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. 2024 Oct 9;128(41):10247–10257. doi: 10.1021/acs.jpcb.4c03393

Adsorption and Morphology Analysis of Bovine Serum Albumin on a Micropillar-Enhanced Quartz Crystal Microbalance

Siqi Ji 1, Ilia Chiniforooshan Esfahani 1, Ruibo Yang 1, Hongwei Sun 1,*
PMCID: PMC11492313  PMID: 39380463

Abstract

graphic file with name jp4c03393_0008.jpg

The adsorption of bovine serum albumin (BSA), a widely used blood plasma protein, onto poly(methyl methacrylate) (PMMA) surface is a fundamental phenomenon attracting increasing interests in molecular biology, cell culture, immunology, diagnostics, and vaccinology. The nanostructured PMMA surfaces have shown a considerable effect on the BSA adsorption process. However, the effect of microstructures (e.g., micropillars) on BSA adsorption has seldom been studied. This research reports on the development of an acoustic resonance based method to explore the adsorption of BSA proteins on PMMA micropillars in terms of surface coverage, apparent binding constants, and pH-induced morphology variation. A theoretical model is developed to understand the frequency changes of QCM induced by BSA adsorption by taking into consideration the effects of the hydrodynamic force and an equivalent BSA/liquid layer formed on the micropillar surface. In addition, it was found that the resonance of micropillars with a quartz crystal microbalance (QCM) substrate significantly influenced BSA adsorption on micropillar surfaces.

1. Introduction

Bovine serum albumin (BSA) is the most abundant blood plasma protein, universally used as a blocking agent, enzyme stabilizer, protein concentration standard, and protein nanoparticles.1−4 For example, the enzyme-linked immunosorbent assay (ELISA) uses BSA to cover empty areas between antibodies to prevent nonspecific binding of antigens to the microtiter well plate.1 BSA is also well-known to be an enzyme stabilizer, and enzyme–BSA hydrophobic interaction during heating is a major determinant of the extent of enzyme stabilization.2 In addition, BSA is a preferred standard for protein assays due to its inert nature in many different biochemical reactions and ability to obtain obvious signal response.3 The BSA nanoparticles have been fabricated to facilitate drug delivery due to their nontoxic, nonimmunogenic, biocompatible, and biodegradable properties and their high binding capacity of various drugs.4 In the fields of biosensors and biochips, BSA is widely chosen to study protein adsorption due to its convenience, simplicity, and affordability.5

Protein adsorption is a substantial phenomenon in food biotechnology, nutritional science, drug delivery, medical implants, and bioelectronics.5−8 BSA is one of the most relevant proteins usually adsorbed first on biomaterials' surface in blood contact.9 On the other hand, poly(methyl methacrylate) (PMMA) is one of the frequently applied biocompatible materials in bone cement, dental fillers, and contact and intraocular lens.10−15 For instance, PMMA was applied in ophthalmology to treat cataracts or use as contact lenses due to its transparency, light weight, and anti-inflammatory properties.16,17 Another important aspect in protein adsorption is the behavior of the quantity, structure, and orientation of the adsorbed protein adlayer18,19 that is affected by substrate material, atom species, and pH values of the interface liquid; protein concentrations; protein–protein interactions; roughness topography; wettability; and surface modification.20−25

The influence of nanometer-scale surface roughness on protein adsorption has been widely studied in recent years.26−29 It was found that the adsorption of biotinylated bovine serum albumin (b-BSA) increased up to 6-fold on plasma-treated nanostructured PMMA surfaces compared to untreated surfaces.26 In the random sequential adsorption (RSA) model, nanoscale surface protrusions not only increase the contact surface area but also reduce steric hindrance between adsorbing proteins, leading to an increase in surface coverage.27 With root-mean-square roughness (Sq) values between 2 and 33 nm on oxidized tantalum surfaces, BSA adsorption mass was found to increase with roughness values by up to about 30%.28 Further studies show that both the amount and rate of BSA adsorption are linearly related to Sq values below 2.7 nm on nanorough titanium oxide surfaces.29 Our previous study showed significant sensitivity enhancement from BSA adsorption onto PMMA micropillar surface modified acoustic wave devices due to the coupling effect.30 However, there is a lack of understanding of BSA adsorption characteristics, such as surface coverage, BSA adsorption orientation, and its conformational structure.

