A novel urease-assisted ratiometric fluorescence sensing platform based on pH-modulated copper-quenched near-infrared carbon dots and methyl red-quenched red carbon dots for selective urea monitoring.

A novel and sensitive fluorescence ratiometric method is developed for urea detection based  on the pH-sensitive response of two fluorescent carbon dot (CD) systems: R-CDs/methyl red (MR) and NIR-CDs/Cu2+. The sensing mechanism involves breaking down urea using the enzyme urease, releasing ammonia and increasing pH. At higher pH, the fluorescence of NIR-CDs is quenched due to the enhanced interaction with Cu2+, while the fluorescence of R-CDs is restored as the acidic MR converts to its basic form, removing the inner filter effect. The ratiometric signal (F608/F750) of the R-CDs/MR and NIR-CDs/Cu2+ intensities changed in response to the pH induced by urea hydrolysis, enabling selective and sensitive urea detection. Detailed spectroscopic and morphological investigations confirmed the fluorescence probe design and elucidated the sensing mechanism. The method exhibited excellent sensitivity (0.00028 mM LOD) and linearity range (0.001 - 8.0 mM) for urea detection, with successful application in milk samples for monitoring adulteration, demonstrating negligible interference and high recovery levels (96.5% to 101.0%). This ratiometric fluorescence approach offers a robust strategy for selective urea sensing in complicated matrices.

Immunoinformatics design and synthesis of a multi-epitope vaccine against Helicobacter pylori based on lipid nanoparticles.

Helicobacter pylori (H. pylori) is responsible for various chronic or acute diseases, such as stomach ulcers, dyspepsia, peptic ulcers, gastroesophageal reflux, gastritis, lymphoma, and stomach cancers. Although specific drugs are available to treat the bacterium's harmful effects, there is an urgent need to develop a preventive or therapeutic vaccine. Therefore, the current study aims to create a multi-epitope vaccine against H. pylori using lipid nanoparticles. Five epitopes from five target proteins of H. pylori, namely, Urease, CagA, HopE, SabA, and BabA, were used. Immunogenicity, MHC (Major Histocompatibility Complex) bonding, allergenicity, toxicity, physicochemical analysis, and global population coverage of the entire epitopes and final construct were carefully examined. The study involved using various bioinformatic web tools to accomplish the following tasks: modeling the three-dimensional structure of a set of epitopes and the final construct and docking them with Toll-Like Receptor 4 (TLR4). In the experimental phase, the final multi-epitope construct was synthesized using the solid phase method, and it was then enclosed in lipid nanoparticles. After synthesizing the construct, its loading, average size distribution, and nanoliposome shape were checked using Nanodrop at 280 nm, dynamic light scattering (DLS), and atomic force microscope (AFM). The designed vaccine has been confirmed to be non-toxic and anti-allergic. It can bind with different MHC alleles at a rate of 99.05%. The construct loading was determined to be about 91%, with an average size of 54 nm. Spherical shapes were also observed in the AFM images. Further laboratory tests are necessary to confirm the safety and immunogenicity of the multi-epitope vaccine.

Immobilized Urease Vector System Based on the Dynamic Defect Regeneration Strategy for Efficient Urea Removal.

The clearance of urea poses a formidable challenge, and its excessive accumulation can cause various renal diseases. Urease demonstrates remarkable efficacy in eliminating urea, but cannot be reused. This study aimed to develop a composite vector system comprising microcrystalline cellulose (MCC) immobilized with urease and metal-organic framework (MOF) UiO-66-NH2, denoted as MCC@UiO/U, through the dynamic defect generation strategy. By utilizing competitive coordination, effective immobilization of urease into MCC@UiO was achieved for efficient urea removal. Within 2 h, the urea removal efficiency could reach up to 1500 mg/g, surpassing an 80% clearance rate. Furthermore, an 80% clearance rate can also be attained in peritoneal dialyzate from patients. MCC@UiO/U also exhibits an exceptional bioactivity even after undergoing 5 cycles of perfusion, demonstrating remarkable stability and biocompatibility. This innovative approach and methodology provide a novel avenue and a wide range of immobilized enzyme vectors for clinical urea removal and treatment of kidney diseases, presenting immense potential for future clinical applications.

