ABSTRACT
Figure 28.1 Schematic illustration of (A, B) direct-write and (C, D) indirect-write DPN. Patterning of (A) single or (B) multiple biomolecules with direct-write DPN. Indirect-write DPN encompasses (C) the patterning of template molecules and (D) then adsorption of targeted biomolecules. Although considerable success has been achieved with direct-
write DPN, the search for optimal writing conditions, specifically for new biomolecules, is tedious. Therefore, an alternative approach, that is, indirect-write DPN, is introduced. Typically, in indirect-write DPN, one kind of molecule was first deposited on substrates by DPN, which acts as templates to adsorb the targeted biomolecules, as shown in Fig. 28.1C,D. For example, the previous report of protein patterning was achieved by indirect-write DPN [17]. In their work, 16-mercaptohexadecanoic acid (MHA) was first patterned on a gold substrate, followed by passivation of unpatterned regions with 11-mercaptoundecyl-tri (ethylene glycol). Proteins including immunoglobulin G (IgG) and lysozyme were then selectively adsorbed on MHA-patterned areas to form protein nanoarrays. It is known that various interactions, such as physical
adsorption, electrostatic force, and chemical bonding [18], could be used to govern the selective adsorption of targeted molecules onto patterned templates. Therefore, the judicious choice of combination of templates and biomolecules will permit the patterning of a large library of biomolecules, which is the remarkable advantage of indirect-write DPN. In the following sections, the application of direct-and indirect-write DPN for patterning biomolecules will be introduced. 28.3 Applications in Biological Systems
Creating DNA or oligonucleotide patterns at the nanometer scale is important, considering their wide range of applications, such as genome sequencing [19]. With the advent of parallel DPN [20], it is now possible to fabricate DNA nanoarrays on a chip scale. The direct write of oligonucleotides on metals and insulates [11] by DPN was first achieved by Demers et al. in 2002. In their work, they have identified several key factors that facilitate DNA patterning. First, the AFM tip modified with 3’-aminopropyltrimethoxysilane (APTMS) is critical for coating of DNA. Second, the judicious choice of the DNA-substrate combination is essential for transfer of DNA. The DNA functionalized with thiol groups was patterned on a gold substrate. While in the case of an insulating silicon oxide (SiO2) substrate, the substrate was modified with 3’-mercaptopropyltrimethoxysilane (MPTMS) and the DNA was modified with 5’-terminal acrylamide groups instead of thiol. Feature sizes ranging from many micrometers to sub-100 nm were realized. Moreover, the resulting DNA patterns exhibit specific-binding properties, which are used to direct assembly of complementary DNA-modified gold nanoparticles. It is worthwhile to mention that patterning of multiple-DNA inks was also achieved. In later studies, the orthogonal assembly was used to assemble complementaryDNA-modified nanoparticles onto DNA nanopatterns [21, 22]. In a recent study [23], Chung et al. demonstrated that DPN can be used to interface a DNA-directed nanoparticle assembly, which could be used to produce tunnel junction circuits. Their strategy could be
extended to biosensors based on recognition processes. Indeed, Li et al. applied a similar approach to functionalize electrical gaps with capture single-stranded DNA (ssDNA) by DPN, which was used for the multiplexed target ssDNA detection by measuring the electrical gap resistance change when the target ssDNA was introduced [24]. A lowest detection limit of 10 pM was achieved. Most recently, agarose-assisted DPN was developed to improve the patterning quality of oligonucleotides and proteins [25]. Nyamjav et al. have succeeded in fabrication of DNA nanopatterns by indirect-write DPN [26]. In their work, DPN was first used to generate surface templates composed of positively and negatively charged regions by patterning polyelectrolyte inks on a SiO2substrate. Then molecular combing was employed to stretch and align DNA on these templates. Positioning long-stretched DNA on surface templates was also achieved. 28.3.2 ProteinsProteins are one of the most important biological molecules. Proteins are the building blocks for cells and functional units responsible for most of the biological processes within cells. Protein nanoarrays are important as they offer platforms for proteomics, address fundamental problems regarding cell-substrate interactions and cell migration, and answer questions related to biorecognition. The high resolution and registration capabilities of DPN make it a suitable tool to fabricate large-area protein nanoarrays with a controlled size. The Mirkin group first reported the generation of protein nanoarrays by indirect-write DPN [17]. In their work, protein nanoarrays were achieved by adsorption of proteins on DPN-patterned MHA gold surfaces via high affinity of proteins toward carboxylic acid-terminated monolayers. Biological recognition processes were demonstrated by screening antibody-antigen reactions based on the height change measured with AFM. Importantly, these protein arrays were used to study the cellular adhesion at submicrometer scales. Most recently, Wu et al. described another indirect method to generate protein nanoarrays with DPN by using Ni (II) ion templates to immobilize proteins via a specific metal-protein interaction [27]. In brief, a nitrilotriacetic acid (NTA)-terminated self-assembled monolayer was first formed on glass slides. Ni (II) was then patterned
on NTA via DPN or microcontact printing. Finally protein arrays were formed by immersing the patterned Ni (II) glass slides in the 6His-tagged protein solution. The chelating chemistry was also employed in the work reported by Kim et al. [28]. Interestingly, by taking advantage of this specific chelating chemistry between Ni (II) and histidine, His-tagged proteins can be selectively immobilized onto NTA/Ni(II) patterns from cell lysates. Apart from other advantages mentioned by the authors, the most significant one is the realization of selective protein immobilization without purification of the cell lysates. The above examples demonstrate the versatility of indirect-write DPN in generating protein nanoarrays. Besides indirect-write DPN, protein nanoarrays could also be easily fabricated with direct-write DPN. The success of the first report of direct-patterning proteins involves the proper chemical modification of both AFM tips and substrates [12, 13]. For example, in order to achieve the satisfactory adsorption of proteins onto tips and easily transfer the adsorbed proteins to substrates, Lim et al. modified a Si3N4 AFM tip with 2-[methoxypoly(ethyleneoxy)propyl]trimethoxysilane (Si-PEG) [13]. The silicon oxide substrate was modified with aldehyde group-terminated silanes to promote the protein transfer. In addition to the early efforts, the research community had witnessed a few exciting development for the direct write of proteins by DPN. In one of the reports, Wu et al. fabricated a porous multilayer-coated AFM tip, which shows capability to adsorb aqueous fluorescent protein solutions and retain their activity for an extended period of time [29]. Figure 28.2A shows the procedure for preparation of such a porous tip. The thickness and pore size of the porous film may be easily adjusted by controlling the cycle of the layer-by-layer process. Importantly, the pore structures provide a large-volume ink reservoir for DPN experiments. His-enhanced green fluorescent protein (EGFP) nanopatterns were successfully fabricated with these porous tips, as shown in Fig. 28.2B. In another report, Senesi et al. developed matrix-assisted DPN, in which agarose was used a matrix that acts as a carrier to encapsulate various biomolecules, for example, protein [25]. The authors showed that agarose was an effective matrix to control the deposition process of biomolecules in DPN, thus providing additional control in the DPN process apart from the control of tip-substrate contact time and humidity.
