Integrated triple signal amplification strategy for ultrasensitive electrochemical detection of gastric cancer-related microRNA utilizing MoS2-based nanozyme, hybridization chain reaction, and horseradish peroxidase.

Early diagnosis and treatment of gastric cancer (GC) play a vital role in improving efficacy, reducing mortality and prolonging patients' lives. Given the importance of early detection of gastric cancer, an electrochemical biosensor was developed for the ultrasensitive detection of miR-19b-3p by integrating MoS2-based nanozymes, hybridization chain reaction (HCR) with enzyme catalyzed reaction. The as-prepared MoS2-based nanocomposites were used as substrate materials to construct nanoprobes, which can simultaneously load probe DNA and HCR initiator for signal amplification. Moreover, the MoS2-based nanocomposites are also employed as nanozymes to amplify electrochemical response. The presence of miR-19b-3p induced the assembly of MoS2-based nanoprobes on the electrode surface, which can activate in-situ HCR reaction to load a large number of horseradish peroxidase (HRP) for signal amplification. Coupling with the co-catalytic ability of HRP and MoS2-based nanozymes, the designed electrochemical biosensor can detect as low as 0.7 aM miR-19b-3p. More importantly, this biosensor can efficiently analyze miR-19b-3p in clinical samples from healthy people and gastric cancer patients due to its excellent sensitivity and selectivity, suggesting that this biosensor has a potential application in early diagnosis of disease.

Fabrication of a Heptapeptide-Modified Poly(glycidyl Methac-Rylate) Nanosphere for Oriented Antibody Immobilization and Immunoassay.

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and ß-2-Microglobulin (ß2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-ß2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-ß2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0-5.3 µg/mL, proving its great potential in immunoassay applications.

Enhanced Immobilization of Enzymes on Plasma Micro-Nanotextured Surfaces and Microfluidics: Application to HRP.

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 µL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability.

Atomic-scale strain engineering of atomically resolved Pt clusters transcending natural enzymes.

Strain engineering plays an important role in tuning electronic structure and improving catalytic capability of biocatalyst, but it is still challenging to modify the atomic-scale strain for specific enzyme-like reactions. Here, we systematically design Pt single atom (Pt1), several Pt atoms (Ptn) and atomically-resolved Pt clusters (Ptc) on PdAu biocatalysts to investigate the correlation between atomic strain and enzyme-like catalytic activity by experimental technology and in-depth Density Functional Theory calculations. It is found that Ptc on PdAu (Ptc-PA) with reasonable atomic strain upshifts the d-band center and exposes high potential surface, indicating the sufficient active sites to achieve superior biocatalytic performances. Besides, the Pd shell and Au core serve as storage layers providing abundant energetic charge carriers. The Ptc-PA exhibits a prominent peroxidase (POD)-like activity with the catalytic efficiency (Kcat/Km) of 1.50 × 109 mM-1 min-1, about four orders of magnitude higher than natural horseradish peroxidase (HRP), while catalase (CAT)-like and superoxide dismutase (SOD)-like activities of Ptc-PA are also comparable to those of natural enzymes. Biological experiments demonstrate that the detection limit of the Ptc-PA-based catalytic detection system exceeds that of visual inspection by 132-fold in clinical cancer diagnosis. Besides, Ptc-PA can reduce multi-organ acute inflammatory damage and mitigate oxidative stress disorder.

Biosensor Based on ZIF-67-HRP and MWCNTs Nanocomposite Modified Glass Carbon Electrode for the Detection of Luteolin in Vegetables.

Luteolin has various pharmacological properties, including anti-inflammatory, antioxidant, and antitumor characteristics. Due to its potential value in drugs and functional foods, it is important to develop an efficient method for detecting luteolin. In this work, the poor selectivity of existing luteolin nonenzymatic sensors was solved by translating the enzyme-catalyzed reaction from bulk solution to the surface of a horseradish peroxidase (HRP) modified electrode through an electrocatalytic oxidation process. Here, we modified the surface of a glassy carbon electrode (GCE) with metal-organic frameworks (MOFs; ZIF-67 here, abbreviated as ZIF), functional nanomaterials, and HRP and finally covered it with Nafion (NF). In this case, luteolin acts as a hydrogen donor, and the electrode acts as a hydrogen acceptor; the oxidation reaction occurs on the electrode surface. The use of ZIF-67 ensured the conformational stability of HRP to ensure the selectivity and anti-interference property, and SDS-dispersed multiwalled carbon nanotubes (MWCNTs) enhanced the electrode conductivity. The use of NF avoids shedding of the electrode material during the testing process. A UV-vis spectrophotometer was used to study the selectivity of luteolin by HRP and the compatibility between HRP and ZIF. The materials were characterized and analyzed by scanning electron microscopy and transmission electron microscopy. Due to the synergistic effect of these nanomaterials, the linear range of NF/ZIF-HRP/MWCNTs-SDS/GCE was 1.0 × 10-2 to 6.0 µM, with detection limits of 25.3 nM (S/N = 3). The biosensor showed long-term stability and reproducibility, with a relative standard deviation of 4.2% for the peak current (n = 5). Finally, the biosensor was successfully used to detect luteolin in carrots, celery, and cauliflower.

