Surface Plasmon Resonance in Living Cell Sensing : SPR Responses in Cell-Exosome Interactions

Surface plasmon resonance (SPR) is a label free technique to study surface interactions. It is based on photon-plasmon coupling. Laser light is directed through a prism and reflects form a metal surface, often gold. At certain conditions, photons turn into plasmons, which then propagate on the metal surface. The refractive index (RI) of the medium close to the metal surface alters the conditions when plasmons can be generated. By changing the incident angle of the light, photon-plasmon coupling can be matched. Thus, change in the SPR sensogram peak angular position (PAP) indicates change in the RI of the sample. Traditionally, SPR has been used to investigate biomolecule dissociation / association kinetics. Recently, it has gained popularity in living cell sensing. Exosomes are 30-100 nm size lipid bilayer structured vesicles, which are excreted by nearly all cells. They play a role in cell-cell communications. Exosomes carry selected cargo from the cells of origin, including mRNA, miRNA, dsDNA and proteins, and they are directed to specific cells, which internalize them. This initiates responses in the recipient cells. The aim of the study was to harvest exosomes from prostate cancer (LNCaP) cells and use SPR as a novel method to detect exosome internalization by these cells. Adhesion proteins were tested in their efficiency to promote confluent cell monolayer formation on SPR gold substrate sensor surface. Nanoparticle tracking analysis (NTA) showed that exosome purification by ultracentrifugation was successful. It was also found that gold substrate supports confluent LNCaP cell monolayer formation. Adhesion proteins did not shorten the incubation time on gold substrate, but helped the cells remain on the sensor during the SPR experiment. Prostate and platelet exosomes were tested on whether they are internalized by LNCaP cells. Control samples with plain medium and PEI/DNA nanoparticles were used. PEI/DNA particles are nonviral gene delivery vectors, which are known to permeate into cells. The SPR results showed RI increase caused 0.9 ° change in the SPR sensogram with the PEI/DNA sample and no change with the medium sample. Exosomes showed more complex responses, both increasing the PAP approximately 0.1 °. Prostate exosome sensogram returned to baseline after sample rinsing, which did not occur with platelet exosomes. It was concluded that SPR shows a response in cell-exosome interactions, which is most likely because of exosome internalization.Pintaplasmoniresonanssi (Surface Plasmon Resonance, SPR) on leimavapaa tekniikka pintailmiöiden tutkimiseen, joka perustuu fotoni-plasmoni pariutumiseen. Laservalo heijastuu prisman läpi metallipinnasta, joka on yleisimmin kultaa. Tietyissä olosuhteissa fotonit muuttuvat plasmoneiksi, jotka etenevät metallipinnalla. Metallin lähellä olevan väliaineen taitekerroin (Refractive Index, RI) muuttaa olosuhteita, jossa plasmoneita voi syntyä. Muuttamalla valon heijastekulmaa, voidaan aikaansaada fotoni-plasmoni pariutuminen. Näin muutos SPR sensogrammin maksimikulman sijainnissa (Peak Angular Position, PAP) kertoo näytteen taitekertoimen muutoksesta. Perinteisesti SPR:ää on käytetty biomolekyylien sitoutumis- / vapautumiskinetiikan tutkimiseen. Viimeaikoina se on kasvattanut suosiotaan elävien solujen tutkimuksessa. Eksosomit ovat 30-100 nm kokoisia lipidikaksoiskerroksisia vesikkeleitä, joita erittävät lähestulkoon kaikki solut. Ne toimivat solujen välisessä kommunikaatiossa ja sisältävät muun muassa lähtösoluista peräisin olevaa mRNA:ta, miRNA:ta, dsDNA:ta ja proteiineja. Eksosomit kohdennetaan tietyille soluille, jotka ottavat ne sisäänsä, ja niiden soluun otto aiheuttaa vasteen näissä soluissa. Tutkimuksen tavoite oli kerätä eksosomeja eturauhassyöpäsoluista (LNCaP solut) ja käyttää SPR:ää uutena menetelmänä havaitsemaan eksosomien soluun ottoa näillä samoilla soluilla. Kokeilimme adheesioproteiinien tehokkuutta edistää yhdenmukaisen solukerroksen muodostumista kultapinnoitteisen SPR sensorin pinnalle. Nanopartikkeli seuranta-analyysi (Nanoparticle Tracking Analysis, NTA) osoitti, että eksosomien eristys ultrasentrifugaatiolla onnistui. Totesimme myös, että LNCaP solut kykenevät kasvamaan yhdenmukaisena kerroksena kultapinnoitteisen sensorin päälle. Adheesioproteiinit eivät lyhentäneet inkubaatioaikaa kultapinnalla, mutta auttoivat soluja pysymään kiinni sensorissa SPR kokeen aikana. Testasimme LNCaP -solujen kykyä ottaa sisäänsä eturauhas- ja verihiutale-eksosomeja. Verrokkinäytteinä käytimme liuosta, joka ei sisältänyt näytettä ja PEI/DNA nanopartikkeleita sisältävää liuosta. PEI/DNA partikkelit ovat ei-viraalisia geenivektoreita, joiden tiedetään kulkeutuvan solujen sisään. Tulokset osoittivat taitekertoimen muutoksen aiheuttavan 0,9 ° muutoksen SPR sensogrammissa PEI/DNA näytteellä. Näytettä sisältämätön liuos ei aiheuttanut lainkaan muutosta, kun taas eksosomit osoittivat monitahoisia muutoksia sensogrammeissa. Molemmat eksosominäytteet nostivat PAP arvoa 0,1 °. Eturauhaseksosomien sensogrammi palasi lähtöarvoon, kun näyte huuhdeltiin pois. Tätä ei tapahtunut verihiutaleeksosomeilla. Totesimme että SPR havaitsee solujen ja eksosomien vuorovaikutusten aiheuttamia muutoksia, jotka todennäköisesti johtuvat eksosomien soluun otosta

