Enzyme Horseradish Peroxidase Research Articles

CMOS-Based Redox-Type Label-Free ATP Image Sensor for In Vitro Sensitive Imaging of Extracellular ATP.

Adenosine 5′-triphosphate (ATP) plays a crucial role as an extracellular signaling molecule in the central nervous system and is closely related to various nerve diseases. Therefore, label-free imaging of extracellular ATP dynamics and spatiotemporal analysis is crucial for understanding brain function. To decrease the limit of detection (LOD) of imaging extracellular ATP, we fabricated a redox-type label-free ATP image sensor by immobilizing glycerol-kinase (GK), L-α-glycerophosphate oxidase (LGOx), and horseradish peroxidase (HRP) enzymes in a polymer film on a gold electrode-modified potentiometric sensor array with a 37.3 µm-pitch. Hydrogen peroxide (H2O2) is generated through the enzymatic reactions from GK to LGOx in the presence of ATP and glycerol, and ATP can be detected as changes in its concentration using an electron mediator. Using this approach, the LOD for ATP was 2.8 µM with a sensitivity of 77 ± 3.8 mV/dec., under 10 mM working buffers at physiological pH, such as in in vitro experiments, and the LOD was great superior 100 times than that of the hydrogen ion detection-based image sensor. This redox-type ATP image sensor may be successfully applied for in vitro sensitive imaging of extracellular ATP dynamics in brain nerve tissue or cells.

Unraveling autoimmune and neurodegenerative diseases by amperometric serological detection of antibodies against aquaporin-4

This work reports the first electroanalytical bioplatform to date for the determination of antibodies against aquaporin-4 (AQP4-Abs), whose serum level is considered as relevant biomarker for certain autoimmune diseases. The bioplatform relies on the use of magnetic microparticles modified with the biotinylated protein for the capture of specific antibodies. The captured IgGs are enzymatically labelled with a secondary antibody conjugated to the horseradish peroxidase (HRP) enzyme. Amperometric transduction is performed using the H2O2/hydroquinone (HQ) system, which results in a cathodic current variation directly proportional to the concentration of the target antibodies. The evaluation of the analytical and operational characteristics of the developed bioplatform shows that it is competitive in terms of sensitivity with the only biosensor reported to date as well as with the commercially available ELISA kits. The achieved limit of detection value is 8.8 pg mL−1. In addition, compared to ELISA kits, the developed bioplatform is advantageous in terms of cost and point of care operation ability. The bioplatform was applied to the analysis of control serum samples with known AQP4-Abs contents as well as of sera from healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) and Alzheimer (AD) diseases, providing results in agreement with the ELISA methodology.

HRP modified pulse laser ablated ZnO nanostructure for H2O2 sensing

Biosensor for the detection of hydrogen peroxide (H2O2) has been prepared by immobilizing horseradish peroxidase (HRP) enzyme using physical adsorption technique on zinc oxide (ZnO) nanostructures. The (002) oriented ZnO nanostructures as confirmed by X-ray diffraction, were successfully grown on indium tin oxide (ITO) coated glass substrate by pulsed laser ablation (PLA) without using any catalyst. The Nafion solution was added onto HRP/ ZnO/ ITO bio-electrode to form a tight membrane on the surface before carrying out bio-sensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared bio-electrode HRP/ZnO/ITO is highly sensitive to the detection of H2O2 over a wide range of concentration with a linear range from 2.5 μM to 100 μM with the limit of detection 0.2 μM and sensitivity of 0.034 µA/ µM cm2. The higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. A relatively low value of the enzyme’s kinetic parameter (Michaelis-Menten constant, Km) of 0.166 μM indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in bio-sensing, clinical, food and beverage industry.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

A recombinant formulation of silk fibroin containing the arginine–glycine–aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal–epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

Carboxylic acid-tethered polyaniline as a generic immobilization matrix for electrochemical bioassays.

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL-1)-1cm-2, with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40ngmL-1) at - 0.1V (vs. Ag/AgCl), and a low detection limit of 2pgmL-1. The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H2O2, respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM-1cm-2), and linear ranges (0.1-5, 0.1-6, 0.1-7mM) with a detection limit of 10, 1, and 8µM for glucose, uric acid, and H2O2, respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H2O2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H2O2. Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H2O2.

