Urea Concentration Range Research Articles

Investigating the Impact of Deposition Parameters on the Biosensing Performance of Zinc Oxide Nanostructured Thin Films Fabricated via Vapor Phase Transport

Despite huge advancements in biosensing technologies in the last few years, there remains a gap in comprehending the intricate relationship between growth parameters and the corresponding biosensing response characteristics. The present work investigates the correlation between the physical properties of ZnO thin films and their biosensing response to address this gap and further fabricate a urea sensor based on the optimized conditions. The Vapor Phase Transport (VPT) method was used to grow ZnO thin films, with biosensing performance observed to be highly dependent on growth conditions. Under optimal conditions, ZnO films demonstrated biosensing-friendly properties such as low stress, strong carrier mobility for electron transfer, and a large surface area for effective biomolecule loading. The prepared bioelectrode (Urs-GLDH/ZnO/Pt/Si) showed excellent performance in detecting urea with a high sensitivity of 41 μAmM−1cm−2 over a wide range of urea concentrations (5–200 mg dl−1 or 0.83–33.33 mM). The urea sensor also exhibited a low limit of detection (LOD) of 1.82 mg dl−1, a high shelf life lasting for 12 weeks, and superior selectivity. Thus, the present study not only aims at enhancing our understanding of the fundamental properties of ZnO thin films and their relation to processing conditions, but also emphasises their potential for enhanced biosensing applications.

Exploiting the Advantages of Ag/ITO/Enzyme Trapped Gel Layers to Develop a Highly Sensitive and Selective Fiber Optic Plasmonic Urea Sensor

The fabrication and characterization of a surface plasmon resonance (SPR)-based urea biosensor, with thin silver (Ag), ITO (In2O3: SnO2), and enzyme-trapped gel over an unclad portion of plastic-clad silica fiber as a sensing element, is represented. The working principle is to identify changes in the refractive index of the enzyme (urease) entrapped gel layer following the interaction with the incoming analyte. This interaction causes swelling and shrinkage of the gel layer, which alters the effective refractive index of the sensing layer. The wavelength interrogation method is used, and the optimized sensor probe is characterized by urea samples having different pH values. Scanning electron microscopy confirmed the uniformity of the silver layer over the unclad core of the fiber. The sensor operates from 0 to 160 mM of urea concentrations to cover the physiological concentration range of blood urea normally present in the human body. The sensitivity and limit of detection (LOD) offered by the sensor are marked 0.59387 nm/mM near zero concentration of the urea sample and 0.56 mM, respectively, along with the provisions of high stability, remote sensing, and online monitoring of urea. The proposed sensor has proven to be one of a kind due to its fast response time.

Exploring the role of preferential solvation in the stability of globular proteins through the study of ovalbumin interaction with organic additives

<abstract> <p>The impact of denaturing and stabilizing osmolytes on protein conformational dynamics has been extensively explored due to the significant contribution of protein solvation to the stability, function, malfunction and regulation of globular proteins. We studied the effect of two nonspecific organic molecules, urea, which is a conventional denaturant, and dimethyl sulfoxide (DMSO), which is a multilateral organic solvent, on the stability and conformational dynamics of a non-inhibitory serpin, ovalbumin (OVA). A differential scanning microcalorimetry (DSC) experimental series conducted in the phosphate buffer solutions containing 0–30% of additives revealed the destabilizing impact of both urea and DMSO in a mild acidic media, manifested in the gradual decrease of thermal unfolding enthalpy and transition temperature. These findings differ from the results observed in our study of the mild alkaline DMSO buffered solutions of OVA, where the moderate stabilization of OVA was observed in presence of 5–10% of DMSO. However, the overall OVA interaction patterns with urea and DMSO are consistent with our previous findings on the stability and conformational flexibility of another model globular protein, α-chymotrypsin, in similar medium conditions. The obtained results could be explained by preferential solvation patterns. Positive preferential solvation of protein by urea in urea/water mixtures mainly weakens the hydrophobic interactions of the protein globule and eventually leads to the disruption of the tertiary structure within the whole range of urea concentrations. Alternatively, under certain experimental conditions in DMSO/water mixtures, positive preferential solvation by water molecules can be observed. We assume that the switch to the positive preferential solvation by DMSO, which is shown to have a soft maximum around 20–30% DMSO, could be shifted towards lower additive concentrations due to the intrinsic capability of ovalbumin OVA to convert into a heat-stable, yet flexible set of conformations that have increased the surface hydrophobicity, characteristic to molten-globule-like states.</p> </abstract>