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds

Label-free characterization and real-time monitoring of cell uptake of extracellular vesicles

Extracellular vesicles (EVs) have the ability to function as molecular vehicles and could therefore be harnessed to deliver drugs to target cells in diseases such as cancer. The composition of EVs determines their function as well as their interactions with cells, which consequently affects the cell uptake efficacy of EVs. In this study, we present two novel label-free approaches for studying EVs; characterization of EV composition by time-gated surface-enhanced Raman spectroscopy (TG-SERS) and monitoring the kinetics and amount of cellular uptake of EVs by surface plasmon resonance (SPR) in real-time. Using these methods, we characterized the most abundant EVs of human blood, red blood cell (RBC)- and platelet (PLT)-derived EVs and studied their interactions with prostate cancer cells. Complementary studies were performed with nanoparticle tracking analysis for concentration and size determinations of EVs, zeta potential measurements for surface charge analysis, and fluorophore-based confocal imaging and flow cytometry to confirm EV uptake. Our results revealed distinct biochemical features between the studied EVs and demonstrated that PLT-derived EVs were more efficiently internalized by PC-3 cells than RBC-derived EVs. The two novel label-free techniques introduced in this study were found to efficiently complement conventional techniques and paves the way for further use of TG-SERS and SPR in EV studies.Peer reviewe

Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the anti-tumor immunity and efficacy against melanoma

