Carboxyl-terminated dendrimer-coated bioactive interface for protein microarray: high-sensitivity detection of antigen in complex biological samples.
Protein microarrays are promising tools that can potentially enable high throughput proteomic screening in areas such as disease diagnosis and drug discovery. A critical aspect in the development of protein microarrays is the optimization of the array's surface chemistry to achieve the high sensitivity required for detection of proteins in cell lysate and other complex biological mixtures. In the present study, a high-density antibody array with minimal nonspecific cellular protein adsorption was prepared using a glass surface coated with a poly(propyleneimine) dendrimer terminated with carboxyl group (PAMAM-COOH). The carboxyl-terminated dendrimer-modified surface has almost similar nonspecific cellular protein adsorption when compared to an inert PEG-modified surface. In addition, the multiple functional sites available for reaction on the dendrimer surface facilitated high-density immobilization of antibodies and efficient capture of bioanalytes. Various molecules were tested for their ability to block or deactivate the reactive carboxyl surface after antibody immobilization to further reduce the nonspecific binding. A short oligoethylene glycol (NH2-d4-PEG-COOH), was found to significantly improve the signal-to-noise ratio of the assay, resulting in higher sensitivity. The properties and functional qualities of the various surfaces were characterized by contact angle and AFM measurements. Nonspecific protein adsorption and protein immobilization as a function of dendrimer generations and sensitivity of antigen capturing from a buffer (1 pM) as well as from the complex cell lysate (10 pM) system were examined. Our detailed experimental studies demonstrated a facile method of preparing surfaces with high protein loading and low nonspecific protein binding for the development of high sensitivity protein microarrays.
Ajikumar
Parayil Kumaran
PK
MEBCS Program, Singapore-MIT Alliance, National University of Singapore, 4 Engineering Drive 3, Singapore 117546. pkaji@mit.edu
Ng
Jin Kiat
JK
Tang
Yew Chung
YC
Lee
Jim Yang
JY
Stephanopoulos
Gregory
G
Too
Heng-Phon
HP
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000941:Antigens / Q000032:analysis*; D045744:Cell Line, Tumor; D002474:Cell-Free System; D050091:Dendrimers / Q000737:chemistry*; D051379:Mice; D011073:Polyamines / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D040081:Protein Array Analysis; D040901:Proteomics; D012680:Sensitivity and Specificity
D000906:Antibodies; D000941:Antigens; D050091:Dendrimers; C104700:PAMAM Starburst; D011073:Polyamines; D011092:Polyethylene Glycols