Advanced microarray technologies for clinical diagnostics

Anke Pierik

Research output: ThesisPhd Thesis 2 (Research NOT TU/e / Graduation TU/e)

268 Downloads (Pure)

Abstract

DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains multiple copies of one oligonucleotide, called the capture probe, which has a sequence complementary to one single specific target molecule. A DNA microarray assay is based on the preferential binding between a DNA target molecule with its complementary probe present on the microarray surface. Provided appropriate conditions, these two single stranded DNA molecules with complementary DNA sequences form a double stranded molecule during a process called hybridization. When the target molecules are labeled with e.g. a fluorophore, the fluorescent microarray pattern achieved upon incubation of the sample with the microarray provides information on the presence of the target sequences in the sample. The key advantage of microarrays is their large degree of parallelism per sample due to the simultaneous reaction between multiple capture probes and target sequences. This thesis focuses on low density DNA arrays that are limited to a few hundred spots for diagnostic use, for the rapid detection of a constrained and specific set of target sequences. One major application of low density microarrays lies in the detection and identification of pathogens in the field of infectious diseases. So, high density arrays containing as high as millions of different spots that allows for the screening and detection of a much higher number of targets simultaneously, usually applied for studying gene expression studies, are beyond the scope of this thesis. The transfer of microarray-based assays from the research lab into the clinic has taken more time than anticipated and it faces a number of technical and regulatory challenges. These are especially related to quality control and data reproducibility caused by a lack of fundamental understanding of the biochemical processes involved. In this thesis, a number of these challenges are addressed and investigated, and potential routes for improvement are identified. This work focuses on the improvement of microarray technology in order to enable a faster implementation of microarray-based assays into clinical practice. Inkjet printing is a suitable technology for manufacturing low density microarrays. It is, however, an open-loop process and does therefore not provide any feedback on the quality of manufactured microarrays. E.g. missing spots are unacceptable since this could lead to potentially false negative results. Moreover, microarrays are generally being considered as high complexity products by the Clinical Laboratory Improvement Amendments (CLIA), or as high risk applications by the Food and Drug Administration (FDA). Therefore specific quality control measures on microarrays for clinical diagnostics are needed. In chapter 2, a closed-loop inkjet printing system equipped with an optical droplet detection system to investigate failure mechanisms of the printing process is described. It was found that of all microarrays analyzed, in 1.6 % jetting failed at some point in time. In 14 % of these cases, these failures could have been detected in advance by detection of changing droplet velocities during jetting. Real time analysis of droplet characteristics and closed-loop control can enable a higher yield of the microarray manufacturing process. The improved production process as explained in chapter 2 is the basis for the microarray experiments carried out for the results in experimental chapters 3-5. Many different surface chemistries exist that are applied to graft the DNA probes onto the microarray surface. A lack of understanding on the characteristics of DNA immobilization and hybridization as separate processes hampers the improvement in reproducibility of the use of microarrays. Immobilization and hybridization rates of oligonucleotides equipped with specific tails on amine-functionalized surfaces by using 254 nm UV-light are studied. This surface chemistry is attractive because of its ease-of-use, robustness and low cost. A method was developed that enables a systematic study of the immobilization and subsequent hybridization processes independently. It was found that immobilization efficiencies are greatly influenced both by the UV dose applied and by the composition, as well as the length of the specific tails attached to the