Cascade Enzymes Confined in DNA Nanoanchors for Antitumor Therapy.

Cascade-enzyme reaction systems have emerged as promising tools for treating malignant tumors by efficiently converting nutrients into toxic substances. However, the challenges of poor localized retention capacity and utilization of highly active enzymes often result in extratumoral toxicity and reduced therapeutic efficacy. In this study, we introduced a cell membrane-DNA nanoanchor (DNANA) with a spatially confined cascade enzyme for in vivo tumor therapy. The DNANAs are constructed using a polyvalent cholesterol-labeled DNA triangular prism, ensuring high stability in cell membrane attachment. Glucose oxidase (GOx) and horseradish peroxidase (HRP), both modified with streptavidin, are precisely confined to biotin-labeled DNANAs. Upon intratumoral injection, DNANA enzymes efficiently colonize the tumor site through cellular membrane engineering strategies, significantly reducing off-target enzyme leakage and the associated risks of extratumoral toxicity. Furthermore, DNANA enzymes demonstrated effective cancer therapy in vitro and in vivo by depleting glucose and producing highly cytotoxic hydroxyl radicals in the vicinity of tumor cells. This membrane-engineered cascade-enzyme reaction system presents a conceptual approach to tumor treatment.

Ultrasensitive Electrochemical Biosensor for Rapid Screening of Chemicals with Estrogenic Effect.

Estrogenic chemicals are widely distributed and structurally diverse. They primarily disrupt estrogen-related metabolism in animals or humans by mimicking the agonistic receptor effects of natural estrogens, thereby influencing the transcription of estrogen receptors to regulate their quantity and sensitivity. This disruption of estrogen-related metabolism can lead to estrogen-related effects, posing risks to biological health, emphasizing the urgent need for simple and effective methods to screen compounds with estrogenic effects. Herein, a new electrochemical biological effect biosensor based on human estrogen receptor α (hERα) is developed, which uses hERα as the biorecognition element and employs the electroactive horseradish peroxidase (HRP) labeled 17ß-estradiol (E2) multifunctional conjugate HRP-E2 as the signal-boosting element and ligand competition agent. Based on the specific ligand-receptor interaction principle between the target and nuclear receptor, by allowing the test compound to compete with HRP-E2 conjugate for binding to hERα and testing the electrocatalytic signal of the conjugate that fails to bind to the hERα estrogen receptor, rapid screening and quantitative detection of chemical substances with estrogenic effect have been achieved. The biosensor shows a wide linear range of 40 pM to 40 nM with a detection limit of 17 pM (S/N = 3) for E2, and the detection limit is 2 orders of magnitude better than that of the previously reported sensors. The biosensor based on ligand-receptor binding can not only quantitatively analyze the typical estrogen E2, but also evaluate the relative estrogen effect strength of other estrogen compounds, which has good stability and selectivity. This electrochemical sensing platform displays its promising potential for rapid screening and quantitative detection of chemicals with estrogenic effects.

Glow-type luminol chemiluminescence based on a supramolecular enhancer of cyclodextrin.

