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Mechanochemical Immobilisation of Metathesis Catalysts in a Metal–Organic Framework
Spekreijse, Jurjen ; Öhrström, Lars ; Sanders, Johan P.M. ; Bitter, Harry ; Scott, Elinor L. - \ 2016
Chemistry-A European Journal 22 (2016)43. - ISSN 0947-6539 - p. 15437 - 15443.
heterogeneous catalysis - immobilization - mechanochemistry - metal–organic frameworks - metathesis
A simple, one-step mechanochemical procedure for immobilisation of homogeneous metathesis catalysts in metal–organic frameworks was developed. Grinding MIL-101-NH2(Al) with a Hoveyda–Grubbs second-generation catalyst resulted in a heterogeneous catalyst that is active for metathesis and one of the most stable immobilised metathesis catalysts. During the mechanochemical immobilisation the MIL-101-NH2(Al) structure was partially converted to MIL-53-NH2(Al). The Hoveyda–Grubbs catalyst entrapped in MIL-101-NH2(Al) is responsible for the observed catalytic activity. The developed synthetic procedure was also successful for the immobilisation of a Zhan catalyst.
Biofunctionalized nanoporous aluminum oxide culture chips : for capture and growth of bacteria
Debrassi, A. - \ 2016
University. Promotor(en): Han Zuilhof; Willem de Vos; Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789462576179 - 218 p.
aluminium oxide - porous media - unimolecular films - immobilization - bacteria - binding - antibodies - aluminiumoxide - poreus medium - unimoleculaire films - immobilisatie - bacteriën - binden - antilichamen
Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis.
After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2.
Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated.
In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface.
With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system.
In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide.
In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.
New applications of the interaction between diols and boronic acids
Duval, F.L. - \ 2015
University. Promotor(en): Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462574717 - 131
antilichamen - immobilisatie - boorzuur - biomarkers - vloeistofchromatografie - antibodies - immobilization - boric acid - liquid chromatography
Florine Duval - New applications of the interaction between diols and boronic acids – Summary
Chapter 1 introduces the theory and known applications of the interaction between boronic acids and diols, and explains the context of this thesis. Diagnosis of depression was the initial goal of this multidisciplinary project. The focus of the PhD project was the development of a strategy to immobilize antibodies on the surface of a chip in such a way that very low concentrations (~ 1 pM) of biomarkers for depression could be detected in urine. To achieve this, the immobilization of antibodies using boronic acids seemed promising.
However, preliminary experiments and further insights revealed the many challenges that this immobilization strategy faces, giving rise to Chapter 2. This chapter discusses several important points that need to be taken into account when one plans to immobilize antibodies via boronic acids: choice of the boronic acid structure and spacer to attach it to the surface, use of an antifouling polymer, choice of an antibody with suitable glycosylation, optimization of the conditions for antibody immobilization...
One big issue for antibody immobilization using boronic acids is the reversibility of the reaction between boronic acids and diols, hence the possible release of the antibody from the surface.
Chapter 3 describes the design and synthesis of boronic acid-containing linkers that would enable the oriented and irreversible immobilization of antibodies. Two linkers were designed with an amine for surface attachment, a boronic acid for capturing antibodies via the N-glycans in their Fc chain, and a diazirine for irreversible immobilization upon UV irradiation while maintaining antibody orientation. From a diazirine building-block that was obtained in three steps, the first linker was synthesized in four steps and the second linker was synthesized in three steps. Diol-functionalized silica was used for the chromatography of two boronic acid-containing intermediates, this method being novel (to the best of our knowledge) and likely based on boronic acid-diol interactions. High-resolution mass spectrometry, through matching exact masses, matching isotope patterns and observation of species corresponding to the esterification of boronic acids with MeOH, confirmed that both linkers were synthesized successfully.
During the synthesis of boronic acid-containing linkers, it was difficult to see which spots on TLC plates corresponded to boronic acids. To solve this problem, a new TLC staining method based on the reaction between boronic acids and alizarin was developed.
Chapter 4 presents this work in detail. After optimization experiments, 1 mM alizarin in acetone was shown to be the preferred staining solution. When the TLC plate was briefly dipped in this solution, allowed to dry in ambient air and observed under 365 nm light, bright yellow fluorescent spots were observed where boronic acids were present. Phenylboronic acid was detected at a concentration as low as 0.1 mM. A range of boronic acids and derivatives was successfully detected, and boron-free compounds resulted in no or very weak fluorescence. The staining method was further tested in the monitoring of three reactions involving boronic acids, and provided clear information about the consumption or formation of boronic acid-containing compounds.
