Signal Amplification by Spatial Concentration for Immunoassay on Cellulose Media.

Immunoassay is one of the most common bioanalytical techniques from lab-based to point-of-care settings. Over time, various approaches have been developed to amplify signals for greater sensitivity. However, the need for effective, versatile, and simple signal amplification methods persists yet. This paper presents a novel signal amplification method for immunoassay that utilizes spatial concentration of a cellulose-based plate possessing sensor transducers, specifically gold nanoparticles. By modifying the dimensions of the plate, the density of nanoparticles increased, resulting in intensified color signals. The coating material, polydopamine, which is utilized to protect the gold nanoparticles. Chemical changes in nanocomposites are characterized using scanning electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. The application of this method to colorimetric quantification demonstrated great consistency across various concentrations of nanoparticles, with better reliability at lower concentration ranges. A model immunoassay is designed to evaluate the analytical performance. As a result, this method successfully corrected a false-negative result with a lowered Kd of 0.509 pmol per zone. This method shows strong signal enhancement capability that can correct false-negative signals in the immunoassays, with potential benefits including versatility, simplicity, low cost, and the ability to operate multiple plates simultaneously.

One-step sensing of foodborne pathogenic bacteria using a 3D paper-based device.

Managing food contamination from bacteria has been an ongoing issue in the public health and industrial fields. Enzymatic substrates possessing optical properties, e.g. fluorescence or color manifestation, are widely exploited in pathogenic/non-pathogenic bacteria culture methods. Recently, various chromogenic substrates have been utilized in the development of point-of-care diagnostic tools. Herein, four types of chromogenic substrates were exploited to develop paper-based sensors for major foodborne pathogens. We designed a compact sized three-dimensional paper device with a simple user interface. By inserting functional layers in the middle of multilayers, pre-lysis and pH regulation steps were excluded and the analysis time was subsequently reduced, while only one sample droplet was needed for the whole analysis process. After the enzymatic reactions had proceeded, target-specific colors appeared. When it was combined with enrichment, 101 cfu mL-1 of pathogens were successfully detected in 4-8 hours, while those in milk samples were readily sensed in 12 hours. The proposed bacteria sensor exhibited great advantages of low cost, portability and simple operation, while showing a respectable limit-of-detection as low as 101 cfu mL-1 and below. Significantly, we emphasize that it takes fewer steps than existing methods and provides a reduced analysis time owing to the layer functionalization.

Probabilistic ecological risk assessment of heavy metals using the sensitivity of resident organisms in four Korean rivers.

The environment has been continuously exposed to heavy metals by various routes, from both natural and artificial sources. In particular, heavy metals in water can affect aquatic organisms adversely, even at very low concentrations, and can lead to the disturbance of the ecosystem balance and biodiversity. Ecological risk assessments are conducted to protect the environment from such situations, primarily by deriving the predicted no-effect concentration (PNEC) from the species sensitivity distribution (SSD). This study developed the SSDs based on the species living in Korean freshwater for four heavy metals including cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn). The species compositions of the SSDs were examined, and three types of PNECs were derived by applying different assessment factors (AF). In addition, the occurrence and concentrations of heavy metals in Korean rivers were investigated, and the ecological risk assessment was carried out to compare the SSDs with the environmental concentrations. The SSDs were developed using a sufficient number of species, but the missing data of plants and insects provided an incomplete species composition. The results show that Cd and Pb in the environmental concentrations of rivers would not cause any risk to aquatic organisms from the derived PNEC. However, some organisms might be adversely affected by the concentrations of Zn, and a small amount of risk was expected under the conservative PNEC. The distribution of Cu in the rivers was not considered to be safe for aquatic organisms because the average environmental concentrations potentially affected the proportion of the SSD, and the environmental concentrations exceeded the PNECs. The concentrations of Cu and Zn in industrial waters indicated a considerable risk to aquatic organisms, and the probability of exceeding the PNECs appeared to be quite high. Therefore, this study indicates that additional actions and parallel field studies are required based on the risk posed to aquatic organisms by Cu and Zn in four Korean rivers.

