Ultra-sensitive detection of extremely low level biological analytes using electrochemical signal amplification and biosensor
First Claim
1. A signal amplification sandwich structure for amplifying, detecting and/or quantifying an analyte or multiple different analytes in a fluid sample, wherein said structure consists of:
- (a) a first outer layer comprising a magnetic particle conjugated with a plurality of a first analyte binding material for binding the analyte;
(b) an inner layer comprising said analyte; and
(c) a second outer layer comprising a nonmagnetic particle conjugated with a plurality of a second analyte binding material for binding said analyte that is a matched pair with the first analyte binding material, and the nonmagnetic particle is also conjugated on its outer structure or temporarily filled in its inner structure with a plurality of an electrochemically detectable oligonucleotide tag in greater amounts than said analyte from said inner layer,wherein;
(i) said electrochemically detectable oligonucleotide tags are for signal amplification, wherein the majority of nucleotides within said oligonucleotide tags are guanine, wherein said nucleotides within said oligonucleotide tags are selected from the group consisting of guanine, adenine, and thymine, and wherein the combination of said nucleotides produces a unique oligonucleotide tag that is used to amplify, detect and/or quantify said analyte or multiple different analytes;
(ii) analyte amplification performance of said signal amplification sandwich structure can be tuned to meet the desired limit of detection by adjusting one or more of the following parameters;
(a) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle;
(b) the number of guanines per electrochemically detectable oligonucleotide tag;
(c) the size of the nonmagnetic particle; and
(d) the surface area of the nonmagnetic particle for conjugating electrochemically detectable oligonucleotide tags;
(iii) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle ranges from 10 2 to 1013, the number of guanines per electrochemical detectable oligonucleotide tag ranges from 10 to 400, wherein the nonmagnetic particles are spherical and/or nonspherical, the diameter of spherical nonmagnetic particles ranges from 1 to 400 micrometers, the surface area of nonspherical nonmagnetic particles has an equivalent surface area of spherical nonmagnetic particles with ranges from 1 to 400 micrometers, and the surface of the nonmagnetic particles is smooth, rough, porous, or extended with attachments to other nonmagnetic particles; and
(iv) no optically detectable tags are used for amplification, detection or quantification.
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Abstract
This invention allows ultra-low levels of virtually any biological analyte to be detected and quantified rapidly, simply and inexpensively with an electrochemical biosensor using a novel electrochemical signal amplification technique. The invention amplifies detection signals from low level analytes using an innovative sandwich ELISA structure that replaces optical labels with a massive amount of electrochemically detectable guanine rich oligonucleotide tags. Selective binding is achieved with matched pairs of either commercial or custom analyte binding materials such as monoclonal antibodies or single strand DNA. The guanine tags are eluted from the sandwich structures and hybridize with complementary cytosine rich oligonucleotide recognition probes attached to the surface of a biosensor working electrode. An electrochemical technique generates a signal in proportion to the guanine level on the working electrode which is also proportional to the analyte level in the sample. Magnetic separation and a nanosensor are used to improve the signal-to-noise ratio for measuring analyte levels 1,000,000 times lower than enzyme-linked immunosorbent assay (ELISA).
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Citations
20 Claims
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1. A signal amplification sandwich structure for amplifying, detecting and/or quantifying an analyte or multiple different analytes in a fluid sample, wherein said structure consists of:
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(a) a first outer layer comprising a magnetic particle conjugated with a plurality of a first analyte binding material for binding the analyte; (b) an inner layer comprising said analyte; and (c) a second outer layer comprising a nonmagnetic particle conjugated with a plurality of a second analyte binding material for binding said analyte that is a matched pair with the first analyte binding material, and the nonmagnetic particle is also conjugated on its outer structure or temporarily filled in its inner structure with a plurality of an electrochemically detectable oligonucleotide tag in greater amounts than said analyte from said inner layer, wherein; (i) said electrochemically detectable oligonucleotide tags are for signal amplification, wherein the majority of nucleotides within said oligonucleotide tags are guanine, wherein said nucleotides within said oligonucleotide tags are selected from the group consisting of guanine, adenine, and thymine, and wherein the combination of said nucleotides produces a unique oligonucleotide tag that is used to amplify, detect and/or quantify said analyte or multiple different analytes; (ii) analyte amplification performance of said signal amplification sandwich structure can be tuned to meet the desired limit of detection by adjusting one or more of the following parameters;
(a) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle;
(b) the number of guanines per electrochemically detectable oligonucleotide tag;
(c) the size of the nonmagnetic particle; and
(d) the surface area of the nonmagnetic particle for conjugating electrochemically detectable oligonucleotide tags;(iii) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle ranges from 10 2 to 1013, the number of guanines per electrochemical detectable oligonucleotide tag ranges from 10 to 400, wherein the nonmagnetic particles are spherical and/or nonspherical, the diameter of spherical nonmagnetic particles ranges from 1 to 400 micrometers, the surface area of nonspherical nonmagnetic particles has an equivalent surface area of spherical nonmagnetic particles with ranges from 1 to 400 micrometers, and the surface of the nonmagnetic particles is smooth, rough, porous, or extended with attachments to other nonmagnetic particles; and (iv) no optically detectable tags are used for amplification, detection or quantification. - View Dependent Claims (2)