Although TLC is useful to synthetic chemists, analysis of reaction mixtures by HPLC is sometimes necessary for obtaining more accurate information or for optimization of preparative HPLC conditions.
Chapter 5 presents the development and applicability of a method for the on-line HPLC detection of boronic acids using alizarin. After optimization experiments at an HPLC flow rate of 0.40 mL/min, the HPLC-separated analytes were mixed post-column with a solution of 75 μM alizarin and 0.1% triethylamine in ACN, which was delivered at a flow rate of 0.60 mL/min. The reaction between alizarin and boronic acids occurred in a reaction coil of dimensions of 3.5 m × 0.25 mm at a temperature of 50 °C, resulting in fluorescent complexes that were detected as positive peaks by a fluorescence detector (lexc 469 nm and lem 610 nm). The method enabled the selective detection of various boronic acids and derivatives, with a limit of detection of phenylboronic acid of 1.2 ng or 1 μM. It could successfully monitor the progress of two organic reactions involving boronic acid-containing compounds, and provided useful insights into the course of the reactions.
Chapter 6 provides a reflexion about the work presented in this thesis, suggestions for future research, and a general conclusion.
Surface characterization and antifouling properties of nanostructured gold chips for imaging surface plasmon resonance biosensing
Joshi, S. ; Pellacani, P. ; Beek, T.A. van; Zuilhof, H. ; Nielen, M.W.F. - \ 2015
Sensors and Actuators B: Chemical 209 (2015). - ISSN 0925-4005 - p. 505 - 514.
mass-spectrometry - nonspecific adsorption - zwitterionic polymers - organic monolayers - films - spr - functionalization - immobilization - microscopy - proteins
Surface Plasmon Resonance (SPR) optical sensing is a label-free technique for real-time monitoring of biomolecular interactions. Recently, a portable imaging SPR (iSPR) prototype instrument, featuring a nanostructured gold chip, has been developed. In the present work, we investigated the crucial first steps, prior to eventual use of the nanostructured iSPR chip, i.e., its surface modification, in-depth surface characterization and the antifouling performance. Results were compared with conventional flat (i)SPR gold chips having the same surface chemistries, viz. different types of polyethylene glycol and zwitterionic polymers. Characterization of the (i)SPR chips before and after surface modification was performed using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA), X-ray photoelectron spectroscopy (XPS) and direct analysis in real time high resolution mass spectrometry (DART-HRMS). The antifouling properties were then studied using the nanostructured chip in the portable iSPR instrument and the flat gold chip in conventional SPR setup. The zwitterionic polymer surface chemistries showed the best antifouling properties. Comparison of the nanostructured iSPR chips with conventional flat (i)SPR gold chips showed that the latter perform slightly better in terms of surface modification as well as antifouling properties. The portable iSPR instrument is almost as sensitive as conventional iSPR (IBIS) and nine times less sensitive than conventional SPR (Biacore 3000). The nanostructured iSPR chip, along with the portable instrument, offers the advantage of about ten-fold reduction in instrument size, weight and costs compared to conventional (i)SPR instruments using flat gold, thus making it highly interesting for future biosensing applications.
The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase—A means of separating amino acids for synthesizing biobased chemicals
Teng, Y. ; Scott, E.L. ; Sanders, J.P.M. - \ 2014
Biotechnology Progress 30 (2014)3. - ISSN 8756-7938 - p. 681 - 688.
nitrogen-containing chemicals - bulk chemicals - electrodialysis - biorefinery - biomass - immobilization - chromatography - alginate
Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate a-decarboxylase (GAD, Type I, EC 18.104.22.168) was applied to specifically convert Glu into c-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme’s initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.
Orientation of llama antibodies strongly increases sensitivity of biosensors
Trilling, A.K. ; Hesselink, T. ; Houwelingen, A. van; Cordewener, J.H.G. ; Jongsma, M.A. ; Schoffelen, S. ; Hest, J.C.M. van; Zuilhof, J.T. ; Beekwilder, J. - \ 2014
Biosensors and Bioelectronics 60 (2014). - ISSN 0956-5663 - p. 130 - 136.
autodisplayed z-domains - click chemistry - immobilization - coli
Sensitivity of biosensors depends on theorientation of bio-receptors on the sensor surface.The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups)to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics.