Size and Surface Charge of Engineered Poly(amidoamine) Dendrimers Modulate Tumor Accumulation and Penetration: A Model Study Using Multicellular Tumor Spheroids.

An enormous effort has been put into designing nanoparticles (NPs) with controlled biodistributions, prolonged plasma circulation times, and/or enhanced tissue targeting. However, little is known about how to design NPs with precise distributions in the target tissues. In particular, understanding NP tumor penetration and accumulation characteristics is crucial to maximizing the therapeutic potential of drug molecules carried by the NPs. In this study, we employed poly(amidoamine) (PAMAM) dendrimers, given their well-controlled size (<10 nm) and surface charge, to understand how the physical properties of NPs govern their tumor accumulation and penetration behaviors. We demonstrate for the first time that the size and surface charge of PAMAM dendrimers control their distributions in both a 3D multicellular tumor spheroid (MCTS) model and a separate extracellular matrix (ECM) model, which mimics the tumor microenvironment. Smaller PAMAM dendrimers not only diffused more rapidly in the ECM model but also efficiently penetrated to the MCTS core compared to their larger counterparts. Furthermore, cationic, amine-terminated PAMAM dendrimers exhibited the greatest accumulation in MCTS compared to either charge-neutral or anionic dendrimers. Our findings indicate that the size and surface charge of PAMAM dendrimers may tailor their tumor accumulation and penetration behaviors. These results suggest that controlled tumor accumulation and distinct intratumoral distributions can be achieved by simply controlling the size and surface charge of dendrimers, which may also be applicable for other similarly sized NPs.

Advancements in Nanogels for Enhanced Ocular Drug Delivery: Cutting-Edge Strategies to Overcome Eye Barriers.

Nanomedicine in gel or particle formation holds considerable potential for enhancing passive and active targeting within ocular drug delivery systems. The complex barriers of the eye, exemplified by the intricate network of closely connected tissue structures, pose significant challenges for drug administration. Leveraging the capability of engineered nanomedicine offers a promising approach to enhance drug penetration, particularly through active targeting agents such as protein peptides and aptamers, which facilitate targeted release and heightened bioavailability. Simultaneously, DNA carriers have emerged as a cutting-edge class of active-targeting structures, connecting active targeting agents and illustrating their potential in ocular drug delivery applications. This review aims to consolidate recent findings regarding the optimization of various nanoparticles, i.e., hydrogel-based systems, incorporating both passive and active targeting agents for ocular drug delivery, thereby identifying novel mechanisms and strategies. Furthermore, the review delves into the potential application of DNA nanostructures, exploring their role in the development of targeted drug delivery approaches within the field of ocular therapy.

Paper-Based Substrate for a Surface-Enhanced Raman Spectroscopy Biosensing Platform-A Silver/Chitosan Nanocomposite Approach.

Paper is a popular platform material in all areas of sensor research due to its porosity, large surface area, and biodegradability, to name but a few. Many paper-based nanocomposites have been reported in the last decade as novel substrates for surface-enhanced Raman spectroscopy (SERS). However, there are still limiting factors, like the low density of hot spots or loss of wettability. Herein, we designed a process to fabricate a silver-chitosan nanocomposite layer on paper celluloses by a layer-by-layer method and pH-triggered chitosan assembly. Under microscopic observation, the resulting material showed a nanoporous structure, and silver nanoparticles were anchored evenly over the nanocomposite layer. In SERS measurement, the detection limit of 4-aminothiophenol was 5.13 ppb. Furthermore, its mechanical property and a strategy toward further biosensing approaches were investigated.

Fluidic timers for time-dependent, point-of-care assays on paper.

