Insight into the kinetically and thermodynamically controlled biosynthesis of silver nanoparticles

Abstract

The mechanism of nanoparticles (NPs) synthesis is a crucial prerequisite for the engineering of desirable material properties and is elusive due to the difficulty of studying the synthesis path and also due to lack of published work. The present study moves beyond the current conventional study of characterisation of NPs synthesis. It focuses on study of classical kinetics of metal NP using silver as a model metal to perform the required experiments, it aimed at understanding the crystallisation process which is the major root for the synthesis of metal NP. The authors have chosen biological approach to explain the process of synthesis of metal NP using inductively coupled plasma – optical emission spectroscopy that directly analyses the concentration of metal NPs. Alpha‐amylase solution, when incubated with silver nitrate solution, turned brown at a specific concentration of enzyme and substrate, which shows the formation of silver NPs. Inductively coupled plasma data and dynamic light scattering spectra were studied to understand the kinetics and the kinetics model was obtained. The enthalpy (ΔH *), activation energy (ΔE *), and equilibrium constant (K) were calculated for understanding the thermodynamics of the reaction. The process of NP synthesis is dependent on the kinetics of the reaction, and other process parameters limit the thermodynamics of the process.

Nomenclature

ΔE

activation energy

ΔH

enthalpy

R

gas constant

K ₀

frequency factor

K

equilibrium constant

k

rate constant

1 Introduction

Silver nanoparticles (AgNPs) are an arch product from the field of nanotechnology, among all the noble metal nanoparticles (NP). The components having a role in the reduction of metal ions depends on the organism and the mode of synthesis. The biological approach for the synthesis of NPs includes microbes, enzymes, and plant. These are considered to be an eco‐friendly alternative to chemical as well as physical methods of synthesis [1]. The green synthetic approach of AgNPs includes solvent medium, reducing agent, and substances for stability [2]. The aggregation ratio may alter the physical properties of NPs, even though the NPs of identical size are present in the colloidal solution [3].

The mechanism for the formation of metal NPs is due to the action of enzymes used in the reaction mixture [4]. The alpha‐amylase is composed of 12 molecules of cysteine along with 2 exposed thiol group which are away from the catalytic sites and are responsible for the synthesis of AgNPs [5]. The thiol group present in cysteine interacts with the metal ion and enables the reduction of Ag⁺ to Ag⁰ [6] and cysteine (–SH) functional group exposed on the surface, gives stability to the AgNP [7].

The mechanism to control the biosynthesis of NPs is crystallisation, which is a combination of two‐stage process of growth and nucleation. The non‐valent form of metal is insoluble in the liquid aggregates slowly into an embryo [8]. Growth of the embryo and increase in number atoms ensure the stability of the formed embryo. The driving force for the initiation of nucleation is the reduction of overall Gibbs free energy. It controls the final product, size distribution, and phase transfer, and the free solution combines to obtain a thermodynamically stable cluster [9]. Segregation of metal atom in solution reduces the overall Gibbs free energy, and its concentration dictates the difference in Gibbs free energy per unit volume. When the aggregation of metal atoms increases, the nuclei grow and form primary particles [10].

The formation, as well as the growth of NPs, is a complex chemical process in which nucleation occurs after the solution reaches constant concentration [11]. The synthesis of nano‐crystals is essential to analyse the process of growth. Due to the large surface area of NPs, the surface‐to‐volume ratio becomes quite high, and the excess surface energy becomes vital in tiny particles [12].

Therefore, a non‐thermodynamic equilibrium solution leads to a process that allows the formation of larger particles and hence helps in the growth of crystals [13]. The enzymes serve as a set of vital tools which have diverse structures, the additional attraction to use enzymes is its commercial availability in the purest form with different chemical, biochemical as well as biological functions [14].

The effect of pH on the reaction kinetics of the enzyme‐catalysed reaction was studied, and it demonstrated that pH explains the nature of the enzyme compounds formed by the reaction [15]. The effects of temperature on enzyme‐catalysed reactions are the reaction is slow at low temperature; the reaction velocity increases rapidly with temperature and reaches a maximum value at the optimal temperature [16]. The essential idea of this principle is that positive results obtained at a short time and high temperature before enzyme inactivation can be used to predict the results at a long time and low temperature [17]. This phenomenon is similar to the thermodynamic restrictions of the temperature and pressure to reaction velocity constants in chemical reaction kinetics [18].