Dual-enhanced enzyme cascade hybrid hydrogel for the construction of optical biosensor.

The biomimetic enzyme cascade system plays a key role in biosensing as a sophisticated signal transduction and amplification strategy. However, constructing a regulated enzyme cascade sensing system remains challenging due to the mismatch of multiple enzyme activities and poor stability. Herein, we design an efficient dual-enhanced enzyme cascade hybrid system (UFD-DEC) containing DNA-controlled nanozymes (Fe-cdDNA) and enzyme (urease) via combining the electrostatic contact effect with the hydrogel-directed confinement effect. Precise modulation of Fe-cdDNA nanozyme by DNA offers a means to control its catalytic efficiency. This regulated UFD-DEC system accelerates the reaction rate and provides remarkable stability compared with the free enzyme system. Benefiting from the plasticity properties of hydrogels, a "lab-in-a-tube" platform was constructed by encapsulating UFD-DEC in a microcentrifuge tube. Such a UFD-DEC-based hydrogel tube exhibits sufficient adaptability to profile urea when used in conjunction with a smartphone-assisted image processing algorithm, which on-site delivers urea information with a detection limit of 0.12 mmol L-1. This customizable and inexpensive miniaturized biosensor platform for monitoring urea may facilitate point-of-care testing applications.

A Multienzyme Logic H+ and Na+ Biotransducer.

Sodium ions and protons regulate various fundamental processes at the cell and tissue levels across all biological kingdoms. It is therefore pivotal for bioelectronic devices, such as biosensors and biotransducers, to control the transport of these ions through biological membranes. Our study explores the regulation of proton and sodium concentrations by integrating an Na+-type ATP synthase, a glucose dehydrogenase (GDH), and a urease into a multienzyme logic system. This system is designed to operate using various chemical control input signals, while the output current corresponds to the local change in proton or sodium concentrations. Therein, a H+ and Na+ biotransducer was integrated to fulfill the roles of signal transducers for the monitoring and simultaneous control of Na+ and H+ levels, respectively. To increase the proton concentration at the output, we utilized GDH driven by the inputs of glucose and nicotinamide adenine dinucleotide (NAD+), while recorded the signal change from the biotransducer, together acting as an AND enzyme logic gate. On the contrary, we introduced urease enzyme which hydrolyzed urea to control the decrease in proton concentration, serving as a NOT gate and reset. By integrating these two enzyme logic gates we formed a simple multienzyme logic system for the control of proton concentrations. Furthermore, we also demonstrate a more complex, Na+-type ATP synthase-urease multienzyme logic system, controlled by the two different inputs of ADP and urea. By monitoring the voltage of the peak current as the output signal, this logic system acts as an AND enzyme logic gate. This study explores how multienzyme logic systems can modulate biologically important ion concentrations, opening the door toward advanced biological on-demand control of a variety of bioelectronic enzyme-based devices, such as biosensors and biotransducers.

Urine osmolality assessment through the integration of urea hydrolysis and impedance measurement.

We present the development and validation of an impedance-based urine osmometer for accurate and portable measurement of urine osmolality. The urine osmolality of a urine sample can be estimated by determining the concentrations of the conductive solutes and urea, which make up approximately 94% of the urine composition. Our method utilizes impedance measurements to determine the conductive solutes and urea after hydrolysis with urease enzyme. We built an impedance model using sodium chloride (NaCl) and urea at various known concentrations. In this work, we validated the accuracy of the impedance-based urine osmometer by developing a proof-of-concept first prototype and an integrated urine dipstick second prototype, where both prototypes exhibit an average accuracy of 95.5 ± 2.4% and 89.9 ± 9.1%, respectively in comparison to a clinical freezing point osmometer in the hospital laboratory. While the integrated dipstick design exhibited a slightly lower accuracy than the first prototype, it eliminated the need for pre-mixing or manual pipetting. Impedance calibration curves for conductive and non-conductive solutes consistently yielded results for NaCl but underscored challenges in achieving uniform urease enzyme coating on the dipstick. We also investigated the impact of storing urine at room temperature for 24 hours, demonstrating negligible differences in osmolality values. Overall, our impedance-based urine osmometer presents a promising tool for point-of-care urine osmolality measurements, addressing the demand for a portable, accurate, and user-friendly device with potential applications in clinical and home settings.

