A Multidisciplinary Experiment to Characterize Antifouling Biocompatible Interfaces via Quantification of Surface Protein Adsorption

Abstract

Novel biomaterial development is a rapidly growing field that is crucial because biomaterial fouling, due to rapid and irreversible protein adsorption, leads to cellular responses and potentially detrimental consequences such as surface thrombosis, biofilm formation, or inflammation. Therefore, biomaterial technology’s fundamentals, like material biocompatibility, are critical in undergraduate education. Exposing undergraduate students to biomaterials and biomedical engineering through interdisciplinary experiments allows them to integrate knowledge from different fields to analyze multidisciplinary results. In this practical laboratory experiment, undergraduate students will characterize surface properties (contact and sliding angle measurements) for the antifouling polydimethylsiloxane (PDMS) polymer using a goniometer and a smartphone, as well as quantify protein adsorption on antifouling surfaces via a colorimetric assay kit to develop their understanding of antifouling surface characteristics, UV–vis spectroscopy, and colorimetric assays. The antifouling PDMS polymer is prepared by silicone oil infusion and compared to untreated control PDMS. The polymer hydrophobicity was demonstrated by static water contact angles of ~99° and 102° for control and antifouling PDMS surfaces, respectively. The control PDMS sliding angle (>90°) was significantly reduced to 9° after antifouling preparation. After 24 h incubation of polymer samples in a 200 mg/mL bovine serum albumin (BSA) solution, the surface adsorbed BSA was quantified using a colorimetric assay. The adsorbed protein on the fouling PDMS controls (29.1 ± 7.0 μg/cm²) was reduced by ~79% on the antifouling PDMS surface (6.2 ± 0.9 μg/cm²). Students will gain experience in materials science, biomedical engineering, chemistry, and biology concepts and better understand the influence of material properties on biological responses for biomaterial interfaces.

Graphical Abstract

1. INTRODUCTION

Within the realm of biomedical science and engineering, “fouling” is the word used to describe a situation in which substances adhere to material surfaces. The issue of surface fouling affects many real-world applications such as water resource equipment, medical device implants, or marine applications.^1–3 For example, in 2002, the International Maritime Organization reported that a ship’s hull that is unprotected from fouling could gather 150 kg of fouling material per square meter in less than six months, which can result in >6000 tons of fouling on a very large crude carrier ship. It is a known fact that ships can travel faster with better fuel efficiency when their hulls are free of fouling materials such as algae, mollusks, and barnacles. Just a small amount of fouling on the ship’s hull can lead to a 50% increase in fuel consumption.^4,5 While antifouling surfaces can save billions of dollars for shipping, air transportation, and other industries every year, they can make a significant impact on human healthcare as well. In almost every aspect of healthcare, biomaterials play crucial roles, and yet numerous challenges need to be addressed even after many years of bench and clinical studies. Most recently, there has been significant progress toward developing biomaterials and biomedical devices to address biocompatibility challenges such as surface fouling.^1,2 Biomaterials and medical device surfaces are prone to fouling.^2,6–8 Biofouling can be described as the attachment of biomolecules and cells such as proteins, platelets, microorganisms, or even our body’s cells on the material surface. Protein adsorption on biomaterial surfaces is the very first incident that occurs when medical devices are implanted in the body (Figure 1).^9,10 In some cases, cellular adhesion to the proteins/biomaterials can lead to severe clinical challenges such as blood clotting, bacterial infection, or fibrous encapsulation, which can limit the medical device function or lead to life-threatening risks.^11–14 One of the leading problems of indwelling materials and devices is surface thrombosis, which is orchestrated by initial protein adsorption to the surface upon exposure to blood. For example, when a patient undergoes open-heart surgery, the blood flows through a cardiopulmonary bypass machine. Proteins present in the blood will rapidly adhere to surfaces of the cardiopulmonary bypass circuitry (tubing, oxygenator) and can lead to unwanted clot formation.¹⁵ In contrast, protein adsorption for some biomedical applications, such as biodegradable wound dressings or orthopedic implants (e.g., artificial hip or knee joints), is considered beneficial because the adsorbed proteins can facilitate skin repair in the wound healing process and bone growth that leads to an improved attachment to the implant surface.¹⁶ Therefore, understanding protein adsorption on material surfaces and correlating with material properties is one measure of biocompatibility, especially with regards to its intended medical device application.

