Bosch etching for the creation of a 3D nanoelectroporation system for high throughput gene delivery

Abstract

In order to create a high-throughput electroporation based cell transfection system, it is required that each cell has localized delivery and minimal membrane damage to ensure optimal transfection and longevity post-biomolecule delivery. To meet these requirements, a three-dimensional (3D) nanochannel device was fabricated on a Si platform due its ease of etching, wide industrial availability, and mechanical stability. The device is designed to shoot desired biomolecules into a seated array of target cells to achieve the high-throughput of bulk electroporation, but with greatly reduced cell mortality. To accomplish this, a wafer-scale Bosch etching process was optimized to etch a 3D array of channels consisting of larger microchannels feeding into smaller nanochannels that cells are ultimately seated on for transfection. The microchannel array consists of 50 μm wells spaced 50 μm apart, which are etched from the “back side.” The wafer is then flipped over to etch the smaller 650 nm channels on the “front side.” In the creation of the 3D silicon device, other feature sizes were explored, and their Bosch etching was characterized for comparison. The results show that when etching samples with the same feature sizes, but different densities, there was no relation between feature density and etch rate for our recipe. However, when etching features, or more specifically, circular channels of different sizes (650 nm–150 μm), the results show a positive correlation with etch rate (1.10–4.06 μm/min). Standard deviations indicate very uniform etching with an average value of 0.1 μm/min across all etches. After optimization, the 3D Si device was tested to ensure successful cell seating and transfection via electroporation, using fluorescence as the tool of evaluation. Fluorescent imaging (postelectroporation) indicates a transfection efficiency of approximately 70% with a cell viability of roughly 90%.

Recent Advances in Microscale Electroporation

“…Nonendocytic uptake of larger cargos, such as plasmids, into the cell improved dosage control and enabled more rapid transcription compared to delivery with bulk EP. Early studies used 2D NEP devices for precise and uniform delivery of macromolecules. − Higher-throughput 3D NEP systems have since been developed using microchannel , and nanochannel arrays (Figure A). ,,− The configuration of these devices is similar to previously described membrane devices, except that the subcellular channels here are more orderly. Subcellular channels are accessible from a single microchamber for parallelized cell to subcellular channel pairing.…”

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

“…iv) DAPI stain showing cell seating on porous array (scale bar = 100 µm).Reproduced with permission. [164] Copyright 2015, AIP Publishing. sensors, [79] and MEMS capacitive force sensors.…”

“…Porous substrates can consist of commercially available membranes with random pore distribution, [151][152][153][154][155][156][157][158][159] or uniform arrays of pores on silicon chips. [160][161][162][163][164][165][166][167][168][169][170] Like nanostructures, cells seal around the pores which limits the electric field exposure to discrete regions of each cell. Nanostructures may generate tighter seals at the membrane-substrate interface, but it has been shown that prior coating of substrates with extracellular matrix proteins can significantly enhance this seal in porous substrate-based methods to achieve high efficiency electroporation.…”

“…Pulse frequencies have been reported from 1-200 Hz. The number of pulses applied per train range from 1-2400 pulses, while the number of trains is generally one and HeLa cervical epithelial Poly-L-Lysine [151] or Fibronectin [151,154] >95% [151] >80% [151] [ 151,153,154,159] HL-60 Promyeloblast, suspended >90% 65% a) [155] HT1080 Connective tissue Fibronectin [152] 50% [153] [ 152,153,159] Jurkat T lymphocyte, suspended Poly-L-Lysine or Fibronectin, [151] PEG-Silane [163] >95% [151] 75% [151] [ 151,163] KG1a Promyeloblast, suspended PEG-Silane 96% [163] K562 Lymphoblast, suspended PEG-Silane [163] 92% [163] 83.4% [163] [ 163,164] MDA-MB231 Mammary epithelial Fibronectin >99% [152] 70% [152] [ 152,158] NK-92 Natural killer, suspended 90% 74% [165] Mouse NIH3T3 Embryonic fibroblast Poly-l-lysine or fibronectin [151] >95% [151] 75% [151] […”

Controllable Large-Scale Transfection of Primary Mammalian Cardiomyocytes on a Nanochannel Array Platform

“…The nanochannel array chip was made using a silicon wafer via clean room techniques (Figure S1, Supporting Information) . A standard double‐side polished wafer (4 in., 500 μm in thickness) was first thinned to 250 μm using wet etching (45% KOH, 80 °C).…”