Optimization of immobilized urease enzyme on porous polymer for enhancing the stability, reusability and enzymatic kinetics using response surface methodology.

The study examines the immobilization of the urease enzyme on a range of High Internal Phase Emulsion (polyHIPE) materials, assessing characteristics, efficiency, and performance. It also investigates the impact of polyHIPE type, quantity, incubation time, and various parameters on the process and enzyme activity. Surface morphology and functional groups of polyHIPE materials were determined through scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FT-IR) analyses, revealing significant alterations after modification with polyglutaraldehyde (PGA). The maximum immobilization efficiency of 95% was achieved by adding PGA to polyHIPE materials with an incubation period of 15 h. The optimized conditions for immobilized enzyme using a Box-Behnken design (BBD) of response surface methodology (RSM) were as follows: temperature (40.8 °C), pH (7.1) and NaCl concentration (0.007 g/L). Furthermore, the immobilized enzyme demonstrated remarkable reusability, retaining 75% of its initial activity after six cycles, and sustained shelf-life stability, retaining over 40% activity after 10 days at room temperature. Kinetic analyses revealed that immobilized urease exhibited higher affinity for the substrate, but lower rate of substrate conversion compared to the free enzyme. These findings offer valuable insights into optimizing urease immobilization processes and enhancing urease stability and activity, with potential applications in various fields, including biotechnology and biocatalysis.

In Situ Measurement of Urea Concentration With an In-Fiber SPR-MZI Sensor.

A fiber-optic urea sensor based on surface plasmon resonance (SPR) and Mach-Zehnder interference (MZI) combined principle was designed and implemented. By plating gold film on the single-mode-no-core-thin-core-single-mode fiber structure, we successfully excited both SPR and MZI, and constructed two parallel detection channels for simultaneously measurement of urea concentration and temperature. Urease was immobilized on the gold film by metal-organic zeolite skeleton (ZIF-8), which can not only fix a large number of urease to improve measurement sensitivity of urea, but also protect urease activity to ensure the sensor stability. Experimental results indicate that the designed urea sensor with temperature compensation function can detect urea solution with concentration of 1-9 mM, and the sensitivity is 1.4 nm/mM. The proposed measurement method provides a new choice for monitoring urea concentration in the field of medical diagnosis and human health monitoring.

Phase-separated droplets swim to their dissolution.

Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzyme's substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior "dialytaxis", and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion through the Marangoni effect. These results could potentially suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots.

Dynamic changes in the aggregation-depolymerization behavior of Ovomucin-Complex and its binding to urease during in vitro simulated gastric digestion.

Ovomucin-Complex extracted from egg white is expected to have a barrier function similar to gastric mucin. In this study, the dynamic changes in structure, rheological properties and binding ability of Ovomucin-Complex during in vitro simulated gastric digestion were investigated. The results from HPLC and CLSM showed that extremely acidic pH (pH = 2.0) promoted Ovomucin-Complex to form aggregation. Acid-induced aggregation may hinder its binding to pepsin, thus rendering Ovomucin-Complex resistant to pepsin. Consequently, most of the polymer structure and weak gel properties of Ovomucin-Complex retained after simulated gastric digestion as verified by HPLC, CLSM and rheological measurement, although there was a small breakdown of the glycosidic bond as confirmed by the increased content of reducing sugar. The significantly reduced hydrophobic interactions of Ovomucin-Complex were observed under extremely acidic conditions and simulated gastric digestion compared with the native. Noticeably, the undigested Ovomucin-Complex after simulated gastric digestion showed a higher affinity (KD = 5.0 ± 3.2 nm) for urease - the key surface antigen of Helicobacter pylori. The interaction mechanism between Ovomucin-Complex and urease during gastric digestion deserves further studies. This finding provides a new insight to develop an artificial physical mucus barrier to reduce Helicobacter pylori infection.

Profuse color-evolution based aptasensor for mucin 1 detection utilizing urease-mediated color mixing of the mixed pH indicator.

