NBT (Nitro Blue Tetrazolium) staining method is a commonly used technique for detecting enzyme activity in biochemical reactions, particularly when assessing the hydroxylation effects of tyrosinase on proteins. Tyrosinase is a copper enzyme that catalyzes the hydroxylation of substrates such as tyrosine and DOPA, playing a crucial role in melanin synthesis and certain biosynthetic pathways. The use of NBT staining can indirectly reflect the effects of tyrosinase on its substrates.

NBT staining uses nitroblue tetrazolium (NBT) as a qualitative analysis method to specifically detect quinones. In the presence of excess glycine as a reducing agent, quinones and related compounds undergo catalytic redox cycling at alkaline pH. Under the conditions of nitroblue tetrazolium and oxygen, the tetrazolium is reduced to formaldehyde. This property of quinolone compounds is employed for the specific staining of quinone proteins, separating them using SDS-PAGE and then transferring them to nitrocellulose. Previous studies have utilized NBT as a reagent in staining experiments for yolk proteins containing DOPA groups and for the enzyme tyrosinase containing 6-hydroxydopa from bovine serum.

The NBT staining method is based on the reduction of NBT to form a deep blue insoluble product in a reducing environment. Mfp (Mussel foot protein) are rich in dopaminergic groups and positively charged amino acids. The dopaminergic groups not only possess strong anti-inflammatory and antioxidant properties but also confer powerful underwater adhesion to Mfps. Therefore, when Mfp proteins are catalyzed by tyrosinase, they produce quinone intermediates that further promote the reduction of NBT. As a result, after the substrate is hydroxylated by tyrosinase, if reactive reducing substances are generated, they can lead to the coloration of NBT, with the intensity of the color being related to the degree of hydroxylation reaction.

Material and regents

Materials and reagents: mushroom tyrosinase, NBT, PVDF membrane, Image J software.

Procedures:

(1) To process the image, first open the corresponding image data with Image J, and convert the image under “Image > Type > 8-bit” to achieve 256 levels of grayscale.

(2) Normalize the background grayscale of the entire image to eliminate the influence of the background. Use “Process > Subtract Background”, with the default value set to 50.

(3) Set the background to black and the stripes to white. This restores the appearance of the image when illuminated and makes it easier for us to select later: “Edit > Invert”.

(4) Set the measurement parameters by going to “Analyze > Set Measurements, then select Area, Mean gray value, and Integrated density”.

(5) Set the measurement unit to "single pixel" by going to “Analyze > Set Scale, changing the Unit of length from inches to pixels”.

(6) Select the objects and areas to be measured, ensuring that the selection box size is consistent for different objects, and measure the integrated optical density.

(7) Analyze the measured gray integrated optical density data using graphing and statistical software, comparing differences between groups.

Fig. 1 NBT staining for the measurement of hydroxylation of DOPA

Fig.2 Image J analysis of the gray value of NBT staining

Based on the measured grayscale values, the values of different groups were statistically analyzed. BSA protein serves as the negative control group, Post-hydro is the fusion protein after the hydroxylation reaction, Pre-hydro is the fusion protein that has not undergone hydroxylation treatment, and Cell-Tak is the positive control (a commercially available tissue cell adhesion agent).

The images and results from NBT staining show that the zwii-Mfp5-D51 fusion protein successfully underwent a hydroxylation reaction after treatment with tyrosinase. Compared to the control group and the non-hydroxylated protein group, the grayscale value of the hydroxylated protein was significantly higher (94907 > 33943). However, the positive control group had an even higher grayscale value, reaching 203756. The study indicates that Cell-Tak, as an extracellular matrix bio-coating, is a specially formulated protein solution that can be used to treat surfaces of various materials, allowing cells to adhere to biological materials such as plastics, glass, and metals. Its main component is also a polyphenolic protein extracted from Mytilus edulis. Compared to our fusion protein, the primary differences could lie in the protein concentration and the ratio of polyphenolic groups, which provides potential directions for the optimization of our product in the future.