This article describes an integrated approach to tracking the end point of a time-based assay that is conducted on an analytical device made out of paper. The timing mechanism is built directly into a paper-based analytical device and does not require starting, stopping, reset buttons, batteries, or maintenance; the timer simply starts once the sample is added to the device. These "fluidic timers" are composed of paraffin wax and a signaling feature (e.g., a dye). The timing function is made possible by the specific time required for a liquid sample to wick through predefined regions in the device. This time period can be anywhere between 1 min and 2 h and is controlled by the quantity of wax present in the timer. Because both the fluidic timers and paper-based assays depend on the wicking rate of the sample, the fluidic timers automatically calibrate themselves (relative to the assay) to account for differences in wicking rates that are caused by variations in humidity. Fluidic timers are 97% accurate (with respect to the time required for the assay) and provide slightly better accuracy than an external timer when used to track an assay that measured the level of glucose in a sample.

Metering the capillary-driven flow of fluids in paper-based microfluidic devices.

This article describes an exceedingly simple and low-cost method for metering the capillary-driven flow rate of fluids within three-dimensional (3D) microfluidic, paper-based analytical devices (microPADs). Initial prototypes of 3D microPADs control the spatial distribution of fluids within a device, but they provide little control over how quickly (or slowly) fluids move within the device. The methods described in this article provide control over when and how quickly a fluid is distributed into detection zones. These methods are inexpensive (the metering regions are composed of paraffin wax), the devices are easy to fabricate, and they are capable of controlling the flow of fluids to detection zones with precise time delays (e.g., +/-6% of the total wicking time). We anticipate that this type of precise control over fluid distribution rates will be useful particularly for point-of-care assays that require multiple steps (where each step requires that the reagents interact for a defined period of time) or for simultaneously displaying the results of multiple different assays on a single device.

Paper-Based Diagnostic System Facilitating Escherichia coli Assessments by Duplex Coloration.

Laboratory support for low-resource regions is a rising global issue. As microbiological contamination is closely associated with other issues like food safety, water supply sustainability, and public health, bacterial assessments in this setting need to be improved. Herein, we demonstrate a paper-based diagnostic device for point-of-need testing, in which fecal-indicating Escherichia coli and highly pathogenic E. coli are detected by duplex coloration. This device was functionalized by mixing different chromogenic substrates that reflect each bacterial enzymatic phenotype. In the final part of the paper, we describe this microbiological diagnostic system tested with bacteria-contaminated food samples. The device sensitivity was shown to have greatly reduced the total analysis time (below to 4 h) when combined with an enrichment amplification procedure. Notably, this paper device successfully detected 10 cfu/mL of target bacteria in a contaminated milk sample. Our diagnostic system shows acceptable accuracy, short analysis time, and a user-friendly interface, thereby eliminating demands for high-end equipment and a highly trained staff. We expect that this diagnostic system will be a sustainable solution in supporting microbiological or clinical laboratories in low-income countries.

Volumetric interpretation of protein adsorption: ion-exchange adsorbent capacity, protein pI, and interaction energetics.

Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI=11) and net-negatively-charged HSA (pI=5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation-exchange resin, -0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI=7.0) in simultaneous adsorption competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quaternary ammonium sepharose (Q; a positively-charged, strong anion exchanger, +0.22 mmol/mL exchange capacity) or to octadecyl sepharose (ODS; a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into or onto which IgG adsorbs in adsorption competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy of adsorption Delta Gads( degrees ) of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net Delta Gads( degrees ). Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not exhibit ion-exchange properties, and surfaces with strong Lewis acid/base functional groups that exhibit ion-exchange properties in the conventional chemistry sense of ion-exchange.

Volumetric interpretation of protein adsorption: kinetic consequences of a slowly-concentrating interphase.