Mucin 1 is a significant tumor marker, and developing portable and cost-effective methods for its detection is crucial, especially in resource-limited areas. Herein, we developed an innovative approach for mucin 1 detection using a visible multicolor aptasensor. Urease-encapsulated DNA microspheres were used to mediate multicolor change facilitated by the color mixing of the mixed pH indicator, a mixed methyl red and bromocresol green solution. Distinct color changes were exhibited in response to varying mucin 1 concentrations. Notably, the color mixing of the mixed pH indicator was used to display various hues of colors, broadening the range of color variation. And color tonality is much easier to differentiate than color intensity, improving the resolution with naked-eyes. Besides, the variation of color from red to green (a pair of complementary colors) enhanced the color contrast, heightening sensitivity for visual detection. Importantly, the proposed method was successfully applied to detect mucin 1 in real samples, demonstrating a clear differentiation of colors between the samples of healthy individuals and breast cancer patients. The use of a mixed pH indicator as a multichromatic substrate offers the merits of low cost, fast response to pH variation, and plentiful color-evolution. And the incorporation of calcium carbonate microspheres to encapsulate urease ensures stable urease activity and avoids the need for extra urease decoration. The color-mixing dependent strategy opens a new way for multicolor detection of MUC1, characterized by vivid color changes.

Deterioration phenomenon of Pb-contaminated aqueous solution remediation and enhancement mechanism of nano-hydroxyapatite-assisted biomineralization.

Modern metallurgical and smelting activities discharge the lead-containing wastewater, causing serious threats to human health. Bacteria and urease applied to microbial-induced carbonate precipitation (MICP) and enzyme-induced carbonate precipitation (EICP) are denatured under high Pb2+ concentration. The nano-hydroxyapatite (nHAP)-assisted biomineralization technology was applied in this study for Pb immobilization. Results showed that the extracellular polymers and cell membranes failed to secure the urease activity when subjected to 60 mM Pb2+. The immobilization efficiency dropped to below 50% under MICP, whereas it due to a lack of extracellular polymers and cell membranes dropped to below 30% under EICP. nHAP prevented the attachment of Pb2+ either through competing with bacteria and urease or promoting Ca2+/Pb2+ ion exchange. Furthermore, CO32- from ureolysis replaced the hydroxyl (-OH) in hydroxylpyromorphite to encourage the formation of carbonate-bearing hydroxylpyromorphite of higher stability (Pb10(PO4)6CO3). Moreover, nHAP application overcame an inability to provide nucleation sites by urease. As a result, the immobilization efficiency, when subjected to 60 mM Pb2+, elevated to above 80% under MICP-nHAP and to some 70% under EICP-nHAP. The findings highlight the potential of applying the nHAP-assisted biomineralization technology to Pb-containing water bodies remediation.

Thermal accelerated urease-driven hyaluronan-targeted melanin nano-missile for bio-radar detection and chemodrug-free phototherapy.

Polymer-based nanomotors are attracting increasing interest in the biomedical field due to their microscopic size and kinematic properties which support overcoming biological barriers, completing cellular uptake and targeted blasting in limited spaces. However, their applications are limited by the complex viscous physiological environment and lack of sufficient biocompatibility. This manuscript firstly reports a natural melanin nano-missile of MNP@HA-EDA@Urease@AIE PS (MHUA) based on photothermally accelerated urease-driven to achieve chemodrug-free phototherapy. Compared to conventional nano-missiles that only provide driving force, this photothermally accelerated urease-driven nanomotor is independent of chemodrug to maximise biocompatibility, and achieve ideal therapeutic effect through targeted PTT/PDT. In particular, the thermal effect can not only boost the catalytic activity of urease but also achieve ideally anti-tumor effect. In addition, guided by and AIE PS, the nanomotor can generate 1O2 to achieve PDT and be traced in real time serving as an effective fluorescent bio-radar for intracellular self-reporting during cancer treatment. Finally, the targeting ability of MUHA is provided by hyaluronan. Taken together, this MHUA platform provides a simple and effective strategy for target/fluorescence radar detective-guided PTT/PDT combination, and achieves good therapeutic results without chemodrug under thermal accelerated strategy, providing a new idea for the construction of chemodrug-free nanomotor-therapy system.

Simple enzyme based fluorimetric biosensor for urea in human biofluids.