oligonucleotides. Hybridization is mainly influenced by the length of the oligonucleotides, and much less by the composition of the tail as observed for immobilization. This means that within the UV-window as applied in this study, no significant UV-damage was found. The standard microarray procedure runs as follows: microarrays are incubated with the sample, followed by a washing step to remove non-specific binding, then dried and scanned. This process flow implies that only a single measurement (end) point is used to evaluate target and capture molecule interactions. Since these interactions depend on many factors, including the preceding steps and the composition of the mixture itself, a complete hybridization measurement would provide additional and more reliable data. This means, however, that measurements should be done in high background signals requiring the deployment of methods that enable background suppression. Chapter 4 describes a method that measures real time hybridization and melting curves based on the repeated flow of the solution containing the targets through a porous microarray substrate. During each hybridization cycle, background was suppressed by pumping the liquid through the substrate. This method, using the Human Papilloma Virus (HPV) assay as a model assay, was evaluated with clinical samples and benchmarked against a method commonly used in the clinic (Reverse Line Blot). Both methods show comparable results, while the flow-through method also enabled the identification of cross-hybridizations by the analysis of the binding and melting kinetics. This method can contribute to identification of false positive signals. The Polymerase Chain Reaction (PCR) is a method to amplify the concentration of specific DNA target sequences and is often performed prior to a microarray hybridization. Sample preparation variations including this amplification step contribute to a lower reproducibility of microarrays. In the real time array PCR concept the steps of real time amplification (qPCR) and detection are integrated, thereby combining the advantages of both methods: the high multiplexing capabilities of microarray based assays and the quantitative characteristics of real time PCR. In this procedure the hybridization of the formed amplicons on the microarray surface is monitored, during each annealing step of the amplification process. Consequently an amplification curve on the microarray surface is obtained from which quantitative information on the target input concentration can be derived for a much higher number of targets compared to qPCR. After the amplification, an additional hybridization and melting curve step can be performed to further assess the specificity. The detection of low signals within high background levels in relatively short times is the greatest technical challenge of this concept. This was achieved by the use of a confocal fluorescence scanner that significantly reduced background levels. A proof of concept study was carried out with a prototype instrument that was designed and built from standard components known from the optical storage technology. In order to minimize costs of goods, standard products (reagents and disposables) were also used for the biochemical processes of amplification and hybridization as much as possible. Amplification and detection were performed in a single and closed chamber, minimizing risks on cross-contamination and reducing manual steps, making this method especially suited for diagnostic testing. As low as 10 copies of a target sequence could be detected using this method. Chapter 6 contains a mathematical analysis of the real time array PCR process which was carried out to provide insights in the biochemical processes taking place. Models of qPCR and Langmuir adsorption were combined into a new model describing the different steps in the real time array protocol. Reasonably good agreement between the model and the experiments was found. Using this result for the experimental array-based assay, a number of measures have been identified and tested to increase the overall amplification efficiency which resulted in a similar performance compared to bulk qPCR.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Department of Chemical Engineering and Chemistry
Supervisors/Advisors
  • Broer, Dirk J., Promotor
Award date19 Dec 2011
Place of PublicationEindhoven
Publisher
Print ISBNs978-94-6191-088-2
DOIs
Publication statusPublished - 2011

