Optical Waveguide Lightmode Spectroscopy Research Articles

Proteolytic Degradation of Bovine Submaxillary Mucin (BSM) and Its Impact on Adsorption and Lubrication at a Hydrophobic Surface.

The effects of proteolytic digestion on bovine submaxillary mucin (BSM) were investigated in terms of changes in size, secondary structure, surface adsorption, and lubricating properties. Two proteases with distinctly different cleavage specificities, namely trypsin and pepsin, were employed. SDS-PAGE analysis with staining for proteins and carbohydrate moieties showed that only the unglycosylated terminal regions of BSM were degraded by the proteases. Size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses indicated that tryptic digestion mainly led to the reduction in size, whereas pepsin digestion rather caused an increase in the size of BSM. Less complete cleavage in terminal peptide regions by pepsin and subsequent aggregation were possibly responsible for the increased size. Far-UV circular dichroism (CD) spectra of the protease-treated BSM showed a slight change in the secondary structure owing to the removal of terminal domains, but the overall random coil conformation adopted by the central glycosylated domain remained dominant and essentially unchanged. Surface adsorption properties as characterized by optical waveguide lightmode spectroscopy (OWLS) showed that tryptic digestion of BSM resulted in a decrease in the adsorbed mass, but pepsin digestion led to a slight increase in the adsorbed mass onto a hydrophobic surface compared to intact BSM. This is in agreement with the partial preservation of peptide segments in the terminal regions after pepsin digestion as confirmed by SEC and DLS studies. Despite a contrast in the adsorbed amount of the protease-treated BSMs onto the surface, both proteases substantially deteriorated the lubricating capabilities of BSM at a hydrophobic interface. The present study supports the notion that the terminal domains of BSM are critical to the adsorption and lubricating properties of BSM at hydrophobic interfaces.

Incubator proof miniaturized Holomonitor to in situ monitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: validity of the measured parameters and their corrections.

The in situ observation of cell movements and morphological parameters over longer periods of time under physiological conditions is critical in basic cell research and biomedical applications. The quantitative phase-contrast microscope applied in this study has a remarkably small size, therefore it can be placed directly into a humidified incubator. Here, we report on the successful application of this M4 Holomonitor to observe cancer cell motility, motility speed, and migration in the presence of the green tea polyphenol, epigallocatechin gallate, as well as to monitor the adhesion of preosteoblast cells on nanostructured titanate coatings, relevant for biomedical applications. A special mechanical stage was developed to position the sample into that range of the optical arrangement where digital autofocusing works with high reproducibility and precision. By in-depth analyzing the obtained single cell morphological parameters, we show that the limited vertical resolution of the optical setup results in underestimated single cell contact area and volume and overestimated single cell averaged thickness. We propose a simple model to correct the recorded data to obtain more precise single cell parameters. We compare the results with the kinetic data recorded by a surface sensitive optical biosensor, optical waveguide lightmode spectroscopy.

Label-free immunosensor for monitoring vitellogenin as a biomarker for exogenous oestrogen compounds in amphibian species

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL−1) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1–10 µg mL−1. During the competitive measurement, 100 ng mL−1 Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5–50 ng mL−1. We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples.

Sequential and competitive adsorption of peptides at pendant PEO layers

Earlier work provided direction for development of responsive drug delivery systems based on modulation of the structure, amphiphilicity, and surface density of bioactive peptides entrapped within pendant polyethylene oxide (PEO) brush layers. In this work, we describe the sequential and competitive adsorption behavior of such peptides at pendant PEO layers. Three cationic peptides were used for this purpose: the arginine-rich, amphiphilic peptide WLBU2, a peptide chemically identical to WLBU2 but of scrambled sequence (S-WLBU2), and the non-amphiphilic peptide poly-l-arginine (PLR). Optical waveguide lightmode spectroscopy (OWLS) was used to quantify the rate and extent of peptide adsorption and elution at surfaces coated with PEO. UV spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to quantify the extent of peptide exchange during the course of sequential and competitive adsorption. Circular dichroism (CD) was used to evaluate conformational changes after adsorption of peptide mixtures at PEO-coated silica nanoparticles. Results indicated that amphiphilic peptides are able to displace adsorbed, non-amphiphilic peptides in PEO layers, while non-amphiphilic peptides were not able to displace more amphiphilic peptides. In addition, peptides of greater amphiphilicity dominated the adsorption at the PEO layer from mixtures with less amphiphilic or non-amphiphilic peptides.

