Wanda Guedens

Protein Engineering For Directed Immobilization

Much effort has been put into the optimization of the functional activity of proteins. For biosensors this protein functional optimization will increase the biosensors sensitivity and/or selectivity. However, the strategy chosen for the immobilization of the proteins to the sensor surface might be equally important for the development of sensor surfaces that are optimally biologically active. Several studies published in recent years show that the oriented immobilization of the bioactive molecules improves the sensors properties. In this review, we discuss the state of the art of the different protein immobilization strategies that are commonly used today with a special focus on biosensor applications. These strategies include nonspecific immobilization techniques either by physical adsorption, by covalent coupling, or by specific immobilization via site-specifically introduced tags or bio-orthogonal chemistry. The different tags and bio-orthogonal chemistry available and the techniques to site-specifically introduce these groups in proteins are also discussed.

An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation

In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

Detection of Lung Cancer through Metabolic Changes Measured in Blood Plasma

Introduction: Low‐dose computed tomography, the currently used tool for lung cancer screening, is characterized by a high rate of false‐positive results. Accumulating evidence has shown that cancer cell metabolism differs from that of normal cells. Therefore, this study aims to evaluate whether the metabolic phenotype of blood plasma allows detection of lung cancer. Methods: The proton nuclear magnetic resonance spectrum of plasma is divided into 110 integration regions, representing the metabolic phenotype. These integration regions reflect the relative metabolite concentrations and were used to train a classification model in discriminating between 233 patients with lung cancer and 226 controls. The validity of the model was examined by classifying an independent cohort of 98 patients with lung cancer and 89 controls. Results: The model makes it possible to correctly classify 78% of patients with lung cancer and 92% of controls, with an area under the curve of 0.88. Important moreover is the fact that the model is convincing, which is demonstrated by validation in the independent cohort with a sensitivity of 71%, a specificity of 81%, and an area under the curve of 0.84. Patients with lung cancer have increased glucose and decreased lactate and phospholipid levels. The limited number of patients in the subgroups and their heterogeneous nature do not (yet) enable differentiation between histological subtypes and tumor stages. Conclusions: Metabolic phenotyping of plasma allows detection of lung cancer, even in an early stage. Increased glucose and decreased lactate levels are pointing to an increased gluconeogenesis and are in accordance with recently published findings. Furthermore, decreased phospholipid levels confirm the enhanced membrane synthesis.

Versatile post-polymerization functionalization of poly(p-phenylene vinylene) copolymers containing carboxylic acid substituents: development of a universal method towards functional conjugated copolymers

A novel conjugated copolymer of poly(2-methoxy-5-(3,7-dimethyloctyloxy)-1,4-phenylene vinylene) (MDMO-PPV) and poly(1,4-(2-(5-carboxypentyloxy)-5-methoxyphenylene) vinylene) (CPM-PPV), which can be used as a platform for further post-polymerization functionalization, is presented. This copolymer is soluble in common organic solvents, such as CHCl3, CH2Cl2 and THF. Post-polymerization functionalization of the acid-groups of this copolymer has been successfully performed via an optimized DCC/DMAP-procedure with the desired alcohol. Several examples have been prepared to demonstrate the versatility of the developed functionalization methodology. PPV-copolymers have been prepared with vinyl groups, propynylphenyl groups, methacrylate groups, ATRP initiator groups, dithiocarbamate initiator groups and propargyl groups. The latter group can be utilized to do “click” chemistry, which has been demonstrated with an azide-functionalized reagent. In all cases, analytical data are consistent with a quantitative conversion of the functional groups. It is demonstrated that no substantial degradation of the conjugated system occurs even after consecutive post-polymerization functionalization reactions.

Metabolic phenotyping of human blood plasma: a powerful tool to discriminate between cancer types?