Highly selective and sensitive detection of cadmium ions by horseradish peroxidase enzyme inhibition using a colorimetric microplate reader and smartphone paper-based analytical device

With the knowledge of the extent of damage caused by cadmium ions (Cd2+), it becomes imperative to detect the presence of Cd2+ in drinking water with a rapid, efficient, and selective analytical method. Herein, a facile and novel colorimetric method for detecting cadmium ions, based on the irreversible inhibition of horseradish peroxidase (HRP) enzyme was established. Alternatively, a low-cost and affordable paper-based analytical device integrated with a smartphone platform was further designed to visualize, on-site and quantitative detection of Cd2+. The method developed for Cd2+ detection is of great interest to ensure a rapid diagnosis of water quality. Under the optimal conditions, the limit of detection of cadmium ions was 0.1 ppb, which is much lower than the permissible limit of WHO (5 ppb) for human exposure. Interference measurements showed that the method exhibited good selectivity. The proposed method was successfully applied as a rapid screening for cadmium ions monitoring in drinking water.

Bioinspired Integration of Naturally Occurring Molecules towards Universal and Smart Antibacterial Coatings

AbstractSurface‐independent smart antibacterial coatings with on‐demand antibiotics release profiles have attracted increasing attention in medical instruments, antibiotics delivery platforms, and implanted devices. Although several approaches have been well documented, the facile preparation of antibacterial coatings onto universal substrates with high antibiotics loading efficiencies, tunable drug release profiles, and excellent antibacterial performances is still met with many challenges, especially related to the redundant coating material synthesis and selective substrate fabrication process. To resolve this issue, the authors report a facile and robust fabrication method towards smart antibacterial coating surfaces on various substrates via a one‐pot integration of two types of naturally occurring building blocks, aminoglycosides and protocatechualdehyde in the presence of plant enzyme horseradish peroxidase. The simultaneous dynamic imine bonds formation and enzymatic polymerization within the system during the one‐pot reaction enable the facile fabrication of diverse surface‐independent smart antibacterial coatings, which can demonstrate several promising features including high drug loading and dynamic (i.e., pH‐responsive) properties, tunable and on‐demand antibiotics release behaviors, and efficient antibacterial activities both in vitro and in vivo. This work may offer new opportunities towards the robust and universal antibacterial coating materials by the bioinspired integration of all natural building blocks.

High-sensitive photometric microplate assay for tumor necrosis factor-alpha based on Fe@BC nanozyme

Optical immunoassay for the detection of protein biomarker tumor necrosis factor-α (TNF-α) is reported based on core–shell Fe@BC structure nanozyme as label. The nanozyme is composed of Fe nanoparticles as core and carbon layer as shell with element B doped into the carbon shell. The nanozyme displays good peroxidase-like activity that can oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to oxTMB in the presence of H2O2, generating blue colored compound. The detection of TNF-α was following the ELISA protocol by replacing traditional enzyme horseradish peroxidase (HRP) with nanozyme Fe@BC. The assay can be finished by about 2 h and has wide linear range for TNF-α from 5 pg/mL to 1 ng/mL with limit of detection of 2 pg/mL. The assay was applied for the detection of TNF-α in human serum samples with detection results comparable to commercial ELISA.

NADH Regeneration Promoted by Solar Light Using Gold Nanoparticles/Layered Double Hydroxides as Novel Photocatalytic Nanoplatforms

AbstractNicotinamide adenine dinucleotide NAD(P)H is a prolific cofactor in oxidoreductases biochemistry, but its high cost and the stoichiometric amounts motivates significant research for establishing efficient solutions for regenerating NAD(P)H from NAD(P)+. This study presents nanoparticles of gold/layered double hydroxides (AuNPs/LDH) as nanoplatforms with highly efficient photocatalytic response for solar‐light promoted NADH regeneration. The novel photocatalysts are obtained via self‐assembling of ZnAlLDH and AuNPs by using the manifestation of the “structural memory” of the anionic clay in gold salt solution. The chemical composition, structural, optical and nanotextural features of AuNPs/LDH have been assessed by transmission electron microscopy (TEM), energy dispersive X‐Ray Spectroscopy (EDX), powder X‐ray diffraction (XRD), and UV‐Vis Spectroscopy (UV‐Vis). It was found that, at pH 8 and in the presence of flavin mononucleotide (FMN) as a mediator and using water as an electron donor, the regeneration the NADH under solar light is defined by a rate of 20.5 μM/h. Furthermore, the regenerated NADH cofactor is tested in presence of Horseradish Peroxidase (HRP) enzyme to promote the hydrogen peroxide reduction.