Synthesis of galacto-oligosaccharides with ß-galactosidases
Warmerdam, A. - \ 2013
University. Promotor(en): Remko Boom, co-promotor(en): Anja Janssen. - S.l. : s.n. - ISBN 9789461735621 - 171
oligosacchariden - bèta-galactosidase - productie - synthese - immobilisatie - bioreactoren - lactose - oligosaccharides - beta-galactosidase - production - synthesis - immobilization - bioreactors
Galacto-oligosaccharides (GOS) are generally enzymatically synthesized with β-galactosidases. GOS are of interest because of their prebiotic effects on human health. They are mainly applied in infant nutrition, because of their resemblance to human milk oligosaccharides, but they are also applied in e.g. dairy products and beverages.
β-Galactosidases synthesize GOS from lactose through transgalactosylation: instead of only using water as acceptor (as in hydrolysis), they can use carbohydrates as acceptor. In this way, GOS with a degree of polymerization up to ten can be formed. The ratio of hydrolysis over transgalactosylation depends on the substrate concentration, temperature, and the source of the enzyme.
A β-galactosidase preparation from Bacillus circulans, called Biolacta N5, is known to produce high GOS yields compared to enzymes from other sources. The aim of this thesis was to obtain more insight on the mechanism of GOS production with Biolacta N5 and to investigate how the GOS production process can be optimized.
Biolacta N5 consists of four β-galactosidase isoforms, β-gal-A, β-gal-B, β-gal-C, and β-gal-D, which were purified and characterized in chapter 2. At low substrate concentrations, these isoforms differ in hydrolysis and transgalactosylation activity. β-Gal-D seems the best isoform for GOS production, followed by β-gal-C and β-gal-B, and β-gal-A showed the least GOS formation. By studying the thermodynamics of lactose conversion with isothermal titration calorimetry (ITC), the differences in behavior were confirmed, although the interpretation of the results of ITC was quite difficult dealing with a complex mixture of reactions. In contrast to the selectivity at low substrate concentrations, the selectivity of the isoforms hardly differed at high lactose concentrations. These conditions are usually used for industrial GOS production. Only β-gal-A produced slightly more galactose. The initial GOS formation rates indicated that β-gal-A and β-gal-B are the best isoforms for GOS production.
In chapter 3, the effect of high concentrations was further studied on the behavior of the complete Biolacta N5 preparation. High concentrations of reacting and non-reacting carbohydrates were added to the oNPG activity assay with Biolacta N5. Small carbohydrates were found to act as acceptor in the reaction, which resulted in an increased reaction rate. The rate of the limiting step of the reaction, i.e. the binding of the galactose residue with the acceptor, is increased, and therewith the release of the product is faster. At the same time, the additives cause molecular crowding, which results in a higher affinity between the enzyme and the substrate.
In chapter 4, a kinetic model was developed to quantify the effects of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of the β-galactosidases from B.circulans, Aspergillus oryzae and Kluyveromyces lactis. Using multiple substrates simultaneously yields more information than using only lactose or oNPG, because of the competition between the substrates. Three main differences were found that explain why Biolacta N5 produces higher GOS yields than other β-galactosidases: (i) it had a higher reaction rate constant of using lactose or oligosaccharides as substrate relative to water as acceptor (so it had a very low relative hydrolysis rate); and (ii) it also had a high reaction rate with galactose as acceptor, whereas (iii) the other two enzymes are strongly inhibited by galactose. The reaction rate constants indicate that β-gal-A is the most active isoforms in GOS production; however, also its hydrolysis rate is highest. Many of the rate constants increase with increasing molecular weight of the isoforms.
Chapter 5 reports on the stability of Biolacta N5 at various temperatures in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. Samples were taken in time and analyzed for oNPG converting activity. The oNPG converting activity was corrected for the presence of lactose, glucose, galactose, and oligosaccharides with the mechanistic model from chapter 4. The stability, expressed with the half-life time, of the enzyme was found to strongly increase with initial lactose concentrations. At high substrate concentration, higher temperatures can be used for GOS production than was presumed feasible based on stability measurements in diluted solutions.
Biolacta N5 is still active after one batch run of GOS production, but in a batch process the enzyme is wasted after the reaction. For this reason, the use of immobilized enzyme in a continuous packed bed reactor (PBR) was investigated in terms of productivity in chapter 6. The carbohydrate composition of the product in both systems was comparable. The half-life time of the immobilized enzyme at a lactose concentration of 33% (w/w) and 50ºC was approximately 90 days. The enzymatic productivity using immobilized enzyme in a PBR may be six times higher than that using free enzyme in a batch reactor. When striving for an equal volumetric productivity of both systems, the volume of a PBR can be much smaller than that of a batch reactor, depending on the enzyme dosage and running time of the one batch.