Fingerprint

Microarrays
Amplification
Assays
Polymerase chain reaction
DNA
Oligonucleotides
Molecules
Printing
Melting
Surface chemistry
Quality control
Substrates
Chemical analysis

Cite this

Pierik, A. (2011). Advanced microarray technologies for clinical diagnostics. Eindhoven: Technische Universiteit Eindhoven. https://doi.org/10.6100/IR724484
Pierik, Anke. / Advanced microarray technologies for clinical diagnostics. Eindhoven : Technische Universiteit Eindhoven, 2011. 168 p.
@phdthesis{b4acfb1e81da416eb9830db985f1abb0,
title = "Advanced microarray technologies for clinical diagnostics",
abstract = "DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains multiple copies of one oligonucleotide, called the capture probe, which has a sequence complementary to one single specific target molecule. A DNA microarray assay is based on the preferential binding between a DNA target molecule with its complementary probe present on the microarray surface. Provided appropriate conditions, these two single stranded DNA molecules with complementary DNA sequences form a double stranded molecule during a process called hybridization. When the target molecules are labeled with e.g. a fluorophore, the fluorescent microarray pattern achieved upon incubation of the sample with the microarray provides information on the presence of the target sequences in the sample. The key advantage of microarrays is their large degree of parallelism per sample due to the simultaneous reaction between multiple capture probes and target sequences. This thesis focuses on low density DNA arrays that are limited to a few hundred spots for diagnostic use, for the rapid detection of a constrained and specific set of target sequences. One major application of low density microarrays lies in the detection and identification of pathogens in the field of infectious diseases. So, high density arrays containing as high as millions of different spots that allows for the screening and detection of a much higher number of targets simultaneously, usually applied for studying gene expression studies, are beyond the scope of this thesis. The transfer of microarray-based assays from the research lab into the clinic has taken more time than anticipated and it faces a number of technical and regulatory challenges. These are especially related to quality control and data reproducibility caused by a lack of fundamental understanding of the biochemical processes involved. In this thesis, a number of these challenges are addressed and investigated, and potential routes for improvement are identified. This work focuses on the improvement of microarray technology in order to enable a faster implementation of microarray-based assays into clinical practice. Inkjet printing is a suitable technology for manufacturing low density microarrays. It is, however, an open-loop process and does therefore not provide any feedback on the quality of manufactured microarrays. E.g. missing spots are unacceptable since this could lead to potentially false negative results. Moreover, microarrays are generally being considered as high complexity products by the Clinical Laboratory Improvement Amendments (CLIA), or as high risk applications by the Food and Drug Administration (FDA). Therefore specific quality control measures on microarrays for clinical diagnostics are needed. In chapter 2, a closed-loop inkjet printing system equipped with an optical droplet detection system to investigate failure mechanisms of the printing process is described. It was found that of all microarrays analyzed, in 1.6 {\%} jetting failed at some point in time. In 14 {\%} of these cases, these failures could have been detected in advance by detection of changing droplet velocities during jetting. Real time analysis of droplet characteristics and closed-loop control can enable a higher yield of the microarray manufacturing process. The improved production process as explained in chapter 2 is the basis for the microarray experiments carried out for the results in experimental chapters 3-5. Many different surface chemistries exist that are applied to graft the DNA probes onto the microarray surface. A lack of understanding on the characteristics of DNA immobilization and hybridization as separate processes hampers the improvement in reproducibility of the use of microarrays. Immobilization and hybridization rates of oligonucleotides equipped with specific tails on amine-functionalized surfaces by using 254 nm UV-light are studied. This surface chemistry is attractive because of its ease-of-use, robustness and low cost. A method was developed that enables a systematic study of the immobilization and subsequent hybridization processes independently. It was found that immobilization efficiencies are greatly influenced both