WO2020194303A1 - Nanopore fabrication - Google Patents

Nanopore fabrication Download PDF

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Publication number
WO2020194303A1
WO2020194303A1 PCT/IL2020/050356 IL2020050356W WO2020194303A1 WO 2020194303 A1 WO2020194303 A1 WO 2020194303A1 IL 2020050356 W IL2020050356 W IL 2020050356W WO 2020194303 A1 WO2020194303 A1 WO 2020194303A1
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Prior art keywords
membrane
layer
light
laser
spot
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PCT/IL2020/050356
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French (fr)
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WO2020194303A9 (en
Inventor
Amit Meller
Tal Gilboa
Adam ZREHEN
Arik GIRSAULT
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Technion Research & Development Foundation Limited
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Application filed by Technion Research & Development Foundation Limited filed Critical Technion Research & Development Foundation Limited
Priority to EP20777890.3A priority Critical patent/EP3947730A4/en
Publication of WO2020194303A1 publication Critical patent/WO2020194303A1/en
Priority to US17/482,904 priority patent/US20220009034A1/en
Publication of WO2020194303A9 publication Critical patent/WO2020194303A9/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0039—Inorganic membrane manufacture
    • B01D67/0053—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0039—Inorganic membrane manufacture
    • B01D67/0053—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes
    • B01D67/006—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/009—After-treatment of organic or inorganic membranes with wave-energy, particle-radiation or plasma
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/10—Supported membranes; Membrane supports
    • B01D69/108—Inorganic support material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/02—Inorganic material
    • B01D71/0215—Silicon carbide; Silicon nitride; Silicon oxycarbide
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/02—Inorganic material
    • B01D71/024—Oxides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/02—Inorganic material
    • B01D71/024—Oxides
    • B01D71/025—Aluminium oxide
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00023—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems without movable or flexible elements
    • B81C1/00087—Holes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/34—Use of radiation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/42—Details of membrane preparation apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/04—Characteristic thickness

Definitions

  • the present invention is in the field of nanopore fabrication.
  • ssNPs solid-state nanopores
  • the development of synthetic solid-state nanopores (ssNPs) as a substitute for biological channels remains a major focus in nanotechnology given their greater flexibility in terms of size, shape, surface properties, and cross-device compatibility. While traditionally the principal mode of single-molecule detection was based on ionic resistive pulsing measurements, a rapidly growing trend in the nanopore community has been towards “electro-optical” sensing. The simultaneous measurement of the electrical (ion current) and fluorescence signal (photon emission) extends the scope of biomolecular targets for nanopores and opens up new applications since both multiple fluorophore colors and varying photon intensities can be acquired to obtain specific information on the molecule of interest.
  • ssNPs can be applied to DNA sequencing, DNA barcoding, epigenetic modification analysis, DNA methylation quantification and polypeptide discrimination.
  • electro-optical sensing brings its own set of fabrication challenges. Nanopores must be prepared in a way such that their position can be readily identified in situ. Furthermore, the peripheral structure heavily impacts the background noise and fluorescent signal of a translocating molecule.
  • CBD which uses an applied voltage to induce randomly accumulating material defects, is nonetheless comparatively less flexible and efficient at localizing nanopore formation.
  • Recent attempts to do so relied on the principle that nanopores preferentially form at the hotspot of an infrared (IR) laser or at the thinnest membrane cross-section.
  • IR infrared
  • milling or lithographic steps were implemented upstream of CBD as a preparatory step to direct nanopore formation.
  • IR laser was complicated by the need to simultaneously control the applied voltage and laser power, as the IR laser only enhanced the local DC field necessary for dielectric breakdown and did not independently form nanopores.
  • Fast, highly-reproducible, in situ methods of fabricating nanopores and nanopore arrays are greatly needed.
  • the present invention provides systems comprising a light source, a membrane and a system to direct and focus light from the light source to a spot on the membrane.
  • Methods of light- induced thinning and etching a membrane and generating a nanopore in a membrane are also provided.
  • Membranes comprising a nanopore with a Gaussian curve shaped cross- section are provided as well.
  • a method of thinning a membrane comprising a first layer comprising an index of refraction of greater than 2.0, the method comprising shining focused light on a spot on the first layer, thereby thinning the membrane.
  • a method of thinning a membrane comprising shining pulsed laser light on a spot on the membrane, thereby thinning the membrane.
  • the focused light is laser light and the laser light is at a wavelength of between 300 and 600 nm.
  • the pulsed laser light is at a wavelength of between 300 and 600 nm.
  • the focused light is within tlR5 ⁇ i p ? ⁇ 2 S/S£3 ⁇ 4F g reen spectrum.
  • the light comprises an intensity of at least 100 pW.
  • the laser light is continuous-wave laser light or pulsed laser light.
  • the membrane comprises a first layer comprising an index of refraction of greater than 2.0.
  • the index of refraction is greater than 2.20.
  • the first layer comprises silicon nitride (SiNx).
  • the first layer is a SiNx layer comprising an average silicon to nitrogen ratio of greater than 0.75.
  • the average silicon to nitrogen ratio is greater than
  • the membrane is a freely standing membrane, covered by an aqueous solution on both sides.
  • the membrane comprises a second layer refractory to thinning by the focused light when not layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, and wherein the second layer is layered onto the first layer.
  • the second layer is a layer of metal oxide and wherein the metal oxide is titanium oxide (Ti02), aluminum oxide (A102) or hafnium oxide (Hf02).
  • the membrane does not comprise a thickness of less than 20 nm.
  • the membrane comprises a thickness of less than 20 nm.
  • the membrane is immersed in ultrapure water or salt buffer comprising an alkaline pH.
  • ⁇ SL3 ⁇ 4* j 20 (4?i3 ⁇ 43 ⁇ 4ing to some embodiments, the membrane is at room terfpcf(3 ⁇ 4l?£3 ⁇ 4?/u 5 p?5asure.
  • the method further comprises measuring photoluminescent (PL) intensity from the spot on the membrane.
  • PL photoluminescent
  • the PL intensity is inversely proportional to the thickness of the spot on the membrane, and the thinning is halted at a desired thickness based on a measured PL intensity.
  • the thinning comprises forming a pore through the membrane.
  • the pore is a nanopore.
  • the membrane is covered in an aqueous solution and the method further comprising measuring ionic current through the membrane; optionally, wherein an increase in ionic current through the membrane indicates the pore has been formed in the membrane.
  • the spot in the membrane comprises a thickness of at least 40 nm before the shining and the pore can be produced though the spot in the membrane in less than 20 seconds.
  • the thinning comprises widening a pore through the membrane.
  • the membrane is covered in an aqueous solution and an increase in ionic current through the membrane is proportional to a widening of the pore.
  • the method is for producing a pore of a given diameter, wherein the focused light is automatically shut off at a predetermined current.
  • a system comprising: a. a light source;
  • a membrane comprising a first layer comprising an index of refraction of greater than 2.0;
  • the membrane is in an optically accessible flow cell. 'YSt 2 * j 20 (4?l3 ⁇ 43 ⁇ 4ing to some embodiments, the membrane is a freel ⁇ 3 ⁇ 4o?MP 20 u?v9 ⁇ ?urane, covered by an aqueous solution on both sides.
  • the index of refraction is greater than 2.20.
  • the system further comprises a photodetector, wherein the photodetector: a. is capable of measuring low light intensities and/or measuring at high temporal resolution;
  • b. is an avalanche photodiode, a photo -multiplier tube or a CMOS camera; or
  • c. is configured to detect emissions from the spot on the membrane.
  • the light source is at least one of: a. a solid-state or gas lasers configured to emit within the purple, blue or green spectrum;
  • a solid-state laser configured to emit at between 300-600 nanometers (nm);
  • d. configured to produce light at an intensity of at least 100 micro-watts (pW) at the spot on the membrane
  • e. configured to produce light at an intensity of at least 1 milliwatts (mW) at the spot on the membrane.
  • the system further comprises an imaging sensor, optionally, wherein the imaging sensor is selected from an electron multiplying CCD camera, a CMOS camera and a sCMOS camera.
  • the first layer comprises SiNx and comprises a silicon to nitrogen ratio of greater than 0.75.
  • the silicon to nitrogen ratio is greater than 0.80.
  • the membrane does not comprise a thickness of less than 20 nm, comprises a thickness of less than 20 nm, is at room temperature and pressure, or a combination thereof.
  • the membrane is immersed in ultrapure water or salt buffer at an alkaline pH. 'YS ) -r3 ⁇ 4 j 20 (4?l3 ⁇ 43 ⁇ 4ing to some embodiments, the system further compril ⁇ T /i v 3 ⁇ 4?®?Si 0 uSu3 ⁇ 4 and an apparatus configured to pass an electric current between the two electrodes, wherein one electrode is positioned on one side of the membrane and a second electrode is positioned on another side of the membrane, optionally, further comprising a current detector configured to measure current between the two electrodes.
  • the membrane further comprises a second layer layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, optionally wherein the metal oxide is Ti02, A102, or Hf02.
  • a membrane comprising a nanopore, wherein the membrane comprises a first layer comprising an index of refraction of greater than 2.0 and wherein the nanopore comprises a varying diameter and a Gaussian curve shaped cross-section.
  • the index of refraction is greater than 2.20.
  • the nanopore increases in diameter from one side of the membrane to the other, and wherein the increasing diameter follows a Gaussian curve.
  • the membrane produces a lower optical background at the nanopore than a nanopore in the membrane without a Gaussian curve shaped cross-section or not produced by a method of the invention.
  • the first layer comprises SiNx and wherein the SiNx comprises a silicon to nitrogen ratio of greater than 0.75.
  • the silicon to nitrogen ratio is greater than 0.8.
  • the membrane further comprises a second layer layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, optionally wherein the metal oxide is Ti02, A102 or Hf02.
  • the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from an interface of the first layer with the second layer to an exposed surface of the first layer and a second Gaussian curve shaped cross-section increasing in diameter from the interface to an exposed surface of the second layer.
  • the membrane comprises with different indexes of refraction.
  • FIGS 1A-D Laser thinning of freestanding SiN x .
  • (1C) Photoluminescence emission during laser-exposure, measured in counters per second. The laser is activated at t 0 seconds.
  • ID Images of the 42 x 42 pm 2 membrane under white- light illumination before etching (i). After 300 seconds of laser exposure, a thin region is visible as a contrasted spot (ii).
  • FIG. 1 Photoluminescence (PL) intensity calibration as a function of SiN x thickness.
  • the PL in counts per second (CPS) was measured by the APD during laser- exposure (488 nm, 30 pW) for 6 chips of different membrane thickness. Prior to the PL measurements, the membrane thickness was measured by ellipsometry. Thicker membranes result in higher PL.
  • FIG. 3A-E Thickness characterization of a laser-etched spot.
  • (3D) Blue curve- TEM thickness map Purple curve- normalized photoluminescence (PL) scanned in the x direction with a 30 nm step size. m u 1 ated normalized PL based on a convolution of ⁇ T u3 ⁇ 4?Pa9/i ⁇ 93 ⁇ 4 iited Gaussian, representing the laser beam, with the TEM thickness map.
  • FIGS 4A-B Nanopore fabrication by laser-etching.
  • (4A) Measured photoluminescence (PL) and ionic current during laser-exposure (red and grey curves, respectively). The PL sharply increases when the laser is activated (i). Pore formation is signaled by an increase in current (ii). Following ⁇ 20 s of pore growth under continued laser- exposure, the laser is deactivated, and the PL returns to zero (iii). Turning off the laser causes a conductivity decrease, resulting in a coincident drop in current which stabilizes over time.
  • (4B) Principle of calcium (Ca 2+ ) activators used for verifying the creation of a nanopore (top panels). The entire membrane is illuminated by a 488 nm laser.
  • FIG. 5A-N Noise and functionality of a laser-etched nanopore.
  • 5A TEM image showing a nanopore with a diameter of 6.5 nm. Compared to the peripheral membrane, the nanopore is very bright, owing to an unobstructed electron beam path.
  • 5B Three examples of measured photoluminescence (red curve) and ionic current (grey curve) during laser- exposure. A nanopore was formed after (top) 400 s (middle) 920 s and (bottom) 140 s.
  • 5C Power spectral density (PSD) plot of a nanopore for an applied bias of 300 mV. The inlet shows the corresponding current-voltage (IV) curve for this nanopore, with a linear fitting (R 2 > 0.99).
  • the trans chamber was biased to 300 mV to drive translocation of 300 pM 5054 bp dsDNA from cis to trans.
  • the size of the pore is 3.1 + 0.3 nm based on the current blockage level/molecular ruler model.
  • (5E) A concatenated ionic current trace showing sample dsDNA translocation events.
  • (5F-H) Translocations of 5054 bp DNA at different applied voltages: 300 mV (red), 450 mV (orange) and 600 mV (yellow).
  • FIGS 6A-D Localized laser-etching of freestanding SiN x .
  • (6A) Laser-etched T- shape array of 9 thin regions spaced 1500 ⁇ 50 nm center-to-center. The top and vertical bars were etched with a laser intensity of ⁇ 30 mW and 45 mW, respectively, for 4 minutes each. Next to the T is a lithography-fabricated thin region (20 ⁇ 2 nm) for comparison.
  • (6B) Zoom in of just the T.
  • FIGS 7A-E Nanopore fabrication by laser-etching.
  • (7A) Schematic illustration of the electro-optical apparatus used for laser-assisted nanopore drilling.
  • Chips with refractive index of 2.3 and initial thickness of 38-42 nm were immersed in 1M KC1 buffer with pH of 7 or 10 heated to 90°C in a temperature- controlled hot water bath.
  • the SiN x thicknesses of 4 different chips were measured using ellipsometry after 2, 5, 30 and 60 minutes (pH 7 - blue triangles, pHIO - green tringles).
  • the low refractive index chip (2.15) could not be drilled at these laser intensities even after 10 minutes. Illuminating the chips with the red laser (640 nm) did not result in thinning or drilling in either case.
  • (8D) Average PL values for four different types of chip (n 2.15, 2.2, 2.3, 2.43) of similar thickness (44-46 nm). Lower refractive index corresponds to higher PL (dark grey marks). The results are fitted to exponential curves (dark grey solid line). Light grey triangles present the ratio of the red- band emission over the total emission which increases as the refractive index increases. This is asscociated with a red-shift of the PL at the higher Si:N ratio. Each measurement was repeated using 4 chips of each type.
  • FIG. 9 Normalized PL traces of membrane thinning using different laser intensities (1.9 to 7 mW) and the four pH values (pH 4 - red, pH 7 - green, pH 10 - blue, pH 12 - purple). While acidic and neutral buffers (pH 4 and 7) hardly change the PL behavior even when the intensity is increased, the hydroxyl-rich solutions (pH 10 and 12) increase the effectiveness of the etching, producing thinner membranes at high speed.
  • FIGS 11A-D Characterization of the ultra-fast drilling process.
  • the PL decays over time as the membrane is thinned until pore formation, signaled by an increase in the electrical current.
  • the inset shows the current- voltage (IV) curve for this nanopore after the buffer was changed to pH 7 and the open pore current stabilized.
  • FIGS 13A-C Fast drilling of nanopores array using a focused laser beam.
  • 13A Top: laser intensity time trace during nanopore array drilling. The laser power is switched automatically on and off for each drilling event, followed by controlled movement of the piezo stage to the next coordinate.
  • 13B Histograms of the drilling time and the change of current for each pore, and Gaussian fitting for each histogram (13.6+ 3.1 seconds, 1.9 + 0.9 nA)
  • 13C Wide-field illumination images of the entire membrane using 488 nm laser.
  • Ca +2 activated fluorophores are used for verifying the creation and position of the nanopore array.
  • Ca +2 passes through the pore and binds to Fluo- ⁇ -9 3 ⁇ 4S9 i? g 3 ⁇ 49 3 a fluorescence spot at the thin region (middle panel).
  • Th?? p Z3 ⁇ 4?93 ⁇ 4 pp 5S?f ⁇ hen the bias is reversed to -300 mV (left panel).
  • the membrane position is outlined by a white dashed line.
  • the histogram in the right panel describes the normalized intensity distribution of the 25 pores.
  • Grey curve is a Gaussian fit (0.97+ 0.18).
  • FIG. 14A Cartoon of drilling in a membrane comprising SiN x and Ti0 2 .
  • the bottom layer is the SiN x and the top layer is the T1O2.
  • Laser light shown on the silicon layer catalyzes drilling in the titanium layer which had previously been refractory to drilling. Acidic pH on the titanium side inhibits this catalyzation, whereas neutral or alkaline pH is permissive.
  • (14B) Line graph of the atomic fraction of each component of the membrane at the site of drilling as a function of time.
  • the present invention in some embodiments, provides systems for light-induced etching a membrane and/or producing a nanopore in a membrane. Methods of thinning and etching a membrane are also provided, as are membranes comprising a nanopore with a Gaussian curve shaped cross-section.
  • the present invention is based on the unexpected finding that the inventors could perform purely photo-chemical solid-state nanometer-scale pore fabrication with unparalleled in situ control over the nanopore position.
  • Local SiNx thinning and subsequent pore formation are performed at any arbitrary point along the membrane by simply positioning the membrane at the tightly focused laser spot.
  • the T was made next to a lithography-fabricated thin region (20 + 2 nm) used for thickness calibration and to further prove that the produced contrast is due to thinning (Fig. 6A).
  • the etch time of ⁇ 4 minutes per spot was more than enough to produce the visible contrast; under TEM inspection, we found that a ⁇ 20 nm nanopore had formed in one of the spots (Fig. 6D).
  • the etch rate is also highly controllable. To show this, we varied the laser intensity in constructing the T: ⁇ 30 mW and 45 mW for the top and vertical bars, respectively.
  • nanopores With a blue or green laser and a membrane in water, nanopores can be formed optically in as little as one minute in 45 nm thick SiN x , using a purple laser or increasing pH allowed for significantly faster pore formation.
  • the technique is rapid and highly automatable— it can be monitored by the PL intensity and ionic current— it can be used to construct vast nanopore arrays for massively parallel optical sensing.
  • nanopore ? ⁇ 3 ⁇ 4* /’ p ieced s so quickly, it would not be necessary to compri’i ⁇ i ⁇ i ⁇ /S ⁇ lness of the supporting membrane, as might be necessary using thickness-limited strategies such as CBD.
  • these nanopores benefit from significantly improved spatial resolution, reduced background PL and improved mechanical robustness.
  • both the etching and nanopore drilling kinetics can be accelerated by orders of magnitudes using higher Si to N ratio membranes, measured as a slight increase in their index of refraction.
  • a change in the index of refraction of SiN x membranes from -2.20 to -2.40 corresponded to a transition from a practically non drilling membrane, even after nearly an hour of exposure, to nearly instantaneous nanopore formation.
  • drilling yielded single 5 nm pores in 15 seconds from a starting 45 nm thick substrate.
  • pulsed laser light was superior to continuous- wave laser light for thinning and drilling. Shone with the same average power and at the same wavelength, pulsed laser light could thin and drill faster than continuous-wave laser light. Even some membranes that were resistant to etching with a continuous-wave laser could be etched with a pulsed laser.
  • a system comprising, a light source, a membrane, and a system to direct and focus light from the light source to a spot on the membrane.
  • a system comprising a light source, an area configured to receive a free-standing membrane, and a system to direct and focus light from the light source to a spot on a received free-standing membrane.
  • the light source produces coherent light. In some embodiments, the light source produces collimated light. In some embodiments, the light source produces coherent and collimated light. In some embodiments, the light source produces a coherent and collimated light beam. In some embodiments, the light source is a laser or light emitting diode (LED). In some embodiments, the light source is a laser. In some ! 3 ⁇ 4 3 ⁇ 4 ⁇ ? the laser is a solid-state laser. In some embodiments, E£T/II ⁇ 20/0 ⁇ ) 356 aser In some embodiments, the laser is a wave laser. In some embodiments, the laser is a continuous-wave laser. In some embodiments, the laser is a pulsed laser. In some embodiments, a pulsed laser is a pico-second pulsed laser. In some embodiments, the laser light is continuous-wave laser light. In some embodiments, the laser light is pulsed laser light.
  • the light source is a monochromatic light source. In some embodiments, the light source is configured to produce monochromatic light. In some embodiments, the light source produces purple, blue and/or green light. In some embodiments, the light source produces purple light. In some embodiments, purple light is violet light. In some embodiments, the light source produced blue light. In some embodiments, the light source produced green light. In some embodiments, the light source produced blue and/or green light. In some embodiments, purple light comprises a wavelength between 380 and 420 nm. In some embodiments, purple light comprises a wavelength between 380 and 450 nm. In some embodiments, purple light comprises a wavelength between 400 and 420 nm.
  • purple light comprises a wavelength between 400 and 450 nm.
  • blue light comprises a wavelength between 380 and 490 nm.
  • blue light comprises a wavelength between 420-490 nm.
  • blue light comprises a wavelength between 450-490 nm.
  • blue light comprises a wavelength between 380 and 500 nm.
  • blue light comprises a wavelength between 420- 500 nm.
  • blue light comprises a wavelength between 450-500 nm.
  • blue light comprises a wavelength between 380 and 520 nm.
  • blue light comprises a wavelength between 420-520 nm.
  • blue light comprises a wavelength between 450-520 nm. In some embodiments, green light comprises a wavelength between 500 and 580 nm. In some embodiments, green light comprises a wavelength between 520 and 580 nm. In some embodiments, green light comprises a wavelength between 500 and 560 nm. In some embodiments, green light comprises a wavelength between 520 and 560 nm. In some embodiments, the light source is configured to emit light at a wavelength between 380 and 580 nm. In some embodiments, the light source is configured to emit light at a wavelength between 300 and 580 nm. the light source is configured to emit light at a wavelength between 380 and 600 nm.
  • the light source is configured to emit light at a WO ? ⁇ ?v!Y .yi wccn 300 and 600 nm.
  • tSS 1 (iii3 ⁇ 4 02 9gi? l 0 3 ⁇ 4 6 at a wavelength of between 300 and 600 nm.
  • the power of the light source is at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 25, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 milliwatts (mW).
  • the power of the light source is at least 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 micro-watts (pW).
  • pW micro-watts
  • the power of the light source is at least 1 pW. In some embodiments, the power of the light source is at least 10 pW.
  • the power of the light source is at least 100 pW. In some embodiments, the power of the light source is between 1 pW and 50 mW, 1 pW and 45 mW, 1 pW and 40 mW, 1 pW and 35 mW, 1 pW and 30 mW, 1 pW and 25 mW, 1 pW and 20 mW, 1 pW and 15 mW, 1 pW and 1 mW, 1 pW and 5 mW, 1 pW and 1 mW, 10 pW and 50 mW, 10 pW and 45 mW, 10 pW and 40 mW, 10 pW and 35 mW, 10 pW and 30 mW, 10 pW and 25 mW, 10 pW and 20 mW, 10 pW and 15 mW, 10 pW and 10 mW, 10 pW and 5 mW, 10 pW and 1 mW, 100 pW
  • the power of the light source is the intensity of the light at a spot on the membrane.
  • the light source is configured 'Yv9 1 ?/t?uujy ⁇ ?)!;* intensity of light at a spot on the membrane.
  • spot is on the first layer.
  • the first spot is on the first layer.
  • the second spot is on the second layer.
  • a pulsed laser produces pulse widths of between 50-150 picoseconds (ps). In some embodiments, pulsed laser light comprises pulse widths of between 50-150 ps. In some embodiments, a pulsed laser produces pulse widths of at least 20, 30, 40, 50, 60, 70, 80, 90 100, 110, 120, 130, 140, or 150 ps. Each possibility represents a separate embodiment of the invention. In some embodiments, pulsed laser light comprises pulse widths of at least 20, 30, 40, 50, 60, 70, 80, 90 100, 110, 120, 130, 140, or 150 ps. Each possibility represents a separate embodiment of the invention.