As an important biomarker for renal related diseases, detection of urea is playing a vital role in human biofluids on clinical diagnosis concern. In this work, a synthetic salicyaldehyde based imine fluorophore was synthesized using sonication method and conjugated with urease which was used as fluorescent biosensor for the detection of urea in serum samples. This enzyme based biosensor has shown a good selectivity and sensitivity towards urea with the linear range from 2 to 80 mM and the detection limit of 73 µM. The sensing response obtain is highly agreeing with existing analytical technique for urea detection which strongly recommends this biosensor for clinical application.

Synthetic DNA-based Swimmers Driven by Enzyme Catalysis.

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

Honeycomb-Inspired Surface-Enhanced Raman Scattering Microarray for Large-Area Automated Testing of Urease in Saliva Samples.

Surface-enhanced Raman scattering (SERS) technology, as an important analytical tool, has been widely applied in the field of chemical and biomedical sensing. Automated testing is often combined with biochemical analysis technologies to shorten the detection time and minimize human error. The present SERS substrates for sample detection are time-consuming and subject to high human error, which are not conducive to the combination of SERS and automated testing. Here, a novel honeycomb-inspired SERS microarray is designed for large-area automated testing of urease in saliva samples to shorten the detection time and minimize human error. The honeycomb-inspired SERS microarray is decorated with hexagonal microwells and a homogeneous distribution of silver nanostars. Compared with the other four common SERS substrates, the optimal honeycomb-inspired SERS microarray exhibits the best SERS performance. The RSD of 100 SERS spectra continuously collected from saliva samples is 6.56%, and the time of one detection is reduced from 5 min to 10 s. There is a noteworthy linear relationship with a R2 of 0.982 between SERS intensity and urease concentration, indicating the quantitative detection capability of the urease activity in saliva samples. The honeycomb-inspired SERS microarray, combined with automated testing, provides a new way in which SERS technology can be widely used in biomedical applications.

In vitro enzymatic, in silico ADME and molecular docking based analysis for the identification of novel bis-indole containing triazine-thiazole hybrids derivatives as promising urease inhibitors.

The current study details a sequence of sequential reactions for synthesizing bis-indole-based triazine bearing thiazole derivatives. Several steps were involved in the synthesis of bis-indole-based triazine bearing thiazole derivative. The synthetic reactions were monitored via thin-layer chromatography (TLC). Synthesized compounds were characterized using various spectroscopic techniques, including 1H NMR, 13C NMR, and HR-EIMS. The inhibitory activity against urease enzyme of these synthesized compounds was compared with that of thiourea, a standard drug (IC50 = 9.30 ± 0.20 µM). A range of inhibitory potencies were observed for the synthesized compounds, ranging from moderate to excellent, as follows (IC50 = 5.10 ± 0.40 µM to 29.80 ± 0.20 µM). Analyzing the structure-activity relationship (SAR) provided insight into the results, showing that different substituents had different effects on aromatic rings. Several compounds displayed outstanding inhibitory properties (among those tested were 1, 2, 4, 5, and 6 with IC50 = 6.30 ± 0.80, 5.10 ± 0.40, 5.90 ± 0.50, 8.20 ± 0.10, 8.90 ± 0.60 µM, respectively). Anti-urease evaluation of all the synthesized derivatives was conducted in which the selected compounds have shown remarkable potency compared with the standard drug thiourea (IC50 = 9.30 ± 0.20 µM). Molecular docking analysis was carried out for investigating the better binding sites and distance of the derivatives. Moreover, the drug-like properties were explored by the ADME attributes of the synthesized analogs.

Machine Learning-Driven Classification of Urease Inhibitors Leveraging Physicochemical Properties as Effective Filter Criteria.

