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Identification of Biomolecules using Thermoplastic Nanofluidic Devices for Applications in Single-Molecule Sequencing
Rathnayaka, Chathurika
Rathnayaka, Chathurika
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Abstract
We are developing an innovative Single-Molecule Sequencing (SMS) strategy that consists of enzymatically cleaving intact RNAs using a processive enzyme to generate individual ribonucleotide monophosphates (rNMPs). This can be achieved using a processive enzyme, such as exoribonuclease 1 (XRN-1). We have recently shown that this enzyme can be tethered to a solid support and processively clip in the 5’ → 3’ direction an RNA strand into its constituent rNMPs when activated by the cofactor, Mg2+. The released rNMPs are then electrokinetically transported through a nanochannel one-at-a-time with the electrophoretic travel time through a nanometer column used to identify each rNMP. Therefore, a thorough understanding of the electrophoretic properties of the rNMPs through nanochannels made from thermoplastics that determine their molecular-dependent mobility will allow high identification accuracy of the rNMPs. In this study, the electrophoretic properties of the rNMPs were investigated in different nanochannel materials. Here, we thermally fusion bonded different plastic substrates containing nanochannels (100 nm × 100 nm, width × depth, and 100 μm in length) with a cover plate made from COC and the change in the electroosmotic flow was investigated by varying UV/O3 dosing time, which changed the amount of surface charge, after device assembly. Nano-electrophoresis of the rNMPs labeled with an ATTO-532 dye reporter were tracked using an epifluorescence microscope. Micro-electrophoresis of ATTO-532 tagged rNMPs were investigated but could not achieve baseline resolution for the rCMP/rAMP couple. Nanoscale electrophoresis of the dye-labeled rNMPs were explored in both poly(methyl methacrylate) (PMMA) and COC nanochannel devices and higher resolution (1.5) was achieved with the COC nanochannel device for all four rNMPs compared to PMMA. The results acquired for COC nanoscale electrophoresis indicated high identification accuracy (99%) of the rNMPs. Furthermore, we were able to separate the methylated rNMPs from their non-methylated counterparts, which will provide insight for identifying epitranscriptomal modifications using our SMS strategy. Our final exonuclease time of flight (X-TOF) nano sensor will utilize label-free rNMPs by using resistive pulse sensing (RPS) that used in-plane nanopores. Therefore, we investigated a simple method for tailoring the size of in-plane nanopores for sensing label-free rNMPs. The nano sensor consists of 2 in-plane pores that flank a nanometer flight tube (length = 5 μm; width × depth = 50 × 50 nm) fabricated in thermoplastics via replication technology. We could reduce the width and depth of the in-plane nanopores from ~30 × 30 nm to ~17 × 10 nm during the thermal fusion bonding (TFB) process, which placed a cover plate over the imprinted substrate under a controlled pressure and temperature to form enclosed nanofluidic devices. Increased pressures during TFB caused the size of the in-plane pore to be reduced. The in-plane nanopores fabricated with different TFB pressures were utilized to detect single λ-DNA molecules via RPS, which showed a higher current amplitude in devices bonded at higher TFB pressures. Using this method, we also showed the ability to tune the pore size to detect single rNMP molecules. Translocation of rNMPs through in-plane pores were initially explored using O2 plasma treated PMMA devices and 1× NEBuffer 3 at pH 7.9; low event frequency was observed due to a combination of ion exclusion and electroosmostic forces arising from surface carboxy groups. The surface carboxylic acid groups generated via O2 plasma activation was modified with ethanolamine via EDC/NHS coupling chemistry. Ethanolamine modification of thermoplastics was characterized by sessile water contact angle measurements and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The surface charge and electroosmotic flow (~10 fold) were found to be reduced upon ethanolamine modification of the PMMA surface. The event frequency of the dual in-plane nanopore sensor (60 events/s for 10 nM rAMPs) increased significantly upon ethanolamine modification. The average ToF, current blockage amplitude, and dwell time for rAMPs was 4.14 ± 0.97 ms, 425.89 ±175.89 pA, and 0.31 ±0.26 ms, respectively. Furthermore, we investigated geometrical effects on the sampling efficiency of rNMPs using the dual in-plane nanopore sensor. We showed increased capture efficiency with tapered geometries via both experimental and COMSOL simulations. We utilized the dual in-plane nanopore device with 5 µm and 10 µm long nano flight tubes and showed an increase in identification accuracy with increasing length of the nano-flight tube. Moreover, we generated scatter plots to identify rNMPs based on two variables and PCA plots showed correlation of each factor (peak amplitude, TOF, dwell time) in the identification of rNMPs. Furthermore, ablation of the PMMA substrate was demonstrated upon activation with UV/O3 light, which was not seen with COP nanofluidic devices. In addition, dual in-plane nanopore devices were fabricated in COP using injection molding, which showed the ability to be used in label-free identification of rNMPs based upon unique molecular-dependent TOFs. In addition, we studied the electrokinetic identification of peptides using thermoplastic nanochannels, which will be utilized in single molecule peptide fingerprinting. We performed nanoscale electrophoresis of peptides using different electrophoretic conditions, such as electric field strength, and material effects including modified surfaces. We used O2 plasma activated PMMA/COC, UV/O3 activated COC/COC, and ethanolamine modified PMMA/COC hybrid devices to perform nanoscale electrophoresis at different electroosmotic flow conditions. We also showed efficient identification of several peptides using free solution nanoscale electrophoresis via their molecular-dependent mobilities with efficiencies 99.99% in unmodified PMMA/COC and ethanolamine modified PMMA/COC nanofluidic devices.
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2022-05-31
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University of Kansas
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Analytical chemistry, Bioengineering, Nanoscience, Nanochannels, Nanofluidics, Nanopores, Peptide finger printing, RNA sequencing, Thermoplastic