Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring.

Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

Aptamers targeting different functional groups of 17ß-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 17ß-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 (R(th)) 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 R(th) change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear R(th) shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, R(th) 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.

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.

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.

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.

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.