Biosensing at Drop Scale Assisted by Electrowetting at Moderate Frequency

Sequential and competitive adsorption of bovine serum albumin and β-lactoglobulin, and their resistance to exchange with α-lactalbumin and β-casein

Natural proteins such as bovine serum albumin (BSA) are readily extracted from biological fluids and widely used in various applications such as drug delivery and surface coatings. It is standard practice to dope BSA proteins with an amphipathic stabilizer, most commonly fatty acids, during purification steps to maintain BSA conformational properties. There have been extensive studies investigating how fatty acids and related amphiphiles affect solution-phase BSA conformational properties, while it is far less understood how amphipathic stabilizers might influence noncovalent BSA adsorption onto solid supports, which is practically relevant to form surface coatings. Herein, we systematically investigated the binding interactions between BSA proteins and different molar ratios of caprylic acid (CA), monocaprylin (MC), and methyl caprylate (ME) amphiphiles-all of which have 8-carbon-long, saturated hydrocarbon chains with distinct headgroups-and resulting effects on BSA adsorption behavior on silica surfaces. Our findings revealed that anionic CA had the greatest binding affinity to BSA, which translated into greater solution-phase conformational stability and reduced adsorption-related conformational changes along with relatively low packing densities in fabricated BSA adlayers. On the other hand, nonionic MC had moderate binding affinity to BSA and could stabilize BSA conformational properties in the solution and adsorbed states while also enabling BSA adlayers to form with higher packing densities. We discuss physicochemical factors that contribute to these performance differences, and our findings demonstrate how rational selection of amphiphile type and amount can enable control over BSA adlayer properties, which could lead to improved BSA protein-based surface coatings.

In recent years, Fe3O4 nanoparticles have been widely developed as adsorbents for various applications. However, their hydrophobic surface limits their application in polar systems, necessitating surface modifications to enhance adsorption. In this study, gallic acid was employed to modify Fe3O4 nanoparticles, as its carboxyl and phenolic hydroxyl groups can provide sites for protein adsorption. The research aims to determine the optimal pH, adsorption capacity, isotherm models, and thermodynamic parameters for bovine serum albumin (BSA) adsorption, as a model protein, onto gallic acid-modified and unmodified Fe3O4 nanoparticles using batch experiments. The experiments varied pH (3.6–5.6), initial BSA concentrations (0.50–1.75 mg/L), and temperatures (30–50 °C). FTIR analysis confirmed successful modification through the presence of C=O stretching bonds, while BSA adsorption was indicated by the appearance of amide groups and transmission electron microscopy (TEM) observations. Maximum adsorption was achieved at pH 4.8, in the vicinity of BSA isoelectric point. The Langmuir isotherm model best described the adsorption process for all adsorbents. Thermodynamic analysis showed that BSA adsorption was spontaneous and endothermic with increased randomness, as evidenced by negative Gibbs free energy, along with positive enthalpy and entropy values. Surface modification with gallic acid enhanced BSA adsorption capacity to 68.49 mg/g compared to 34.84 mg/g for unmodified Fe3O4 at 50 °C.

Protein-encapsulated nanoclusters (NCs) are emerging as a versatile platform for in-vivo imaging and other biomedical applications due to their ultrasmall size and excitation in the near-infrared region. Encapsulation may however affect protein structure, size, charge, and its interaction with lipid membranes. In this study, bulk characterization methods along with surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy were employed to study the secondary structure of bovine serum albumin (BSA) with blue-emitting Au8NCs at the air/water and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) lipid/water interfaces. With this approach, the difference in the adsorption behavior between native BSA and BSA with an increasing number of blue-emitting NCs was investigated under different pH conditions. At pH 7, at which both BSA and the lipid are negatively charged, protein molecules are found to associate with the DPPG monolayer via hydrophobic interactions with no preferential orientation across the lipid monolayer. At pH 3, adsorption of BSA at the DPPG monolayer occurs mainly due to electrostatic interactions between the negatively charged lipid headgroups and the positively charged protein, resulting in a uniform orientation of the protein across the lipid monolayer. Complimentary bulk studies by circular dichroism and particle size measurements show that the encapsulation of Au8NCs is associated with the loss of BSA helicity, which makes BSA-encapsulated Au8NCs prone to oligomerization, especially at a high content of Au8NCs at one BSA protein. The results indicate that the hydrodynamic diameter of BSA with Au8NCs strongly depends on the molar fraction of gold, the pH, and the storage time. A prolonged storage of Au8NCs@BSA at pH 7 increases the rate of protein oligomerization.