The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAd´s immunogenicity might be important for their efficacy. Therefore, we developed a versatile and rapid system to adsorb tumor-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response towards the tumor-epitopes. By studying the model epitope SIINFEKL we demonstrated that the peptide-coated OAd (PeptiCRAd) retains its infectivity and the cross-presentation of the modified-exogenous epitope on MHC-I is not hindered. We then showed that the SIINFEKL-targeting PeptiCRAd achieves a superior anti-tumor efficacy and increases the percentage of anti-tumor CD8+ T-cells and mature epitope-specific dendritic cells in vivo. PeptiCRAds loaded with clinically relevant tumor epitopes derived from tyrosinase-related protein 2 (TRP-2) and human gp100 could reduce the growth of primary-treated tumors and secondary-untreated melanomas, promoting the expansion of antigen-specific T-cell populations. Finally, we tested PeptiCRAd in humanized mice bearing human melanomas. In this model, a PeptiCRAd targeting the human melanoma-associated antigen A1 (MAGE-A1) and expressing granulocyte and macrophage colony-stimulating factor (GM-CSF) was able to eradicate established tumors and increased the human MAGE-A1-specific CD8+ T-cell population. Herein we show that the immunogenicity of OAds plays a key role in their efficacy and it can be exploited to direct the immune response system towards exogenous tumor epitopes. This versatile and rapid system overcomes the immunodominance of the virus and elicits a tumor-specific immune response, making PeptiCRAd a promising approach for clinical testing.Peer reviewe

Real-Time Label-Free Targeting Assessment and in Vitro Characterization of Curcumin-Loaded Poly-lactic-co-glycolic Acid Nanoparticles for Oral Colon Targeting

The exploitation of curcumin for oral disease treatment is limited by its low solubility, poor bioavailability, and low stability. Surface-functionalized poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) have shown promising results to ameliorate selective delivery of drugs to the gastro-intestinal tract. In this study, curcumin-loaded PLGA NPs (C-PLGA NPs) of about 200 nm were surface-coated with chitosan (CS) for gastro-intestinal mucosa adhesion, wheat germ agglutinin (WGA) for colon targeting or GE11 peptide for tumor colon targeting. Spectrometric and zeta potential analyses confirmed the successful functionalization of the C-PLGA NPs. Real-time label-free assessment of the cell membrane-NP interactions and NP cell uptake were performed by quartz crystal microbalance coupled with supported lipid bilayers and by surface plasmon resonance coupled with living cells. The study showed that CS-coated C-PLGA NPs interact with cells by the electrostatic mechanism, while both WGA- and GE11-coated C-PLGA NPs interact and are taken up by cells by specific active mechanisms. In vitro cell uptake studies corroborated the real-time label-free assessment by yielding a curcumin cell uptake of 7.3 ± 0.3, 13.5 ± 1.0, 27.3 ± 4.9, and 26.0 ± 1.3 μg per 104 HT-29 cells for noncoated, CS-, WGA-, and GE11-coated C-PLGA NPs, respectively. Finally, preliminary in vivo studies showed that the WGA-coated C-PLGA NPs efficiently accumulate in the colon after oral administration to healthy Balb/c mice. In summary, the WGA- and GE11-coated C-PLGA NPs displayed high potential for application as active targeted carriers for anticancer drug delivery to the colon.Peer reviewe

Surface Plasmon Resonance-Based Cellular Assays in Pharmaceutical Research : Interpretation of the Label-Free Signals