Determination of aflatoxin M1 in milk samples by an OWLS-based immunosensor

An immunoreaction-based method was investigated for the detection of aflatoxin M1 (AFM1), which is the hydroxylated metabolite of aflatoxin B1 (AFB1). This mycotoxin may be found in milk and milk products obtained from livestock that have ingested contaminated feed. Quantitative analysis of AFM1 was carried out using indirect (competitive) immunoassay method, which can be used for low weight molecules. The real-time measurement was done with Optical Waveguide Lightmode Spectroscopy (OWLS) technique. After the optimization of the chemical and biochemical parameters (determination of the optimal concentration of the immobilized AFM1-protein conjugate, determination of the AFM1 antibody content of the samples, etc.) real samples were also examined. Three kinds of milk sample preparation methods (filtration, centrifugation, size exclusion centrifugation) and two dilution rates (100 and 200 fold) were compared. The presented competitive immunoassay method showed the best results when 100 fold diluted filtered ...

Label-free in situ optical monitoring of the adsorption of oppositely charged metal nanoparticles.

The mechanism of alternating deposition of oppositely charged gold nanoparticles (AuNPs) was investigated by optical waveguide lightmode spectroscopy (OWLS). OWLS allows monitoring of the kinetics of layer-by-layer (LbL) adsorption of positively and negatively charged nanoparticles in real time without using any labels so that the dynamics of layer formation can be revealed. Positively charged NPs that are already deposited on a negatively charged glass substrate strongly facilitate the adsorption of the negatively charged particles. The morphology of the adsorbed layer was also investigated with atomic force microscopy (AFM). AFM revealed that the interaction between oppositely charged particles results in the formation of NP clusters with sizes varying between 100 and 6000 NPs. The cluster size distribution is found to be an exponentially decaying function, and we propose a simple theory to explain this finding.

Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria

Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12ngcm−2 (0.08pmolcm−2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1CFUmL−1) over a large linear working range (10–106CFUmL−1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Binding interactions of bacterial lipopolysaccharide and the cationic amphiphilic peptides polymyxin B and WLBU2

Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and rapid/strong binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB). Analysis of the interfacial behavior of mixtures of LPS and peptide using air–water interfacial tensiometry and optical waveguide lightmode spectroscopy strongly suggests insertion of intact LPS vesicles by the peptide WLBU2 without vesicle destabilization. In contrast, dynamic light scattering (DLS) studies show that LPS vesicles appear to undergo peptide-induced destabilization in the presence of PmB. Circular dichroism spectra further confirm that WLBU2, which shows disordered structure in aqueous solution and substantially helical structure in membrane-mimetic environments, is stably located within the LPS membrane in peptide-vesicle mixtures. We therefore expect that presentation of WLBU2 at an interface, if tethered in a fashion which preserves its mobility and solvent accessibility, will enable the capture of bacteria and endotoxin without promoting reintroduction of endotoxin to the circulating blood, thus minimizing adverse clinical outcomes. On the other hand, our results suggest no such favorable outcome of LPS interactions with polymyxin B.

Comparative determination of two probiotics by QCM and OWLS-based immunosensors

The regular consumption of foods containing probiotic bacteria has beneficial physiological effects on the health and the digestion system. There is a need for novel analytical approaches for the determination of these bacteria that are faster than the classical plate counting method. For this purpose, two label-free biosensors were investigated and presented in this paper: Quartz Crystal Microbalance (QCM) and Optical Waveguide Lightmode Spectroscopy (OWLS) based direct immunosensors were developed for real-time direct detection of probiotic bacteria in fermented dairy products. Bifidobacterium bifidum O1356 and Lactobacillus acidophilus O1132 were detected by polyclonal anti-B. bifidum IgG and anti-L. acidophilus IgG immobilized on the sensors' surface. Sulfo-LC-SPDP cross linking agent was used to bind antibodies to the gold surface of the QCM's AT-cut quartz wafer. Concerning OWLS, antibodies were covalently bound to the amino groups of the silanized surface of the waveguide by glutaraldehyde. The dynamic measuring range was found between 1.0E+3 and 5.0E+5CFUmL(-1) in 100 fold diluted fermented milk products by QCM and with OWLS. Considering the current legislation of the probiotic content in probiotic products, the two developed immunosensors can be applied for rapid quantification of L. acidophilus and B. bifidum in fermented milk. These examinations offer effective alternatives to the microbiological plate counting method.