  • a pulsed laser produces pulse widths of at most 50, 60, 70, 80, 90 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 ps.
  • pulsed laser light comprises pulse widths of at most 50, 60, 70, 80, 90 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 ps.
  • pulsed laser light comprises pulse widths of at most 50, 60, 70, 80, 90 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 ps.
  • Each possibility represents a separate embodiment of the invention.
  • a pulsed laser produces pulse widths or pulsed laser light comprises pulse widths of between 40-200, 40-180, 40- 160, 40-150, 10-140, 10-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 50-200, 50-180, 50- 160, 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-200, 60-180, 60- 160, 60-150, 60-140, 60-130, 60-120, 60- 110, 60-100, 60-90, 60-80, 60-70, 70-200, 70-180, 70- 160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-200, 80-180, 80- 160, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 80-
  • pulsed laser light comprises a repetition rate of up to 80 MHz.
  • pulsed laser light comprises a repetition rate of up to 20, 30, 40, 50, 60, 70, 75, 80, 90 or 100 MHz.
  • pulsed laser light comprises a repetition rate of up to 20, 30, 40, 50, 60, 70, 75, 80, 90 or 100 MHz.
  • Each possibility represents a separate the invention.
  • pulsed laser light comprises a repetition rate of not more than 40, 50, 60, 70, 80, 90 or 100 MHz.
  • pulsed laser light comprises a repetition rate of not more than 40, 50, 60, 70, 80, 90 or 100 MHz.
  • a pulsed laser pulses with a repetition rate or pulsed laser light comprises a repetition rate of between 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40- 80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100 or 80-90 MHz.
  • Each possibility represents a separate embodiment of the invention.
  • the terms“film” and“membrane” are used interchangeably and refer to a thin water-impermeable layer of material.
  • the membrane is ion- impermeable.
  • the membrane is insulated.
  • the membrane is electrically insulated.
  • the membrane is mechanically robust.
  • mechanically robust refers to physical strength.
  • the membrane comprises a tensile strength of at least 100, 150, 200, 250, 275, 300, 325, 350, 375, 400, 450 or 500 mega pascals (MPa). Each possibility represents a separate embodiment of the invention.
  • the membrane comprises a tensile strength of at least 300 mega pascals (MPa).
  • the membrane is chemically inert.
  • chemically inert comprises difficult to etch by organic solvent or aqueous based acids or bases.
  • the membrane is free-standing.
  • the membrane is freely standing.
  • a free-standing membrane is immersed in an aqueous solution.
  • a free-standing membrane is surrounded on both sides by an aqueous solution.
  • a free-standing membrane is covered by aqueous solution on both sides. In some embodiments, both sides are two drillable sides.
  • both sides the side upon which the light is shone and the side upon which light exits when a pore is drilled do not need to be covered by the aqueous solution. Rather, the spot where the light is shone needs to be covered by the aqueous solution, and if the laser spot is considered three dimensionally to pass through the membrane, the spot on the opposite side of the membrane must also be covered by the aqueous solution.
  • a laser spot has a thickness (-300 nm) that is thicker than the width of the membrane. Thus, the“spot” on the membrane actually is a three-dimensional spot that extends through the entire width of the membrane.
  • a free-standing membrane comprises both sides of the spot on the membrane covered by an aqueous solution. In some embodiments, a free-standing membrane comprises access of an aqueous solution over both sides of the spot on the membrane.
  • the laser spot on the first side of the membrane is a first spot. In some embodiments, the laser spot on the second side of the membrane is a second spot. In some embodiments, the spot is a first spot.
  • the spot is a second spot. In some embodiments, the spot is a first spot and a second spot. In some embodiments, a free-standing membrane comprises access of an aqueous solution over a first spot on the membrane. In some embodiments, a free-standing membrane comprises access of an aqueous solution over a second spot on the membrane. In some embodiments, the first spot and the second spot are on opposite sides of the membrane. In some embodiments, the first spot and the second spot are positioned such that a line passing through the membrane passes through the first spot and the second spot. In some embodiments, a free-standing membrane comprises an aqueous solution over a first spot on the membrane. In some embodiments, a free-standing membrane comprises an aqueous solution over a second spot on the membrane.
  • the membrane comprises a first layer.
  • the membrane comprises silicon.
  • the first layer comprises silicon.
  • the membrane or first layer is silicon based.
  • the membrane or first layer comprises silicon nitride (SiN x ).
  • SiN x silicon nitride
  • the terms“SiN x ” and“Si x N” are used interchangeably and refer to silicon nitride.
  • The“x” refers to the ratio of silicon to nitride which is variable in the substance.
  • the silicon nitride is amorphous silicon nitride.
  • amorphous silicon nitride is one with a variable ratio of silicon to nitride.
  • membrane comprises a second layer.
  • the film comprises a metal oxide.
  • the second layer is a layer of metal oxide.
  • the metal oxide is selected from aluminum oxide (AIO2), titanium oxide (TiCh), silicon oxide (SiCh) and hafnium oxide (HGO2).
  • the metal oxide is selected from aluminum oxide (AIO2), titanium oxide (TiCh), and hafnium oxide (HfCh).
  • the metal oxide is TiCh .
  • membrane or second layer comprises amorphous silicon nitride.
  • the silicon nitride is amorphous silicon nitride.
  • the membrane or second layer comprises silicon nitride, titanium dioxide or a combination ⁇ n ⁇ RL ⁇ u ⁇ ie embodiments, the membrane comprises more thcE££iit3 ⁇ 4i j PP. 51 3 ⁇ 4 5 P,ome embodiments, the membrane comprises a layer of SiN x . In some embodiments, the membrane comprises a layer of T1O2. In some embodiments, the membrane comprises a layer of T1O2 layered on a layer of SiN x . In some embodiments, the second layer is layered on the first layer. In some embodiments, the second layer is a dielectric layer.
  • the term“layer” refers to a thin flat continuous piece of material.
  • the layer comprises a metallic layer having plasmonic properties.
  • the layer comprises a metal.
  • the metal is selected from gold, silver, copper, aluminum, titanium, hafnium and a combination thereof.
  • the metallic layer comprises at least one layer of metal.
  • the metallic layer comprises more than one layer of metal.
  • the more than one layer of metal is layered one on top of the other to create one combined metallic layer.
  • the layer comprises a metal oxide.
  • the layer is a silicon layer.
  • the layer is a layer that comprises silicon.
  • the layer is a silicon nitride layer.
  • a layer is deposited by Atomic Layer Deposition (ALD). In some embodiments, one layer is deposited upon another by ALD.
  • ALD Atomic Layer Deposition
  • the second layer is refractory to etching. In some embodiments, the second layer is refractory to thinning. In some embodiments, the second layer is refractory when not layered on the first layer. In some embodiments, the second layer when layered on the first layer can be etched or thinned. In some embodiments, the second layer is layered on the first layer.
  • the membrane is deposited on a silicon wafer.
  • the membrane is formed from a silicon wafer.
  • the wafer is a crystal orientation wafer.
  • the wafer is thicker in regions that lack a nanopore.
  • the wafer comprises a diameter of at least 1, 10, 50, 75, 100 or 200 mm. Each possibility represents a separate embodiment of the invention.
  • the wafer comprises a thickness of at least 50, 100, 150, 200, 250, 300, 350 or 400 pm. Each possibility represents a separate embodiment of the invention.
  • the membrane or first layer has a universal thickness. In some embodiments, the membrane or first layer has a constant thickens across its entire area. In some embodiments, the membrane or first layer has a variable thickness. In some embodiments, the membrane or first layer is thinner in the area of the nanopore. In some ! 3 ⁇ 4 3 ⁇ 4 ⁇ ? the membrane or first layer does not comprise a t h i c 3 ⁇ 4 ⁇ ' 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nm. Each possibility represents a separate embodiment of the invention.
  • the membrane or first layer does not comprise a thickness of greater than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nm.
  • the membrane or first layer at the spot where the light focuses comprises a thickness of between 1-100, 1-75, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-100, 5-75, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-100, 10-75, 10-50, 10- 45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-100, 15-75, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-100, 20-75, 20-50, 20-45, 20-40, 20-35, 20-30, or 20-25 nm.
  • the membrane or first layer at the spot where the light focuses comprises a thickness of between 10 and 50 nm. In some embodiments, the membrane or first layer at the spot where the light focuses does not comprise a thickness of less than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane or first layer at the spot where the light focuses does not comprise a thickness of more than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nm. Each possibility represents a separate embodiment of the invention.
  • the membrane comprises a high index of refraction.
  • the first layer comprises a high index of refraction.
  • the silicon nitride membrane comprises a high index of refraction.
  • a high index of refraction is an index at and/or above 1.8, 1.9, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35 or 2.4. Each possibility represents a separate embodiment of the invention.
  • a high index of refraction is an index greater than 1.8, 1.9, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35 or 2.4. Each possibility represents a separate embodiment of the invention.
  • a high index of refraction is an index greater than 2.2. In some embodiments, a high index of refraction is an index greater than 2.0. In some embodiments, the second layer does not comprise a high index of refraction. In some embodiments, the second layer comprises an index of refraction below a high index of refraction.
  • the SiN x membrane comprises a silicon to nitride ratio of at least 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 or 1.25. Each possibility represents a separate embodiment of the invention.
  • the SiN x membrane comprises a silicon to nitride ratio of at least 0.8.
  • the SiN x membrane comprises ⁇ 91 ⁇ o ⁇ ⁇ ? ⁇ riclc ratio of at least 0.75.
  • the SiN?9J/A3 ⁇ 43 ⁇ 4?9/.°.3 ⁇ 4A3 ⁇ 45 rises a silicon to nitride ratio of greater than 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 or 1.25.
  • the SiN x membrane comprises a silicon to nitride ratio of greater than 0.8.
  • the SiN x membrane comprises a silicon to nitride ratio of greater than 0.75.
  • the membrane comprises a first layer and a second layer wherein the two layers have different refraction indexes, and/or different ratios of silicon to nitride.
  • the second layer that is refractory to etching/thinning by focused light.
  • the second layer is an inert layer.
  • the membrane comprises a dielectric layer.
  • the second layer is refractory /inert when not layered on a silicon nitride layer.
  • a metal oxide layer is refractory and/or inert.
  • a dielectric layer is refractory and/or inert.
  • the metal oxide layer is selected from a T1O2, an aluminum oxide (AIO2) and a hafnium oxide (HfCh) layer.
  • the metal oxide is TiCh.
  • the inert/refractory layer is layered onto a layer of silicon nitride.
  • the layer of silicon nitride sensitizes the refractory /inert layer to etching/thinning.
  • the silicon nitride layer catalyzes the etching/thinning of the refractory/inert layer when focused light is shown on the silicon nitride layer and/or the refractory/inert layer.
  • the membrane comprises a plurality of layers with different refraction indexes, and/or different ratios of silicon to nitride.
  • thinning/etching can be done in only particular layers at a time.
  • the membrane is a free-standing membrane.
  • the membrane is immersed in an aqueous solution.
  • the membrane is at least partially immersed in an aqueous solution.
  • the spot on the membrane is immersed in an aqueous solution.
  • immersed comprises an aqueous solution on both sides of the membrane.
  • the same aqueous solution is on both sides of the membrane.
  • different atjutu ua ?i3 ⁇ 4? ons are on each side of the membrane.
  • eml? ⁇ i3 ⁇ 4?ft! ⁇ P3 ⁇ 4 3 fj*st ⁇ em further comprises a first and second liquid reservoir.
  • the first and second liquid reservoirs are separated by the membrane.
  • the system is configured to receive a free-standing membrane.
  • the system comprises an area configured to receive a free-standing membrane.
  • the area is a receiving area.
  • the area comprises at least one dimension configured for the placement of a free-standing membrane.
  • the area comprises a receptacle for an aqueous solution.
  • a receptacle is configured to receive an aqueous solution.
  • a receptacle is configured to hold an aqueous solution.
  • the receptacle is configured such that a received aqueous solution covers a spot on a received membrane.
  • the receptacle is configured such that a received aqueous solution covers a first spot on a received membrane. In some embodiments, the receptacle is configured such that a received aqueous solution covers a first spot and a second spot on a received membrane. In some embodiments, the area comprises two receptacles. In some embodiments, a first receptacle is configured such that a received aqueous solution covers a spot on one side of a received membrane and a second receptacle is configured such that a received aqueous solution covers the spot on the opposite side of a received membrane.
  • a first receptacle is configured such that a received aqueous solution covers a first spot on a received membrane and a second receptacle is configured such that a received aqueous solution covers a second spot on a received membrane.
  • a first receptacle and second receptacle receive different aqueous solutions.
  • the aqueous solution is water. In some embodiments, the water is ultrapure water. In some embodiments, the aqueous solution is a salt buffer. In some embodiments, the aqueous solution comprises neutral pH. In some embodiments, the aqueous solution comprises alkaline pH. In some embodiments, the aqueous solution comprises neutral or alkaline pH. In some embodiments, alkaline pH is pH above 8. In some embodiments, alkaline pH is a pH between 8 and 10. In some embodiments, alkaline pH is a pH between 8 and 12. In some embodiments, alkaline pH is a pH between 10 and 12.
  • the aqueous solution is at most at room temperature. In some embodiments, the membrane is at most at room temperature. In some embodiments, the spot on the membrane is at most at room temp. In some embodiments, the aqueous solution is at most at room pressure. In some embodiments, the membrane is at most at room pressure. In some embodiments, the spot on the membrane is at most at room pressure. In some ! 3 ⁇ 4 3 ⁇ 4 ⁇ ? room temperature is 25 degrees Celsius. In someP ⁇ li S ⁇ uPuSSPi ⁇ Oom temperature is 20 degrees Celsius. In some embodiments, room temperature is between 20 and 25 degrees Celsius. In some embodiments, room pressure is 1 atmosphere. It will be understood by a skilled artisan that the method of drilling is advantageous as it does not require high temperature and/or high pressure in order to achieve drilling.
  • the membrane is in an optically accessible flow cell.
  • the system further comprises an optically accessible flow cell.
  • optically accessible comprises optically accessible by a high numerical aperture objective.
  • the system further comprises a high numerical aperture objective.
  • the high numerical aperture objective is in a microscope.
  • the high numerical aperture objective is configured to focus light to a spot on the membrane.
  • the high numerical aperture objective is configured to focus light to a first spot on the membrane.
  • the system further comprises a microscope.
  • the microscope is a confocal microscope.
  • the microscope is for positioning the membrane so the light is focused on the spot.
  • the lens of the microscope focuses the light from the light source.
  • the system that directs and focuses light is a system of mirrors.
  • the system that directs and focuses light is a microscope.
  • the system that directs and focuses the light is a system of reflective metal surfaces.
  • the system is configured to direct and focus light from the light source.
  • a spot on the membrane is a predetermined spot.
  • a spot on the membrane is a diffraction limited spot.
  • a spot is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 spots.
  • a spot is part of an array of spots.
  • the array of spots in a geometric configuration. Geometric configurations include squares, rectangles, circles, ovals, triangles, bowties, rods, cylinders, ellipses, disks, rhombuses and any other shape that may be found by one skilled in the art for nanopore arrays.
  • the membrane comprises a second spot.
  • the second spot is on an opposite side of the membrane from the first spot.
  • the second spot is on the inverse side of the membrane from the first spot.
  • the second spot is positioned such that a line passing through the first spot also passed through the second spot.
  • the second spot is membrane where light shined on the first spot exists t?£T ⁇ Si?ii3 ⁇ 4a3i53 ⁇ 4?' tome embodiments, the second spot is the spot on the membrane where light shined on the first spot would exit the membrane if the light generated a pore through the membrane.
  • the first spot and the second spot are a single spot that passes through the width of the membrane.
  • the system further comprises a sensor.
  • the sensor is a photodetector.
  • the sensor is capable and/or configured to measure low light intensities.
  • the sensor is capable and/or configured to measure at high temporal resolution.
  • the sensor is configured to detect at the spot on the membrane.
  • the sensor is configured to detect emissions from the spot on the membrane.
  • the sensor is configured to detect at the membrane.
  • the sensor is configured to detect emissions from the membrane.
  • the sensor is configured to detect photoluminescent intensity (PL).
  • the sensor is configured to detect light.
  • the sensor is an electron detector.
  • the photodetector is a photodiode. In some embodiments, the photodiode is an avalanche photodiode. In some embodiments, the photodetector is a photo-multiplier tube. In some embodiments, the photodetector is a CMOS camera.
  • the sCMOS scientific CMOS
  • the system further comprises a means to induce movement of a molecule from one side of the membrane to the other side of the membrane when there is a pore through the membrane. In some embodiments, the system further comprises a means to induce a current through the membrane. In some embodiments, the system further comprises a means to induce a current from one side of the membrane to the other side. In some embodiments, the system further comprises a means to induce movement of a molecule or create a charge from a first reservoir to a second reservoir. In some embodiments, the means to induce movement comprises a means of inducing an electrical current from one side of the membrane to the other side. In some embodiments, one side of the membrane to the other is from the first reservoir to the second reservoir.
  • the means 'Yi ⁇ ?5?uZi3 ⁇ 4?5vement comprises a negative electrode on one side or ⁇ > ( I ⁇ t3 ⁇ 4 i nowadays i /' i ⁇ ! ⁇ .3 ⁇ 4 i ⁇ no ⁇ and a positive electrode on the second side or in the second reservoir.
  • the means is an apparatus configured to pass an electric current between two electrodes.
  • the system further comprises a current detector.
  • the current detector is configured to measure current between the two electrodes.
  • the current detector is configured to measure current between one side of the membrane and the other.
  • the current detector is configured to measure current between a first reservoir and a second reservoir.
  • the current detector and/or the sensor are configured to shut off the light source upon a particular measurement.
  • the particular measurement is indicative of thinning to a desired thickness.
  • the particular measurement is indicative of the formation of a nanopore.
  • the particular measuring is indicative of a nanopore reaching a predetermined diameter.
  • the particular measurement is a predetermined threshold of PL intensity.
  • PL intensity is inversely proportional to the thickness of the spot on said membrane.
  • the sensor is configured to shut off the light source based on a measured PL intensity.
  • the particular measurement is an increase in ionic current.
  • an increase in ionic current through the membrane indicates the formation of a pore in the membrane.
  • a threshold ionic current indicates a particular diameter of pore has been formed.
  • the particular measurement is a visual measurement of the pore and/or its diameter.
  • the system of the invention comprises the set up depicted in Figure 1A. In some embodiments, the system of the invention comprises the set up depicted in Figure 7A. In some embodiments, the system of the invention comprises a portion of the set up depicted in Figures 1A and/or 7A.
  • the system of the invention is for thinning a membrane. In some embodiments, the system of the invention is for controlled thinning of a membrane. In some embodiments, the system of the invention is for forming a pore through the membrane. In some embodiments, the forming a pore is in situ forming a pore at a predetermined spot. In some embodiments, the forming a pore is forming an array of pores. In some embodiments, the pore is a nanopore.
  • the system is for forming at least 1, 2 , , 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 nanopores. Each possibility represents a separate embodiment of the invention. In some embodiments, the system is for producing a plurality of nanopores.
  • the array comprises dimensions of 5 x 5, 5 x 10, 5 x 15, 5 x 20, 5 x 25, 5 x 30, 5 x 35, 5 x 40, 5 x 45, 5 x 50, 10 x 10, 10 x 15, 10 x 20, 10 x 25, 10 x 30, 10 x 35, 10 x 40, 10 x 45, 10 x 50, 15 x 15, 15 x 20, 15 x 25, 15 x 30, 15 x 35, 15 x 40, 15 x 45, 15 x 50, 20 x 20, 20 x 25, 20 x 30, 20 x 35, 20 x 40, 20 x 45, 20 x 50, 25 x 25, 25 x 30, 25 x 35, 25 x 40, 25 x 45, 25 x 50, 30 x 30, 30 x 35, 30 x 40, 30 x 45, 30 x 50, 35 x 35, 35 x 40, 35 x 45, 35 x 50, 40 x 40, 40 x 45, 40 x 50, 45 x 45, 45, 45, 30
  • the nanopores are separated by about 1 pm. In some embodiments, the nanopores in the array are separate by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 pm. Each possibility represents a separate embodiment of the invention.
  • a method of thinning a membrane comprising shining focused light on a spot on a membrane, thereby thinning the membrane.
  • a method of thinning a membrane comprising shining pulsed laser light on a spot on the membrane, thereby thinning the membrane.
  • the method is for light- induced thinning. In some embodiments, the method is for controlled thinning. In some embodiments, the method is for thinning in situ on a membrane. In some embodiments, the method is for thinning at a predetermined spot on a membrane. In some embodiments, the method is for rapid thinning. In some embodiments, thinning comprises producing a pore through the membrane. In some embodiments, thinning is to a thickness of less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nm. Each possibility represents s separate embodiment of the invention. In some embodiments, thinning is to a thickness of less than 5 nm.
  • thinning is to a thickness of less than 3 nm. In some embodiments, thinning is a thinning of at least 50, 60, 70, 75, 80, 85, 90, 95, 97, 99 or 100% of the membrane thickness. Each possibility represents s separate embodiment of the invention. In some embodiments, thinning is a thinning of at least 75% of the membrane thickness. It will be understood that thinning of 100% produces a hole through the membrane. ⁇ 9 3 ⁇ 4?«' 1 embodiments, the thinning does not comprise a p ⁇ 99 ⁇ !M93 ⁇ 4]iP. 5, l? 56 aome embodiments, the thinning does not comprise a pre -patterning step.
  • pretreatment is a pre -patterning.
  • pre-patterning is by a method other than a method of the invention. Any method of pre-patterning known in the art is envisioned.
  • the method is devoid of chemical etching.
  • the method is devoid of dielectric breakdown.
  • the method is devoid of electrochemical anodization.
  • the method is devoid of metal-assisted chemical etching.
  • the method is devoid of electron beam lithography (EBL) etching.
  • the method is devoid of reactive ion etching (RIE).
  • the method is devoid of metal deposition fabrication.
  • the method is devoid of ion-track etching. In some embodiments, the method is devoid of focused electron beam (e-Beam) lithography. In some embodiments, the method is devoid of focused ion beam (FIB) lithography.
  • e-Beam focused electron beam
  • FIB focused ion beam
  • the focused light is laser light. In some embodiments, the focused light is within the purple spectrum. In some embodiments, the focused light is purple light. In some embodiments, the focused light is within the blue spectrum. In some embodiments, the focused light is blue light. In some embodiments, the focused light is within the green spectrum. In some embodiments, the focused light is green light. In some embodiments, the focused light is light focused from a light source. In some embodiments, the light is focused by a system for focusing the light and directing it to a spot on the membrane. In some embodiments, the light comprises a wavelength of between 300-600 nm. In some embodiments, the laser light is continuous-wave laser light. In some embodiments, the laser light is pulsed laser light. In some embodiments, shinning pulsed laser light is pulsing laser light on a spot on the membrane.
  • the light has an intensity of at least 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 pW. Each possibility represents a separate embodiment of the invention.
  • the light has an intensity of at least 1 pW.
  • the light has an intensity of at least 10 pW.
  • the light has an intensity of at least 100 pW.
  • the light has an intensity of at least 1000 pW.
  • the power of the light is between 1 pW and 50 mW, 1 pW and 45 mW, 1 pW and 40 mW, 1 pW and 35 mW, 1 pW and 30 mW, 1 pW and 25 mW, 1 pW and 20 mW, 1 pW and 15 mW, 1 pW and 1 mW, 1 pW and 5 mW, 1 pW and 1 mW, 10 pW and 50 mW, 10 pW and 45 mW, 10 pW and 40 mW, 10 pW and 35 mW, 10 pW and 30 mW, 10 pW and 25 mW, 10 pW and 20 mW, 10 pW and 15 mW, 10 pW and 100 pW and 50
  • the method further comprises measuring PL intensity from the membrane. In some embodiments, the measuring PL intensity is from the spot on the membrane. In some embodiments, the method further comprises stopping thinning bases on the measured PL intensity. In some embodiments, the stopping occurs when the PL intensity reaches a predetermined threshold. In some embodiments, the predetermined threshold is when a pore is formed in the membrane. In some embodiments, the predetermined threshold is when a pore has reached a predetermined diameter. In some embodiments, the PL intensity is inversely proportional to the thickness of the spot on the membrane. In some embodiments, the thinning is stopped at a predetermined thickness.
  • the thinning comprises forming a pore through the membrane at the spot.
  • the pore is a nanopore.
  • the nanopore comprises a diameter not greater than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm. Each possibility represents a separate embodiment of the invention.
  • the nanopore comprises a diameter not greater than 5 nm.
  • the nanopore comprises a diameter of about 5 nm.
  • the nanopore comprises a diameter between 0.5 and 10, 0.5 and 15, 0.5 and 20, 1 and 10, 1 and 15, 1 and 20, 3 and 10, 3 and 15, 3 and 20, 5 and 10, 5 and 15, or 5 and 20 nm.
  • -, c selected by monitoring the PL intensity and or Op £J c iJ3 ⁇ 4 2 P?Si?3 ⁇ 4?£ g the nanopore formation.
  • the diameter of the nanopore can be selected by altering the light intensity, wavelength, or duration, or the pH of the solution. A skilled artisan can optimize the variable parameters to generate a nanopore of the desired diameter in the desired time.
  • the nanopore comprises a Gaussian shape. In some embodiments, the nanopore comprises a varying diameter. In some embodiments, the nanopore comprises a Gaussian curve shaped cross-section. In some embodiments, the thinning produces a nanowell adjacent to the nanopore. In some embodiments, the thinning produces a nanowell without a nanopore. In some embodiments, the thinning produces a nanowell in one layer and an adjacent nanopore in a second layer. As used herein, the term “nanowell” refers to a passage through the membrane. A nanowell may also be referred to as a nanoslot. In some embodiments, the nanowell comprises a Gaussian shape.
  • the nanowell comprises a varying diameter. In some embodiments, the nanowell comprises a Gaussian curve shaped cross-section. In some embodiments, the nanopore and/or nanowell produce a low optical background. In some embodiments, the membrane produces a low optical background. In some embodiments, the low optical background is lower than the optical background of a nanopore and/or nanowell that does not have a Gaussian curve shape or a Gaussian curve shaped cross-section. In some embodiments, the low optical background is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97 or 99% lower. Each possibility comprises a separate embodiment of the invention.
  • a membrane comprising a first and second layer comprises an interface of the first and second layer.
  • the interface is the place wherein the second layer is layered on the first layer.
  • the membrane comprises a first layer and a second layer and the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the first layer.
  • the membrane comprises a first layer and a second layer and the nanopore comprises a second Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the second layer.
  • the nanopore comprises two regions each with a Gaussian curve shaped cross-section.
  • the first Gaussian curve shaped cross-section is in the first layer.
  • the second Gaussian curve shaped cross-section is in the second layer.
  • the method further comprises measurin g ?X(3 ⁇ 4l3 ⁇ 43 ⁇ 49AP?3 ⁇ 4Jugh the membrane.
  • the spot of the membrane is immersed in an aqueous solution and the ionic current is measured from the solution on one side of the membrane to the solution on the other side of the membrane.
  • an increase in ionic current through the membrane or from one side to the other, or from one reservoir to the other indicates the formation of a pore in the membrane.
  • formation of the pore in the membrane is formation of the pore at the spot in the membrane.
  • the spot is a predetermined spot.
  • the increase in ionic current is a sudden increase in ionic current.
  • the increase in ionic current is not gradual.
  • the increase is at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500% increase in ionic current.
  • a gradual increase in ionic current corresponds to the widening of the diameter of the nanopore.
  • the thinning is stopped at a predetermined current and/or diameter.
  • the method is for rapid formation of a nanopore.
  • a pore can be produced at a spot in the membrane with a thickness of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm. Each possibility represents a separate embodiment of the invention.
  • a pore can be produced at a spot in the membrane with a thickness of at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm. Each possibility represents a separate embodiment of the invention.
  • a pore can be produced at a spot in the membrane with a thickness of at most 50 nm.
  • a pore can be produced at a spot in the membrane with a thickness of at most 100 nm. In some embodiments, a pore can be produced in less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 seconds. Each possibility represents a separate embodiment of the invention. In some embodiments, a pore can be produced through a spot in the membrane with a thickness of at least 40 nm in less than 20 seconds.
  • a membrane comprising a nanopore and/or nanowell, wherein the nanopore and/or nanowell is produced by a method of the invention.
  • a membrane comprising a nanopore and/or nanowell, wherein the nanopore and/or nanowell comprises a varying diameter and a Gaussian curve shaped cross-section.
  • a nanopore and/or nanowell producetf ⁇ J ⁇ ii ⁇ MifS ⁇ uf the invention comprises a varying diameter.
  • the varying diameter is a Gaussian shape.
  • the varying diameter corresponds to a Gaussian shaped cross-section of the nanowell and/or nanopore.
  • the nanopore and/or nanowell produced by a method of the invention comprises a Gaussian curve shaped cross-section.
  • the nanopore and/or nanowell increases in diameter from one side of the membrane to the other.
  • the nanopore and/or nanowell has a larger diameter on the side of the membrane upon which the light was shown.
  • the increasing diameter from one side to the other follows a Gaussian curve.
  • the Gaussian curve comprises a full width at half maximum of one half of the wavelength of the focused light used to generate the nanopore and/or nano well.
  • the nanopore, nanowell and/or membrane produces a low optical background.
  • the low optical background is lower than the background of a membrane comprising a nanopore and/or nanowell that does not comprise a Gaussian shape or cross-section. In some embodiments, the low optical background is lower than the background of a membrane not produced by the method of the invention.
  • a membrane comprising a first and second layer comprises an interface of the first and second layer.
  • the interface is the place wherein the second layer is layered on the first layer.
  • the membrane comprises a first layer and a second layer and the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the first layer.
  • the membrane comprises a first layer and a second layer and the nanopore comprises a second Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the second layer.
  • the nanopore comprises two regions each with a Gaussian curve shaped cross-section.
  • the first Gaussian curve shaped cross-section is in the first layer.
  • the second Gaussian curve shaped cross-section is in the second layer.
  • the membrane is a membrane as described hereinabove.
  • the nanopore is a nano-scale aperture.
  • the nanopore is as described hereinabove.
  • the nanowell is a nanowell as described hereinabove.
  • the membrane comprises a plurality of layers. In some embodiments, at least two layers comprise nanopores and/or nanowells of different '3 ⁇ 4n3 ⁇ 4*. 2 ⁇ / 1 3 ⁇ 4 ⁇ ?3 ⁇ 4 embodiments, one layer comprises a nanowell and a s ⁇ J/S ⁇ JE j S/ ⁇ SA ⁇ p rises a nanopore.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • Nanopore chips were fabricated from a 4” (100 mm) double-side polished, 350 pm thick silicon wafers coated with 500 nm of thermal Si0 2 (Silicon Valley Microelectronics, CA USA). 50 nm thick low-stress silicon nitride (SiN x ) layers were deposited on both sides using low pressure chemical vapor deposition (LPCVD) with ⁇ ⁇ II' i 1 ? ⁇ ! 1 ?/ S1H2CI2 gas ratios, resulting in different refractive ind?v£T ⁇ 3 ⁇ 4? g ? ⁇ ? ) ?l ) uS 2.15 to 2.43.
  • LPCVD low pressure chemical vapor deposition
  • the refractive index was then measured by ellipsometry (Film Sense, FS-1).
  • Direct-write photolithography (MicroWriter ML3, DMO) was used to pattern the windows and dice lines on the resist.
  • a hard mask was created using reactive ion etch (RIE, Diener Electronic) followed by buffered oxide etch (BOE) to remove the S1O2 and expose the Si layer.
  • RIE reactive ion etch
  • BOE buffered oxide etch
  • the wafer was then immersed in KOH at 65 °C for up to 20 hours followed by a second round of BOE to open up a freestanding SiN x membrane.
  • Each chip was cleaned by piranha before usage (3: 1 EbS0 4 :El 2 0 2 ), vacuum dried, and mounted onto a Teflon holder with Ecoflex 5 (Smooth-ON, Reynolds Advanced Materials) silicone rubber. The chip was then placed in a Teflon cell equipped with a quartz cover-slide bottom. The position of the cell was controlled using a 3D nanopositioner stage (Physik Instrumente, P-561.3CD). Alternatively, nanopore chips were fabricated from a 4” silicon wafer coated with 500 nm S1O2 and 50 nm low-stress amorphous SiN x .
  • a hard mask was RIE-etched into the SiN x followed by HF etching to remove the S1O2, and then through- etching of Si with KOH.
  • the free-standing membranes were 40-45 nm thick.
  • Chip assembly Chips were first cleaned by piranha (3: 1 H 2 S0 4 :H 2 0 2 ). They were then glued onto a custom-made Teflon insert, immersed in buffer (1 M KC1, 40 mM Tris- HC1, 1 mM EDTA, pH 7.5), and placed in a Teflon cell with a quartz cover-slide bottom. The cell was mounted onto a 3D nanopositioner located above the microscope objective. The setup was shielded by a grounded copper box and placed on a vibration-isolating optical table.
  • Fig. 1A-D the full photoluminescence is roughly 3 orders in magnitude larger.
  • an ND3 filter is positioned in the emission pathway during etching and before the excitation pathway during profiling.
  • the photoluminescence count is a summation of the red (>650 nm) and green (550-650 nm) channels.
  • Ionic current was measured by cis/trans -immersed Ag/AgCl electrodes connected to a high-bandwidth amplifier (Axon 200B) sampled at 125 kHz (DAQ NI-6211) and filtered at 10 kHz. Photon counting was sampled at 500 kHz (DAQ NI-6602). The two cards were u 9 l3 ⁇ 43 ⁇ 4ltaneously via a hardware connection and were fully p - ⁇ /i ⁇ 1 ⁇ 293S ⁇ ®3 v3 ⁇ 4?stom Lab VIEW software.
  • Ionic current was measured by cis/trans- immersed Ag/AgCl electrodes connected to a high-bandwidth amplifier (Axon 200B) sampled at 125 kHz (DAQ NI-6514) and filtered at 10 kHz. The two cards were triggered simultaneously via a hardware connection and were fully controlled by custom Lab VIEW software.
  • TEM imaging High-resolution images were acquired with a FEI Titan Themis Cs- Correct HR-S/TEM.
  • STEM scanning transmission electron microscopy
  • Composition analysis Chemical mapping of the SiN x membranes was performed using energy dispersive X-ray imaging (EDS, Dual Bruker XFlash6) and scanning transmission electron microscopy (STEM) based on core-loss electron energy loss spectroscopy (EELS). The EDS quantification was done using Velox (Thermo Fisher) and EELS quantification was done using the Digital Micrograph software (Gatan). 'YS > ⁇ 3 ⁇ 4? j ° ⁇ l?Ap3 ⁇ 4 metry measurements. Performed with model Fi ⁇ ? T ⁇ fii?/l? ® ⁇ 9a ( v 3 ⁇ 4 ⁇ ngth
  • Nanopores were allowed to equilibrate at a low probing voltage (0.1 to 0.3 V) in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA, pH 7.5) for at least 10 minutes to obtain a stable open pore current prior to adding homemade 5054 bp dsDNA. Events were monitored using an Axon 200B filtered at 100 kHz and custom Lab VIEW software.
  • the chip was immersed in a pH 7 buffer (in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA) and 300 mV was applied until the open pore current stabilized. 300 bp dsDNA was added to the cis chamber at a concentration of 1 nM. Translocation events were monitored and recorded using an Axon 200B filtered at 100 kHz and custom Lab VIEW software. An offline analysis program was used to analyze each event separately to extract the amplitude block and dwell time for each translocation.
  • Nanopores were kept dry in air for up to 10 days prior to performing the experiment. These nanopores were cleaned by Dynasolve 185 to remove PDMS and then made hydrophilic by piranha (3:1 HiSO ⁇ HiCh). Nanopores were allowed to equilibrate at a low probing voltage (0.1 to 0.3 V) in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA, pH 7.0) for at least 10 minutes to obtain a stable open pore current prior to adding 0.007 pg/m ⁇ di-ubiquitin.
  • buffer 1 M KC1, 40 mM Tris-HCl, 1 mM EDTA, pH 7.0
  • home-purified proteins such as Ubiquitin, eIF4A, Albumin, etc. where suspended in the buffer and translocation events were monitored using an Axon 200B filtered at 100 kHz and custom Lab VIEW software.
  • Table 2 Refractive index measurements for the different solutions.
  • Figure 5A gives an example TEM image of a 6.5 nm nanopore formed in under 5 minutes, which was a typical fabrication time in a 40-45 nm thick membrane based on >30 trials (100% yield).
  • i 0 and i B are the open and blocked pore current levels, respectively, l is the local membrane thickness, d the pore diameter, s the solution conductivity and a is the analyte ⁇ 3 ⁇ 4 3 ⁇ 4)>-!303 e mo n s tratc the extent by which the conductance mode?9I(3 ⁇ 4??33 ⁇ 4® 5 83 ⁇ 4 j uS ted, we calculated the effective thickness for a pore with an OPC of 11 ⁇ 0.7 nS and a diameter of 3.2 ⁇ 0.3 nm. Remarkably, we get an effective thickness of 4-6 nm, which is up to 11 times smaller than the surrounding membrane and is consistent with our observation that the membrane gradually thins to the point of nanopore formation.
  • Such ultrathin architectures are highly desirable due to their larger conductance and hence improved spatial resolution and have therefore been the subject of much research.
  • Table 1 Table of nanopores fabricated by laser-etching.
  • the table is ordered according to the open pore current (OPC) from smallest to largest.
  • OPC open pore current
  • the open pore current (OPC) was recorded 1-2 minutes after the laser was turned off.
  • FIG. 5C shows the power spectral density (PSD) plot of a nanopore for an applied bias of 300 mV after allowing the nanopore to stabilize in KC1 buffer. Similar to TEM-drilled nanopores, two sources of noise dominate the PSD: high-frequency background noise associated with the chip capacitance, and low-frequency flicker noise with 1/P dependency. At an applied voltage of 300 mV, these nanopores typically exhibit an IRMS in the range of 100-200 pA.
  • PSD power spectral density
  • Amorphous SiN x films are typically produced using a chemical vapor deposition (CVD) process, tuned to form silicon-rich membranes with respect to stoichiometric S13N4, resulting in low-stress thin films.
  • the Si:N ratio (denoted x) only slightly alters the material’s refractive index, but it greatly affects the abundance of the Si dangling bonds, and in turn the photoluminescence (PL) spectrum produced by the film, as the latter involves photo-activated electron excitation and relaxation.
  • PL photoluminescence
  • Material composition analysis is not routinely performed as part of the LPCVD process because it involves delicate elemental spectroscopy.
  • the material’s index of refraction is often used as a proxy for the Si:N composition, in which a higher index of refraction corresponds to a higher content of Si over N in SiN x membranes.
  • small changes in the index of refraction correspond to significantly different Si:N compositions, preventing fine control of the Si:N ratio, and resulting in significant batch-to- a 3 ⁇ 43 ⁇ 4$h. While these variations may be too small to affecP£3 ⁇ 4IL2020/05035 ⁇ eam nanopore drilling methods, we readily detect their effect on laser-based drilling as reported here.
  • n ⁇ is the refractive index of pure Si and nm is the refractive
  • the high refractive index chip was easily drilled with the 488 nm laser (in this case the laser power was decreased since higher power resulted in immediate current overload) as well as while the low refractive index chip could not be J/niZil ⁇ vv 2 u/°m9 ⁇ 3 ⁇ 4f the three wavelengths.
  • Example 4 SiNx membrane etching is accelerated under alkaline conditions
  • the etching mechanism involves a photochemical reaction.
  • the laser-induced temperature rise in the water-submersed thin-film appears to be less critical than electronic excitation.
  • the enhancement in etch rate can be related to the generation of electron-hole pairs within the SiN x surface and charge transfer at the liquid-solid interface.
  • the dissolution rate of a silica-like material is expected to be strongly affected by pH since the hydroxyl ion is a catalyst for the hydrolysis that underlies the dissolution process.
  • Example 5 Ultra-fast nanopore drilling in Si-rich membranes ⁇ 9. ong dependency of thinning on pH and material comp ⁇ Tulir?tL nowadayst/wu9 3 ⁇ 4 fd us to further analyze and characterize this process in order to achieve controlled, ultra-fast, nanopore drilling at low laser intensities.
  • a chip with refractive index of 2.3 in high pH buffer (pH 10), exposed it to a 488 nm laser of 7 mW for varying durations, and created a thickness map of the exposed region using EELS.
  • the thickness maps presented in Figure 11A show that longer illumination results in increased thinning. This is further demonstrated by comparing the line scans in the middle of each thinned region (Fig. 11B-C).
  • Integrating over this line scan shows a similar trend to the PL measurement during thinning, indicating that the PL can be used to approximate changes in thickness and membrane composition (Fig. 11D).
  • High magnification TEM images revealed that an exposure of ⁇ 15 seconds was enough to create a ⁇ 5 nm pore in the center of the Gaussian shaped thin region (see left inset in Fig. HD). However, further irradiation of the surface under these conditions resulted in multiple pores, as can be seen at ⁇ 20 s or at longer time points.
  • An OPC gradient threshold e.g. 0.65 nA over 100 ms
  • FIG. 12A A drilling trace using a similar chip (refractive index of 2.3), pH 10 buffer, and 488 nm laser with intensity of 6 mW in which pore formation was detected electrically in less than 15 seconds is presented in Figure 12A.
  • we immediately changed the buffer to a pH 7 buffer added DNA sample (300 bp) and measured translocations (Fig. 12A-B).
  • a scatter plot of the dsDNA translocation events and concatenated ionic current trace showing sample translocation events are presented in Figure 12A-B.
  • a pulsed laser source specifically a pico second laser (PicoQuant LDH series) was used to shine blue and green pulsed laser light on a membrane as described hereinabove (10-50 nm thickness). Pulse widths in the range 50 ps to 150 ps, with a repetition rate of up to 80 MHz were tested.
  • NA numerical aperture water immersion objective
  • the pulsed laser successfully drilled nanopores with an average intensity (power) of 5 mW or less. In comparison, at the same intensity and similar wavelengths a continues-wave laser took a longer time to drill. In where the continuous-wave laser resulted in no dri 11 i iP g i i utcs
  • the pulsed laser was still able to produce thinning of the membrane. This demonstrates the superiority of the pulsed laser of the continuous-wave laser for thinning and drilling.
  • Example 6 An automated in-situ fabrication of nanopore arrays using direct laser etching.
  • the piezo stage moved the membrane to the first coordinates, and the laser intensity was increased to 7 mW. Once the change in current increased above the current gradient threshold the laser was switched off and the piezo stage moved to the next coordinates (Fig. 13A, top), where the laser was switched on again. In this way the drilling process could stop immediately when a predetermined increase in current was detected (Fig. 13A, bottom).
  • Figures 13A-B display an example of an array of 25 nanopores drilled in ⁇ 6.5 minutes.
  • the top panel in Figure 13A displays time traces of the laser intensity.
  • the PL and current are shown in the bottom panel (red and blue lines, respectively).
  • Histograms of the drilling time and the change in the total current for each drilled pore are presented in Figure 13B.
  • the nanopore array was inspected by wide-field fluorescence microscopy using Ca 2+ -activated fluorophores.
  • a pH bias can be applied across the membrane surfaces (Fig. 14A, right panel),, where the T1O 2 side is held at low pH and the SiN x is held at high pH, in order to prevent laser etching of T1O 2 and produce a free-standing T1O 2 membrane (Fig. 14B).
  • Fig. 14A right panel
  • the T1O 2 side is held at low pH and the SiN x is held at high pH
  • Fig. 14B free-standing T1O 2 membrane
  • the T1O 2 layer can be conveniently made ultra-thin ( ⁇ 3 nm) by tuning the ALD deposition process, and generally photoluminescence monitoring allows for monitoring of local thinning in real time. This allows for distinct etching in distinct layers with different indexes of refraction.

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Abstract

Systems comprising a light source, thin membrane immersed in an aqueous solution and a system to direct and focus light from the light source to a spot on the membrane are provided. Methods of thinning and etching a membrane are also provided, as are membranes comprising a nanopore with a Gaussian curve shaped cross-section.

Description

WO 2020/194303 NANOPORE FABRICATION PCT/IL2020/050356
CROSS REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of priority from U.S. Provisional Patent Application No. 62/823,065, filed on March 25, 2019, entitled “NANOPORE FABRICATION”, the contents of which are incorporated by reference herein in their entirety.
FIELD OF INVENTION
[002] The present invention is in the field of nanopore fabrication.
BACKGROUND OF THE INVENTION
[003] The development of synthetic solid-state nanopores (ssNPs) as a substitute for biological channels remains a major focus in nanotechnology given their greater flexibility in terms of size, shape, surface properties, and cross-device compatibility. While traditionally the principal mode of single-molecule detection was based on ionic resistive pulsing measurements, a rapidly growing trend in the nanopore community has been towards “electro-optical” sensing. The simultaneous measurement of the electrical (ion current) and fluorescence signal (photon emission) extends the scope of biomolecular targets for nanopores and opens up new applications since both multiple fluorophore colors and varying photon intensities can be acquired to obtain specific information on the molecule of interest. Specifically, by selective fluorescent labelling of the analyte of interest, researchers have shown that ssNPs can be applied to DNA sequencing, DNA barcoding, epigenetic modification analysis, DNA methylation quantification and polypeptide discrimination. Although superior to strictly electrical sensing with respect to the amount of encodable information, electro-optical sensing brings its own set of fabrication challenges. Nanopores must be prepared in a way such that their position can be readily identified in situ. Furthermore, the peripheral structure heavily impacts the background noise and fluorescent signal of a translocating molecule.
[004] In the first decade of nanopore sensing, the controlled focusing of an ion or electron beam, as by transmission electron microscopy (TEM), was the only practical method for with nanometric dimensions. As these methods utihzt^T/I^PaS^Pu^uVing pore drilling, it followed that they were inherently slow, expensive, and importantly, produced un-hydrated surfaces that must be further treated to permit water passage and subsequent resistive pulse sensing. More recently, controlled dielectric breakdown (CBD) emerged as a powerful, low-cost alternative to TEM because it could create nanopores in freestanding silicon nitride (SiNx) directly in solution and could be almost fully automated. CBD, which uses an applied voltage to induce randomly accumulating material defects, is nonetheless comparatively less flexible and efficient at localizing nanopore formation. Recent attempts to do so relied on the principle that nanopores preferentially form at the hotspot of an infrared (IR) laser or at the thinnest membrane cross-section. Thus, in the latter case, milling or lithographic steps were implemented upstream of CBD as a preparatory step to direct nanopore formation. Using an IR laser, on the other hand, was complicated by the need to simultaneously control the applied voltage and laser power, as the IR laser only enhanced the local DC field necessary for dielectric breakdown and did not independently form nanopores. Fast, highly-reproducible, in situ methods of fabricating nanopores and nanopore arrays are greatly needed.
SUMMARY OF THE INVENTION
[005] The present invention provides systems comprising a light source, a membrane and a system to direct and focus light from the light source to a spot on the membrane. Methods of light- induced thinning and etching a membrane and generating a nanopore in a membrane are also provided. Membranes comprising a nanopore with a Gaussian curve shaped cross- section are provided as well.
[006] According to a first aspect, there is provided a method of thinning a membrane comprising a first layer comprising an index of refraction of greater than 2.0, the method comprising shining focused light on a spot on the first layer, thereby thinning the membrane.
[007] According to another aspect, there is provided a method of thinning a membrane, the method comprising shining pulsed laser light on a spot on the membrane, thereby thinning the membrane.
[008] According to some embodiments, the focused light is laser light and the laser light is at a wavelength of between 300 and 600 nm.
[009] According to some embodiments, the pulsed laser light is at a wavelength of between 300 and 600 nm.
z 'YS) ±¾j 20(4?l¾¾ing to some embodiments, the focused light is within tlR5^ip?^2S/S£¾Fgreen spectrum.
[011] According to some embodiments, the light comprises an intensity of at least 100 pW.
[012] The method of the invention, wherein the light comprises an intensity of between 1 and 45 mW.
[013] According to some embodiments, the laser light is continuous-wave laser light or pulsed laser light.
[014] According to some embodiments, the membrane comprises a first layer comprising an index of refraction of greater than 2.0.
[015] According to some embodiments, the index of refraction is greater than 2.20.
[016] According to some embodiments, the first layer comprises silicon nitride (SiNx).
[017] According to some embodiments, the first layer is a SiNx layer comprising an average silicon to nitrogen ratio of greater than 0.75.
[018] According to some embodiments, the average silicon to nitrogen ratio is greater than
0.8.
[019] According to some embodiments, the membrane is a freely standing membrane, covered by an aqueous solution on both sides.
[020] According to some embodiments, the membrane comprises a second layer refractory to thinning by the focused light when not layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, and wherein the second layer is layered onto the first layer.
[021] According to some embodiments, the second layer is a layer of metal oxide and wherein the metal oxide is titanium oxide (Ti02), aluminum oxide (A102) or hafnium oxide (Hf02).
[022] According to some embodiments, the membrane does not comprise a thickness of less than 20 nm.
[023] According to some embodiments, the membrane comprises a thickness of less than 20 nm.
[024] According to some embodiments, the membrane is immersed in ultrapure water or salt buffer comprising an alkaline pH. ^SL¾*j 20(4?i¾¾ing to some embodiments, the membrane is at room terfpcf(¾l?£¾?/u5p?5asure.
[026] According to some embodiments, the method further comprises measuring photoluminescent (PL) intensity from the spot on the membrane.
[027] According to some embodiments, the PL intensity is inversely proportional to the thickness of the spot on the membrane, and the thinning is halted at a desired thickness based on a measured PL intensity.
[028] According to some embodiments, the thinning comprises forming a pore through the membrane.
[029] According to some embodiments, the pore is a nanopore.
[030] According to some embodiments, the membrane is covered in an aqueous solution and the method further comprising measuring ionic current through the membrane; optionally, wherein an increase in ionic current through the membrane indicates the pore has been formed in the membrane.
[031] According to some embodiments, the spot in the membrane comprises a thickness of at least 40 nm before the shining and the pore can be produced though the spot in the membrane in less than 20 seconds.
[032] According to some embodiments, the thinning comprises widening a pore through the membrane.
[033] According to some embodiments, the membrane is covered in an aqueous solution and an increase in ionic current through the membrane is proportional to a widening of the pore.
[034] According to some embodiments, the method is for producing a pore of a given diameter, wherein the focused light is automatically shut off at a predetermined current.
[035] According to another aspect, there is provided a system comprising: a. a light source;
b. a membrane comprising a first layer comprising an index of refraction of greater than 2.0;
c. an apparatus to direct and focus light from the light source to a spot on the layer.
[036] According to some embodiments, the membrane is in an optically accessible flow cell. 'YSt2*j 20(4?l¾¾ing to some embodiments, the membrane is a freel^¾o?MP20 u?v9 ±?urane, covered by an aqueous solution on both sides.
[038] According to some embodiments, the index of refraction is greater than 2.20.
[039] According to some embodiments, the system further comprises a photodetector, wherein the photodetector: a. is capable of measuring low light intensities and/or measuring at high temporal resolution;
b. is an avalanche photodiode, a photo -multiplier tube or a CMOS camera; or
c. is configured to detect emissions from the spot on the membrane.
[040] According to some embodiments, the light source is at least one of: a. a solid-state or gas lasers configured to emit within the purple, blue or green spectrum;
b. a solid-state laser configured to emit at between 300-600 nanometers (nm);
c. a continuous-wave laser or a pulsed laser;
d. configured to produce light at an intensity of at least 100 micro-watts (pW) at the spot on the membrane; and
e. configured to produce light at an intensity of at least 1 milliwatts (mW) at the spot on the membrane.
[041] According to some embodiments, the system further comprises an imaging sensor, optionally, wherein the imaging sensor is selected from an electron multiplying CCD camera, a CMOS camera and a sCMOS camera.
[042] According to some embodiments, the first layer comprises SiNx and comprises a silicon to nitrogen ratio of greater than 0.75.
[043] According to some embodiments, the silicon to nitrogen ratio is greater than 0.80.
[044] According to some embodiments, the membrane does not comprise a thickness of less than 20 nm, comprises a thickness of less than 20 nm, is at room temperature and pressure, or a combination thereof.
[045] According to some embodiments, the membrane is immersed in ultrapure water or salt buffer at an alkaline pH. 'YS)-r¾j 20(4?l¾¾ing to some embodiments, the system further compril^T/iv¾?®?Si0uSu¾ and an apparatus configured to pass an electric current between the two electrodes, wherein one electrode is positioned on one side of the membrane and a second electrode is positioned on another side of the membrane, optionally, further comprising a current detector configured to measure current between the two electrodes.
[047] According to some embodiments, the membrane further comprises a second layer layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, optionally wherein the metal oxide is Ti02, A102, or Hf02.
[048] According to another aspect, there is provided a thinned membrane produced by a method of the invention.
[049] According to another aspect, there is provided a membrane comprising a nanopore, wherein the membrane comprises a first layer comprising an index of refraction of greater than 2.0 and wherein the nanopore comprises a varying diameter and a Gaussian curve shaped cross-section.
[050] According to some embodiments, the index of refraction is greater than 2.20.
[051] According to some embodiments, the nanopore increases in diameter from one side of the membrane to the other, and wherein the increasing diameter follows a Gaussian curve.
[052] According to some embodiments, the membrane produces a lower optical background at the nanopore than a nanopore in the membrane without a Gaussian curve shaped cross-section or not produced by a method of the invention.
[053] According to some embodiments, the first layer comprises SiNx and wherein the SiNx comprises a silicon to nitrogen ratio of greater than 0.75.
[054] According to some embodiments, the silicon to nitrogen ratio is greater than 0.8.
[055] According to some embodiments, the membrane further comprises a second layer layered on the first layer, wherein the second layer is a dielectric layer or a layer of metal oxide, optionally wherein the metal oxide is Ti02, A102 or Hf02.
[056] According to some embodiments, the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from an interface of the first layer with the second layer to an exposed surface of the first layer and a second Gaussian curve shaped cross-section increasing in diameter from the interface to an exposed surface of the second layer. 'YSt2*j 20(4?l¾¾ing to some embodiments, the membrane comprises with different indexes of refraction.
[058] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[059] Figures 1A-D: Laser thinning of freestanding SiNx. (1A) Schematic of the confocal setup. SC- SiNx chip; PS-piezo stage OL- objective lens; DM- dichroic mirror; LP- low pass filter; TL- tube lens PH- pinhole. The emission pathway is switchable between the APD and EMCCD. (IB) Focusing of a ~45 mW 488 nm laser on the membrane results in photoluminescence emission, which is recorded by the APD in the >550 nm range. (1C) Photoluminescence emission during laser-exposure, measured in counters per second. The laser is activated at t = 0 seconds. (ID) Images of the 42 x 42 pm2 membrane under white- light illumination before etching (i). After 300 seconds of laser exposure, a thin region is visible as a contrasted spot (ii).
[060] Figure 2. Photoluminescence (PL) intensity calibration as a function of SiNx thickness. The PL in counts per second (CPS) was measured by the APD during laser- exposure (488 nm, 30 pW) for 6 chips of different membrane thickness. Prior to the PL measurements, the membrane thickness was measured by ellipsometry. Thicker membranes result in higher PL.
[061] Figures 3A-E. Thickness characterization of a laser-etched spot. (3A) TEM images at 195x, 39000x and 75000x (left to right). The lighter region corresponds with higher transmittance and thus thinner material. (3B) TEM images of laser-etched thin regions. (Upper left) TEM image at 7500x of two thin regions corresponding to 2 different laser exposure durations: 1 and 3 minutes (left to right). (Upper right) TEM image at 21000x of one thin region after 1 minute of laser exposure. (Lower) TEM image at 16500X of a thin region after 1 minute of laser exposure (left), and a zoom-in at 75000x (right). (3C) TEM thickness map of an etched spot in nanometers. (3D) Blue curve- TEM thickness map. Purple curve- normalized photoluminescence (PL) scanned in the x direction with a 30 nm step size. m u 1 ated normalized PL based on a convolution of ^Tu¾?Pa9/i^9¾ iited Gaussian, representing the laser beam, with the TEM thickness map. (3E) Laser-etching wavelength dependency. Free-standing 40-45 nm thick SiNx were subjected to ~45 mW 488 (blue), 532 (green), and 645 (red) laser intensities for 2, 4 and 6 minutes. Consequently, the membranes were imaged using a light microscope in transmission mode. The grayscale intensity values are shown as arbitrary greyscale units, obtained by averaging the pixel values at the center of the etched regions. Experiments were performed in triplicates. A higher pixel intensity corresponds to greater light transmittance and thus a thinner membrane region.
[062] Figures 4A-B. Nanopore fabrication by laser-etching. (4A) Measured photoluminescence (PL) and ionic current during laser-exposure (red and grey curves, respectively). The PL sharply increases when the laser is activated (i). Pore formation is signaled by an increase in current (ii). Following ~20 s of pore growth under continued laser- exposure, the laser is deactivated, and the PL returns to zero (iii). Turning off the laser causes a conductivity decrease, resulting in a coincident drop in current which stabilizes over time. (4B) Principle of calcium (Ca2+) activators used for verifying the creation of a nanopore (top panels). The entire membrane is illuminated by a 488 nm laser. At -300 mV, Ca2+ is driven away from the pore. At +300 mV, Ca2+ is driven through the pore where it binds to Fluo-4 resulting in detectable fluorescence at >510 nm. The bottom panels show calcium activators applied to laser-drilled pores. The bias is repeatedly switched between positive and negative to validate the presence of a nanopore.
[063] Figure 5A-N. Noise and functionality of a laser-etched nanopore. (5A) TEM image showing a nanopore with a diameter of 6.5 nm. Compared to the peripheral membrane, the nanopore is very bright, owing to an unobstructed electron beam path. (5B) Three examples of measured photoluminescence (red curve) and ionic current (grey curve) during laser- exposure. A nanopore was formed after (top) 400 s (middle) 920 s and (bottom) 140 s. (5C) Power spectral density (PSD) plot of a nanopore for an applied bias of 300 mV. The inlet shows the corresponding current-voltage (IV) curve for this nanopore, with a linear fitting (R2 > 0.99). (5D) Scatter plot of dsDNA translocation events. The trans chamber was biased to 300 mV to drive translocation of 300 pM 5054 bp dsDNA from cis to trans. The size of the pore is 3.1 + 0.3 nm based on the current blockage level/molecular ruler model. (5E) A concatenated ionic current trace showing sample dsDNA translocation events. (5F-H) Translocations of 5054 bp DNA at different applied voltages: 300 mV (red), 450 mV (orange) and 600 mV (yellow). (5F) Scatter plot of normalized translocation event blockage ^9 iSi°uwi??¾me. (5G) Translocation dwell-time histograms s h o w w¾g i\ dent time constants of 500 ± 25, 175 ± 3, and 135 ± 4 ps from lowest to highest voltage. (5H) Sample concatenated translocation events at each voltage. (51) Scatter plot of normalized translocation event blockage versus dwell time of translocations of 5054 bp at 300 mV. (5J) Normalized translocation blockage histogram fitted by two Gaussians: <IB>=0.57 (green curve) and <IB>=0.7 (red curve) (5K) Sample concatenated translocation events. (5L) Scatter plot of di-ubiquitin (K63 -linked di-Ub) translocation events at pH 7. The trans chamber was biased to 300 mV to drive translocation of 0.007 pg/mΐ di-Ub from cis to trans. (5M) A concatenated ionic current trace showing sample di-Ub translocation events. (5N) Translocations of di-ubiquitin (K63 -linked Di-Ub) at 300 mV. (left) Translocation dwell- time histogram showing a decay time constant of 139 ± 10 ps. (right) Normalized translocation blockage histogram fitted by a Gaussian: <IB>=0.77. N=326 events.
[064] Figures 6A-D. Localized laser-etching of freestanding SiNx. (6A) Laser-etched T- shape array of 9 thin regions spaced 1500 ± 50 nm center-to-center. The top and vertical bars were etched with a laser intensity of ~30 mW and 45 mW, respectively, for 4 minutes each. Next to the T is a lithography-fabricated thin region (20 ± 2 nm) for comparison. (6B) Zoom in of just the T. (6C) TEM image of the T, showing a difference in brightness for the top and vertical bars, corresponding with a difference in thickness. (6D) Nanopore formed in one of the thin regions.
[065] Figures 7A-E. Nanopore fabrication by laser-etching. (7A) Schematic illustration of the electro-optical apparatus used for laser-assisted nanopore drilling. (7B-D) Laser thinning in three membranes with different indices of refraction: n=2.20, 2.29, 2.42 for 7B, 7C and 7D, respectively. Laser intensity is equal in all cases. Left: photoluminescence (PL) and current traces during the etching time. A sudden incline in the current trace indicates pore formation. For the n=2.20 membrane no pore was formed even after 2500 seconds, where for the n=2.29,2.42 membranes pores were formed after 140 and 120 seconds respectively. Right: images of the membrane before and after thinning. In all cases a black spot (indicated by a black arrow) appears in the later images, where less light was reflected, indicating a thinner region. The formation of the thin regions corresponds to the PL traces (left) which decrease in all cases regardless of pore formation. These experiments were reproduced more than 100 times each. (7E) Chips with refractive index of 2.3 and initial thickness of 38-42 nm were immersed in 1M KC1 buffer with pH of 7 or 10 heated to 90°C in a temperature- controlled hot water bath. The SiNx thicknesses of 4 different chips were measured using ellipsometry after 2, 5, 30 and 60 minutes (pH 7 - blue triangles, pHIO - green tringles). Ύ w u<l?ll/! i ps were immersed in 1 M KC1, pH 10 buffer at room^Tipt^a^i^SAu6 their thickness was measured as well (red triangles). After an hour immersion at 90 °C we observed a change in thickness of about 1 nm only.
[066] Figures 8A-D. (8A) Material composition as a function of the refractive index measured using EDS (triagles) and EELS (circles). Red curve represents the theoretical model with reported theoretical valus of n¥=3.86 n3/4=1.99. Higher refractive index indicates higher precentage of silicon in the membrane. Fitting each of the measurements with the theoretical model resulted in the following parameter values: n¥=3.9949 n3/4=1.9638 for the EELS and n¥=3.6827 n3/4=1.7401 for the EDS (solid black lines). (8B) Low loss peaks measured using HR-EELS with the monochromated zero loss peak (ZLP) of 0.17 eV (left panel). The measurement was repeated for two different chips (n=2.15 - green curve, n=2.42 - blue curve). Zoomed in plot of the 0-8 eV region in presented in the right panel and was used to estimate the material bandgap. The energies of the laser wavelengths available in our sensing apparatus is presented using red (640 nm), green (532 nm), and blue (488 nm) vertical lines. (8C) Measured photoluminescence (PL) and ionic current during laser- exposure (red and grey curves, respectively) of different wavelengths. The high refractive index (2.43) chip was easily drilled using a 488 and 532 nm lasers in less than a minute or roughly two minutes, respectively. In contrast, the low refractive index chip (2.15) could not be drilled at these laser intensities even after 10 minutes. Illuminating the chips with the red laser (640 nm) did not result in thinning or drilling in either case. (8D) Average PL values for four different types of chip (n=2.15, 2.2, 2.3, 2.43) of similar thickness (44-46 nm). Lower refractive index corresponds to higher PL (dark grey marks). The results are fitted to exponential curves (dark grey solid line). Light grey triangles present the ratio of the red- band emission over the total emission which increases as the refractive index increases. This is asscociated with a red-shift of the PL at the higher Si:N ratio. Each measurement was repeated using 4 chips of each type.
[067] Figure 9. Normalized PL traces of membrane thinning using different laser intensities (1.9 to 7 mW) and the four pH values (pH 4 - red, pH 7 - green, pH 10 - blue, pH 12 - purple). While acidic and neutral buffers (pH 4 and 7) hardly change the PL behavior even when the intensity is increased, the hydroxyl-rich solutions (pH 10 and 12) increase the effectiveness of the etching, producing thinner membranes at high speed.
[068] Figures 10A-B. Membrane thinning as a function of the solution pH. (10A)
Reflected white light image of a chip (n=2.3) before laser exposure (488 nm) and after exposing it for one minute at different pH levels and 1 M KC1 (each measurement was ^ ¾>¾i?Su ¾*¾?uies). (10B) The normalized intensity is the change if?Jffii?t®?P^w¾££6light according to (signal-background)/background for each condition (4 different pH levels) after exposing the chip for two minutes as a function of the laser intensity. Solid lines for each pH imply a linear dependence.
[069] Figures 11A-D. Characterization of the ultra-fast drilling process. (11A) STEM thickness map of a thin region created using increasing laser exposure durations. Conditions: 488 nm, 34 mW, 1 M KC1, pHlO. A longer exposure duration results in a thinner membrane. (11B) STEM thickness map of a thinned region created using increasing laser exposure durations. Conditions: 488 nm, 7 mW, 1M KC1, pH 10. Longer exposure duration results in thinner membrane. (11C) x-line scan in the middle of the thickness map for each exposure duration. (11D) Normalized, integrated thickness for x-line scans taken in the middle of the thickness maps for each exposure duration (circles), and the PL trace as a function of time (grey). Inset - high magnification TEM images for selected thin regions. After laser exposure of 15 s one ~6 nm pore was created in the middle of the Gaussian shaped thin region. Longer exposures resulted in the formation of multiple pores.
[070] Figures 12A-B. DNA translocations in a fast-drilled nanopore. (12A)
Photoluminescence (PL) and current traces of a pore drilled in ~15 seconds. The laser was turned on at t = 0, as can be indicated by an abrupt PL increase. The PL decays over time as the membrane is thinned until pore formation, signaled by an increase in the electrical current. Conditions: 1 M KC1, pH 10, 300 mV, 488nm wavelength with intensity of 7 mW. The inset shows the current- voltage (IV) curve for this nanopore after the buffer was changed to pH 7 and the open pore current stabilized. (12B) Concatenated dsDNA (300 bp) translocation events with a zoomed in plot of an event, and the scatter plot of the blocked current (IB = Ibiocked / Iopen) vs. the dwell time tD of all the events (N=560).
[071] Figures 13A-C. Fast drilling of nanopores array using a focused laser beam. (13A) Top: laser intensity time trace during nanopore array drilling. The laser power is switched automatically on and off for each drilling event, followed by controlled movement of the piezo stage to the next coordinate. Bottom: Photoluminescence (PL, red) and current (blue) traces of an array of 25 pores drilled in ~6 minutes. When the current rapidly increases, the laser is automatically turned off and the PL drops. (13B) Histograms of the drilling time and the change of current for each pore, and Gaussian fitting for each histogram (13.6+ 3.1 seconds, 1.9 + 0.9 nA) (13C) Wide-field illumination images of the entire membrane using 488 nm laser. Calcium (Ca+2) activated fluorophores are used for verifying the creation and position of the nanopore array. At 300 mV Ca+2 passes through the pore and binds to Fluo- ^ -9 ¾S9 i?g¾93a fluorescence spot at the thin region (middle panel). Th??pZ¾?9¾pp5S?f^hen the bias is reversed to -300 mV (left panel). The membrane position is outlined by a white dashed line. The histogram in the right panel describes the normalized intensity distribution of the 25 pores. Grey curve is a Gaussian fit (0.97+ 0.18).
[072] Figures 14A-B. (14A) Cartoon of drilling in a membrane comprising SiNx and Ti02. The bottom layer is the SiNx and the top layer is the T1O2. Laser light shown on the silicon layer catalyzes drilling in the titanium layer which had previously been refractory to drilling. Acidic pH on the titanium side inhibits this catalyzation, whereas neutral or alkaline pH is permissive. (14B) Line graph of the atomic fraction of each component of the membrane at the site of drilling as a function of time.
DETAILED DESCRIPTION OF THE INVENTION
[073] The present invention, in some embodiments, provides systems for light-induced etching a membrane and/or producing a nanopore in a membrane. Methods of thinning and etching a membrane are also provided, as are membranes comprising a nanopore with a Gaussian curve shaped cross-section.
[074] The present invention is based on the unexpected finding that the inventors could perform purely photo-chemical solid-state nanometer-scale pore fabrication with unparalleled in situ control over the nanopore position. Local SiNx thinning and subsequent pore formation are performed at any arbitrary point along the membrane by simply positioning the membrane at the tightly focused laser spot. To illustrate this, we constructed an evenly spaced, nanoscale-accurate (1500 + 50 nm center-to-center) T-shape of 9 thin regions in a SiNx membrane in just 36 minutes (Fig. 6A-C). The T was made next to a lithography-fabricated thin region (20 + 2 nm) used for thickness calibration and to further prove that the produced contrast is due to thinning (Fig. 6A). The etch time of ~4 minutes per spot was more than enough to produce the visible contrast; under TEM inspection, we found that a ~20 nm nanopore had formed in one of the spots (Fig. 6D). The etch rate is also highly controllable. To show this, we varied the laser intensity in constructing the T: ~30 mW and 45 mW for the top and vertical bars, respectively.
[075] With a blue or green laser and a membrane in water, nanopores can be formed optically in as little as one minute in 45 nm thick SiNx, using a purple laser or increasing pH allowed for significantly faster pore formation. Given that the technique is rapid and highly automatable— it can be monitored by the PL intensity and ionic current— it can be used to construct vast nanopore arrays for massively parallel optical sensing. Notably, as nanopore , ?ίί¾* /’ pieced s so quickly, it would not be necessary to compri’i^i^i^/S^^lness of the supporting membrane, as might be necessary using thickness-limited strategies such as CBD. Furthermore, as a consequence of the inverted-Gaussian etch profile, these nanopores benefit from significantly improved spatial resolution, reduced background PL and improved mechanical robustness.
[076] The inventors further found that both the etching and nanopore drilling kinetics can be accelerated by orders of magnitudes using higher Si to N ratio membranes, measured as a slight increase in their index of refraction. Specifically, a change in the index of refraction of SiNx membranes from -2.20 to -2.40 corresponded to a transition from a practically non drilling membrane, even after nearly an hour of exposure, to nearly instantaneous nanopore formation. Indeed, when the solution pH was raised to alkaline pH ultra-fast pore formation was observed, even when using a low laser power that could not etch the material at normal pH. Following optimization, drilling yielded single 5 nm pores in 15 seconds from a starting 45 nm thick substrate. Such ultra-fast drilling can be utilized for preparing massive nanopore arrays at any arbitrary position, limited only by light diffraction. As a proof-of-principle, we designed a fully automated feed-back controlled protocol for drilling a 25-nanopore array in about 7 minutes without any user-intervention.
[077] Lastly, it was unexpectedly found that pulsed laser light was superior to continuous- wave laser light for thinning and drilling. Shone with the same average power and at the same wavelength, pulsed laser light could thin and drill faster than continuous-wave laser light. Even some membranes that were resistant to etching with a continuous-wave laser could be etched with a pulsed laser.
Systems of the invention
[078] By a first aspect, there is provided a system comprising, a light source, a membrane, and a system to direct and focus light from the light source to a spot on the membrane.
[079] By another aspect, there is provided a system comprising a light source, an area configured to receive a free-standing membrane, and a system to direct and focus light from the light source to a spot on a received free-standing membrane.
[080] In some embodiments, the light source produces coherent light. In some embodiments, the light source produces collimated light. In some embodiments, the light source produces coherent and collimated light. In some embodiments, the light source produces a coherent and collimated light beam. In some embodiments, the light source is a laser or light emitting diode (LED). In some embodiments, the light source is a laser. In some ! ¾ ¾·? the laser is a solid-state laser. In some embodiments, E£T/II^20/0^)356aser In some embodiments, the laser is a wave laser. In some embodiments, the laser is a continuous-wave laser. In some embodiments, the laser is a pulsed laser. In some embodiments, a pulsed laser is a pico-second pulsed laser. In some embodiments, the laser light is continuous-wave laser light. In some embodiments, the laser light is pulsed laser light.
[081] In some embodiments, the light source is a monochromatic light source. In some embodiments, the light source is configured to produce monochromatic light. In some embodiments, the light source produces purple, blue and/or green light. In some embodiments, the light source produces purple light. In some embodiments, purple light is violet light. In some embodiments, the light source produced blue light. In some embodiments, the light source produced green light. In some embodiments, the light source produced blue and/or green light. In some embodiments, purple light comprises a wavelength between 380 and 420 nm. In some embodiments, purple light comprises a wavelength between 380 and 450 nm. In some embodiments, purple light comprises a wavelength between 400 and 420 nm. In some embodiments, purple light comprises a wavelength between 400 and 450 nm. In some embodiments, blue light comprises a wavelength between 380 and 490 nm. In some embodiments, blue light comprises a wavelength between 420-490 nm. In some embodiments, blue light comprises a wavelength between 450-490 nm. In some embodiments, blue light comprises a wavelength between 380 and 500 nm. In some embodiments, blue light comprises a wavelength between 420- 500 nm. In some embodiments, blue light comprises a wavelength between 450-500 nm. In some embodiments, blue light comprises a wavelength between 380 and 520 nm. In some embodiments, blue light comprises a wavelength between 420-520 nm. In some embodiments, blue light comprises a wavelength between 450-520 nm. In some embodiments, green light comprises a wavelength between 500 and 580 nm. In some embodiments, green light comprises a wavelength between 520 and 580 nm. In some embodiments, green light comprises a wavelength between 500 and 560 nm. In some embodiments, green light comprises a wavelength between 520 and 560 nm. In some embodiments, the light source is configured to emit light at a wavelength between 380 and 580 nm. In some embodiments, the light source is configured to emit light at a wavelength between 300 and 580 nm. the light source is configured to emit light at a wavelength between 380 and 600 nm. In some embodiments, the light source is configured to emit light at a WO ?ίί ?v!Y .yi wccn 300 and 600 nm. In some embodiments, tSS1(iii¾029gi?l 0¾6 at a wavelength of between 300 and 600 nm.
[082] In some embodiments, the power of the light source is at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 25, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 milliwatts (mW). Each possibility represents a separate embodiment of the invention. In some embodiments, the power of the light source is at least 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 micro-watts (pW). Each possibility represents a separate embodiment of the invention. In some embodiments, the power of the light source is at least 1 pW. In some embodiments, the power of the light source is at least 10 pW. In some embodiments, the power of the light source is at least 100 pW. In some embodiments, the power of the light source is between 1 pW and 50 mW, 1 pW and 45 mW, 1 pW and 40 mW, 1 pW and 35 mW, 1 pW and 30 mW, 1 pW and 25 mW, 1 pW and 20 mW, 1 pW and 15 mW, 1 pW and 1 mW, 1 pW and 5 mW, 1 pW and 1 mW, 10 pW and 50 mW, 10 pW and 45 mW, 10 pW and 40 mW, 10 pW and 35 mW, 10 pW and 30 mW, 10 pW and 25 mW, 10 pW and 20 mW, 10 pW and 15 mW, 10 pW and 10 mW, 10 pW and 5 mW, 10 pW and 1 mW, 100 pW and 50 mW, 100 pW and 45 mW, 100 pW and 40 mW, 100 pW and 35 mW, 100 pW and 30 mW, 100 pW and 25 mW, 100 pW and 20 mW, 100 pW and 15 mW, 100 pW and 10 mW, 100 pW and 5 mW, 100 pW and 1 mW, 200 pW and 50 mW, 200 pW and 45 mW, 200 pW and 40 mW, 200 pW and 35 mW, 200 pW and 30 mW, 200 pW and 25 mW, 200 pW and 20 mW, 200 pW and 15 mW, 200 pW and 10 mW, 200 pW and 5 mW, 200 pW and 1 mW, 300 pW and 50 mW, 300 pW and 45 mW, 300 pW and 40 mW, 300 pW and 35 mW, 300 pW and 30 mW, 300 pW and 25 mW, 300 pW and 20 mW, 300 pW and 15 mW, 300 pW and 10 mW, 300 pW and 5 mW, 300 pW and 1 mW, 400 pW and 50 mW, 400 pW and 45 mW, 400 pW and 40 mW, 400 pW and 35 mW, 400 pW and 30 mW, 400 pW and 25 mW, 400 pW and 20 mW, 400 pW and 15 mW, 400 pW and 10 mW, 400 pW and 5 mW, 400 pW and 1 mW, 500 pW and 50 mW, 500 pW and 45 mW, 500 pW and 40 mW, 500 pW and 35 mW, 500 pW and 30 mW, 500 pW and 25 mW, 500 pW and 20 mW, 500 pW and 15 mW, 500 pW and 10 mW, 500 pW and 5 mW, or 500 pW and 1 mW. Each possibility represents a separate embodiment of the invention. A person skilled in the art will appreciate that as the wavelength of the light is decreases the power can be decreased without a deleterious effect on the ability of the system to etch. Thus, a purple light at a lower power can etch at the same rate as a green light at a higher power, for non-limiting example. In some embodiments, the power of the light source is the intensity of the light at a spot on the membrane. In some embodiments, the light source is configured 'Yv91?/t?uujy·?)!;* intensity of light at a spot on the membrane. In some spot is on the first layer. In some embodiments, the first spot is on the first layer. In some embodiments, the second spot is on the second layer.
[083] In some embodiments, a pulsed laser produces pulse widths of between 50-150 picoseconds (ps). In some embodiments, pulsed laser light comprises pulse widths of between 50-150 ps. In some embodiments, a pulsed laser produces pulse widths of at least 20, 30, 40, 50, 60, 70, 80, 90 100, 110, 120, 130, 140, or 150 ps. Each possibility represents a separate embodiment of the invention. In some embodiments, pulsed laser light comprises pulse widths of at least 20, 30, 40, 50, 60, 70, 80, 90 100, 110, 120, 130, 140, or 150 ps. Each possibility represents a separate embodiment of the invention. In some embodiments, a pulsed laser produces pulse widths of at most 50, 60, 70, 80, 90 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 ps. Each possibility represents a separate embodiment of the invention. In some embodiments, pulsed laser light comprises pulse widths of at most 50, 60, 70, 80, 90 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 ps. Each possibility represents a separate embodiment of the invention. In some embodiments a pulsed laser produces pulse widths or pulsed laser light comprises pulse widths of between 40-200, 40-180, 40- 160, 40-150, 10-140, 10-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 50-200, 50-180, 50- 160, 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-200, 60-180, 60- 160, 60-150, 60-140, 60-130, 60-120, 60- 110, 60-100, 60-90, 60-80, 60-70, 70-200, 70-180, 70- 160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-200, 80-180, 80- 160, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-200, 90-180, 90- 160, 90-150, 90-140, 90-130, 90-120, 90-110, 90-100, 100-200, 100-180, 100- 160, 100-150, 100-140, 100-130, 100-120, 100-110, 100- 200, 110-180, 110-160, 110-150, 110-140, 110-130, 110-120, 120-200, 120-180, 120-160, 120-150, 120-140, 120-130, 130-200, 130-180, 130-160, 130-150, 130-140, 140-200, 140- 180, 140-160, 140-150, 150-200, 150-180, or 150-160. Each possibility represents a separate embodiment of the invention.
[084] In some embodiments, a pulsed laser pulses with a repetition rate of up to 80 mega Hertz (MHz). In some embodiments, pulsed laser light comprises a repetition rate of up to 80 MHz. In some embodiments, a pulsed laser pulses with a repetition rate of up to 20, 30, 40, 50, 60, 70, 75, 80, 90 or 100 MHz. Each possibility represents a separate embodiment of the invention. In some embodiments, pulsed laser light comprises a repetition rate of up to 20, 30, 40, 50, 60, 70, 75, 80, 90 or 100 MHz. Each possibility represents a separate the invention. In some embodiments, a pulsed laser ]^^iL v??uP(?¾p¾ition rate of not more than 40, 50, 60, 70, 80, 90 or 100 MHz. Each possibility represents a separate embodiment of the invention. In some embodiments, pulsed laser light comprises a repetition rate of not more than 40, 50, 60, 70, 80, 90 or 100 MHz. Each possibility represents a separate embodiment of the invention. In some embodiments, a pulsed laser pulses with a repetition rate or pulsed laser light comprises a repetition rate of between 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40- 80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100 or 80-90 MHz. Each possibility represents a separate embodiment of the invention.
[085] As used herein, the terms“film” and“membrane” are used interchangeably and refer to a thin water-impermeable layer of material. In some embodiments, the membrane is ion- impermeable. In some embodiments, the membrane is insulated. In some embodiments, the membrane is electrically insulated. In some embodiments, the membrane is mechanically robust. In some embodiments, mechanically robust refers to physical strength. In some embodiments, the membrane comprises a tensile strength of at least 100, 150, 200, 250, 275, 300, 325, 350, 375, 400, 450 or 500 mega pascals (MPa). Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane comprises a tensile strength of at least 300 mega pascals (MPa). In some embodiments, the membrane is chemically inert. In some embodiments, chemically inert comprises difficult to etch by organic solvent or aqueous based acids or bases. In some embodiments, the membrane is free-standing. In some embodiments, the membrane is freely standing. In some embodiments, a free-standing membrane is immersed in an aqueous solution. In some embodiments, a free-standing membrane is surrounded on both sides by an aqueous solution. In some embodiments, a free-standing membrane is covered by aqueous solution on both sides. In some embodiments, both sides are two drillable sides. In some embodiments, both sides the side upon which the light is shone and the side upon which light exits when a pore is drilled. It will be understood by a stilled artisan that the sides of the membrane that are not being drilled do not need to be covered by the aqueous solution. Rather, the spot where the light is shone needs to be covered by the aqueous solution, and if the laser spot is considered three dimensionally to pass through the membrane, the spot on the opposite side of the membrane must also be covered by the aqueous solution. A laser spot has a thickness (-300 nm) that is thicker than the width of the membrane. Thus, the“spot” on the membrane actually is a three-dimensional spot that extends through the entire width of the membrane. ^9 ?;¾{L91¾2 spot is on both sides of the membrane and both sides o ΐrί ϊ7.I p3i* ?u (i1 n'i n ^ilrs cd in an aqueous solution. In some embodiments, a free-standing membrane comprises both sides of the spot on the membrane covered by an aqueous solution. In some embodiments, a free-standing membrane comprises access of an aqueous solution over both sides of the spot on the membrane. In some embodiments, the laser spot on the first side of the membrane is a first spot. In some embodiments, the laser spot on the second side of the membrane is a second spot. In some embodiments, the spot is a first spot. In some embodiments, the spot is a second spot. In some embodiments, the spot is a first spot and a second spot. In some embodiments, a free-standing membrane comprises access of an aqueous solution over a first spot on the membrane. In some embodiments, a free-standing membrane comprises access of an aqueous solution over a second spot on the membrane. In some embodiments, the first spot and the second spot are on opposite sides of the membrane. In some embodiments, the first spot and the second spot are positioned such that a line passing through the membrane passes through the first spot and the second spot. In some embodiments, a free-standing membrane comprises an aqueous solution over a first spot on the membrane. In some embodiments, a free-standing membrane comprises an aqueous solution over a second spot on the membrane.
[086] In some embodiments, the membrane comprises a first layer. In some embodiments, the membrane comprises silicon. In some embodiments, the first layer comprises silicon. In some embodiments, the membrane or first layer is silicon based. In some embodiments, the membrane or first layer comprises silicon nitride (SiNx). As used herein, the terms“SiNx” and“SixN” are used interchangeably and refer to silicon nitride. The“x” refers to the ratio of silicon to nitride which is variable in the substance. In some embodiments, the silicon nitride is amorphous silicon nitride. In some embodiments, amorphous silicon nitride is one with a variable ratio of silicon to nitride.
[087] In some embodiments, membrane comprises a second layer. In some embodiments the film comprises a metal oxide. In some embodiments, the second layer is a layer of metal oxide. In some embodiments, the metal oxide is selected from aluminum oxide (AIO2), titanium oxide (TiCh), silicon oxide (SiCh) and hafnium oxide (HGO2). In some embodiments, the metal oxide is selected from aluminum oxide (AIO2), titanium oxide (TiCh), and hafnium oxide (HfCh). In some embodiments, the metal oxide is TiCh. In some embodiments, membrane or second layer comprises amorphous silicon nitride. In some embodiments, the silicon nitride is amorphous silicon nitride. In some embodiments, the membrane or second layer comprises silicon nitride, titanium dioxide or a combination ^^n^RLίϊί^u ΐie embodiments, the membrane comprises more thcE££iit¾ijPP.51 ¾5P,ome embodiments, the membrane comprises a layer of SiNx. In some embodiments, the membrane comprises a layer of T1O2. In some embodiments, the membrane comprises a layer of T1O2 layered on a layer of SiNx. In some embodiments, the second layer is layered on the first layer. In some embodiments, the second layer is a dielectric layer.
[088] As used herein, the term“layer” refers to a thin flat continuous piece of material. In some embodiments, the layer comprises a metallic layer having plasmonic properties. In some embodiments, the layer comprises a metal. In some embodiments, the metal is selected from gold, silver, copper, aluminum, titanium, hafnium and a combination thereof. In some embodiments, the metallic layer comprises at least one layer of metal. In some embodiments, the metallic layer comprises more than one layer of metal. In some embodiments, the more than one layer of metal is layered one on top of the other to create one combined metallic layer. In some embodiments, the layer comprises a metal oxide. In some embodiments, the layer is a silicon layer. In some embodiments, the layer is a layer that comprises silicon. In some embodiments, the layer is a silicon nitride layer. In some embodiments, a layer is deposited by Atomic Layer Deposition (ALD). In some embodiments, one layer is deposited upon another by ALD.
[089] In some embodiments, the second layer is refractory to etching. In some embodiments, the second layer is refractory to thinning. In some embodiments, the second layer is refractory when not layered on the first layer. In some embodiments, the second layer when layered on the first layer can be etched or thinned. In some embodiments, the second layer is layered on the first layer.
[090] In some embodiments, the membrane is deposited on a silicon wafer. In some embodiments, the membrane is formed from a silicon wafer. In some embodiments, the wafer is a crystal orientation wafer. In some embodiments, the wafer is thicker in regions that lack a nanopore. In some embodiments, the wafer comprises a diameter of at least 1, 10, 50, 75, 100 or 200 mm. Each possibility represents a separate embodiment of the invention. In some embodiments, the wafer comprises a thickness of at least 50, 100, 150, 200, 250, 300, 350 or 400 pm. Each possibility represents a separate embodiment of the invention.
[091] In some embodiments, the membrane or first layer has a universal thickness. In some embodiments, the membrane or first layer has a constant thickens across its entire area. In some embodiments, the membrane or first layer has a variable thickness. In some embodiments, the membrane or first layer is thinner in the area of the nanopore. In some ! ¾ ¾·? the membrane or first layer does not comprise a t h i c ¾ ί' 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane or first layer does not comprise a thickness of greater than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane or first layer at the spot where the light focuses comprises a thickness of between 1-100, 1-75, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-100, 5-75, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-100, 10-75, 10-50, 10- 45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-100, 15-75, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-100, 20-75, 20-50, 20-45, 20-40, 20-35, 20-30, or 20-25 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane or first layer at the spot where the light focuses comprises a thickness of between 10 and 50 nm. In some embodiments, the membrane or first layer at the spot where the light focuses does not comprise a thickness of less than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the membrane or first layer at the spot where the light focuses does not comprise a thickness of more than 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nm. Each possibility represents a separate embodiment of the invention.
[092] In some embodiments, the membrane comprises a high index of refraction. In some embodiments, the first layer comprises a high index of refraction. In some embodiments, the silicon nitride membrane comprises a high index of refraction. In some embodiments, a high index of refraction is an index at and/or above 1.8, 1.9, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35 or 2.4. Each possibility represents a separate embodiment of the invention. In some embodiments, a high index of refraction is an index greater than 1.8, 1.9, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35 or 2.4. Each possibility represents a separate embodiment of the invention. In some embodiments, a high index of refraction is an index greater than 2.2. In some embodiments, a high index of refraction is an index greater than 2.0. In some embodiments, the second layer does not comprise a high index of refraction. In some embodiments, the second layer comprises an index of refraction below a high index of refraction.
[093] In some embodiments, the SiNx membrane comprises a silicon to nitride ratio of at least 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 or 1.25. Each possibility represents a separate embodiment of the invention. In some embodiments, the SiNx membrane comprises a silicon to nitride ratio of at least 0.8. In some embodiments, the SiNx membrane comprises ^91 }º} ·? ^riclc ratio of at least 0.75. In some embodiments, the SiN?9J/A¾¾?9/.°.¾A¾5rises a silicon to nitride ratio of greater than 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 or 1.25. Each possibility represents a separate embodiment of the invention. In some embodiments, the SiNx membrane comprises a silicon to nitride ratio of greater than 0.8. In some embodiments, the SiNx membrane comprises a silicon to nitride ratio of greater than 0.75. It will be appreciated by a skilled artisan that increasing the refraction index and/or the ratio of silicon to nitride will increase the rate at which etching/thinning of the membrane proceeds. Further, a lower laser power, or a high wavelength of light could be used in combination with a membrane of higher index or ratio and not proceed more slowly as the two changes would offset.
[094] In some embodiments, the membrane comprises a first layer and a second layer wherein the two layers have different refraction indexes, and/or different ratios of silicon to nitride. In some embodiments, the second layer that is refractory to etching/thinning by focused light. In some embodiments, the second layer is an inert layer. In some embodiments, the membrane comprises a dielectric layer. In some embodiments, the second layer is refractory /inert when not layered on a silicon nitride layer. In some embodiments, a metal oxide layer is refractory and/or inert. In some embodiments, a dielectric layer is refractory and/or inert. In some embodiments, the metal oxide layer is selected from a T1O2, an aluminum oxide (AIO2) and a hafnium oxide (HfCh) layer. In some embodiments, the metal oxide is TiCh. In some embodiments, the inert/refractory layer is layered onto a layer of silicon nitride. In some embodiments, the layer of silicon nitride sensitizes the refractory /inert layer to etching/thinning. In some embodiments, the silicon nitride layer catalyzes the etching/thinning of the refractory/inert layer when focused light is shown on the silicon nitride layer and/or the refractory/inert layer. In some embodiments, the membrane comprises a plurality of layers with different refraction indexes, and/or different ratios of silicon to nitride. A skilled artisan will appreciate that by modulating the intensity of light, the wavelength of the light, or the pH of the aqueous solution, thinning/etching can be done in only particular layers at a time.
[095] In some embodiments, the membrane is a free-standing membrane. In some embodiments, the membrane is immersed in an aqueous solution. In some embodiments, the membrane is at least partially immersed in an aqueous solution. In some embodiments, the spot on the membrane is immersed in an aqueous solution. In some embodiments, immersed comprises an aqueous solution on both sides of the membrane. In some embodiments, the same aqueous solution is on both sides of the membrane. In some embodiments, different atjutu ua ?i¾? ons are on each side of the membrane. In some eml?^i¾?ft!^P¾3fj*st< em further comprises a first and second liquid reservoir. In some embodiments, the first and second liquid reservoirs are separated by the membrane.
[096] In some embodiments, the system is configured to receive a free-standing membrane. In some embodiments, the system comprises an area configured to receive a free-standing membrane. In some embodiments, the area is a receiving area. In some embodiments, the area comprises at least one dimension configured for the placement of a free-standing membrane. In some embodiments, the area comprises a receptacle for an aqueous solution. In some embodiments, a receptacle is configured to receive an aqueous solution. In some embodiments, a receptacle is configured to hold an aqueous solution. In some embodiments, the receptacle is configured such that a received aqueous solution covers a spot on a received membrane. In some embodiments, the receptacle is configured such that a received aqueous solution covers a first spot on a received membrane. In some embodiments, the receptacle is configured such that a received aqueous solution covers a first spot and a second spot on a received membrane. In some embodiments, the area comprises two receptacles. In some embodiments, a first receptacle is configured such that a received aqueous solution covers a spot on one side of a received membrane and a second receptacle is configured such that a received aqueous solution covers the spot on the opposite side of a received membrane. In some embodiments, a first receptacle is configured such that a received aqueous solution covers a first spot on a received membrane and a second receptacle is configured such that a received aqueous solution covers a second spot on a received membrane. In some embodiments, a first receptacle and second receptacle receive different aqueous solutions.
[097] In some embodiments, the aqueous solution is water. In some embodiments, the water is ultrapure water. In some embodiments, the aqueous solution is a salt buffer. In some embodiments, the aqueous solution comprises neutral pH. In some embodiments, the aqueous solution comprises alkaline pH. In some embodiments, the aqueous solution comprises neutral or alkaline pH. In some embodiments, alkaline pH is pH above 8. In some embodiments, alkaline pH is a pH between 8 and 10. In some embodiments, alkaline pH is a pH between 8 and 12. In some embodiments, alkaline pH is a pH between 10 and 12.
[098] In some embodiments, the aqueous solution is at most at room temperature. In some embodiments, the membrane is at most at room temperature. In some embodiments, the spot on the membrane is at most at room temp. In some embodiments, the aqueous solution is at most at room pressure. In some embodiments, the membrane is at most at room pressure. In some embodiments, the spot on the membrane is at most at room pressure. In some ! ¾ ¾·? room temperature is 25 degrees Celsius. In someP^li S^uPuSSPi^ Oom temperature is 20 degrees Celsius. In some embodiments, room temperature is between 20 and 25 degrees Celsius. In some embodiments, room pressure is 1 atmosphere. It will be understood by a skilled artisan that the method of drilling is advantageous as it does not require high temperature and/or high pressure in order to achieve drilling.