BACKGROUND: Chemiluminescence (CL) bioassay is one of the most advanced and used detection method in clinical diagnosis and biomedical research because of the advantages of low background, easy operation, and wide-field imaging without a light source or microscope. The luminol/hydrogen peroxide/horseradish peroxidase (luminol/H2O2/HRP) system is the most popular CL system, but its application in high-throughput imaging detection is challenged due to its low luminescence efficiency and flash-type emission which is difficult in ensuring the reproducibility and consistency of detection results. RESULTS: We reported a glow-type CL system of luminol@CD/H2O2/HRP by using a supramolecular enhancer of cyclodextrin (CD). This luminol@CD/H2O2/HRP system exhibited a luminescence lifetime of 41 min for sensitive and accurate imaging analysis. The long-lasting CL emission was attributed to the formation of a 1:1 host-guest complex between luminol and CD, which could stabilize the emitter and effectively reduce nonradiative relaxation. The formation of luminol@CD complex was determined through NMR experiments and theoretical analysis. Under optimum conditions, the luminol@CD/H2O2/HRP system showed higher sensitivity and much better precision than classical luminol/H2O2/HRP system for imaging detection of HRP. Especially, this glow-type luminol@CD/H2O2/HRP system realized CL imaging of microwell arrays on microfluidic chips. In addition, the luminol@CD/H2O2/HRP system was successfully applied for point-of-care detection of 17ß-estradiol based on a competitive mechanism of host-guest recognition. SIGNIFICANCE: An efficient CL system is crucial for obtaining reproducible and consistent results for accurate detection. Our luminol@CD/H2O2/HRP system emitted strong and persistent luminescence, resulting in reliability and efficiency at both CL macroscopic and microscopic imaging detection. We expected the luminol@CD/H2O2/HRP CL system to be applied in various detection fields.

Immobilization of HRP enzyme on polymeric microspheres and its use in decolourisation of organic dyes.

In this study, horseradish peroxidase (HRP) enzyme was immobilized on Pd(II) containing polymeric microspheres by adsorption method and used for the decolourisation of Methyl Orange (MO) and Rhodamine B (RB) dyes. The synthesized microspheres were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy-Energy Dispersive X-ray (SEM/EDX), Thermal Gravimetric Analysis (TGA). The effects of pH, dye concentration, temperature, and H2O2 concentration on the decolourisation of MO and RB were determined. According to the results of various parameters studied, when 2-AEPS-napht-HRP support was used, MO and RB were biodegraded to 69.72% and 80.65%, respectively, within 60 min. When 2-AEPS-napht-Pd-HRP support was used, MO and RB were biodegraded to 58.35% and 90.81%, respectively, under optimum conditions. When the reproducibility results of the immobilized supports were examined, it was observed that they remained efficient during the first five reusability cycles and even reached 65% decolourisation efficiency after the 9th reuse. The immobilized enzyme (2AEPS-npht-HRP and 2AEPS-npht-Pd-HRP) showed remarkable resistance to higher temperatures compared to the free enzyme.

Enhancing the sensitivity of immunomagnetic assay for 3-phenoxybenzonic acid: a novel approach utilizing magnetosome-nanobody complexes and gold nanoparticles probes.

The 3-phenoxybenzoic acid (3-PBA) residues in environment are posing a significant challenge to our daily lives. To establish a more sensitive and rapid detection method, anti-3-PBA nanobodies (Nbs) were immobilized onto magnetosomes (bacterial magnetic nanoparticles, termed as BMPs), forming a robust BMP-Nb complex. The 3-PBA derivative was labeled with horseradish peroxidase (HRP) and further associated with gold nanoparticles (AuNPs) to create a highly sensitive probe (3-PBA-HRP-AuNP). An innovative immunoassay that combined BMP-Nb complex with 3-PBA-HRP-AuNP was developed for determinaton of 3-PBA. This method enabled the determination of 3-PBA with a half-maximum signal inhibition concentration (IC50) of 1.03 ng/mL, which was more sensitive than that of using 3-PBA-HRP as tracer with an IC50 of 2.18 ng/mL. The reliability of the assay was evidenced by the quantitative recovery of 3-PBA from water and soil samples ranging from 76.85 to 95.64%. The 3-PBA residues determined by this assay in actual water samples were between < LOD and 2.54 ng/mL and were between < LOD and 11.25 ng/g (dw) in real soils, respectively, which agreed well with those of liquid chromatography mass spectrometry (LC-MS). Collectively, the BMP-Nb and 3-PBA-HRP-AuNP-based immunoassay provides a powerful tool for the precise detection of 3-PBA residues in environment matrices, reinforcing our capacity to monitor and mitigate potential ecological and health impacts associated with this prevalent pollutant.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Glucose oxidase, horseradish peroxidase and phenothiazine dyes-co-adsorbed carbon felt-based amperometric flow-biosensor for glucose.

