Karel Knez

Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices

The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.

Real-time monitoring of DNA hybridization and melting processes using a fiber optic sensor

In this paper a fiber optic surface plasmon resonance (FO-SPR) sensor was used to analyze the melting process of DNA linked to silica nanoparticles. Real-time monitoring of a DNA melting process has rarely been studied using surface plasmon resonance (SPR), since most commercial SPR setups do not allow for dynamic and accurate temperature control above 50 °C. The FO-SPR sensor platform, with silica nanobead signal amplification, allows sensing inside a standard PCR thermocycler, which makes high resolution DNA melting curve analysis possible. This innovative combination was used to characterize the hybridization and melting events between DNA immobilized on the sensor surface and DNA probes on silica nanoparticles. At optimized hybridization conditions complementary DNA strands of different lengths could be distinguished. While the real-time FO-SPR analysis of DNA hybridization did not result in significant variances, the analysis of DNA melting determined the exact length of overlap and the matching Gibbs energy.

Nucleic Acids for Ultra-Sensitive Protein Detection

Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of “personalized medicine”. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given.

Spherical nucleic acid enhanced FO-SPR DNA melting for detection of mutations in Legionella pneumophila.

A home-built fiber optic surface plasmon resonance platform (FO-SPR) was applied to directly screen PCR amplified DNA for mutations. The FO-SPR sensor was used for real-time monitoring of DNA duplex melting during high resolution temperature cycling. The signal of the DNA melting was enhanced by means of gold nanoparticle labels. This FO-SPR genetic assay allowed for detection of single-point mutations (SNP) in less than 20 min. The concept was demonstrated for the analysis of 9 different serogroups of the bacterium Legionella pneumophila, a common human pathogen responsible for atypical pneumonia. FO-SPR allowed us to detect genetic mutations inhibiting PCR, which could lead to amplification bias when molecular diagnostics are applied for L. pneumophila detection. All serogroups were found to display unique melting temperatures, indicating that mutations have accumulated in the target sequence. In a next step, clinical samples of L. pneumophila were analyzed using the FO-SPR sensor. This technology was proven to be reliable for the detection of mutations for those samples that previously displayed ambiguous qPCR quantification results. When these results were benchmarked, FO-SPR results were found to be consistent with Sanger sequencing but not with fluorescence based DNA melting. The presented results convincingly advocate the advantages of FO-SPR as a high resolution and fast genetic screening tool that can compete with the current standard techniques for SNP detection.

Real-Time Monitoring of Solid-Phase PCR Using Fiber-Optic SPR

8 ] More recently, PCR related research has advanced to include solid-phase PCR (SP-PCR) because it holds sig-nifi cant promise for applications in high-throughput DNA sequencing and large-scale single-nucleotide polymorphism analysis, circumventing some of the short-comings of con-ventional PCR in these applications. In spite of its poten-tial, SP-PCR is often hampered by relatively low yields of amplicon and subsequent low effi ciencies that are seen in a typical reaction.

Affinity Comparison of p3 and p8 Peptide Displaying Bacteriophages Using Surface Plasmon Resonance

Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of koff and kon values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (Kd) in the femtomolar range.

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

A simple double-bead sandwich assay for protein detection in serum using UV–vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)(-1) and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.

Multiplexed protein detection using an affinity aptamer amplification assay

AbstractAffinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer–target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time. FigureMultiplexed affinity probe capillary electrophoresis (APCE) is enhanced through the use of PCR affinity aptamer amplification, improving both the limits of detection as well as the resolution of a typical APCE assay.

Assay design considerations for use of affinity aptamer amplification in ultra-sensitive protein assays using capillary electrophoresis

Affinity probe capillary electrophoresis (APCE) holds promise to be a powerful tool in diagnostic applications that rely on the selective and sensitive detection of proteins. Aptamers can serve to unlock the full potential of the technology since their use opens up possibilities to unite the immuno-sensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification. To enable routine use of aptamer APCE, careful selection of the experimental conditions is crucial. Here it is shown how customization and careful consideration of the experimental parameters can significantly improve assay performance.