Cell-based assays have become an integral part of pharmaceutical research and drug development. They offer researchers not only a means to investigate basic cellular functions and different disease states, but also a way to study drug action in cells. Nowadays, during the long journey that new drug candidates take from an early discovery to a final product, all drug candidates undergo a preclinical test phase where their properties are evaluated using various cell-based assays. While perhaps the most common and important goal for these cell-based assays is to ensure that the investigated molecules are not harmful to cells, cell-based assays are also used to investigate the biological activity, mechanisms of action, and possible interactions, such as interaction with other drugs. With the traditionally used cell-based assays it is a common practice to alter the investigated pharmaceuticals or cells, or both, to produce a measurable signal, such as radioactivity, chemiluminescence, or fluorescence. However, there are well-known issues with this strategy, not the least that by subjecting the pharmaceuticals and/or cells to these modifications, an artificial system is created where the cells or pharmaceuticals under investigation are no longer in their original state. This can have a profound impact on the properties of the pharmaceuticals or behavior of the cells, thereby resulting in erroneous readouts. Because of this, different label-free cell-based assays are an attractive and logical alternative. Label-free technologies have been applied in the field of drug research and discovery since the 1980s. During the turn of the last millennium an increasing number of studies have implemented these methods to measure cellular activity, such as receptor activation. Nowadays, some of the commercial label-free devices are designed specifically with cell-based assays in mind. Of course, these technologies are not without their own challenges. The most prominent of these stems from the inherit property of these technologies: they are not sensitive to just a single predefined cellular event or response, but instead they measure cell activity as a whole. While well-controlled experimental design can mitigate the contribution of non-relevant cell responses to the measured label-free signals, advanced signal analysis methods have made, and can be expected to continue to make, label-free cell-based assays even more powerful tools for cell studies and help researchers to better understand the effect of different cellular activities on the label-free signals. Amongst the different label-free techniques, optical methods have been widely adopted for cell-based assays. While resonant waveguide grating (RWG) has been mainly utilized in the investigation of membrane receptors, namely G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), surface plasmon resonance (SPR) has been applied in the study of a variety of cellular processes, such as cell adhesion, detachment, spreading, contraction, cytoskeleton rearrangement, cell-to-cell contacts, monolayer formation and cell death. Surprisingly, studies on GPCRs with SPR are relatively low in number, even though they should be of high interest as around 35% of all current drugs mediate their effect via these receptors. Although SPR has already been shown to be a powerful tool for studying various cellular processes, its wider use in life sciences is hampered by the challenge in interpreting the contribution of the cell responses of interest and other non-relevant responses interfering with the measured signals. A variety of NPs are used as drug carriers, especially for biological drugs, in order to protect them from degradation and guide them to diseased cells or other desired location within the human body, while EVs are naturally produced nanosized structures that function as message carriers between cells. Because of the inherent property of EVs to penetrate cellular barriers, there is a growing interest in using them as “natural” and personalized drug carriers. This thesis investigates the use of SPR in the context of cellular uptake studies of nanoparticles (NPs) and extracellular vesicles (EVs), and GPCR-mediated signaling. Various approaches to analyze real-time label-free SPR signals are implemented in order to improve the correlation between the measured SPR responses and cell activities. The results show, for the first time, how SPR can be used to measure cellular uptake of NPs and EVs. However, because NP and EV uptake lead to ambiguous SPR signals, different signal analysis strategies are investigated and implemented to provide meaningful SPR responses. Three strategies proved to be effective: (1) varying the measurement temperature allowed to investigate possible cell entry mechanisms of different NPs in HeLa cells; (2) correcting the SPR angle response by removing the contribution of total internal reflection (TIR) angle