Biofunctionalised surfaces for molecular sensing

Dissertation

Sanna Auer

Research output: ThesisDissertationCollection of Articles

Abstract

In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab'-fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assembled monolayers on gold. The molecules were immobilized on surfaces via reactive thiol- or disulphide groups. On surfaces assembled of proteins, the non-specific binding was minimized by hydrophilic polymer molecules and on surfaces assembled of oligonucleotides by means of lipoate molecules embedded on the surface in between the biomolecules, respectively. With these highly sensitive biomolecular surfaces, a nanomolar detection of small sized molecules such as the 3,4-methylenedioxymethamphetamine (MDMA) drug was achieved. MDMA was analysed from a difficult sample matrix of diluted saliva. Improved orientation of surface immobilized Fab'-fragments leading to a higher sensitivity was shown with surfaces constructed of cys-tagged avidins: Fab'-fragments immobilized via thiol-biotinylation to a surface constructed of cys-tagged avidins bound almost ten times the amount of antigen when compared to a conventional surface constructed of non-oriented wild-type avidins. Polymer molecules embedded in between the biomolecules efficiently reduced non-specific binding. Selective neuronal cell attachment was also shown with polymer and neuronal-specific antibody molecules physisorbed on cell culture plates. Only the differentiated neuronal cells attached to surfaces physisorbed with neuronal-specific antibodies, while the non-differentiated neurospheres did not. Selective surfaces were also developed for oligonucleotide sequences. Lipoate-based molecules efficiently reduced the non-specific binding of proteins and non-complementary DNA. A nanomolar detection range was achieved for single-stranded, breast cancer-specific polymerase chain reaction (PCR) products. First, the shorter single-stranded PCR-products were analysed and a nanomolar detection range was achieved in buffer. In the following study, the DNA-surfaces were analysed in the presence of diluted serum. Even in diluted serum matrix, nanomolar concentrations of longer single- stranded sequences could be analysed due to the efficient blocking of non-specific binding of serum proteins. It was found that sensitive detection surfaces for biomolecular recognition can be achieved, when optimal function of the biomolecules is ensured by immobilizing the molecules on surfaces in an oriented manner towards the analyte. Efficient reduction of non-specific binding is also important in reaching highly sensitive label-free detection. The surfaces were also found to be effective in selective neuronal stem cell attachment.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Tampere University of Technology (TUT)
Supervisors/Advisors
  • Vikholm-Lundin, Inger, Supervisor, External person
Award date14 Jun 2013
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-8001-9
Electronic ISBNs978-951-38-8002-6
Publication statusPublished - 2013
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Molecules
Biomolecules
Avidin
Immunoglobulin Fab Fragments
Oligonucleotides
N-Methyl-3,4-methylenedioxyamphetamine
Polymers
Polymerase chain reaction
Stem cells
Reaction products
Sulfhydryl Compounds
Biosensors
Gold
Labels
Immunoglobulin Fragments
Antibodies
DNA-Binding Proteins
Surface plasmon resonance
Self assembled monolayers
Tissue engineering

Keywords

  • antibody
  • Fab -fragment
  • cysteine tagged avidin
  • neuronal cells
  • DNA hybridisation
  • gold surface
  • immobilisation
  • surface plasmon resonance
  • non-specific binding

Cite this

Auer, S. (2013). Biofunctionalised surfaces for molecular sensing: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Auer, Sanna. / Biofunctionalised surfaces for molecular sensing : Dissertation. Espoo : VTT Technical Research Centre of Finland, 2013. 120 p.
@phdthesis{ef3e91199527432797d78af5f80b9fee,
title = "Biofunctionalised surfaces for molecular sensing: Dissertation",
abstract = "In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab'-fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assembled monolayers on gold. The molecules were immobilized on surfaces via reactive thiol- or disulphide groups. On surfaces assembled of proteins, the non-specific binding was minimized by hydrophilic polymer molecules and on surfaces assembled of oligonucleotides by means of lipoate molecules embedded on the surface in between the biomolecules, respectively. With these highly sensitive biomolecular surfaces, a nanomolar detection of small sized molecules such as the 3,4-methylenedioxymethamphetamine (MDMA) drug was achieved. MDMA was analysed from a difficult sample matrix of diluted saliva. Improved orientation of surface immobilized Fab'-fragments leading to a higher sensitivity was shown with surfaces constructed of cys-tagged avidins: Fab'-fragments immobilized via thiol-biotinylation to a surface constructed of cys-tagged avidins bound almost ten times the amount of antigen when compared to a conventional surface constructed of non-oriented wild-type avidins. Polymer molecules embedded in between the biomolecules efficiently reduced non-specific binding. Selective neuronal cell attachment was also shown with polymer and neuronal-specific antibody molecules physisorbed on cell culture plates. Only the differentiated neuronal cells attached to surfaces physisorbed with neuronal-specific antibodies, while the non-differentiated neurospheres did not. Selective surfaces were also developed for oligonucleotide sequences. Lipoate-based molecules efficiently reduced the non-specific binding of proteins and non-complementary DNA. A nanomolar detection range was achieved for single-stranded, breast cancer-specific polymerase chain reaction (PCR) products. First, the shorter single-stranded PCR-products were analysed and a nanomolar detection range was achieved in buffer. In the following study, the DNA-surfaces were analysed in the presence of diluted serum. Even in diluted serum matrix, nanomolar concentrations of longer single- stranded sequences could be analysed due to the efficient blocking of non-specific binding of serum proteins. It was found that sensitive detection surfaces for biomolecular recognition can be achieved, when optimal function of the biomolecules is ensured by immobilizing the molecules on surfaces in an oriented manner towards the analyte. Efficient reduction of non-specific binding is also important in reaching highly sensitive label-free detection. The surfaces were also found to be effective in selective neuronal stem cell attachment.",
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publisher = "VTT Technical Research Centre of Finland",
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Auer, S 2013, 'Biofunctionalised surfaces for molecular sensing: Dissertation', Doctor Degree, Tampere University of Technology (TUT), Espoo.

