Bacterial Adhesion Research Articles

Factors influencing initial bacterial adhesion to antifouling surfaces studied by single-cell force spectroscopy

Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. Escherichia coli adhesion is mediated by appendages, including type-1 fimbriae and curli amyloid fibers. Antifouling surfaces prevent the adhesion of bacteria to combat biofilm formation. Here, we used single-cell force-spectroscopy to study the interaction between E.coli and glass or two antifouling surfaces: the tripeptide DOPA-Phe(4F)-Phe(4F)-OMe and poly(ethylene glycol) polymer-brush. Our results indicate that both antifoulants significantly deter E.coli initial adhesion. By using two mutant strains expressing no type-1 fimbriae or curli amyloids, we studied the adhesion mechanism. Our results suggest that the bacteria adhere to different antifoulants via separate mechanisms. Finally, we show that some bacteria adhere much better than others, illustrating how the variability of bacterial cultures affects biofilm formation. Our results emphasize how additional study at the single-cell level can enhance our understanding of bacterial adhesion, thus leading to novel antifouling technologies.

A facile method to construct ZIF-8 MOFs on contact lens for high antibiotics loading and self-defensive release

Eye trauma, ophthalmic surgery and contact lens (CL) wearing cause corneal infection and bacterial biofilms formation, which further lead to corneal ulcer and perforation. In view of the low bioavailability of antibiotic eye drops and the clinical problems that cause bacterial resistance, we used pH sensitive zeolitic imidazolate framework-8 metal–organic frameworks (ZIF-8 MOFs) to load broad-spectrum antibacterial drugs levofloxacin (LEVO). After the surface modification by hyaluronic acid (HA), the nanomaterial surface was negatively charged. A simple amination treatment was used to endow CL surface with amino groups to combine the negatively charged nano drug loading system through electrostatic forces. The bacteriostatic ring of ZIF-8@LEVO@HA-PEI modified CL (ZLHP-CL) was 34 mm, which could be owing to the high drug loading capacity of ZIF-8 (0.65 mg/cm2). The bactericidal function was further confirmed by shake flask method and live/dead bacterial staining assay against S. aureus, S. epidermidis and E. coli. It is worth mentioning that ZLHP-CL showed significant pH sensitivity to the bacterial infection, which released drug rapidly at pH 5.5 and hardly released drug at pH 7.4. In the bacterial biofilm’s formation experiment, it was found that ZLHP-CL obviously inhibited bacterial adhesion and biofilms formation. Furthermore, it was proved in infectious keratitis animal that ZLHP-CL could kill the bacteria and reduce the inflammation reaction, which showed advantages in corneal tissue repair and light transmittance maintenance. In this work, based on nanotechnology, a facile CL drug loading technology is developed, which can be used to sustained release of various drugs for ophthalmic diseases treatment.

Surface roughness associated with bacterial adhesion on dental resin-based materials.

This study investigates the surface quality and bacterial adhesion properties of various dental materials, including indirect composites, veneering composites, direct composites, polyether ether ketone (PEEK), and two millable polymethyl methacrylate (PMMA). Material specimens were processed following manufacturer instructions, initially evaluated for surface roughness and Streptococcus sanguinis (S. sanguinis) adhesion. Subsequently, toothbrushing simulation was employed to simulate aging, and changes in material surfaces were assessed via roughness measurements and bacterial adhesion testing. Prior to simulated aging, direct and indirect composites exhibited the lowest roughness values. However, after the simulated toothbrushing, veneering composites displayed the highest roughness levels. Both PMMA materials demonstrated the highest S. sanguinis adhesion levels, both before and after artificial aging. Interestingly, the indirect composite material showed a reduction in bacterial adhesion following toothbrushing simulation. Surprisingly, this study did not reveal a clear correlation between roughness and bacterial adhesion.

A low-shrinkage-stress and anti-bacterial adherent dental resin composite: physicochemical properties and biocompatibility.