Common methods for evaluating protein adsorption include ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry (SE), surface plasmon resonance (SPR), optical waveguide lightmode spectroscopy (OWLS), and atomic force microscopy (AFM).31−42

For instance, UV spectroscopy detects the protein concentration in a solution before and after adsorption by identifying the 280 nm wave radiated from the aromatic amino acids tyrosine and tryptophan in proteins, and the “Beer–Lambert law” is used to quantify the concentration. This method relies on the solution having enough target amino acids and no other UV-absorbing components, such as bound nucleotide cofactors and heme.31 IR spectroscopy is another optical technology for identifying protein adsorption by wavelength-dependent attenuation of the infrared light intensity. However, because of the complex structures of proteins compared to diatomic molecules, the theoretical analysis of IR spectroscopy has always been difficult and nonstraightforward.32 XPS detects the nitrogen photoelectron peak from X-ray scattering with proteins and then determines the protein film thickness by assuming depth homogeneity.33−35 SE records the change in polarization of elliptically polarized light reflected from the protein’s adsorbed surface. From the changes in the ellipsometry angles and refractive index, the optical thickness of the protein film can be deduced. At a coverage above 100 ng/cm2, the relative errors in the measured thickness and refractive index of the protein films were less than 5%. SE can be applied to both transparent and nontransparent samples. However, both XPS and SE cannot be applied for protein adsorption with a low surface coverage.

SPR can measure the mass of protein adsorption by the refractive index changes sensed by the surface evanescent wave.36 SPR is excited at a certain angle of incident using monochromatic and plane-polarized light that is directed through a quartz prism at the interface between the quartz and a thin (∼50 nm) layer of gold. Protein adsorption can change the medium refractive index (decay length ∼200 nm) and cause a proportional change in the angle of incident, which can be recorded by SPR. SPR has a high sensitivity (∼0.01 ng/cm2); however, it can only be used for metal surfaces, which limits its usage in biomedical applications.37 OWLS uses a similar evanescent field as SPR; however, it has a unique optical grating to excite the guided modes of a planar waveguide. The incident plane-polarized laser diffracts from the grating and starts to propagate via internal reflections inside the waveguide, thus generating an evanescent field. This evanescent field is used to probe the optical properties of the solution or protein adsorption in the vicinity of the surface, with sensitivity reported as 0.5 ng/cm2.38−40 OWLS is not as commonly used as SPR due to its complex components; however, it has higher angular precision and requires no calibration.41

AFM is commonly used to obtain the 3D surface topography of the protein adsorbed film by tapping mode with a nanometer-size tip. It has both contact and noncontact modes for surface scanning purposes. The limitation is the size and geometry of the tip when scanning high-aspect-ratio roughness structures.42

The techniques discussed above have their own specialty and advantages. However, a more portable, easy-to-operate/-analyze, and low-cost biosensor is necessary for quick and efficient protein adsorption measurements. Acoustic wave devices, such as the quartz crystal microbalance (QCM), have become an emerging technique to measure protein adsorption in real time.43 Because of its advantages of being simple, cost-effective, and highly sensitive, QCM is one of the most common sensors for applications such as mass adsorption, polymer structure, liquid viscosity and density, chemical interaction, and cell binding strength analysis occurring on the substrate surface.44,45 QCM is made of an AT-cut quartz crystal plate with both sides coated with a chromium adhesion layer followed by a gold film serving as the electrode.46,47 By oscillating in thickness shear mode (TSM), QCM sensors operating at around 10 MHz can detect mass variations of less than 10 ng/cm2 due to their intrinsic piezoelectric properties.48−52 Applying overtones of oscillation resonance, the mass sensitivity can be improved to 0.5 ng/cm2 in liquid media.53

In recent years, BSA adsorption on different surfaces has been extensively studied using QCM across various surface topographies and modifications, and this technique has been proven to be a flexible and attractive analytical tool.23,28,54−60 By quantifying surface mass densities, QCM can evaluate the impact of nanoscale rough surface morphologies on BSA and other protein adsorptions, and as a result, the end-on or side-on adsorption orientations can be determined.28,54,55 The investigation into BSA adsorption on self-assembled monolayers on QCM sensors revealed that adsorption orientations are influenced by different chemical groups.23,59,60 Additionally, QCM has been applied to study the selectivity of BSA adsorption on surfaces modified by molecularly imprinted polymers (MIPs) that retain the shape and functional group memory of template proteins.56−58 However, this analysis required the combination of QCM and ellipsometry to obtain accurate thickness measurements of the adsorbed protein layer. The other studies can only provide qualitative analyses of the effects of surface modifications on BSA adsorption regarding the amount, orientation, and selectivity.

