Introduction

Hydrogen peroxide (H2O2) is formed as a by-product of oxidative metabolism in organisms. Naturally existing peroxidases in the biological system play key role in the catalytic degradation of H2O2 [1]. H2O2 concentration is regulated by peroxidases, which are generally generated from kidney and human red blood cells [2]. Even though peroxidases regulate the concentration of H2O2 but still they required ambient conditions (pH, temperature etc) for their functioning [3]. Evaluation and monitoring of H2O2 is also critical in many other fields such as food production, pulp, paper bleaching, clinical, biological, chemical, sterilization processes etc. H2O2 plays an important role in the field of bioanalysis as well as food security and environmental protection [4]. Therefore, it is practically important to determine and monitor the concentration of hydrogen peroxide [5]. H2O2 can be determine through multiple different routes. Most important of existing routes are enzymatic and nanomaterials based [6]. Out of these two ways nanomaterials-based detection is gaining more interest due to certain limitations associated with the enzymes such as less stability, low shelf life, high cost, and delicate handling [7].

Nanomaterials have received significant attention in the past decades due to their unique tuneable optical properties, large surface areas, structure dependent properties and high catalytic efficiency [8, 9]. In this context, a number of materials including metal oxide nanoparticles, noble metal nanoparticles, carbon nanotubes, bimetallic alloy nanoparticles, magnetic nanoparticles, polyoxometalates and conjugated polymers have been used as peroxidase mimics [10,11,12].

On the other hand, Ionic liquids (ILs) are much attractive and environmentally friendly solvents because of their negligible vapour pressure, wide temperature range stability, tuneable properties through appropriate modification of the cations & anions and the ability to dissolve a number of materials [13, 14]. ILs have been actively explored as an excellent media for the preparation and stabilization of nanoparticles for a variety of applications [1]. Combining ionic liquid with nanoparticles makes it possible to achieve catalytic reactions that are not possible to conduct in common solvents. The observations in the present study will provide a new avenue for the development of highly sensitive analytical system.

Various techniques based on chromatographic [15], spectrophotometric [16,17,18,19], chemiluminescence [20] and electrochemical methods [21,22,23] have been utilized in order to develop low-cost reliable monitoring methods of hydrogen peroxide (H2O2). Compared with other methods, colorimetric biosensing is one of the simplest and cost-effective method used for H2O2 detection [24, 25]. Owing to its simplicity, easy operation and sensitivity, colorimetric analysis are widely employed for field analysis and point-of-care diagnosis [26].

Iron nanoparticles were used as promising peroxidase mimics for optical determination of H2O2. The doping of bismuth was performed based on the fact that doping increases the shelf-life, mobility, separation from purified solution, and passivation of the zerovalent particles [27]. The prepared materials were characterized using FTIR, SEM and XRD. After characterization, ionic liquid a known versatile material was coated onto iron nanoparticles to enhance its catalytic efficiency, stability, well dispersibility, and rapid separation in comparison to other peroxidase mimics for colorimetric detection of hydrogen peroxide (H2O2). The NPs was optimized with respect of pH, NPs concentration, TMB and H2O2 for maximum peroxidase activity. Stability and kinetic assay was performed for IL coated iron particles. Moreover, the hydrogen peroxide detection and interference study were also evaluated.

Materials and methods

Reagents and materials

All the chemicals including iron (II) sulfate (FeSO4.7H2O, 99%), Bismuth nitrate (Bi(NO3)3.5H2O, 99.95%), “3,3′,5,5′-tetramethylbenzidine” (TMB), L-Ascorbic acid, acetic acid CH3COOH (99.9–100.5%) and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich (https://www.sigmaaldrich.com/technical-service-home/product-catalog.html). Hydrogen peroxide (H2O2, 35%) was obtained from MerchKGaA (https://www.merckgroup.com/en). 1-methylimidazole C4H6N2(99.2%) was purchased from ACROS (http://www.acros.com/). Phosphate buffer saline (PBS, pH -7.4) was from bio World. Dopamine was obtained from Alfa Aesar (https://www.alfa.com/en/). Urea (98%) was received from DAEJUNG (http://www.daejungchem.co.kr/eng/product/search.asp). Cuvettes (Disposable) of 10 mM were purchased from Kartell having capacity volume of 2 mL. All chemicals were of analytical grade and were used without further purifications. All the solutions were made in deionized water from ELGA PURELAB ® Ultra water deionizer.

