Veronique Vermeeren

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.

Heat-Transfer Resistance at Solid–Liquid Interfaces: A Tool for the Detection of Single-Nucleotide Polymorphisms in DNA

In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low- to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA.

Structural and optical properties of DNA layers covalently attached to diamond surfaces

Label-free detection of DNA molecules on chemically vapor-deposited diamond surfaces is achieved with spectroscopic ellipsometry in the infrared and vacuum ultraviolet range. This nondestructive method has the potential to yield information on the average orientation of single as well as double-stranded DNA molecules, without restricting the strand length to the persistence length. The orientational analysis based on electronic excitations in combination with information from layer thicknesses provides a deeper understanding of biological layers on diamond. The pi-pi* transition dipole moments, corresponding to a transition at 4.74 eV, originate from the individual bases. They are in a plane perpendicular to the DNA backbone with an associated n-pi* transition at 4.47 eV. For 8-36 bases of single- and double-stranded DNA covalently attached to ultra-nanocrystalline diamond, the ratio between in- and out-of-plane components in the best fit simulations to the ellipsometric spectra yields an average tilt angle of the DNA backbone with respect to the surface plane ranging from 45 degrees to 52 degrees . We comment on the physical meaning of the calculated tilt angles. Additional information is gathered from atomic force microscopy, fluorescence imaging, and wetting experiments. The results reported here are of value in understanding and optimizing the performance of the electronic readout of a diamond-based label-free DNA hybridization sensor.

Topographical and Functional Characterization of the ssDNA Probe Layer Generated Through EDC-Mediated Covalent Attachment to Nanocrystalline Diamond Using Fluorescence Microscopy

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

DNA Sensors with Diamond as a Promising Alternative Transducer Material

Bio-electronics is a scientific field coupling the achievements in biology with electronics to obtain higher sensitivity, specificity and speed. Biosensors have played a pivotal role, and many have become established in the clinical and scientific world. They need to be sensitive, specific, fast and cheap. Electrochemical biosensors are most frequently cited in literature, often in the context of DNA sensing and mutation analysis. However, many popular electrochemical transduction materials, such as silicon, are susceptible to hydrolysis, leading to loss of bioreceptor molecules from the surface. Hence, increased attention has been shifted towards diamond, which surpasses silicon on many levels.

Aptamers targeting different functional groups of 17β-estradiol

Aptamers, short synthetic ssDNA or RNA molecules with a specific three-dimensional structure, are promising recognition elements in biosensor technology. In vitro generation of aptamers with high sensitivity and specificity toward a broad range of analytes has been achieved using the systematic evolution of ligands by exponential enrichment (SELEX) process. This iterative pathway of aptamer generation consists of sequential positive and counterselection steps. The present research aimed to select two sets of ssDNA aptamers which both are able to bind to different functional groups on the cyclopentanoperhydrophenanthrene ring of 17β-estradiol (E2). By repetitively switching between positive selection steps using E2 as target molecule and counterselection steps with nortestosterone as countermolecule, aptamers were successfully selected against the hydroxylated aromatic A ring of E2. Additionally, an aptamer which binds the upper segments of the B, C and D ring of the cyclopentanoperhydrophenanthrene ring of E2 was generated after repetitively swapping between positive selection steps with E2 as target molecule and counterselection steps with dexamethasone as countermolecule. Epitope specificity of the aptamers was demonstrated by evaluating their binding responses toward a number of steroid hormones structurally related to E2. The selected aptamers with affinities for different functional groups of E2 can potentially be applied to develop a cross-reactive aptasensor. This aptasensor introduces a promising tool for the future of in-field real-time monitoring of a wide range of steroid hormones.

Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

Conventional neonatal diagnosis of phenylketonuria is based on the presence of abnormal levels of phenylalanine in the blood. However, for carrier detection and prenatal diagnosis, direct detection of disease-correlated mutations is needed. To speed up and simplify mutation screening in genes, new technologies are developed. In this study, a heat-transfer method is evaluated as a mutation-detection technology in entire exons of the phenylalanine hydroxylase (PAH) gene. This method is based on the change in heat-transfer resistance (Rth) upon thermal denaturation of dsDNA (double-stranded DNA) on nanocrystalline diamond. First, ssDNA (single-stranded DNA) fragments that span the size range of the PAH exons were successfully immobilized on nanocrystalline diamond. Next, it was studied whether an Rth change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear Rth shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, Rth was shown to detect prevalent single-nucleotide polymorphisms, c.473G>A (R158Q), c.932T>C (p.L311P), and c.1222C>T (R408W), correlated with phenylketonuria, displaying an effect related to the different melting temperatures of homoduplexes and heteroduplexes.

Construction of helper plasmid-mediated dual-display phage for autoantibody screening in serum

M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA.21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown.

Evolution towards the implementation of point-of-care biosensors

Health care, food quality control, and environmental management often rely on the detection of abnormal molecules, in the body, in food, and in the environment, respectively. More and more, these fields are moving towards point-of-care detection, since increased analysis speed, and hence, decreased cost, are becoming important determining factors for funding. In parallel, but also to allow for this speedy and on-site analysis, the scientific world has evolved into the ‘nano’-scale. This was made possible by the dawn of bio-electronics: a scientific field coupling the achievements in molecular biology with the advances in electronics. The goal of this field is to interrogate the functional activity of bioreceptor molecules, i.e. the recognition and/or metabolization of their targets, with electronics to increase the detection speed for, and sensitivity to, certain pathogens, pollutants, and genetic mutations. To enable the electronic interrogation of these bioreceptor molecules, they need to be attached to, or embedded into, a solid support or transducer with a favourable orientation and density, ensuring the retention of their biological functionality. The resulting analytical device is called a biosensor. The transducer ‘translates’ the biological recognition event between the receptor molecule and its target into a readable signal. Many biosensors have become established in the clinical and scientific world. However, still few of them have made it to point-of-care applications. The success and applicability of biosensors as point-of-care tools is based on five requirements. They need to be sensitive, specific, fast, cheap, and portable. Electronic biosensors based on (semi-)conductive transducers are hence preferred for point-of-care applications, since they are fast in signal generation and cheap to produce. Many popular semiconductive transduction materials, such as silicon (Si) and germanium (Ge), however, are susceptible to hydrolysis, leading to a loss of bioreceptor molecules from the surface, and hence, to instability of the sensor platform. This negatively influences the sensitivity and specificity of the sensor. This explains our increased attention towards diamond, which surpasses Si and Ge on many levels. It can be made into a semiconductor, preferred for electronic applications, it is chemically and mechanically very stable, it can be functionalized with bioreceptor molecules (DNA, aptamers, antibodies, whole cells), and it is biocompatible since it is only composed of carbon (C).

Head-On Immobilization of DNA Fragments on CVD-Diamond Layers

Synthetic diamond is regarded as a promising material for biosensors: it forms a stable platform for genetic assays and its biocompatibility opens the possibility for in vivo sensing. In this study the use of a thymidine linker for covalent DNA attachment was evaluated. Contact angle measurements provided a qualitative test of the initially oxidized surface. X-ray photoemission spectroscopy was used for further analysis of the oxides and for monitoring the effect of subsequent chemical treatments. The presence of FITC-labelled DNA was confirmed by confocal fluorescence microscopy. Enzyme linked immunosorbent assays indicated that this DNA was merely adsorbed on the diamond surface instead of covalently bound.