Modulating electronic structure of ternary NiMoV LDH nanosheet array induced by doping engineering to promote urea oxidation reaction

The NiMoV LDH/NF electrode realizes high-efficiency catalysis for UOR in view of the rapid kinetics, high intrinsic activity and robust durability in variable urea concentration and pH values conditions. • Mo and V dopants in NiMoV LDH optimizes the electronic structure to facilitate the in-situ conversion of Ni 3+ species. • Co-doping of Mo and V atoms in NiMoV LDH enhances the adsorption energy of Ni site to urea molecule during UOR process. • 1.40 V is required to achieve 100 mA cm −2 in urea oxidation for NiMoV LDH in alkaline. • The NiMoV LDH electrode is suitable for a wide range of urea concentrations and pH changes. Fabrication of advance electrocatalysts for urea oxidation reaction (UOR) is of great significance for purifying urea-enriched wastewater and producing hydrogen. Here, a doping engineering strategy is adopted to obtain a ternary NiMoV layered double hydroxide (LDH) nanosheet array supported on the three-dimensional (3D) nickel foam substrate. The synergistic effect brought by the unique 2D/3D hierarchical structure could expose more active sites and accelerate charge and mass transfer. In addition, experimental and theoretical results confirm that Mo and V dopants are capable of modifying the local electronic structure of Ni sites to optimize the adsorption energy for urea molecules. Therefore, the as-prepared NiMoV LDH/NF electrode realizes high-efficiency catalysis for UOR in view of the rapid kinetics, high intrinsic activity and robust durability. Specifically, NiMoV LDH/NF electrode presents a low potential of 1.40 V to deliver 100 mA cm −2 for UOR, which is about 260 mV less for its urea-free counterpart in alkaline electrolyte. When anodic NiMoV LDH/NF electrode and cathodic Pt/C electrode are integrated into a two-electrode system for water electrolysis in variable urea concentration and pH values conditions, urea-assisted electrolysis water system exhibit the lower potential (≤1.63 V) than that of conventional water electrolysis at 100 mA cm −2 . This work shows that the local electronic structure adjustment of the active site caused by the heteroatom doping effect could improve the electrocatalytic oxidation performance of some small molecules.

Highly Stable Potentiometric (Bio)Sensor for Urea and Urease Activity Determination.

There is growing interest for bioanalytical tools that might be designed for a specific user, primarily for research purposes. In this perspective, a new, highly stable potentiometric sensor based on glassy carbon/polyazulene/NH4+-selective membrane was developed and utilized for urease activity determination. Urease–urea interaction studies were carried out and the Michaelis–Menten constant was established for this enzymatic reaction. Biofunctionalization of the ammonium ion-selective sensor with urease lead to urea biosensor with remarkably good potential stability (drift coefficient ~0.9 mV/h) and short response time (t95% = 36 s). The prepared biosensor showed the Nernstian response (S = 52.4 ± 0.7 mV/dec) in the urea concentration range from 0.01 to 20 mM, stable for the experimental time of 60 days. In addition, some insights into electrical properties of the ion-to-electron transducing layer resulting from impedance spectroscopy measurements are presented. Based on the RCQ equivalent circuits comparison, it can be drawn that the polyazulene (PAz) layer shows the least capacitive behavior, which might result in good time stability of the sensor in respect to response as well as potential E0. Both the polyazulene-based solid-contact ion selective electrodes and urea biosensors were successfully used in trial studies for determination of ammonium ion and urea in human saliva samples. The accuracy of ammonium ion and urea levels determination by potentiometric method was confirmed by two reference spectrophotometric methods.