Figure 1.

Simplified schematic of response to a foreign surface that comes in contact with blood. (a) First response is protein adsorption to the surface of the biomaterial, which consequently leads to (b) platelet activation and cellular adhesion to the surface.

There is a wide range of medical devices that would benefit from the development of antifouling surfaces to overcome the challenges of unwanted adsorbed proteins, cells, or tissues. Antifouling materials have been shown to decrease blood clots on cardiopulmonary bypass machine surfaces and also reduce bacterial adhesion on a wide range of implants (e.g., intravascular or urinary catheters). The surface properties of a material are vital to the protein adsorption process (e.g., surface roughness, wettability). Biofouling and protein adsorption to the surface are the first irreversible responses for any foreign surface coming in contact with the physiological environment.^17,18 Protein adsorption to the surface is influenced by many factors; some of which are related to the surface properties including surface topography, wettability, heterogenicity, and chemical composition. Protein adsorption is also related to protein properties such as concentration, affinity to adhere to the surface, mobility, and unfolding rate.¹⁹ Therefore, translational research has aimed at developing material surfaces with antifouling properties that can prevent the adhesion of molecules and biofouling on the surface for medical device applications where this is unwanted.²⁰ It is necessary to be able to measure the amount of protein adsorption to different surfaces as an initial evaluation of biocompatibility.

One approach to creating antifouling biomaterial interfaces is the development of superhydrophobic surfaces based on lotus leaves, which essentially uses the concept of trapped air pockets on the material surface to prevent surface fouling. There have been practical laboratory experiments designed to explore superhydrophobicity and its properties.^7,21 Over time, however, these air pockets could potentially collapse and lose the superhydrophobicity. That is why the development of slippery, liquid-infused porous surfaces (SLIPs), which are inspired by the mucus membrane in the gastrointestinal tract, is a simple and beneficial alternative.²² The SLIPs polymers are prepared by infusing the polymer with an antifouling lubricant. One common and simple technique to develop a group of SLIPs is to use silicone oil as the antifouling surface lubricant for infusing the polydimethylsiloxane (PDMS) polymer. Silicone oil has been FDA approved for several biomedical applications, and its nontoxic nature makes it a preferred choice for antifouling biomedical applications.²³ While silicone oil leaching and durability of the SLIPs materials are important factors to consider for real biomedical device applications,^24,25 maintaining a slippery nature on the surface for these durations is more than adequate for the students to be able to conduct the characterization experiments described in this manuscript. Although silicone oil is one of the most common lubricant oils used for SLIPs, other reagents used for generating slippery surfaces include n-hexadecane and perfluoropolyethers.^26,27 The PDMS polymer also has been used in a wide variety of biomedical applications such as scaffolds fabrication,²⁸ extracorporeal circulation (ECC) circuits,²⁹ and intravascular and Foley catheters due to its many advantages such as nontoxicity to human cells, acceptable mechanical properties, transparency, and low manufacturing cost. However, the PDMS surface is prone to fouling in a physiological environment.³⁰ To combat this disadvantage, the silicone-based SLIPs surfaces are highly efficient in decreasing fouling, and reducing protein adsorption and bacterial adhesion, making them a promising candidate to enhance the biocompatibility of medical device surfaces.^31–33 These materials exhibit an ultralow fouling interface by employing capillary forces to produce a low adhesion liquid lubricating layer between the material surface and the exterior physiological environment. The silicone oil can be infused into PDMS polymers matrix, creating the antifouling interface that is resistant to biofouling and enhances the biocompatibility of medical devices.

