Toward Multiprotein Nanoarrays Using Nanografting and DNA Directed Immobilization of Proteins

Abstract

Atomic force microscopy nanografting was utilized to prepare DNA nanopatches of different sizes (200 x 200 to 1000 x 1000 nm(2)) onto which DNA-protein conjugates can be anchored through DNA-directed immobilization. Height measurements were used to assess the binding of the proteins as well as their subsequent interaction with other components, such as antibodies. The results indicate that nanografted patch arrays are well suited for application in biosensing and could enable the fabrication of multifeature protein nanoarrays.

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…The fact that the nanografted patch arrays are potentially capable to be employed in biosensing applications was evident by the observed supreme specificity toward biomolecular recognition in addition to reduced nonspecific binding. [113] …”

Miniaturized Aptamer-Based Assays for Protein Detection

“…A tip-assisted AFM-based nanolithography technique has been used to fabricate DNA nanoarrays with high surface density (1-2 × 10 13 molecules/cm 2 ): using Si cantilevers (NSC36B Mikromasch (Mikromasch, Innovative Solutions Bulgaria Ltd., Sofia, Bulgaria), spring constant: 0.6 N/m) multiple nanografting assembled monolayers (NAM) of thiol-modified single-stranded DNA (ssDNA), named cF9, were prepared by serial AFM-based nanografting inside a self-assembled monolayer (SAM) of a top oligo ethylene glycol terminated alkanethiol, TOEG ((1-mercaptoundec-11-yl)hexa(ethyleneglycol), HS-(CH2)11-(OCH2CH2)6-OH from Sigma Aldrich) on ultraflat gold surfaces [22] following standard protocols reported earlier [9,23]. The DNA patches were obtained promoting the replacement of the TOEG molecules with the oligonucleotides by the AFM tip scanning an area of 1 µm × 1 µm or less at high force (about 100 nN) in the presence of a solution of thiolated ssDNA sequences (5 µM in TE buffer 1 M NaCl) at a scan rate of 2 Hz.…”

“…By using AFM topographic imaging, we monitored height profile changes over DNA-aptamers loaded by DDI on cF9 nanografted arrays (Figure 4: left, upper patches; right, black profile) vs. different THR concentrations in the binding buffer (Figure 4 left, mid and lower images; Figure 4 right, red and green profiles correspond to 12 nM and 1000 nM THR, respectively), due to the conformation change of the aptamer upon binding the ligand. In all these steps the TOEG SAM embedding the patches is used as a reference level to monitor topographic height changes [9]. Differential height changes are shown in Figure 5a.…”

“…Both of these platforms have been designed and developed to allow for the sensitive detection of biomolecules in small sample volumes. In the case of nanoarrays, we capitalize here on the exploitation of AFM-based lithography, nanografting [6][7][8], and AFM topographic imaging in physiological environment successfully demonstrated in the work of Bano et al for the detection of multiple proteins in standardized human serum [9], and successively by Bosco and Ganau et al, for the ultimate integration with a device for cell sorting, to measure the secretome of few selected cells [10]. By AFM nanografting we confined nanospots of thiol-modified single-stranded DNA monolayers inside a bio-repellent, self-assembled monolayer (SAM) of oligoethyleneglycol-terminated alkanethiols.…”

“…The density of the DNA-confined SAM can be tuned via nanografting, and adjusted to the steric requirements of the biorecognition elements. On such active DNA nanospots, the aptamer-cDNA conjugate is then loaded through Watson-Crick base-pairing, in a process known as DNA-directed immobilization [9]. On the other side, an electrochemical impedance-based biosensor, with a three-electrode design developed in our group for the recognition of nucleic acids [11,12], are tested towards protein detection through modified aptamers.…”