Chapter 7 discusses various alternatives for process optimization. One option for a higher GOS productivity is to use an enzyme preparation that contains only β-gal-A and β-gal-B. A somewhat higher oligosaccharide yield can be obtained when initially using a mixture of lactose with a better acceptor molecule. This results in a changed oligosaccharide composition and less lactose in the final product. The sustainability of GOS production in a PBR with immobilized enzyme and 33% (w/w) lactose seems to be similar in terms of exergy to that in a batch reactor with free enzyme and 60% (w/w) lactose.
Oriented antibodies as versatile detection element in biosensors
Trilling, A.K. - \ 2013
University. Promotor(en): Han Zuilhof, co-promotor(en): Jules Beekwilder. - S.l. : s.n. - ISBN 9789461735683 - 168
biosensoren - oppervlakten - oppervlaktechemie - oriëntatie - antilichamen - lama's - antistoftesten - mycobacterium tuberculosis - unimoleculaire films - immobilisatie - biosensors - surfaces - surface chemistry - orientation - antibodies - llamas - antibody testing - unimolecular films - immobilization
The aim of this thesis is to explore orientation of detection elements on biosensor
surfaces. To this end, different strategies were combined such as surface chemistry and protein functionalization, with the aim to generate a platform for oriented immobilization of antibodies
in biosensors. Chapter 2 investigates the formation of organic monolayers on
oxide-free copper. Detailed studies were performed to characterize the monolayers
and proof its quality. Apart from being the first oxide-free monolayers on copper
reported thus far, further functionalization was successfully investigated.
Chapter 3 gives an overview about approaches used to orient antibodies on surfaces.
It also summarizes methods used to characterize the orientation of immobilized
antibodies in a more direct manner.
In chapter 4 a set of detection elements for tuberculosis bacteria is described. These
are variable domains of llama heavy-chain antibodies, known as VHH proteins. A
number of VHHs, selected by phage display, were expressed by Escherichia coli bacteria
and characterized for binding towards Mycobacterium tuberculosis bacteria.
Specificity of VHHs was investigated and the antigen was identified.
In chapter 5 the impact of orientation on the analyte binding capacity was studied
by SPR as model biosensor. Established techniques (NH2 coupling, biotyinylation)
were used to immobilize VHHs, and a comparison between oriented and random
immobilized VHHs was made. The effect of molecular weight, epitope number and
affinity of the target analyte was investigated.
In chapter 6, a novel coupling chemistry was used to immobilize VHHs, and in this case the same chemistry could be used for oriented and random immobilization. VHHs were engineered and functionalized with a non-natural amino acid to bear either one or five azide groups. Azide groups served as unique chemical handles on the VHHs and were used to click proteins onto a cyclooctyne-modified surface in an oriented and random approach. Spectacular effects on biosensor sensitivity were observed when VHHs were immobilized in an oriented manner.
Finally, in chapter 7, the main results of this thesis are summarized and remaining
problems as well as ideas for future research are discussed.
Mineralization and herbage recovery of animal manure nitrogen after application to various soil types
Shah, G.M. ; Rashid, M.I. ; Shah, G.A. ; Groot, J.C.J. ; Lantinga, E.A. - \ 2013
Plant and Soil 365 (2013)1-2. - ISSN 0032-079X - p. 69 - 79.
uncomposted poultry litter - organic-matter - different texture - plant uptake - slurry - decomposition - ryegrass - carbon - immobilization - microorganisms
Background and aim - Typical values of plant available nitrogen (N) from animal manures are provided in fertilizer recommendation schemes. However, only a few attempts have been made thus far to study the variation in these values among contrasting soil types. The objective of this study was to examine the interactions between animal manure and soil types on N mineralization and total plant N recovery (shoots¿+¿roots) during one growing season. Methods - A pot experiment was conducted in a greenhouse during a growth period of 180 days. Experimental treatments included solid cattle manure (SCM), cattle slurry (CS) and poultry manure (PM), all applied to sandy, clay and peat soils sown with perennial ryegrass. Total N application rate was 120 kg ha-1. Results - There were clear interactions (P¿¿sandy¿>¿clay. In case of the peat soil, net mineralization of the applied organic N was on average 90 % from PM, 39 % from SCM and 26 % from CS. However, in the clay soil a positive net N mineralization occurred only from PM (42 %). Besides, significant proportions of the applied mineral N from SCM (17 %) and CS (35 %) were immobilized in this soil type. Consequently, apparent total plant N recovery was highest in the peat soil with values of 80, 57 and 50 % from PM, CS and SCM, respectively. In contrast, these values were only 57, 28 and 15 % for the clay soil. Conclusions - It is concluded that wide variations do exist in the extent of net N mineralization and plant N recovery from a given animal manure type when applied to diverging soil types. This indicates the need for more soil-specific manure fertilizer recommendations.