Characterization:

UV − Vis absorption spectra were recorded using a double beam Perkin Elmer UV-Vis spectrophotometer Lambda-25 (UV-25, Perkin Singapore) with 10 mm disposable cuvettes having 2 mL capacity and a bandwidth setting of 1 nm at a scan speed of 960 nm/min in the range of 400 to 800 nm. The crystal structures of Zero-Valent Iron nanoparticle and Bismuth doped iron (Bi-nZVI) nanoparticle were investigated using powder X-ray diffraction MPD XP’ERT PRO™ diffractometer of PANALYTICAL equipped with monochromatic Cu-Kα (λ = 0.15418 nm) over the 2θ range of 20° - 80°. Fourier transform Infrared (FTIR) spectra were recorded to determine the functional groups attached to Zero-Valent Iron nanoparticle (nZVI) and Bismuth doped nanoparticle using Thermo Fisher Scientific (Nicolet 6700) spectrometer over the wave range of 600–4000 cm−1 with resolution of 8 cm−1. Surface morphology of Zero-Valent Iron (nZVI) nanoparticles and Bismuth doped nanosized zero-Valent Iron (Bi-nZVI) nanoparticles was examined using the Scanning electron microscope (SEM, Vega 3, LMU, Tescan). Images have been taken at different magnifications at accelerated voltage of 10 kV.

Synthesis of iron nanoparticles

These were synthesized based on the following reaction.

$$ \mathbf{4}{\mathbf{Fe}}^{\mathbf{2}+}+\mathbf{3}{{\mathbf{BH}}_{\mathbf{4}}}^{-}+\mathbf{9}{\mathbf{H}}_{\mathbf{2}}\mathbf{O}\to \mathbf{4}{\mathbf{Fe}}^{\mathbf{0}}+\mathbf{3}{\mathbf{H}}_{\mathbf{2}}{{\mathbf{BO}}_{\mathbf{3}}}^{-}+\mathbf{12}{\mathbf{H}}^{+}+\mathbf{6}{\mathbf{H}}_{\mathbf{2}} $$

Sodium borohydride (NaBH4, 140 mM) was added drop wise into ferrous sulphate (FeSO4.7H2O, 20 mM) through titration followed by simultaneous formation of black particles of nZVI to collect jet-black iron nanoparticles. The particles were washed with deionized (DI) water and anhydrous ethanol three times each and dried in Oven at 90o C. After drying, the powder particles were calcined in furnace to 400 °C with a ramp of 5 °C/min.

The protocol for synthesis of bismuth doped iron nanoparticles particles is provided in the Electronic Supporting Material (ESM).

Synthesis of ionic liquid

The 1 H-3-Methylimidazolium acetate was synthesized using the modified protocol reported by Qian et al. [28]. 1-methylimidazole (0.01 mol) was neutralized with acetic acid (0.01 mol) in two neck flasks under cooling. The sample was stirred under cooling followed by stirring for 6 h at room temperature. The prepared ionic liquid after subjecting to rotary evaporator was characterized using NMR Bruker Avance (500 MHz).

$$ {\displaystyle \begin{array}{l}{}^1\mathrm{H}\ \mathrm{NMR}\ \mathrm{spectroscopy}\ \left(\mathrm{Bruker},500\ \mathrm{MHz},\mathrm{DMSO}-d6\right);\delta :7.60\ \left(\mathrm{s},1\mathrm{H},\mathrm{C}2-\mathbf{H}\right),\\ {}7.12\ \left(\mathrm{s},1\mathrm{H},\mathrm{C}4-\mathbf{H}\right),6.90\ \left(\mathrm{s},1\mathrm{H},\mathrm{C}5-\mathbf{H}\right),4.35\ \left(\mathrm{s},1\mathrm{H},\mathrm{N}2-\mathbf{H}\right),3.65\ \left(\mathrm{s},3\mathrm{H},\mathrm{N}3-\mathrm{C}\mathbf{H}3\right),\\ {}1.92\ \left(\mathrm{s},3\mathrm{H},\mathrm{C}\mathbf{H}3\mathrm{COO}\right)\end{array}} $$

