Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.
The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Meihua
M
Lin
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Ping
P
Song
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Guobao
G
Zhou
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Xiaolei
X
Zuo
Department of Chemistry, King Saud University, Riyadh, Saudi Arabia.
Ali
A
Aldalbahi
Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, China.
Xiaoding
X
Lou
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.|Kellogg College, University of Oxford, Oxford, UK.|UCB Pharma, Slough, UK.
Jiye
0000-0002-9628-8680
J
Shi
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.|School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Chunhai
0000-0002-7171-7338
C
Fan
D000779:Anesthetics, Local / Q000032:analysis; D000818:Animals; D001360:Avidin / Q000737:chemistry; D001560:Benzidines / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D003042:Cocaine / Q000032:analysis; D004247:DNA / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods; D009369:Neoplasms / Q000175:diagnosis; D009696:Nucleic Acids / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results
D000779:Anesthetics, Local; D001560:Benzidines; D009696:Nucleic Acids; D011506:Proteins; C083986:avidin-horseradish peroxidase complex; D001360:Avidin; C021758:3,3',5,5'-tetramethylbenzidine; D006046:Gold; D004247:DNA; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D003042:Cocaine