The effect of uniform capture molecule orientation on biosensor sensitivity : dependence on analyte properties
Trilling, A.K. ; Harmsen, M.M. ; Ruigrok, V.J. ; Zuilhof, H. ; Beekwilder, J. - \ 2013
Biosensors and Bioelectronics 40 (2013)1. - ISSN 0956-5663 - p. 219 - 226.
mouth-disease virus - domain antibody fragments - chain variable domain - passive-immunization - immobilization - protein - llama - immunoglobulin - biotinylation - strategies
Uniform orientation of capture molecules on biosensors has been reported to increase sensitivity. Here it is investigated which analyte properties contribute to sensitivity by orientation. Orientation of capture molecules on biosensors was investigated using variable domains of llama heavy-chain antibodies (VHHs) as capture molecule, and a surface plasmon resonance (SPR) chip as biosensor. Two VHHs were tested in this study: one recognizing foot-and-mouth disease virus (FMDV) and another recognizing the 16 kDa heat-shock protein of Mycobacterium tuberculosis. SPR chips with randomly immobilized biotinylated VHHs were compared to streptavidin-coated SPR chips, on which similar quantities of oriented biotinylated VHHs were non-covalently immobilized. Analytes that differ in molecular weight, epitope number and epitope affinity were compared using the FMDV-recognizing VHH. When binding of intact FMDV particles (146 S; 8200 kDa) or pentameric FMDV coat protein aggregates (12 S; 282 kDa) was detected, a modest (1–2-fold) increase in sensitivity was observed. When a 26-residue peptide (3 kDa) containing the epitope for VHH recognition was tested, much larger effects of capture molecule orientation (14-fold) on signal were observed. A 20–227-fold improvement was also observed when the epitope peptide was covalently linked to bovine serum albumin (67 kDa) or R-phycoerythrin (240 kDa). The results indicate that orientation of the capture molecule hardly affects high-affinity interactions, while it leads to strong improvements in sensitivity for lower-affinity interactions
Bioconjugation of Protein-Repellent Zwitterionic Polymer Brushes Grafted from Silicone Nitride
Nguyen, A.T. ; Baggerman, J. ; Paulusse, J.M.J. ; Zuilhof, H. ; Rijn, C.J.M. van - \ 2012
Langmuir 28 (2012)1. - ISSN 0743-7463 - p. 604 - 610.
biosensor applications - poly(ethylene glycol) - antibody microarrays - surface - adsorption - well - membranes - plasma - immobilization - strategies
A new method for attaching antibodies to protein-repellent zwitterionic polymer brushes aimed at recognizing microorganisms while preventing the nonspecific adsorption of proteins is presented. The poly(sulfobetaine methacrylate) (SBMA) brushes were grafted from a-bromo isobutyryl initiator-functionalized silicon nitride (SixN4, x = 3) surfaces via controlled atom-transfer radical polymerization (ATRP). A trifunctional tris(2-aminoethyl)amine linker was reacted with the terminal alkylbromide of polySBMA chains. N-Hydroxysuccinimide (NHS) functionalization was achieved by reacting the resultant amine-terminated polySBMA brush with bifunctional suberic acid bis(N-hydroxysuccinimide ester). Anti-Salmonella antibodies were subsequently immobilized onto polySBMA-grafted SixN4 surfaces through these NHS linkers. The protein-repellent properties of the polySBMA-grafted surface after antibody attachment were evaluated by exposing the surfaces to Alexa Fluor 488-labeled fibrinogen (FIB) solution (0.1 g·L–1) for 1 h at room temperature. Confocal laser scanning microscopy (CLSM) images revealed the minimal adsorption of FIB onto the antibody-coated polySBMA in comparison with that of antibody-coated epoxide monolayers and also bare SixN4 surfaces. Subsequently, the interaction of antibodies immobilized onto polySBMA with SYTO9-stained Salmonella solution without using blocking solution was examined by CLSM. The fluorescent images showed that antibody-coated polySBMA efficiently captured Salmonella with only low background noise as compared to antibody-coated monolayers lacking the polymer brush. Finally, the antibody-coated polySBMA surfaces were exposed to a mixture of Alexa Fluor 647-labeled FIB and Salmonella without the prior use of a blocking solution to evaluate the ability of the surfaces to capture bacteria while simultaneously repelling proteins. The fluorescent images showed the capture of Salmonella with no adsorption of FIB as compared to antibody-coated epoxide surfaces, demonstrating the potential of the zwitterionic layer in preventing the nonspecific adsorption of the proteins during the detection of bacteria in complex matrices.
