Patent for “Methods and systems for seperating biomolecules from mixtures containing them”

Nanotechnology – Fernando Patolsky, Vadim KRIVITSKY, Ramot at Tel Aviv University Ltd

Search Patent for “Methods and systems for seperating biomolecules from mixtures containing them”

Abstract for “Methods and systems for seperating biomolecules from mixtures containing them”

“A system consists of a substrate and multiple nanostructures that are arranged on it. A minimum of one portion of the nanostructures has a capturing moiety that is covalently attached on its surface. At least another portion of nanostructures, which may be the same as or different from the first, features a light activatable moiety that is covalently attached onto its surface. The capturing moiety selectively interacts and produces a light-activatable moiety upon exposure to light. This reactive moiety interferes with the interaction between the analyte and the capturing moiety. Systems with a substrate and multiple nanostructures that are generally aligned vertically to it, at least one of these nanostructures being branch nanostructures, are further provided. The systems can be used to extract and, optionally, identify the analyte in a sample.

Background for “Methods and systems for seperating biomolecules from mixtures containing them”

The present inventors have successfully developed and practiced a system that is composed of multiple elongated nanostructures with a porous and/or rough surface. Modified by a capturing moiety which selectively interacts and activates an analyte and by a light activatable moiety which, when activated, causes a release of analytes. This allows for direct extraction and optional further analysis of analytes in liquid mixtures containing the same.

The present invention provides an integrated light-controlled system for filtering and selectively separating liquid mixtures. It also allows for controlled release platforms that can be used for direct analysis of complex liquid mixtures like whole blood or other complex biosamples.

“Accordingly to one aspect of some embodiments the present invention, there is a system consisting of a substrate and a multitude of nanostructures arranged upon the substrate at a density at least 100,000 nanostructures/1 cm2. Each nanostructure in the first portion of the microstructures featuring an analyte-capable moiety, and each nanostructure within the second portion of the macrostructures featuring an analyte-capable moiety, wherein the capturing molecule selectively interacts with the analy with the analyte.

“Accordingly to at least one embodiment of any of these embodiments, at minimum one of the nanostructures has the light-activatable and the capturing moiety covalently attached on a surface of such a structure.”

“Activating the light-activatable moiet is, according to some embodiments herein, exposing the system at a wavelength that generates a reactive moiety.”

“Accordingly to some embodiments herein, the reactive moieties is such that it induces a release at least 20% of the molecules of analyte which interact with the capturing moieties within 10 minutes.

“According some of the embodiments herein, analyte can be a biomarker.”

“According some embodiments, the capturing moiety and the analyte are first members of an affinity couple.”

“According to some of any of the embodiments described herein, the affinity pair is selected from an antigen-antibody pair, a receptor-ligand pair, an enzyme-substrate pair, a streptavidin-biotin pair, a protein-cofactor pair, a protein-protein pair, and pairs of complementary oligonucleotides, or of complementary oligonucleotide-peptide nucleic acid.”

“According some embodiments herein, the capturing moie is an antibody. The analyte selectively interacts the antibody with it.”

“According some of the embodiments herein, an analyte can be considered an antigen.”

“According some embodiments herein, the analyte and the capturing moiety have a pH-dependent constant for dissociation.”

“Accordingly to some embodiments herein, the light activatable moiety produces, upon exposure to light, an reactive moiety that causes a change of protons concentration near the at least one portion of the nanostructures that feature the capturing moiety.”

“According some embodiments herein, the light activatable moiety generates protons upon exposure to light.”

“According some embodiments herein, the elongated Nanostructures are generally parallel one to the other.”

“According some embodiments, the elongated microstructures are generally aligned vertically with the substrate,”

“The elongated nanostructures can be described as nanowires if they are used in some of the embodiments.”

“According some embodiments, the average length of nanostructures ranges between 10 nm and 500 microns.”

“According some embodiments, the average diameter of nanostructures ranges between 10 nm and 30 microns.”

“According some embodiments, the average inter-distance between nanostructures ranges between 10 nm and 10000 nm.

“The nanostructures are silicon, according to some embodiments herein.”

“According some of the embodiments herein, at most a portion of nanostructures comprise branched nanostructures.”

“Accordingly to one aspect of the present invention, there is provided a system for extracting analyte out of a liquid that contains it.