Antifouling surfaces are characterized by static and sliding water contact angle experiments.^34,35 In static water contact angle measurements, a water droplet comes in contact with the surface in an atmospheric environment, and the contact angle (θ) is measured at the line tangent to the three-phase point (gas–liquid–solid). Conventionally, for a water droplet, if θ > 90°, the surface is considered hydrophobic, and if θ < 90°, the surface is considered hydrophilic (Figure 2a,b).^35,36 Static water contact angle measurements, while ideal for surfaces where the liquid–solid interface does not change, do not encompass dynamic interfaces. Therefore, advancing and receding contact angle measurements, the angle measurements between the droplet and solid surface when water is added or removed (or alternatively performed with a tilting stage if available), respectively, could be a potential measurement. Advancing contact angle tells the maximum contact angle the surface can have and the receding contact angle tells the minimum. The difference between advancing and receding contact angles is contact angle hysteresis, which occurs due to topographical heterogeniety of a surface.^37,38 Contact angle hysteresis cannot be measured directly but through the measurement of advancing and receding contact angles. In a sliding angle experiment, a 10–20 μL water droplet is placed on a flat polymer surface mounted on a sliding stage, and the stage is slowly tilted until the water droplet rolls off the surface.⁶ When a water droplet is placed on an unmodified polymer, the water droplet will remain stagnant on the surface even when the material is placed vertically, exhibiting a sliding angle of >90°. In contrast, when a water droplet is placed on the antifouling SLIPs polymers, it will immediately roll off the surface, exhibiting a sliding angle of <10° (Figure 2c,d).⁷ A low sliding angle is related to low contact angle hysteresis. While contact angle and sliding angle measurements do not holistically characterize a surface’s fouling or antifouling nature, they both are indicative of a surface’s wettability and can be used to further understand its surface properties and topography.³⁷ These phenomena are explained by Thomas Young, Cassie–Baxter, and Wenzel wetting states and general kinetics of material interfaces, where the reduction in interactions between a solid surface and liquid droplets on a very hydrophobic material reduces the total free energy leading to an increase in the contact angle of the droplets. This phenomenon leads to the well-known effect of SLIPs materials being repellant to fouling.^23,39

Figure 2.

Schematic representation of contact angle and sliding angle of different surfaces. (a) Hydrophobic surface with static water contact angle θ > 90°, and (b) a hydrophilic surface with θ < 90°. (c) Unmodified surface exhibiting sliding angle of water droplet at >90°. (d) Antifouling slippery, liquid-infused porous surface (SLIPs) surface exhibiting sliding angle of water droplet at <10°.

2. EXPERIMENTAL OVERVIEW

This hands-on laboratory experiment adapts recent research in developing antifouling SLIPs interfaces for medical device applications to an undergraduate laboratory course where the students will explore basic surface characterization methods, such as contact angle and sliding angle measurements, and will utilize an absorbance-based protein detection kit to quantify protein adsorption on surfaces. The following sections describe the sequence of the experimental activities for the students including the antifouling PDMS sample preparation using the silicone SLIPs technology, surface characterization, BSA protein exposure and the Pierce 660 nm protein quantification assay, and the corresponding results (Figure 3). Serum albumin is the most abundant blood plasma protein with a normal concentration in the blood of 34 to 54 mg/mL, high stability and water solubility, and is known to adsorb on a variety of biomaterial and medical device surfaces (within seconds). The polymer samples will be exposed to a model protein, bovine serum albumin (BSA). Students will make comparisons between the control PDMS and antifouling SLIPs PDMS polymer samples in terms of both surface wettability properties and in vitro protein adsorption, allowing them to make connections between these biocompatibility concepts and the ultimate impact these properties can have on biomaterials and medical device interfaces.

Figure 3.

Schematics showing step-by-step two session plan for this hands-on undergraduate laboratory experiment aimed at comparing control PDMS and antifouling SLIPs PDMS. In the first session, students will measure the water contact angle and sliding angle (using a smart phone) of samples. In the second session, students will expose the samples to a protein solution followed by quantification of protein adsorption on the surfaces via a colorimetric assay and UV–vis spectroscopy. Dashed boxes indicate steps that can be performed by the teaching assistant (TA) or instructor prior to the sessions if time constraints exist for the course.

3. LEARNING OBJECTIVE

The main objective for this laboratory hands-on experiment design is to provide students with an authentic insight into one of the most challenging problems of medical devices: biofouling and protein adsorption on the surface. These experiments will be conducted utilizing a simple silicone-based antifouling SLIPs polymer and the corresponding unmodified PDMS control. When the students have completed the experimental and analytical studies in the present laboratory experiment, it is expected that they will have observed, understood, and gained hands-on experience with the following:

• Measuring and comparing water contact and sliding angle of sample polymers.