Recently, a PMMA micropillar array has been attached onto a quartz substrate (QCM-P), where the micropillars and the QCM substrate form a coupled 2-degree-of-freedom system.61 As a result, the QCM-P system can achieve around 27-fold improvement in sensitivity over traditional QCM, which is comparable to the sensitivity of the SPR technique.30,62−64 Our previous study showed the Krimholtz–Leedom–Matthaei (KLM) circuit based model can be modified to take into consideration the effect of coupled micropillar and QCM on the frequency responses of the sensor in a liquid environment.65 This research focuses on the further development of the KLM model for analyzing the surface coverage and morphology of BSA protein adsorbed on PMMA micropillar surfaces.

2. Methods

2.1. Modified KLM Model for QCM-P

The total impedance (Z) of the Krimholtz–Leedom–Matthaei (KLM) model, a three-port (six-terminal) equivalent circuit, for analyzing the QCM-P device can be described as:66−71

2.1. 1

where ZL represents the load impedance induced by any extra structures/mass on top of QCM and Z0 is the quartz characteristic impedance. α is the acoustic phase shift of the QCM-P device. K and C0 are the electromechanical coupling factor and static capacitance of the quartz plate, respectively. ω is the operating angular resonance frequency.

Figure 1a,b illustrates a micropillar element operating in the pure PBS buffer solution with or without BSA adsorption.65,70,72

Figure 1.

Figure 1

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Force analysis of a differential element of a micropillar operating in (a) PBS and (b) PBS with BSA adsorption.

The load impedance ZL of the micropillars with or without BSA in phosphate-buffered saline (PBS) solution is calculated as:65

2.1. 2

where τ̂0 and v0 are the shear stress and velocity of QCM’s top surface, respectively. N is the number of micropillars per unit area (m2) of the QCM surface, κ is the Timoshenko shear coefficient of the micropillar, A is the cross-sectional area of the micropillar, G is the complex shear modulus of the micropillar (G = G′ + jG″), G′ and G″ are the real and imaginary parts of the shear modulus of micropillars, u0 is the substrate displacement at the bottom of micropillar, ρ is the density of the micropillar material, and h is the residual layer thickness (1–2 μm) due to the nanoimprint technology.61u(z) represents the micropillar displacement at different pillar location (z) that is solved based on Newton’s second law70,72 and the Euler Bernoulli beam equation73 as:

2.1. 3
2.1. 4

where Fhydro is the hydrodynamic force acting on the micropillar sidewall during their vibration,65 ρp is the density of the micropillar, E is the elastic modulus, and I is the second moment of the micropillar’s cross-sectional area. u0 is the micropillar displacement at z = 0. The hydrodynamic loading Fhydro is calculated by:

2.1. 5

where ρeq is the equivalent density of PBS (with or without an attached BSA layer) in the vicinity of the micropillar and d is the diameter of the micropillar. Γ(ω) is the hydrodynamic function of vibrating beams with a circular cross section in the liquid and is given by74

2.1. 6

where Re is the Reynolds number of the micropillar in the liquid and K0 and K1 are modified Bessel functions of the third kind.74 Substituting ZL in eq 1 gives the total impedance (Z) as a function of ω and the resonance frequency peak, which can be identified.

2.2. Binding Parameters

Kinetic parameters such as the apparent binding constant (Ka) and association and dissociation rate constants (k1 and k–1) could be obtained from Figure 3 based on the frequency decrease with time following an exponential decay function, given by eqs 7–9.75,76

2.2. 7
2.2. 8
2.2. 9

where Δf(t) is the frequency decrease value changing with time t, Δfmax is the maximum frequency change value, τ is the relaxation time or the reciprocal of the BSA adsorption rate (s–1), and c is the BSA molar concentration.

Figure 3.

Figure 3

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Frequency changes of (a) QCM-F and (b, c) QCM-Ps with micropillar heights of 14.50 and 8.48 μm due to BSA adsorption.

2.3. Experimental Study

The 10 MHz QCM sensors were purchased from Fortiming (MA). PMMA with a molecular weight of 77K was provided by MicroChem (MA). BSA powder (CAS No. 9048-46-8) was purchased from Sigma-Aldrich (MO), dissolved in PBS solution, and further diluted into different concentrations. Biotechnology-grade phosphate-buffered saline (PBS) tablets (Cat. No. 97062-732) were purchased from VWR International (PA). The PBS tablet was dissolved in 100 mL of DI water to make 1× PBS solution. According to the data sheet, the 1× PBS solution contains 137 mM sodium chloride, 2.7 mM potassium chloride, and 10 mM phosphate buffer. Hydrochloric acid (HCl) was obtained from Millipore Sigma (MA) for revising the pH value of the PBS solution.