shift allowed the quantification of the uptake efficacy of functionalized NPs into HT-29 colorectal adenocarcinoma cells; (3) comparing the responses measured with two different wavelengths corrected for the large response variation observed across different experiments for EV uptake in PC-3 prostate cancer cells. In addition, multiple label-free SPR parameters from the full SPR curves were analyzed after stimulating different GPCR subtypes, signaling primarily via Gs-, Gq- or Gi-coupled pathways. It was revealed that by combining two key label-free parameters, unique label-free signal profiles for each pathway could be generated, which makes it possible to recognize the pathway coupling of the receptor subtypes from the label-free responses alone, which has not been possible before. Collectively, these results demonstrate how SPR can be effectively utilized in cell-based assays to investigate NP and EV uptake and cell signaling. Cell-based SPR provides high information content, and with the help of the developed and used analysis methods, the otherwise hard-to-interpret label-free signals provide meaningful information on these cell responses.Soluja on kasvatettu laboratorio-oloissa jo yli puoli vuosisataa solutoimintojen ja lääkkeiden vaikutusmekanismien tutkimiseksi. Nykyään eri tutkimusmenetelmiä on runsaasti ja elävillä soluilla tehtävät kokeet ovat keskeinen osa farmaseuttista tutkimusta ja lääkekehitystä. On kuitenkin hyvin tavallista, että lääkkeiden vaikutuksia tutkittaessa soluja tai lääkeaineita joudutaan muokkaamaan eri tavoin. Solut voidaan esimerkiksi muuntaa tuottamaan tiettyä merkkiproteiinia tai tutkittavaan lääkeaineeseen voidaan lisätä fluoresoiva molekyyli. Nämä muokkaukset ovat tarpeellisia, koska käytetyt mittalaitteet perustuvat näiden merkkimolekyylien havainnointiin. Tämä lähestymistapa voi kuitenkin olla ongelmallinen, koska muokkaukset voivat vaikuttaa siihen, miten solut tai tutkittavat lääkkeet käyttäytyvät. Tämä voi puolestaan johtaa tulosten virheellisiin tulkintoihin. Leimavapaita mittatekniikoita on käytetty lääketutkimuksessa jo pitkään, ja vuosituhannen vaihteen jälkeen niiden käyttö elävien solujen tutkimuksessa alkoi yleistyä. Optiseen havainnointiin perustuvat reaaliaikaiset leimavapaat tekniikat, kuten pintaplasmoniresonanssi (surface plasmon resonance, SPR), tarjoavat selviä etuja verrattuna perinteisiin yleisesti käytössä oleviin solututkimusmenetelmiin. Nimensä mukaisesti ne mittaavat soluja reaaliajassa, mutta merkittävintä on niiden kyky mitata soluvasteita ilman leima-aineita. Toisin kuin leima-aineisiin perustuvat menetelmät, optinen leimavapaa havainnointi on herkkä monille eri solumuutoksille. Näin myös ennalta odottamattomat soluvasteet voidaan havainnoida. Toisaalta näiden epäspesifisten optisten signaalien tulkinta on haastavaa. Tässä väitöskirjassa tutkittiin, kuinka SPR-tekniikkaa voidaan hyödyntää solumittauksissa. Vaikka vastaavilla menetelmillä on jo aikaisemmin tutkittu runsaasti muun muassa reseptoreiden toimintaa, leimavapailla menetelmillä ei ole tutkittu nanopartikkelien soluun ottoa. Soluun oton tutkiminen on tärkeää, koska nanopartikkelit ovat nousseet merkittävään rooliin modernissa lääketutkimuksessa ja kehityksessä. Tämä johtuu siitä, että yhä suurempi osa uusista kehitteillä olevista lääkkeistä on suuria molekyylejä, kuten proteiineja tai DNA- tai RNA-pohjaisia lääkeaineita. Rakenteensa takia ne eivät kulkeudu passiivisesti solujen sisään, joten ne on pakattava erilaisiin nanopartikkeleista valmistettuihin lääkekuljettimiin. Synteettisten nanopartikkelien lisäksi työssä tutkittiin solun ulkoisten vesikkelien soluun ottoa. Näistä vesikkeleistä on povattu uudentyyppisiä lääkekuljettimia, koska niillä on kyky läpäistä solukalvo ja solujen luonnollisesti erittäminä rakenteina ne eivät aiheuta puolustusreaktiota elimistössä. Työn keskeisenä osana oli lisäksi leimavapaiden signaalien tulkinta. Koska solujen tuottamat SPR-signaalivasteet ovat moninaisia ja epäspesifisiä, työssä tutkittiin erilaisten signaalianalyysimenetelmien kykyä tuottaa oleellista tietoa soluvasteista. Tulokset osoittivat, että oikeanlaisella analyysillä saadaan tietoa eri nanopartikkelien ja solun ulkoisten vesikkelien soluun oton tehokkuudesta, ja myös mahdollisesti niiden soluun oton mekanismeista. Lisäksi soluista tai näytteistä johtuvaa signaalivaihtelua kyettiin korjaamaan tulosten vertailun mahdollistamiseksi. Osaltaan nämä tulokset saavutettiin käyttämällä SPR-laitetta, joka kykenee mittaamaan useampaa leimavapaata parametria samanaikaisesti. Tätä SPR-laitteen ominaisuutta hyödynnettiin myös G-proteiinikytkentäisten reseptorien aktivaation tulkitsemisessa. Tulokset osoittivat, että tarkastelemalla useampaa kuin yhtä parametria samanaikaisesti, G-proteiinikytkentäiset reseptorit voidaan tunnistaa niiden G-proteiininalatyypin mukaan. Tässä ei ole onnistuttu yhtä luotettavasti aiemmin edes muilla leimavapailla menetelmillä