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3. A device for amplifying, detecting and/or quantifying an analyte or multiple different analytes in a fluid sample, wherein said device consists of:
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(a) a magnetic separation unit configured to form a first outer layer and inner layer of signal amplification sandwich structures, (b) an analyte amplification unit configured to form a second outer layer of signal amplification sandwich structures, (c) a tag discharge unit configured to discharge electrochemically detectable oligonucleotide tags from signal amplification sandwich structures, and (d) an electrochemical detection unit with at least one biosensor working electrode configured to measure detection signals from the electrochemically detectable oligonucleotide tags, wherein said device employs the units from step (a), step (b), step (c) and step (d), collectively referred to as the device units, to form one or more signal amplification sandwich structures for amplifying detection signals from said analyte or multiple different analytes in a fluid sample, wherein said signal amplification sandwich structure consists of a first outer layer comprising a magnetic particle conjugated with a plurality of a first analyte binding material for binding said analyte, an inner layer comprising said analyte, and a second outer layer comprising a nonmagnetic particle conjugated with a plurality of a second analyte binding material for binding said analyte that is a matched pair with the first analyte binding material and the nonmagnetic particle is also conjugated on its outer structure or temporarily filled in its inner structure with a plurality of an electrochemically detectable oligonucleotide tag in greater amounts than said analyte from said inner layer, wherein; (i) said electrochemically detectable oligonucleotide tags are for signal amplification, wherein the majority of nucleotides within said oligonucleotide tags are guanine, wherein said nucleotides within said oligonucleotide tags are selected from the group consisting of guanine, adenine, and thymine, and wherein the combination of said nucleotides produces a unique oligonucleotide tag that is used to amplify, detect and/or quantify said analyte or multiple different analytes; (ii) analyte amplification performance of said signal amplification sandwich structure can be tuned to meet the desired limit of detection by adjusting one or more of the following parameters;
(a) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle;
(b) the number of guanines per electrochemically detectable oligonucleotide tag;
(c) the size of the nonmagnetic particle; and
(d) the surface area of the nonmagnetic particle for conjugating electrochemically detectable oligonucleotide tags;(iii) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle ranges from 102 to 1013, the number of guanines per electrochemical detectable oligonucleotide tag ranges from 10 to 400, wherein the nonmagnetic particles are spherical and/or nonspherical, the diameter of spherical nonmagnetic particles ranges from 1 to 400 micrometers, the surface area of nonspherical nonmagnetic particles has an equivalent surface area of spherical nonmagnetic particles with ranges from 1 to 400 micrometers, and the surface of the nonmagnetic particles is smooth, rough, porous, or extended with attachments to other nonmagnetic particles; and (iv) no optically detectable tags are used for amplification, detection or quantification. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for amplifying, detecting and/or quantifying an analyte or multiple different analytes in a fluid sample, wherein said method consists of the following steps performed sequentially:
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(a) providing the fluid sample that may contain non-specific materials and an analyte or multiple different analytes; (b) providing one or more sets of magnetic particles, wherein each set comprises a plurality of a magnetic particle conjugated with a plurality of a first analyte binding material to create analyte-magnetic particle complexes if said analyte or said multiple different analytes are present; (c) providing one or more sets of nonmagnetic particles, wherein each set comprises a plurality of a nonmagnetic particle conjugated with a plurality of a second analyte binding material that is a matched pair with the first analyte binding material and is also conjugated with a plurality of a second electrochemically detectable oligonucleotide tag in greater amounts than said analyte to create electrochemically detectable oligonucleotide tag-nonmagnetic particle-analyte-magnetic particle structures if said analyte is present; and (d) providing one or more working electrodes, wherein each working electrode is associated with said analyte or said group of multiple different analytes that may be present in said sample and wherein each working electrode is conjugated with a plurality of an oligonucleotide recognition probe to bind with associated electrochemically detectable oligonucleotide tags, and an electrochemical detection technique produces electrochemical signals on each working electrode in proportion to the quantity of said analyte or said group of multiple different analytes if said analyte or said group of multiple different analytes is present in the fluid sample; wherein said method employs one or more signal amplification sandwich structures for amplifying detection signals from the analyte or multiple different analytes in the fluid sample, wherein said structure consists of a first outer layer comprising a magnetic particle conjugated with a plurality of a first analyte binding material for binding said analyte, an inner layer comprising said analyte, and a second outer layer comprising a nonmagnetic particle conjugated with a plurality of a second analyte binding material