• Utilizing the Beer–Lambert law and UV–vis spectrometry to quantify protein concentrations in solution.

• Utilizing serial dilutions to create a calibration curve which is used to quantify the amounts of unknown (e.g., protein amount adsorbed on polymer surfaces).

• Correlating material surface properties, such as hydrophobicity and water sliding angle, to the effects on protein adsorption and their fouling behavior.

4. MATERIALS AND METHODS

4.1. Materials

Silicone oil with the viscosity of 45–55 cSt; bovine serum albumin, heat shock treated; phosphate buffer saline, 1X powder, pH 7.4; 10% sodium dodecyl sulfate (SDS) solution; ethanol; Pierce 660 nm protein assay reagent; and ionic detergent compatibility reagent for Pierce 660 nm protein assay reagent were purchased from Fisher Scientific, Pittsburgh, PA. PDMS sheet; platinum-cured silicone sheet 12 × 12 × 0.02″ was obtained from McMaster Carr, IL. The Pierce reagent solution was prepared by dissolving one packet (1 g) of Ionic Detergent Compatibility Reagent in 20 mL of the Pierce 660 nm Protein Assay Reagent.

4.2. Sample Preparation and Surface Characterization

The PDMS sheet was rinsed with water and ethanol prior to the experiment. The cleaned PDMS samples were completely submerged in silicone oil for 24 h to produce the antifouling PDMS (via the antifouling SLIPs technology). If there is a time constraint for the course, the TA or instructor may prepare the antifouling PDMS via silicone oil infusion prior to the laboratory period. Samples were prepared by cutting 2 cm × 1 cm rectangular samples of antifouling PDMS. For control samples, the clean PDMS sheet was also cut to 2 cm × 1 cm rectangular samples.

4.3. Surface Characterization

The untreated control and antifouling PDMS samples were analyzed for their surface properties to obtain static water contact angle measurements using a contact angle goniometer (Ossila, UK). For the contact angle measurement, a 10 μL water droplet was put on the surface of the sample and the contact angle of two positions of the droplet was measured at least five times and the average of the total measurements was considered as the contact angle for each sample. Similar undergraduate experiments have been reported in literature that explore the wettability and surface characteristics of physically or chemically modified substrates.^40,41 An alternative method that can be used to measure the contact angle is by using a smartphone.⁴² With this method, a 10 μL water droplet would be put on the surface of the sample and a mounted smartphone camera would zoom in and take a picture of the droplet. The picture would be imported into ImageJ, a digital imaging analysis software, and the static water contact angle could be measured. For further characterization of advanced concepts, dynamic contact angle measurements (advancing and receding) could also be measured by adding or taking away water from the droplet and measuring the contact angle. For the sliding angle measurements, the polymer samples were placed on a smartphone screen and a 10 μL water droplet was placed on the sample surface. The phone was slowly tilted until the droplet began to slide off the surface and the angle reported by the digital protractor smartphone app was recorded.

4.4. Protein Adsorption Characterization

To create a calibration curve, a stock solution of BSA in PBS was prepared with a concentration of 2000 μg/mL and serially diluted to lower concentrations (i.e., 1000, 500, 250, and 125 μg/mL). All the BSA solutions were sonicated for 20 min to ensure complete dissolution. BSA standard solutions (0.1 mL) were mixed with 1.5 mL of the Pierce reagent solution and incubated for 5 min in dark at room temperature before measuring the absorbance at 660 nm using a UV–vis spectrometer (Cary 60, Agilent Technologies, Santa Clara, CA). The absorbance values obtained were used to plot the standard calibration curve.