Preparation of ionic liquid coated nanoparticles

The preparation of ionic liquid modified iron nanoparticles was carried out as follow: Firstly, nZVI (5 mg) was added into the protic IL 1-H-3- Methylimidazolium acetate (HMIM OAc) (1 mL) and mechanically grinded for 30 min in the pestle-mortal until a well dispersed ionic liquid coated nZVI was achieved. A similar procedure was employed to prepare ionic liquid coated bismuth doped nano Zero-Valent Iron (Bi-nZVI).

Peroxidase like-catalytic activity of ionic liquid coated nanoparticles

The peroxidase like activity was evaluated through colorimetric detection of H2O2 using TMB as a substrate. The procedure was carried out as follow; 35 μL of stock solution of dispersed IL coated NPs, 200 μL of TMB (12 mM), 100 μL of Ionic Liquid and 565 μL of phosphate buffered saline were mixed. Subsequently, 100 μL of H2O2 (6 mM) was added into the reaction solution and was incubated for 30 min to detect the optical change. The resultant solution was used for the UV-Vis spectra measurement at a wavelength of 652 nm to observe a quantitative relationship between IL coated NPs, TMB and varying concentration of H2O2.

Results and discussions

FTIR is the most effective technique for qualitative analysis of functional groups attached to the surface of zero-valent iron nanophase (Fig. S1). The peak present in the range of 910–930 cm−1 and a sharp peak observed at 945 cm−1 corresponds to the stretching vibration of hydroxyl groups adsorbed on the surface of the iron nanoparticles and of the doped iron nanoparticles. A peak shift was observed with doping the nano zero-valent iron particles. The contribution of metal is confirmed from the appearance of ν(M = Fe) band at 1000–1100 cm−1. Disappearance of peak is shown in doped Fe nanoparticle at 1270 cm−1 conform the successful doping of Bi atoms into Fe. The FTIR investigation confirms the doping of atom on the surface of iron nanoparticles.

XRD analysis was carried out to investigate structural analysis of the Bi/Fe0 and Fe0 and both materials were found to be highly crystalline (Fig. S2). The diffraction peak at 2θ = 44.9o, 63.3o in Bi/Fe0 and Fe0 are assigned to Fe0[29, 30]. The XRD pattern showing peaks at 2θ = 30o and 2θ = 35o may be attributed to oxides of iron (i.e., magnetite and maghemite) formed due to partial oxidation of the Bi/Fe0 and Fe0 before their use in batch experiments. The XRD pattern of Bi/Fe0 shows new peaks at 2θ = 25.9o and 55.2o (not found in Feo) that corresponds to Bi crystal phase (Fig. 1). The Debye Scherrer approach based on the X-ray line broadening (is shown in Eq. (1)) was performed for approximation of the average crystal size.

$$ D=\frac{K\lambda}{\beta Cos\theta} $$
(1)
Fig. 1
figure 1

a Dispersion of nZVI in (a) phosphate buffered saline; (b) Ionic Liquid, b Dispersion of Bi-nZVI in (a) phosphate buffered saline; (b) Ionic Liquid, c Absorption spectra and optical inset for Ionic Liquid coated nZVI in the presence of (a) TMB (1.2 mM), H2O2 (0.3 mM) and 100 μL of IL in 2 mL of reaction medium; (b) control in the presence of only TMB; (c) control in the presence of H2O2, d Absorption spectra and optical inset for Ionic Liquid coated Bi-nZVI in the presence of (a) TMB (1.2 mM), H2O2 (0.3 mM) and 100 lL of μL in 2 mL of reaction medium; (b) control in the presence of only TMB; (c) control in the presence of H2O2

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Where D is an average crystalline size and K is constant equal to 0.89 and lambda (λ) is wavelength of X-ray diffraction = 0.154 nm, β is full width at half-maximum (FWHM) of peak, and θ is the angle of diffraction [31]. The average crystallite sizes were calculated to be 4.95 and 3.40 nm for Fe0 and Bi/Fe0, respectively. This suggests that coupling metallic Bi reduces the crystal size of Fe0.