by the UV dose applied and by the composition, as well as the length of the specific tails attached to the oligonucleotides. Hybridization is mainly influenced by the length of the oligonucleotides, and much less by the composition of the tail as observed for immobilization. This means that within the UV-window as applied in this study, no significant UV-damage was found. The standard microarray procedure runs as follows: microarrays are incubated with the sample, followed by a washing step to remove non-specific binding, then dried and scanned. This process flow implies that only a single measurement (end) point is used to evaluate target and capture molecule interactions. Since these interactions depend on many factors, including the preceding steps and the composition of the mixture itself, a complete hybridization measurement would provide additional and more reliable data. This means, however, that measurements should be done in high background signals requiring the deployment of methods that enable background suppression. Chapter 4 describes a method that measures real time hybridization and melting curves based on the repeated flow of the solution containing the targets through a porous microarray substrate. During each hybridization cycle, background was suppressed by pumping the liquid through the substrate. This method, using the Human Papilloma Virus (HPV) assay as a model assay, was evaluated with clinical samples and benchmarked against a method commonly used in the clinic (Reverse Line Blot). Both methods show comparable results, while the flow-through method also enabled the identification of cross-hybridizations by the analysis of the binding and melting kinetics. This method can contribute to identification of false positive signals. The Polymerase Chain Reaction (PCR) is a method to amplify the concentration of specific DNA target sequences and is often performed prior to a microarray hybridization. Sample preparation variations including this amplification step contribute to a lower reproducibility of microarrays. In the real time array PCR concept the steps of real time amplification (qPCR) and detection are integrated, thereby combining the advantages of both methods: the high multiplexing capabilities of microarray based assays and the quantitative characteristics of real time PCR. In this procedure the hybridization of the formed amplicons on the microarray surface is monitored, during each annealing step of the amplification process. Consequently an amplification curve on the microarray surface is obtained from which quantitative information on the target input concentration can be derived for a much higher number of targets compared to qPCR. After the amplification, an additional hybridization and melting curve step can be performed to further assess the specificity. The detection of low signals within high background levels in relatively short times is the greatest technical challenge of this concept. This was achieved by the use of a confocal fluorescence scanner that significantly reduced background levels. A proof of concept study was carried out with a prototype instrument that was designed and built from standard components known from the optical storage technology. In order to minimize costs of goods, standard products (reagents and disposables) were also used for the biochemical processes of amplification and hybridization as much as possible. Amplification and detection were performed in a single and closed chamber, minimizing risks on cross-contamination and reducing manual steps, making this method especially suited for diagnostic testing. As low as 10 copies of a target sequence could be detected using this method. Chapter 6 contains a mathematical analysis of the real time array PCR process which was carried out to provide insights in the biochemical processes taking place. Models of qPCR and Langmuir adsorption were combined into a new model describing the different steps in the real time array protocol. Reasonably good agreement between the model and the experiments was found. Using this result for the experimental array-based assay, a number of measures have been identified and tested to increase the overall amplification efficiency which resulted in a similar performance compared to bulk qPCR.",
author = "Anke Pierik",
year = "2011",
doi = "10.6100/IR724484",
language = "English",
isbn = "978-94-6191-088-2",
publisher = "Technische Universiteit Eindhoven",
school = "Department of Chemical Engineering and Chemistry",

}

Pierik, A 2011, 'Advanced microarray technologies for clinical diagnostics', Doctor of Philosophy, Department of Chemical Engineering and Chemistry, Eindhoven. https://doi.org/10.6100/IR724484

Advanced microarray technologies for clinical diagnostics. / Pierik, Anke.