Each control and antifouling PDMS sample was submerged in a 200 mg/mL BSA solution in PBS for 24 h at room temperature. If there is a time constraint for the course, the TA or instructor may prepare the samples in the BSA solution prior to the laboratory period. The samples were removed and briefly rinsed in a fresh PBS solution by gently dipping the samples in fresh PBS solution two to three times to remove the loosely adhered protein. These samples were transferred to a new microcentrifuge tube containing 1% sodium dodecyl sulfate (SDS) solution followed by 20 min sonication. The SDS is a synthetic surfactant that has a hydrophobic hydrocarbon tail combined with a hydrophilic polar head-group, giving the compound an amphiphilic property, which enables the SDS solution to detach the BSA protein from the silicone sheet surface. In a fume hood, the resulting solution from each sample (0.1 mL) was added to 1.5 mL of Pierce reagent solution while protected from light and incubated for 5 min. To measure the amount of adhered protein on the surface of the samples, 660 nm wavelength absorbance measurement was carried out by using a UV–vis spectrometer.

4.5. Statistical Analysis

Each student collected the data with a sample size n ≥ 3. Significance statistical difference was determined using a two-tailed Student’s t test with a hypothesis of unequal variance and p = 0.05.

5. HAZARDS

Students are required to review the SDS for the reagents in advance. Students are expected to follow safety regulations for the lab they work in and wear proper personal protective equipment (PPE). For this experiment, safety glasses or goggles, gloves, and lab coats are required. Although ionic detergent compatibility reagent (IDCR) is not considered hazardous by the OSHA Hazard Communication Standard, it is recommended to avoid breathing any dust. The Pierce 660 nm Protein Assay is considered hazardous by the OSHA Hazard Communication Standard as explained in the SDS. It may cause damage to organs. Do not breathe vapor. Do not eat, drink, or smoke when using this product. Harmful if inhaled, absorbed through the skin, or swallowed. It causes eye and skin irritation, and contains material that can cause targeted organ toxicity. Wash hands thoroughly after handling.

6. RESULTS AND DISCUSSION

6.1. Student Experiments

This experiment is designed to be a two-session educational laboratory work for undergraduate students as described above (Figure 3). A student guide and instructor notes are provided in the Supporting Information. In this experiment, students will be working in groups to prepare the control and antifouling PDMS samples and characterize the surface properties in Session 1. Furthermore, antifouling and control PDMS samples will be incubated in BSA solution for another 24 h prior to the quantification experiment using the colorimetric Pierce 660 nm Protein Detection Kit and UV–vis spectroscopy in Session 2. After obtaining the data, students will work in their groups to calculate the amount of protein adsorbed on the samples, perform statistical analysis, and analyze the results to draw conclusions on how the surface properties characterized in Session 1 have impacts on the biocompatibility and protein adsorption properties observed in Session 2.

6.2. Contact Angle and Sliding Angle Measurements

The static water contact angle slightly increased from 99.04° ± 1.8° on control PDMS to 102.54° ± 2.1° on antifouling PDMS samples (Table 1). Although there is not a considerable difference between control and antifouling samples, this behavior is hypothesized to relate to the Wenzel State of rough surfaces where the silicone oil infuses into the matrix of the polymer and subsequently decreases the surface roughness. According to the Wenzel equation,⁴³ a smoother surface on a hydrophobic matrix will have a smaller contact angle.⁴⁴ Nevertheless, the contact angle measurements for both test groups exhibited the hydrophobic nature of PDMS polymer which acts in favor of protein binding affinity to the surface.⁴⁵

Table 1.

Comparison of Control and Antifouling PDMS Surface Properties

	Sample Group
Surface Characterization	Control PDMS	Antifouling PDMS
Contact angle,a (degrees)	99.04 ± 1.8	102.54 ± 2.1
Sliding angle,a (degrees)	>90	8.67 ± 1.1

^aAll experiments were carried out in triplicate, and the results are presented as mean ± standard deviation.

Sliding angle measurements are a crucial analysis of material surfaces that can reveal the antifouling properties and facilitate biocompatibility evaluation for biomedical applications. It is well-known that the liquid-infused SLIPs polymer matrices can use the advantage of capillary forces between the polymeric matrix and the liquid lubricant which is infused in the polymer matrix, resulting in reduced surface tension which consequently repels liquids from the surface. Sliding angle measurements revealed a significant reduction of sliding angle from more than 90° for control PDMS to less than 10° (Table 1). This slippery nature demonstrates the potential for significant antifouling properties that are imparted on the PDMS surfaces, which leads to significant reductions in protein adsorption.⁴⁶