Modulation of Protein Adsorption by Poloxamer 188 in Relation to Polysorbates 80 and 20 at Solid Surfaces

Poloxamer 188 (BASF Pluronic® F68) is widely used as a shear-protective excipient to enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. However, little is known in any quantitative sense of its effect on protein adsorption and aggregation. Optical waveguide lightmode spectroscopy was used here to compare the adsorption kinetics exhibited by poloxamer 188, and polysorbates 80 and 20, in the presence and absence of a model protein (chicken egg white lysozyme) and in separate experiments, a recombinant protein (human granulocyte colony-stimulating factor) at hydrophilic, silica-titania surfaces. Experiments were performed in sequential and competitive adsorption modes, enabling the adsorption kinetic patterns to be interpreted in a fashion revealing the dominant mode of surfactant-mediated stabilization of protein in each case. Kinetic results showed that polysorbates 80 and 20 are able to inhibit protein adsorption only by their preferential location at an interface to which they show sufficient affinity, and not by formation of less surface active, protein-surfactant complexes. On the other hand, poloxamer 188 is able to inhibit protein adsorption by entering into formation of protein-surfactant complexes of low adsorption affinity (i.e., high colloidal stability), and not by its preferential location at the interface.

Blood protein repulsion after peptide entrapment in pendant polyethylene oxide layers

A number of sufficiently small peptides have been shown to integrate into polyethylene oxide (PEO) brush layers in accordance with their amphiphilicity and ordered structure. Those results have suggested that responsive drug delivery systems based on peptide-loaded PEO layers can be controlled by modulation of solution conditions and peptide amphiphilicity. However, the presence of entrapped peptide may compromise the protein repulsive character of the PEO layer, and in this way reduce the viability of a medical device coating based on such an approach. Nisin is a cationic, amphiphilic, and antimicrobial peptide that has been shown to integrate into PEO brush layers. In this work, the preferential location of fibrinogen at PEO-coated, nisin-loaded layers was investigated in nisin-fibrinogen sequential adsorption experiments using detection of fluorescein isothiocyanate labeled fibrinogen, detection of changes in zeta potential, and measurement of adsorption and elution kinetics by optical waveguide lightmode spectroscopy. Results from each technique indicate that the presence of entrapped nisin does not affect fibrinogen interaction with the PEO layer. In addition, entrapment of blood solutes within PEO layers contacted with 25% equine plasma in phosphate-buffered saline was reduced by the prior entrapment of nisin within the layer.

Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface

Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5–85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic light scattering (DLS), showed a slight increase after heating at high temperature (80°C). Optical waveguide lightmode spectroscopy (OWLS) studies showed facile adsorption of BSM onto poly(dimethylsiloxane) (PDMS) surface (>180ng/cm2) at room temperature due to its amphiphilic characteristics. Adsorbed mass of BSM was noticeably reduced after heating at 80°C, possibly resulting from its aggregation. BSM showed excellent lubricity at self-mated sliding contacts between PDMS at room temperature or lower (friction coefficient≈0.02), even when BSM solution was pre-heated up to 120min at 80°C. Gradual degradation of lubricity of BSM was observed with increasing temperature, but it was also reversibly recovered with decreasing temperature. Structural and functional stability of BSM against heating is proposed to originate from heavy glycosylation and lack of higher degree of protein structure in BSM.

Bulk and surface sensitivity of a resonant waveguide grating imager

We report the assessment of the sensitivity of a microplate-compatible resonant waveguide grating imager. The sensitivity to bulk refractive index changes was determined using a serial dilution of glycerol solution with the help of a refractometer. The surface sensitivity was examined using layer-by-layer polyelectrolyte films in conjunction with optical waveguide lightmode spectroscopy and characterized by the binding of acetazolamide to immobilized carbonic anhydrase under microfluidics. The results suggest that the imager has a limit of detection down to 2.2 × 10−6 for refractive index change and 0.078 ng/cm2 for the adsorbed mass.

New Antifouling Platform Characterized by Single-Molecule Imaging

Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm2). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others.