Urea as a green thermodynamic inhibitor of sII gas hydrates

This work is devoted to a systematic study of urea CO(NH2)2 as a promising and green gas hydrate inhibitor. The thermodynamic stability of sII gas hydrates forming from a binary gas mixture 4.34% C3H8 + 95.66% CH4 (mol%) in the presence of urea was analyzed. The hydrate equilibrium conditions were measured in a wide range of temperatures (267 – 294 K), pressures (0.9 – 9.4 MPa), and urea concentrations (0 – 50 mass%). The urea decreases the equilibrium temperature of sII hydrates formation, i.e., CO(NH2)2 acts as a thermodynamic hydrate inhibitor (THI). Powder X-ray diffraction at 173 K revealed the phases of sII gas hydrate, hexagonal ice, and tetragonal P4¯21m phase I urea. The determined parameter of sII hydrate unit cell (17.17 Å) indicates the non-inclusion of urea into the hydrate framework. An empirical correlation was proposed to describe the hydrate equilibrium temperature depression ΔTh depending on pressure and urea concentration. A linear relationship between ΔTh and a decrease in the ice freezing point ΔTice in urea aqueous solutions confirms the thermodynamic consistency of our data. Inhibition power of urea at 20 mass% and 6 MPa is 2 times less than that of methanol and is close to that for monoethylene glycol (MEG). In the range, up to 45 mass% urea is more effective THI than diethylene glycol (DEG). Urea is a less toxic compound compared to methanol and glycols, and also more cost-effective than MEG and DEG. Despite the medium anti-hydrate activity, urea can be considered as a safer for the environment hydrate inhibitor. Its application is justified if a shift of the hydrate equilibrium curve by no more than 10 – 12 K is required.

Performance analysis of photonic crystal based biosensor for the detection of bio-molecules in urine and blood

Photonic crystal based sensing technology is the latest technology for various bio-sensing applications. A 2D photonic crystal (PhC) hexagonal shaped ring resonator based biosensor is simulated and designed for the detection of bio-molecules such as glucose, bilirubin, urea and albumin in blood and urine over different concentration ranges. The biosensor structure is created using line and point defect in order to form a ring waveguide and a bus waveguide above the ring structure. If the concentration of bilirubin, glucose, albumin and urea in blood and urine is varied, the refractive index is varied, which in turn changes the output response of the sensor [1]. This principle can be used to identify the aberrant value range of the glucose, bilirubin, albumin and urea in blood and urine. Finite Difference Time Domain (FDTD) tool called Lumericals is used for the modeling and simulation of the biosensor. The resonant wavelength shift, sensitivity, Full-Width Half-Maximum (FWHM) and quality factor (Q-factor) for different concentrations of glucose, bilirubin, albumin and urea are observed and tabulated. The Q-factor obtained for the normal range of glucose, bilirubin, albumin and urea concentration in blood and urine is 310, sensitivity is 400 nm and the resonant wavelength is around 1518.8 nm with the efficiency of 86%.

Diffusion NMR Measurements: What can we Learn about the Compactness of Disordered Proteins?

Intrinsically disordered proteins (IDPs) play a prominent role in normal cell physiology and pathological conditions. However, experimental characterization of complex conformational equilibria in IDPs remain a major challenge. Hydrodynamic radius Rh is a measurable parameter which reports on compactness of an IDP chain. We used the double stimulated echo NMR pulse sequence to obtain Rh data for several disordered proteins in a range of urea concentration from 0 to 10 M. Dioxan molecule was used as an internal standard to account for changes in solvent viscosity.

High stability potentiometric urea biosensor based on enzyme attached nanoparticles

Highly stable new urea biosensor was prepared by using poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) nanoparticles, which was used as a nano-carrier for urease enzyme. The urea biosensor was prepared by an ammonium selective electrode along with the urease immobilized nanoparticles. Synthesized nanoparticles were characterized by SEM and attachment of the enzyme onto the nanoparticles was evaluated by EDX analysis. Potentiometric response of the biosensor was examined as a function of the different concentrations of urea solution, and linear concentration range of urea was demonstrated in the range of 0.01 mM-500 mM. The detection limit was also investigated and measured as 0.77 µM. Response time of the biosensor was also determined as 30 s. Repeatability of the urea biosensor system was found to be very high, and it was demonstrated that response of the urea sensor was protected by 95.7% at the end of the 170th reuse. This biosensor system protected 83% of its initial activity at the end of the 90 day storage. Additionally, interference profile of the urea biosensor was tested against the common interferents such as ascorbic acid (AA), uric acid (UR), glucose (GL) and sodium ions (Na+), and the response of these interfering agents was found to be as negligible. Also, applicability of the synthesized urea sensor for detection of urea from artificial human serum was demonstrated.