for binding said analyte that is a matched pair with the first analyte binding material and the nonmagnetic particle is also conjugated on its outer structure or temporarily filled in its inner structure with a plurality of an electrochemically detectable oligonucleotide tag in greater amounts than said analyte from said inner layer, wherein; (i) said electrochemically detectable oligonucleotide tags are for signal amplification, wherein the majority of nucleotides within said oligonucleotide tags are guanine, wherein said nucleotides within said oligonucleotide tags are selected from the group consisting of guanine, adenine, and thymine, and wherein the combination of said nucleotides produces a unique oligonucleotide tag that is used to amplify, detect and/or quantify said analyte or said group of multiple different analytes; (ii) analyte amplification performance of said signal amplification sandwich structure can be tuned to meet the desired limit of detection by adjusting one or more of the following parameters;
(a) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle;
(b) the number of guanines per electrochemically detectable oligonucleotide tag;
(c) the size of the nonmagnetic particle; and
(d) the surface area of the nonmagnetic particle for conjugating electrochemically detectable oligonucleotide tags;(iii) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle ranges from 102 to 1013, the number of guanines per electrochemical detectable oligonucleotide tag ranges from 10 to 400, wherein the nonmagnetic particles are spherical and/or nonspherical, the diameter of spherical nonmagnetic particles ranges from 1 to 400 micrometers, the surface area of nonspherical nonmagnetic particles has an equivalent surface area of spherical nonmagnetic particles with ranges from 1 to 400 micrometers, and the surface of the nonmagnetic particles is smooth, rough, porous, or extended with attachments to other nonmagnetic particles; and (iv) no optically detectable tags are used for amplification, detection or quantification. - View Dependent Claims (18, 19)
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20. A method for amplifying an analyte or multiple different analytes in a fluid sample, wherein said method consists of the following steps performed sequentially:
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(a) providing the fluid sample that may contain non-specific materials and an analyte or multiple different analytes; (b) providing one or more sets of magnetic particles, wherein each set comprises a plurality of a magnetic particle conjugated with a plurality of a first analyte binding material to create analyte-magnetic particle complexes if said analyte or said multiple different analytes are present; (c) providing one or more sets of nonmagnetic particles, wherein each set comprises a plurality of a nonmagnetic particle conjugated with a plurality of a second analyte binding material that is a matched pair with a first analyte binding material and is also conjugated with a plurality of a second electrochemically detectable oligonucleotide tag in greater amounts than said analyte to create electrochemically detectable oligonucleotide tag-nonmagnetic particle-analyte-magnetic particle structures if said analyte is present; and (d) said electrochemically detectable oligonucleotide tag-nonmagnetic particle-analyte-magnetic particle structures are magnetically immobilized and said electrochemically detectable oligonucleotide tags are unbound from said structures, and washed and delivered as electrochemically detectable oligonucleotide tags for amplifying detection signals from said analytes in said fluid sample, wherein said method employs one or more signal amplification sandwich structures for amplifying detection signals from the analyte or multiple different analytes in the fluid sample, wherein said structure consists of a first outer layer comprising a magnetic particle conjugated with a plurality of a first analyte binding material for binding said analyte, an inner layer comprising said analyte, and a second outer layer comprising a nonmagnetic particle conjugated with a plurality of a second analyte binding material for binding said analyte that is a matched pair with the first analyte binding material and the nonmagnetic particle is also conjugated on its outer structure or temporarily filled in its inner structure with a plurality of an electrochemically detectable oligonucleotide tag in greater amounts than said analyte from said inner layer, wherein; (i) said electrochemically detectable oligonucleotide tags are for signal amplification, wherein the majority of nucleotides within said oligonucleotide tags are guanine, wherein said nucleotides within said oligonucleotide tags are selected from the group consisting of guanine, adenine, and thymine, and wherein the combination of said nucleotides produces a unique oligonucleotide tag that is used to amplify said analyte or a group of multiple different analytes; (ii) analyte amplification performance of said signal amplification sandwich structure can be tuned to meet the desired limit of detection by adjusting one or more of the following parameters;
(a) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle;
(b) the number of guanines per electrochemically detectable oligonucleotide tag;
(c) the size of the nonmagnetic particle; and
(d) the surface area of the nonmagnetic particle for conjugating electrochemically detectable oligonucleotide tags;(iii) the number of electrochemically detectable oligonucleotide tags per nonmagnetic particle ranges from 102 to 1013, the number of guanines per electrochemical detectable oligonucleotide tag ranges from 10 to 400, wherein the nonmagnetic particles are spherical and/or nonspherical, the diameter of spherical nonmagnetic particles ranges from 1 to 400 micrometers, the surface area of nonspherical nonmagnetic particles has an equivalent surface area of spherical nonmagnetic particles with ranges from 1 to 400 micrometers, and the surface of the nonmagnetic particles is smooth, rough, porous, or extended with attachments to other nonmagnetic particles; and (iv) no optically detectable tags are used for amplification.
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Specification