TY - JOUR
T1 - A Three-Arm Scaffold Carrying Affinity Molecules for Multiplex Recognition Imaging by Atomic Force Microscopy
T2 - The Synthesis, Attachment to Silicon Tips, and Detection of Proteins
AU - Manna, Saikat
AU - Senapati, Subhadip
AU - Lindsay, Stuart
AU - Zhang, Peiming
N1 - Publisher Copyright:
© 2015 American Chemical Society.
PY - 2015/6/17
Y1 - 2015/6/17
N2 - We have developed a multiplex imaging method for detection of proteins using atomic force microscopy (AFM), which we call multiplex recognition imaging (mRI). AFM has been harnessed to identify protein using a tip functionalized with an affinity molecule at a single molecule level. However, many events in biochemistry require identification of colocated factors simultaneously, and this is not possible with only one type of affinity molecule on an AFM tip. To enable AFM detection of multiple analytes, we designed a recognition head made from conjugating two different affinity molecules to a three-arm linker. When it is attached to an AFM tip, the recognition head would allow the affinity molecules to function in concert. In the present study, we synthesized two recognition heads: one was composed of two nucleic acid aptamers, and the other one composed of an aptamer and a cyclic peptide. They were attached to AFM tips through a catalyst-free click reaction. Our imaging results show that each affinity unit in the recognition head can recognize its respective cognate in an AFM scanning process independently and specifically. The AFM method was sensitive, only requiring 2 to 3 μL of protein solution with a concentration of ∼2 ng/mL for the detection with our current setup. When a mixed sample was deposited on a surface, the ratio of proteins could be determined by counting numbers of the analytes. Thus, this mRI approach has the potential to be used as a label-free system for detection of low-abundance protein biomarkers.
AB - We have developed a multiplex imaging method for detection of proteins using atomic force microscopy (AFM), which we call multiplex recognition imaging (mRI). AFM has been harnessed to identify protein using a tip functionalized with an affinity molecule at a single molecule level. However, many events in biochemistry require identification of colocated factors simultaneously, and this is not possible with only one type of affinity molecule on an AFM tip. To enable AFM detection of multiple analytes, we designed a recognition head made from conjugating two different affinity molecules to a three-arm linker. When it is attached to an AFM tip, the recognition head would allow the affinity molecules to function in concert. In the present study, we synthesized two recognition heads: one was composed of two nucleic acid aptamers, and the other one composed of an aptamer and a cyclic peptide. They were attached to AFM tips through a catalyst-free click reaction. Our imaging results show that each affinity unit in the recognition head can recognize its respective cognate in an AFM scanning process independently and specifically. The AFM method was sensitive, only requiring 2 to 3 μL of protein solution with a concentration of ∼2 ng/mL for the detection with our current setup. When a mixed sample was deposited on a surface, the ratio of proteins could be determined by counting numbers of the analytes. Thus, this mRI approach has the potential to be used as a label-free system for detection of low-abundance protein biomarkers.
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U2 - 10.1021/jacs.5b03079
DO - 10.1021/jacs.5b03079
M3 - Article
C2 - 25996033
AN - SCOPUS:84935013576
SN - 0002-7863
VL - 137
SP - 7415
EP - 7423
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 23
ER -