size of the N1 formed were continuously monitored using DLS probe during each injection. It was revealed from the DLS study that 10 nM in 1 mL THF is the concentration, which gives optimum sized organic nanoparticles for receptor 1. Prepared N1 were kept under sonication for another 5 minutes by keeping the temperature with in ± 5 °C of the ambient temperature. The whole experimentation also perceived that the concentration above or below does not result in organic nanoparticles formation. When concentration higher than optimum is used it caused larger sized organic nanoparticles, which agglomerated and settled down on the beaker surface, even in presence of continuous sonication and stirring.

Recognition studies

The recognition studies were performed by recording the fluorescence spectra of well shaken and properly sonicated solutions at 25 ± 1 °C. 200 nM of N1 was added in 350 nM of various biomolecules in aqueous media, having total volume of 5 mL taken in volumetric flasks. The volumetric flasks were shaken properly and allowed to stand for half an hour before recording the spectra. Titrations of N1 was performed with NADH by adding subsequent amount of NADH (350 nM) solution to the volumetric flasks containing N1 (200 nM) in aqueous medium. Possible interference due to different biomolecules for NADH estimation was evaluated by making solutions of N1 (200 nM) with and without other interfering biomolecules (350 nM). Effect of ionic strength was also evaluated by recording the spectrum at different concentration of TBA salt of perchlorate (0–100 equiv.). The pH titrations were also performed to explore the effect of pH to on the recognition behaviour of N1 by varying the acidity and basicity of the solution.

Theoretical studies

The geometry of the complex was optimized using DMol3 package^41,42 with GGA-DFT, using double numerical plus polarization (DNP) as basic set. All electrons of the system were treated with BLYP^43,44 local functions for the exchange-correlation potential. The whole set of calculations were run with water as solvent.

Conclusion

Organic nanoparticles of dipodal receptor were prepared and characterized using NMR, CHN analysis. Size of organic nanoparticles formed was characterized using DLS studies. The prepared ONPs were tested for determination of biomolecules on the basis of their structure and it was perceived that N1 responded selectively for determination of NADH in aqueous media, without having any interference from any of the potential interferents, having a detection limit of 96 nM. Hence, the prepared organic nanoparticles of receptor 1 can be used as sensor for determination of NADH in aqueous samples of both environmental and medical concern.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10209f

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