Surface morphology of synthesized nanoparticles have been investigated using Scanning electron microscopy(SEM) is shown in Fig. S3. Results indicate that the synthesized nanoparticles are almost spherical and uniform in size. These structures tend to form a chain like aggregate due to the magnetic attractive force between particles, increasing the available surface area of reaction [32].

Peroxidase-like activity of ionic liquid coated nanoparticle

The peroxidase-like activity was investigated by the catalytic oxidation of peroxidase substrate TMB in the presence of H2O2.Considering the importance of ionic liquid for biomedical application, Ionic liquid coated nanoparticles have recently gained importance for its peroxidase like catalytic activity due to its stabilizing nature and serve as a modulator for reaction medium [33]. To assess the stabilizing behaviour of IL, the synthesized nanoparticles were dispersed in phosphate buffered saline in the presence and absence of ionic liquid. It was observed that presence of ionic liquid limits the particle agglomeration showing well dispersibility is shown in Fig. 1a and b. These results suggest that IL stabilize the nanoparticles forming an ion layer around the metal nanoparticles with enhanced catalytic activity. There were various methods reported in literature subjected to the stabilizing nature of nanoparticle with enhanced catalytic activity, but they faced many limitations, changed the surface properties and did not allow to absorb other molecules. Our study suggested that ionic liquid coated nanoparticles didn’t face such limitations thereby enhancing the catalytic activity many folds. The peroxidase-like activity of ionic liquid coated nZVI nanoparticle and ionic liquid coated Bismuth doped nZVI nanoparticle has been shown in Fig. 1c and d. Moreover, the nZVI nanoparticle as alone showed very low activity as compared to ionic liquid coated nZVI nanoparticle (the result is not presented in the manuscript). The Ionic coated nZVI nanoparticles generated intense blue green color in aqueous solutions in the presence of H2O2 as an analyte using chromogenic substrate TMB with strongly distance-dependent optical properties. The peroxidase like catalytic activity of IL coated nZVI nanoparticles can be attributed to the metal centre [34]. Adsorption of peroxidase substrate “3,3′,5,5′-tetramethylbenzidine” (TMB) on the surface of IL coated nZVI nanoparticles shows π-π and hydrogen bonding interactions. Interaction of H2O2 molecules with metal centres iron ions and conversion to •OH radicals helped in catalysing and oxidation of colorless TMB [34].

Zero-valent or metallic iron acted as a two-electron donor through the redox pair of Fe+2/Fe0 that can directly reduce dissolved molecular oxygen in aqueous solutions to hydrogen peroxide providing alternative pathway of inducing Fenton oxidation is shown in Eq. (2). The practical applicability of nZVI lies in the fact that it can easily get oxidized to +2 and + 3 oxidation states [35].

$$ {\mathrm{Fe}}^0\left(\mathrm{s}\right)+{\mathrm{O}}_2\to {\mathrm{Fe}}^{2+}+{\mathrm{H}}_2{\mathrm{O}}_2 $$
(2)

Hydrogen peroxide oxidize zero-valent iron into ferrous iron if added exogenously [36]:

$$ {\mathrm{Fe}}^0\left(\mathrm{s}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{Fe}}^{2+}+2{\mathrm{O}\mathrm{H}}^{-} $$
(3)

nZVI is the reactive reagent. Oxidation of nZVI to ferrous (Fe2+) and ferric iron (Fe3+) helps in releasing the electron to become available for the reduction of other compounds [35].

$$ {\mathrm{Fe}}^{2+}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{Fe}}^{3+}+{\mathrm{O}\mathrm{H}}^{-}+\bullet \mathrm{OH} $$
(4)

