TY - JOUR
T1 - Isolation of thermally sensitive protein-binding oligonucleotides on a microchip
AU - Hilton, John P.
AU - Olsen, Timothy
AU - Kim, Jinho
AU - Zhu, Jing
AU - Nguyen, Thai Huu
AU - Barbu, Mihaela
AU - Pei, Renjun
AU - Stojanovic, Milan
AU - Lin, Qiao
N1 - Publisher Copyright:
© 2015, Springer-Verlag Berlin Heidelberg.
PY - 2015/10/1
Y1 - 2015/10/1
N2 - This paper presents a microfluidic chip capable of isolating thermally sensitive protein-binding aptamer candidates. The chip makes use of bead-immobilized target molecules and DNA (deoxyribonucleic acid) sequences to enable a simplified chip design, in which affinity selection and PCR (polymerase chain reaction) amplification of target-binding sequences occur in temperature-controlled microchambers. Using pressure-driven flow, buffer containing single-stranded DNA molecules with randomized sequences is cycled through a series of affinity selection and PCR amplification steps on microbeads. Successive introduction of the sample to each chamber effects a process of competition whereby DNA strands with weak binding strength to target molecules are rejected in favor of strongly binding sequences. Using bead-based PCR, the amplification step was miniaturized and integrated with affinity selection, resulting in significant reductions in process time and reagent use. As a demonstration, temperature-dependent selection and amplification of single-stranded oligonucleotides that bind to human Immunoglobulin E (IgE) was performed in 4 h, a 20-fold reduction in process time as compared to conventional methods that would require approximately a week. Fluorescent binding assays then demonstrated that the desired temperature specificity was imparted to the aptamer candidates within just one round of selection, and within two rounds the aptamer candidates exhibited enhanced affinity toward IgE.
AB - This paper presents a microfluidic chip capable of isolating thermally sensitive protein-binding aptamer candidates. The chip makes use of bead-immobilized target molecules and DNA (deoxyribonucleic acid) sequences to enable a simplified chip design, in which affinity selection and PCR (polymerase chain reaction) amplification of target-binding sequences occur in temperature-controlled microchambers. Using pressure-driven flow, buffer containing single-stranded DNA molecules with randomized sequences is cycled through a series of affinity selection and PCR amplification steps on microbeads. Successive introduction of the sample to each chamber effects a process of competition whereby DNA strands with weak binding strength to target molecules are rejected in favor of strongly binding sequences. Using bead-based PCR, the amplification step was miniaturized and integrated with affinity selection, resulting in significant reductions in process time and reagent use. As a demonstration, temperature-dependent selection and amplification of single-stranded oligonucleotides that bind to human Immunoglobulin E (IgE) was performed in 4 h, a 20-fold reduction in process time as compared to conventional methods that would require approximately a week. Fluorescent binding assays then demonstrated that the desired temperature specificity was imparted to the aptamer candidates within just one round of selection, and within two rounds the aptamer candidates exhibited enhanced affinity toward IgE.
KW - Aptamers
KW - Microfluidics
KW - Proteins
KW - SELEX
KW - Temperature dependence
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U2 - 10.1007/s10404-015-1604-2
DO - 10.1007/s10404-015-1604-2
M3 - Article
AN - SCOPUS:84942989031
SN - 1613-4982
VL - 19
SP - 795
EP - 804
JO - Microfluidics and Nanofluidics
JF - Microfluidics and Nanofluidics
IS - 4
ER -