Time-dependent energetics of blood-protein adsorption are interpreted in terms of a slowly-concentrating three-dimensional interphase volume initially formed by rapid diffusion of protein molecules into an interfacial region spontaneously formed by bringing a protein solution into contact with a physical surface. This modification of standard adsorption theory is motivated by the experimental observation that interfacial tensions of protein-containing solutions decrease slowly over the first hour to a steady-state value while, over this same period, the total adsorbed protein mass is constant (for lysozyme, 15 kDa; alpha-amylase, 51 KDa; albumin, 66 kDa; prothrombin, 72 kDa; IgG, 160 kDa; fibrinogen, 341 kDa studied in this work). These seemingly divergent observations are rationalized by the fact that interfacial energetics (tensions) are explicit functions of solute chemical potential (concentration), not adsorbed mass. Hence, rates of interfacial tension change parallel a slow interphase-concentration effect whereas solution depletion detects a constant interphase composition within the timeframe of experiment. A straightforward mathematical model approximating the perceived physical situation leads to an analytic formulation that is used to compute time-varying interphase volume and protein concentration from experimentally-measured interfacial tensions. Derivation from the fundamental thermodynamic adsorption equation verifies that protein adsorption from dilute solution is controlled by a partition coefficient at equilibrium, as is observed experimentally at steady state. Implications of the alternative interpretation of adsorption kinetics on biomaterials and biocompatibility are discussed.

Surface energy effects on osteoblast spatial growth and mineralization.

While short-term surface energy effects on cell adhesion are relatively well known, little is revealed as regards its later stage effects on cell behavior. We examined surface energy effects on osteoblastic cell growth and mineralization by using human fetal osteoblastic (hFOB) cells cultured on plasma-treated quartz (contact angle, theta=0 degrees) and octadecyltrichlorosilane (OTS)-treated quartz (theta=113 degrees). hFOB cells formed a homogeneous cell layer on plasma-treated quartz, while those cultured on OTS-treated quartz produced randomly distributed clump-like structures that were filled with cells (confirmed by confocal microscopy). Mineral deposition by hFOB cells was spatially homogeneous when cultured on hydrophilic surfaces. Furthermore, cells on hydrophilic surfaces exhibited increased mineralized area as well as enhanced mineral-to-matrix ratio (assessed by Fourier transform infrared spectroscopy), relative to cells on hydrophobic surfaces. Experiments using other types of osteoblast-like cells (MC3T3-E1, MG63, and SAOS-2) revealed more or less similar effects in spatial growth morphology. It was concluded that hydrophilic surfaces induce homogeneous spatial osteoblastic cell growth and mineral deposition and enhance the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization relative to hydrophobic surfaces. Our data suggest that surface energy effects on osteoblastic cell differentiation, especially mineralization, may be correlated with surface energy dependent changes in spatial cell growth.

Volumetric interpretation of protein adsorption: competition from mixtures and the Vroman effect.

A Vroman-like exchange of different proteins adsorbing from a concentrated mixture to the same hydrophobic adsorbent surface is shown to arise naturally from the selective pressure imposed by a fixed interfacial-concentration capacity (w/v, mg/mL) for which protein molecules compete. A size (molecular weight, MW) discrimination results because fewer large proteins are required to accumulate an interfacial w/v concentration equal to smaller proteins. Hence, the surface region becomes dominated by smaller proteins on a number-or-mole basis through a purely physical process that is essentially unrelated to protein biochemistry. Under certain conditions, this size discrimination can be amplified by the natural variation in protein-adsorption avidity (quantified by partition coefficients P) because smaller proteins (MW<50 kDa) have been found to exhibit characteristically higher P than larger proteins (MW<50 kDa). The standard depletion method is implemented to measure protein-adsorption competition between two different test proteins (i and j) for the same hydrophobic octyl sepharose adsorbent particles. SDS-gel electrophoresis is used as a multiplexing, separation-and-quantification tool for this purpose. Identical results obtained using sequential and simultaneous competition of human immunoglobulin G (IgG, protein j) with human serum albumin (HSA, protein i) demonstrates that HSA was not irreversibly adsorbed to octyl sepharose over a broad range of competing solution concentrations. A clearly observed exchange of HSA for IgG or fibrinogen (Fib) shows that adsorption of different proteins (i competing with j) to the same hydrophobic surface is coupled whereas adsorption among identical proteins (i or j adsorbing from purified solution) is not coupled. Interpretive theory shows that this adsorption coupling is due to competition for the fixed surface capacity. Theory is extended to hypothetical ternary mixtures using a computational experiment that illustrates the profound impact size-discrimination has on adsorption from complex mixtures such as blood.