Eindhoven : Technische Universiteit Eindhoven, 2011. 168 p.

Research output: ThesisPhd Thesis 2 (Research NOT TU/e / Graduation TU/e)

TY - THES

T1 - Advanced microarray technologies for clinical diagnostics

AU - Pierik, Anke

PY - 2011

Y1 - 2011

N2 - DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains multiple copies of one oligonucleotide, called the capture probe, which has a sequence complementary to one single specific target molecule. A DNA microarray assay is based on the preferential binding between a DNA target molecule with its complementary probe present on the microarray surface. Provided appropriate conditions, these two single stranded DNA molecules with complementary DNA sequences form a double stranded molecule during a process called hybridization. When the target molecules are labeled with e.g. a fluorophore, the fluorescent microarray pattern achieved upon incubation of the sample with the microarray provides information on the presence of the target sequences in the sample. The key advantage of microarrays is their large degree of parallelism per sample due to the simultaneous reaction between multiple capture probes and target sequences. This thesis focuses on low density DNA arrays that are limited to a few hundred spots for diagnostic use, for the rapid detection of a constrained and specific set of target sequences. One major application of low density microarrays lies in the detection and identification of pathogens in the field of infectious diseases. So, high density arrays containing as high as millions of different spots that allows for the screening and detection of a much higher number of targets simultaneously, usually applied for studying gene expression studies, are beyond the scope of this thesis. The transfer of microarray-based assays from the research lab into the clinic has taken more time than anticipated and it faces a number of technical and regulatory challenges. These are especially related to quality control and data reproducibility caused by a lack of fundamental understanding of the biochemical processes involved. In this thesis, a number of these challenges are addressed and investigated, and potential routes for improvement are identified. This work focuses on the improvement of microarray technology in order to enable a faster implementation of microarray-based assays into clinical practice. Inkjet printing is a suitable technology for manufacturing low density microarrays. It is, however, an open-loop process and does therefore not provide any feedback on the quality of manufactured microarrays. E.g. missing spots are unacceptable since this could lead to potentially false negative results. Moreover, microarrays are generally being considered as high complexity products by the Clinical Laboratory Improvement Amendments (CLIA), or as high risk applications by the Food and Drug Administration (FDA). Therefore specific quality control measures on microarrays for clinical diagnostics are needed. In chapter 2, a closed-loop inkjet printing system equipped with an optical droplet detection system to investigate failure mechanisms of the printing process is described. It was found that of all microarrays analyzed, in 1.6 % jetting failed at some point in time. In 14 % of these cases, these failures could have been detected in advance by detection of changing droplet velocities during jetting. Real time analysis of droplet characteristics and closed-loop control can enable a higher yield of the microarray manufacturing process. The improved production process as explained in chapter 2 is the basis for the microarray experiments carried out for the results in experimental chapters 3-5. Many different surface chemistries exist that are applied to graft the DNA probes onto the microarray surface. A lack of understanding on the characteristics of DNA immobilization and hybridization as separate processes hampers the improvement in reproducibility of the use of microarrays. Immobilization and hybridization rates of oligonucleotides equipped with specific tails on amine-functionalized surfaces by using 254 nm UV-light are studied. This surface chemistry is attractive because of its ease-of-use, robustness and low cost. A method was developed that enables a systematic study of the immobilization and subsequent hybridization processes independently. It was found that immobilization efficiencies are greatly influenced both by the UV dose applied and by the composition, as well as the length of the specific tails attached to the oligonucleotides. Hybridization is mainly influenced by the length of the oligonucleotides, and much less by the composition of the tail as observed for immobilization. This means that within the UV-window as applied in this study, no significant UV-damage was found. The standard microarray procedure runs as follows: microarrays are incubated with the sample, followed by a washing step to remove non-specific binding, then dried and scanned. This process flow implies that only a single measurement (end) point is used to evaluate target and capture molecule interactions. Since these interactions depend on many factors, including the preceding steps and the composition of the mixture itself, a complete hybridization measurement would provide additional and more reliable data. This means, however, that measurements should be done in high background signals requiring the deployment of methods that enable background suppression. Chapter 4 describes a method that measures real time hybridization and melting curves based on the repeated flow of the solution containing the targets through a porous microarray substrate. During each hybridization cycle, background was suppressed by pumping the liquid through the substrate. This method, using the Human Papilloma Virus (HPV) assay as a model assay, was evaluated with clinical samples and benchmarked against a method commonly used in the clinic (Reverse Line Blot). Both methods show comparable results, while the flow-through method also enabled the identification of cross-hybridizations by the analysis of the binding and melting kinetics. This method can contribute to identification of false positive signals. The Polymerase Chain Reaction (PCR) is a method to amplify the concentration of specific DNA target sequences and is often performed prior to a microarray hybridization. Sample preparation variations including this amplification step contribute to a lower reproducibility of microarrays. In the real time array PCR concept the steps of real time amplification (qPCR) and detection are integrated, thereby combining the advantages of both methods: the high multiplexing capabilities of microarray based assays and the quantitative characteristics of real time PCR. In this procedure the hybridization of the formed amplicons on the microarray surface is monitored, during each annealing step of the amplification process. Consequently an amplification curve on the microarray surface is obtained from which quantitative information on the target input concentration can be derived for a much higher number of targets compared to qPCR. After the amplification, an additional hybridization and melting curve step can be performed to further assess the specificity. The detection of low signals within high background levels in relatively short times is the greatest technical challenge of this concept. This was achieved by the use of a confocal fluorescence scanner that significantly reduced background levels. A proof of concept study was carried out with a prototype instrument that was designed and built from standard components known from the optical storage technology. In order to minimize costs of goods, standard products (reagents and disposables) were also used for the biochemical processes of amplification and hybridization as much as possible. Amplification and detection were performed in a single and closed chamber, minimizing risks on cross-contamination and reducing manual steps, making this method especially suited for diagnostic testing. As low as 10 copies of a target sequence could be detected using this method. Chapter 6 contains a mathematical analysis of the real time array PCR process which was carried out to provide insights in the biochemical processes taking place. Models of qPCR and Langmuir adsorption were combined into a new model describing the different steps in the real time array protocol. Reasonably good agreement between the model and the experiments was found. Using this result for the experimental array-based assay, a number of measures have been identified and tested to increase the overall amplification efficiency which resulted in a similar performance compared to bulk qPCR.