BACKGROUND Accumulating evidence has shown that cancer cell metabolism differs from that of normal cells. However, up to now it is not clear whether different cancer types are characterized by a specific metabolite profile. Therefore, this study aims to evaluate whether the plasma metabolic phenotype allows to discriminate between lung and breast cancer. PATIENTS AND METHODS The proton nuclear magnetic resonance spectrum of plasma is divided into 110 integration regions, representing the metabolic phenotype. These integration regions reflect the relative metabolite concentrations and were used to train a classification model in discriminating between 80 female breast cancer patients and 54 female lung cancer patients, all with an adenocarcinoma. The validity of the model was examined by permutation testing and by classifying an independent validation cohort of 60 female breast cancer patients and 81 male lung cancer patients, all with an adenocarcinoma. RESULTS The model allows to classify 99% of the breast cancer patients and 93% of the lung cancer patients correctly with an area under the curve (AUC) of 0.96 and can be validated in the independent cohort with a sensitivity of 89%, a specificity of 82% and an AUC of 0.94. Decreased levels of sphingomyelin and phosphatidylcholine (phospholipids with choline head group) and phospholipids with short, unsaturated fatty acid chains next to increased levels of phospholipids with long, saturated fatty acid chains seem to indicate that cell membranes of lung tumors are more rigid and less sensitive to lipid peroxidation. The other discriminating metabolites are pointing to a more pronounced response of the body to the Warburg effect for lung cancer. CONCLUSION Metabolic phenotyping of plasma allows to discriminate between lung and breast cancer, indicating that the metabolite profile reflects more than a general cancer marker. CLINICAL TRIAL REGISTRATION NUMBER NCT02362776.

Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation

Site-specific functionalization of nanobodies after introducing bioorthogonal groups offers the possibility to biofunctionalize surfaces with a uniformly oriented layer of nanobodies. In this paper, expressed protein ligation (EPL) was used for site-specific alkynation of the model nanobody NbBcII10. In contrast to EPL constructs, which are typically expressed in the cytoplasm, nanobodies are expressed in the periplasm where its oxidizing environment ensures a correct folding and disulfide bond formation. Different pathways were explored to express the EPL constructs in the periplasm but simultaneously, the effect of cytoplasmic expression on the functionality of NbBcII10 was also evaluated. By using Escherichia coli SHuffle®T7 cells, it was demonstrated that expression of the EPL complex in the cytoplasm was readily established and that site-specifically mono-alkynated nanobodies can be produced with the same binding properties as the non-modified NbBcII10 expressed in the periplasm. In conclusion, this paper shows that periplasmic expression of the EPL complex is quite challenging, but cytoplasmic expression has proven to be a valuable alternative.

Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

Silver(I) complex formation with 1,4-diaminobutane and 1,5-diaminopentane: A potentiometric study in 1.0 m KNO3

Abstract By means of potentiometric pH measurements, the acidity constants of 1,4-diaminobutane and 1,5-diaminopentane have been determined in 1.0 M K N O3 at 25.0°C. Combined pH/pAg data were used at the same temperature in aqueous 1.0 M K N O3 to study the complexation of Ag(I) with the diamines. Protonated and non-protonated m ononuclear and polynuclear complexes were detected. No evidence was found for the existence of hydroxo complexes. The resulting overall stability constants have been compared with previously determined stability constants for Ag(I)-ethylenediamine and Ag(I)-l,3-propylenediamine complexes. Some relationships concerning the length of the CH2 backbone have been identified.

High Electronic Conductance through Double-Helix DNA Molecules with Fullerene Anchoring Groups

Determining the mechanism of charge transport through native DNA remains a challenge as different factors such as measuring conditions, molecule conformations, and choice of technique can significantly affect the final results. In this contribution, we have used a new approach to measure current flowing through isolated double-stranded DNA molecules, using fullerene groups to anchor the DNA to a gold substrate. Measurements were performed at room temperature in an inert environment using a conductive AFM technique. It is shown that the π-stacked B-DNA structure is conserved on depositing the DNA. As a result, currents in the nanoampere range were obtained for voltages ranging between ±1 V. These experimental results are supported by a theoretical model that suggests that a multistep hopping mechanism between delocalized domains is responsible for the long-range current flow through this specific type of DNA.

Identification of metabolic phenotypes in childhood obesity by 1H NMR metabolomics of blood plasma

Aim: To identify the plasma metabolic profile associated with childhood obesity and its metabolic phenotypes. Materials & methods: The plasma metabolic profile of 65 obese and 37 normal-weight children was obtained using proton NMR spectroscopy. NMR spectra were rationally divided into 110 integration regions, which reflect relative metabolite concentrations, and were used as statistical variables. Results: Obese children show increased levels of lipids, N-acetyl glycoproteins, and lactate, and decreased levels of several amino acids, α-ketoglutarate, glucose, citrate, and cholinated phospholipids as compared with normal-weight children. Metabolically healthy children show lower levels of lipids and lactate, and higher levels of several amino acids and cholinated phospholipids, as compared with unhealthy children. Conclusion: This study reveals new valuable findings in the field of metabolomics and childhood obesity. Although validation should be performed, the proof of principle looks promising and justifies a deeper investigation of the diagnostic possibilities of proton NMR metabolomics in follow-up studies. Trial registration: NCT03014856. Registered January 9, 2017.