G5 Artikkeliväitöskirja
Quantification of Proteins and Cells – Luminometric Nonspecific Particle-Based Methods




Alaotsikko: Luminometric Nonspecific Particle-Based Methods
Julkaisun tekijät: Sari Pihlasalo
Kustantaja: Annales Universitatis Turkuensis
Paikka: Painosalama Oy, Turku
Julkaisuvuosi: 2011
ISBN: 978-951-29-4553-5
eISBN: 978-951-29-4554-2

Tiivistelmä
New  luminometric  particle-based  methods  were  developed  to  quantify  protein  and  to 

count cells. The developed methods rely on the interaction of the sample with nano- or 

microparticles  and  different  principles  of  detection.  In  fluorescence  quenching,  time-

resolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation 

fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the 

particles. Depending on the system, the addition of the analyte increases or decreases the 

luminescence.  In  the  dissociation  method,  the  adsorbed  protein  protects  the  Eu(III) 

chelate on the surface of the particles from dissociation at a low pH. The experimental 

setups  are  user-friendly  and  rapid  and  do  not  require  hazardous  test  compounds  and 

elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg 

bovine  serum  albumin  in  a  sample)  was  20-500-fold  better  than  in  most  sensitive 

commercial methods. The quenching method exhibited low protein-to-protein variability 

and the dissociation method insensitivity to the assay contaminants commonly found in 

biological samples. Less than ten eukaryotic cells were detected and quantified with all 

the developed methods under optimized assay conditions. Furthermore, two applications, 

the  method  for  detection  of  the  aggregation  of  protein  and  the  cell  viability  test,  were 

developed by utilizing the TR-LRET method. The detection of the aggregation of protein 

was allowed at a more than 10,000 times lower concentration, 30 µg/L, compared to the 

known  methods  of  UV240  absorbance  and  dynamic  light  scattering.  The  TR-LRET 

method was combined with a nucleic acid assay with cell-impermeable dye to measure 

the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

Last updated on 2019-20-07 at 17:19