“According some embodiments, extracting involves contacting the liquid with a system and exposing it to light at a wavelength that produces the reactive moiety.”

“According some of the embodiments herein, the liquid can be considered a biological sample.”

“According some embodiments, the system also includes a sensing element/system in fluid communication with it.”

“According some embodiments, the sensing element/system comprises a nanostructure that is covalently attached to the capturing moie. It is designed so that, upon interaction between the capturing and analyte, an detectable signal is generated which indicates the presence or level of the analyte.”

“According some of the embodiments herein, this system can be used to extract the analyte form a liquid sample containing the same and determine the presence and/or levels of the analyte within the liquid sample.”

“Accordingly to one aspect of the present invention, there is provided a method for extracting an analyte out of a liquid sample containing the same. The method comprises:

“Contact the liquid sample with any of the claims 1-20 to obtain a system that has the analyte attracted to the nanostructures.

“Exposed the system to light at the wavelength that generates reactive moiety.”

“According some embodiments, the method involves, during the exposure, contacting the system using an aqueous solutions to obtain an analyte-containing aqueous solution.”

“According some embodiments, the exposing takes place for a time that ranges between 1 to 1000 or 1 to 500 or 1 to 100 seconds.

“According some embodiments, the method further comprises, after contacting the liquid sample with system, washing system with an aqueous (also known herein as sensing solution or sensing buffer).

“According some embodiments, the time for which the liquid is contacted with the system can be between 1 and 180 minutes or 10 to 120 minutes.

“Accordingly to some embodiments herein, the light activatable moiety is such, that within 10 minutes, at least 20% are desorbed from the adsorbed analyte compounds.”

“According some of the embodiments herein, the liquid can be considered a biological sample.”

“According some of the embodiments herein, the concentration of analyte is less than 1mM or lower that 1?M or lower that 1 nM or lower still than 1pM.”

“Accordingly to one aspect of the present invention, there is a method for determining the presence or level of an analyte within a liquid sample. The method comprises:

“Subjecting the liquid as described in the respective embodiments or any combination thereof;

“Contact the aqueous solution after exposure with a sensing device configured for identifying or determining the presence and/or levels of the analyte.”

“According some of the embodiments herein, the sensing system or element is as described in the respective embodiments.”

“Accordingly to one aspect of some embodiments, the present invention provides a system consisting of a substrate and a plurality of nanostructures that are aligned generally vertically with the substrate at a density at least 100,000 nanostructures/1 cm2, at most a portion of these nanostructures being branched.

“Each of the branched, nanostructures can be viewed as a separate entity according to any one of the embodiments herein. Each branch may contain between 10 and 1,500,000 branches.”

“According some embodiments, the average length of each branch ranges independently from 5 to 40000nm.”

“According some embodiments, the average diameter of the branched microstructures is between 2 and 200 nm.”

“According some embodiments, every nanostructure in at most a portion the nanostructures has an capturing moiety covalently attached on a surface thereof. The capturing moiety selectively interacts and interacts with an analyte, according to some of the embodiments herein. Each nanostructure of at least one portion of the nanostructures is covalently attached with a light activatable moiety according to some embodiments.

“Accordingly to some embodiments herein, the system can be used to extract the analyte out of a liquid sample containing the same and optionally to release the analyte upon light exposure and further analyze it using a sensing element, or system, as described herein.”

“Unless otherwise stated, all technical terms and/or scientific terms herein have the same meanings as those commonly understood by an ordinary skilled person in the art to the invention. While methods and materials that are similar or identical to the ones described herein may be used for the practice and testing of embodiments, the following exemplary methods and/or material can be used. The patent specification will govern in the event of a conflict. The examples, materials, and methods are intended to be illustrative and not necessarily limitative.

Implementation of the method/or system according to the invention may involve performing selected tasks manually, automatically or a combination of both. Furthermore, depending on the actual instrumentation and equipment, various tasks can be executed by hardware, software, firmware, or a combination thereof using an Operating System.

“For example, hardware that performs selected tasks according the embodiments of this invention could be implemented in a chip or circuit. Select tasks according to embodiments could be implemented using a variety of software instructions that are executed by any computer running an appropriate operating system. One or more of the tasks described in an exemplary embodiment are executed by a data processor. This could be a computing platform that executes a number of instructions. Optionally, the processor may include a volatile memory to store instructions and/or information and/or non-volatile storage such as a magnetic hard drive and/or removable media for storing data. A network connection can be provided. Optionally, a display and/or an input device for the user such as a keyboard and mouse are provided.

