51 research outputs found
Biofunctionalised surfaces for molecular sensing
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 cystagged 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 neuronalspecific antibodies, while the non-differentiated neurospheres did not.Selective surfaces were also developed for oligonucleotide sequences. Lipoatebased molecules efficiently reduced the non-specific binding of proteins and noncomplementary DNA. A nanomolar detection range was achieved for singlestranded, 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.Monilla sovellusalueilla, kuten bioantureissa, tietylle analyytille herkät biomolekyylit kiinnitetään kiinteälle pinnalle, mikä mahdollistaa hyvin pienten analyyttipitoisuuksien määrittämisen. Tällaisia sovellusalueita ovat esimerkiksi sairauksien merkkiaineiden määritys, huumausaineiden tai ympäristömyrkkyjen määritys ja kudosteknologia.Tämä väitöskirja käsittelee biomolekyylien (vasta-aineiden ja Fab´-fragmenttien, avidiinien ja deoksiribonukleiinihappo (DNA) -koettimien) kiinnittämistä kultapinnoille bioantureissa. Tunnistavien molekyylien kiinnittämistapaa ja pitoisuutta biomolekulaarisilla pinnoilla tutkittiin yksityiskohtaisesti nanomolaarisen herkkyyden saavuttamiseksi myös vaikeista lähtömateriaaleista. Lisäksi tutkittiin, miten nämä pinnat soveltuvat kantasoluista erilaistettujen hermosolujen tartuttamiseen. Reaaliaikainen määritys ilman leima-aineita tehtiin pintaplasmoniresonanssin (SPR) avulla. Tutkimuksessa käytetyt itseasettuvat kalvot muodostettiin biomolekyyleistä ja hylkivistä molekyyleistä kultapinnoille. Molekyylit kiinnitettiin pinnoille tioli- tai disulfidiryhmien kautta. Proteiinipinnoilla epäspesifistä sitoutumista vähennettiin hydrofiilisten polymeerien avulla ja DNA-koetinpinnoilla vastaavasti lipoaattipohjaisten molekyylien avulla, jotka oli asetettu pinnoilla biomolekyylien väliin.Biomolekulaaristen pintarakenteiden avulla pystyttiin mittaamaan myös pienikokoinen 3,4-dimetyleenidioksimetyyliamfetamiini (MDMA) -huumausaine nanomolaarisessa pitoisuudessa. MDMA pystyttiin määrittämään myös laimennetusta sylkinäytteestä. Kysteiinimuokattujen avidiinipintojen avulla pystyttiin parantamaan Fab´-fragmenttien orientaatiota pinnoilla, mikä johti tavoiteltuihin, korkeampiin antigeenivasteisiin. Tioli-ryhmiin biotinyloituja Fab´-fragmentteja pystyttiin kiinnittämään kysteiinimuokattuihin avidiinipintoihin kymmenkertainen määrä verrattuna villityypin ei-orientoituihin avidiinipintoihin. Biomolekyylien väliin pinnoille kiinnitetyt polymeerimolekyylit ehkäisivät tehokkaasti epäspesifistä sitoutumista. Kun hermosolujen kasvatuslevyille kiinnitettiin polymeeriä ja hermosoluille spesifisiä vastaainemolekyylejä, levyille saatiin tarttumaan valikoidusti vain kantasoluista erilaistuneita hermosoluja. Kantasoluista erilaistumattomat solut eivät kiinnittyneet vastaainepolymeeripinnoille.Valikoivia pintoja kehitettiin myös DNA-koettimille. Proteiinien ja ei-pariutuvan DNA:n epäspesifinen sitoutuminen DNA-koetinpinnoille pystyttiin ehkäisemään tehokkaasti lipoaattipohjaisten molekyylien avulla. Yksijuosteiset rintasyöpäspesifiset DNA-juosteet pystyttiin tunnistamaan nanomolaarisella herkkyydellä. Ensin tutkittiin lyhyiden yksijuosteisten DNA-näytteiden tunnistusta puskuriliuoksessa saavuttaen nanomolaarinen herkkyys. Seuraavaksi DNA-pintojen toiminnallisuutta tutkittiin seerumiin laimennetuilla näytteillä. Myös pidempiä yksijuosteisia DNA-näytteitä pystyttiin määrittämään nanomolaarisina pitoisuuksina seerumilaimennoksesta, koska lipoaattipohjaiset molekyylit estivät tehokkaasti seerumin proteiinien epäspesifisen sitoutumisen pinnoille.Biomolekyylien määritykseen pystytään tekemään herkästi tunnistavia pintoja, kunhan biomolekyylien optimaalinen toiminta varmistetaan kiinnittämällä biomolekyylit pinnoille siten, että analyytin tunnistavat osat ovat orientoituneet analyyttiä kohden. Myös epäspesifisen sitoutumisen estäminen pinnoille on tärkeää korkean herkkyyden saavuttamiseksi leimavapaissa mittauksissa. Vasta-aine-polymeeripinnat todettiin hyvin toiminnallisiksi myös haluttaessa tartuttaa pinnoille valikoiden vain hermosoluja
Rapid and sensitive detection of norovirus antibodies in human serum with interferometry biosensor
CMOS-Integrated Film Bulk Acoustic Resonators for Label-Free Biosensing
The throughput is an important parameter for label-free biosensors. Acoustic resonators like the quartz crystal microbalance have a low throughput because the number of sensors which can be used at the same time is limited. Here we present an array of 64 CMOS-integrated film bulk acoustic resonators. We compare the performance with surface plasmon resonance and the quartz crystal microbalance and demonstrate the performance of the sensor for multiplexed detection of DNA
A Model of the Roles of Essential Kinases in the Induction and Expression of Late Long-Term Potentiation
The induction of late long-term potentiation (L-LTP) involves complex
interactions among second messenger cascades. To gain insights into these
interactions, a mathematical model was developed for L-LTP induction in the CA1
region of the hippocampus. The differential equation-based model represents
actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II
(CAMKII) in the vicinity of the synapse, and activation of transcription by CaM
kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic
weight. Simulations suggest that steep, supralinear stimulus-response
relationships between stimuli (elevations in [Ca2+]) and kinase activation are
essential for translating brief stimuli into long-lasting gene activation and
synaptic weight increases. Convergence of multiple kinase activities to induce
L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply
with the number of tetanic electrical stimuli. The model simulates tetanic,
theta-burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to
synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of
MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to
delineate mechanisms underlying L-LTP induction and expression. For example,
the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to
inhibit ERK activation. The model also appears useful to clarify similarities
and differences between hippocampal L-LTP and long-term synaptic strengthening
in other systems.Comment: Accepted to Biophysical Journal. Single PDF, 7 figs include
Detection of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) by displacement of antibodies
Applying a surface-sensitive fluorescence method to fast on-site detection of cocaine in saliva
AbstractFast and sensitive detection of cocaine in saliva is realized utilizing a surface-sensitive fluorescence measurement platform. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation (TIR) and supercritical angle fluorescence detection (SAF), which results in extreme surface sensitivity in the measurements. The molecular recognition takes place in a scheme, where cocaine molecules to be detected generate a displacement of the labelled anti-cocaine antibodies from the cocaine-BSA-conjugate molecules immobilized on a surface. The results with untreated saliva spiked with cocaine demonstrate that by monitoring the dissociation process in real time, cocaine concentrations down to 1 ng/mL can be detected within 60s
Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators
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