Development of surface plasmon resonance-based sensor for detection of silver nanoparticles in food and the environment
Rebe-Raz, S. ; Leontaridou, M. ; Bremer, M.G.E.G. ; Peters, R.J.B. ; Weigel, S. - \ 2012
Analytical and Bioanalytical Chemistry 403 (2012)10. - ISSN 1618-2642 - p. 2843 - 2850.
heavy-metals - metallothionein - immobilization - biosensor - ions - nanosilver - proteins
Silver nanoparticles are recognized as effective antimicrobial agents and have been implemented in various consumer products including washing machines, refrigerators, clothing, medical devices, and food packaging. Alongside the silver nanoparticles benefits, their novel properties have raised concerns about possible adverse effects on biological systems. To protect consumer’s health and the environment, efficient monitoring of silver nanoparticles needs to be established. Here, we present the development of human metallothionein (MT) based surface plasmon resonance (SPR) sensor for rapid detection of nanosilver. Incorporation of human metallothionein 1A to the sensor surface enables screening for potentially biologically active silver nanoparticles at parts per billion sensitivity. Other protein ligands were also tested for binding capacity of the nanosilver and were found to be inferior to the metallothionein. The biosensor has been characterized in terms of selectivity and sensitivity towards different types of silver nanoparticles and applied in measurements of real-life samples—such as fresh vegetables and river water. Our findings suggest that human MT1-based SPR sensor has the potential to be utilized as a routine screening method for silver nanoparticles, that can provide rapid and automated analysis dedicated to environmental and food safety monitoring.
Pronounced effects of accute endurance exercise on gene expression in resting and exercising human skeletal muscle
Catoire, M. ; Mensink, M.R. ; Boekschoten, M.V. ; Hangelbroek, R.W.J. ; Muller, M.R. ; Schrauwen, P. ; Kersten, A.H. - \ 2012
PLoS One 7 (2012)11. - ISSN 1932-6203
messenger-rna expression - resistance exercise - protein-synthesis - nuclear receptor - metabolism - nur77 - immobilization - cytoscape - recovery - biopsies
Regular physical activity positively influences whole body energy metabolism and substrate handling in exercising muscle. While it is recognized that the effects of exercise extend beyond exercising muscle, it is unclear to what extent exercise impacts non-exercising muscles. Here we investigated the effects of an acute endurance exercise bouts on gene expression in exercising and non-exercising human muscle. To that end, 12 male subjects aged 44–56 performed one hour of one-legged cycling at 50% Wmax. Muscle biopsies were taken from the exercising and non-exercising leg before and immediately after exercise and analyzed by microarray. One-legged cycling raised plasma lactate, free fatty acids, cortisol, noradrenalin, and adrenalin levels. Surprisingly, acute endurance exercise not only caused pronounced gene expression changes in exercising muscle but also in non-exercising muscle. In the exercising leg the three most highly induced genes were all part of the NR4A family. Remarkably, many genes induced in non-exercising muscle were PPAR targets or related to PPAR signalling, including PDK4, ANGPTL4 and SLC22A5. Pathway analysis confirmed this finding. In conclusion, our data indicate that acute endurance exercise elicits pronounced changes in gene expression in non-exercising muscle, which are likely mediated by changes in circulating factors such as free fatty acids. The study points to a major influence of exercise beyond the contracting muscle
Copper-Free Click Biofunctionalization of Silicon Nitride Surfaces via Strain-Promoted Alkyne-Azide Cycloaddition Reactions
Manova, R.K. ; Pujari, S.P. ; Weijers, C.A.G.M. ; Zuilhof, H. ; Beek, T.A. van - \ 2012
Langmuir 28 (2012)23. - ISSN 0743-7463 - p. 8651 - 8663.
