Nanoparticle-Assisted Immunoassays for Point-of-Care Testing - With Specific Interest in Minimally Interference-Prone Assays for Cardiac Troponin I

Cardiac troponins (cTn) I and T are the current golden standard biochemical markers in the diagnosis and risk stratification of patients with suspected acute coronary syndrome. During the past few years, novel assays capable of detecting cTn‐concentrations in >50% of apparently healthy individuals have become readily available. With the emerging of these high sensitivity cTn assays, reductions in the assay specificity have caused elevations in the measured cTn levels that do not correlate with the clinical picture of the patient. The increased assay sensitivity may reveal that various analytical interference mechanisms exist. This doctoral thesis focused on developing nanoparticle‐assisted immunometric assays that could possibly be applied to an automated point‐of‐care system. The main objective was to develop minimally interference‐prone assays for cTnI by employing recombinant antibody fragments. Fast 5‐ and 15‐minute assays for cTnI and D‐dimer, a degradation product of fibrin, based on intrinsically fluorescent nanoparticles were introduced, thus highlighting the versatility of nanoparticles as universally applicable labels. The utilization of antibody fragments in different versions of the developed cTnI‐assay enabled decreases in the used antibody amounts without sacrificing assay sensitivity. In addition, the utilization of recombinant antibody fragments was shown to significantly decrease the measured cTnI concentrations in an apparently healthy population, as well as in samples containing known amounts of potentially interfering factors: triglycerides, bilirubin, rheumatoid factors, or human anti‐mouse antibodies. When determining the specificity of four commercially available antibodies for cTnI, two out of the four cross‐reacted with skeletal troponin I, but caused crossreactivity issues in patient samples only when paired together. In conclusion, the results of this thesis emphasize the importance of careful antibody selection when developing cTnI assays. The results with different recombinant antibody fragments suggest that the utilization of antibody fragments should strongly be encouraged in the immunoassay field, especially with analytes such as cTnI that require highly sensitive assay approaches.

Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Relay card for capacitance measurements

In this bachelor's thesis a relay card for capacitance measurements was designed, built and tested. The study was made for the research and development laboratory of VTI Technologies, which manufactures capacitive silicon micro electro mechanical accelerometers and pressure sensors. As the size of the sensors is decreasing the capacitance value of the sensors also decreases. The decreased capacitance causes a need for new and more accurate measurement systems. The technology used in the instrument measuring the capacitance dictates a framework how the relay card should be designed, thus the operating principle of the instrument must be known. To achieve accurate results the measurement instrument and its functions needed to be used correctly. The relay card was designed using printed circuit board design methods that minimize interference coupling to the measurement. The relay card that was designed in this study is modular. It consists of a separate CPU card, which was used to control the add-on cards connected to it. The CPU card was controlled from a computer through a serial bus. Two add-on cards for the CPU card were designed in this study. The first one was the measurement card, which could be used to measure 32 capacitive sensors. The second add-on card was the MUX card, which could be used to switch between two measurement cards. The capacitance measurements carried out through the MUX card and the measurement cards were characterized with a series of test measurements. The test measurement data was then analysed. The relay card design was confirmed to work and offer accurate measurement results up to a measurement frequency of 10 MHz. The length of the measurement cables limited the measurement frequency.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Taajuusmuuttajiakoskevat EMC-standardit

Taajuusmuuttajat aiheuttavat toimintansa seurauksena runsaasti laajakaistaisia sähkömagneettisia häiriöitä. Häiriöt etenevät sekä johtumalla että säteilemällä ja ne ovat seurausta pääasiassa taajuusmuuttajien toimintaan perustuvista nopeista kytkentäilmiöistä sekä puolijohdekomponenttien epälineaarisuuksista. Sähkömagneettisille häiriöille määritellään suurimmat sallitut tasot useissa EMC-standardeissa. Taajuusmuuttajia koskevia EMC-standardia on neljä; tuotestandardi EN 61800-3, harmonisia virtoja pienjänniteverkoissa käsittelevä EN 61000-3-12 sekä yleiset standardit EN 61000-6-1, -6-2 ja EN 61000-6-3, -6-4. Tämän diplomityön tarkoituksena on tutkia ja koota yhteen edellä mainittujen standardien sisältämät vaatimukset. Työn pääpaino on kohdistettu häiriöpäästöille asetettuihin vaatimuksiin, sillä häiriöiden sietoon liittyvien vaatimusten täyttäminen ei taajuusmuuttajien kohdalla yleensä tuota ongelmia.

Performance Evaluation of Six-Sectored Configuration in Hexagonal WCDMA (UMTS) Cellular Network Layout

The objective of this master's thesis is to evaluate the optimum performance of sixsectored hexagonal layout of WCDMA (UMTS) network and analyze the performance at the optimum point. The maximum coverage and the maximum capacity are the main concern of service providers and it is always a challenging task for them to achieve economically. Because the optimum configuration of a network corresponds to a configuration which minimizes the number of sites required to provide a target service probability in the planning area which in turn reduces the deployment cost. The optimum performance means the maximum cell area and themaximum cell capacity the network can provide at the maximum antenna height satisfying the target service probability. Hexagon layout has been proven as the best layout for the cell deployment. In this thesis work, two different configurations using six-sectored sites have been considered for the performance comparison. In first configuration, each antenna is directed towards each corner of hexagon, whereas in second configurationeach antenna is directed towards each side of hexagon. The net difference in the configurations is the 30 degree rotation of antenna direction. The only indoor users in a flat and smooth semi-urban environment area have been considered for the simulation purpose where the traffic distribution is 100 Erl/km2 with 12.2 kbps speech service having maximum mobile speed of 3 km/hr. The simulation results indicate that a similar performance can be achieved in both the configurations, that is, a maximum of 947 m cellrange at antenna height of 49.5 m can be achieved when the antennas are directed towards the corner of hexagon, whereas 943.3 m cell range atantenna height of 54 m can be achieved when the antennas are directed towards the side of hexagon. However, from the interference point of view the first configuration provides better results. The simulation results also show that the network is coverage limited in both the uplink and downlink direction at the optimum point.