6.3. Colorimetric Evaluation and UV–vis Spectroscopy

The Pierce 660 nm Protein Assay method is a colorimetric assay that enables detection of the BSA present in the SDS solution using a UV–vis spectrophotometer.⁸ The proprietary dye–metal complex in the Pierce Assay Reagent will bind to the protein in the solution, resulting in an increase of the absorbance of the dye at the wavelength of 660 nm. The Pierce Reagent dye is reddish-brown initially and gradually changes to green upon reaction with higher concentrations of proteins. This color change results from the deprotonation of the dye at low pH, accelerated by interactions with positively charged amino acid groups in protein structure.⁴⁷ Therefore, according to Beer–Lambert’s law, the absorbance of the can be correlated to the amount of protein in the solution. This phenomenon is visually observable and quantifiable with UV–vis spectroscopy. While the reagent itself has a reddish-brown color, it gradually changes to dark green when comes in contact with BSA solution and the intensity of the green color is proportional to the amount of protein in the solutions (Figure 4a,b). Using the Beer–Lambert’s Law^48,49 (eq 1) with a known BSA concentration (c, μg/mL), absorbance (A), and cuvette light path length (b = 1 cm), the absorptivity coefficient (ε) of BSA-Pierce dye can be obtained from the slope of the BSA calibration curve, mL cm⁻¹ μg⁻¹:

$$A=\epsilon bc$$ (1)

Figure 4.

(a) BSA calibration curve solutions after reaction with Pierce reagent exhibit different colors from brown to green with increasing protein content of the solution, and (b) different absorbance on 660 nm, which makes it possible to plot a calibration curve for absorption–concentration relation of BSA solutions reacting with Pierce 660 protein detection reagent.

When the reagent solution is added to different BSA solutions with a range of concentration 125–2000 μg/mL, the binding of a proprietary dye–metal complex to protein causes an increase in the absorbance of the dye at 660 nm and with the known concentrations and obtained absorptions, a calibration curve can be obtained (Figure 5a).⁵⁰ The results obtained from this experiment are in correlation with previously reported results on BSA detection with the Pierce 660 nm kit.

Figure 5.

(a) Representative Beer’s Law calibration curve plot obtained from the range of 125–2000 μg/mL standard BSA solutions. (b) Example of final quantification of the amount of BSA adsorbed on control and antifouling PDMS samples (n = 3). The statistically significant differences between the adsorbed BSA concentration values on the surface of control PDMS and antifouling PDMS are depicted by ** for p ≤ 0.01.

This experiment aims to quantitate the amount of protein adsorbed on the surface of control and antifouling PDMS polymer samples. Students can also observe the difference in the amount of the protein that is detached from samples through the experiment even before UV–vis quantification. The students can observe the relationship between protein concentration and absorbance using the Beer–Lambert’s Law using the data obtained from UV–vis spectroscopy for both known standard BSA concentration solutions and the unknown solutions from the polymer samples (Table 2). After obtaining ε from the calibration curve data, the concentration of an unknown BSA concentration can be calculated for the control and antifouling PDMS samples. Although the Beer–Lambert’s Law is the fundamental equation for this calculation, the equation of the calibration curve in the standard linear form was used for more accurate calculations of the concentrations, where y = absorbance and x = concentration (μg/mL) (eq 2):

$$y\text{ = 0}.0008x\text{ + 0}.0871$$ (2)

Table 2.

Quantitative Data Obtained from UV–vis Spectroscopy and Calculated from Beer–Lambert’s Law

Experiment	Sample #	Absorbance (660 nm)	Concentration (μg/mL)	Adsorbed BSA (μg/cm2)
Standard curve	1	1.7	2000	n/a
	2	1.0	1000
	3	0.5	500
	4	0.3	250
	5	0.2	125
Control PDMS	1	0.32	291.1	36.4
	2	0.27	228.6	28.6
	3	0.23	178.6	22.3
			Avg	29.1
			STDEV	7.0
Antifouling PDMS	1	0.13	53.6	6.7
	2	0.12	41.1	5.1
	3	0.13	53.6	6.7
			Avg	6.2
			STDEV	0.9

The total amount of BSA that was adsorbed on the polymer sample (in μg) using eq 3, where M is the mass of protein in 1 mL of unknown solution (μg), and V is the initial volume of the unknown sample (1 mL):

$$M\text{ = }xV$$ (3)

Normalizing the amount of BSA adsorbed on the samples enables a comparison of the control versus antifouling PDMS surfaces, where M_f is the final normalized mass of protein (μg/cm²), and S is surface area (cm²); 8 cm² in this experiment (eq 4):

$$M_{\text{f}}=\frac{M}{S}$$ (4)

The students will calculate a mean and standard deviation for each sample type and then plot a bar graph (Figure 5b) to visualize the effects of the control versus antifouling surfaces on surface protein adsorption.