Bovine serum albumin adsorption and desorption rates on solid surfaces with varying surface properties

Swellable poly (ethylene glycol dimethacrylate-hydroxyethyl methacryate) [poly(EGDMA-HEMA] microbeads, in the size range of 150–200 μm, were produced by a modified suspension copolymerization of EGDMA and HEMA. Cibacron Blue F3GA was attached, then Zn(II) ions were incorporated within the microbeads by chelating with the Cibacron Blue F3GA molecules. The maximum amount of Cibacron Blue F3GA attachment was 16.5 μmol/g polymer. Different amounts of Zn(II) ions (0.4–32.4 mg/g polymer were incorporated on the microbeads by changing the initial concentration of Zn(II) ions and medium pH. Bovine serum albumin (BSA) adsorption onto unmodified, Cibacron Blue F3GA-attached and Cibacron Blue F3GA-Zn(II)-derivatized microbeads was investigated. The non-specific adsorption of BSA was very low (0.051 mg/g polymer). Cibacron Blue F3GA attachment significantly increased the BSA adsorption (60.5 mg/g polymer). The maximum BSA adsorption (122 mg/g polymer) was observed at pH 6.0 when Cibacron Blue F3GA-Zn(II)-derivatized microbeads were used. Desorption of BSA was achieved by using 25 mM EDTA (pH 4.9). High desorption ratios (up to 95% of the adsorbed BSA) were observed. It was possible to reuse Cibacron Blue F3GA-Zn(II)-derivatized microbeads without significant losses in the adsorption capacities.

We previously studied the mechanism underlying the adsorption of oral bacteria on the surfaces of dental prosthetic materials such as ceramics and resins in vitro. The aim of the present study was to examine bovine serum albumin (BSA) adsorption on crown composite resin surfaces by means of zeta potential. We measured the zeta potentials of resins alone, BSA alone, and resins after BSA adsorption. Eight resins were pulverized into powders (300 - 1000 nm). All experiments were conducted in 10 mM sodium chloride solution (pH 6.5). BSA was dissolved in 10 mM NaCl with a concentration of 2.0 × 10-5 mol/l. An adsorption assay was performed for one hour at 37°C under continuous rotation (6 rpm). The zeta potentials of both resins and BSA were negative, with BSA itself less negative than the resins themselves as an absolute value (p

Properties of protein adsorption onto pore surface during microfiltration: Effects of solution environment and membrane hydrophobicity

Liquid-liquid extraction techniques are one of the major tools in chemical engineering, analytical chemistry, and biology, especially in a system where two immiscible liquids have an interface solutes exchange between the two liquid phases along the interface up to a point where the concentration ratios in the two liquids reach their equilibrium values [1]. Solutes including nucleic acids and proteins of interests can be extracted from one liquid phase to the other immiscible liquid phase as a preparation step for many analytical processes. There are several advantages in miniaturizing the liquid-liquid extraction methods to on-chip level extraction. Usual advantages of miniaturization are the reduction in the sample size and portability. In addition, transport phenomena is faster in Micro-systems than in ordinary size systems, and therefore, one may expect that liquid-liquid extraction takes less time to achieve in miniaturized devices. It is due to shorter diffusion time in micro scale as well as high surface to volume ratio of Microsystems. Electrowetting on dielectric (EWOD) digital microfluidics is an efficient platform to process droplet based analytical processes [2]. Nanoliter (nL) or smaller volume of aqueous liquid droplets can be generated and transported on a chip by EWOD process. In addition to the high surface to volume ratio, high chemical potential can be expected in droplet based extraction when the droplets are in motion. In this paper, we propose to use room temperature ionic liquid (RTIL) as a second liquid phase for extraction, which forms immiscible interface with aqueous solutions. Properties of RTIL can be tailored by choice of cation, anion and substituents. RTIL has been investigated as replacements for the organic solvents and various “task-specific” ionic liquid are being developed which exhibit many attractive properties such as very low vapor pressure, high thermal stability [3]. We recently published EWOD properties of various RTILs toward microfluidic applications [4]. To demonstrate liquid-liquid micro extraction on chip, we fabricated and tested EWOD digital microfluidic devices. Fig. 1 shows (a) top and (b) cross sectional views of EWOD device. Two model extraction systems were tested. One is organic dye extracted from RTIL (1-butyl-3-methylimidazolium bis(trifluoromethanesulfonylimide or BMIMNTf2) to water and the other is iodine (I2) extracted from water to BMIMNTf2. The later model experiment is demonstrated in Fig. 2. Droplets of aqueous solution and BMIMNTf2 solution were generated on chip reservoir then transported for extraction and separated by EWOD actuation. When an aqueous solution and BMIMNTf2 solution join together, they created an interface, since water and BMIMNTf2 are immiscible. Extraction of I2 was done along the interface. After successful extraction, two immiscible liquid phases were separated by EWOD actuation and formed two separate droplets. From the result shown in Fig 2 (g), it is expected that extraction performance at the interface of moving droplet would be enhanced compared to the stationary droplet, because a moving interface prevent the chemical equilibrium, thus more chemical extraction potential can be provided with a moving interface than at a stationary interface. This demonstration is the first step toward total analysis system. The presented result opens the way to on-chip micro extraction, which will be readily integrated with other sample preparation microfluidic components and detection components. Currently, micro extraction systems for larger molecules such as nucleic acids, proteins and biological cells are being developed for further analytical applications.