“BRIEF DESCRIPTION ABOUT THE VIEWS OF MANY DRAWINGS”

“Some embodiments are described in this document, but only as an example. Refer to the accompanying drawings for details. It is important to mention the drawings in detail. The details are intended to be used as examples and for illustrative purposes only. The description given with the drawings illustrates how embodiments may be used.

“In the drawings:

“FIG. “FIG.

“FIG. 2 shows SEM images of SiNWF, SiNWF in gold nanoparticles are deposited onto the surface of the nanowires (left), and the 3D branched SiNW matrix.

“FIGS. 3A-B present SE(FIG. 3A and BSE (FIG. 3B) Images of SiNWF where gold nanoparticles have been deposited on the surface of the nanowires.”

“FIGS. 4A-D show SE images of Branched Si Nanowires Forest, (BSiNWF), at different magnifications.

“FIGS. 5A-B show the data for adsorption CA 15-3 on anti?CA 15-3- or HTPS-modified siNW forest. The solution contained 5.35 pM antigen. These data are expressed as a function time and percents (%) of the antigen present in the solution. 5A and the concentration of the adsorbed Antigen out of the Total Antigen in the Solution (FIG. 5B).”

“FIG. “FIG.

“FIGS. 7A-B show the desorption of CA15-3 from modified SiNW forests devices upon application of light (HPTS excitation), in percents (%) the desorbed Antigen (FIG. 7A and the concentration of desorbed antigen (FIG. 7B, showing that more than 80 percent of the adsorbed antibody are released after 10 minutes of radiation.

“FIG. 8 shows the original signals that were generated by the modified SiNW Forest device during desorption of the CA 15-3 solution.”

“FIG. “FIG. The calibration curve is plotted log-log in the inset.

“FIG. “FIG.

“FIG. “FIG. 13 shows the desorption kinetics for hemoglobin exposed to light (dark grey squares) or dark (light gray circles).

“FIG. 14 shows comparative plots showing improved hemoglobin adsorption to SiNW branched forest surfaces modified with anti-hemoglobin, HPTS, and compared to SiNW forests similarly modified.”

“FIG. “FIG.

“FIG. “FIG. 16 shows a graph that illustrates the desorption kinetics for eGFP absorbed onto SiNW Forest surface modified with anti?eGFP, HTPS. This graph was obtained after contact with a blood sample containing eGFP. It is shown in light gray circles (light gray rectangles) and dark gray rectangulars (dark gray rectangles).

“DESCRIPTION OF EMBODIMENTS SPECIFIC TO THE INVENTION”

“The present invention, in some embodiments thereof, relates to separation/extraction of analytes, and, more particularly, but not exclusively, to systems usable in, and methods for, selectively separating analytes of interest (e.g., biomolecules such as biomarkers) from a liquid mixture containing same, for example, from a biological sample.”

“Before I explain at least one embodiment, it is important to understand that the invention does not have to be limited to the details of construction or the arrangement of components and/or methods described in the following description and/or illustrated by the drawings and/or examples. The invention can be used in other ways or embodied in different forms.

The present inventors developed a method that uses a system with an extremely large surface area and the ability to selectively absorb analytes in a liquid sample. They also created a fast release system that allows for the rapid removal of the absorbed substances from the system.

The system is built on dense forests. “The system is based on a dense?forest?

The system is composed of many elongated nanostructures, with a porous or rough surface. It’s modified by a capturing moiety which selectively interacts to an analyte and by a light activated moiety which, when activated induces the release of the analyte. This allows for a direct extraction of analytes from fluid mixtures that contain them.

“In exemplary embodiments, the separation of analytes-of-interest (e.g., biomarkers) from raw biosamples is performed using a roughness-controlled, optionally branched, silicon nanowire forest of ultra-large binding surface area, modified by a capturing moiety that is selective towards the analyte, followed by the fast release of target proteins in a controlled liquid media, using drastic pH change near silicon nanowire surface. The pH change is caused by illumination of light-activatable moiety (e.g., photo-acid/photo-base) which is covalently bonded to nanowires surface.”