An AgNP-deposited commercial electrochemistry test strip as a platform for urea detection

We developed an inexpensive, portable platform for urea detection via electrochemistry by depositing silver nanoparticles (AgNPs) on a commercial glucose test strip. We modified this strip by first removing the enzymes from the surface, followed by electrodeposition of AgNPs on one channel (working electrode). The morphology of the modified test strip was characterized by Scanning Electron Microscopy (SEM), and its electrochemical performance was evaluated via Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). We evaluated the performance of the device for urea detection via measurements of the dependency of peak currents vs the analyte concentration and from the relationship between the peak current and the square root of the scan rates. The observed linear range is 1–8 mM (corresponding to the physiological range of urea concentration in human blood), and the limit of detection (LOD) is 0.14 mM. The selectivity, reproducibility, reusability, and storage stability of the modified test strips are also reported. Additional tests were performed to validate the ability to measure urea in the presence of confounding factors such as spiked plasma and milk. The results demonstrate the potential of this simple and portable EC platform to be used in applications such as medical diagnosis and food safety.

Spatiotemporal Urea Distribution, Sources, and Indication of DON Bioavailability in Zhanjiang Bay, China

In marine environments, urea is an important component of the biogeochemical cycle of nitrogen. The autochthonous and allochthonous sources (rivers, aquaculture, waste water input, etc.) of urea play a key role in urea cycles in adjacent coastal waters. Because urea is a specific marker to trace the sewage fluxes in coastal waters, we investigated urea associated with terrestrial source input and coastal water in Zhanjiang Bay (ZJB) during the time from November 2018 to July 2019, and the spatiotemporal urea distribution and the bioavailability of dissolved organic nitrogen (DON) based on urea concentration in the ZJB were explored. The results showed that the urea enrichment in coastal water was mainly due to discharge from urban sewage systems, rivers, and coastal aquaculture. The concentration of urea ranged from 1.14 to 5.53 μmol·L−1, and its mean value was 3.13 ± 1.02 μmol·L−1 in the ZJB. The urea concentration showed a significantly different seasonal variation in the ZJB (p < 0.05), and the highest and lowest concentrations were found in November 2018 and April 2019, respectively. Its high value appeared in the north and northeast of the ZJB, which were polluted by coastal aquaculture and agriculture fertilizer utilization. The range of urea concentration of terrestrial source inputs in the ZJB was 1.31–10.29 μmol·L−1, and the average urea concentration reached 3.22 ± 0.82 μmol·L−1. Moreover, the total urea flux surrounding the ZJB was 2905 tons·year−1. The seasonal terrestrial source of urea flux contributions had significant seasonal variation in wet, normal, and dry seasons (p < 0.05). The ZJB was subjected to a large flux of urea by estuaries and sewage outlet discharges. The seasonal urea concentration in all stations (>1 μmol·L−1) indicated that urea in the ZJB may have a bioavailable DON source. As a bioavailable nitrogen source, the ability of terrestrial source-derived urea to increase eutrophication should not be ignored in ZJB.

Urea detection by ZnO-MWCNT nanocomposite sensor

Plants, animals and compost are the main cause to enable urea in the environment. Beyond a certain level urea creates various problems. Thus for the environmental and biological protection it is required to detect the concentration of urea in various samples. In the present investigation zinc oxide (ZnO)-multiwalled carbon nanotube (MWCNT) nanocomposites were prepared by sol–gel technique for urea sensing. The fabricated sensor was characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), and energy dispersive x-ray spectroscopy (EDX). The sensing property for detecting urea was studied by resistance measurements in the urea concentration range of 0–200 ppm and in the varying temperature of 0–300 °C. Results have indicated that ZnO-MWCNT nanocomposite based sensor has higher sensing properties as compared to ZnO and MWCNT alone. ZnO-2 wt% MWCNT exhibited highest response value for 100 ppm urea concentration at 150 °C.

Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering.

Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0–10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0–5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7–10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.

Enhanced electron transfer properties of NiO thin film for the efficient detection of urea

Ur/NiO matrix is used efficiently for the detection of Urea. NiO thin film is deposited using rf magnetron sputtering. Variation in processing pressure (10–40 mT) and growth temperature (room temperature to 500 °C) during film deposition alter its defect profile, thus tailoring the charge transfer characteristics. The studies reveal a relationship between biosensing characteristics with various properties of NiO matrix (structural, electrical etc.). The Ur/NiO/ITO/glass biosensor demonstrate very high sensitivity (66.7 μA/(mM)) with rapid time of response (5 s) and good linearity over a wide range of urea concentration (0.83–16.65 Mm). Lower Michaelis–Menten constant value (Km = 0.34 mM) ensures high affinity of Urease for urea. High electron transfer characteristics and long shelf life (20 weeks) are the key features of the fabricated urea biosensor.