AB - DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains multiple copies of one oligonucleotide, called the capture probe, which has a sequence complementary to one single specific target molecule. A DNA microarray assay is based on the preferential binding between a DNA target molecule with its complementary probe present on the microarray surface. Provided appropriate conditions, these two single stranded DNA molecules with complementary DNA sequences form a double stranded molecule during a process called hybridization. When the target molecules are labeled with e.g. a fluorophore, the fluorescent microarray pattern achieved upon incubation of the sample with the microarray provides information on the presence of the target sequences in the sample. The key advantage of microarrays is their large degree of parallelism per sample due to the simultaneous reaction between multiple capture probes and target sequences. This thesis focuses on low density DNA arrays that are limited to a few hundred spots for diagnostic use, for the rapid detection of a constrained and specific set of target sequences. One major application of low density microarrays lies in the detection and identification of pathogens in the field of infectious diseases. So, high density arrays containing as high as millions of different spots that allows for the screening and detection of a much higher number of targets simultaneously, usually applied for studying gene expression studies, are beyond the scope of this thesis. The transfer of microarray-based assays from the research lab into the clinic has taken more time than anticipated and it faces a number of technical and regulatory challenges. These are especially related to quality control and data reproducibility caused by a lack of fundamental understanding of the biochemical processes involved. In this thesis, a number of these challenges are addressed and investigated, and potential routes for improvement are identified. This work focuses on the improvement of microarray technology in order to enable a faster implementation of microarray-based assays into clinical practice. Inkjet printing is a suitable technology for manufacturing low density microarrays. It is, however, an open-loop process and does therefore not provide any feedback on the quality of manufactured microarrays. E.g. missing spots are unacceptable since this could lead to potentially false negative results. Moreover, microarrays are generally being considered as high complexity products by the Clinical Laboratory Improvement Amendments (CLIA), or as high risk applications by the Food and Drug Administration (FDA). Therefore specific quality control measures on microarrays for clinical diagnostics are needed. In chapter 2, a closed-loop inkjet printing system equipped with an optical droplet detection system to investigate failure mechanisms of the printing process is described. It was found that of all microarrays analyzed, in 1.6 % jetting failed at some point in time. In 14 % of these cases, these failures could have been detected in advance by detection of changing droplet velocities during jetting. Real time analysis of droplet characteristics and closed-loop control can enable a higher yield of the microarray manufacturing process. The improved production process as explained in chapter 2 is the basis for the microarray experiments carried out for the results in experimental chapters 3-5. Many different surface chemistries exist that are applied to graft the DNA probes onto the microarray surface. A lack of understanding on the characteristics of DNA immobilization and hybridization as separate processes hampers the improvement in reproducibility of the use of microarrays. Immobilization and hybridization rates of oligonucleotides equipped with specific tails on amine-functionalized surfaces by using 254 nm UV-light are studied. This surface chemistry is attractive because of its ease-of-use, robustness and low cost. A method was developed that enables a systematic study of the immobilization and subsequent hybridization processes independently. It was found that immobilization efficiencies are greatly influenced both by the UV dose applied and by the composition, as well as the length of the specific tails attached to the oligonucleotides. Hybridization is mainly influenced by the length of the oligonucleotides, and much less by the composition of the tail as observed for immobilization. This means that within the UV-window as applied in this study, no significant UV-damage was found. The standard microarray procedure runs as follows: microarrays are incubated with the sample, followed by a washing step to remove non-specific binding, then dried and scanned. This process flow implies that only a single measurement (end) point is used to evaluate target and capture molecule interactions. Since these interactions depend on many factors, including the preceding steps and the composition of the mixture itself, a complete hybridization measurement would provide additional and more reliable data. This means, however, that measurements should be done in high background signals requiring the deployment of methods that enable background suppression. Chapter 4 describes a method that measures real time hybridization and melting curves based on the repeated flow of the solution containing the targets through a porous microarray substrate. During each hybridization cycle, background was suppressed by pumping the liquid through the substrate. This method, using the Human Papilloma Virus (HPV) assay as a model assay, was evaluated with clinical samples and benchmarked against a method commonly used in the clinic (Reverse Line Blot). Both methods show comparable results, while the flow-through method also enabled the identification of cross-hybridizations by the analysis of the binding and melting kinetics. This method can contribute to identification of false positive signals. The Polymerase Chain Reaction (PCR) is a method to amplify the concentration of specific DNA target sequences and is often performed prior to a microarray hybridization. Sample preparation variations including this amplification step contribute to a lower reproducibility of microarrays. In the real time array PCR concept the steps of real time amplification (qPCR) and detection are integrated, thereby combining the advantages of both methods: the high multiplexing capabilities of microarray based assays and the quantitative characteristics of real time PCR. In this procedure the hybridization of the formed amplicons on the microarray surface is monitored, during each annealing step of the amplification process. Consequently an amplification curve on the microarray surface is obtained from which quantitative information on the target input concentration can be derived for a much higher number of targets compared to qPCR. After the amplification, an additional hybridization and melting curve step can be performed to further assess the specificity. The detection of low signals within high background levels in relatively short times is the greatest technical challenge of this concept. This was achieved by the use of a confocal fluorescence scanner that significantly reduced background levels. A proof of concept study was carried out with a prototype instrument that was designed and built from standard components known from the optical storage technology. In order to minimize costs of goods, standard products (reagents and disposables) were also used for the biochemical processes of amplification and hybridization as much as possible. Amplification and detection were performed in a single and closed chamber, minimizing risks on cross-contamination and reducing manual steps, making this method especially suited for diagnostic testing. As low as 10 copies of a target sequence could be detected using this method. Chapter 6 contains a mathematical analysis of the real time array PCR process which was carried out to provide insights in the biochemical processes taking place. Models of qPCR and Langmuir adsorption were combined into a new model describing the different steps in the real time array protocol. Reasonably good agreement between the model and the experiments was found. Using this result for the experimental array-based assay, a number of measures have been identified and tested to increase the overall amplification efficiency which resulted in a similar performance compared to bulk qPCR.

U2 - 10.6100/IR724484

DO - 10.6100/IR724484

M3 - Phd Thesis 2 (Research NOT TU/e / Graduation TU/e)

SN - 978-94-6191-088-2

PB - Technische Universiteit Eindhoven

CY - Eindhoven

ER -

Pierik A. Advanced microarray technologies for clinical diagnostics. Eindhoven: Technische Universiteit Eindhoven, 2011. 168 p. https://doi.org/10.6100/IR724484