Micropatterning at single-molecule resolution is a powerful approach for engineering advanced functional materials, including multiplexed bioassays for high-throughput applications. It enables researchers to investigate nanoscale mechanical and biomolecular interactions underlying biological processes such as receptor–ligand binding and motor protein activity. The ability to scale these experiments further facilitates the analysis of complex samples and the characterization of molecular heterogeneity. Consequently, there is a growing need for precise, accessible patterning techniques that can deliver rapid, cost-effective, and high-throughput results for research and development. 

As described in our latest eBook, DMD-based 2D photopatterning is a highly effective technique for high-resolution maskless micropatterning. Choi and colleagues recently demonstrated the potential of this approach in a study published in Small (2026), where they fabricated complex arrays of functional oligonucleotides with precise spatial control. The authors subsequently employed single-molecule force spectroscopy to validate these micropatterned constructs for high-throughput nanoscale biomechanical characterization and biomolecular analysis. 

Methods

Azide-functionalized coverslips were prepared for surface micropatterning by first immobilizing base oligonucleotides modified with DBCO-PEG13, which react with azide groups via copper-free click chemistry. Target oligonucleotides were then hybridized to the surface-bound strands and covalently patterned using 3-cyanovinylcarbazole (CNVK), a photoactive crosslinker that forms covalent bonds upon UV illumination. 

Micropatterning was carried out using a Polygon 1000 DMD module (Mightex) coupled to a 365 nm UV light source (Mightex) and integrated with a Nikon microscope equipped with a 20× objective. This configuration enabled high-resolution spatial patterning of oligonucleotides on the substrate. After UV exposure, non-crosslinked oligonucleotides were removed by denaturation and washing with formamide. The patterning process could then be repeated with additional target sequences to generate complex, multiplexed patterns. Using this approach, the authors achieved patterning resolutions as fine as 390 nm with a 20× objective. 

To demonstrate the functionality of the patterned surfaces, DNA-functionalized beads were introduced and captured through complementary DNA hybridization. The beads were tethered to DNA constructs containing sequences complementary to those immobilized during the micropatterning process, enabling selective binding to the patterned regions (Fig. 1). 

Figure 1. Surface micropatterning workflow. In the first step, base oligos were covalently conjugated to the surface, followed by hybridization with patterning oligos using CNVK and UV-coupled Mightex Polygon DMD for site-specific crosslinking. Oligos not exposed to UV were denatured and washed before repeating the process for multiple targets. Beads were then added and arranged based on the sequence of the complimentary crosslinked oligos. Figure adapted from publication. 

Main findings and conclusion

Because the lateral spacing between beads must be optimized for single-molecule force spectroscopy, the ability to digitally generate and modify patterns with a DMD offers a significant advantage over traditional photomask-based lithography. The authors demonstrated this flexibility by patterning hexagonal and square lattices with varying inter-bead distances. In addition, the programmability and precision of the method were showcased by patterning beads into the shapes of various letters and numbers, as well as by creating multiplexed patterns using multiple oligonucleotide targets and fluorescent bead populations. 

Single-molecule force spectroscopy was performed using magnetic tweezers, a technique in which magnetic beads are tethered to large DNA constructs known as “nanoswitches” and manipulated using an external magnetic field. Biomolecular interactions can be studied by attaching two complementary targets to different sites on the DNA nanoswitch and measuring the change in length between the paired state, which forms a looped structure, and the unpaired state, which returns the nanoswitch to its linear configuration (Fig. 2). This approach was used to measure the unzipping force required to separate DNA oligonucleotide pairs with varying guanine–cytosine (GC) content, which is positively correlated with binding strength. Similar experiments were also performed using hydrodynamic force spectroscopy, in which a flow cell replaces the magnetic stage, yielding results consistent with those obtained using magnetic tweezers. Overall, these findings demonstrate both the versatility of the methodology and the value of employing complementary techniques to generate robust measurements. 

Figure 2. Magnetic tweezer experiments conducted by bringing magnets to the sample with DNA nanoswitches and unzipping oligos with varying GC content (A). Force-extension curves of the DNA nanoswitch constructs with the left panel (green) showing switches with no unzipping oligos and the right panel (yellow) showing a loop size of 0.65 µm and 48% GC content (B). Hydrodynamic force spectroscopy with the same DNA nanoswitches used in the magnetic tweezer experiments where the positions of patterned beads are monitored under flow, which creates hydrodynamic forces (C). Figure adapted from publication. 

In conclusion, the authors presented a high-throughput workflow for rapid and precise surface micropatterning of biomacromolecules, along with a proof-of-concept study demonstrating its feasibility for single-molecule force spectroscopy. The versatile and programmable nature of DMD-based patterning enabled the fabrication of complex molecular architectures with nanoscale precision while supporting rapid design iterations and multiplexing. As the demand for scalable and customizable patterning techniques continues to grow, DMD-based photopatterning is poised to become an increasingly valuable tool for next-generation biofabrication, molecular diagnostics, and single-molecule analysis. 

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Michael Yitayew, PhD    Applications Scientist at Mightex