Printed flexible bifunctional electrochemical urea-pH sensor based on multiwalled carbon nanotube/polyaniline electronic ink

Urea concentration and pH are two crucial parameters in food and clinical analysis. Traditional analytical methods of urea and pH determination are unsuitable for field and involve complex instrumentation or and enzyme-based assays. In this study, we used screen printing technology to prepare an electrochemical sensor with a carbon electrode modified by a multiwalled carbon nanotube/polyaniline (MWCNT/PANi) composite for the simultaneous detection of urea and pH. Urea was detected by a simple current–potential (I–V) experiment and its concentration level on the MWCNT/PANi-modified screen-printed carbon electrode (SPCE) surface was determined by cyclic voltammetry. The MWCNT/PANi-modified SPCE had a linear response (R2 = 0.99902), lower detection limit, higher selectivity, and higher sensitivity than reported biosensors. Specifically, the detection limit was 10 µM and the sensitivity was 0.38 mA mM−1 cm−2 in the urea concentration range of 10–50 µM. Chronoamperometry was applied to investigate the changes in voltage on the MWCNT/PANi-modified SPCE with varying solution pH. The sensor exhibited excellent linearity (R2 = 0.99089) and an average sensitivity of 20.63 mV/pH over a wide pH range of 2–11. Thus, the MWCNT/PANi-modified SPCE has a promising field application as a simple, bifunctional non-enzymatic sensor.

LED-Based Portable Optical Biosensor for Measurement of Serum Urea Levels Using Urease Immobilized Agarose-Guar Gum Composite Film

A simple, reliable, low cost, and portable biosensor has been developed for direct measurement of urea levels in serum samples. It is based on hydrolysis of urea by immobilized urease, generating ammonia that is estimated by modified Berthelot method using simple optical detection system. The immobilization of urease was achieved in agarose guar-gum composite film for the first time which offered an excellent retention of enzyme activity (90%–95%) with negligible leaching and good operational stability. The optical detection system involving LED light, optical fiber, and phototransistor provides simple and portable measurement system. The sensor shows linear response in the wide range of urea concentration from 0.025 to 10 mM with reusability for 10 cycles and superior storage stability of 180 days. The fabricated biosensor was successfully employed for measurement of normal and abnormal urea levels in serum samples with accuracy of 96% using very small sample volume ( $15~\mu \text{l}$ ). It showed an excellent correlation ( $r^{2}=0.99$ ) for determination of urea levels with that of using Siemens autoanalyzer.

RETRACTED ARTICLE: Effect of Urea on the Morphology of Fe3O4 Magnetic Nanoparticles and Their Application in Potentiometric Urea Biosensors

The effect of different concentrations of urea on the morphology of iron oxide (Fe3O4) magnetic nanoparticles was studied. Fe3O4 magnetic nanoparticles were fabricated by the coprecipitation method. The morphology, crystallinity, compositional purity, and emission characteristics were tested by the techniques of scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and Raman characterization. The drop-casting technique was successfully used to fabricate a potentiometric urea biosensor producing initially isopropanol and chitosan solution, consisting of Fe3O4 nanoparticles, on a glass fiber filter. To measure the developed biosensor’s voltage signal from the functionalized nanoparticles, a copper wire was utilized. The Fe3O4 nanoparticle surface functionalization was performed through the electrostatic immobilization of urease with the Fe3O4-chitosan (CH) nanobiocomposite. The presented urea biosensor measured a wide logarithmic range of urea concentration of 0.1–80 mM with a sensitivity of 42 mV/decade, and indicated a fast response time of approximately 12 s. The developed urea biosensor showed enhanced sensitivity, stability, reusability, and specificity. All experimental results demonstrate the application potential of the developed urea sensor for the monitoring of urea concentrations in human serum, drugs, and food industry-related samples.

Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK193–446) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ]222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε292) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0–7.0M. A coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax, the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies.

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0-6 М at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.

Unveiling the folding mechanism of the Bromodomains

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism.