Microcontact printing with aminosilanes: creating biomolecule micro- and nanoarrays for multiplexed microfluidic bioassays

Abstract

Microfluidic systems integrated with protein and DNA micro- and nanoarrays have been the most sought-after technologies to satisfy the growing demand for high-throughput disease diagnostics. As the sensitivity of these systems relies on the bio-functionalities of the patterned recognition biomolecules, the primary concern has been to develop simple technologies that enable biomolecule immobilization within microfluidic devices whilst preserving bio-functionalities. To address this concern, we introduce a two-step patterning approach to create micro- and nanoarrays of biomolecules within microfluidic devices. First, we introduce a simple aqueous based microcontact printing (μCP) method to pattern arrays of (3-aminopropyl)triethoxysilane (APTES) on glass substrates, with feature sizes ranging from a few hundred microns down to 200 nm (for the first time). Next, these substrates are integrated with microfluidic channels to then covalently couple DNA aptamers and antibodies with the micro- and nanopatterned APTES. As these biomolecules are covalently tethered to the device substrates, the resulting bonds enable them to withstand the high shear stresses originating from the flow in these devices. We further demonstrated the flexibility of this technique, by immobilizing multiple proteins onto these APTES-patterned substrates using liquid-dispensing robots to create multiple microarrays. Next, to validate the functionalities of these microfluidic biomolecule microarrays, we perform (i) aptamer-based sandwich immunoassays to detect human interleukin 6 (IL6); and (ii) antibody-based sandwich immunoassays to detect human c-reactive protein (hCRP) with the limit of detection at 5 nM, a level below the range required for clinical screening. Lastly, the shelf-life potential of these ready-to-use microfluidic microarray devices is validated by effectively functionalizing the patterns with biomolecules up to 3 months post-printing. In summary, with a single printing step, this aminosilane patterning technique enables the creation of functional microfluidic micro- and nano-biomolecule arrays, laying the foundation for high-throughput multiplexed bioassays.

Microfluidic methods for aptamer selection and characterization

“…One innovation that has dramatically lessened the time- and labor-intensiveness of aptamer technologies is the incorporation of novel microfluidic strategies; no single advancement has led to as much improvement in speed, throughput, and automation in aptamer selection and characterization. (Microfluidics have also led to notable advances in aptamer-based sensing (aptasensors), especially involving electrochemical detection 38–41 and low-cost printed or paper-based devices; 42–44 however, microfluidic applications of aptamers will not be covered here. Aptasensors have been recently reviewed elsewhere.…”

Generation of Well-Defined Micro/Nanoparticles via Advanced Manufacturing Techniques for Therapeutic Delivery

“…Soft lithography is another advanced manufacturing technique that has been explored for producing micro/nano particles with controllable features [ 115 , 116 , 117 , 118 ]. Typically, the soft lithography technique employs a soft polymer stamp to print a monolayer of materials (i.e., polyelectrolytes, DNAs, proteins, nanoparticles, etc.)…”

Plasma-Assisted Large-Scale Nanoassembly of Metal–Insulator Bioplasmonic Mushrooms

“…To immobilize the capture IgGs onto the Au NI and NM substrates, we exploited the microcontact printing (µCP) technique (more details in the SI document): a simple method that involves transfer of biomolecules from a PDMS stamp onto higher surface energy substrates in controlled microscale features with high accuracy and reproducibility. 50,51 Figure 3 (Figure 3(b,c)). A complex pattern (OIST university logo) of IgGs was also successfully printed on NMs (Figure 3c), while a less uniform transfer of antibodies on the Au NIs substrate was achieved (Figure 3e).…”