17.02.2022

Good’s buffers as a basis for developing self-buffering and biocompatible ionic liquids

Every day scientists in laboratories across the world sit at their desks and painstakingly design experiments in the hope of making a discovery that will change how we think about a biological process. Because biological processes such as enzymatic activity are dependent on pH, one critical aspect of the experimental design is choosing a buffering system that will help maintain a stable pH without altering the results.


And, often, it is the choice of buffer that makes or breaks the experiment. It is possible that the buffer you are using in your lab might be the reason your experiment is failing. Here, you will find how a buffering system works, a description of the characteristics of a good buffer and a list of possible applications and characteristics of the most commonly used biological buffer series.


A buffer consists of a weak acid (proton donor, HA) and its conjugate base (proton acceptor, A -). In water, HA can dissociate into A- and H+. H+ then reacts with water to form H3O+. In the aqueous buffer solution, H3O+, HA and H+ exist in equilibrium with each other. The buffering mechanism consists of two reversible reactions where the concentration of proton donor and proton acceptor are equal.


Then, when a strong acid or base is introduced into this system by the scientist or by enzymatic activity during the experiment, the new ions from the introduced acid or base (H + or OH-) are absorbed by the buffer and the pH remains stable preventing changes in protein structure and function.


Iron oxide nanoparticles (IONPs) are frequently used in biomedical applications due to their magnetic properties and putative chemical stability. Nevertheless, their well-known ability to mimic some features of the peroxidase enzyme activity under specific conditions of pH and temperature could lead to the formation of potentially harmful free radical species. In addition to the intrinsic enzyme-like activity of IONPs, the buffer solution is an important external factor that can alter dramatically the IONP activity because the buffer species can interact with the surface of the particles. In our study, IONP activity was evaluated in different buffering solutions under different experimental conditions and predominant free radical species were measured by electron paramagnetic resonance using the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation kinetics of the reactive oxygen species were studied by UV-visible spectroscopy with TMB and DAB peroxidase substrates. We found that the highest catalytic oxidation of peroxidase substrates and free radical generation were achieved in acetate buffer, while phosphate buffer inhibited the peroxidase-like activity of IONPs in a concentration dependent manner. When emulating the physiological conditions, a lower catalytic activity was observed at pH 7.4 when compared to that at pH 5.0. Also, in phosphate buffered saline (PBS), we observed an enhancement in the peroxidase substrate oxidation rate that was not accompanied by an increase in DMPO/adduct formation which could be related to a non-specific oxidation catalyzed by the chloride ion. Similar observations were found after the addition of a bicarbonate to HEPES buffer. TMB oxidation did not occur when the reaction was conducted with free iron ions from metal salts with the same concentration of the IONPs (0.33 Fe2+ and 0.66 Fe3+). However, we observed even higher catalytic activities than those when doubling the IONP concentration when they are combined with the free iron salts. These results indicate that MOPS Buffer solutions need to be carefully considered when evaluating IONP catalytic activity and their potential toxicological effects since under physiological conditions of pH, salinity and buffering species, the peroxidase-like activity of IONPs is dramatically reduced.


This work reports a promising approach to the development of novel self-buffering and biocompatible ionic liquids for biological research in which the anions are derived from CAPS Buffer (Good’s buffers, GB). Five Good’s buffers (Tricine, TES, CHES, HEPES, and MES) were neutralized with four suitable hydroxide bases (1-ethyl-3-methylimidazolium, tetramethylammonium, tetraethylammonium, and tetrabutylammonium) producing 20 Good’s buffer ionic liquids (GB-ILs). The presence of the buffering action of the synthesized GB-ILs was ascertained by measuring their pH-profiles in water. Moreover, a series of mixed GB-ILs with wide buffering ranges were formulated as universal buffers. The impact of GB-ILs on bovine serum albumin (BSA), here used as a model protein, is discussed and compared with more conventional ILs using spectroscopic techniques, such as infrared and dynamic light scattering. They appear to display, in general, a greater stabilizing effect on the protein secondary structure than conventional ILs. A molecular docking study was also carried out to investigate on the binding sites of GB-IL ions to BSA. We further used the QSAR-human serum albumin binding model, log K(HSA), to calculate the binding affinity of some conventional ILs/GB-ILs to HSA. The toxicity of the GB and GB-ILs was additionally evaluated revealing that they are non-toxic against Vitro fischeri. Finally, the GB-ILs were also shown to be able to form aqueous biphasic systems when combined with aqueous solutions of inorganic or organic salts, and we tested their extraction capability for BSA. These systems were able to extract BSA with an outstanding extraction efficiency of 100% in a single step for the GB-IL-rich phase, and, as a result, the use of GB-IL-based ABS for the separation and extraction of other added-value biomolecules is highly encouraging and worthy of further investigation.