Volumetric interpretation of protein adsorption: Partition coefficients, interphase volumes, and free energies of adsorption to hydrophobic surfaces.

The solution-depletion method of measuring protein adsorption is implemented using SDS gel electrophoresis as a separation and quantification tool. Experimental method is demonstrated using lysozyme (15kDa), alpha-amylase (51kDa), human serum albumin (66kDa), prothrombin (72kDa), immunoglobulin G (160kDa), and fibrinogen (341kDa) adsorption from aqueous-buffer solution to hydrophobic octyl-sepharose and silanized-glass particles. Interpretive mass-balance equations are derived from a model premised on the idea that protein reversibly partitions from bulk solution into a three-dimensional (3D) interphase volume separating the physical-adsorbent surface from bulk solution. Theory both anticipated and accommodated adsorption of all proteins to the two test surfaces, suggesting that the underlying model is descriptive of the essential physical chemistry of protein adsorption. Application of mass balance equations to experimental data quantify partition coefficients P, interphase volumes V(I), and the number of hypothetical layers M occupied by protein adsorbed within V(I). Partition coefficients quantify protein-adsorption avidity through the equilibrium ratio of interphase and bulk-solution-phase w/v (mg/mL) concentrations W(I) and W(B), respectively, such that P identical withW(I)/W(B). Proteins are found to be weak biosurfactants with 45

Volumetric interpretation of protein adsorption: mass and energy balance for albumin adsorption to particulate adsorbents with incrementally increasing hydrophilicity.

The solution-depletion method of measuring human serum albumin (HSA) adsorption to surface-modified glass-particle adsorbents with incrementally increasing hydrophilicity is implemented using SDS gel electrophoresis as a separation and quantification tool. It is shown that adsorbent capacity for albumin measured in interfacial-concentration units (mg/mL) decreases monotonically with increasing surface energy (water wettability) to detection limits near an adsorbent-particle water adhesion tension tau(0)=30 dyne/cm (nominal water contact angle theta=65( composite function)) and that albumin does not adsorb to (concentrate within the surface region of) more hydrophilic adsorbents. These adsorbed-mass measurements corroborate predictions based on interfacial energetics and are consistent with AFM measurement of protein-surface adhesion. Interpretive mass-balance equations are derived from a model premised on the idea that protein reversibly partitions from bulk solution into a three-dimensional (3D) interphase volume separating the physical adsorbent surface from bulk solution. Theory is shown to both anticipate and accommodate experimental results for all test adsorbents, suggesting that the underlying model is descriptive of the essential physical chemistry of albumin adsorption to surfaces spanning the full range of observable water wetting. In particular, application of theory to experimental data shows that the free-energy cost of dehydrating the surface region by protein displacement upon adsorption increases with increasing adsorbent hydrophilicity in a manner that controls ultimate capacity for protein. It is concluded that a simple, three-component free-energy rule adequately describes protein adsorption from aqueous solution, at least for materials bearing varying surface concentrations of anionic (not cationic) functional groups. IMPACT STATEMENT: This work yields detailed insights into the physical chemistry of protein adsorption by elucidating relationships among adsorbent surface energy, capacity to adsorb the protein human serum albumin, and the energy required to displace vicinal water from the interface.

Electrophoretic implementation of the solution-depletion method for measuring protein adsorption, adsorption kinetics, and adsorption competition among multiple proteins in solution.