Enzyme prodrug therapy: cytotoxic potential of paracetamol turnover with recombinant horseradish peroxidase

Targeted cancer treatment is a promising, less invasive alternative to chemotherapy as it is precisely directed against tumor cells whilst leaving healthy tissue unaffected. The plant-derived enzyme horseradish peroxidase (HRP) can be used for enzyme prodrug cancer therapy with indole-3-acetic acid or the analgesic paracetamol (acetaminophen). Oxidation of paracetamol by HRP in the presence of hydrogen peroxide leads to N-acetyl-p-benzoquinone imine and polymer formation via a radical reaction mechanism. N-acetyl-p-benzoquinone imine binds to DNA and proteins, resulting in severe cytotoxicity. However, plant HRP is not suitable for this application since the foreign glycosylation pattern is recognized by the human immune system, causing rapid clearance from the body. Furthermore, plant-derived HRP is a mixture of isoenzymes with a heterogeneous composition. Here, we investigated the reaction of paracetamol with defined recombinant HRP variants produced in E. coli, as well as plant HRP, and found that they are equally effective in paracetamol oxidation at a concentration ≥ 400 µM. At low paracetamol concentrations, however, recombinant HRP seems to be more efficient in paracetamol oxidation. Yet upon treatment of HCT-116 colon carcinoma and FaDu squamous carcinoma cells with HRP–paracetamol no cytotoxic effect was observed, neither in the presence nor absence of hydrogen peroxide.Graphic abstract

Laser-induced graphene electrodes for electrochemical ion sensing, pesticide monitoring, and water splitting.

Laser-induced graphene (LIG) has shown to be a scalable manufacturing route to create graphene electrodes that overcome the expense associated with conventional graphene electrode fabrication. Herein, we expand upon initial LIG reports by functionalizing the LIG with metallic nanoparticles for ion sensing, pesticide monitoring, and water splitting. The LIG electrodes were converted into ion-selective sensors by functionalization with poly(vinyl chloride)-based membranes containing K+ and H+ ionophores. These ion-selective sensors exhibited a rapid response time (10-15s), near-Nernstian sensitivity (53.0mV/dec for the K+ sensor and - 56.6mV/pH for the pH sensor), and long storage stability for 40days, and were capable of ion monitoring in artificial urine. The pesticide biosensors were created by functionalizing the LIG electrodes with the enzyme horseradish peroxidase and displayed a high sensitivity to atrazine (28.9nA/μM) with negligible inference from other common herbicides (glyphosate, dicamba, and 2,4-dichlorophenoxyacetic acid). Finally, the LIG electrodes also exhibited a small overpotential for hydrogen evolution reaction and oxygen evolution reaction. The oxygen evolution reaction tests yielded overpotentials of 448mV and 995mV for 10mA/cm2 and 100mA/cm2, respectively. The hydrogen evolution reaction tests yielded 35mV and 281mV for the corresponding current densities. Such a versatile LIG platform paves the way for simple, efficient electrochemical sensing and energy harvesting applications.

Determination of miRNA derived from exosomes of prostate cancer via toehold-aided cyclic amplification combined with HRP enzyme catalysis and magnetic nanoparticles

MicroRNAs (miRNAs) play a significant role in tumorigenesis and tumor development. Exosomal microRNA-141 (miRNA-141, miR-141) has been reported to be overexpressed in prostate cancer (PCa) and has become a potential biomarker for the diagnosis of PCa. Herein, a novel fluorescent biosensor based on toehold-aided cyclic amplification combined with horseradish peroxidase (HRP) enzyme catalysis and magnetic nanoparticles (MNPs) was designed for determination of the exosomes-derived microRNA-141 (miRNA-141, miR-141). The synergy of HRP enzyme catalysis and toehold mediated strand display reaction (TSDR) increase the sensitivity of the method, and the good separation ability of MNPs ensures the specificity of the method. Therefore, under the optimized experimental conditions, the highly sensitive and specific detection of miRNA-141 can be realized, and the detection limit is as low as 10 fM. More importantly, the biosensor successfully determinates the exosomal miR-141 in the plasma of patients with PCa.