Polymerisation shrinkage and biofilm accumulation are the two main problems associated with dental resin composites (DRCs) that induce secondary caries, which can cause restoration failure. Polymerisation shrinkage can lead to microleakage gaps between the tooth and the DRCs, causing the aggregation of bacteria and development of secondary caries. Reducing the shrinkage stress (SS) and improving the resistance to bacterial adhesion have always been the focus of this field in modifying DRCs. A thiol-ene resin system can effectively reduce the polymerisation SS via its step-growth mechanism for delaying the gel point. Fluorinated compounds can reduce the surface free energies, thereby reducing bacterial adhesion. Thus, in this study, a range of mass fractions (0, 10, 20, 30, and 40 wt%) of a fluorinated thiol-ene resin system were added to a fluorinated dimethacrylate resin system/tricyclo decanedimethanol diacrylate to create a fluorinated methacrylate-thiol-ene ternary resin matrix. DRCs were prepared using the obtained ternary resin matrix, and their physical and chemical properties, effect on bacterial adhesion, and biocompatibility were investigated. The results demonstrated that the volumetric shrinkage and SS of the DRCs were reduced with no reduction in conversion degree even after the thiol-ene resin system was added. All DRC-based fluorinated resin systems exhibited an excellent anti-bacterial adhesion effect, as evidenced by the colony-forming unit counts, live/dead bacterial staining, and crystal violet staining tests against Streptococcus mutans (S. mutans). The genetic expressions associated with the bacterial adhesion of S. mutans were substantially affected after being cultured with fluorinated DRCs. All fluorinated DRCs demonstrated good biocompatibility through the in vitro cytotoxicity test and live/dead staining images of the L-929 cells. The above results illustrate that the DRCs based on the fluorinated methacrylate-thiol-ene resin matrix can be potentially applied in clinical practice due to their low SS and anti-bacterial adhesion effect.

Surface characteristics and bacterial adhesion on single-shade composite resins: A comparative in vitro study of one-step versus multi-step polishing techniques.

This study evaluated the effects of two polishing systems, OptraGloss (G; one-step) and OptiDisc (D; multi-step), on surface roughness (SR), contact angle (CA), surface free energy (SFE), and bacterial adhesion on three single-shade composite resins: Omnichroma (O), ZenChroma (Z), and Charisma Diamond One (C). Data for SR, CA, SFE, and adhesion of Streptococcus mutans (S. mutans) and Streptococcus mitis (S. mitis) were analyzed using one-way ANOVA, Tukey post-hoc tests, and Pearson correlation (α=0.05). Multi-step polishing groups (OD, ZD, and CD) exhibited significantly lower SR (0.18, 0.18, and 0.29 µm, respectively) compared to OG (0.46 µm), ZG (0.30 µm), and CG (0.44 µm) (p<0.05). The highest CA was observed in ZG (91.6º). S. mitis adhesion was greater than S. mutans in all groups except OG. A significant correlation was observed between SR and the adhesion of S. mutans (r=0.693, p<0.001). Polishing systems applied to single-shade composite resins did not impact the SFE but affected SR, and bacterial adhesion.

Nanotopography and oral bacterial adhesion on titanium surfaces: in vitro and in vivo studies.

The present study aimed to evaluate the influence of titanium surface nanotopography on the initial bacterial adhesion process by in vivo and in vitro study models. Titanium disks were produced and characterized according to their surface topography: machined (Ti-M), microtopography (Ti-Micro), and nanotopography (Ti-Nano). For the in vivo study, 18 subjects wore oral acrylic splints containing 2 disks from each group for 24 h (n = 36). After this period, the disks were removed from the splints and evaluated by microbial culture method, scanning electron microscopy (SEM), and qPCR for quantification of Streptococcus oralis, Actinomyces naeslundii, Fusobacterium nucleatum, as well as total bacteria. For the in vitro study, adhesion tests were performed with the species S. oralis and A. naeslundii for 24 h. Data were compared by ANOVA, with Tukey's post-test. Regarding the in vivo study, both the total aerobic and total anaerobic bacteria counts were similar among groups (p > 0.05). In qPCR, there was no difference among groups of bacteria adhered to the disks (p > 0.05), except for A. naeslundii, which was found in lower proportions in the Ti-Nano group (p < 0.05). In the SEM analysis, the groups had a similar bacterial distribution, with a predominance of cocci and few bacilli. In the in vitro study, there was no difference in the adhesion profile for S. oralis and A. naeslundii after 24 h of biofilm formation (p > 0.05). Thus, we conclude that micro- and nanotopography do not affect bacterial adhesion, considering an initial period of biofilm formation.