[099] In some embodiments, the membrane is in an optically accessible flow cell. In some embodiments, the system further comprises an optically accessible flow cell. In some embodiments, optically accessible comprises optically accessible by a high numerical aperture objective. In some embodiments, the system further comprises a high numerical aperture objective. In some embodiments, the high numerical aperture objective is in a microscope. In some embodiments, the high numerical aperture objective is configured to focus light to a spot on the membrane. In some embodiments, the high numerical aperture objective is configured to focus light to a first spot on the membrane. In some embodiments, the system further comprises a microscope. In some embodiments, the microscope is a confocal microscope. In some embodiments, the microscope is for positioning the membrane so the light is focused on the spot. In some embodiments, the lens of the microscope focuses the light from the light source. In some embodiments, the system that directs and focuses light is a system of mirrors. In some embodiments, the system that directs and focuses light is a microscope. In some embodiments, the system that directs and focuses the light is a system of reflective metal surfaces. In some embodiments, the system is configured to direct and focus light from the light source.
[0100] In some embodiments, a spot on the membrane is a predetermined spot. In some embodiments, a spot on the membrane is a diffraction limited spot. In some embodiments, a spot is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 spots. Each possibility represents a separate embodiment of the invention. In some embodiments, a spot is part of an array of spots. In some embodiments, the array of spots in a geometric configuration. Geometric configurations include squares, rectangles, circles, ovals, triangles, bowties, rods, cylinders, ellipses, disks, rhombuses and any other shape that may be found by one skilled in the art for nanopore arrays.
[0101] In some embodiments, the membrane comprises a second spot. In some embodiments, the second spot is on an opposite side of the membrane from the first spot. In some embodiments, the second spot is on the inverse side of the membrane from the first spot. In some embodiments, the second spot is positioned such that a line passing through the first spot also passed through the second spot. In some embodiments, the second spot is membrane where light shined on the first spot exists t?£T±Si?ii¾a3i5¾?' tome embodiments, the second spot is the spot on the membrane where light shined on the first spot would exit the membrane if the light generated a pore through the membrane. In some embodiments, the first spot and the second spot are a single spot that passes through the width of the membrane.
[0102] In some embodiments, the system further comprises a sensor. In some embodiments, the sensor is a photodetector. In some embodiments, the sensor is capable and/or configured to measure low light intensities. In some embodiments, the sensor is capable and/or configured to measure at high temporal resolution. In some embodiments, the sensor is configured to detect at the spot on the membrane. In some embodiments, the sensor is configured to detect emissions from the spot on the membrane. In some embodiments, the sensor is configured to detect at the membrane. In some embodiments, the sensor is configured to detect emissions from the membrane. In some embodiments, the sensor is configured to detect photoluminescent intensity (PL). In some embodiments, the sensor is configured to detect light. In some embodiments, the sensor is an electron detector. In some embodiments, the photodetector is a photodiode. In some embodiments, the photodiode is an avalanche photodiode. In some embodiments, the photodetector is a photo-multiplier tube. In some embodiments, the photodetector is a CMOS camera.
[0103] In some embodiments, the sensor is an imaging sensor. In some embodiments, the system comprises a photodetector, an imaging sensor or both. In some embodiments, the imaging sensor is an electron microscope. In some embodiments, the imaging sensor is an electron multiplying charge-coupled device (CCD) camera. In some embodiments, the imaging sensor is a complementary metal oxide semiconductor (CMOS) camera. In some embodiments, the imaging sensor is a scientific CMOS (sCMOS) camera.
[0104] In some embodiments, the system further comprises a means to induce movement of a molecule from one side of the membrane to the other side of the membrane when there is a pore through the membrane. In some embodiments, the system further comprises a means to induce a current through the membrane. In some embodiments, the system further comprises a means to induce a current from one side of the membrane to the other side. In some embodiments, the system further comprises a means to induce movement of a molecule or create a charge from a first reservoir to a second reservoir. In some embodiments, the means to induce movement comprises a means of inducing an electrical current from one side of the membrane to the other side. In some embodiments, one side of the membrane to the other is from the first reservoir to the second reservoir. In some embodiments, the means 'Yi^ ?5?uZi¾?5vement comprises a negative electrode on one side or ^ΐ> (Iίt¾i?!i/'i^!^.¾i·noίΐ and a positive electrode on the second side or in the second reservoir. In some embodiments, the means is an apparatus configured to pass an electric current between two electrodes. In some embodiments, the system further comprises a current detector. In some embodiments, the current detector is configured to measure current between the two electrodes. In some embodiments, the current detector is configured to measure current between one side of the membrane and the other. In some embodiments, the current detector is configured to measure current between a first reservoir and a second reservoir.
[0105] In some embodiments, the current detector and/or the sensor are configured to shut off the light source upon a particular measurement. In some embodiments, the particular measurement is indicative of thinning to a desired thickness. In some embodiments, the particular measurement is indicative of the formation of a nanopore. In some embodiments, the particular measuring is indicative of a nanopore reaching a predetermined diameter. In some embodiments, the particular measurement is a predetermined threshold of PL intensity. In some embodiments, PL intensity is inversely proportional to the thickness of the spot on said membrane. In some embodiments, the sensor is configured to shut off the light source based on a measured PL intensity. In some embodiments, the particular measurement is an increase in ionic current. In some embodiments, an increase in ionic current through the membrane indicates the formation of a pore in the membrane. In some embodiments, a threshold ionic current indicates a particular diameter of pore has been formed. In some embodiments, the particular measurement is a visual measurement of the pore and/or its diameter.
[0106] In some embodiments, the system of the invention comprises the set up depicted in Figure 1A. In some embodiments, the system of the invention comprises the set up depicted in Figure 7A. In some embodiments, the system of the invention comprises a portion of the set up depicted in Figures 1A and/or 7A.
[0107] In some embodiments, the system of the invention is for thinning a membrane. In some embodiments, the system of the invention is for controlled thinning of a membrane. In some embodiments, the system of the invention is for forming a pore through the membrane. In some embodiments, the forming a pore is in situ forming a pore at a predetermined spot. In some embodiments, the forming a pore is forming an array of pores. In some embodiments, the pore is a nanopore. Ύ9 ¾?«'1 ,' C embodiments, the system is for forming at least 1, 2 , , 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 nanopores. Each possibility represents a separate embodiment of the invention. In some embodiments, the system is for producing a plurality of nanopores. In some embodiments, the array comprises dimensions of 5 x 5, 5 x 10, 5 x 15, 5 x 20, 5 x 25, 5 x 30, 5 x 35, 5 x 40, 5 x 45, 5 x 50, 10 x 10, 10 x 15, 10 x 20, 10 x 25, 10 x 30, 10 x 35, 10 x 40, 10 x 45, 10 x 50, 15 x 15, 15 x 20, 15 x 25, 15 x 30, 15 x 35, 15 x 40, 15 x 45, 15 x 50, 20 x 20, 20 x 25, 20 x 30, 20 x 35, 20 x 40, 20 x 45, 20 x 50, 25 x 25, 25 x 30, 25 x 35, 25 x 40, 25 x 45, 25 x 50, 30 x 30, 30 x 35, 30 x 40, 30 x 45, 30 x 50, 35 x 35, 35 x 40, 35 x 45, 35 x 50, 40 x 40, 40 x 45, 40 x 50, 45 x 45, 45 x 50, or 50 x 50 pm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanopores are separated by about 1 pm. In some embodiments, the nanopores in the array are separate by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 pm. Each possibility represents a separate embodiment of the invention.
Methods of use
[0109] By another aspect, there is provided a method of thinning a membrane, the method comprising shining focused light on a spot on a membrane, thereby thinning the membrane.
[0110] By another aspect, there is provided a method of thinning a membrane, the method comprising shining pulsed laser light on a spot on the membrane, thereby thinning the membrane.
[0111] In some embodiments, the method is for light- induced thinning. In some embodiments, the method is for controlled thinning. In some embodiments, the method is for thinning in situ on a membrane. In some embodiments, the method is for thinning at a predetermined spot on a membrane. In some embodiments, the method is for rapid thinning. In some embodiments, thinning comprises producing a pore through the membrane. In some embodiments, thinning is to a thickness of less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nm. Each possibility represents s separate embodiment of the invention. In some embodiments, thinning is to a thickness of less than 5 nm. In some embodiments, thinning is to a thickness of less than 3 nm. In some embodiments, thinning is a thinning of at least 50, 60, 70, 75, 80, 85, 90, 95, 97, 99 or 100% of the membrane thickness. Each possibility represents s separate embodiment of the invention. In some embodiments, thinning is a thinning of at least 75% of the membrane thickness. It will be understood that thinning of 100% produces a hole through the membrane. Ύ9 ¾?«'1 embodiments, the thinning does not comprise a p±99<!M9¾]iP.5,l?56aome embodiments, the thinning does not comprise a pre -patterning step. In some embodiments, pretreatment is a pre -patterning. In some embodiments, pre-patterning is by a method other than a method of the invention. Any method of pre-patterning known in the art is envisioned. In some embodiments, the method is devoid of chemical etching. In some embodiments, the method is devoid of dielectric breakdown. In some embodiments, the method is devoid of electrochemical anodization. In some embodiments, the method is devoid of metal-assisted chemical etching. In some embodiments, the method is devoid of electron beam lithography (EBL) etching. In some embodiments, the method is devoid of reactive ion etching (RIE). In some embodiments, the method is devoid of metal deposition fabrication. In some embodiments, the method is devoid of ion-track etching. In some embodiments, the method is devoid of focused electron beam (e-Beam) lithography. In some embodiments, the method is devoid of focused ion beam (FIB) lithography.
[0113] In some embodiments, the focused light is laser light. In some embodiments, the focused light is within the purple spectrum. In some embodiments, the focused light is purple light. In some embodiments, the focused light is within the blue spectrum. In some embodiments, the focused light is blue light. In some embodiments, the focused light is within the green spectrum. In some embodiments, the focused light is green light. In some embodiments, the focused light is light focused from a light source. In some embodiments, the light is focused by a system for focusing the light and directing it to a spot on the membrane. In some embodiments, the light comprises a wavelength of between 300-600 nm. In some embodiments, the laser light is continuous-wave laser light. In some embodiments, the laser light is pulsed laser light. In some embodiments, shinning pulsed laser light is pulsing laser light on a spot on the membrane.
[0114] In some embodiments, the light has an intensity of at least 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 pW. Each possibility represents a separate embodiment of the invention. In some embodiments, the light has an intensity of at least 1 pW. In some embodiments, the light has an intensity of at least 10 pW. In some embodiments, the light has an intensity of at least 100 pW. In some embodiments, the light has an intensity of at least 1000 pW. In some embodiments, the power of the light is between 1 pW and 50 mW, 1 pW and 45 mW, 1 pW and 40 mW, 1 pW and 35 mW, 1 pW and 30 mW, 1 pW and 25 mW, 1 pW and 20 mW, 1 pW and 15 mW, 1 pW and 1 mW, 1 pW and 5 mW, 1 pW and 1 mW, 10 pW and 50 mW, 10 pW and 45 mW, 10 pW and 40 mW, 10 pW and 35 mW, 10 pW and 30 mW, 10 pW and 25 mW, 10 pW and 20 mW, 10 pW and 15 mW, 10 pW and
Figure imgf000029_0001
100 pW and 50
100 pW and 40 mW, 100 pW and 35 mW, 100 pW and 30 mW, 100 pW and 25 mW, 100 pW and 20 mW, 100 pW and 15 mW, 100 pW and 10 mW, 100 pW and 5 mW, 100 pW and 1 mW, 200 pW and 50 mW, 200 pW and 45 mW, 200 pW and 40 mW, 200 pW and 35 mW, 200 pW and 30 mW, 200 pW and 25 mW, 200 pW and 20 mW, 200 pW and 15 mW, 200 pW and 10 mW, 200 pW and 5 mW, 200 pW and 1 mW, 300 pW and 50 mW, 300 pW and 45 mW, 300 pW and 40 mW, 300 pW and 35 mW, 300 pW and 30 mW, 300 pW and 25 mW, 300 pW and 20 mW, 300 pW and 15 mW, 300 pW and 10 mW, 300 pW and 5 mW, 300 pW and 1 mW, 400 pW and 50 mW, 400 pW and 45 mW, 400 pW and 40 mW, 400 pW and 35 mW, 400 pW and 30 mW, 400 pW and 25 mW, 400 pW and 20 mW, 400 pW and 15 mW, 400 pW and 10 mW, 400 pW and 5 mW, 400 pW and 1 mW, 500 pW and 50 mW, 500 pW and 45 mW, 500 pW and 40 mW, 500 pW and 35 mW, 500 pW and 30 mW, 500 pW and 25 mW, 500 pW and 20 mW, 500 pW and 15 mW, 500 pW and 10 mW, 500 pW and 5 mW, or 500 pW and 1 mW. Each possibility represents a separate embodiment of the invention. In some embodiments, the power of the light is between 100 pW and 45 mW.
[0115] In some embodiments, the method further comprises measuring PL intensity from the membrane. In some embodiments, the measuring PL intensity is from the spot on the membrane. In some embodiments, the method further comprises stopping thinning bases on the measured PL intensity. In some embodiments, the stopping occurs when the PL intensity reaches a predetermined threshold. In some embodiments, the predetermined threshold is when a pore is formed in the membrane. In some embodiments, the predetermined threshold is when a pore has reached a predetermined diameter. In some embodiments, the PL intensity is inversely proportional to the thickness of the spot on the membrane. In some embodiments, the thinning is stopped at a predetermined thickness.
[0116] In some embodiments, the thinning comprises forming a pore through the membrane at the spot. In some embodiments, the pore is a nanopore. In some embodiments, the nanopore comprises a diameter not greater than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanopore comprises a diameter not greater than 5 nm. In some embodiments, the nanopore comprises a diameter of about 5 nm. In some embodiments, the nanopore comprises a diameter between 0.5 and 10, 0.5 and 15, 0.5 and 20, 1 and 10, 1 and 15, 1 and 20, 3 and 10, 3 and 15, 3 and 20, 5 and 10, 5 and 15, or 5 and 20 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the diameter of Ύ,O \ 02 ( ) / 194303 a h |-,c selected by monitoring the PL intensity and or Op£JciJ¾2P?Si?¾?£ g the nanopore formation. In some embodiments, the diameter of the nanopore can be selected by altering the light intensity, wavelength, or duration, or the pH of the solution. A skilled artisan can optimize the variable parameters to generate a nanopore of the desired diameter in the desired time.
[0117] In some embodiments, the nanopore comprises a Gaussian shape. In some embodiments, the nanopore comprises a varying diameter. In some embodiments, the nanopore comprises a Gaussian curve shaped cross-section. In some embodiments, the thinning produces a nanowell adjacent to the nanopore. In some embodiments, the thinning produces a nanowell without a nanopore. In some embodiments, the thinning produces a nanowell in one layer and an adjacent nanopore in a second layer. As used herein, the term “nanowell” refers to a passage through the membrane. A nanowell may also be referred to as a nanoslot. In some embodiments, the nanowell comprises a Gaussian shape. In some embodiments, the nanowell comprises a varying diameter. In some embodiments, the nanowell comprises a Gaussian curve shaped cross-section. In some embodiments, the nanopore and/or nanowell produce a low optical background. In some embodiments, the membrane produces a low optical background. In some embodiments, the low optical background is lower than the optical background of a nanopore and/or nanowell that does not have a Gaussian curve shape or a Gaussian curve shaped cross-section. In some embodiments, the low optical background is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97 or 99% lower. Each possibility comprises a separate embodiment of the invention.
[0118] In some embodiments, a membrane comprising a first and second layer comprises an interface of the first and second layer. In some embodiments, the interface is the place wherein the second layer is layered on the first layer. In some embodiments, the membrane comprises a first layer and a second layer and the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the first layer. In some embodiments, the membrane comprises a first layer and a second layer and the nanopore comprises a second Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the second layer. In some embodiments, the nanopore comprises two regions each with a Gaussian curve shaped cross-section. In some embodiments, the first Gaussian curve shaped cross-section is in the first layer. In some embodiments, the second Gaussian curve shaped cross-section is in the second layer. Ύ9, 2"y\ embodiments, the method further comprises measuring?X(¾l¾¾9AP?¾Jugh the membrane. In some embodiments, the spot of the membrane is immersed in an aqueous solution and the ionic current is measured from the solution on one side of the membrane to the solution on the other side of the membrane. In some embodiments, an increase in ionic current through the membrane or from one side to the other, or from one reservoir to the other, indicates the formation of a pore in the membrane. In some embodiments, formation of the pore in the membrane is formation of the pore at the spot in the membrane. In some embodiments, the spot is a predetermined spot. In some embodiments, the increase in ionic current is a sudden increase in ionic current. In some embodiments, the increase in ionic current is not gradual. In some embodiments, the increase is at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500% increase in ionic current. Each possibility represents a separate embodiment of the invention. In some embodiments, after a pore has been formed a gradual increase in ionic current corresponds to the widening of the diameter of the nanopore. In some embodiments, the thinning is stopped at a predetermined current and/or diameter.
[0120] In some embodiments, the method is for rapid formation of a nanopore. In some embodiments, a pore can be produced at a spot in the membrane with a thickness of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, a pore can be produced at a spot in the membrane with a thickness of at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, a pore can be produced at a spot in the membrane with a thickness of at most 50 nm. In some embodiments, a pore can be produced at a spot in the membrane with a thickness of at most 100 nm. In some embodiments, a pore can be produced in less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 seconds. Each possibility represents a separate embodiment of the invention. In some embodiments, a pore can be produced through a spot in the membrane with a thickness of at least 40 nm in less than 20 seconds.
Nanopores/nanowells
[0121] By another aspect, there is provided a membrane comprising a nanopore and/or nanowell, wherein the nanopore and/or nanowell is produced by a method of the invention.
[0122] By another aspect, there is provided a membrane comprising a nanopore and/or nanowell, wherein the nanopore and/or nanowell comprises a varying diameter and a Gaussian curve shaped cross-section. Ύ9, l°?j 0/! 4¾^1C embodiments, a nanopore and/or nanowell producetf^J^ii^MifS^uf the invention comprises a varying diameter. In some embodiments, the varying diameter is a Gaussian shape. In some embodiments, the varying diameter corresponds to a Gaussian shaped cross-section of the nanowell and/or nanopore. In some embodiments, the nanopore and/or nanowell produced by a method of the invention comprises a Gaussian curve shaped cross-section. In some embodiments, the nanopore and/or nanowell increases in diameter from one side of the membrane to the other. In some embodiments, the nanopore and/or nanowell has a larger diameter on the side of the membrane upon which the light was shown. In some embodiments, the increasing diameter from one side to the other follows a Gaussian curve. In some embodiments, the Gaussian curve comprises a full width at half maximum of one half of the wavelength of the focused light used to generate the nanopore and/or nano well.
[0124] In some embodiments, the nanopore, nanowell and/or membrane produces a low optical background. In some embodiments, the low optical background is lower than the background of a membrane comprising a nanopore and/or nanowell that does not comprise a Gaussian shape or cross-section. In some embodiments, the low optical background is lower than the background of a membrane not produced by the method of the invention.
[0125] In some embodiments, a membrane comprising a first and second layer comprises an interface of the first and second layer. In some embodiments, the interface is the place wherein the second layer is layered on the first layer. In some embodiments, the membrane comprises a first layer and a second layer and the nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the first layer. In some embodiments, the membrane comprises a first layer and a second layer and the nanopore comprises a second Gaussian curve shaped cross-section increasing in diameter from the interface of the first layer with the second layer to an exposed surface of the second layer. In some embodiments, the nanopore comprises two regions each with a Gaussian curve shaped cross-section. In some embodiments, the first Gaussian curve shaped cross-section is in the first layer. In some embodiments, the second Gaussian curve shaped cross-section is in the second layer.
[0126] In some embodiments, the membrane is a membrane as described hereinabove. In some embodiments, the nanopore is a nano-scale aperture. In some embodiments, the nanopore is as described hereinabove. In some embodiments, the nanowell is a nanowell as described hereinabove. In some embodiments, the membrane comprises a plurality of layers. In some embodiments, at least two layers comprise nanopores and/or nanowells of different '¾n¾*.2ί/1¾ί?¾ embodiments, one layer comprises a nanowell and a s^J/S^JEjS/^SA^prises a nanopore.
[0127] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
[0128] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
[0129] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0130] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Ύ92i A n a 1 objects, advantages, and novel features of the will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
[0132] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
[0133] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Materials and Methods
[0134] Chip fabrication. Nanopore chips were fabricated from a 4” (100 mm) double-side polished, 350 pm thick silicon wafers coated with 500 nm of thermal Si02 (Silicon Valley Microelectronics, CA USA). 50 nm thick low-stress silicon nitride (SiNx) layers were deposited on both sides using low pressure chemical vapor deposition (LPCVD) with Ύίί ^II' i 1?^!1?/ S1H2CI2 gas ratios, resulting in different refractive ind?v£T^¾?g?<?g )?l)uS 2.15 to 2.43. The refractive index was then measured by ellipsometry (Film Sense, FS-1). Next, direct-write photolithography (MicroWriter ML3, DMO) was used to pattern the windows and dice lines on the resist. A hard mask was created using reactive ion etch (RIE, Diener Electronic) followed by buffered oxide etch (BOE) to remove the S1O2 and expose the Si layer. The wafer was then immersed in KOH at 65 °C for up to 20 hours followed by a second round of BOE to open up a freestanding SiNx membrane. Each chip was cleaned by piranha before usage (3: 1 EbS04:El202), vacuum dried, and mounted onto a Teflon holder with Ecoflex 5 (Smooth-ON, Reynolds Advanced Materials) silicone rubber. The chip was then placed in a Teflon cell equipped with a quartz cover-slide bottom. The position of the cell was controlled using a 3D nanopositioner stage (Physik Instrumente, P-561.3CD). Alternatively, nanopore chips were fabricated from a 4” silicon wafer coated with 500 nm S1O2 and 50 nm low-stress amorphous SiNx. To create freestanding membranes, a hard mask was RIE-etched into the SiNx followed by HF etching to remove the S1O2, and then through- etching of Si with KOH. The free-standing membranes were 40-45 nm thick.
[0135] Chip assembly. Chips were first cleaned by piranha (3: 1 H2S04:H202). They were then glued onto a custom-made Teflon insert, immersed in buffer (1 M KC1, 40 mM Tris- HC1, 1 mM EDTA, pH 7.5), and placed in a Teflon cell with a quartz cover-slide bottom. The cell was mounted onto a 3D nanopositioner located above the microscope objective. The setup was shielded by a grounded copper box and placed on a vibration-isolating optical table.
[0136] Optical setup. A previously described custom-built confocal microscope was modified for this study: Two collimated laser lines are focused onto a diffraction-limited laser spot at the membrane surface. The emitted light is collected by the same objective (NA = 1.15), focused onto a spatial pinhole to reject out-of-focus light, passed through an ND- filter and directed onto two spectrally separated APDs for two-color imaging.
[0137] In Fig. 1A-D, the full photoluminescence is roughly 3 orders in magnitude larger. In order not to damage the APDs, an ND3 filter is positioned in the emission pathway during etching and before the excitation pathway during profiling. The photoluminescence count is a summation of the red (>650 nm) and green (550-650 nm) channels.
[0138] Ionic current was measured by cis/trans -immersed Ag/AgCl electrodes connected to a high-bandwidth amplifier (Axon 200B) sampled at 125 kHz (DAQ NI-6211) and filtered at 10 kHz. Photon counting was sampled at 500 kHz (DAQ NI-6602). The two cards were u9l¾¾ltaneously via a hardware connection and were fullyp-^/i±½93S^®3v¾?stom Lab VIEW software.