Potential of unglycosylated horseradish peroxidase variants for enzyme prodrug cancer therapy

Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA). However, the plant-derived enzyme is out of bounds for medical purposes due to its foreign glycosylation pattern and resulting rapid clearance and immunogenicity. In this study, we generated recombinant, unglycosylated HRP variants in Escherichia coli using random mutagenesis and investigated their biochemical properties and suitability for cancer treatment. The cytotoxicity of the HRP-IAA enzyme prodrug system was assessed in vitro with HCT-116 human colon, FaDu human nasopharyngeal squamous cell carcinoma and murine colon adenocarcinoma cells (MC38). Extensive cytotoxicity was shown in all three cancer cell lines: the cell viability of HCT-116 and MC38 cells treated with HRP-IAA was below 1% after 24 h incubation and the surviving fraction of FaDu cells was ≤ 10% after 72 h. However, no cytotoxic effect was observed upon in vivo intratumoral application of HRP-IAA on a MC38 tumor model in C57BL/6J mice. However, we expect that targeting of HRP to the tumor by conjugation to specific antibodies or antibody fragments will reduce HRP clearance and thereby enhance therapy efficacy.

Hydrogen-Bonding-Induced H-Aggregation of Charge-Transfer Complexes for Ultra-Efficient Second Near-Infrared Region Photothermal Conversion

Hydrogen-Bonding-Induced H-Aggregation of Charge-Transfer Complexes for Ultra-Efficient Second Near-Infrared Region Photothermal Conversion

A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits

The collateral cleavage function is only a corollary of programmable nuclease activity of certain Cas effectors, such as Cas12 and Cas13, but it can be utilised to amplify fluorescence signals in various CRISPR/Cas-based biosensing systems. In this work, this special signal amplification capability of CRISPR/Cas12a ribonucleoproteins has been employed to increase the sensitivity of a broad class of commercial ELISA systems with undisclosed chemistry except for the use of horseradish peroxidase (HRP), a common signal reporting molecule. We demonstrated that such ELISA systems with HRP on the detection antibody, can be amplified by 2 orders of magnitude using an example commercial IFN-γ ELISA kit where the detection limit was decreased from 312.5 pg/mL to 1.2 pg/mL. The detection range was simultaneously increased from 2 orders to 3 orders of magnitude. Our CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. The CES-Amplifier can be directly integrated into commercial ELISA kits to replace their original last step, without any additional changes of the ELISA kit reagents or setup. In this way, our CES-Amplifier provides a versatile and affordable approach for expanding CRISPR/Cas-based biosensing to a wide range of non-nucleic acid analytes.

Mg and Ni nano-hydrotalcites modified with gold nanoparticles as platform of enzymatic electrochemical sensors for H2O2 detection

The immobilization of Horseradish Peroxidase enzyme over Magnesium (Mg-nHT) and Nickel (Ni-nHT) nano Hydrotalcite clays were synthesized by the co-precipitation method, and later modified with gold nanoparticles through in-situ synthesis and delamination-restructuration, is reported. The synthesized materials were characterized by Fourier-Transform Infrared Spectroscopy, X-ray diffraction, Scanning Electronic Microscopy, Flame Atomic Absorption Spectroscopy and Cyclic Voltammetry. Results show that the size of the synthetized materials is 26 nm for the gold nanoparticles, and 38.92 nm and 8.44 nm for Mg-AlnHT and Ni-AlnHT, respectively. The clays were used to modify the surface of graphite electrodes (GE); the inclusion of Au nanoparticles in the Hydrotalcites structure improved the enzyme-electrode electric communication. Figures of merit for detection of H2O2 by Differential-Pulse Voltammetry at HRP/Mg-AlnHT/GE, HRP/Ni-AlnHT/GE, HRP-AuNps/Mg-AlnHT/GE and HRP-AuNps/Ni-AlnHT/GE modified electrodes were determined, and respective detection limits of 0.012 μmolL−1, 0.014 μmolL−1, 0.009 μmolL−1, and 0.010 μmolL− 1 were obtained.