To further investigate the catalytic mechanism of iron nanoparticle, it was observed that Fe particles treated with reducing agent NaBH4 increases the activity of the nanoparticles thereby increasing the proportion of Fe2+ ions. This suggests that Fe2+ions may play a dominant role in the catalytic peroxidase-like activity of iron nanoparticle. Literature reports that Fe2+/Fe3+ions in solution (Fenton’s reagent) are known to catalyse the breakdown of hydrogen peroxide [37]. Furthermore, nZVI particle can induce •OH radical production by catalysing decomposition of H2O2 as an intermediate via Fenton-type reaction and then •OH captures a H+ from hydrogen donor such as TMB. A possible mechanism for the iron particle involves (i) Adsorption of H2O2 on the surface of iron particle thereby activated by the bound Fe3+to generate the •OH, and the generated •OH was stabilized by Fe particle via partial electron exchange interaction (ii) TMB was oxidized by •OH to form a blue green color product. The blue green signal comes from the charge-transfer complex, consisting of a cation free radical and TMB. Fe3+ Ions in a complex forms are readily reduced, and the reduced Fe2+reacts with H2O2 to generate •OH radicals in the way of Fenton reaction [6, 38].

In the same context, IL coated bismuth doped nZVI provided less blue green color, optical signal generated was very less as compared to the strong optical signal observed for IL coated nZVI is shown in Fig. 1d. The possible reason for the low catalytic activity may be in the doping of nZVI with dopant bismuth. Bismuth was observed to decrease the reactivity of nZVI toward hydrogen peroxide (H2O2) by stabilizing its surface as reported for nNi-Fe catalytic activity [39].

It can be concluded from the experimental output that IL is not only a powerful stabilizing agent but also modulate and enhance the biomimetic properties of nanomaterials (Fig. 1). The control experiments illustrated that absorbance of pure ionic liquid in buffer medium was negligible as compared to the absorbance of IL coated nanoparticles is shown in Fig. 2. Moreover, the enhanced catalytic activity by IL coating over simple iron nanoparticle was due to structural moiety of 1-h-3-methylimidazolium acetate ionic liquid having pi electrons both in imidazolium cation and carboxylate anion in its structure was employed. The positive effect of IL on the catalysis process is possibly associated with their great solvation power [40], presence of large number of cations and anions and the weak interactions with the oxidized products. Moreover, the acidic hydrogen of cationic part of IL play an important role in the decomposition of H2O2 into OH radical that further catalyze the oxidation of peroxidase substrate TMB. Similarly, ionic liquid was expected to increase the conductivity of the interaction medium along with subsequent enhancement of the oxidation of chromogenic substrate TMB in the presence of H2O2 (Fig. S4). Therefore, it can be concluded that IL not only act as efficient stabilizing agent but also increased the catalytic activity of Fe nanoparticles without changing surface reactivity. However, the use of such materials has two limitations: without IL, NPs shows less activity and besides to get good results, specific ionic liquids having aromaticity in its structure are needed.

Fig. 2
figure 2

a Absorption spectra and optical inset for Ionic Liquid coated nZVI in the presence of (a) TMB (1.2 mM), H2O2 (0.3 mM) and 100 μL of IL in 2 mL of reaction medium; (b) control in the presence of only TMB; (c) control in the presence of H2O2, b Pure Ionic Liquid in the presence of (a) TMB (1.2 mM), H2O2 (0.3 mM) in 2 mL of reaction medium; (b) control in the presence of only TMB; (c) control in the presence of H2O2

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Optimization of experimental conditions

In order to achieve the optimal performance for hydrogen peroxide detection, the effects of TMB, H2O2, amount of nanoparticles on the absorbance have been investigated in Fig. 3. The catalytic activity of IL coated NPs was dependent on such factors. The maximum peroxidase like activity was found under following optimal conditions: pH 7.4, 25 °C, 5 min incubation time, 35 μL IL coated NPs, 12 mM TMB and 6 mM H2O2.

Fig. 3
figure 3

Optimization of volume of (a) Ionic Liquid coated nZVI (stock dispersion 5 mg/mL) in the presence of TMB (1.2 mM) and H2O2 (0.3 mM), (b) Concentration of TMB

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Stability and kinetic assay of ionic liquid coated nanoparticles

Ionic liquid mediated nanoparticle shows excellent stability than natural enzymes. It is reported in literature that enzymatic products change to a colourless solution in buffer thereby loses activity significantly with the passage of time. Beyond high catalytic performance, IL coated NPs exhibited unique features due to its stabilizing nature for an extremely long period of time thereby not only stabilizing the nanomaterial but also enhancing the long-term stability of TMB. Moreover, our method suggested the advantage of stabilizing properties of ionic liquid mediated nanoparticles for performing highly stable artificial enzyme analysis.