[0139] Alternatively, A custom-made three-color confocal set up was used for the electro- optical measurements as depicted in Figure 7A. The lasers were focused to a diffraction- limited spot at the membrane surface using a high NA objective (Zeiss Apochromat 63x/1.15). The emitted light was collected using the same objective, filtered using the appropriate long pass and notch filters (Semrock) and focused on either an EMCCD camera (ANDOR, iXon 887) or a 100 pm pinhole (Thorlabs). Light passing through the pinhole was collimated and split using a dichroic mirror (Semrock) with center wavelengths of l=650 nm and focused onto two APDs (Perkin Elmer SPCM-AQR-14). The emitted light was attenuated using an ND3 filter during thinning and drilling to protect the APDs. Photon counting from the APDs was sampled at 500 kHz (DAQ NI-6602). The photoluminescence presented throughout the paper is a summation of the red (>650 nm) and green (550-650 nm) channels. Ionic current was measured by cis/trans- immersed Ag/AgCl electrodes connected to a high-bandwidth amplifier (Axon 200B) sampled at 125 kHz (DAQ NI-6514) and filtered at 10 kHz. The two cards were triggered simultaneously via a hardware connection and were fully controlled by custom Lab VIEW software.
[0140] TEM imaging. High-resolution images were acquired with a FEI Titan Themis Cs- Correct HR-S/TEM. The relative thickness map (RTM) was automatically generated using the Gatan Digital Micrograph® EFTEM technique by first acquiring an unfiltered and a zero-loss image from the same region under identical conditions. The RTM was then computed using the Poisson statistics of inelastic scattering: t/X=-ln(Io/lt), where Io is the zero-loss intensity and It is the total intensity. To obtain the true thickness, t/l is multiplied by the mean free path (110 nm) in silicon nitride (Si:N 3:4). The low loss energy spectrum was measured in scanning transmission electron microscopy (STEM) in increments of 20 nm and was used to automatically generate relative thickness maps using Digital Micrograph software (Gatan).
[0141] Composition analysis: Chemical mapping of the SiNx membranes was performed using energy dispersive X-ray imaging (EDS, Dual Bruker XFlash6) and scanning transmission electron microscopy (STEM) based on core-loss electron energy loss spectroscopy (EELS). The EDS quantification was done using Velox (Thermo Fisher) and EELS quantification was done using the Digital Micrograph software (Gatan). 'YS> ±¾?j°^l?Ap¾ metry measurements. Performed with model Fi^ ?T^fii?/l?®{9a( v¾^ngth
Ellipsometer (Film Sense).
[0143] Calcium indicator experiments. The setup and protocol exactly follow standard procedures with a Fluo-4 and CaCh concentration of 500 nM and 500 mM, respectively.
[0144] DNA Translocation experiments. Nanopores were allowed to equilibrate at a low probing voltage (0.1 to 0.3 V) in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA, pH 7.5) for at least 10 minutes to obtain a stable open pore current prior to adding homemade 5054 bp dsDNA. Events were monitored using an Axon 200B filtered at 100 kHz and custom Lab VIEW software.
[0145] Alternatively, for the translocation experiment, the chip was immersed in a pH 7 buffer (in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA) and 300 mV was applied until the open pore current stabilized. 300 bp dsDNA was added to the cis chamber at a concentration of 1 nM. Translocation events were monitored and recorded using an Axon 200B filtered at 100 kHz and custom Lab VIEW software. An offline analysis program was used to analyze each event separately to extract the amplitude block and dwell time for each translocation.
[0146] Protein Translocation Experiments. In some cases, nanopores were kept dry in air for up to 10 days prior to performing the experiment. These nanopores were cleaned by Dynasolve 185 to remove PDMS and then made hydrophilic by piranha (3:1 HiSO^HiCh). Nanopores were allowed to equilibrate at a low probing voltage (0.1 to 0.3 V) in buffer (1 M KC1, 40 mM Tris-HCl, 1 mM EDTA, pH 7.0) for at least 10 minutes to obtain a stable open pore current prior to adding 0.007 pg/mΐ di-ubiquitin. Commercially available, as well as home-purified proteins such as Ubiquitin, eIF4A, Albumin, etc. where suspended in the buffer and translocation events were monitored using an Axon 200B filtered at 100 kHz and custom Lab VIEW software.
[0147] Automated nanopore drilling: Custom software (LabVIEW) was used to automatically drill either a single or multiple pores according to the input coordinates (x,y) list and a current gradient threshold. 150mV potential was applying across the membrane and the current was monitored in real time. The piezoelectric stage was moved to each coordinate, where the laser was switched on until the current threshold was reached. The laser was then switched off, stopping the drilling. As a preliminary stage to this process, the laser was focused at the (x=0,y=0) coordinate at low intensity. The nanopores array was validated using Ca+2 based imaging. 'YS) ¾?j 0 |i?f3c periments: Four 1 M KC1 buffers with different pH^vZ&^iSvP pi^ared: 20mM Sodium acetate (SodAc) for pH 4, 20 mM Tris for pH 7, 20mM Sodium bicarbonate (SodBic) for pH 10, and KOH based buffer for pH 12. The buffers’ refractive index was measured using a refractometer (Rudolph, J257) and was found to be similar for the four solutions (Table 2). For measuring the dependency of etching in pH, the membrane was immersed in each buffer solution and was exposed to 2 minutes of laser illumination of increasing intensities. The chip was washed (Milli-Q) and dried before exchanging the buffer. White light images of the membrane were taken before and after the laser exposure using an EMCCD camera and were used to compute the change in reflection through each etched spot (ImageJ and MATLAB). Laser intensity was controlled using a ND filter and monitored using a power meter (Thorlabs).
[0149] To confirm that the pH dependence measurements are not biased by different buffer indices, we measured them using a refractometer (Rudolph, J257). The measurements were performed at similar temperature to the thinning experiment (22°C). The results in Table 2 show similar values for all solutions, indicating that the buffer did not play a role in the PL measurements.
[0150] Table 2: Refractive index measurements for the different solutions.
Figure imgf000038_0001
Example 1: Laser- Etching of Freestanding SiNx
[0151] We first developed a procedure for etching freestanding SiNx with a continuous-wave blue (488 nm) solid-state laser. It begins by assembling a Si-supported SiNx membrane (typically 40-45 nm thick) in an optically accessible flow cell, which is then mounted on top of a high NA microscope objective in a homebuilt confocal setup (Fig. 1A). The setup is equipped with an EMCCD for widefield viewing and an avalanche photodiode (APD) detector for high temporal resolution sensing of the photoluminescence (PL) intensity. For alignment, we set the blue laser at low intensity (40 pW) to prevent unintentional etching and bring the membrane into focus of the laser spot. Once aligned, the laser intensity is increased to full power (~45 mW) for the etching step, but in cases of a high index of refraction and/or high pH values, lower laser intensities were applied.
[0152] Notably, we observed that under high laser intensity, a bright PL emission was visible by our EMCCD camera (Fig. IB). The measured PL intensity exhibited a decay over time Ύ9 w'J'Viain'lng a near plateau level after roughly 300 s (Fig. 1C). ?&T5¾?±¾?P(®u°u¾t the decay in PL is not due to mechanical drift and is in fact irreversible: momentarily switching off the laser beam and then switching it on again showed that the PL level retuned to the same level at which the laser was switched off (and not to the initial level). Furthermore, widefield optical inspection of the membrane revealed a darkened spot at the point where the material was illuminated by the laser (Fig. ID). The material darkened proportionally to the PL reduction, and this spot could not be revived by solvent or acid cleaning.
[0153] As contrast under white-light illumination typically indicates a difference in material thickness, to further characterize this phenomenon we fabricated a series of freestanding SiNx membranes from the same stock material, using reactive ion etching (RIE) to obtain different final thicknesses. Accurate thickness measurements were made by ellipsometery after performing a careful calibration using a factory- supplied model specimen. The chips were then mounted in our optical setup and the PL level was determined under otherwise identical conditions. Our results, summarized in Figure 2, show a linear relationship between PL and the SiNx membrane thickness. We note that, as expected, the measured PL intensity varies sharply with the distance between the objective lens and the membrane and reaches a maximum value when the laser spot is centered in the z direction on the membrane. Hence the measurements shown in Figure 2 involved careful maximization of each PL read in the z direction. Measurements were performed using an attenuated laser (30 pW) to avoid etching of the membrane and remain constant over time. The error bars reflect the standard deviation in the PL intensity over 1 minute of measurement.
[0154] We next imaged the samples by Transmission Electron Microscopy (TEM) to determine whether the darkened membrane spot caused by the laser was in fact due to material removal and not a type of laser-induced chemical reaction or adsorption process. Indeed, the TEM images reveal that the material had thinned at the position of the laser focus (Fig. 3A-B). Moreover, the TEM images show that the material thins non-uniformly: the thickness profile closely follows the intensity point-spread function (PSF) of the laser beam used to induce thinning, where etching occurs fastest at the center (Fig. 3C). See Methods for a description on making the TEM thickness map.
[0155] After establishing that the laser etches SiNx, we formulated a relationship between measured PL and etch depth which is consistent with the TEM analysis. After two minutes of laser exposure, we lowered the laser intensity to prevent etching and scanned in the x direction with a 30 nm step size while measuring PL intensity. The generated ID PL profile and TEM thickness map were both normalized and overlaid on the same graph (Fig. 3D, ΎRa?5??(ί?ά¾?a6 curves, respectively). As can be seen, the PL curve ^ir i Nx thickness, deviating slightly because of practical limitations of the optical setup. We simulated a PL curve based on a convolution of a Gaussian PSF, representing the laser beam, with the TEM thickness map (Fig. 3D, red curve). Overlaying the modelled data on the same graph shows a tight fit with the PL measurement, with a PSF full width at half maximum (FWHM) of 325 ± 15 nm. This compares favorably with the diffraction-limited FWHM of 330 ± 20 nm for PL emission collected by an objective lens with a numerical aperture (NA) of 1.15. Therefore, we can reliably use the PL measurement to infer the membrane thickness.
[0156] We found that the etch rate is significantly reduced at low laser intensity and is practically undetectable for 488 nm laser intensities < 1 mW over the course of our measurements. The etch rate for the 488 nm laser at an intensity of ~45 mW was found to be up to 25 nm/minute. Interestingly, red laser (645 nm) induced no appreciable membrane thinning over a similar timescale, while green laser (532 nm) focused on the membrane at the same power as the 488 nm laser, resulted in roughly an order of magnitude less thinning, indicating that the etching mechanism is dependent not only on the laser intensity but also on its wavelength (Fig. 3E). This is consistent with a previous study which did not report any membrane thinning despite using a comparable laser power (~45 mW, 785 nm). Interestingly, we found that etching also proceeds in ultrapure water (18.2 MW x cm) and not just in KC1 buffer. Although slow SiNx etching in water has been reported before in the literature, it required the use of sub- or super-critical water with temperatures in the 200°C range and a pressure of 10 MPa. Our finding that the 532 nm laser produced much less SiNx thinning than the 488 nm laser at the same power suggests that the etch process is likely not temperature-activated but rather follows a wavelength-dependent photochemical etching. It is known that differences in laser-etching rates is a consequence of differences in spatial- electron hole pair density, which is a function of their respective absorption coefficient for a particular material.
Example 2: Nanopore Fabrication and Validation
[0157] Based on our observation that a ~45 milliwatt-intensity blue laser etches SiNx, we attempted to fabricate nanopores by progressively thinning the membrane until the point of nanopore formation. For this, we monitored the ionic current across the membrane, applying a 300 mV transmembrane potential via cis/trans -immersed AgCl electrodes connected to an Axon 200B amplifier. We simultaneously measure the PL as a way to track the fabrication progress. An example experiment with concurrent ionic and PL feedback is given in Figure 4A. In this example, we observed an increase in ionic current after roughly 145 seconds, ^vii?9?0w24aS?ibute to the formation of an ionic passageway throu^FJ/i^P^/S^S?^ The current continues to rise until the laser is deactivated, which we associate with nanopore growth. Notably, upon pore formation the ~45 mW laser also causes an increase in electrolyte conductivity hence, turning the laser off causes the current to drop. The open pore conductance then stabilizes over the next few minutes, usually deviating at most 2 nS from its initial value. The final conductance level increases with the time that the laser is kept on after the initial formation of the pore.
[0158] We first validated that a thoroughfare path was truly made in the membrane and that the measured current was not caused by surface charging or some other effect. To do so, we loaded the cis side chamber with calcium (Ca2+) and the trans chamber with Fluo-4, and illuminated the entire membrane at 488 nm while monitoring it with a CCD (Fig. 4B, upper panel). For there to be a path through the membrane, the fluorescence signal should sharply increase when the applied cis/trans bias is positive, as the Ca2+ would be driven through and activate Fluo-4. Indeed, as shown in Figure 4B (lower panel), we observed a fluorescent signal at the exact position where the material was etched. We next sought to corroborate our calcium-imaging data with TEM data. After a laser drilling experiment, we allowed the OPC to stabilize for over 15 minutes. We then immersed the SiNx chip in ultrapure water to remove salt residue. Figure 5A gives an example TEM image of a 6.5 nm nanopore formed in under 5 minutes, which was a typical fabrication time in a 40-45 nm thick membrane based on >30 trials (100% yield).
[0159] As we show, by choosing a current threshold for laser shutoff, we are able to reproducibly fabricate both small (1 nm), medium (5 nm), and large (over 10 nm) nanopores according to the sensing requirement (Fig. 5B, Table 1). Given that our nanopore fabrication strategy is markedly different than existing techniques such as CBD or TEM-drilling, we cannot expect that the standard conductance model for pore size determination applies; in particular, this model assumes an effective nanopore height equivalent to or one third of the membrane thickness, depending on the method employed. Instead, we can reliably estimate the nanopore diameter according to the translocation blockage level using a molecular ruler of known dimensions, such as dsDNA (2.2 ± 0.1 nm), and the following equations:
Figure imgf000041_0001
where i0 and iB are the open and blocked pore current levels, respectively, l is the local membrane thickness, d the pore diameter, s the solution conductivity and a is the analyte Ύ¾ ¾)>-!303 e mo n s tratc the extent by which the conductance mode?9I(¾??3¾®58¾j uSted, we calculated the effective thickness for a pore with an OPC of 11 ± 0.7 nS and a diameter of 3.2 ± 0.3 nm. Remarkably, we get an effective thickness of 4-6 nm, which is up to 11 times smaller than the surrounding membrane and is consistent with our observation that the membrane gradually thins to the point of nanopore formation. Such ultrathin architectures are highly desirable due to their larger conductance and hence improved spatial resolution and have therefore been the subject of much research.
[0160] Table 1: Table of nanopores fabricated by laser-etching.
Figure imgf000042_0001
The table is ordered according to the open pore current (OPC) from smallest to largest. In order to show that a wide distribution of pore sizes is possible, the laser was kept on following pore creation to expand the pore. The open pore current (OPC) was recorded 1-2 minutes after the laser was turned off.
[0161] We next evaluated the noise characteristics of laser-etched nanopores. Figure 5C shows the power spectral density (PSD) plot of a nanopore for an applied bias of 300 mV after allowing the nanopore to stabilize in KC1 buffer. Similar to TEM-drilled nanopores, two sources of noise dominate the PSD: high-frequency background noise associated with the chip capacitance, and low-frequency flicker noise with 1/P dependency. At an applied voltage of 300 mV, these nanopores typically exhibit an IRMS in the range of 100-200 pA. To assess ionic current rectification, which occurs due to a geometric or surface charge asymmetry along the axis of current flow, we varied the potential from -300 to +300 mV ίaΐ salt concentrations in the cis/trans chambers. The res iP near
(R2 > 0.99), indicating minimal rectification and therefore a symmetric geometry (Fig. 5C, inset). This suggests that the laser-induced etch mechanism occurs on both sides of the membrane equally to produce a very thin hourglass -shaped nanopore.
[0162] Finally, we validated the functionality of laser-etched nanopores by performing extensive sets of DNA and proteins translocation experiments. First, we added 300 pM 5054 bp dsDNA, produced and purified in house, to the cis chamber filled with KC1 buffer. Upon biasing the trans chamber at +300 mV, the initially stable open pore was interrupted by current blockage events of 1.4-2.2 nA or 0.42-0.62 of the open pore current (Fig. 5D-E). For a pore of this small size (3.2 + 0.3 nm), we can expect a significant fraction of events to be collisions, as has been established by both theory and experiment. Therefore, to determine whether there are any full translocations, we performed an additional two translocation experiments at 450 and 650 mV and compared the dwell times of the three experiments. As can be seen in Figure 5F-H, there is an obvious decrease in average dwell time with increasing voltage, indicating that a distinct portion of the events are successful translocations and not collisions. In a subsequent experiment using another pore, the Gaussian fitting clearly delineates two populations corresponding to two event types: short and low blockage/shallow events representing translocations, and long and high blockage/deep events representing collisions (Fig. 5I-K). The short and shallow events, though fewer in number, appear at the expected ratio relative to the long and deep events assuming that the DNA polymer behaves the same as it does with TEM-drilled nanopores. Nevertheless, both nanopores studied generated sufficient events to produce a statistically reliable result.
[0163] We further challenged our nanopore fabrication method to the purpose of detecting one of the smallest protein molecules (K63-linked di-ubiquitin, ~17 kDa), which compared to DNA, poses exceptional spatial and temporal resolution requirements. As has been demonstrated with TEM-drilled pores, one way to reduce the protein translocation rate is to use a buffer pH close to the isoelectric point (pi) of the protein. Therefore, for di-ubiquitin (di-Ub) with a pi of 6.7, we adjusted the KC1 buffer to an experimentally determined pH value of 7. Using a nanopore with an OPC of 7-7.2 nA, we observed shallow (0.2 of the OPC) and mainly short (40-200 ps) events upon the addition of di-Ub to the cis chamber (Fig. 5L-N), expected for this pH value. This set of experiments proves that these nanopores are suitable not just for DNA studies but also small and compact proteins such as di-Ub. Moreover, we note that many of the ssNPs used for translocation experiments were over 10 dry in air and made hydrophilic prior to the experimenP^dfelP^^^high stability of laser-etched nanopores.
Example 3: The SiNx membrane etching rate strongly depends on the Si:N ratio
[0164] We have shown direct, in-situ laser-based membrane-thinning and fabrication of ssNPs in the range of just a few nanometers in freestanding silicon nitride (SiNx) membranes (x = 0.75 for stoichiometric S13N4; wherein just a mW-intensity laser and a confocal microscope was necessary for nanopore fabrication at any arbitrary position and in any quantity. However, the physical process governing laser-drilling in thin, water-immersed membranes, particularly in the absence of any dielectric breakdown application, remains obscure. Specifically, the relative contributions of direct heating versus polarization of the thin membrane by the laser light, remain unclear.
[0165] Amorphous SiNx films are typically produced using a chemical vapor deposition (CVD) process, tuned to form silicon-rich membranes with respect to stoichiometric S13N4, resulting in low-stress thin films. The Si:N ratio (denoted x) only slightly alters the material’s refractive index, but it greatly affects the abundance of the Si dangling bonds, and in turn the photoluminescence (PL) spectrum produced by the film, as the latter involves photo-activated electron excitation and relaxation. We hypothesized that materials composed of slightly different Si:N ratios would result in dramatically altered laser drilling characteristics. To check this hypothesis, we systematically fabricated a series of SiNx films with different Si:N ratios. We characterized the material properties for each batch, including the Si:N composition and energy bandgaps using electron energy-loss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDS), and studied the laser drilling mechanism under various excitation wavelengths and solution pH. Our results point to a highly Si:N composition- and pH-dependent mechanism that is clearly photo-activated. Importantly, we show that at high Si:N ratios and alkaline conditions, we can drill functional nanopores in < 10 s at laser excitation powers that are roughly an order of magnitude smaller than those employed in previous reports. This enables controlled in-situ laser fabrication of nanopore arrays with arbitrary patterns within minutes.
[0166] Material composition analysis is not routinely performed as part of the LPCVD process because it involves delicate elemental spectroscopy. Instead, the material’s index of refraction is often used as a proxy for the Si:N composition, in which a higher index of refraction corresponds to a higher content of Si over N in SiNx membranes. Notably, however, small changes in the index of refraction correspond to significantly different Si:N compositions, preventing fine control of the Si:N ratio, and resulting in significant batch-to- a¾¾$h. While these variations may be too small to affecP£¾IL2020/05035^eam nanopore drilling methods, we readily detect their effect on laser-based drilling as reported here. We produced four wafer batches using the same LPCVD instrument which had slightly different Si:N compositions, characterized by their indices of refraction (2.15, 2.20, 2.29, and 2.42, as measured by an ellipsometer). To monitor the SiNx membrane thinning prior to pore creation, we used a custom-made confocal microscope equipped with multiple laser excitation lines and two spectrally-resolved emission channels coupled to two avalanche photodiodes (APDs; see Figure 7A and Materials and Methods). After positioning the membrane at the focus of the laser spot, we measured the PL intensity time-trace during laser irradiation. We typically observed a fast PL intensity reduction followed by a slower decay associated with the gradual decrease in membrane thickness and the formation of a Gaussian-shaped etch profile. The ion current and PL were simultaneously monitored during laser irradiation, and pore formation was signaled by an abrupt jump in the ion current. We also inspected each nanochip before and after the laser process under white light illumination to locate visible thinning of the membrane (see Materials and Methods).
[0167] We first compare the membrane thinning and NP drilling kinetics of two 45 nm thick SiNx membranes (488 nm, 6 mW measured at sample plane) having slightly different indices of refraction (n = 2.20 and n =2.29, Fig. 7B and 7C). Although the difference in the reflectivity of the different batches (calculated as R = (ns— nw)2 / (ns + nw)2 where ns and nw are the SiNx and water indices, respectively) is less than 2%, they were affected differently by laser irradiation: the membrane with the higher index of refraction formed a thinned area and a pore through the 45 nm thick membrane within 2 minutes, while the membrane with a slightly lower index of refraction did not form a pore even after >40 minutes of continuous irradiation, and displayed significantly higher initial PL. Inspecting these chips under white light illumination (right-hand experiments were performed at pH 7 in high salt (Tris-HCl buffer, 1 M KC1) and reproduced many times (N >100 times).
[0168] The striking difference in the thinning and drilling time between the two chip types, which only differed slightly in their Si:N compositions, prompted us to hypothesize that the nanopore drilling process is photo-activated. Attempts to thermally induce membrane etching by suspending the membranes in the same buffer (Tris-HCl pH 7, 1 M KC1) at 90°C for over 60 minutes produced negligible or no etching at all of either SiNx membranes as measured by ellipsometry (Fig. 7E). This may suggest that the drilling process requires an electronic transition in the Si-rich membranes, which cannot be provided by heating alone. Indeed, nanochips with an even higher index of refraction ( n = 2.42) could be drilled in less Ύίίi <J,f ic s at even lower excitation laser power (2.8 mW, F¾9^U)?°A¾2¾p¾g to drill the n =2.42 chips at 6 mW laser power resulted in near instantaneous (less than a second) formation of a large pore, which was hard to control.