Effects of temperature and concentration on mechanism and kinetics of thermally induced deposition from coffee extracts

AbstractProduction of soluble (instant) coffee powders typically involves extraction of roasted coffee by water followed by evaporation in order to concentrate extracts before spray or freeze drying to produce dry coffee powder. In the course of evaporation, deposition of dissolved material from coffee extracts is a major cause of fouling at the heat exchange surfaces of evaporators. Therefore, in order to improve the design and optimization of evaporation processes of coffee extracts, better understanding of the deposition mechanism and kinetics is needed. In this study, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the initial formation of nanometer scale deposits on surfaces exposed to coffee extracts. OWLS measurements were complemented by light scattering from extract solutions, gravimetry of macroscopic deposits, and scanning electron microscopy imaging of deposited layers. Primary molecular-scale layers of about 1 mg m−2 were rapidly formed in the first stage of deposition, even at ambient temperature, followed by the secondary deposition with kinetics strongly dependent on temperature. Secondary deposition rates were low and largely independent of the extract concentration at ambient temperature, but became strongly dependent on the extract concentration at elevated temperatures. In particular, activation energies for the deposition between 25°C and 70°C were much higher for the original extract (13.3 mass %, solids) than for diluted extracts (up to 1.3 mass %, solids). Furthermore, heating of the original extracts above 60°C resulted in rapid aggregation of suspended macromolecules into large clusters, while only gradual aggregation was observed in diluted extracts.

Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging

Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins – wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) – were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV–vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used.

ToF-SIMS analysis of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) ultrathin adlayers

Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb2O5 surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL-PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL-PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL-PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL-PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum.

Build-up of an antimicrobial multilayer coating on a textile support based on a methylene blue–poly(cyclodextrin) complex

The aim of this work was to develop an antibacterial multilayer coating activated with methylene blue (MB) and based on chitosan (CHT) and cyclodextrin polyelectrolyte (polyCD) onto a non-woven polyethylene terephthalate (PET) textile support. The MB-free and MB-loaded systems were built-up by applying the dip-coating technique, alternating soak cycles of the PET textile preliminarily modified with carboxylate groups in CHT and in polyCD or polyCD/MB complex solutions. The layer-by-layer assembly build-up was followed by optical waveguide lightmode spectroscopy on the one hand and by gravimetry once it was applied on the textile substrate on the other hand. Two chitosan grades were used, low molecular weight (CHT-L) and medium molecular weight (CHT-M). The influence of the molar ratio CD/MB in the polyCD solutions was varied and finally the system underwent a post reticulation with genipin. Such parameters influences were investigated with regard to the loading capacity in MB of the systems, the release kinetics profiles of MB in pure water, phosphate buffer and MEM media, and the degradation of the self-assembled coating in the same media. Finally, biological and microbiological tests were performed to demonstrate the cytocompatibility of the systems and their ability to display a sustained antibacterial effect of the device through the MB prolonged release.

In-situ and label-free optical monitoring of the adhesion and spreading of primary monocytes isolated from human blood: Dependence on serum concentration levels

Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface. Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5ml mini-incubator, controlling both temperature and CO2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica–titania), when the serum level of the culturing medium was 0.0–0.01%. Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell–substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely noninvasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics.

Cleaning requirements for silica‐coated sensors used in optical waveguide lightmode spectroscopy

Optical waveguide lightmode spectroscopy (OWLS), based on the incoupling of laser light into a waveguide sensor by an optical grating, allows for the in situ measurement of protein adsorption. Few reports have described cleaning methods for the surfaces of such sensors, and in this investigation, we compare common methods for cleaning of silica surfaces in relation to their effectiveness for cleaning silica‐coated waveguide sensors used in OWLS. For this purpose, atomic force microscopy (AFM) analysis of surface morphology and OWLS detection of protein adsorption kinetics were used to evaluate waveguide sensors before and after cleaning. While AFM line scans showed a substantial increase in average waveguide peak‐to‐valley height after RCA cleaning relative to all other methods tested, chemical etching owing to the alkaline component of the rolling circle amplification method rendered the waveguide unusable for detection of protein adsorption with OWLS. A revised method, based on replacement of the alkaline step with immersion in sodium dodecyl sulfate, was not only effective at cleaning OWLS waveguides off‐the‐shelf but also showed excellent protein adsorption reproducibility after ex situ cleaning. Moreover, the revised method showed excellent reproducibility when applied in situ, between repeated adsorption‐elution cycles. Copyright © 2013 John Wiley & Sons, Ltd.