In the example experiment performed by students under supervision, the amount of protein adsorbed on the surface of control samples is 29.1 ± 7.0 μg/cm², while this number is 6.2 ± 0.9 μg/cm² for an antifouling surface, which exhibits the ability of the SLIPs technology to reduce the amount of protein adsorbed on the surface by ~79%. Although there are materials reported in the literature that exhibit more robust reductions in protein adsorption,⁵¹ the aim for this experimental protocol is to introduce the concepts of antifouling surfaces to undergraduate students and utilize a simple protein quantification method to explore the protein adsorption aspects of biocompatibility of medical device interfaces. Finally, with the accomplishments achieved in both sessions of this study, students can correlate the rational relation between surface properties of an antifouling material (i.e., a very low sliding angle) to its adsorbed protein content and compare these results to a control surface that is prone to biofouling due to its high contact angle and very high sliding angle.

6.4. Observations and Discussion

This hands-on experiment was conducted by a group of eight students composed of four students from National Science Foundation’s Research Experience for Undergraduate (NSF-REU) program, two undergraduate students (UG), and two graduate students (Master of Science program in biomedical engineering) under the supervision of the instructor. After a 30 min introductory session about the laboratory experiment, students were asked to complete the pre-experiment survey in Box 1, to identify their knowledge prior to participation in the experiment. The post-experiment survey in Box 2, was designed to be completed by the students after they were done with the experiment sessions. A brief questionnaire in the post-experiment survey was designed to identify students’ understanding of the experiment and its impact on their knowledge and excitement about the field of biomedical engineering and protein surface interaction research.

Box 1. Pre-experiment Survey.

On a scale of 1 to 5, where 1 is completely disagree and 5 is completely agree, mark the statements in the following survey. Please add your comments where needed.

1. I know what proteins are, and I can explain their impact on medical devices.

2. I know what biofouling means.

3. I can explain a facile technique to downregulate protein adhesion to medical devices.

4. I know about static water contact angle and sliding angle measurements and its easy for me to explain it to someone who has never heard of it.

5. I know at least one technique to identify proteins in a solution and quantify their concentration.

6. I know about UV–vis spectroscopy technique and I can easily calculate the concentration of an unknown solution if I am given the absorbance wavelength of the material

7. I have seen a UV calibration curve before and I know how I can use them to calculate the molar absorptivity of a particular solution.

Box 2. Post-experiment Survey.

On a scale of 1 to 5, where 1 is completely disagree and 5 is completely agree, mark the statements in the following survey. Please add your comments where needed.

1. This laboratory experiment gave me an insight about protein fouling problem in medical devices. Can you briefly explain how protein adsorption can impact medical devices?

2. The hands-on experiment designed in this study gave me an idea about water contact angle and sliding angle values and their relationship to the surface property.

3. By the end of this laboratory experiment I have better understanding about how UV–vis spectroscopy works and how I can use it in research.

4. The time that I spent doing this hand-on experiment was beneficial for me to obtain basic understandings about experiments and increased my confidence in working in a lab.