self-assembled monolayers - terminated monolayers - organic monolayers - one-step - functionalization - chemistry - films - immobilization - microarrays - dna
Cu-free "click" chemistry is explored on silicon nitride (Si3N4) surfaces as an effective way for oriented immobilization of biomolecules. An omega-unsaturated ester was grafted onto Si3N4 using UV irradiation. Hydrolysis followed by carbodiimide-mediated activation yielded surface-bound active succinimidyl and pentafluorophenyl ester groups. These reactive surfaces were employed for the attachment of bicyclononyne with an amine spacer, which subsequently enabled room temperature strain-promoted azide alkyne cycloaddition (SPAAC). This stepwise approach was characterized by means of static water contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy. The surface-bound SPAAC reaction was studied with both a fluorine-tagged azide and an azide-linked lactose, yielding hydrophobic and bioactive surfaces for which the presence of trace amounts of Cu ions would have been problematic. Additionally, patterning of the Si3N4 surface using this metal-free click reaction with a fluorescent azide is shown. These results demonstrate the ability of the SPAAC as a generic tool for anchoring complex molecules onto a surface under extremely mild, namely ambient and metal-free, conditions in a clean and relatively fast manner.
Nanopatterned submicron pores as a shield for nonspecific binding in surface plasmon resonance-based sensing
Rebe-Raz, S. ; Marchesini, G.R. ; Bremer, M.G.E.G. ; Colpo, P. ; Garcia, C.P. ; Guidetti, G. ; Norde, W. ; Rossi, F. - \ 2012
The Analyst 137 (2012)22. - ISSN 0003-2654 - p. 5251 - 5259.
biosensor immunoassays - protein adsorption - gold surfaces - blood-plasma - milk - immobilization - reduction - matrix - serum - size
We present a novel approach to tackle the most common drawback of using surface plasmon resonance for analyte screening in complex biological matrices – the nonspecific binding to the sensor chip surface. By using a perforated membrane supported by a polymeric gel structure at the evanescent wave penetration depth, we have fabricated a non-fouling sieve above the sensing region. The sieve shields the evanescent wave from nonspecific interactions which interfere with SPR sensing by minimizing the fouled area of the polymeric gel and preventing the translocation of large particles, e.g. micelles or aggregates. The nanopatterned macropores were fabricated by means of colloidal lithography and plasma enhanced chemical vapor deposition of a polyethylene oxide-like film on top of a polymeric gel matrix commonly used in surface plasmon resonance analysis. The sieve was characterized using surface plasmon resonance imaging, contact angle, atomic force microscopy and scanning electron microscopy. The performance of the sieve was studied using an immunoassay for detection of antibiotic residues in full fat milk and porcine serum. The non-fouling membrane presented pores in the 92–138 nm range organized in a hexagonal crystal lattice with a clearance of about 5% of the total surface. Functionally, the membrane with the nanopatterned macropores showed significant improvements in immunoassay robustness and sensitivity in untreated complex samples. The utilization of the sensor built-in sieve for measurements in complex matrices offers reduction in pre-analytical sample preparation steps and thus shortens the total analysis time.
The impact of metal transport processes on bioavailability of free and complex metal ions in methanogenic granular sludge
Bartacek, J. ; Fermoso, F.G. ; Vergeldt, F. ; Gerkema, E. ; Maca, J. ; As, H. van; Lens, P.N.L. - \ 2012
Water Science and Technology 65 (2012)10. - ISSN 0273-1223 - p. 1875 - 1881.
afvalwaterbehandeling - bioreactoren - anaërobe omstandigheden - korrelslib - biologische beschikbaarheid - metalen - toxiciteit - kernspintomografie - waste water treatment - bioreactors - anaerobic conditions - granular sludge - bioavailability - metals - toxicity - magnetic resonance imaging - magnetic-resonance - dynamics - immobilization - biofilm - nickel
Bioavailability of metals in anaerobic granular sludge has been extensively studied, because it can have a major effect on metal limitation and metal toxicity to microorganisms present in the sludge. Bioavailability of metals can be manipulated by bonding to complexing molecules such as ethylenediaminetetraacetate (EDTA) or diethylenetriaminepentaacetate (DTPA). It has been shown that although the stimulating effect of the complexed metal species (e.g. [CoEDTA]2-) is very fast, it is not sustainable when applied to metal-limited continuously operated reactors. The present paper describes transport phenomena taking place inside single methanogenic granules when the granules are exposed to various metal species. This was done using magnetic resonance imaging (MRI). The MRI results were subsequently related to technological observations such as changes in methanogenic activity upon cobalt injection into cobalt-limited up-flow anaerobic sludge blanket (UASB) reactors. It was shown that transport of complexed metal species is fast (minutes to tens of minutes) and complexed metal can therefore quickly reach the entire volume of the granule. Free metal species tend to interact with the granular matrix resulting in slower transport (tens of minutes to hours) but higher final metal concentrations.
