Sample preconcentration in open microchannels: Combinations with MALDI and nano-ESI mass spectrometry and computer simulations
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
In this thesis a novel concept for preconcentration of biomolecules in open microchannels is presented. The preconcentration is based on electromigration of charged analytes, and detection is performed with matrix-assisted laser desorption/ionization (MALDI) or nano-electrospray ionization (nESI) mass spectrometry (MS).
Analysis of minute volumes of low-concentration samples is an important and challenging task within several fields of chemistry, biology and medicine. In bioanalytical chemistry in particular, sample pretreatment procedures including preconcentration must frequently be applied. Due to the often small available sample volumes, it is advantageous to perform these pretreatments in microfluidic devices. Moreover, since MS in many cases is the detection method of choice, there is a requirement for developing suitable interfacing techniques between the microchip and MS.
In Paper I, the preconcentration concept is presented; silicon microchips with parallel open channels were used. The channels have a rectangular shape and are 1 cm long, 50-150 µm wide and 50 µm deep, yielding a total channel volume of 25-75 nL. By supplying sample to the channel and applying a voltage over the channel length, charged analytes will migrate towards the oppositely charged electrode and become concentrated. In Paper I, detection was performed by using the open microchannel directly as a MALDI-target. To achieve this, matrix solution was added to the channel after the preconcentration with electrospray matrix deposition. Using this approach, preconcentration of cytochrome c was achieved, and the lowest initial protein concentration successfully detected after preconcentration was 1 nM. The trypsin digest of cytochrome c was also analyzed, and the peptides were preconcentrated at different ends of the channel based on charge.
Other means of coupling the preconcentration to MS, by extracting a nanovolume of the preconcentrated sample from the open channel, are presented in Paper II. The extracted samples could either be analyzed directly using nESI- or MALDI-MS, or subjected to further pretreatment (such as enzymatic digestion) in a nanodroplet under a fluorocarbon (FluorinertPTMP) liquid lid prior to MS-analysis. Furthermore, in Paper II the method was applied on an amyloid beta cell culture. This resulted in that peptides not detectable without preconcentration easily could be detected with MALDI-MS in nanodroplets extracted from the microchannels after preconcentration.
Paper III includes theoretical simulations of the preconcentration procedure obtained using the electrophoresis simulator GENTRANS. The experimental results from Paper I are compared to simulations of similar systems, and simulations of an isoelectric focusing (IEF) procedure for proteins or peptides in a mixture of amino acids, are presented. The IEF procedure is to be used in the open microchannels in future experimental work.
Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , vi, 41 p.
TRITA-CHE-Report, ISSN 1654-1081 ; 2014:12
Preconcentration, open microchannel, microchip, MALDI, electrospray ionization, nanoelectrospray, mass spectrometry, computer simulation
IdentifiersURN: urn:nbn:se:kth:diva-144872ISBN: 978-91-7595-101-0OAI: oai:DiVA.org:kth-144872DiVA: diva2:714978
2014-05-23, E2, Lindstedtsvägen 3, KTH, Stockholm, 10:00 (English)
Ilag, Leopold, DocentOttosson Takanen, Jenny, Doktor
Emmer, Åsa, Professor
QC 201405132014-05-132014-04-302014-05-13Bibliographically approved