Peroxidase-like activity of Fe3O4/carbon core-shell nanostructured : effects of carbon shell thickness for application to glucose biosensor

In this study, we present a protocol for synthesis of carbon coated Fe3O4 nanoparticles with core-shell structured nanocomposite (FeC) following a two steps approach. The peroxidase-like acitivity of the synthesized FeC nanocomposite has been evaluated towards replacing of the horseradish peroxidase enzyme (HRP) in hydrogen peroxide enzymatic biosensor. In which, FeC has catalyzed for a redox reaction 5,5'-tetramethylbenzidine (TMB) and H2O2 to produce oxidized state of TMB with as a blue color. Results exhibited that FeC has a high catalytic activity accepting for fabrication of a high selectivity hydrogen peroxide (H2O2) colorimetric sensor with low detection of limit (LoD) of 0.02 mM H2O2. Based on this finding, we have used FeC and combined with glucose oxidase (GOx) enzyme to construct a new colorimetric glucose biosensor with high selectivity.

Reusable OIRD Microarray Chips Based on a Bienzyme-Immobilized Polyaniline Nanowire Forest for Multiplexed Detection of Biological Small Molecules.

Quantitative detection of multiple biological small molecules is critical for health evaluation and disease diagnosis. In this study, a microarray chip featuring a bienzyme-immobilized polyaniline nanowire forest on fluorine-doped tin oxide (bienzyme-PANI/FTO) is developed for this purpose. On such a chip, the target molecules are oxidized under the catalysis of their attached oxidases to produce hydrogen peroxide, which further induces the partial oxidation of local PANI nanowires in the presence of horseradish peroxidase (HRP) enzyme. The redox state change of PANI nanowires is monitored by the oblique incident reflectivity difference (OIRD) technique in a real-time and wireless manner, thus allowing for quantitative analysis of the target molecules. As typical model targets, hydrogen peroxide, glucose, lactic acid, and cholesterol are successfully detected with low detection limits, excellent specificities, and broad detection ranges, all of which fully meet the requirements for clinical analysis of human serum samples. Simultaneous detection of multiple targets on an individual chip is further demonstrated using the OIRD scanning mode. Meanwhile, by simple electrochemical reduction of the PANI nanowires, the chip is reusable for more than eight detection cycles without evident decay in its performance. The detection principle of this chip is also universal to other small molecules, and thus, it shows great promise as a valuable device to analyze biological small molecules.

Effect of Concomitant Hydroxyurea Therapy with Rutin and Gallic Acid: Integration of Pharmacokinetic and Pharmacodynamic Approaches.

Hydroxyurea (HU) is the first-ever approved drug by USFDA for sickle cell anemia (SCA). However, its treatment is associated with severe side effects like myelosuppression. Current studies are focused on the supplementation therapy for symptomatic management of SCA. In the present study, we aimed to explore rutin’s and gallic acid’s potential individually, for concomitant therapy with HU using pharmacokinetic and pharmacodynamic approaches since there is no such precedent till date. In vivo pharmacokinetic studies of HU in rats showed that rutin could be safely co-administered with HU, while gallic acid significantly raised the plasma concentration of HU. Both the phytochemicals did not have any marked inhibitory effect on urease but have considerable effects on horseradish peroxidase enzyme. The experimental phytoconstituents displayed a very low propensity to cause in vitro hemolysis. Gallic acid markedly enhanced the HU-induced decrease in lymphocyte proliferation. A substantial improvement by rutin or gallic acid was observed in HU-induced reduction of the main hematological parameters in rats. Combined treatment of HU with rutin and gallic acid reduced serum levels of both IL-6 and IL-17A. Overall, both rutin and gallic acid are found to have promising phytotherapy potential with HU. Further exploration needs to be done on both candidates for use as phytotherapeutics for SCA.

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards β-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.