5. Instructions that were given to me in written or oral format helped me understand what I was doing.

6. The series of experiments that I did in this study made me more interested in biomedical research subjects.

For the experiment sessions, the students were divided into groups of two and each group did the experiment in two sessions according to the student guide (Supporting Information). A representative example of the data sets obtained by these students under the supervision is provided and discussed above in Sections 6.1–6.4. Throughout the experiments, the students exhibited curiosity and excitement while working with the antifouling SLIPs materials, especially with the immediate results obtained when conducting the water contact angle and sliding angle experiments where they were able to directly observe the dramatic effects between the controls and antifouling samples. However, there were also several portions of the experiments that did require some additional guidance from the instructor. Some of the undergraduate level students were less familiar with proper techniques of using micropipettes, so in a laboratory course setting it would be beneficial to introduce the basics and provide an opportunity for these students to refine their pipetting skills earlier in the course. Students also needed guidance on the preparation of the serial dilutions to prepare the standard BSA solutions for the UV–vis calibration curve. Some groups needed clarification that the serial dilution process gradually dilutes the BSA as they mix and transfer aliquots of the solution from one tube to the next. There was also discussion among the students and instructor with regards to how to properly dilute samples before UV–vis analysis if the absorbance values were too high, and then how to account for the dilution factor in their final calculations. Through discussions with the students, some of these basic laboratory techniques (e.g., pipetting, serial dilutions) were briefly introduced in some of the introductory chemistry and biology lab courses, but they still needed a brief introduction as not all of them had consistently been using these experimental techniques after completing those classes. Some general reminders about proper handling of the polymer samples, BSA solutions, light-sensitive reagents, and hazardous materials also was beneficial to running the experiments smoothly and safely. The experimental techniques were straightforward for undergraduate-level students, and the multidisciplinary nature of the results challenged the students to connect fundamental concepts of biomaterials characterization to the biological results obtained, leading to a better overall understanding of biomaterials characterization and biocompatibility.

Overall the observations during the student experiments and the survey are supportive of the benefits of this laboratory experiment in terms of providing the opportunity for students to revisit and refine basic laboratory techniques as well as exposing students to multidisciplinary experiments that span the fields of materials science, biomedical engineering, chemistry, and biology. According to the results obtained from the pre- and postlab surveys (Figure 6), the impact of the experiment on undergraduate-level students was very promising. For the graduate-level students who did have prior research experience, the experimental techniques were easier to grasp, but they also demonstrated enhancement in their knowledge related to this protein adsorption biomaterials characterization. In addition to the hands-on laboratory aspects described here, this series of experiments would also be a useful tool in a laboratory course to train students on how to prepare technical reports or scientific journal writing. This set of experiments is well suited to expose students to a simple research-style project common in the biomaterials field so that students can gain experience both with the hands-on experimental aspects as well as scientific communication. After completing the experiments, the teams of students could write a communication or full scientific manuscript (providing an appropriate introduction, materials, methods, results, discussion, conclusions, figures, and graphs) based on these experiments that they conducted and the results they obtained. In a course setting, combining this set of experiments with manuscript writing would be appropriate for upper-level undergraduates and especially beneficial for those students considering graduate school.

Figure 6.

Radar graph depicting results of the questions indicated by: (A–C) Q1–7 for pre-experiment survey and (D–F) Q1–6 for post-experiment survey. Students who participated in the experiments answered the survey questions on a scale of 1–5 (depicted on the rings of the radar graphs) where 1 indicates completely disagree and 5 indicates completely agree. The impact of the laboratory experiment is evident in visually comparing the two data sets. While the most impact was observed for the undergraduate-level students, graduate students also showed improvement after participating in the experiment.

7. CONCLUSIONS

This study provides a multidisciplinary laboratory practice for undergraduate students to obtain knowledge via hands-on experiments with polymers and antifouling materials, material science and surface characterizations, and biocompatibility via a simple protein adhesion colorimetric assay to compare adsorption on different polymeric surfaces. Being able to characterize the surface properties of materials is one of the cornerstones of a wide range of engineering sciences. Thus, it is necessary to involve students in these hands-on experiments that can train them about material modifications and surface properties and expose them to applicability toward biocompatibility and protein adsorption issues present in medical devices. Through the experimental activities, the students will learn about fundamentals of antifouling surface modification, surface wettability concepts, protein adsorption behavior on materials surfaces, the effect of antifouling properties on protein adsorption, UV–vis spectroscopy, the Beer–Lambert’s law, and the related calculations and statistical analysis. This multidisciplinary experiment brings some of the latest research advancements in creating biocompatible and protein-resistant materials into the hands of undergraduate students, allowing them to explore and connect the concepts of material science to their implications for medical device applications.