To investigate the peroxidase-like catalytic property of nanoparticle, kinetic parameters were applied for catalytic oxidation of TMB in H2O2 using Line weaver-Burk plots including Michaelis-Menton (Km) and maximum initial velocity (Vm) by changing the concentration of TMB and H2O2. The kinetic parameters measured for IL coated nZVI and literature data are presented in Table 1. The lower Km value suggests that the lower concentration of oxidizing agent (H2O2) is required to achieve maximum catalytic response with IL coated nZVI. The catalytic efficiency of an enzyme is directly related to km value towards the substrate. A lower Km value of IL coated nZVI reveals the synergistic catalytic contribution of IL towards oxidation of substrate. Thus, increasing the affinity of IL coated nanoparticles towards H2O2 decomposition.

Table 1 Kinetic profile of IL based Zero valent iron particles
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Detection of H2O2

Under the optimum experimental condition and based on the peroxidase like-activity of IL coated nZVI, a simple colorimetric method was used for the detection of H2O2.

Keeping in view the color change for the quantitative assay of H2O2, a selective calorimetric method has been used based on the relationship between H2O2 concentration and absorbance intensity at 652 nm as depicted in Fig. 4a (magnified Fig. S5). This method enabled the detection of H2O2 with a linear range of 30–300 μM with a limit of detection of 0.15 μM and standard deviation of 5–7% was calculated (Electronic supporting documents Table S1). Comparison of this study with literature has been provided in Table 2. Figure 4b represents the standard curve plotted based on the absorbance at 652 nm as a function of H2O2 concentration to detect the strong optical changes as depicted in Fig. 4a. It is noteworthy that this method offers a strategy to monitor H2O2 by recording the clear visual change in the blue green color which is completely distinguishable by the unaided eye. In addition, the adsorption of H2O2 on the nano surface generates OH radical which are subsequently involved in the oxidation of TMB to give a blue green color product. The sugars like fructose, lactose, and galactose have completely different structures and hence are not expected to interfere with the signal.

Fig. 4
figure 4

a UV/Vis response of the varying concentrations of H2O2, inset shows the corresponding visual changes in color; b Corresponding calibration curve at wavelength 652 nm

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Table 2 An overview on recently reported nanomaterial-based POx mimics for the detection of hydrogen peroxide
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Interference studies

To explore the selectivity of detection by using IL coated nZVI nanoparticles as peroxidase mimic, the absorbance responses of the sensing system were investigated with potential interfering substances such as dopamine, ascorbic acid, and uric acid. From Fig. 5, it can be found that an obvious blue green colour change was easily distinguished visually induced by H2O2 for glucose analyte. In contrast, absorbance measured for dopamine, ascorbic acid, and uric acid were much weak. However, only the addition of H2O2 can result in a high absorbance, and no obvious absorbance changes were found upon the addition of coexisting substances. Therefore, this assay further demonstrated high selectivity for H2O2 detection.

Fig. 5
figure 5

Selectivity and interfering study of glucose with other analytes (concentration of glucose and other analytes; 6 μM

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Conclusion

In summary, Ionic liquid coated nanoparticles were prepared and investigated as peroxidase mimics. The catalytic oxidation of peroxidase substrate TMB in the presence of H2O2 using the ILNPs was realized as peroxidase mimic that provides a colorimetric assay to detect H2O2.The catalytic mechanism are from hydroxyl radicals, due to the decomposition of H2O2 catalysed by ILNPs. Furthermore, the ionic liquid used not only act as an agent to stabilize the nanoparticle, but also investigated as efficient modulator to synergize artificial enzyme mimic properties of iron nanoparticles. Thus, the analytical platform for the detection of H2O2 used not only confirms that ILNPs possesses an intrinsic peroxidase-like activity but also shows great potential applications in varieties of simple, cost-effective, and easy-to-make biosensors in the future.