[0169] In order to establish the relationship between the SiNx membrane indices of refraction and the Si:N composition, we analyzed the materials using both electron energy loss spectroscopy (EELS) and energy dispersive x-ray spectroscopy (EDS) (see Materials and Methods). Each chip was cleaned using argon plasma before measuring the EELS or EDS spectrum. We employed dual EELS measurements to obtain both low loss and core loss data, in order to estimate both the material thickness (using the low loss spectrum) and the material composition (using the core loss spectrum). The atomic percentage of each material was also measured at the same position using EDS. The thickness estimation indicated similar thicknesses for ah tested chips in the range of 44-46 nm. While systematic differences between EELS and EDS in measuring the Si:N ratio are expected based on previous literature, our results (Fig. 8A) show a consistent trend and agree very well with a previously employed empirical model (red solid lines) predicting that:
3/1 3/1 6/1
Si : N = - . Where n¥ is the refractive index of pure Si and nm is the refractive
4 n - 4 n
index of S13N4 (reported theoretical values are n¥ =3.86, n3/4=1.99). Fitting each of the measurements with the model resulted in the following parameter values: n¥=3.995, n3/4=1.964 for the EELS and n¥=3.683, n3/4=1.740 for the EDS (dashed lines). These measurements indicate that on average, the atomic Si:N ratio ranges from about 0.9 to 1.5 for the range of indices from 2.15 to 2.42 respectively, representing significantly larger Si content as compared to the stoichiometric value of S13N4 (0.75). EELS-based band gap measurements of the chips with refractive indexes of 2.15 and 2.42 showed, as described before, that higher refractive index results in a smaller band gap (Fig. 8B). Using the lower band gap Si-rich chips we were able to drill nanopores using a green (532 nm) laser with intensity of 5 mW in less than 2 minutes (Fig. 8C).
[0170] Specifically, in Figure 8C we compare the drilling characteristics of a chips with different refractive indices (n=2.3 and n=2.43) using three laser wavelengths (488 nm, 532 nm and 640 nm). Each chip was immersed in 1 M KC1 pH 7 solution and was exposed to a focused laser beam of the specified wavelengths and intensities. The current and the PL were measured until a pore was formed or up to 10 minutes. As can be seen in Figure 8C, the high refractive index chip was easily drilled with the 488 nm laser (in this case the laser power was decreased since higher power resulted in immediate current overload) as well as while the low refractive index chip could not be J/niZil· vv2u/°m9^¾f the three wavelengths.
[0171] The fact that Si-rich membranes were much more readily drilled using focused light prompted us to further study their optical properties. We focused our attention on the PL emission of the membranes, as this phenomenon is strictly related to photon absorption and photon emission associated with electron excitation/relaxation (unlike scattering). To avoid inducing any material etching, we reduced the laser power by three orders of magnitude to ~7 pW and measured the PL emission in two spectrally-defined emission bands (550 nm < Chi < 650 nm, Ch2 > 650 nm; see Methods). In Figure 8D we show the total PL emission (dark grey markers and line) measured for these samples, as well as the ratio of the red-band emission over the total emission (light grey markers and line). Our results show significantly lower apparent PL intensities for the higher SiNx indices of refraction: Changing the index of refraction from 2.15 to 2.42 resulted in roughly 7-fold PL reduction in the measured visible band. This result initially appeared to be counterintuitive; the SiNx membrane band- gap energy slightly decreases with increasing Si content, which should allow more efficient electron excitation from the valance to conduction bands prior to their relaxation and the emission of a red-shifted PL. However, as the material becomes successively more Si-rich, the density of the Si dangling bonds is known to increase, providing additional energy relaxation pathways involving lower energy photon emissions. Indeed, we observe a systematic red-shifting of the PL at the higher Si:N ratio (Fig. 8D, light grey curve). Noticeably, the lower energy photons associated with these transitions are expected to be outside of the photon counter measurement band. Consequently, these processes would substantially reduce the apparent PL measured in the visible emission band.
Example 4: SiNx membrane etching is accelerated under alkaline conditions
[0172] The strong dependency of the SiNx thinning and nanopore drilling on the Si:N composition suggests that the etching mechanism involves a photochemical reaction. At low irradiation intensities, and specifically for Si-rich material, the laser-induced temperature rise in the water-submersed thin-film appears to be less critical than electronic excitation. In this regime, the enhancement in etch rate can be related to the generation of electron-hole pairs within the SiNx surface and charge transfer at the liquid-solid interface. At the water interface, the dissolution rate of a silica-like material is expected to be strongly affected by pH since the hydroxyl ion is a catalyst for the hydrolysis that underlies the dissolution process. We therefore hypothesized that the etch rate and subsequent pore formation rates could be further accelerated under alkaline conditions. Ύ9, w4 'ί n cstigatc this possibility, we performed a set of experE£J(li^29ift¾au± 6e the membrane etching rate and pore formation as a function of pH, under different laser irradiation intensities. We performed two complimentary measurements: (1) using white- light microscopy we measured the membrane thinning rate by comparing the transmitted light intensity before and after irradiation of a laser for a fixed length of time. (2) Additionally, we used the PL intensity as a proxy for the etching process and characterized its kinetics (Fig. 9). Our results show a clear and consistent trend: Under acidic pH, the thinning process is slowed down significantly, as evidenced by nearly imperceptible changes in the transmitted light intensity. In fact, only under strong laser intensity could we visually discern thinning at all. The PL kinetics measurements were only weakly dependent on the laser intensity at this pH. In contrast, under alkaline conditions (i.e. pH 10 or 12) the drilling process is highly accelerated. Specifically, we observe membrane thinning even at extremely low laser power irradiation down to just a few mW and the PL kinetics show strong dependency on the laser power.
[0174] To quantify the thinning rate under different conditions, we irradiated the same chip for a fixed length of time at different laser intensities. Then we switched buffers as indicated (see Materials and Methods), and the measurements were repeated several times. In Figure 10A we show typical results of the white light image (100X magnification) at 4 pH values, measured using the same laser intensity. We can clearly observe increased thinning under alkaline conditions (pH 10-12) and little to no thinning at pH 4. To quantify the result, we show in Figure 10B the normalized intensity changes as a function of laser power, measured at t = 120s. Our results can be approximated by a linear dependence on the laser power. From the slopes of the curve we obtain the following ratios for pH 7, 10 and 12 as compared with the pH 4 slope: 2.3+0.24, 8.46+0.6, and 10+0.61 mW 1, respectively. These results indicate that thinning can occur at high pH, even at low laser power. In addition, high pH buffer allows fast initiation of the thinning process, which could provide excellent conditions to drill nanopores at high speed (Fig. 9).
[0175] To confirm that the pH dependence measurements are not biased by different buffer indices, we measured them using a refractometer (Rudolph, J257). The measurements were performed at similar temperature to the thinning experiment (22°C). The results in Table 2 show similar values for all solutions, indicating that the buffer did not play a role in the PL measurements.
Example 5: Ultra-fast nanopore drilling in Si-rich membranes Ύ9. ong dependency of thinning on pH and material comp^Tulir?tL?!t/wu9¾fd us to further analyze and characterize this process in order to achieve controlled, ultra-fast, nanopore drilling at low laser intensities. We first immersed a chip with refractive index of 2.3 in high pH buffer (pH 10), exposed it to a 488 nm laser of 7 mW for varying durations, and created a thickness map of the exposed region using EELS. As expected, the thickness maps presented in Figure 11A show that longer illumination results in increased thinning. This is further demonstrated by comparing the line scans in the middle of each thinned region (Fig. 11B-C). Integrating over this line scan shows a similar trend to the PL measurement during thinning, indicating that the PL can be used to approximate changes in thickness and membrane composition (Fig. 11D). High magnification TEM images revealed that an exposure of ~ 15 seconds was enough to create a ~5 nm pore in the center of the Gaussian shaped thin region (see left inset in Fig. HD). However, further irradiation of the surface under these conditions resulted in multiple pores, as can be seen at ~20 s or at longer time points. An OPC gradient threshold (e.g. 0.65 nA over 100 ms) should be set to automatically shut off the laser at the onset of nanopore formation to minimize the likelihood of multiple pores, as was done for fabricating a nanopore array (Fig. 13A-C).
[0177] A drilling trace using a similar chip (refractive index of 2.3), pH 10 buffer, and 488 nm laser with intensity of 6 mW in which pore formation was detected electrically in less than 15 seconds is presented in Figure 12A. To check the nanopore functionality, we immediately changed the buffer to a pH 7 buffer, added DNA sample (300 bp) and measured translocations (Fig. 12A-B). The pore diameter and effective thickness were calculated according to the average fractional blockage level (0.81 ± 0.01) and conductance (19.1 nS). The calculated results suggest that the pore diameter is 5 ± 0.4 nm with an effective thickness of 5 ± 1 nm. A scatter plot of the dsDNA translocation events and concatenated ionic current trace showing sample translocation events are presented in Figure 12A-B.
[0178] Having used a continuous-wave laser to this point, the ability of a pulsed laser to drill a high refractive index SiNx chip was tested. A pulsed laser source, specifically a pico second laser (PicoQuant LDH series), was used to shine blue and green pulsed laser light on a membrane as described hereinabove (10-50 nm thickness). Pulse widths in the range 50 ps to 150 ps, with a repetition rate of up to 80 MHz were tested. The pulsed laser was coupled to the system using a single-mode optical fiber and focused on the sample using high numerical aperture water immersion objective (NA = 1.15). The pulsed laser successfully drilled nanopores with an average intensity (power) of 5 mW or less. In comparison, at the same intensity and similar wavelengths a continues-wave laser took a longer time to drill. In where the continuous-wave laser resulted in no dri 11 i iPg i i utcs
(i.e. higher wavelengths, lower refractive index) the pulsed laser was still able to produce thinning of the membrane. This demonstrates the superiority of the pulsed laser of the continuous-wave laser for thinning and drilling.
Example 6: An automated in-situ fabrication of nanopore arrays using direct laser etching.
[0179] The ability to quickly form arrays of nanopores placed at any chosen locations is extremely important for future use of nanopores in high-throughput applications including nucleic-acid sequencing and protein identification. Both the means to electrically address each individual nanopore in an array, as well as parallel optical sensing, have been proposed and developed. Taking advantage of the ultra-fast, in situ drilling process presented hereinabove, we developed a simple hardware-controlled system for drilling an arbitrary array of pores. Specifically, drilling was automated by inputting a list of coordinates and a current gradient threshold. After focusing the laser on the membrane at low intensity, 150 mV was applied across the membrane, and the PL and current were measured in real time. After focusing, the piezo stage moved the membrane to the first coordinates, and the laser intensity was increased to 7 mW. Once the change in current increased above the current gradient threshold the laser was switched off and the piezo stage moved to the next coordinates (Fig. 13A, top), where the laser was switched on again. In this way the drilling process could stop immediately when a predetermined increase in current was detected (Fig. 13A, bottom).
[0180] Figures 13A-B display an example of an array of 25 nanopores drilled in ~6.5 minutes. The top panel in Figure 13A displays time traces of the laser intensity. The PL and current are shown in the bottom panel (red and blue lines, respectively). As can be seen, all pores formed in less than 20 s, where small variations in the drilling time are mainly due to changes in the laser focus. Histograms of the drilling time and the change in the total current for each drilled pore are presented in Figure 13B. The nanopore array was inspected by wide-field fluorescence microscopy using Ca2+-activated fluorophores. Upon applying a +300 mV bias, 24 out of 25 fluorescent spots appeared at the expected nanopore locations as Ca2+ ions were drawn through the nanopores and reacted with the Ca2+ indicator dye (Fig. 13C, middle panel). The fluorescent spots disappeared when the opposite voltage bias was applied (Fig. 13C, left panel). To estimate the variation in pore sizes, we integrated the fluorescence intensities of each spot. A histogram of the result is shown in Figure 13C (right) and is well-fit by a Gaussian distribution. ΎR ?R2®cI¾a93 i)eeh reported that atomic layer deposition (ALD)-depc?ilT(iIt¾¾?/95?¾uxide (T1O2) thin membranes produce an extremely low photoluminescence and hence can be used favorably for electro-optical sensing of labelled DNA and proteins. We found that an ALD- deposited T1O2 thin membrane is not appreciably etched even at high laser power (>45 mw using either red, blue, or green lasers) given its very low photon absorption in this range. However, coupling a T1O2 layer to a high index refraction (>ND 2.2) layer such as SiNx enables etching at neutral and high pH (> 7), similar to the etch behavior exhibited by free standing SiNx. In Figure 14A (middle panel), the etching that occurs in a T1O2 membrane on top of a SiNx membrane is depicted. This method can be used to fabricate nanopores directly in T1O2. As can also be seen in Figure 14A, the shape of the etch produced in the membrane is that of a Gaussian curve. This shape was observed when SiNx was thinned alone or with T1O2. Further, the full width at half maximum of the Gaussian curve was found to always be one half of the wavelength of the laser light used to generate the nanopore, thus a given shape can be generated by selecting the wavelength of light used for the drilling.
[0182] In an alternative configuration a pH bias can be applied across the membrane surfaces (Fig. 14A, right panel),, where the T1O2 side is held at low pH and the SiNx is held at high pH, in order to prevent laser etching of T1O2 and produce a free-standing T1O2 membrane (Fig. 14B). Continued laser-etching (at a higher pH) of this free-standing T1O2 membrane results in nanopore generation within the T1O2 layer. The T1O2 layer can be conveniently made ultra-thin (< 3 nm) by tuning the ALD deposition process, and generally photoluminescence monitoring allows for monitoring of local thinning in real time. This allows for distinct etching in distinct layers with different indexes of refraction.
[0183] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

W vOia 2i0ii2i0o/194303 PCT/IL2020/050356
1. A method of thinning a membrane comprising a first layer comprising an index of
refraction of greater than 2.0, the method comprising shining focused light on a spot on said first layer, thereby thinning said membrane.
2. A method of thinning a membrane, the method comprising shining pulsed laser light on a spot on said membrane, thereby thinning said membrane.
3. The method of claim 1, wherein said focused light is laser light and said laser light is at a wavelength of between 300 and 600 nm.
4. The method of claim 2, wherein said pulsed laser light is at a wavelength of between 300 and 600 nm.
5. The method of claim 1, wherein said focused light is within the purple, blue or green spectrum.
6. The method of any one of claims 1 to 5, wherein said light comprises an intensity of at least 100 pW.
7. The method of any one of claims 1 to 6, wherein said light comprises an intensity of between 1 and 45 mW.
8. The method of any one of claims 2 to 7, wherein said laser light is continuous-wave laser light or pulsed laser light.
9. The method of any one of claims 2 to 8, wherein said membrane comprises a first layer comprising an index of refraction of greater than 2.0.
10. The method of any one of claims 1 and 3 to 9, wherein said index of refraction is greater than 2.20.
11. The method of any one of claims 1 and 3 to 10, wherein said first layer comprises silicon nitride (SiNx).
12. The method of claim 11, wherein said first layer is a SiNx layer comprising an average silicon to nitrogen ratio of greater than 0.75.
13. The method of claim 12, wherein said average silicon to nitrogen ratio is greater than
0.8.
14. The method of any one of claims 1 to 13, wherein said membrane is a freely standing membrane, covered by an aqueous solution on both sides.
15. The method of any one of claims 1 and 2 to 14, wherein said membrane comprises a second layer refractory to thinning by said focused light when not layered on said first layer, wherein said second layer is a dielectric layer or a layer of metal oxide, and wherein said second layer is layered onto said first layer. of claim 15, wherein said second layer is a layer of rSii3[/¾¾¾9{?¾3 vv^ierein said metal oxide is titanium oxide (Ti02), aluminum oxide (A102) or hafnium oxide (Hf02).
17. The method of any one of claims 1 to 16, wherein said membrane does not comprise a thickness of less than 20 nm.
18. The method of any one of claims 1 to 16, wherein said membrane comprises a thickness of less than 20 nm.
19. The method of any one of claims 1 to 18, wherein said membrane is immersed in
ultrapure water or salt buffer comprising an alkaline pH.
20. The method of any one of claims 1 to 19, wherein said membrane is at room temperature and pressure.
21. The method of any one of claims 1 to 20, further comprising measuring
photoluminescent (PL) intensity from said spot on said membrane.
22. The method of claim 21, wherein said PL intensity is inversely proportional to the
thickness of said spot on said membrane, and said thinning is halted at a desired thickness based on a measured PL intensity.
23. The method of any one of claims 1 to 22, wherein said thinning comprises forming a pore through said membrane.
24. The method of claim 23, wherein said pore is a nanopore.
25. The method of claim 23 or 24, wherein said first membrane is immersed in an aqueous solution and the method further comprising measuring ionic current through said membrane; optionally, wherein an increase in ionic current through said membrane indicates said pore has been formed in said membrane.
26. The method of any one of claims 23 to 25, wherein said spot in said membrane
comprises a thickness of at least 40 nm before said shining and said pore can be produced though said spot in said membrane in less than 20 seconds.
27. The method of any one of claims 1 to 26, wherein said thinning comprises widening a pore through said membrane.
28. The method of claim 27, wherein said membrane is immersed in an aqueous solution and an increase in ionic current through said membrane is proportional to a widening of said pore.
29. The method of any one of claims 23 to 28, for producing a pore of a given diameter, wherein said focused light is automatically shut off at a predetermined current.
30. A system comprising:
a. a light source; WO 2020/194303 a membrane comprising a first layer comprising an in<?i^/¾¾?¾95¾?£&f greater than 2.0;
c. an apparatus to direct and focus light from said light source to a spot on said layer.
31. The system of claim 30, wherein said membrane is in an optically accessible flow cell.
32. The system of claim 30 or 31, wherein said membrane is a freely standing membrane, covered by an aqueous solution on both sides.
33. The system of any one of claims 30 to 32, wherein said index of refraction is greater than 2.20.
34. The system of any one of claims 30 to 33, further comprising a photodetector, wherein said photodetector:
a. is capable of measuring low light intensities and/or measuring at high
temporal resolution;
b. is an avalanche photodiode, a photo -multiplier tube or a CMOS camera; or c. is configured to detect emissions from said spot on said membrane.
35. The system of any one of claims 30 to 34, wherein said light source is at least one of:
a. a solid-state or gas lasers configured to emit within the purple, blue or green spectrum;
b. a solid-state laser configured to emit at between 300-600 nanometers (nm); c. a continuous-wave laser or a pulsed laser;
d. configured to produce light at an intensity of at least 100 micro-watts (pW) at said spot on said membrane; and
e. configured to produce light at an intensity of at least 1 milliwatts (mW) at said spot on said membrane.
36. The system of any one of claims 30 to 35, further comprising an imaging sensor,
optionally, wherein said imaging sensor is selected from an electron multiplying CCD camera, a CMOS camera and a sCMOS camera.
37. The system of any one of claims 30 to 36, wherein said first layer comprises SiNx and comprises a silicon to nitrogen ratio of greater than 0.75.
38. The system of claim 37, wherein said silicon to nitrogen ratio is greater than 0.80.
39. The system of any one of claims 30 to 38, wherein said membrane does not comprise a thickness of less than 20 nm, comprises a thickness of less than 20 nm, is at room temperature and pressure, or a combination thereof.
40. The system of any one of claims 30 to 39, wherein said membrane is immersed in
ultrapure water or salt buffer at an alkaline pH. ^9.2P?^Vyl¾?±i of any one of claims 30 to 40, further comprising twc?£T^3S2¾^S?m apparatus configured to pass an electric current between said two electrodes, wherein one electrode is positioned on one side of said membrane and a second electrode is positioned on another side of said membrane, optionally, further comprising a current detector configured to measure current between said two electrodes.
42. The system of any one of claims 30 to 41, wherein said membrane further comprises a second layer layered on said first layer, wherein said second layer is a dielectric layer or a layer of metal oxide, optionally wherein said metal oxide is Ti02, A102, or Hf02.
43. A thinned membrane produced by a method of any one of claims 1 to 29.
44. A membrane comprising a nanopore, wherein said membrane comprises a first layer comprising an index of refraction of greater than 2.0 and wherein said nanopore comprises a varying diameter and a Gaussian curve shaped cross-section.
45. The membrane of claim 44, wherein said index of refraction is greater than 2.20.
46. The membrane of claim 44 or 45, wherein said nanopore increases in diameter from one side of the membrane to the other, and wherein said increasing diameter follows a Gaussian curve.
47. The membrane of any one of claims 44 to 46, wherein said membrane produces a lower optical background at said nanopore than a nanopore in said membrane without a Gaussian curve shaped cross-section or not produced by a method of any one of claims 1 to 25.
48. The membrane of any one of claims 44 to 47, wherein said first layer comprises SiNx and wherein said SiNx comprises a silicon to nitrogen ratio of greater than 0.75.
49. The membrane of claim 48, wherein said silicon to nitrogen ratio is greater than 0.8.
50. The membrane of any one of claims 44 to 49, wherein said membrane further comprises a second layer layered on said first layer, wherein said second layer is a dielectric layer or a layer of metal oxide , optionally wherein said metal oxide is Ti02, A102 or Hf02.
51. The membrane of claim 50, wherein said nanopore comprises a first Gaussian curve shaped cross-section increasing in diameter from an interface of said first layer with said second layer to an exposed surface of said first layer and a second Gaussian curve shaped cross-section increasing in diameter from said interface to an exposed surface of said second layer.
52. The membrane of any one of claims 44 to 51, wherein said membrane comprises at least two layers with different indexes of refraction.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113770546A (en) * 2021-10-11 2021-12-10 上海莘芝光电科技有限公司东莞分公司 Process for manufacturing three-dimensional circuit by laser etching and carbonizing plastic surface

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014153047A1 (en) * 2013-03-14 2014-09-25 The Trustees Of Boston University Optoelectronic control of solid-state nanopores
WO2016135656A1 (en) * 2015-02-24 2016-09-01 The University Of Ottawa Localizing nanopore fabrication on a membrane by laser illumination during controlled breakdown

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014153047A1 (en) * 2013-03-14 2014-09-25 The Trustees Of Boston University Optoelectronic control of solid-state nanopores
WO2016135656A1 (en) * 2015-02-24 2016-09-01 The University Of Ottawa Localizing nanopore fabrication on a membrane by laser illumination during controlled breakdown

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MOHAMMADAMIR GHADERI, REINOUD F. WOLFFENBUTTEL: "Design and fabrication of ultrathin silicon-nitride membranes for use in UV-visible airgap-based MEMS optical filters", JOURNAL OF PHYSICS: CONFERENCE SERIES, vol. 757, 30 August 2016 (2016-08-30), pages 3, XP055744863 *
RUBY DELA TORRE; JOSEPH LARKIN; ALON SINGER; AMIT MELLER: "Fabrication and characterization of solid-state nanopore arrays for high-throughput DNA sequencing", NANOTECHNOLOGY, vol. 23, no. 38, 5 September 2012 (2012-09-05), pages 385308, XP020228711 *
TAL GILBOA; ZREHEN ADAM; GIRSAULT ARIK; MELLER AMIT: "Optically-monitored nanopore fabrication using a focused laser beam", SCIENTIFIC REPORTS, vol. 8, no. 1, 27 June 2018 (2018-06-27), pages 1 - 10, XP055500930 *
WU MENG-YUE; KRAPF DIEGO; ZANDBERGEN MATHIJS; ZANDBERGEN HENNY; BATSON PHILIP: "Formation of nanopores in a Si N/ Si O 2 membrane with an electron beam", APPLIED PHYSICS LETTERS, vol. 87, no. 11, 7 September 2005 (2005-09-07), pages 113106, XP012075670 *
YANWEN YUAN; GUANGYUAN LI; HAMED ZARIBAFZADEH; MAR\'IA DE LA MATA; PABLO CASTRO-HARTMANN; QING ZHANG; JORDI ARBIOL; QIHUA XIONG: "Sub-20 nm nanopores sculptured by a single nanosecond laser pulse", ARXIV PREPRINT ARXIV: 1806.08172, 31 December 2018 (2018-12-31), XP081238755 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113770546A (en) * 2021-10-11 2021-12-10 上海莘芝光电科技有限公司东莞分公司 Process for manufacturing three-dimensional circuit by laser etching and carbonizing plastic surface

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