Coomassie Brilliant Blue Staining is a commonly used technique for protein detection and analysis, widely applied in gel electrophoresis experiments such as SDS-PAGE for visual detection of protein bands. Proteins need to be visualized by some method, and Coomassie Brilliant Blue is a dye that can specifically bind to proteins and produce a distinct coloration. In 1970, Fairbanks et al. first systematically reported the use of Coomassie Brilliant Blue staining for post-electrophoretic staining of proteins (Fairbanks, G., et al., 1971).

Coomassie Brilliant Blue (commonly referred to as R-250 or G-250) binds to basic amino acids (such as lysine, arginine, and histidine) and aromatic amino acids (such as tyrosine, tryptophan, and phenylalanine) in protein molecules through electrostatic attraction, hydrophobic interactions, and other means, resulting in the protein exhibiting a blue color. After staining, the protein can be treated with a decolorizing agent to remove unbound dye, leaving only the portion that is bound to the protein colored. The staining sensitivity is quite high, capable of detecting approximately 0.1-0.5 micrograms of protein.

Material and regents:

Coomassie Brilliant Blue R-250 (or G-250) dye solution should be prepared using methanol, glacial acetic acid, and deionized water. The commonly used formula is 0.1% (w/v) Coomassie Brilliant Blue R-250, 45% methanol, and 10% glacial acetic acid. The destaining solution is usually a mixture of 40% methanol and 10% glacial acetic acid.

Procedures:

1. Take 5 microliters of the protein solution from different experimental groups and control groups (negative control, hydroxylation treatment group, non-hydroxylation treatment group, positive control Cell-Tak group) and drop them onto different clean material surfaces using a pipette.

2. Dry at room temperature for 12 hours.

3. Shake in pure water for 0.5 hours and dry at room temperature.

4. Stain with Coomassie Brilliant Blue G-250 for 10 minutes, and compare the adsorption ability of the sample proteins on different matrix surfaces based on the staining intensity.

5. Analyze and quantify the stained images using Image J software.

Fig. 3A Coomassie Brilliant Blue staining for the adhesion test of fusion protein in glass

Fig. 3B Coomassie Brilliant Blue staining for the adhesion test of fusion protein in polystyrene

From the statistics of the images and grayscale values, after hydroxylation, more fusion proteins adhered to the glass surface, resulting in the deepest protein staining among all groups. This also demonstrated that hydroxylation treatment can significantly enhance the adhesion capability of proteins. Similarly, we tested the adhesion capability of Polystyrene using the same method, and the results showed that the hydroxylated fusion proteins exhibited strong adhesion capabilities comparable to the positive control Cell-Tak.

Biological adhesion testing refers to experimental methods used to evaluate the adhesion capabilities between materials, drugs, or surfaces and biological systems (such as cells, tissues, or biological membranes). In fields such as drug design, tissue engineering, and biomaterials science, such tests are crucial for understanding the interactions between materials and the biological environment. Some commonly used adhesion tests include quantitative filter membrane methods, centrifugation methods, titration methods, and staining assays.

Among them, the quantitative filtration method involves placing the sample on a filter paper (simulating the surface of cells or tissues) to allow it to come into full contact with the filter paper. Rinsing or soaking steps can remove the unbound portions, and the remaining adhered amount is calculated by weighing or measuring the concentration. The centrifugation method involves co-culturing the sample with a biological surface (such as a cell monolayer or artificial membrane) and then using centrifugal force to "shake off" the unbound portions, leaving only the adhered parts. During detection, the remaining amount is counted to reflect the strength of adhesion. The titration method is suitable for analyzing the changes in substances during the adhesion process, such as energy consumption, binding amount, or morphological changes. Adhesion status can be inferred from the amount of titration consumed or changes in related properties. The dye assessment method uses dyes such as Coomassie Brilliant Blue or crystal violet, which react with the adhered samples, and the adhesion status is judged based on color intensity (e.g., absorbance, etc.).

The detection principle of biological adhesion tests generally involves the formation of a stable adhesion between the sample and the material surface. Afterward, quantitative or qualitative analysis of the adhesion amount and strength is performed using physical, chemical, or optical methods to evaluate the adhesion strength and capability. In this method, we mainly assess the adhesion ability of our fusion protein zwi-Mfp5-D51 using materials easily obtainable in a laboratory and simple macroscopic approaches. The fusion protein forms a stable adhesion interface between two materials, and then the adhesive effect of the pipette tip is observed through dynamic changes and gravitational effects, allowing us to macroscopic evaluate the adhesion ability of our fusion protein.