The estimation of the intrinsic enzyme‐catalysed reaction velocity by establishing the relationship between kinetics and thermodynamics is a complex process [19]. Therefore, the analysis was performed for the estimation of the combined effects of temperature and time on enzyme‐catalysed reaction velocity. The classical Arrhenius equation is an empirical equation that describes the relationship between the rate constant of the reaction and the temperature of the elementary reaction [20]. The optimum temperature and the enthalpy of denaturalisation of enzymes have been used to study the biochemical process [21]. The kinetics of the enzyme‐catalysed reaction is different from the elementary reaction; therefore, the adaptability of the Arrhenius equation was investigated in detail.

In the nano‐dispersed systems, the dispersivity has effects on thermodynamic and kinetic parameters of chemical reactions [22]. The surface thermodynamic properties of NPs take a distinct effect on thermodynamics and kinetic parameters of chemical reactions in nano‐dispersed systems [23]. Another technique explored for the assurance of surface thermodynamic properties of NPs. The investigation performed by thermal analysis described the influence of the properties on the thermodynamic and kinetic parameters of chemical reaction [24].

The study of thermodynamics focuses on deciding the direction of reaction. The reaction proceeds in terms of spontaneity, as stated in the second law of thermodynamics also the equilibrium constant, which determines the extent of reaction taking place under particular conditions [25]. It has already been reported that the free energy ΔG which derives the chemical reaction needs to be calculated for various reaction pathways. The second law of thermodynamics does not determine the product which gets accumulated in a system [26].

2 Materials and methods

2.1 Chemicals

Alpha‐amylase and silver nitrate (AgNO₃) were procured from Merck, India. Nitric acid was purchased from Rankem, India. Analytical grade chemicals, as well as solvents, were used.

2.2 Biosynthesis of AgNPs

The synthesis of AgNPs was carried out by incubating the enzyme alpha‐amylase (2 mg/ml in Tris‐HCl buffer, pH 8.0) in a freshly prepared solution of AgNO₃ (0.05 M). The experiments were performed in four different sets of temperature, pH, and enzyme–substrate concentration monitored.

2.3 Characterisation

UV–Vis spectral analysis (Shimadzu UV‐1800 UV spectrophotometer) operated at a resolution of 1 nm as a function of reaction time. Scanning electron microscopy (SEM, Joel, Japan, JSM‐6390LV) at an accelerating voltage of 20 kV for understanding the morphology of the NPs. Dynamic light scattering (DLS, Malvern instruments, UK, Nano ZS) was used to determine the average size of biosynthesised NPs. Inductively coupled plasma – optical emission spectroscopy (ICP‐OES, Perkin Elmer, USA; Optical 2100 DV ICP‐OES) was used to determine the concentration of AgNPs synthesised.

2.4 Study of crystallisation kinetics of AgNPs synthesis

The investigation was performed by shifting the temperature of the responses 25, 30, and 37°C, the catalyst substrate proportion just as pH was kept consistent, 2:3, and 8 individually. The expansion in size of particles concerning (time versus size) was concentrated to comprehend the energy of the development of gems and development. The pace of the response was acquired from another diagram (temperature versus rate).

2.5 Study of reaction kinetics of AgNPs synthesis

It was studied to understand the influence of temperature, pH, and the enzyme–substrate concentration on the kinetics of NPs synthesis. Distinctive arrangement of investigations was performed by changing the temperature, pH, and the grouping of the substrate, for example [E: C_A].

The study of the effect of temperature on the kinetics of synthesis of NPs, done by selecting different temperatures: 25, 30, 35, and 37°C and other parameters like pH (7) and enzyme–substrate ratio (2:3) was kept constant.

The examination of the effect of pH on the vitality of amalgamation of NPs wrapped up by picking pH in the extent of 5–8, and the temperature (35°C) similarly as the substrate obsession extent (2:3) was kept relentless.

The study of the effect of substrate concentration on the kinetics of synthesis of NPs done by selecting enzyme–substrate ratio: 1:1, 2:1, 2:3, 2:5 and the temperature (30°C), as well as the pH (7), was kept constant. ICP‐OES analysis was done for all the set of experiments and from the data graphs (time versus concentration) were plotted to obtain the rate of reaction. The highest and lowest rate of reaction was observed from the graph (temperature versus rate), (pH versus rate), and (substrate versus rate).

2.6 Study of order of reaction

The rate of reactions obtained from the various experiments were used to study the order of reaction.

2.7 Study of thermodynamics

The thermodynamics, i.e. energy difference resulting from the free energy given off during the chemical reaction, was studied to know the equilibrium condition of the product. The study was initiated by obtaining the activation energy from the Arrhenius plot (1/T versus lnk), where T is the temperature (Kelvin), and k is the rate constant. Since the reaction is uni‐molecular and there was no change in the number of moles, so it was assumed that there was no change of volume in the solution. Therefore, the enthalpy (ΔH) was considered equal to the activation energy (ΔE). Arrhenius equation

$$K=K_0{\mathrm{e}}^{-E/RT}$$ (1)

was used to obtain the equilibrium constant (K). The frequency factor (K ₀) of the reaction was obtained using the Arrhenius plot.

