Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 μm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Nanoimprint lithography (NIL) fabrication of dual in-plane nanopore sensors.Fabrication of dual in-plane nanopore sensors is shown in Figure S1A.Si wafers that were 100 nm thick and possessed a silicon nitride (Si 3 N 4 ) layer on each side were used for fabricating the Si master mold.Microchannels were fabricated using photolithography and wet-chemical etching.This was done by placing a 1.3 µm thick S1813 photoresist layer spin-coated at 4,000 rpm for 60 s on the Si wafer followed by baking at 115°C for 60 s.Photolithography was performed using the appropriate photomask and a UV exposure station (Quintel) in a class 100 cleanroom.UV exposure was carried out at 130-140 mJ/cm 2 with a post-exposure bake at 95°C for 60 s.The wafer was then subjected to development using a MF319 developer followed by washing with deionized water.
The exposed Si 3 N 4 layer was etched using ICP-DRIE (Plasmalab System 100, Oxford Instruments, Abingdon, UK).The wafer was then placed in a 40 wt% KOH bath with IPA (5 % v/v; 70°C).After 25 min etching to form 5 µm deep microchannels, the wafer was removed from the KOH bath, rinsed with water, and dried with N 2 .Prior to FIB milling, the Si 3 N 4 layer was removed using a dilute HF solution.The nanostructures comprising the sensor were fabricated using FIB (Quanta 3D Dual Beam system, FEI, Hillsboro, OR), which was performed at a beam voltage and current of 30 kV and 10 pA, respectively, in a bitmap mode.
The Si master mold with the appropriate microstructures and nanostructures was used to produce a resin stamp using a UV resin solution (70 wt% TPGDA, 28 wt% TMPTA, and 2 wt% photoinitiator).Drops of the UV-resin were dispensed onto the Si master mold and a flexible PET sheet coated with an adhesive layer (NOA72) was then slightly pressed against the liquid drop and used as a backbone for the resin stamp.Residual resin solution and air bubbles were gently removed.During the curing process, the sample was exposed to a flash-type UV light (250-400 nm) for 20 s at an intensity of ~1.8 W/cm 2 using UV nanoimprint lithography, NIL (Eitre6, Obducat, Lund, Sweden).After UV-curing, the molded UV-resin/PET backbone was demolded from the Si master.See Figure S1B for a process flow diagram.Nanopore devices were thermally imprinted into the appropriate plastic substrate using thermal NIL (Nanonex 2500, Monmouth Junction, NJ). 1 The optimized imprinting conditions were 145C, 300 psi, and 5 min for PMMA nanofluidic devices, and 130C, 300 psi, and 5 min for COP devices.Imprinted nanofluidic devices were then characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM).NIL produced devices were sealed using a COC 8007 cover plate (see Figure S1D) via thermal fusion bonding.Both the substrate and cover plate were exposed to UV light (20 mW/ cm 2 ) for 3 min or an O 2 plasma (1 min) to activate the surface before thermal fusion bonding.The device was assembled using the Nanonex 2500 nanoimprint lithography (NIL) machine.In Figure S2A is shown the relative voltage drop through the dual in-plane nanopore sensor along with the open pore current at an applied voltage of 2.5 V while in Figure S2B is shown the equivalent electrical circuit of the nanofluidic device that consisted of several resistors in series with a capacitor in parallel to each pore resistance.To determine the fluidic circuit's bandwidth, f C , we needed to calculate R T and C T (see equation 3 in the main text).R T was determined from the 3 resistors in series, namely R w1 , R nc , and R w2 , but excluded R p1 and R p2 .The value for each resistive element was deduced from the applied voltage (2.5 V) and the current through the system (8.4 nA) using Ohm's law.The values for the relevant elements are shown in Figure S2C with RT shown as well.For CT, we calculated the capacitance for each pore (CS or Cp1 and Cp2) using CT = 1/C p1 + 1/C p2 ; if C p1 = C p2 , then C T = C p /2. C p for pore 1 or pore 2 was calculated using;

𝑑
where d is the average spacing between the capacitor plates (10 nm), A p is the area of the capacitor plates (4.0 × 10 -15 m 2 ), ε 0 is the permittivity of free space (8.85 × 10 -12 F/m), and ε r is the material's relative dielectric (COP, ε r = 2.2; PMMA, ε r = 3.9).In both cases, the PSDs were performed using 1× NEBuffer 3 (pH = 7.9) with an applied voltage to the device equal to 2.5 V.The data was subjected for both plastics to a 15-point moving average filter.The apparent peak in the PSD at f > 10 4 is due to interaction of the in-plane pore capacitance with that of the Axopatch current amplifier.Finally, the spiking present in the PSD for the PMMA device is likely due to signal aliasing -we are correcting for this in the future using the appropriate anti-aliasing filter.

Figure S1 .
Figure S1.Fabrication steps of nanofluidic devices using NIL.(A) Schematic showing the production of the Si master mold, which used a combination of photolithography (microstructures) and focused ion beam milling for the nanostructures.(B) Process strategy for making resin stamps from the Si master mold using UV-NIL.(C) Thermal NIL for producing finished devices in the appropriate thermoplastic using the resin stamp.(D) Strategy for thermal fusion bonding a low Tg cover plate (COC 8007) to the imprinted device, which possessed a higher Tg than the cover plate.

Figure S2 .
Figure S2.(A) Fluidic circuit showing the relative voltage drop across each element of the circuit (not included is the microfluidic network).The numeric value is deduced from the percentage of voltage drop (V) across each element of the circuit and the applied voltage across the fluidic circuit.The open pore

Figure
Figure S3.(A) High resolution SEM of an in-plane pore taken from the device shown in Figure 1 of the main text.(B) Histogram of baseline current (i.e., open pore current, I 0 ) of dual in-plane nanopore sensors used for rNMP translocation experiments (1× NEBuffer 3 at pH 7.9) and an applied voltage of 2.5 V (n = 34).

Figure S4 .
Figure S4.Power spectral density (PSD) for a COP and PMMA dual in-plane nanopore sensor device.In both cases, the PSDs were performed using 1× NEBuffer 3 (pH = 7.9) with an applied voltage to the device equal to 2.5 V.The data was subjected for both plastics to a 15-point moving average filter.The apparent peak in the PSD at f > 10 4 is due to interaction of the in-plane pore capacitance with that of the Axopatch current amplifier.Finally, the spiking present in the PSD for the PMMA device is likely due to signal aliasing -we are correcting for this in the future using the appropriate anti-aliasing filter.