Pristine graphene based biosensor for biomarker detection and related core particles, materials compositions methods and systems

Patent metadata

Field	Value
Publication number	US-12461102-B2
Application number	US-202218051422-A
Country	US
Kind code	B2
Filing date	Oct 31, 2022
Priority date	Dec 30, 2020
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

Abstract

Provided herein are graphene biosensors and related core particles, compositions methods and systems in which more than one pristine graphene sheet is coated with a coating layer of an organic or inorganic material to provide a core graphene particle, to which detectable components comprising a detectable moiety and a peptide linkage are attached through binding of the peptide linkage.

First claim

The invention claimed is:

1 . A graphene biosensor configured to detect a target biomarker, the graphene biosensor comprising:
a graphene core particle and a detectable moiety capable of emitting a fluorescent signal, the detectable moiety is covalently bonded to the graphene core particle through a peptide linkage comprising a recognition sequence specific for the target biomarker,
wherein the graphene core particle comprises
at least two pristine graphene nanosheets having a pristine graphene nanosheet surface stacked over one another to form a pristine graphene aggregate, and
a coating layer covering at least 50% of a pristine graphene nanosheet surface, said coating layer comprising an organic and/or inorganic compound,

wherein the organic and/or inorganic compound has a solubility in water of at least 10 grams per liter H 2 O at 25 degrees C. and is able to be covalently linked to, or in water electrostatically stably attached to, pristine graphene,
wherein the organic and/or inorganic compound is attached to a pristine graphene nanosheet to form said coating layer,
wherein the pristine graphene of the at least two pristine graphene nanosheets has a carbon content of at least 99%, an oxygen content of less than 1% and is pure, such that the pristine graphene is free of foreign substances and impurities,
wherein the pristine graphene nanosheet and/or the organic and/or inorganic compound of the coating layer is configured to present a functional group capable of binding a corresponding functional group in an aqueous solution,
wherein, the coating layer and the peptide linkage are configured to set a quenching distance between the graphene nanosheets and the detectable moiety in which the graphene nanosheets quench the fluorescent signal of the detectable moiety,
wherein the peptide linkage is further configured to set, upon recognition by the target biomarker of the recognition sequence, an emitting distance between the graphene nanosheets and the detectable moiety wherein the graphene nanosheets do not quench the fluorescent signal of the detectable moiety, and
wherein the target biomarker specific peptide linkage and the detectable moiety thus form a target specific detectable component configured to emit a detectable fluorescent signal upon modification of the peptide linkage by the target biomarker.

2 . The graphene biosensor of claim 1 , wherein said recognition sequence is specific for a biomarker selected from the group consisting of proteases, kinases, arginases, cytokines/chemokines, dioxygenases, esterases, and combinations thereof.

3 . The graphene biosensor of claim 2 , wherein said protease is selected from the group consisting of serine, aspartyl, cysteine, metalloprotease, caspase, urokinase, cathepsin, caspase, and combinations thereof.

4 . The graphene biosensor of claim 1 , wherein said biomarker is indicative of a condition selected from the group consisting of cancerous or precancerous cellular activity, bacterial activity, infection, inflammation, and combinations thereof.

5 . The graphene biosensor of claim 1 , wherein said detectable moiety is a chromophore/luminophore selected from the group consisting of organic dyes, inorganic dyes, fluorophores, phosphophores, luminophores, light absorbing particles, quantum dots, combinations thereof, and the metalated complexes thereof.

6 . The graphene biosensor of claim 5 , wherein said chromophore/luminophore is an organic dye selected from the group consisting of coumarins, pyrene, cyanines, BODIPY dyes, benzenes, N-methylcarbazole, erythrosin B, N-acetyl-L-tryptophanamide, 2,5-diphenyloxazole, rubrene, and N-(3-sulfopropyl) acridinium.

7 . The graphene biosensor of claim 5 , wherein said chromophore/luminophore is an inorganic dye selected from the group consisting of porphyrins, phthalocyanines, chlorins, and metalated chromophores.

8 . The graphene biosensor of claim 7 , wherein said porphyrins are selected from the group consisting of tetra carboxy-phenyl-porphyrin (TCPP) and Zn-TCPP.

9 . The graphene biosensor of claim 5 , wherein said chromophore/luminophore is a fluorophore or phosphor selected from the group consisting of phosphorescent dyes, fluoresceines, rhodamines, and anthracenes.

10 . The graphene biosensor of claim 5 , wherein said quantum dot is selected from the group consisting of CdSe/ZnS core/shell quantum dots, CdTe/CdSe core/shell quantum dots, CdSe/ZnTe core/shell quantum dots, and alloyed semiconductor quantum dots.

11 . The graphene biosensor of claim 1 , wherein said coating layer comprises a polymer monolayer selected from the group consisting of polyethyleneimine, polycaprolactone, polyvinyl alcohol, polyvinylpyrrolidone, (meth)acrylate- and (meth)acrylamide polymers, polystyrene-carboxylic acid, polystyrene-amine, dextran, pullulan, chitosan, alginate, cellulose, and hyaluronic acid, and mixtures or co-polymers thereof.

12 . The graphene biosensor of claim 1 , wherein the graphene core particle has a size of from about 50 nm to about 500 nm.

13 . The graphene biosensor of claim 1 , comprising a plurality of detectable components attached to the graphene core particle via respective peptide linkages.

14 . The graphene biosensor of claim 1 , comprising at least two different detectable moieties attached to said central carrier the graphene core particle via respective peptide linkages that each have a recognition sequence specific for different target biomarkers.

15 . A composition comprising one or more graphene biosensors of claim 1 , in combination with a suitable auxiliary agent.

16 . The composition of claim 15 , wherein the composition is a pharmaceutical composition and the auxiliary agent is a pharmaceutically acceptable auxiliary agent.

17 . A method of detecting the activity of a biomarker in a biological sample comprising:
contacting a biological sample with the graphene biosensor of claim 1 to create a reaction solution; and detecting a change in said reaction solution.

18 . The method of claim 17 further comprising:
exposing said reaction solution to an excitation light source; and
detecting a change in an absorption or emission spectrum of the detectable moiety as a function of activity of said biomarker in said sample.

19 . The method of claim 17 , wherein said change comprises a blue-shift in the absorption or emission maximum of said detectable moiety after said contacting relative to the absorption or emission spectrum prior to said contacting.

20 . The method of claim 17 , wherein said change comprises the appearance of a new visible color or luminescence band relative to the absorption or emission spectrum of said detectable moiety prior to said contacting.

21 . The method of claim 17 , wherein said contacting comprises incubating said sample with said biosensor for a period of time of less than 90 minutes, preferably less than 60 minutes.

22 . The method of claim 17 , wherein said contacting comprises providing a microplate comprising a plurality of microwells therein, one or more of said microwells comprising a plurality of said biosensors distributed therein, and adding said biological sample to said microwells to create respective reaction solutions in each of said microwells.

23 . The method of claim 17 , wherein the detecting is preceded by a heating of the sample and/or of the testing solution.

24 . A system f

Assignees

• Hawkeye Bio Ltd
• Univ Kansas
• Univ Kansas State

Inventors

• Dempsey Paul W
• Hantula Spencer
• Aparicio Cristina-Mihaela Sandu
• Bossmann Stefan H
• Sorensen Christopher M
• Covarrubias-Zambrano Obdulia
• Covarrubias Jose

Classifications

• G01N33/551

  the carrier being inorganic · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• G01N33/533

  with fluorescent label · CPC title

• B82Y30/00

  Nanotechnology for materials or surface science, e.g. nanocomposites · CPC title