The 3D nanostructure system described herein in certain embodiments serves as an on chip filter that has an ultra-large binding area and an optionally reversible light controlled release of adsorbed marker molecules. This allows for the selective collection and separation of biomarkers. It also removes unwanted components from blood cells and proteins. Light controlled filter separation systems can be integrated with downstream sensors, such as SiNW-based arrays of sensor arrays, to allow for multiplex, real time and ultrasensitive detection (sensing), of biomarkers.

The methodology described herein has the following advantages over current practiced methods: It allows for rapid separation without the need to use time-consuming processes like centrifugation or affinity columns; it is cost-effective to manufacture and operate; it is multiplex?capable of performing multi-bio-molecular separat; it can release analytes in controlled liquid media; the method features desalting capabilities. The methodology performs sample preconcentration which is especially useful for biomarkers with low abundance; both the system as well as the methodology have high selectivity

The system of the present embodiments is also known as a selective capture system or a selective extraction, separation and/or filtering device. It selectively absorbs and/or binds an analyte from a mixture and can release the absorbed substances upon illumination. Optionally, the capturing, separation, or filtering system may be in fluid communication to a sensing element, system, or device, which determines the composition, presence, and/or level, of the released analyte.

“Systems and methods of the present embodiments are useful for quick and direct analysis of samples. They can also be used to perform blood tests and detect biomarkers for chronic disease.

“The systems and procedures described herein are easy to operate and can be set up as a lab on a chip. This allows for the use of, for example at the point-of-care, laboratory personnel and/or equipment without any additional cost.”

“The separation/capturing/filtering system:”

“Accordingly to one aspect of some embodiments, the present invention provides a system consisting of a substrate and a plurality of nanostructures arranged on that substrate at a density at or below 100,000 or at minimum 200,000 or at most 300,000 or at the least 400,000 or at the least 500,000 or at the least 1,000,000 or at the least 2,500,000 nanostructures per cm2.

“According some embodiments, each nanostructure of at least one portion of the Nanostructures features an capturing moiety covalently attach to a surface thereof. Each nanostructure of at least one second portion of these nanostructures features light-activatable moiety of covalently attach to a substrate thereof.”

“Nanostructures Forest:”

“According to at least one of the embodiments herein, at most a portion (e.g. at minimum 50% or at best 60% or at lowest 70% or at least 80% or at least 90% or at least 95% or at least 99%) of the nanostructures is elongated.

“Elongated nanostructure” is defined herein. A three-dimensional body made from a solid substance and having at least one cross-sectional dimension. In some embodiments, this may be less that 200 nanometers or less then 150 nanometers. Or less about 100 nanometers. Any shape can be used for the cross-section of an elongated nanostructure, including but not limited to: rectangular, square, rectangular and elliptical. Both regular and irregular shapes can be included.”

“In some embodiments the nanostructure is shaped like a hollow tube. Preferably, it is completely hollow along its longitudinal length. This is also known as the?nanotube? “, or a?nanotubular arrangement?

“The nanotubes may be single-walled, multi-walled or a combination of both.”

“In some embodiments, the average inner diameter of nanotubes ranges between 0.5 nanometers and 200 nanometers. Or, from 1 to 100 nanometers or 1 to 50 nanometers.

“Multi-walled nanotubes can have interval distances ranging from 0.5 to 200 nanometers in certain embodiments. Or from 1 to 100 nm or 50 nm.

“In some embodiments, the average length of nanostructures is between 0.1 and 500 microns. Or 1 to 200 microns.

“In some embodiments described herein, the average diameter of nanostructures ranges between 10 nm and 30 microns. This includes any intermediate subranges or values.

“In most of the embodiments herein, the elongated Nanostructures are generally parallel to one another.”

“In some embodiments, the elongated microstructures are generally aligned vertically with the substrate.”

“In some embodiments, the elongated microstructures are parallel to one another and align generally vertically with the substrate.”

“Some embodiments of this invention have an average inter-distance of 10 nm-10000 nm or 10 nm-106000 nm or 10 nm-105000 nm.

“The elongated nanostructures in the present embodiments are collectively called?nanopillars?” Or as ‘nanowires’?

Summary for “Methods and systems for seperating biomolecules from mixtures containing them”