Bacterial adhesion and surface properties of computer-aided design-computer-aided manufacturing restorative materials.

This study aimed to evaluate the surface properties and bacterial adhesion of computer-aided design-computer-aided manufacturing (CAD-CAM) restorative materials. Four CAD-CAM resin-based blocks (Vita Enamic, Shofu block HC, Cerasmart [CS] and Lava Ultimate [LU]) and a leucite-reinforced glass ceramic block (IPS Empress CAD) were used in the present study. Specimens prepared with dimensions of 10 × 10 × 1 mm were polished. Surface characteristics were assessed with hydrophobicity and surface free energy (SFE) analysis. Surface roughness was measured using a profilometer, and elemental and topographic evaluations were performed with SEM-EDX analysis. After being kept in artificial saliva for 1 h, Streptococcus mutans (S. mutans) and Streptococcus mitis (S. mitis) were incubated separately in 5% CO2 atmosphere at 37°C for 24 h. The adhered bacteria were counted as ×108 CFU/mL. Surface roughness, contact angle and SFE measurement values were found to be in the range of 0.144-0.264 Ra, 28.362°-70.074° and 39.65-63.62 mN/m, respectively. The highest adhered amount of S. mutans was found in CS and the lowest in LU, while there was no significant difference between the amounts of adhered S. mitis. Despite differences in the surface properties of the materials used for the study, the materials exhibited identical properties with respect to bacterial adhesion.

Enhanced Bacterial and Biofilm Adhesion Resistance of ALD Nano-TiO2 Coatings Compared to AO Coatings on Titanium Abutments.

The study was intended to compare the surface properties and the bacterial and biofilm adhesion resistance of two potential antibacterial nanometer titanium dioxide (nano-TiO2) coatings on dental titanium (Ti) abutments prepared by atomic layer deposition (ALD) and the anodic oxidation (AO) techniques. Nano-TiO₂ coatings were developed using ALD and AO techniques and applied to Ti surfaces. The surface properties and the bacterial and biofilm adhesion resistance of these coatings were evaluated against commonly used Ti and Zirconia (ZrO₂) surfaces. The chemical compositions, crystalline forms, surface topography, roughness and hydrophilicity were characterized. The antibacterial performance was assessed by the scanning electron microscope (SEM), the Colony-forming unit (CFU) assay and the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay using in vitro models of Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans), and Porphyromonas gingivalis (P. gingivalis) in both single- and mixed-species bacterial compositions. ALD-prepared nano-TiO₂ coatings resulted in a dense, smooth, and less hydrophilic surface with an anatase phase, significantly reducing the adhesion of the three bacteria by over 50%, comparable to ZrO₂. In contrast, AO-prepared coatings led to a less hydrophilic surface, characterized by various nano-sized pores within the oxide film. This alteration, however, had no impact on the adhesion of the three bacteria. The adhesion patterns for mixed-species bacteria were generally consistent with single-species results. ALD-prepared nano-TiO₂ coatings on Ti abutments demonstrated promising antibacterial properties comparable to ZrO₂ surfaces, suggesting potential in preventing peri-implantitis. However, the bacterial and biofilm adhesion resistance of AO-produced nano-TiO₂ coatings was limited.

Smart zwitterionic coatings with precise pH-responsive antibacterial functions for bone implants to combat bacterial infections.

Hydrophilic antifouling coatings based on zwitterionic polymers have been widely applied for the surface modification of bone implants to combat biofilm formation and reduce the likelihood of implant-related infections. However, their long-term effectiveness is significantly limited by the lack of effective and precise antibacterial activity. Here, a pH-responsive smart zwitterionic antibacterial coating (PSB/GS coating) was designed and robustly fabricated onto titanium-base bone implants by using a facile two-step method. First, dopamine (DA) and a poly(sulfobetaine methacrylate-co-dopamine methacrylamide) (PSBDA) copolymer were deposited on implants via mussel-inspired surface chemistry, resulting in a hydrophilic base coating with abundant catechol residues. Next, an amino-rich antibiotic, gentamicin sulfate (GS), was covalently linked to the coating through the formation of acid-sensitive Schiff base bonds between the amine groups of GS and the catechol residues present in both the zwitterionic polymer and the DA component. During the initial implantation period, the hydrophilic zwitterionic polymers demonstrated the desired anti-fouling properties that could effectively reduce protein and bacterial adhesion by over 90%. With time, the bacterial proliferation led to a decrease in the microenvironment pH value, resulting in the hydrolysis of the acid-sensitive Schiff base bonds, thereby releasing GS on demand and effectively enhancing the anti-biofilm properties of coatings. Benefiting from this synergistic antifouling and smart antibacterial activities, the PSB/GS coating exerted an excellent anti-infective activity in both in vivo preoperative and postoperative infection rat models. This proposed facile yet effective coating strategy is expected to provide a promising solution to combat bone implant-related infections.