3 Results and discussion

The enzyme alpha‐amylase catalysed the formation of AgNPs by acting as a sole reducing agent. The enzymatic reaction produced a visible colour change, from colourless to brown, which showed the reduction of Ag+ to NPs due to the surface plasmon resonance (SPR). The metal NPs having free electrons give rise to SPR absorption band; the plasmon represents the collective oscillation of a free charge in metal while positive electrical charge remains fixed and the free electron is free to move around it. The free electrons of the metal and the vibration of an electron in metal vibrate collectively and give rise to surface plasmon.

The various concentration of AgNO₃ (constant enzyme concentration) was used to see the influence of substrate and enzyme concentration on reaction kinetics (Fig. 1) and proper change in colour was observed in 2:3 [E : C _A] concentration ratio. The visible spectrum of solution was recorded using UV spectrophotometer at different periods, and there was a noticeable increase in the absorption at 418–420 nm (Fig. 2).

Fig. 1.

Various Concentrations of enzyme and substrate [E: C_A]

(a) 1:1, (b) 1:2, (c) 2:3, (d) 2:5

Fig. 2.

UV–Vis spectroscopy plot (Abs. versus time) (enzyme substrate reaction)

According to various studies, the morphology of the synthesised crystals depends on the distance of NP formation from thermodynamic equilibrium. It is reported that the increase in the driving force for crystallisation leads to the formation of polyhedrons nanostructures.

It was also reported that at high pH, the product was formed of both spherical‐ and rod‐shaped. The SEM images showed a change in the shape of particles from 0 to 24 h (Fig. 3).

Fig. 3.

Surface morphology (SEM) of the synthesized nanoparticles

(a) 0 hr, (b) 6 hr, (c) 12 hr, (d) 24 hr

The Fourier‐transform infrared (FTIR) spectra of AgNPs (Fig. 4) showed peak at 1384.89 cm⁻¹ and alpha‐amylase (Fig. 5) showed a peak at 1373.32 cm⁻¹, arising from N–H binding and C–N stretching vibrations as well as conformational shifts. The absorption band appears in the range of 3000–3400 cm⁻¹. It is the intermolecular hydrogen bond which emerges from amide and hydroxide gatherings of amylase.

Fig. 4.

FTIR spectra of AgNP

Fig. 5.

FTIR spectra of alpha‐amylase

The accurate measurement of the size and distribution of synthesised NPs was performed through DLS analysis. The DLS spectra showed an increase in particle size concerning time. The particle ranged from 100 to 200 nm, in the interval of every 2 h (Fig. 6). Since the study does not include control of size, therefore, no further experiments were performed to decrease the size of particles. The information obtained from dynamic light dispersing demonstrated an expansion in the size of NPs. It was observed from the crystallisation kinetics plot (Fig. 7) that the rate of change of the size of particles increases with increase in temperature. Process parameters of the reaction were maintained until the reaction reaches equilibrium.

Fig. 6.

Size of the NPs analysed by DLS

(a) 6hr, (b) 12hr, (c) 24hr

Fig. 7.

Influence of temperature on the size of NPs synthesis

The different temperature selected for the reaction was 25, 30, and 37°C, and the enzyme–substrate ratio was kept constant, [E : C _A] = 2:3 and the pH of the reaction was kept 8 throughout the reaction. From the graph (Fig. 8), the rate of the reaction was observed to be 0.69 g/ml/h.

Fig. 8.

Influence of temperature on rate of change of NPs size

The vitality of reaction was concentrated to fathom the temperature effect on the vitality of the mix of NPs. The ICP‐OES information stated that the grouping of NPs being integrated increments till the 12th hour of the response and from that point forward, the response arrives at harmony and consequently at 24th hour there was no expansion in the convergence of the item. As compared together to understand the influence of temperature on the kinetics of synthesis of NPs, the concentration of NPs increases with increase in temperature and after reaching an optimum level the concentration decreases (Fig. 9). It is evident that the temperature affects the kinetics of NPs synthesis and the highest rate of reaction (0.1065 g/ml/h) was at 30°C, pH8, 2:3 [E : C _A] and lowest rate of the reaction (0.0078 g/ml/h) was at 25°C, pH 8, 2:3 [E : C _A] (Fig. 10).

Fig. 9.

Influence of temperature on kinetics of NPs synthesis

Fig. 10.