Material and regents:

Pipette tips, centrifuge tube lids, fusion protein solution

Procedures：

1. Select appropriate pipette tips and centrifuge tube caps for the laboratory, handle them in a clean manner to ensure no contamination.

2. Take different specifications of tips (PTFE) to pick up 1 mg/mL of the modified and unmodified recombinant proteins, BSA, and Cell-Tak, and adhere them to the centrifuge tube caps (polypropylene), allowing them to dry at room temperature for 1 to 3 hours.

3. Slowly rotate the centrifuge tube cap and dynamically observe the macroscopic adhesion of the sample proteins.

Fig. 4 adhesion test by pipette tips adhesion

In the experiment groups, there are four different groups including 1: BSA，2: Cell-Tak, 3: zwi-Mfp5-D51 fusion protein(Hydroxylated), 4: wi-Mfp5-D51 fusion protein(non-Hydroxylated).

From the tested images and videos, it can be seen that the hydroxylated fusion protein firmly adhered the pipette tips to the centrifuge tube lid, maintaining adhesion under the force of gravity, while the tips from other groups fell off. Therefore, through this method, we can quickly and easily assess and confirm the adhesion effects of certain biological solution samples, laying the groundwork for further functional tests.

Antibacterial activity testing is an important experimental method for evaluating the inhibitory or lethal effects of a substance on the growth of bacteria and other microorganisms, widely used in the screening and research of new antibacterial agents, drugs, antibiotics, and more. Among them, the bacterial growth curve method and optical density OD600 method are one of the most commonly used evaluation means. Bacteria can continue to proliferate under suitable culture conditions, manifesting typical changes in growth curves, including lag phase, logarithmic phase, stationary phase, and death phase. If a sample with antibacterial activity is added to the cultivation system, its growth rate or final density will be inhibited, and this change can be observed and quantified by continuously monitoring the turbidity of bacteria (i.e., optical density, usually OD600). The OD600 detection technology refers to estimating the concentration of bacteria or microbial suspensions by measuring the optical density (Optical Density, OD) of the sample at a wavelength of 600 nm. This technology is widely applied in fields such as microbiology, molecular biology, and bioengineering for monitoring cell growth, fermentation processes, and bacterial activity. The background of OD600 measurement originated from the development of early optical detection technologies. With the popularity of spectrophotometers, scientists discovered that by determining a sample's absorption or scattering of light at specific wavelengths, they could indirectly estimate the concentration or turbidity of biological cells in a suspension. The OD600 value has an approximately linear relationship with cell concentration (within a certain range), making it a simple and rapid method for measuring cell density.

Growth curve method: Regularly measure the absorbance (OD600) of the bacterial liquid culture medium to depict the trend of bacterial growth over time (growth curve). If the sample has an inhibitory effect on the bacteria, the growth curve will rise more slowly during the logarithmic phase, and the final "height" will also be reduced.

Material and regents:

Common liquid culture media such as LB

Antimicrobials for testing (drugs, material extracts, etc.)

Target bacteria (e.g., Escherichia coli, Bacillus subtilis, etc.)

Microplate or small glass tubes

Spectrophotometer or microplate reader (capable of measuring 600 nm)

Various sterile tips, pipettes, and other basic sterile consumables

Procedures：

1. To investigate the antibacterial effect of the fusion protein coating, the Escherichia coli and Bacillus subtilis bacterial solutions were diluted to 1×10^6 CFU/mL.

2. Glass slides and stainless steel pieces coated with proteins (with different concentrations of coating proteins: 0 µg/mL, 750 µg/mL, 3000 µg/mL) were added to a 12-well plate, each well containing 1 mL of bacterial solution, and incubated at 37 degrees Celsius with 220 rpm for 20 hours.