This study compared bovine serum albumin (BSA) adsorption onto octacalcium phosphate (OCP) materials prepared from two wet preparations in the absence (w-OCP) and presence (c-OCP) of gelatin. Raman spectroscopy was used to analyze the BSA adsorption onto OCPs in a 150 mM Tris-HCl buffer containing 0.5 mM calcium and inorganic phosphate (Pi) ions at pH 7.4 and at 37°C. The degree of supersaturation of the supernatants after the adsorption was determined by measuring the ion composition. The results showed that BSA adsorption onto w-OCP was higher than that for c-OCP. The calcium ion concentration of the supernatant decreased for both w-OCP and c-OCP, whereas the Pi ion concentration increased, approaching OCP equilibria at different saturation levels. BSA adsorbed even onto c-OCP, which included a small amount of gelatin during c-OCP preparation. These results indicate that the biodegradability of w-OCP and c-OCP may be modulated through interactions with serum proteins.

Intrinsic porosity of calcium phosphate cements and its significance for drug delivery and tissue engineering applications

To meet the increasing demand of efficient cooling performance in small scale, this paper presents a digital microfluidics (DMF) microscale liquid cooling system which works on the principle of electrowetting on dielectric (EWOD). In EWOD DMF, fluids are handled drop-wise by external electric field. When the dispensed liquid droplet arrives at the hotspot by EWOD DMF operation, it picks up heat and removes heat when it leaves. This process can be repeated for a series of droplets by using a completely automated LabVIEW controlled system connected to the PCB package. With the help of indium tin oxide (ITO) thin film resistance temperature detectors (RTD) and pre-calibrated temperature coefficient of resistance (TCR) data, the temperatures of the hotspot before and after the residence of liquid droplet (i.e., cooling) can be recorded for different frequencies (dwelling time period of droplet on the hotspot) of the drop motion and varying heater power. Future work will involve RTD resistance data collection to plot the heat flux and the temperature difference (before and after cooling) for different frequencies of drop motion. Although the primary focus is to study single phase cooling, the DI water drop will experience considerable evaporation resulting in higher cooling performance. The single phase cooling studies will help in establishing a robust platform for future two-phase cooling analysis in which evaporation effects will be considered.

Adsorption of bovine serum albumin on fused silica: Elucidation of protein–protein interactions by single-molecule fluorescence microscopy

In this study, a novel titania-supported spherical porous hydroxyapatite (sHAp + TiO2) was prepared by compounding minute, uniquely shaped spherical porous hydroxyapatites (sHAp) with TiO2. Adsorption of the representative protein bovine serum albumin (BSA) was evaluated and simulated body fluid immersion experiments were carried out. Minimal adsorption of BSA to TiO2 was observed, and 33% and 25% adsorption to sHAp + TiO2 and sHAp were observed, respectively. The reduction in BSA concentration observed in the sHAp and sHAp + TiO2 solutions is presumably due to adsorption to HAp. It is highly likely that adsorption of the acidic protein BSA occurred at the sHAp Ca2+ site. In the simulated body fluid immersion experiments, there was greater expression of hydroxyapatite (HAp) on the surface of sHAp than there was on the surface of sHAp + TiO2. In the case of TiO2 alone, no HAp was produced, even after immersion for 3 days. These results suggest a relationship between BSA adsorption and the osteoconductivity of materials.

Triethoxysilyl functionalized hyperbranched polsiloxysilanes at the focal (FT-HBPSs) and terminal (TT-HBPSs) positions were synthesized to investigate adsorption behavior onto a silicon wafer surface. The surface of the silicon wafer adsorbed with the HBPSs was characterized by X-ray photoelectron spectroscopy, atomic force microscopy (AFM), static and dynamic water contact angle measurements. The AFM images indicated the formation size of dot-like structures were approximately 200 nm. The presence of vinyl terminal groups of FT-HBPSs permitted conversion of the surface from a non-polar hydrocarbon to a polar hydroxylated or carboxylated structures. After the polarity was changed, the surface properties were also studied using the above surface analysis techniques. The dynamic contact angle measurement indicated that the silicon wafer surface modified by FT-HBPSs was more hydrophilic in water than TT-HBPS. This behavior can be explained by the difference of connecting points between HBPS and the silicon wafer surface.