Pelilliset orientaatiot ongelmallisen digipelaamisen hahmottamisen tukena

Tässä opinnäytetyössä tarkastelemme pelillisiä menetelmiä osana ennalta ehkäisevää työskentelyä peliongelmien parissa. Työn tarkoituksena on jatkokehittää lautapeliä, jonka kehitystyössä on hyödynnetty yhteiskehittämisen menetelmiä, sekä sosiaalipedagogista näkökulmaa. Alkuperäinen virtuaalilautapeli on kehitetty yhteistyössä Metropolian Connext -hankkeen kanssa. Lautapelit ohjausmenetelminä ovat helposti lähestyttäviä niiden pelillisyyden ja toiminnallisen luonteen vuoksi, jolloin tekeminen on mielekästä ja pelillä on selkeät tavoitteet, raamit, ja tarkoitus. Tässä opinnäytetyössä kehitettävä lautapelipohja on tuotettu yhteiskehittämismenetelmällä Metropolian opiskelijoiden ohjausryhmistä, ja tämän opinnäytetyön tarkoituksena on jatkokehittää lautapelistä menetelmä, jota sosiaalialan ammattilaiset voivat hyödyntää ohjaustilanteiden tukena liittyen ongelmalliseen pelaamisen. Ongelmallinen digipelaaminen on yleistyvä ongelma yhteiskunnassa digipelien kehittyessä vaikuttavimmiksi yhdessä teknologian kehittymisen kanssa. Keskusteleminen aiheesta saattaa olla hankalaa, mikäli ohjaustyön ammattilaisella ei ole omaa kokemusta pelaamisesta, tai ongelmallisesta digipelaamisesta. Tässä opinnäytetyössä käytettävän virtuaalilautapelin tarkoitus on toimia ongelmallisen digipelaamisen keskustelun herättelijänä, sekä toimia turvallisena alustana tutustua ongelmallisen digipelaamisen maailmaan. Virtuaalilautapelissä hyödynnetään case työskentelymallia, jonka avulla pelaajat (virtuaalilautapelin käyttäjät) voivat tarinallisten sisältöjen avulla löytää samaistumiskohtia omasta arjestansa. Case tehtävissä on pyritty huomioimaan myös potentiaalisten käyttäjien monimuotoisuus, sekä elämänkaaren kehitysvaiheen tyypilliset kehityskulut. Opinnäytetyössämme iteratiivisessa osiossa tarkastelemme myös testiryhmien avulla ongelmalliseen digipelaamiseen liittyviä käsityksiä ongelmallisen digipelaamisen tunnistamisesta, samaistuttavuudesta, sekä luonteesta. Ongelmallinen digipelaaminen on terminologisesti hankala, joten testiryhmien kanssa on käytetty termiä ”ongelmallinen videopelaaminen”, joka on yleiskielellisempi termi samasta aiheesta. Virtuaalinen lautapeli on pelattavissa osoitteessa www.seppo.io pelikoodilla 262FECIn this thesis, we look at gamification methods as part of preventive work on problematic digital gaming. The purpose of the work is to further develop a board game, in the development work of which the methods of co-development have been utilized, as well as the social pedagogical perspective. The virtual board game was originally developed in collaboration with the Metropolia Connext project. Board games as methods of tutelage are easily approachable due to their playfulness and functional nature, which makes participating sensible and the game has clear goals, frameworks, and purpose. The board game template developed in this thesis has been produced using the co-development method from Metropolia's student guidance groups, and the purpose of this thesis is to further develop a board game method that can be used by social professionals to support tutelage situations related to problematic digital gaming. Problematic digital gaming is a widespread problem in society as digital games become more influential along with the development of technology. It can be difficult to discuss a topic if the tutor does not have their own experience of gaming or problematic digital gaming. The purpose of the virtual board game used in this thesis is to stimulate the discussion regarding problematic digital gaming, and to serve as a safe platform to get to know the world of problematic digital gaming. The virtual board game utilizes a case-based working model, which allows players (users of the virtual board game) to find identities in their own daily lives through storytelling content. Case assignments have also sought to consider the diversity of potential users, as well as the typical development costs of the life cycle development phase. In the iterative section of our thesis, we also use test groups to examine the perceptions of problematic digital gaming related to the identification, identity, and nature of problematic digital gaming. Problematic digital gaming is terminologically cumbersome, so the term “problem video gaming” has been used with test groups, which is a more general term of the same topic. The virtual board game can be played at www.seppo.io with the game code 262FE

