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Avidin Nucleic Acid Nano ASsembly (ANANAS) as a tool to enhance the performance of competitive ELISA detection of low mol weight analytes
Margherita Morpurgo, Assistant Professor, University of Padova
The Avidin-Nucleic Acids Nano-Assembly (ANANAS) is a supra-molecular poly-avidin (400 avidins/particle) nanoparticle obtained upon a nature-driven self-assembly process1. The ANANAS (Ø = 100 nm) can overcome the maximum 4-ligand limit of classic avidin biotin technology. Recently, this system was shown to be capable of improving by 2-3 orders of magnitude the sensitivity of ELISA detection of high MW compounds in sandwich formats2.
In this work we evaluated the use of ANANAS for the colorimetric immunoenzymatic detection of low MW analytes in competitive configuration. To this end, we used the 2'(4-hydroxyazo- benzene)benzoic acid (HABA) as a model for low MW analytes. Rabbit polyclonal anti-HABA antibodies and a HABA-BSA conjugate were developed and used for competitive assay development. The sensitivity of the ANANAS-based detection was compared to that generated by using a commercial “monomeric” HRP-avidin conjugate.
Our preliminary results show that the analytical sensitivity increases by about 10-fold when using the ANANAS, allowing to reach a detection limit in the range of 50 ppb. This sensitivity may be good enough for many applications and could be further improved by using monoclonal antibodies. This tool can also be integrated within technological platforms others than ELISA (SPR, microbalance etcc.) to improve their own performance.
Detection of estrogenic substances via aptamers
Katrijn Vanschoenbeek, Student, Hasselt University
Different chemical substances in environmental samples interfere with the endocrine system by binding to the estrogen receptor, potentially leading to adverse health effects. An aptasensor offers a valuable tool for the direct detection of these substances.
17ß-estradiol is used as a proof-of-principle target molecule. Initially, a SELEX-procedure was carried out. After 5 selection rounds, 19 candidate aptamer clones were sequenced. A search for sequence-match showed a recurrent motif of 8 nucleotides in 9 clones. Via Mfold, the secondary structures of these motif containing sequences were predicted and revealed similar three-way junction structures. The 9 sequences were further analyzed via ELISA whereby binding of the aptamer to its target was visualized via qPCR. These bindingstudies showed that the aptamers not only bind 17ß-estradiol but also the structurally related nortestestosteron, whereas they do not bind the structurally related dexamethasone. This offers more information about the specific chemical components of 17ß-estradiol which are necessary for aptamer binding.
In a next phase, binding kinetics of the selected aptamers will be investigated via SPR and aptamers against other estrogenic substances will be selected.
The different aptamers will eventually be integrated as receptor molecules in a biosensing device for the in field detection of estrogen-like substances.
The development of an all-round sensor platform for biomarker detection in medical diagnostics
Natalie Bon, Student, Hasselt University
The goal of this project is to develop an all-round sensor for biomarker detection in medical diagnostics. Within this stage of the project the focus is on the detection of C-reactive protein (CRP), because this is one of the proteins that are tested to diagnose cardiovascular diseases (CVD) in human. In a previous study, a diamond-based immunosensor, using impedance as read-out, was developed for label-free and real-time CRP detection. This sensor attained a clinically relevant sensitivity in buffer.
To reach physiologically relevant sensitivity in patient samples, the signal-to-noise-ratio of our system is being improved.
First both receptor molecules and surface blockers are covalently coupled to the sensor surface, because this results in a more stable impedance signal.
Second, the best, in house produced anti-CRP antibodies will be used as receptor molecules, they are currently being selected.
Finally the CRP detection signal will be enhanced by using magnetic nanoparticles.
In conclusion the optimized sensor will increase the capability to discriminate between healthy controls and patients at risk for CVD within point-of-care compatible limits.
Using an impedimetric, real-time, and label-free DNA-sensor based on nanocrystalline diamond to detect SNPs
Veronique Vermeeren, , Hasselt University
Recently, attention is being focused on the development of miniaturized, automated molecular techniques to improve the speed of DNA analysis. Label-free detection techniques based on electronic read-out principles, such as Electrochemical Impedance Spectroscopy (EIS), are being favoured, because they are cheap, fast, real-time, and they don’t require target labelling.
Our goal was to develop a DNA-sensor based on nanocrystalline diamond (NCD) combined with EIS, allowing real-time mutation detection. NCD allows covalent immobilization of ssDNA, and is a stable semiconductor [1].
Distinction between DNA with and without a single nucleotide polymorphism (SNP) was investigated during the denaturation of a perfect DNA duplex and of 2 duplexes, each with a SNP at a different position. The SNPs are mutations leading to Phenylketonuria (PKU).
We could reproducibly identify the denaturation time constants for the above mentioned DNA duplexes. All time constants are in the minute scale, with the denaturation time constant for the DNA duplex without a SNP being longer than that of the DNA duplexes with a SNP. The denaturation time constants correlate well with the calculated melting temperatures of the corresponding duplexes. This was confirmed with thermal denaturation.
Thus, our approach can be used for a fast mutation identification [2].
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