Investigation of the structure and morphology of modified materials based on polyvinyl chloride

The use of polyvinyl chloride in practice is associated with many difficulties due to the hydrophobic nature of the polymer surface. This can lead to great adverse consequences when used in technological processes of the chemical industry and hydrometallurgy. Due to its high hydrophobicity and low surface free energy, PVC has a low degree of wettability with water, resulting in low ion sorption capacity, as well as undesirable protein absorption and bacterial adhesion. The aim of the work was to increase the hydrophilicity of the surface of industrially produced powdered PVC by modifying it with various organic amines and, as a result, hydrophilic ion-exchange materials of various structures and morphology will be obtained on its basis. The course of the reactions was studied by potentiometric titration, IR spectroscopy, and diffractionometry. The correlation between the morphology and chemical properties of the products obtained in the studied reactions and the formation of new covalent bonds in the structure of the polymer matrix was investigated using IR spectroscopy and diffractionometry. The influence of the nature of the modifying reagent, solvent and temperature on the modification process has been established.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

The intriguing strategies of Tannerella forsythia's host interaction.

Tannerella forsythia, a member of the "red complex" bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O-glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia, including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management.

Опсонизация бактерий изменяет адгезионные взаимодействия между эндотелиоцитами и нейтрофилами в модели экспериментальной септицемии

Опсонизация бактерий изменяет адгезионные взаимодействия между эндотелиоцитами и нейтрофилами в модели экспериментальной септицемии

Effect of anti-biofilm peptide CRAMP-34 on the biofilms of Acinetobacter lwoffii derived from dairy cows.

Dairy mastitis is one of the most common diseases in dairy farming, and the formation of pathogenic bacteria biofilms may be an important reason why traditional antibiotic therapy fails to resolve some cases of dairy mastitis. We isolated and identified three strains of A. lwoffii were with strong biofilm forming ability from dairy cow mastitis samples from Chongqing dairy farms in China. In order to investigate the effect of novel anti-biofilm peptide CRAMP-34 on A.lwoffii biofilms, the anti-biofilm effect was evaluated by crystal violet staining, biofilms viable bacteria counting and confocal laser scanning microscopy (CLSM). In addition, transcriptome sequencing analysis, qRT-PCR and phenotypic verification were used to explore the mechanism of its action. The results showed that CRAMP-34 had a dose-dependent eradicating effect on A. lwoffii biofilms. Transcriptome sequencing analysis showed that 36 differentially expressed genes (11 up-regulated and 25 down-regulated) were detected after the intervention with the sub-inhibitory concentration of CRAMP-34. These differentially expressed genes may be related to enzyme synthesis, fimbriae, iron uptake system, capsular polysaccharide and other virulence factors through the functional analysis of differential genes. The results of subsequent bacterial motility and adhesion tests showed that the motility of A.lwoffii were enhanced after the intervention of CRAMP-34, but there was no significant change in adhesion. It was speculated that CRAMP-34 may promote the dispersion of biofilm bacteria by enhancing the motility of biofilm bacteria, thereby achieving the effect of eradicating biofilms. Therefore, these results, along with our other previous findings, suggest that CRAMP-34 holds promise as a new biofilm eradicator and deserves further research and development.

Safety assessment of five candidate probiotic lactobacilli using comparative genome analysis.

Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.

A natural multifunction and multiscale hierarchical matrix as a drug-eluting scaffold for biomedical applications.