3. The OD600 absorbance of the different wells was measured at different time points.

Fig. 5 Antimicrobial Effect Testing of Coated Glass and Coated Stainless Steel

By continuously cultivating glass and stainless steel material samples coated with fusion proteins on a porous plate in a co-culture system with Escherichia coli and Bacillus subtilis, we observed through the intuitive turbidity of the bacterial solution and the specific OD600 absorbance values over time that the coated protein on the surface of the glass material exhibited a more significant antibacterial effect compared to the stainless steel material group. Notably, in the 3 mg/mL concentration group, the growth of both bacterial strains was significantly inhibited. Therefore, glass materials exhibit better coating compatibility.

With the increasing demand for clean surfaces and anti-fouling coatings in industrial production and daily life, developing efficient, environmentally friendly, and durable anti-fouling technologies has become a research focus. Peptides, due to their high designability, biodegradability, and good biocompatibility, are widely used in biomedical materials and environmental protection. In particular, dual-ion peptides (peptide molecules with both positive and negative charges) can achieve efficient surface modification through charge interactions. Commonly used anti-fouling testing methods include contact angle measurement, protein adsorption tests, and stain simulation tests. Among them, stain simulation tests use common stains found in daily or industrial environments, such as grease, sludge, dust, ink, and food residues, to artificially contaminate and then observe and record the material surface. Stain simulation testing is a method that is closer to actual application environments, with detection methods including visual observation, microscopic imaging, and image analysis software for quantitative measurement of stain area and color intensity.

Colony plating, also known as the plate coating method, is a classic and widely applied microbiological quantitative and qualitative detection method. A liquid culture of a certain dilution is evenly spread on the surface of a solid culture medium, and under suitable conditions, individual viable bacterial cells or clusters grow to form visible independent colonies. Colony counting is widely used in environmental monitoring, food safety, clinical diagnostics, and antimicrobial agent evaluation due to its simplicity, intuitiveness, and low cost. As a qualitative analysis method, it is suitable for rapid screening and guiding further quantitative testing or colony identification. Additionally, with the current advancements in machine learning in image analysis, more automated algorithms and software for quantitatively counting colonies are being developed, and they are expected to be practically applied in future colony count analyses.

This testing method primarily involves the use of material samples prepared with a combined coating protein, and co-culturing with Escherichia coli and Bacillus subtilis. After incubation, the material samples are removed. Bacteria on the surface of the materials are removed through washing, sonication, and other methods, and the analysis and determination of the material's surface anti-fouling properties are achieved through plate coating culture and colony observation.

Material and regents:

Basic microbiological media such as LB liquid medium, LB solid culture plates

48-well cell culture plates

Circular material discs with a diameter of 10mm and a thickness of 1mm (glass, stainless steel, etc.)

0.9% NaCl solution

Ultrasonic cleaning machine

Procedures:

1. First, culture E. coli and B. subtilis in a 48-well plate to an OD600 of 0.6, then place the coated glass slides (10mm glass slides covered with 18 micrograms of recombinant fusion protein) and incubate at 60 rpm for 120 minutes.

2. Next, remove the glass slides and wash the coating three times with 1 mL of sterile 0.9% NaCl solution.

3. Then use an ultrasonic cleaner (5 min) to wash off the bacteria from the coating in 1 mL of sterile 0.9% sodium chloride solution.

4. Finally, spread 100 microliters of bacterial suspension on an LB plate, incubate at 37 degrees Celsius for 24 hours, and observe colony growth.

Fig. 6 Test of stain resistance of coated glass and coated stainless steel materials.

From the analysis of colony images, on the surfaces of glass and stainless steel, compared to the uncoated blank materials, the experimental group with the coated protein (zwi-Mfp5-D51) shows fewer colony numbers. This indicates that our fusion protein, after forming a coating on the surfaces of glass and stainless steel materials, can to some extent reduce the adhesion of bacteria stains such as Escherichia coli and Bacillus subtilis to the material surfaces.

Reference

[1] Paz, M. A., Flückiger, R., Boak, A., Kagan, H. M., & Gallop, P. M. (1991). Specific detection of quinoproteins by redox-cycling staining. The Journal of biological chemistry, 266(2), 689–692.

[2] James G. Cappuccino, Natalie Sherman, Microbiology: A Laboratory Manual, ISBN 0321840224, 9780321840226