To develop an amperometric flow-biosensor for glucose, the stabilizing effect of methylene blue (MB) toward adsorbed glucose oxidase (GOx) on carbon felt (CF) was successfully applied to prepare the GOx-modified CF-based enzyme reactor combined with a horseradish peroxidase (HRP)-modified CF-based H2O2 detector. Upon mixing MB in the GOx-adsorption solution, the O2-dependent GOx-activity was significantly increased with increasing concentration of MB in the GOx-adsorption solution. The GOx-immobilization protocol on CF is very straightforward [i.e., adsorption of the GOx/MB mixed aqueous solution for 5 min under ultrasound (US)-irradiation]. Under the optimized operational conditions (i.e., applied potential, 0 vs. Ag/AgCl; carrier pH, 5.0; carrier flow rate, 4.0 mL min-1), the resulting GOx/MB-CF-reactor and HRP/TN-CF-detector combined amperometric flow-biosensor exhibited sensitive, selective, reproducible and stable cathodic peak current responses to glucose with the following analytical performances: sensitivity, 6.22 µA mM-1; linear range, 0.01 to 1 mM; limit of detection, 9.6 µM (S/N = 3, noise level, 20 nA); sample throughput, 46-96 samples per h for 10-0.1 mM glucose. The developed amperometric flow-biosensor allowed the determination of glucose in beverages and liquors, and the analytical results by the sensor were in fairly good agreement with those by conventional spectrophotometry.

Exploring the Bonding Nature of Iron(IV)-Oxo Species through Valence Bond Calculations and Electron Density Analysis.

Compound I (Cpd I) plays a pivotal role in substrate transformations within the catalytic cycle of cytochrome P450 enzymes (P450s). A key constituent of Cpd I is the iron(IV)-oxo unit, a structural motif also found in other heme enzymes and nonheme enzymes. In this study, we performed ab initio valence bond (VB) calculations, employing the valence bond self-consistent field (VBSCF) and breathing orbital valence bond (BOVB) methods, to unveil the bonding nature of this vital "Fe(IV)═O″ unit in bioinorganic chemistry. Comparisons were drawn with the triplet O2 molecule, which shares some electronic characteristics with iron(IV)-oxo. Additionally, Cpd I models of horseradish peroxidase (HRP) and catalase (CAT) were analyzed to assess the proximal ligand effect on the electronic structure of iron(IV)-oxo. Our VB analysis underscores the significant role of noncovalent resonance effects in shaping the iron(IV)-oxo bonding. The resonance stabilization within the π and σ frameworks occurs to comparable degrees, with additional stabilization resulting from resonance between VB structures from these frameworks. Furthermore, we elucidated the substantial influence of proximal and equatorial ligands in modulating the relative significance of different VB structures. Notably, in the presence of these ligands, iron(IV)-oxo is better described as iron(III)-oxyl or iron(II)-oxygen, displaying weak covalent character but enhanced by resonance effects. Although both species exhibit diradicaloid characters, resonance stabilization in iron(IV)-oxo is weaker than in O2. Further exploration using the Laplacian of electron density shows that, unlike O2, which exhibits a charge concentration region between its two oxygen atoms, iron(IV)-oxo species display a charge depletion region.

Cell Cycle Modulation through Physical Confinement in Micrometer-Thick Hydrogel Sheaths.

Recently, surface engineering of the cell membrane with biomaterials has attracted great attention for various biomedical applications. In this study, we investigated the possibility of modulating cell cycle progression using alginate and gelatin-based hydrogel sheaths with a thickness of ∼1 µm. The hydrogel sheath was formed on cell surfaces through cross-linking catalyzed by horseradish peroxidase immobilized on the cell surface. The hydrogel sheath did not decrease the viability (>95% during 2 days of culture) of the human cervical carcinoma cell line (HeLa) expressing the fluorescent ubiquitination-based cell cycle indicator 2 (HeLa/Fucci2). Coating the HeLa/Fucci2 cells with the hydrogel sheath resulted in a cell cycle arrest in the G2/M phase, which can be caused by the reduced F-actin formation. As a result of this cell cycle arrest, an inhibition of cell growth was observed in the HeLa/Fucci2 cells. Taken together, our results demonstrate that the hydrogel sheath coating on the cell surface is a feasible approach to modulating cell cycle progression.

Multi-walled carbon nanotubes functionalized with a new Schiff base containing phenylboronic acid residues: application to the development of a bienzymatic glucose biosensor using a response surface methodology approach.