The experimental setup includes a four-well flow chamber, oscillator circuits (Discovery-Q Invitrometrix, MA), DAQ system, syringe pump, and PC.64 The schematic and actual experimental setups are shown in Figure 2.

Figure 2.

Figure 2

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(a) Actual and (b) schematic experimental setup for measuring BSA adsorption. (c) SEM images of micropillars on the QCM device.

The micropillars were fabricated on a QCM substrate using nanoimprint lithography (NIL), and the fabrication procedure can be found in previous papers.61 PMMA micropillars with a circular cross section, diameter of 5 μm, heights of 14.50 μm (far from the “critical height”) and 8.48 μm (near the “critical height”), and center-to-center spacing of 16 μm were utilized in this research. It should be pointed out that the two different micropillar heights (14.50 and 8.48 μm) were used in the experiment to show the sensitivity enhancement induced by the coupling effect and at the same time explore the impact of surface morphology on BSA adsorption. BSA concentrations ranged from 0.01 to 0.1 mg/mL.

3. Results and Discussion

3.1. Surface Coverage Measurement

Control experiments were first conducted on a PMMA film coated QCM sensor (QCM-F) to estimate the surface coverage of BSA adsorption. Initially, a stable baseline of frequency was obtained in a PBS solution. Then, PBS solution was pumped into the flow chamber for 3 min. Note that frequency dips in the signal were mainly due to the pressure variations caused by pumping, which returned to the previous baseline after pumping was stopped. Next, BSA solutions with concentrations ranging from 0.01 to 0.1 mg/mL were pumped into the flow chamber for 3 min and then stopped. We then waited until the signal became stable. Finally, pure PBS solution was flowed into the system to remove any weakly bonded or unbonded BSA molecules. The experimental procedures were repeated for the QCM-F and QCM-P sensors. It should be noted that the PBS solutions for these tests were maintained at pH 7.5 to avoid any morphological changes. All experiments were conducted at room temperature of 25 °C. The PBS and BSA solutions were taken out of the refrigerator and warmed to room temperature before use. It is worth noting that the frequency shift of these baselines returning to the previous baseline values proved that there was no temperature change during the whole experiment. Figure 3 presents the frequency shifts of QCM-F and QCM-Ps (H = 14.50 and 8.48 μm) due to BSA adsorption.

As we can see, the QCM-P sensor with a height of 14.5 μm (away from the “critical height”) exhibited a low-frequency response to the BSA adsorption due to a weak coupling effect according to the newly developed KLM model (Supporting Information). Further analysis shows the 14.5 μm QCM-P sensor demonstrated a 1.6-fold sensitivity improvement in comparison to the QCM-F due to the increased surface area. However, QCM-P sensors with the height of 8.48 μm (near the “critical height”) achieved a 6-fold improvement over QCM-F that was mainly due to the coupling effect between micropillars and the QCM substrate. The calculated mass sensitivities of QCM-P sensors are around 2.75 and 0.73 ng/(cm2·Hz) for micropillar heights of 14.5 and 8.48 μm, respectively. In addition, the limits of detection (LODs) of the QCM-P sensors were calculated to be 0.004 and 0.003 mg/mL for micropillar heights of 14.5 and 8.48 μm, respectively. The details of the LOD calculation can be found in the Supporting Information.

Based on Sauerbrey’s theory, the surface coverage (SC) of BSA on the PMMA film surfaces can be estimated by:77

3.1. 10
3.1. 11

where Δf is the frequency shift due to BSA adsorption (Hz), f0 is the fundamental resonant frequency (f0 = 10 MHz), Δm is the additional mass due to adsorbed BSA (g), A is the piezoelectrically active crystal area or the surface area of one parallel electrode (A = 0.2051 cm2) as provided by the manufacturer, ρq is the quartz density (ρq = 2.648 g/cm3),77 μq is the shear modulus of the AT-cut quartz crystal (μq = 2.947 × 1011 g cm–1 s–2),77AB is the projected area of a single BSA protein (AB = 12.57 nm2) assuming a circular shape with a diameter of 4 nm,25,78−85 and mB is the mass of a single BSA protein (mB = 1.108 × 10–19 g).78,79

The morphology of the BSA proteins is shown in Figure 4. The volume of a single BSA protein was estimated to be 108 nm3 based on dielectric dispersion measurements.78,79 The molecular weight and size of a single BSA protein were estimated to be 66,700 Da and 4 × 4 × 14 nm, respectively.25,78−85 Spectroscopic ellipsometry measurements suggested that BSA proteins might take a vertical orientation and densely adhere to the surface.85 BSA proteins have an isoelectric point (IEP) of pH 4.5–5.0,23 which means that the proteins are negatively charged under neutral pH. Consequently, BSA proteins tend to repel each other and form a single layer on the substrate.86−89

Figure 4.