The venerable solution-depletion method is perhaps the most unambiguous method of measuring solute adsorption from solution to solid particles, requiring neither complex instrumentation nor associated interpretive theory. We describe herein an SDS-gel electrophoresis implementation of the solution--depletion method for measuring protein adsorption and protein-adsorption kinetics. Silanized-glass particles with different surface chemistry/energy and hydrophobic sepharose-based chromatographic media are used as example adsorbents. Electrophoretic separation enables quantification of adsorption competition among multiple proteins in solution for the same adsorbent surface, demonstrated herein by adsorption--competition kinetics from binary solution.

Hazard Classification of Household Chemical Products in Korea according to the Globally Harmonized System of Classification and labeling of Chemicals.

OBJECTIVES: This study was conducted to review the validity of the need for the application of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) to household chemical products in Korea. The study also aimed to assess the severity of health and environmental hazards of household chemical products using the GHS. METHODS: 135 products were classified as 'cleaning agents and polishing agents' and 98 products were classified as 'bleaches, disinfectants, and germicides.' The current status of carcinogenic classification of GHS and carcinogenicity was examined for 272 chemical substances contained in household chemical products by selecting the top 11 products for each of the product categories. In addition, the degree of toxicity was assessed through analysis of whether the standard of the Republic of Korea's regulations on household chemical products had been exceeded or not. RESULTS: According to GHS health and environmental hazards, "acute toxicity (oral)" was found to be the highest for two product groups, 'cleaning agents and polishing agents', and 'bleaches, disinfectants, and germicides' (result of classification of 233 household chemical products) at 37.8% and 52.0% respectively. In an analysis of carcinogenicity assuming a threshold of IARC 2B for the substances in household chemical products, we found 'cleaning agents and polishing agents' to contain 12 chemical substances and 'bleaches, disinfectants, and germicides' 11 chemical substances. CONCLUSION: Some of the household chemical products were found to have a high hazard level including acute toxicity and germ cell mutagenicity, carcinogenicity, and reproductive toxicity. Establishing a hazard information delivery system including the application of GHS to household chemical products in Korea is urgent as well.

Volumetric interpretation of protein adsorption: interfacial packing of protein adsorbed to hydrophobic surfaces from surface-saturating solution concentrations.

The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square or hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square or hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM.

Volumetric interpretation of protein adsorption: capacity scaling with adsorbate molecular weight and adsorbent surface energy.

Silanized-glass-particle adsorbent capacities are extracted from adsorption isotherms of human serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa) for adsorbent surface energies sampling the observable range of water wettability. Adsorbent capacity expressed as either mass-or-moles per-unit-adsorbent-area increases with protein molecular weight (MW) in a manner that is quantitatively inconsistent with the idea that proteins adsorb as a monolayer at the solution-material interface in any physically-realizable configuration or state of denaturation. Capacity decreases monotonically with increasing adsorbent hydrophilicity to the limit-of-detection (LOD) near tau(o) = 30 dyne/cm (theta approximately 65 degrees) for all protein/surface combinations studied (where tau(o) identical with gamma(lv)(o) costheta is the water adhesion tension, gamma(lv)(o) is the interfacial tension of pure-buffer solution, and theta is the buffer advancing contact angle). Experimental evidence thus shows that adsorbent capacity depends on both adsorbent surface energy and adsorbate size. Comparison of theory to experiment implies that proteins do not adsorb onto a two-dimensional (2D) interfacial plane as frequently depicted in the literature but rather partition from solution into a three-dimensional (3D) interphase region that separates the physical surface from bulk solution. This interphase has a finite volume related to the dimensions of hydrated protein in the adsorbed state (defining "layer" thickness). The interphase can be comprised of a number of adsorbed-protein layers depending on the solution concentration in which adsorbent is immersed, molecular volume of the adsorbing protein (proportional to MW), and adsorbent hydrophilicity. Multilayer adsorption accounts for adsorbent capacity over-and-above monolayer and is inconsistent with the idea that protein adsorbs to surfaces primarily through protein/surface interactions because proteins within second (or higher-order) layers are too distant from the adsorbent surface to be held surface bound by interaction forces in close proximity. Overall, results are consistent with the idea that protein adsorption is primarily controlled by water/surface interactions.