Influence of temperature on rate of NPs synthesis

The different pH range 5–8, enzyme–substrate ratio (2:3), and temperature 35°C were opted for the reactions. With the increase in pH, the concentration of the NPs synthesis increases, and the rate of synthesis of reaction changes (Fig. 11). The pH affects the kinetics of NPs synthesis, and the highest rate of reaction (0.2784 g/ml/h) was at pH7, and lowest rate of the reaction (0.0573 g/ml/h) was at pH 5 (Fig. 12).

Fig. 11.

Influence of pH on kinetics of NPs synthesis

Fig. 12.

Influence of pH on rate of NPs synthesis

The different concentration selected was 1:1, 2:1, 2:3, 2:5, and the temperature of the reaction was kept 35°C throughout the reaction. The increase in the concentration of substrate showed that the concentration of the NPs synthesis increases and the reaction becomes saturated after a particular concentration of substrate and the rate of reaction changes (Fig. 13). The concentration of substrate affects the kinetics of NPs synthesis, and the highest rate of reaction (0.0492 g/ml/h) was in 2:3, and lowest rate of the reaction (0.005 g/ml/h) was in 1:1 as well as 2:1 (Fig. 14).

Fig. 13.

Influence of concentration on kinetics of NPs synthesis

Fig. 14.

Influence of concentration on rate of NPs synthesis

The reaction initially follows zero‐order because initially there is no much observable change in the rate of the reaction; after a particular time, the rate of reaction increases. Therefore, the inference from the graph (Fig. 15) is that initially, the reaction follows zero order, and then it ends up with first order of reaction. The reaction initially followed zero‐order kinetics because of the high concentration of AgNO₃ in the solution, and as the reaction proceeded, and the substrate got consumed, the reaction became concentration dependent and followed the first‐order kinetics.

Fig. 15.

Rate kinetics model

The study of thermodynamics was initiated to understand whether the reaction takes place or not under a given set of conditions, amount of heat absorbed or liberated during the reaction and to observe the maximum possible extent of reaction. The diagram determines the enactment vitality, and after that, the Arrhenius condition was acquired utilising the information. Equation obtained from graph (Fig. 16) is

$$y=-16071x-55.09$$ (2)

Since, Slope = −(E /R), where E is the activation energy and R is the gas constant

$$E=-16071\times 8.314=-133614.294\mathrm{J}/\mathrm{mol}$$ (3)

Therefore, Activation energy (ΔE) = − 133,614.294 J/mol. In a single molecular reaction (Δn  = 0), the number of moles does not change; there is also no change in the volume of solution. Therefore ΔH  = ΔE, i.e. change in enthalpy is equal to the activation energy. Hence, Enthalpy (ΔH) = − 133,614.294 J/mol. The Arrhenius plot obtains (K ₀) frequency factor, also known as the pre‐exponential factor, which is important in enzyme reactions. Therefore, frequency factor is

$$\left(K_0\right)=\mathrm{EXP}\left(-55.09\right)=1.118535\times {10}^{-24}$$ (4)

The Arrhenius equation obtains an equilibrium constant for the decomposition of the enzyme–substrate complex ES to product P, hence the equilibrium constant of the reaction (K) = 0.0527.

Fig. 16.

Arrhenius plot

The result showed negative enthalpy, which indicates that the reaction is exothermic, and the enthalpy value is the same as the activation energy. The negative activation energy shows that the rate of reaction decreases with increase in temperature. The determined estimation of balance consistent demonstrates that the compound catalysed response is controlled dynamically and not thermodynamically. The K value shows that the middle of the road complex is increasingly intuitive with the substrate, and a restricted measure of substrate is changed over into item. Therefore, the enzyme provides a favourable kinetic environment to the substrate molecules to change. Notwithstanding, the thermodynamics of the response was constrained by different procedure factors, mostly the convergence of the substrate.

4 Conclusion

AgNPs were synthesised using enzyme alpha‐amylase. The synthesis of AgNPs is found to be crystallisation process as supported by the literature. The process engineering parameters studied helped to understand the mechanism of NPs synthesis. The particle size and shape increased with the increase in time and the rate of formation of NP increased with the increase in temperature, pH, and the concentration of substrate. The expansion was observed in the centralisation of item in parameters until a breaking point. From the outcomes, it is construed that the response may have arrived at immersion. The increase in rate is due to an increase in the kinetic energy and the collision between the molecules. The reaction initially followed zero‐order kinetics because of the high concentration of AgNO₃ in the solution, and as the reaction proceeded, and the substrate got consumed, the reaction became concentration dependent and followed first‐order kinetics.