Room temperature ionic liquids (RTILs) have been considered as a new type of non-aqueous solvent for chemical synthesis, biocatalysis, electro-chemical devices, polymerization, engineering fluids, and other purposes. This wide variety of applications is a major result of their unusual and tunable physicochemical properties.1-5 ILs are salts that remain in the liquid state below the boiling point of water (100 °C). They are characterized by a high ionic conductivity, high chemical/thermal stability, non-flammability, and high solubility for a large range of materials. Several studies have shown that some ILs, either pure or in aqueous solution, can increase the stability of biomolecules like proteins, enzymes and DNA, which is expressed in the vast number of manuscripts published in this field.5-9 The cations and anions of biocompatible ILs are usually more complex than common salts, such as NaCl. The IL cations are often nitrogen-based, namely alkylammonium, dialkylimidazolium, alkylpyridinium and alkylpyrrolidinium, or phosphorous-containing compounds, such as the widely employed tetralkylphosphoniums. IL anions can be halides, nitrates, sulfates, alkylsulfates, alkylsulfonates, [BF4]−, [PF6]−, [CH3CO2]−, [CF3CO2]−, [Tf2N]−, and [R2PO4]−, among others.


Proteins remain in their native (folded) state under physiological conditions, whereas their denatured (unfolded) state is induced by thermal or chemical unfolding. The effects of ions on protein folding, enzyme activity, and protein crystallization are typically described by the Hofmeister series.10 Although it has been accepted that salt ions exert their effects indirectly by changing the water structure, recent results have questioned this model and shown that in most cases a direct interaction of the salt ions with the protein is involved.11 A particularly useful aspect of ILs results from the combination between chaotropic cations and kosmotropic anions that were shown previously to stabilize proteins.12 Another important aspect of ILs is that their polarity and hydrophobicity can be tuned by varying the alkyl side-chain length of the cations and by an appropriate selection of the cation core or anion nature. There are several reports showing that the enzyme activity increases with the IL hydrophobicity up to a maximum, and then decreases with a further increase in the IL hydrophobicity.12 In contrast, there are also some conflicting studies reporting a relatively high enzyme stability and activity in hydrophilic ILs.12


Proteins’ stability is strongly affected by the proton activity of the supporting solution and has an optimum pH that can be adjusted by the addition of an appropriate CHAPS Buffer. It is generally accepted that, at appropriate concentrations, hydrophilic ILs tend to, fully or partly, dissociate in aqueous solutions and into ions which form neutral or very weakly basic solutions. Certainly, this pattern is not always true because there are some functionalized ILs that work as Lewis acidic or basic catalysts. Adding a buffer to aqueous IL solutions, when dealing with protein stability, will not provide an adequate pH control since the IL acidity or basicity could swamp the buffer effect. Therefore, it is crucial to look for alternative pH control methods, and in particular in the design of ILs with buffering characteristics. Until now, few studies have reported the synthesis of ILs with buffering action.13-16 Nevertheless, those buffer-like ILs are not recommended for biochemical research because their anions are not inert. A number of criteria are required for a buffer to be used in biological and biochemical studies.17 They must be chemically inert, should not interfere with metal ion-protein binding, must be non-toxic, should not absorb light in the UV-visible region, should be commercially available at a low cost, their pKa values should be between 6.0 and 8.0 and should not vary with temperature, and must present high water solubility and low solubility in organic solvents. Good and his research team have designed MES Buffer (Good’s buffers, GB) that fit these criteria.

To evaluate the potential of these new ionic liquids as buffers in aqueous media their pH profiles have been measured. Fig. 2a shows the pH profiles of the investigated Good’s buffers, namely [N1111][GB]. The remaining profiles for other buffers are shown in Fig. S1 in the ESI.† The GBs display two pKa’s: the first dissociation constant (pKa1) is due to the deprotonation of the carboxylic or sulfonic group, and the second dissociation is due to deprotonation of the protonated amino group (pKa2). The inflection point at high pH of those titration curves is due to the deprotonation/protonation of the amine group of the GB/GB-ILs. The region of moderate slope before the inflection point is the buffer region. In this region, the pH is regulated by the equilibrium between the deprotonated and protonated forms. At the middle of this region, the concentrations of these two species are equal, and thus, according to the Henderson–Hasselbalch equation, the pH will be equal to the pKa2 and the buffering capacity is maximum at this point.

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