Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules

Abstract

We report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. The in-plane pores were fabricated via nanoimprint lithography (NIL) from resin stamps, which were generated from Si masters. We could reduce the size of the in-plane nanopores from 30 to ∼10 nm during the thermal fusion bonding (TFB) step, which places a cover plate over the imprinted polymer substrate under a controlled pressure and temperature to form the relevant nanofluidic devices. Increased pressures during TFB caused the cross-sectional area of the in-plane pore to be reduced. The in-plane nanopores prepared with different TFB pressures were utilized to detect single-λ-DNA molecules via resistive pulse sensing, which showed a higher current amplitude in devices bonded at higher pressures. Using this method, we also show the ability to tune the pore size to detect single-stranded (ss) RNA molecules and single ribonucleotide adenosine monophosphate (rAMP). However, due to the small size of the pores required for detection of the ssRNA and rAMPs, the surface charge arising from carboxylate groups generated during O2 plasma oxidation of the surfaces of the nanopores to make them wettable had to be reduced to allow translocation of coions. This was accomplished using EDC/NHS coupling chemistry and ethanolamine. This simple modification chemistry increased the event frequency from ∼1 s–1 to >136 s–1 for an ssRNA concentration of 100 nM.

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection

“…We demonstrated label-less detection of single rCMP molecules by RPS from dual in-plane nanopores and obtained the molecular ToF from peak pairs, which produced signatures that could be used as a means for identifying molecules. 38 We also demonstrated selective binding of a single CAS9 RNA molecule to a solid phase bioreactor with the processive enzyme XRN1 surface immobilized via EDC/NHS coupling chemistry to the single sub-micron polymer pillar.…”

“…Feature sizes <30 nm have been achieved using NIL. 38 In addition, Uba et al used hot embossing to replicate structures into the thermoplastic PMMA that were ∼18 nm in size both in terms of depth and width. 35 While NIL can produce nanofluidic devices forgoing the need for doing direct photolithography and FIB milling to make each device, 35,39,40 it is a medium scale production method and as such, may not allow for the wide dissemination of nanofluidic devices into the research and commercial sectors.…”

“…27,54 The second device had multiscale structures that spanned the range from several 10's of mm to ∼50 nm. 27,55 It was used for selectively binding single fluorescently labeled RNA molecules to a processive riboexonuclease (XRN1) that was immobilized on a nanoscale solid phase reactor.…”

Coreactant-Free and Direct Electrochemiluminescence from Dual-Stabilizer-Capped InP/ZnS Nanocrystals: A New Route Involving n-Type Luminophore

“…As shown in Scheme , InP/ZnS MPA‑MSA -labeled Ab 2 (Ab 2 |InP/ZnS MPA‑MSA ) was prepared with the assistance of 1-(3-(dimethylamino)­propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS). , The coreactant-free ECL immunosensor with InP MPA‑MSA as the ECL tag and PSA as the analyte, i.e., GCE|ABA-Ab 1 <PSA>Ab 2 |InP/ZnS MPA‑MSA , was fabricated via a typical sandwich-immunoassay procedure by immobilizing Ab 1 onto the GCE surface with p -aminobenzoic acid (ABA) as the linker (Scheme B). , ABA was covalently immobilized onto the GCE surface to form GCE|ABA via electrochemical oxidation . Then, the carboxyl groups of ABA were activated with 20 μL of 100 mg·mL –1 EDC/NHS for 30 min and incubated with 10 mM pH 7.4 phosphate-buffered saline (PBS) containing 10 μg·mL –1 capture antibody (Ab 1 ) for 3 h to form GCE|ABA-Ab 1 .…”

In‐plane Extended Nano‐coulter Counter (XnCC) for the Label‐free Electrical Detection of Biological Particles

“…The chromium layer was removed using a chromium etchant (Sigma-Aldrich). Finally, the extended nano-dimensional features of the XnCC were fabricated by focused ion beam (FIB; Quanta 3D Dual Beam System, FEI) milling using Ga ions [36][37][38][39]. Structures were milled with a beam current of 48 pA and a time of 1 μs.…”