Isothermal Titration Calorimetric Methods Research Articles

Highly Sensitive Label-Free Electrochemical Aptasensor Based on Screen-Printed Electrode for Detection of Cadmium (II) Ions

In this study, a simple but highly sensitive label-free aptasensor based on a screen-printed electrode (SPE) was developed for detection of Cd2+. As crucial biorecognizer, the Cd2+ aptamer (issAP08-Cd2+) was first screened out using the isothermal titration calorimetric method, which showed that the equilibrium dissociation constant (KD) of Cd2+ binding with issAP08-Cd2+ was 2.9 μM. Then, the issAP08-Cd2+, which is a single-stranded DNA with 25 nucleotides, was modified with thiol and assembled on gold screen-printed electrode. After that, the electrochemical aptasensor was used for Cd2+ detection by dropping Cd2+ solution on the SPE. The electrochemical properties were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under the optimal conditions, the change of DPV peak currents density values (Δi) increased linearly with the logarithm of Cd2+ concentrations from 0.1 ng/mL to 1000 ng/mL. The regression coefficient was 0.9914 and the limit of detection (LOD) was 0.05 ng/mL (S/N = 3). This outcome supported the conclusion that the electrochemical aptasensor not only showed good sensitivity and selectivity but also obtained satisfactory reproducibility and stability results and performance on applications in real samples. More importantly, the composition of this aptasensor is very simple, and it has promising potential for rapid field detection of Cd2+.

Mechanistic Investigation of Inclusion Complexes of a Sulfa Drug with α- and β-Cyclodextrins

Molecular encapsulation is extremely important in pharmaceutical and drug delivery science. In this article, one of the most important sulfa drugs, namely, sulfacetamide sodium, has been probed in the solution and solid phases for encapsulation within the cavities of α- and β-cyclodextrins. Various physicochemical techniques were employed to establish the outcome of the work. The isothermal titration calorimetric method was used to evaluate the stoichiometry, association constant, and thermodynamic parameters with high accuracy. The solid inclusion complexes were analyzed by spectroscopic techniques to ascertain the encapsulation of the investigated drug within the cavities of α- and β-cyclodextrins. This phenomenon of drug inclusion is exceedingly significant for its stabilization against external hazards, such as oxidation, sensitization, and photolytic cleavage, for the proficient and accurate regulatory release of an essential amount of drug at the targeted site for a period of time and for the preven...

Host-guest interaction of 3-hydroxyflavone and 7-hydroxyflavone with cucurbit [7]uril: A spectroscopic and calorimetric approach

The modulation of photophysical behaviour of small organic molecules in the presence of macrocycles is one of the most interesting areas of research. In this work we reported the interaction of two biologically active molecules 3-hydroxyflavone and 7-hydroxyflavone with macrocyclic host cucurbit [7]uril in aqueous medium. To investigate the change of photophysical properties of these two flavones, we have used steady state absorption, fluorescence, time resolved fluorescence emission spectroscopy and isothermal titration calorimetric technique. It is observed that on complexation with cucurbit [7]uril, the excited state proton transfer processes in both flavones have been facilitated. Isothermal titration calorimetric method was used in order to investigate the involvement of thermodynamic parameters in complexation between flavone with cucurbit [7]uril. The changes in thermodynamic properties due to the complexation of the flavones molecules with cucurbit [7]urils help to understand about the governing parameters involved in this complexation. The inclusion of flavone molecules inside the cavity of cucurbit [7]uril molecules was studied theoretically to decipher the molecular orientation of flavones in the presence of cucurbit [7]uril. The structure of HOMO and LUMO of the complexes between cucurbit [7]uril with flavones was reported. This study will be helpful to get the knowledge about the modulation of photophysical properties of the flavones molecules on addition of macrocyclic host cucurbit [7]uril. This study will be helpful for the use of cucurbit [7]uril as a potential drug delivery system.

A novel aptasensor for electrochemical detection of ractopamine, clenbuterol, salbutamol, phenylethanolamine and procaterol

β-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of β-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant (Kd) to phenylethanolamine (PHL) was 3.34×10−5molL−1. The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04ng/mL (RAC), 0.35pg/mL (CLB), 1.0pg/mL (PHL), 0.53pg/mL (SAL) and 1.73pg/mL(PRO), respectively, The detection time was 15min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on β-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five β-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-β-agonists (may be one or more) in sample.

Self-structure formation in polyadenylic acid by small molecules: new insights from the binding of planar dyes thionine and toluidine blue O

Thionine and toluidine blue targeting poly(A).

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (∆Hº) and favorable (positive) entropic (∆Sº) contributions to the Gibbs free energy (∆Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity.

Catalytic role of His232 in the mechanism of Pseudomonas putida creatine amidinohydrolase

Creatine amidinohydrolase (creatinase) catalyzes the hydrolysis of creatine to sarcosine and urea. The enzyme has been isolated in several organisms and a study has indicated the presence of creatinase in humans. Structural analysis of the enzyme suggests that conserved histidine at position 232 may act as a general acid/base catalyst. To examine the functional role of this residue, the H232A creatinase variant will be created using site-directed mutagenensis. Analyses of the kinetic mechanisms of the mutant and wild type creatinases will be compared using an isothermal titration calorimetric method to measure the change in enthalpy that accompanies product formation. The ability to rescue partial activity of the H232A mutant using imidazole will also be examined.

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12–20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60–180 min to generate 20–30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba2+ to 18-crown-6 and the binding of cytidine 2′-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12–20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60–180 min to generate 20–30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba 2+ to 18-crown-6 and the binding of cytidine 2 ′-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.

Inhibition of β-Amylase Activity by Calcium, Magnesium and Zinc Ions Determined by Spectrophotometry and Isothermal Titration Calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH=4.8 (sodium acetate 16 mM) and T=300 K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8 mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.

An Isothermal Titration Calorimetric Method to Determine the Kinetic Parameters of Enzyme Catalytic Reaction by Employing the Product Inhibition as Probe

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2′,3′-cyclic monophosphate. Employing the inhibition of product as a probe, the K m, K i, k c, and Δ H m can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis–Menten function in the presence of inhibitors, the relationship between K m and K i can be obtained. Then, the dissociation constant, which is equal to K i, was measured by a regular ITC experiment. Thus, K m and k c can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.