Urease, a pivotal enzyme in nitrogen metabolism, plays a crucial role in various microorganisms, including the pathogenic Helicobacter pylori. Inhibiting urease activity offers a promising approach to combating infections and associated ailments, such as chronic kidney diseases and gastric cancer. However, identifying potent urease inhibitors remains challenging due to resistance issues that hinder traditional approaches. Recently, machine learning (ML)-based models have demonstrated the ability to predict the bioactivity of molecules rapidly and effectively. In this study, we present ML models designed to predict urease inhibitors by leveraging essential physicochemical properties. The methodological approach involved constructing a dataset of urease inhibitors through an extensive literature search. Subsequently, these inhibitors were characterized based on physicochemical properties calculations. An exploratory data analysis was then conducted to identify and analyze critical features. Ultimately, 252 classification models were trained, utilizing a combination of seven ML algorithms, three attribute selection methods, and six different strategies for categorizing inhibitory activity. The investigation unveiled discernible trends distinguishing urease inhibitors from non-inhibitors. This differentiation enabled the identification of essential features that are crucial for precise classification. Through a comprehensive comparison of ML algorithms, tree-based methods like random forest, decision tree, and XGBoost exhibited superior performance. Additionally, incorporating the "chemical family type" attribute significantly enhanced model accuracy. Strategies involving a gray-zone categorization demonstrated marked improvements in predictive precision. This research underscores the transformative potential of ML in predicting urease inhibitors. The meticulous methodology outlined herein offers actionable insights for developing robust predictive models within biochemical systems.

Enhancing milk quality assessment with watermelon (Citrullus lanatus) urease immobilized on VS2-chitosan nanocomposite beads using response surface methodology.

An eco-friendly hydrothermal method synthesized VS2 nanosheets. Several spectroscopic and microscopic approaches (TEM) were used to characterize the produced VS2 nanosheet microstructure. VS2, Chitosan, and nanocomposite were used to immobilize watermelon (Citrullus lanatus) urease. Optimization using the Response Surface Methodology and the Box-Behnken design yielded immobilization efficiencies of 65.23 %, 72.52 %, and 87.68 % for chitosan, VS2, and nanocomposite, respectively. The analysis of variance confirmed the mathematical model's validity, enabling additional research. AFM, SEM, FTIR, Fluorescence microscopy, and Cary Eclipse Fluorescence Spectrometer showed urease conjugation to the matrix. During and after immobilization, FTIR spectra showed a dynamic connectivity of chemical processes and bonding. The nanocomposite outperformed VS2 and chitosan in pH and temperature. Chitosan and VS2-immobilized urease were more thermally stable than soluble urease, but the nanocomposite-urease system was even more resilient. The nanocomposite retained 60 % of its residual activity after three months of storage. It retains 91.8 % of its initial activity after 12 reuse cycles. Nanocomposite-immobilized urease measured milk urea at 23.62 mg/dl. This result was compared favorably to the gold standard p-dimethylaminobenzaldehyde spectrophotometric result of 20 mg/dl. The linear range is 5 to 70 mg/dl, with a LOD of 1.07 (±0.05) mg/dl and SD of less than 5 %. The nanocomposite's ksel coefficient for interferents was exceptionally low (ksel < 0.07), indicating urea detection sensitivity. Watermelon urease is suitable for dairy sector applications due to its availability, immobilization on nanocomposite, and reuse.

Poly(acrylamide)-co-poly(hydroxyethyl)methacrylate-co-poly(cyclohexyl methacrylate) hydrogel platform for stability, storage and biocatalytic applications of urease.

In our present work, an explicit crosslinked thermo-responsive hydrogel platform has been developed, by using polyacrylamide (PAAm), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(cyclohexyl methacrylate) (PCHMA), and then coupled with urease to yield bioconjugates (BCs). Synergic effect of these polymer units provides thermoresponsive nature, optimum crosslinking with desired swelling behaviour, and stability and improved catalytic to Urease in the resultant BCs. Synthesis of the terpolymer has been achieved by employing HEMA (monomer as well as crosslinker), instead of using the conventional crosslinkers, through free radical solution polymerization technique. Various grades of TRPUBs have been fabricated by varying HEMA and CHMA contents while keeping fixed amounts of AAm. Further, the structural analysis of BCs has been done by fourier transform infra-red spectroscopic study and their thermal stabilities have been studied by thermogravimetric analysis. Urea present in TRPUBs has beenanalysed for its hydrolysis atdifferent temperatures viz., 25 °C, 45 °C and 70 °C. Further, the effect of crosslinking, temperature and reaction time on catalytic activities of TRPUBs has been studied. TRPUBs grades have showna maximum swelling capacity up to 5200 %; excellent catalytic activity even at 70 °C; and 85 % activity retention after 18 days storage in buffer medium.