Kris P. F. Janssen

Fiber optic SPR biosensing of DNA hybridization and DNA–protein interactions

In this paper we present a fiber optic surface plasmon resonance (SPR) sensor as a reusable, cost-effective and label free biosensor for measuring DNA hybridization and DNA-protein interactions. This is the first paper that combines the concept of a fiber-based SPR system with DNA aptamer bioreceptors. The fibers were sputtered with a 50nm gold layer which was then covered with a protein repulsive self-assembled monolayer of mixed polyethylene glycol (PEG). Streptavidin was attached to the PEGs carboxyl groups to serve as a versatile binding element for biotinylated ssDNA. The ssDNA coated SPR fibers were first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for a random 37-mer ssDNA. This single stranded DNA showed a 15 nucleotides overlap with the receptor ssDNA on the SPR fiber. A linear calibration curve was observed in 0.5-5 microM range. A negative control test did not reveal any significant non-specific binding, and the biosensor was easily regenerated. In a second assay the fiber optic SPR biosensor was functionalized with ssDNA aptamers against human immunoglobulin E. Limits of detection (2nM) and quantification (6nM) in the low nanomolar range were observed. The presented biosensor was not only useful for DNA and protein quantification purposes, but also to reveal the binding kinetics occurring at the sensor surface. The dissociation constant between aptamer and hIgE was equal to 30.9+/-2.9nM. The observed kinetics fully comply with most data from the literature and were also confirmed by own control measurements.

Zeolite-catalysed conversion of C3 sugars to alkyl lactates

The direct conversion of C3 sugars (or trioses) to alkyl lactates was achieved using zeolite catalysts. This reaction represents a key step towards the efficient conversion of bio-glycerol or formaldehyde to added-value chemicals such as lactate derivatives. The highest yields and selectivities towards the desired lactate product were obtained with Ultrastable zeolite Y materials having a low Si/Al ratio and a high content of extra-framework aluminium. Correlating the types and amounts of acid sites present in the different zeolites reveals that two acid functions are required to achieve excellent catalysis. Bronsted acid sites catalyse the conversion of trioses to the reaction intermediate pyruvic aldehyde, while Lewis acid sites further assist in the intramolecular rearrangement of the aldehyde into the desired lactate ester product. The presence of strong zeolitic Bronsted acid sites should be avoided as much as possible, since they convert the intermediate pyruvic aldehyde into alkyl acetals instead of lactate esters. A tentative mechanism for the acid catalysis is proposed based on reference reactions and isotopically labelled experiments. Reusability of the USY catalyst is demonstrated for the title reaction.

Fast and accurate peanut allergen detection with nanobead enhanced optical fiber SPR biosensor

This paper is the first report of a fiber optic SPR biosensor with nanobead signal enhancement. We evaluated the system with a bioassay for the fast and accurate detection of peanut allergens in complex food matrices. Three approaches of an immunoassay to detect Ara h1 peanut allergens in chocolate candy bars were compared; a label-free assay, a secondary antibody sandwich assay and a nanobead enhanced assay. Although label-free detection is the most convenient, our results illustrate that functionalized nanobeads can offer a refined solution to improve the fiber SPR detection limit. By applying magnetite nanoparticles as a secondary label, the detection limit of the SPR bioassay for Ara h1 was improved by two orders of magnitude from 9 to 0.09 μg/mL. The super paramagnetic character of the nanoparticles ensured easy handling. The SPR fibers could be regenerated easily and one fiber could be reused for up to 35 times without loss of sensitivity. The results were benchmarked against a commercially available polyclonal ELISA kit. An excellent correlation was found between the Ara h1 concentrations obtained with the ELISA and the concentrations measured with the SPR fiber assay. In addition, with the SPR fiber we could measure the samples twice as fast as compared to the fastest ELISA protocol. Since the dipstick fiber has no need for microchannels that can become clogged, time consuming rinsing step could be avoided. The linear dynamic range of the presented sensor was between 0.1 and 2 μg/mL, which is considerably larger than the ELISA benchmark.

Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices

The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.

Degradation of Methylammonium Lead Iodide Perovskite Structures through Light and Electron Beam Driven Ion Migration

Organometal halide perovskites show promising features for cost-effective application in photovoltaics. The material instability remains a major obstacle to broad application because of the poorly understood degradation pathways. Here, we apply simultaneous luminescence and electron microscopy on perovskites for the first time, allowing us to monitor in situ morphology evolution and optical properties upon perovskite degradation. Interestingly, morphology, photoluminescence (PL), and cathodoluminescence of perovskite samples evolve differently upon degradation driven by electron beam (e-beam) or by light. A transversal electric current generated by a scanning electron beam leads to dramatic changes in PL and tunes the energy band gaps continuously alongside film thinning. In contrast, light-induced degradation results in material decomposition to scattered particles and shows little PL spectral shifts. The differences in degradation can be ascribed to different electric currents that drive ion migration. Moreover, solution-processed perovskite cuboids show heterogeneity in stability which is likely related to crystallinity and morphology. Our results reveal the essential role of ion migration in perovskite degradation and provide potential avenues to rationally enhance the stability of perovskite materials by reducing ion migration while improving morphology and crystallinity. It is worth noting that even moderate e-beam currents (86 pA) and acceleration voltages (10 kV) readily induce significant perovskite degradation and alter their optical properties. Therefore, attention has to be paid while characterizing such materials using scanning electron microscopy or transmission electron microscopy techniques.

Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.

Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH(2))-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer-IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.

Selection and Characterization of DNA Aptamers for Egg White Lysozyme

We have selected aptamers binding to lysozyme from a DNA library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The final aptamer had a dissociation constant of 2.8 ± 0.3 nM, 6.1 ± 0.5 nM, and 52.9 ± 9.1 nM as determined by fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis respectively. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamers open up possibilities for the development of aptamer based food and medical diagnostics.

Glyoxylase biomimics: zeolite catalyzed conversion of trioses

Direct conversion of biomass derived trioses such as dihydroxyacetone (DHA) and glyceraldehyde (GLA) to alkyl lactates was carried out using alcohols over various Y type zeolite materials. The conversion takes place through a pyruvic aldehyde intermediate which subsequently undergoes esterification. The screened catalysts can be grouped based on their ability to either form methylglyoxal dialkyl acetal or alkyl lactate. The discussed catalysis shows strong resemblance with the biological lactic acid synthesis routes where glyoxylases effect the reaction. Zeolites may therefore be considered as functional biomimics of these enzymes.

Real-time monitoring of DNA hybridization and melting processes using a fiber optic sensor

In this paper a fiber optic surface plasmon resonance (FO-SPR) sensor was used to analyze the melting process of DNA linked to silica nanoparticles. Real-time monitoring of a DNA melting process has rarely been studied using surface plasmon resonance (SPR), since most commercial SPR setups do not allow for dynamic and accurate temperature control above 50 °C. The FO-SPR sensor platform, with silica nanobead signal amplification, allows sensing inside a standard PCR thermocycler, which makes high resolution DNA melting curve analysis possible. This innovative combination was used to characterize the hybridization and melting events between DNA immobilized on the sensor surface and DNA probes on silica nanoparticles. At optimized hybridization conditions complementary DNA strands of different lengths could be distinguished. While the real-time FO-SPR analysis of DNA hybridization did not result in significant variances, the analysis of DNA melting determined the exact length of overlap and the matching Gibbs energy.