An innovative supramolecular architecture is reported for bienzymatic glucose biosensing based on the use of a nanohybrid made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with a Schiff base modified with two phenylboronic acid residues (SB-dBA) as platform for the site-specific immobilization of the glycoproteins glucose oxidase (GOx) and horseradish peroxidase (HRP). The analytical signal was obtained from amperometric experiments at - 0.050 V in the presence of 5.0 × 10-4 M hydroquinone as redox mediator. The concentration of GOx and HRP and the interaction time between the enzymes and the nanohybrid MWCNT-SB-dBA deposited at glassy carbon electrodes (GCEs) were optimized through a central composite design (CCD)/response surface methodology (RSM). The optimal concentrations of GOx and HRP were 3.0 mg mL-1 and 1.50 mg mL-1, respectively, while the optimum interaction time was 3.0 min. The bienzymatic biosensor presented a sensitivity of (24 ± 2) × 102 µA dL mg-1 ((44 ± 4) × 102 µA M-1), a linear range between 0.06 mg dL-1 and 21.6 mg dL-1 (3.1 µM-1.2 mM) (R2 = 0.9991), and detection and quantification limits of 0.02 mg dL-1 (1.0 µM) and 0.06 mg dL-1 (3.1 µM), respectively. The reproducibility for five sensors prepared with the same MWCNT-SB-dBA nanohybrid was 6.3%, while the reproducibility for sensors prepared with five different nanohybrids and five electrodes each was 7.9%. The GCE/MWCNT-SB-dBA/GOx-HRP was successfully used for the quantification of glucose in artificial human urine and commercial human serum samples.

Arginine-Modified Hemin Enhances G-Quadruplex DNAzyme Peroxidase Activity for High Sensitivity Detection.

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

Probing Phoretic Transport of Oxidative Enzyme-Bound Zn(II)-Metallomicelle in Adenosine Triphosphate Gradient via a Spatially Relocated Biocatalytic Zone.

Although cellular transport machinery is mostly ATP-driven and ATPase-dependent, there has been a recent surge in understanding colloidal transport processes relying on a nonspecific physical interaction with biologically significant small molecules. Herein, we probe the phoretic behavior of a biocolloid [composed of a Zn(II)-coordinated metallomicelle and enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx)] when exposed to a concentration gradient of ATP under microfluidic conditions. Simultaneously, we demonstrate that an ATP-independent oxidative biocatalytic product formation zone can be modulated in the presence of a (glucose + ATP) gradient. We report that both directionality and extent of transport can be tuned by changing the concentration of the ATP gradient. This diffusiophoretic mobility of a submicrometer biocolloidal object for the spatial transposition of a biocatalytic zone signifies the ATP-mediated functional transportation without the involvement of ATPase. Additionally, the ability to analyze colloidal transport in microfluidic channels using an enzymatic fluorescent product-forming reaction could be a new nanobiotechnological tool for understanding transport and spatial catalytic patterning processes. We believe that this result will inspire further studies for the realization of elusive biological transport processes and target-specific delivery vehicles, considering the omnipresence of the ATP-gradient across the cell.

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

Harnessing Water Competition to Drive Enzyme Crosstalk.

In nature, enzymatic pathways often involve compartmentalization effects that can modify the intrinsic activity and specificity of the different enzymes involved. Consequently, extensive research has focused on replicating and studying the compartmentalization effects on individual enzymes and on multistep enzyme "cascade" reactions. This study explores the influence of compartmentalization achieved using molecular crowding on the glucose oxidase/horseradish peroxidase (GOx/HRP) cascade reaction. The crowder tested is methoxy poly(ethylene glycol) (mPEG) that can, depending on conditions, promote GOx and HRP coassociation at the nanoscale and extend their contact time. Low-molecular-weight mPEG (0.35 kDa), but not mPEG of higher molecular weights (5 or 20 kDa), significantly enhanced the cascade reaction where up to a 20-fold increase in the rate of the cascade reaction was observed under some conditions. The combined analyses emphasize the particularity of low-molecular-weight mPEG and point toward mPEG-induced coassociation of HRP and GOx, producing nearest crowded neighbor effects of HRP on GOx, and vice versa. These altered the nanoscale environments of these enzymes, which influenced substrate affinity. Using mPEG to promote protein coassociation is simple and does not chemically modify the proteins studied. This approach could be of interest for more broadly characterizing nearest crowded neighbor effects (i.e., protein-protein interactions) for multiprotein systems (i.e., more than just two), thus making it an interesting tool for studying very complex systems, such as those found in nature.