Nano robotic system for high throughput single cell DNA sequencing

A method for forming a sub-10-nm-gap dual-tip AFM cantilever comprising the steps of:

• using FIB chemical vapor deposition to cut a cantilever and its supporting base from a commercially available cantilever that is pre-coated with metal layers on both sides (top and bottom) to form lead lines and microelectrodes on a pyramid or cone-shaped tip;
• if the cone-shaped tip has a precoated metal layer, using FIB to s separate the pre-coated layer into two parts;
• if the cone-shaped tip does not have a precoated metal layer use FIBCVD to directly write electrodes on the side surface with a line width of about 1 μm and a thickness of about 50-100 nm;
• milling off the cone-shaped tip using FIB to form two small flat surfaces with a side length of 1 μm-3 μm;
• applying FIBCVD or EBID to grow nanotips from the flats and to form a lateral bridge on the top of cylindrical tips;
• and
• using a focused highly energetic electron beam to generate a sub-10-nm gap between the cylindrical nanotips by cutting the bridge into two parts at the center.

A method for forming a sub-10-nm-gap dual-tip AFM cantilever comprising the steps of:

• using PECVD to grow a 50-nm thick silicon dioxide (SiO2) thin film;
• forming several 50-nm cross cylindrical tips with a smallest gap around 15 nm using an EBL system to pattern the tips on a spin-coated photoresist;
• using an e-beam to evaporate about a 45-nm-thickness of Au to develop a pattern;
• pressing as-fabricated metal discs from the top using a ns YAG laser, three layers of materials are used for achieving this, including a 4-μm-thick Al layer as a compressor, a graphite one for ablative coating, and a CaF2/ZeSe glass layer for transparent confinement;
• using photolithography masks with pre-designed patterns together with UV exposure for both the top and bottom side patterning of the contours of a supporting base and cantilevers using spin coating of photoresist, developing and dry etching of a SiO2 layer using ICP;
• creating windows for Si wet etching by using a KOH solution;
• forming pyramid tips after etching while the cantilevers are kept with a greater thickness than the final sizes;
• growing a 50-nm-thick layer of SiO2 using PECVD on the top surface as an insulating layer for the fabrication of lead out electrodes on the supporting bases and microelectrodes on the cantilevers;
• selectively removing the SiO2 on the top of the tips using dry etching with ICP based on UV exposure and developing on a spin coated photoresist layer;
• depositing a 45-nm-thick gold film with an e-beam evaporator, to serve as electrodes;
• using FIBCVD to directly write a nanoelectrode connecting the microelectrodes on the cantilevers and the tips so that the nanoelectrode follows a path along the pyramid side and top surfaces, thus realizing a 3D electrical connection;
• and
• using BOE to etch the SiO2 away followed by Si etching using KOH.

A method for forming a sub-10-nm-gap dual-tip AFM cantilever comprising the steps of:

• vapor depositing an insulating layer of SiO2 on both sides of Si wafers using PECVD;
• depositing a sacrificial layer of Al film patterned with UV exposure, evaporating the film with an electron beam and subjecting the film to a lift off process to from a self-curling mesa that is a SixNy film with intrinsic stress;
• using a UV lithography process to design an exposure mask and ICP etching technology to achieve mesa fabrication;
• patterning an Au bottom electrode with UV exposure and electron-beam evaporation;
• obtaining a nano-gap insulating layer by using ALD deposition technology to create an Al2O3 film;
• using photoresist as a mask and ICP etching to retain a pattern of the Al2O3 film above the bottom electrode;
• using UV exposure, electron beam vapor deposition and lift off techniques to obtain an Au film top electrode pattern;
• using the back jacketing method by pre-etching the backside of the SiO2 layer using photoresist to create etching window patterns;
• using ICP etching of the SiO2 to expose the Si etching material and Acetone to remove the etching window photoresist;
• using PECVD to vaporize a portion of the SiO2 anti-etching protection layer on the substrate surface;
• placing the substrate in a KOH solution at room temperature to etch off the substrate Si;
• using UV lithography together with ICP to etch the SiO2 protective layer in order to transfer the etching window pattern to the top of the substrate;
• using acetone to remove the etched window photoresist;
• using UV exposure to obtain a self-curling release window pattern;
• using NMD solution to etch the sacrificial layer Al to gradually release the curling structure;
• placing the sample with its self-curling structure in acetone solution to remove the photoresist;
• using KOH to etch the silicon substrate at the point where the upper and lower release windows are connected;
• and
• placing the sample in a BOE etching solution to completely etch the exposed SiO2 and then placing it in aqueous solution to rest in order to obtain an AFM probe with a self-curling structure.

A method for nano scale robotic single cell DNA sequencing of a strand of DNA positioned on a slide of an atomic force microscope, which moves an end effector in the form of a cantilever with a tip along the DNA strand, and wherein the spacing between electrodes at the tip is greater than the spacing between the nucleobases of the DNA strand so that the electrical potential difference between the electrodes is the sum of the occupied molecular orbital (HOMO) energy of all of the nucleobases between the two electrodes, comprising the steps of:

• preparing a DNA strand for sequencing;
• adding the energy value of nine T nucleobases to the beginning and end of the DNA strand;
• conducting n-9 measurements as one measurement loop where n is the total number of nucleobases after concatenation of T nucleobases at the beginning and end of the DNA chain,
• adding Gaussian noise to the measurement values to account for contact noise between the electrodes and DNA;
• adding beta noise to the measurement values to account for errors in the end effector motion;
• solving for the DNA sequence with a neural network whose inputs are the position serials of each nucleobase and whose outputs are the HOMO energy values of the nucleobases at the corresponding positions, wherein the loss function for training the neural network is defined as L=∥Ax−d∥22, and the best DNA sequence can be determined by minimizing the loss value;
• and
• repeating loops of measurement where the result after the last measurement is used as the initialization of the next calculation;
• and
• continuing to repeat loops of measurement until the there is no change in the generated DNA sequence.