Sea urchin spines are biogenic single crystals of magnesium calcite that are stiff, strong, damage tolerant and light and have a bicontinuous porous structure. Here, we showed that the removal of their intraskeletal organic matrix materials did not affect the compressive mechanical properties and generated an open porosity. This matrix was able to adsorb and release oxytetracycline, a broad-spectrum antibiotic. The drug-loaded sea urchin matrix induced bacterial cell death after 4 and 8 hours of incubation of both Gram-negative E. coli and Gram-positive S. aureus strains and this process induces an inhibition of bacterial cell adhesion. In conclusion, this study shows that thermally treated sea urchin spines are a compressive resistant and lightweight matrix able to load drugs and with potential use in spine fusion, a challenging application that requires withstanding high compressive loading.

Cu2-xS homojunction coatings empower titanium implants with near-infrared-triggered antibacterial and antifouling properties.

For decades, implant-associated infections (IAIs) caused by pathogenic bacteria have been associated with high failure and mortality rates in implantation surgeries, posing a serious threat to global public health. Therefore, developing a functionalized biomaterial coating with anti-fouling and anti-bacterial functions is crucial for alleviating implant infections. Herein, a near-infrared-responsive anti-bacterial and anti-adhesive coating (Ti-PEG-Cu2-xS) constructed on the surface of titanium (Ti) implants is reported. This coating is composed of nano-Cu2-xS with anti-bacterial activity and super-hydrophilic polyethylene glycol (PEG). Under near-infrared irradiation, the nano-catalyst Cu2-xS on the surface of Ti-PEG-Cu2-xS induces bacterial death by catalyzing the production of singlet oxygen (1O2). The Ti-PEG-Cu2-xS coating can effectively prevent bacterial adhesion and biofilm formation. This coating combines the antibacterial mechanisms of "active attack" and "passive defense", which can kill bacteria and inhibit biofilm formation. The results of in vitro and in vivo experiments have shown that Ti-PEG-Cu2-xS exhibits excellent anti-bacterial properties under near-infrared irradiation and can effectively prevent implant-related infections caused by Escherichia coli (E. coli) ATCC 8739 and Staphylococcus aureus (S. aureus). The antibacterial efficiency of Ti-PEG-Cu2-xS coatings against E. coli was 99.96% ± 0.058% and that of S. aureus was 99.66% ± 0.26%, respectively. In addition, the Ti-PEG-Cu2-xS coating has good blood compatibility and excellent bactericidal ability. Therefore, this multifunctional coating combines a non-adhesive surface strategy and a near-infrared phototherapy sterilization method, effectively blocking the initial attachment and proliferation of bacteria on implants via photothermal/photodynamic effects and providing a promising method for preventing bacterium-induced IAIs.

Smart self-defensive coatings with bacteria-triggered antimicrobial response for medical devices.

Bacterial colonization and biofilm formation on medical devices represent one of the most urgent and critical challenges in modern healthcare. These issues not only pose serious threats to patient health by increasing the risk of infections but also exert a considerable economic burden on national healthcare systems due to prolonged hospital stays and additional treatments. To address this challenge, there is a need for smart, customized biomaterials for medical device fabrication, particularly through the development of surface modification strategies that prevent bacterial adhesion and the growth of mature biofilms. This review explores three bioinspired approaches through which antibacterial and antiadhesive coatings can be engineered to exhibit smart, stimuli-responsive features. This responsiveness is greatly valuable as it provides the coatings with a controlled, on-demand antibacterial response that is activated only in the presence of bacteria, functioning as self-defensive coatings. Such coatings can be designed to release antibacterial agents or change their surface properties/conformation in response to specific stimuli, like changes in pH, temperature, or the presence of bacterial enzymes. This targeted approach minimizes the risk of developing antibiotic resistance and reduces the need for continuous, high-dose antibacterial treatments, thereby preserving the natural microbiome and further reducing healthcare costs. The final part of the review reports a critical analysis highlighting the potential improvements and future evolutions regarding antimicrobial self-defensive coatings and their validation.

Significance of Bacterial Adhesion and Motility—Its Association with Pathogenicity Revealed by Machine Learning

Significance of Bacterial Adhesion and Motility—Its Association with Pathogenicity Revealed by Machine Learning

Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.

Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.