Figure 4

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Schematic of oblate-shaped BSA adsorbed onto the PMMA surface: (a) side view and (b) top view.

For QCM-F analysis, the BSA surface coverage was estimated based on the mass sensitivity of −1.102 Hz/ng (eq 10). As for QCM-P analysis, the frequency change was attributed to both the micropillars' surface area and the substrate area between micropillars (see Table S1 in the Supporting Information). The surface coverage of QCM-P sensors was evaluated based on the developed KLM model, which takes into consideration the coupled resonance between micropillars and the QCM substrate. Table S1 in the Supporting Information reports the experimental and KLM modeling results of QCM-F and QCM-P devices. It can be seen that a good agreement was obtained between measurement and modeling results. The comparison of surface coverage for QCM-F and QCM-P can be found in Figure 5a. As seen, the surface coverage increases with increasing BSA concentrations. There is no difference in the surface coverages between QCM-F and QCM-P.

Figure 5.

Figure 5

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(a) Frequency change and surface coverage with different equivalent densities of PBS and BSA solution. (b) Surface coverage of BSA on QCM-F and QCM-Ps (H = 14.50 and 8.48 μm) with different BSA concentrations.

The comparison of surface coverage for QCM-F and QCM-Ps is illustrated in Figure 5b. As can be seen, surface coverage increased with higher BSA concentrations. There was no significant difference in the surface coverages between QCM-F and QCM-P.

It can be concluded that the orientation of BSA proteins is perpendicular to the surface they were adsorbed onto (Figure 4) and that BSA proteins form a single adsorption layer with concentrations ranging from 0.01 to 0.1 mg/mL.

3.2. Apparent Binding Constants

Figure 6a shows a linear correlation of the relaxation time against various concentrations of BSA by curve fitting from experimental results. The association and dissociation rates, k1 and k–1, based on Figure 6a, are 1.23 × 102 M–1 s–1 and 0.0001 s–1 for the PMMA film, 6.02 × 102 M–1 s–1 and 0.0005 s–1 for PMMA micropillars with a height of 14.50 μm, and 3.545 × 103 M–1 s–1 and 0.0028 s–1 for PMMA micropillars with a height of 8.48 μm, as plotted in Figure 6b. The apparent binding constant Ka is calculated to be 1.23 × 106 M–1 for the film PMMA, 1.20 × 106 M–1 for PMMA micropillars with a height of 14.50 μm, and 1.27 × 106 M–1 for PMMA micropillars with a height of 8.48 μm.

Figure 6.

Figure 6

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(a) The relaxation time vs BSA concentrations on QCM-F and QCM-P (H = 8.48 and 14.50 μm), with linear fitting and R2 results. (b) Kinetic parameters for QCM-F and QCM-P.

As can be seen, despite the PMMA film and micropillars having similar apparent binding constants, the association and dissociation rates of the two surfaces differ significantly. It shows that the association and dissociation rates of BSA on PMMA micropillars with the height near the “critical height” (H = 8.48 μm) are around 28 times higher than those of the PMMA film. On the other hand, the association and dissociation rates of the QCM-P with the micropillar height of 14.5 μm are only 5 times higher than those of the PMMA film surface. One hypothesis is that the vibration of these micropillars (∼10 MHz) may induce significantly faster collisions between BSA molecules and the pillar surface, which in turn increased the association and dissociation rates of BSA.

It is worth noting that these parameters were not reported in the literature due to a lack of studies in physical binding of proteins onto untreated PMMA substrates, especially on vibrating PMMA surfaces. Most papers studied protein–protein or protein–ion binding behaviors,90−94 with only a handful papers studying protein binding onto functionalized surfaces. For instance, Sun and Zhu reported that BSA has an apparent binding constant of 1.12–1.42 × 106 M–1 with an epoxy-functionalized glass substrate.95 It can be seen that the binding constant of BSA to the untreated PMMA surface is of the same magnitude as the reported binding constant.

