Inkjet Printed Silver Electrodes on Macroporous Paper for a Paper-Based Isoelectric Focusing Device

We demonstrate a combined printing process utilizing inkjet printing of silver electrodes and solid-ink technology for printing hydrophobic wax barriers for fabricating paper microfluidic devices with integrated electrodes. Optimized printing parameters are given for achieving conducting silver lines on the top of macroporous chromatography paper down to 250 mu m-300 mu m resolution. Electrical characterization and wicking experiments demonstrate that the printed silver patterns are simultaneously conductive and porous enough to allow reliable capillary wicking across the electrodes. The combined wax and silver printing method is used for fabrication of paper microfluidic isoelectric focusing devices for separation and concentration of proteins. Published by AIP Publishing.Peer reviewe

Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes

This work describes a new nanotechnology-based immobilization strategy for cytochrome P450s (CYPs), the major class of drug metabolizing enzymes. Immobilization of CYPs on solid supports provides a significant leap forward compared with soluble enzyme assays by enabling the implementation of through-flow microreactors for, for example, determination of time-dependent inhibition. Immobilization of the complex CYP membrane-protein system is however particularly challenging as the preservation of the authentic enzyme kinetic parameters requires the full complexity of the lipid environment. The developed strategy is based on the spontaneous fusion of biotinylated fusogenic liposomes with lipid bilayers to facilitate the gentle biotinylation of human liver microsomes that incorporate all main natural CYP isoforms. The same process is also feasible for the biotinylation of recombinant CYPs expressed in insect cells, same as any membrane-bound enzymes in principle. As a result, CYPs could be immobilized on streptavidin-functionalized surfaces, both those of commercial magnetic beads and customized microfluidic arrays, so that the enzyme kinetic parameters remain unchanged, unlike in previously reported immobilization approaches that often suffer from restricted substrate diffusion to the enzyme's active site and steric hindrances. The specificity and robustness of the functionalization method of customized microfluidic CYP assays are also carefully examined.Peer reviewe

Mikrofluidististen analyysilaitteistojen kehittäminen proteiinien karakterisointiin sähkösumutusionisaatio-massaspektrometrialla

The aim of this work was to develop new microfluidic tools for proteomics research by exploiting capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI/MS). The microchips used were made of SU-8 polymer according to a previously developed microfabrication protocol. Three different microchip designs were utilized. A typical design comprised a CE sample injection and separation unit in addition to monolithically integrated ESI emitter with on-chip sheath liquid interface. The applicability of the microchips to rapid protein characterization was demonstrated with human erythropoietin as a model compound. Protein sequencing of both recombinant human erythropoietin (rHuEPO) and endogenous EPO was demonstrated using microchip capillary zone electrophoretic (CZE) separation and ESI/MS detection of enzymatically (off-chip) cleaved peptides. As a result, a number of characteristic peptide sequences were detected enabling confident identification of both rHuEPO (score 123) and endogenous EPO (score 98) by Mascot probability based search engine with confidence limit score of>56. The detected peptides covered 36 % and 48 % of the amino acid sequences of rHuEPO and endogenous EPO, respectively. In addition, few potential glycopeptide assignments were identified with the help of GlycoMod online database. Intact EPO glycoforms were also analyzed by microchip CZE-ESI/MS and capillary isoelectric focusing (cIEF)-ESI/MS, but no confident identification of the intact glycoforms was obtained due to insufficient separation, detection sensitivity and/or data interpretation. With a view to improved protein characterization, two alternative microchip techniques were examined, namely, on-chip proteolytic digestion and specific on-chip derivatization of the amino acid residues prior to online ESI/MS detection. Tentative results of the on-chip proteolytic digestion showed that acid-induced degradation at aspartic acid provides rapid proteolytic degradation provided that the microchip can be heated up to +100°C and the cleavage site is easily accessible. However, further development of the microchip heating process seems as a prerequisite for rapid degradation of more complex proteins. On-chip derivatization of the separated amino acids was implemented by applying the derivatization reagent, 9-fluorenylmethyl chloroformate (Fmoc-Cl), to the ESI emitter along with the sheath liquid. Rapid reaction between Fmoc-Cl and selected amino acids was demonstrated. The reaction seemed to occur fastest toward the amino acids with the smallest side chain structure.Tämän työn tavoitteena oli kehittää uudenlaisia mikrofluidistisia sovelluksia proteomiikkaan hyödyntäen kapillaarielektroforeesi-sähkösumutusionisaatio-massaspektrometriaa (CE-ESI/MS). Työssä käytetyt mikrosirut valmistettiin SU-8-polymeerista aiemmin kehitettyjen mikrovalmistusmenetelmien mukaisesti. Työssä käytettiin kolmea eri mikrosirumallia, jotka tyypillisesti koostuivat ESI-kärkeen monoliittisesti liitetyistä näytteensyöttö- ja CE-erotusyksiköistä. Mikrosirujen soveltuvuutta nopeaan proteiinianalytiikkaan tutkittiin käyttäen malliaineena humaani erytropoietiinia (EPO). Sekä rekombinantti humaani erytropoietiinin (rHuEPO) että endogeenisen EPO:n aminohappojärjestys määritettiin entsymaattisesti hajotettujen aminohapposekvenssien avulla. Molemmat EPO-analogit pystyttiin luotettavasti tunnistamaan kapillaarivyöhyke-elektroforeesin (CZE) ja ESIJMS-detektion perusteella. Todennäköisyysvastaavuuteen perustuvalla Mascot-haulla rHuEPO:n luotettavuusasteeksi saatiin 123 (luotettavuusraja >56) ja sekvenssikattavuudeksi 36 %. Vastaavat luvut endogeeniselle EPO:lle olivat 98 ja 48 %. Aminohapposekvenssien lisäksi mikrosiruanalyysi mahdollisti muutamien potentiaalisten glykopeptidien tunnistamisen GlycoMod-tietokannan avulla. Mikrosiru-CE-ESI/MS-tekniikoita, kapillaarivyöhyke-elektroforeesia (CZE) ja kapillaari-isoelektristä fokusointia (cIEF), testattiin myös glykoproteiinien analyysiin, mutta näiden luotettava tunnistus ei ollut mahdollista tämän työn puitteissa. CE-ESI/MS-tekniikoiden ohella työssä kehitettiin kaksi vaihtoehtoista mikrosirutekniikkaa proteiinianalytiikan helpottamiseksi. Mikrosirulla tapahtuva, happokatalysoitu reaktio yhdistettynä suoraan ESI/MS-detektioon mahdollisti nopean aminohappoketjun pilkkoutumisen asparagiinihapon kohdalla. Korkea lämpötila (+100°C) ja asparagiinihapon esteetön sijainti olivat kuitenkin edellytyksiä nopealle pilkkoutumiselle. Suurempien proteiinien pilkkominen edellyttänee erityisesti mikrosirun lämmitysprosessin tehostamista. Mikrosirulla tapahtuva aminohappojen derivatisointi toteutettiin tuomalla derivatisointireagenssi, 9-fluorenyylimetyylikloroformaatti (Fmoc-Cl), apuliuoksen mukana ESI-kärkeen, mikä mahdollisti nopean reaktion Fmoc-Cl:n ja erotuskanavassa erotettujen aminohappojen välillä. Nopein reaktiokinetiikka havaittiin aminohapoilla, joiden sivuketjun koko oli pienin