Performance of Alcalase formulations in near dry organic media: Effect of enzyme hydration on dipeptide synthesis
Vossenberg, P. ; Beeftink, H.H. ; Nuijens, T. ; Quaedflieg, P.J.L.M. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2012
Journal of Molecular Catalysis. B, Enzymatic 78 (2012). - ISSN 1381-1177 - p. 24 - 31.
industrial protease alcalase - catalyzed peptide-synthesis - controlled water activity - subtilisin carlsberg - precursor dipeptide - support material - solvents - immobilization - transesterification - tripeptide
The use of different Alcalase formulations for protease-catalyzed dipeptide synthesis was investigated by studying the coupling of the carbamoylmethyl ester of N-protected phenylalanine with phenylalanine amide in tetrahydrofuran in the presence of molecular sieves (i.e. under near dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous tetrahydrofuran) of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Repeated use, in the presence of molecular sieves, without intermediate rehydration led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation was only partially reversible by hydration. Alcalase immobilized onto dicalite with glutaraldehyde was found to be the most active in dipeptide synthesis, i.e. the formulation that initially produces the largest amount of product per gram of total formulation per unit of time. Due to its small particle size and its lack of operational stability, this formulation may nevertheless not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads due to its reasonable activity, its seemingly good operational stability, and its size and uniform shape
Surface Functionalization by Strain-Promoted Alkyne-Azide Click Reactions
Manova, R.K. ; Beek, T.A. van; Zuilhof, H. - \ 2011
Angewandte Chemie-International Edition 50 (2011)24. - ISSN 1433-7851 - p. 5428 - 5430.
chemistry - immobilization
Clicks without Cu: There is a growing demand for reproducible site-specific functionalization of surfaces with biomolecules without introduction of unwanted groups or catalysts, as they may interfere with later applications. The title reactions (see picture) could fulfill these requirements, and four recent applications are discussed.
The use of L-lysine decarboxylase as a means to separate amino acids by electrodialysis
Teng, Y. ; Scott, E.L. ; Zeeland, A.N.T. van; Sanders, J.P.M. - \ 2011
Green Chemistry 13 (2011)3. - ISSN 1463-9262 - p. 624 - 630.
nitrogen-containing chemicals - biobased production - enzyme electrode - bulk chemicals - immobilization - alginate - biomass
Amino acids (AA's) are interesting materials as feedstocks for the chemical industry as they contain chemical functionalities similar to conventional petrochemicals. This offers the possibility to circumvent process steps, energy and reagents. AA's can be obtained by the hydrolysis of potentially inexpensive voluminous protein streams derived from biofuel production. However, isolation of the preferred AA is required in order to carry out further transformation into the desired product. Theoretically separation may be achieved using electrodialysis. To increase efficiency, specific modification to a product of industrial interest and removes charged groups of AA's with similar isoelectric points is required. Here, the reaction of L-lysine decarboxylase (LDC) was studied as a means to specifically convert L-lysine (Lys) to 1,5-pentanediamine (PDA) in the presence of L-arginine (Arg) to produce products with different charge thus allowing isolation of products by electrodialysis. Immobilization of LDC in calcium alginate enhanced the operational stability and conversion in mixtures of amino acids was highly specific. At 30 °C the presence of Arg had little effect on the activity of the enzyme although inhibition by the product PDA could be observed. Volumetric productivity was calculated and raw material and transformation costs were estimated for a potential process using a mixture of Arg and Lys.
Patterning of Peptide Nucleic Acids Using Reactive Microcontact Printing
Calabretta, A. ; Wasserberg, D. ; Posthuma-Trumpie, G.A. ; Subramaniam, V. ; Amerongen, A. van; Corradini, R. ; Tedeschi, T. ; Sforza, S. ; Reinhoudt, D.N. ; Marchelli, R. ; Huskens, J. ; Jonkheijm, P. - \ 2011
Langmuir 27 (2011)4. - ISSN 0743-7463 - p. 1536 - 1542.
stereogenic centers - dna biosensors - pna - microarrays - nanoparticle - recognition - immobilization - hybridization - handedness - proteins
PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.