3.3. BSA Morphology Change under Different pH

It was reported that the individual BSA protein may lose a small amount of weight/mass, possibly due to water desorption from its structure when pH value is reduced.25 Under pH 4.5, BSA undergoes a drastic morphology change known as the N–F isoform transition (from normal structure to fast migrating structure) or even E-isoform (fully expanded structure).21,96 Above pH 8, another conformational transition occurs, called the N–B transition (from normal structure to basic structure) or even A-isoform (aged structure).22,96

QCM-P (H = 8.48 μm) and QCM-F were employed to evaluate the morphological change of BSA under different pH values. It should be noted that when the pH value of the BSA solution changes from pH 7.5 to 4.5 (near its IEP point), the BSA proteins tend to agglomerate.23 To prevent BSA agglomeration in the experiment, BSA proteins were first adsorbed onto the PMMA substrate by flowing BSA-rich PBS solution with a pH of 7.5, and then the pH value of the PBS solution was switched to 4.5. After this, the PBS solution with pH 7.5 was reintroduced into the chamber to observe any possible reversible changes. Prior to the BSA morphology experiment, PBS solutions at different pH values were flowed over the sensor surfaces to ensure that the frequency change of QCM sensors was solely from the BSA morphology change. The results are presented in Figure 7.

Figure 7.

Figure 7

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Frequency changes due to pH effect before and after BSA adsorption (a) on QCM-F and (b) on QCM-P.

As we can see, no frequency change was observed under the different pH values of PBS solutions. However, when the PBS solution with BSA was introduced into the chamber, large frequency drops were observed due to the adsorption of BSA on the PMMA surface at pH 7.5. When the pH value of the PBS solution was switched to 4.5, an increase in the frequency was observed for both QCM-F and QCM-P sensors. A total increase of 84 Hz was measured by the QCM-P sensor (H = 8.48 μm), while QCM-F showed a 14 Hz increase. We believe that this is mainly caused by the loss of water from the BSA proteins.

Assuming both PMMA surfaces have the same BSA coverage, we can estimate the size change of individual BSA proteins due to water loss based on the KLM model. The results show that the BSA size becomes 97 nm3 compared to the original size of 108 nm3, indicating a reduction of approximately 10.185%. Table 1 summarizes the experimental and modeling results. More calculation details can be found in the Supporting Information.

Table 1. Comparison of the Frequency Shifts Due to pH Changes from the Experiment with Those of Modeling for QCM-F and QCM-P.

experimental results
  pH 7.5 pH 4.5 difference
QCM-F Δf 124 Hz 110 Hz 14 Hz
QCM-P Δf 732 Hz 648 Hz 84 Hz
modeling results
  pH 7.5 pH 4.5 difference
input density (ρeq) 1010.50 kg/m3 1009.29 kg/m3 1.21 kg/m3
total modeling Δf 733 Hz 650 Hz 83 Hz
surface coverage (SC) 62.14% 62.14%  
BSA size 108 nm3 97 nm3 –10.185%
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As can be seen, a good agreement is obtained between modeling results and experimental results. The developed KLM model is a powerful tool for estimating the mass change of proteins in the range of tens of nanograms. In addition, under pH 4.5, a micropillar-based QCM device demonstrated an ultrahigh mass sensitivity over a traditional QCM device in measuring the mass change of proteins like BSA. The developed KLM model is a promising tool for analyzing protein morphological changes in biological and medical fields.

4. Conclusions

BSA adsorption onto both flat and PMMA micropillar surfaces was studied by the dynamic measurement of an acoustic wave device, QCM. The theoretical model considering hydrodynamic loading effects of the solution combined with the protein in the vicinity of micropillars was utilized to assist with the analysis of the surface coverage, orientation of BSA adsorption, and its nanometer-scaled morphology change due to pH effect. The comparison between model and experimental results proved the capacity and accuracy of this model in predicting the BSA adsorption phenomenon and morphological changes. This acoustic wave device is potentially a powerful tool for investigating protein interaction with different surfaces or surface modification in life science and biotechnology fields.

Acknowledgments

The authors thank MicroChem Corp. (Westborough, MA, USA) for providing the PMMA material and the National Science Foundation for the financial support (NSF ECCS 2130716 and TI 2329826).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.4c03393.

  • The coupling effect near the “critical height”; the limit of detection (LOD) of BSA adsorption on QCM-P sensors; detailed modeling results of BSA adsorption; and detailed modeling results of pH effect (PDF)

The authors declare no competing financial interest.

Supplementary Material

jp4c03393_si_001.pdf (292.3KB, pdf)

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