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Aptamer based strategies for ultrasensitive protein detection
In recent years, the concept of personalized medicine is gaining increased interest. It is envisioned that it soon might be possible to accurately assess medical risk -even before the onset of disease- and to monitor, diagnose and treat patients more effectively based on their individual genomes. Currently however, the vastly increased availability of genetic information is of only limited value to the individual, as the number of known genetic markers providing useful predictive information is extremely limited. Furthermore, the genome is highly complex and understanding it in full is daunting. Indeed, many complex yet common diseases, such as e.g. diabetes and virtually all types of cancer likely involve alarge number of different genes and biological pathways, as well as environmental factors. Finally, even if useful genetic markers are found, it will still be beneficial to better monitor the actual onset of diseasesuch that drastic or costly prophylactic procedures might be postponed as long as possible, without compromising long-term patient survival. Therefore, the importance of detailed molecular information, such as can be provided by proteomic analysis, will remain an invaluable complement to genomic information.
In this view, the monitoring of proteins in the largest reservoir of molecular information in the human body, blood, will be essential. As it circulates the body, the blood comes into contact with all tissues and thus contains the much sought after information that will cater for better, more sensitive and accurate diagnostics. Currently, obtaining large-scale proteomic information from clinical samplesis far from straightforward. Whereas modern genomic detection platformsare applied routinely thanks to the inherent, highly specific, base pairing interactions that exist between complementary oligonucleotides, analogous platforms for protein detection are quickly stunted by the complex biophysics of protein-protein interactions. In this respect, nucleic acid aptamers are small, highly stable oligonucleotides that, through their unique structure, have the ability to recognize biomolecular targets,just like antibodies. Nonetheless they are easier to produce, with far greater reproducibility.
The research presented in this dissertation has combined capillary electrophoresis separation, polymerase chain reaction amplification and surface plasmon resonance with the aim of developing novel assays for the detection of trace amounts of proteins with high sensitivity and specificity. This was made possible thanks to the unique target recognition abilities of DNA aptamers, a unique class ofoligonucleotide affinity binders that allowed powerful signal amplification strategies to be introduced to the field of CE and SPR based protein detection.
To this end, traditional on-column detection in aptamer based affinity probe capillary electrophoresis was replaced by quantitative PCR amplification of aptamers. For this purpose, established models describing oligonucleotide melting behavior were used to design primersequences that could subsequently be used for the amplification of aptamers with high fidelity and sensitivity. The principle was demonstrated forthe detection of highly diluted samples of human immunoglobulin E protein whereby it could be shown that proper modification of the aptamer canlead to a lowering of the detection limit by at least three orders of magnitude. The qPCR assisted affinity probe capillary electrophoresis wassubsequently developed further to enable multiplexed protein detection whilst still maintaining the ultra-low limit of detection also achieved by the single analyte assay. Here, the rational design of the aptamer primer regions was extended to allow for the individual quantification of analytes using affinity probe capillary electrophoresis without the strict necessity to fully resolve all complexes during electrophoresis. The ability to multiplex the assay is demonstrated through the simultaneous detection of three relevant protein analytes, human α-thrombin, human immunoglobulin E and HIV reverse transcriptase 1 at picomolar levels.
The target multiplexing potential of aptamer affinity probe capillary elec- trophoresis was expanded even further thanks to the massiveparallelization capabilities of next-generation sequencing. This way, the aptamer sequences themselves were used as a barcode for their targets. It was shown how aptamers could be modified to make them compatible with a next-generation sequencing workflow. The identification and quantification of individual ap- tamers in a complex mixture was demonstratedand next-generation sequencing was used as a detector in an aptamer affinity probe capillary electrophoresis assay, to allow the successful detection of human immunoglobulin E in a clinical serum sample. This proof-of-concept work has shown how affinity capillary electrophoresis still remains an excellent technology for the isolation of aptamer-target binding events from the experimental background whereas next-generation sequencing enhances the specificity and potential scale of the identificationof target bound aptamers through direct quantitative sequencing.
In the second part of this dissertation, fiber-optic SPR based strategies for protein detection were developed. New strategies were evaluated that use deoxyribonucleic acid as a mediator in the detection process in an attempt to use the highly specific nature of oligonucleotide base pairingand the availability of enzymatic methods for DNA amplification to ultimately enable both more specific- and more sensitive SPR assays for protein detection.
First, it was shown how fiber-optic SPR systems can beemployed for the sequence-specific detection of short DNA oligonucleotides even in the presence of fouling compounds that would otherwise interfere with SPR sensing. The viability of the approach is presented through the application of fiber-optic SPR in the serotyping of the bacterium Legionella pneumophila. The results are particularly interesting since they allow for the sequence specific discrimination of different aptamer sequences, which will ultimately contribute to enable multiplexed fiber-optic SPR based aptamer assays.