Design and evaluation of a multiplexed angular-scanning surface plasmon resonance system employing line-laser optics and CCD detection in combination with multi-ligand sensor chips

An angle-scanning Kretschmann configuration SPR instrument allowing multiplexed analysis is presented. Laser light was guided through optics that converted the collimated light into a line-shaped beam, which was directed to a prism, illuminating the gold sensor surface over a 1 × 10 mm area. The reflected light was led to a CCD detector providing simultaneous readout of individual analysis spots along the laser line at a selected angle (fixed-angle detection) or in scanning-angle mode (width of 35°). Full SPR curve could be measured every 3.6 s for each illuminated spot on the sensor surface. Two in-house manufactured flow cell designs were used for evaluating multiplexed angular-scanning SPR. The first comprised six parallel channels with the laser line perpendicular to the flow direction in order to allow interrogation of the sensor surface in the six channels. Refractive index changes by varying solution composition, and adsorption of different concentrations of albumin to the sensor surface could be correctly monitored simultaneously in each of the channels. In the second flow-cell design the laser line was coinciding with the flow path, allowing recording of SPR curves along a 10-mm length of the sensor surface. Adsorption of layers of positively and negatively charged polyelectrolytes could be consistently measured for sixteen selected positions along the channel. As a proof of principle, several target proteins were immobilized on different positions along the sensor and the binding of various antibodies with these proteins was monitored simultaneously, showing excellent selectivity and reproducibility for probing antibody-protein interactions in a multiplexed fashion

Oncolytic adenoviruses coated with MHC-I tumor epitopes for a new oncolytic vaccine platform

none15noneCapasso, Cristian; Hirvinen, Mari; Garofalo, Mariangela; Romaniuk, Dmitrii; Kuryk, Lukasz; Sarvela, Teea; Vitale, Andrea; Antopolsky, Maxim; Magarkar, Aniket; Viitala, Tapani; Suutari, Teemu; Bunker, Alex; Yliperttula, Marjo; Urtti, Arto; Cerullo, VincenzoCapasso, Cristian; Hirvinen, Mari; Garofalo, Mariangela; Romaniuk, Dmitrii; Kuryk, Lukasz; Sarvela, Teea; Vitale, Andrea; Antopolsky, Maxim; Magarkar, Aniket; Viitala, Tapani; Suutari, Teemu; Bunker, Alex; Yliperttula, Marjo; Urtti, Arto; Cerullo, Vincenz