Biofunctionalised surfaces for molecular sensing : Dissertation. / Auer, Sanna.

Espoo : VTT Technical Research Centre of Finland, 2013. 120 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Biofunctionalised surfaces for molecular sensing

T2 - Dissertation

AU - Auer, Sanna

N1 - Project code: 82444

PY - 2013

Y1 - 2013

N2 - In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab'-fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assembled monolayers on gold. The molecules were immobilized on surfaces via reactive thiol- or disulphide groups. On surfaces assembled of proteins, the non-specific binding was minimized by hydrophilic polymer molecules and on surfaces assembled of oligonucleotides by means of lipoate molecules embedded on the surface in between the biomolecules, respectively. With these highly sensitive biomolecular surfaces, a nanomolar detection of small sized molecules such as the 3,4-methylenedioxymethamphetamine (MDMA) drug was achieved. MDMA was analysed from a difficult sample matrix of diluted saliva. Improved orientation of surface immobilized Fab'-fragments leading to a higher sensitivity was shown with surfaces constructed of cys-tagged avidins: Fab'-fragments immobilized via thiol-biotinylation to a surface constructed of cys-tagged avidins bound almost ten times the amount of antigen when compared to a conventional surface constructed of non-oriented wild-type avidins. Polymer molecules embedded in between the biomolecules efficiently reduced non-specific binding. Selective neuronal cell attachment was also shown with polymer and neuronal-specific antibody molecules physisorbed on cell culture plates. Only the differentiated neuronal cells attached to surfaces physisorbed with neuronal-specific antibodies, while the non-differentiated neurospheres did not. Selective surfaces were also developed for oligonucleotide sequences. Lipoate-based molecules efficiently reduced the non-specific binding of proteins and non-complementary DNA. A nanomolar detection range was achieved for single-stranded, breast cancer-specific polymerase chain reaction (PCR) products. First, the shorter single-stranded PCR-products were analysed and a nanomolar detection range was achieved in buffer. In the following study, the DNA-surfaces were analysed in the presence of diluted serum. Even in diluted serum matrix, nanomolar concentrations of longer single- stranded sequences could be analysed due to the efficient blocking of non-specific binding of serum proteins. It was found that sensitive detection surfaces for biomolecular recognition can be achieved, when optimal function of the biomolecules is ensured by immobilizing the molecules on surfaces in an oriented manner towards the analyte. Efficient reduction of non-specific binding is also important in reaching highly sensitive label-free detection. The surfaces were also found to be effective in selective neuronal stem cell attachment.

AB - In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab'-fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assembled monolayers on gold. The molecules were immobilized on surfaces via reactive thiol- or disulphide groups. On surfaces assembled of proteins, the non-specific binding was minimized by hydrophilic polymer molecules and on surfaces assembled of oligonucleotides by means of lipoate molecules embedded on the surface in between the biomolecules, respectively. With these highly sensitive biomolecular surfaces, a nanomolar detection of small sized molecules such as the 3,4-methylenedioxymethamphetamine (MDMA) drug was achieved. MDMA was analysed from a difficult sample matrix of diluted saliva. Improved orientation of surface immobilized Fab'-fragments leading to a higher sensitivity was shown with surfaces constructed of cys-tagged avidins: Fab'-fragments immobilized via thiol-biotinylation to a surface constructed of cys-tagged avidins bound almost ten times the amount of antigen when compared to a conventional surface constructed of non-oriented wild-type avidins. Polymer molecules embedded in between the biomolecules efficiently reduced non-specific binding. Selective neuronal cell attachment was also shown with polymer and neuronal-specific antibody molecules physisorbed on cell culture plates. Only the differentiated neuronal cells attached to surfaces physisorbed with neuronal-specific antibodies, while the non-differentiated neurospheres did not. Selective surfaces were also developed for oligonucleotide sequences. Lipoate-based molecules efficiently reduced the non-specific binding of proteins and non-complementary DNA. A nanomolar detection range was achieved for single-stranded, breast cancer-specific polymerase chain reaction (PCR) products. First, the shorter single-stranded PCR-products were analysed and a nanomolar detection range was achieved in buffer. In the following study, the DNA-surfaces were analysed in the presence of diluted serum. Even in diluted serum matrix, nanomolar concentrations of longer single- stranded sequences could be analysed due to the efficient blocking of non-specific binding of serum proteins. It was found that sensitive detection surfaces for biomolecular recognition can be achieved, when optimal function of the biomolecules is ensured by immobilizing the molecules on surfaces in an oriented manner towards the analyte. Efficient reduction of non-specific binding is also important in reaching highly sensitive label-free detection. The surfaces were also found to be effective in selective neuronal stem cell attachment.

KW - antibody

KW - Fab -fragment

KW - cysteine tagged avidin

KW - neuronal cells

KW - DNA hybridisation

KW - gold surface

KW - immobilisation

KW - surface plasmon resonance

KW - non-specific binding

M3 - Dissertation

SN - 978-951-38-8001-9

T3 - VTT Science

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Auer S. Biofunctionalised surfaces for molecular sensing: Dissertation. Espoo: VTT Technical Research Centre of Finland, 2013. 120 p.