The previously presented DNA hybridization assay was extended to not only allow sequence specific detection of short DNA sequences but further boost the limits of detection of the fiber-optic SPR sensor by showing how it can be used for the real-time detection of a heterogeneous polymerase chain reaction amplification reaction of a DNA aptamer. This achievement is a significant step towards fiber-optic SPR based ultrasensitive protein detection using aptamers, withlimits of detection well below what is currently considered state-of-the-art. This achievement is a first in SPR based biodetection.
In this view, the monitoring of proteins in the largest reservoir of molecular information in the human body, blood, will be essential. As it circulates the body, the blood comes into contact with all tissues and thus contains the much sought after information that will cater for better, more sensitive and accurate diagnostics. Currently, obtaining large-scale proteomic information from clinical samplesis far from straightforward. Whereas modern genomic detection platformsare applied routinely thanks to the inherent, highly specific, base pairing interactions that exist between complementary oligonucleotides, analogous platforms for protein detection are quickly stunted by the complex biophysics of protein-protein interactions. In this respect, nucleic acid aptamers are small, highly stable oligonucleotides that, through their unique structure, have the ability to recognize biomolecular targets,just like antibodies. Nonetheless they are easier to produce, with far greater reproducibility.
The research presented in this dissertation has combined capillary electrophoresis separation, polymerase chain reaction amplification and surface plasmon resonance with the aim of developing novel assays for the detection of trace amounts of proteins with high sensitivity and specificity. This was made possible thanks to the unique target recognition abilities of DNA aptamers, a unique class ofoligonucleotide affinity binders that allowed powerful signal amplification strategies to be introduced to the field of CE and SPR based protein detection.
To this end, traditional on-column detection in aptamer based affinity probe capillary electrophoresis was replaced by quantitative PCR amplification of aptamers. For this purpose, established models describing oligonucleotide melting behavior were used to design primersequences that could subsequently be used for the amplification of aptamers with high fidelity and sensitivity. The principle was demonstrated forthe detection of highly diluted samples of human immunoglobulin E protein whereby it could be shown that proper modification of the aptamer canlead to a lowering of the detection limit by at least three orders of magnitude. The qPCR assisted affinity probe capillary electrophoresis wassubsequently developed further to enable multiplexed protein detection whilst still maintaining the ultra-low limit of detection also achieved by the single analyte assay. Here, the rational design of the aptamer primer regions was extended to allow for the individual quantification of analytes using affinity probe capillary electrophoresis without the strict necessity to fully resolve all complexes during electrophoresis. The ability to multiplex the assay is demonstrated through the simultaneous detection of three relevant protein analytes, human α-thrombin, human immunoglobulin E and HIV reverse transcriptase 1 at picomolar levels.
The target multiplexing potential of aptamer affinity probe capillary elec- trophoresis was expanded even further thanks to the massiveparallelization capabilities of next-generation sequencing. This way, the aptamer sequences themselves were used as a barcode for their targets. It was shown how aptamers could be modified to make them compatible with a next-generation sequencing workflow. The identification and quantification of individual ap- tamers in a complex mixture was demonstratedand next-generation sequencing was used as a detector in an aptamer affinity probe capillary electrophoresis assay, to allow the successful detection of human immunoglobulin E in a clinical serum sample. This proof-of-concept work has shown how affinity capillary electrophoresis still remains an excellent technology for the isolation of aptamer-target binding events from the experimental background whereas next-generation sequencing enhances the specificity and potential scale of the identificationof target bound aptamers through direct quantitative sequencing.
In the second part of this dissertation, fiber-optic SPR based strategies for protein detection were developed. New strategies were evaluated that use deoxyribonucleic acid as a mediator in the detection process in an attempt to use the highly specific nature of oligonucleotide base pairingand the availability of enzymatic methods for DNA amplification to ultimately enable both more specific- and more sensitive SPR assays for protein detection.
First, it was shown how fiber-optic SPR systems can beemployed for the sequence-specific detection of short DNA oligonucleotides even in the presence of fouling compounds that would otherwise interfere with SPR sensing. The viability of the approach is presented through the application of fiber-optic SPR in the serotyping of the bacterium Legionella pneumophila. The results are particularly interesting since they allow for the sequence specific discrimination of different aptamer sequences, which will ultimately contribute to enable multiplexed fiber-optic SPR based aptamer assays.
The previously presented DNA hybridization assay was extended to not only allow sequence specific detection of short DNA sequences but further boost the limits of detection of the fiber-optic SPR sensor by showing how it can be used for the real-time detection of a heterogeneous polymerase chain reaction amplification reaction of a DNA aptamer. This achievement is a significant step towards fiber-optic SPR based ultrasensitive protein detection using aptamers, withlimits of detection well below what is currently considered state-of-the-art. This achievement is a first in SPR based biodetection.
Date:3 Mar 2008 → 6 Mar 2013
Keywords:BMP-2 Biosensors
Disciplines:Inorganic chemistry, Organic chemistry, Theoretical and computational chemistry, Other chemical sciences
Project type:PhD project