# US 10,309,921 B2 · Label-free electrochemical biosensor

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- Number: US 10,309,921 B2
- Document ID: US10309921B2
- Status: Granted
- Kind: B2
- Application number: 14/636,758
- Filed: 2015-03-03
- Published: 2015-09-03
- Granted: 2019-06-04
- Earliest claimed: 2014-03-03
- Assignees: The Florida International University Board of Trustees
- Applicants: Shekhar Bhansali; Abhay Vasudev Mallari
- Inventors: Shekhar Bhansali; Abhay Vasudev Mallari
- Web page: https://patentgrep.com/patent/US10309921B2

## CPC

- G01N 27/3275: {Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction}
- G01N 27/3271: {Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood (amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001)}
- G01N 27/3272: {Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels (optical biosensors G01N33/52)}
- G01N 33/5438: {Electrodes}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The current invention pertains to electrochemical biosensors. The electrochemical biosensor of the current invention comprises: a) a sensing electrode having attached to its surface a binding agent capable of specifically binding to the analyte to form a binding agent-analyte complex and wherein the binding of the analyte to the binding agent alters the electron transfer properties at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-analyte complexes, and b) a test equipment capable of measuring the electrochemical response at the sensing electrode surface. The binding agent can be a binding protein, an antibody, or an aptamer, and the analyte can be a biomolecule. Accordingly, the current invention provides a method of detecting the presence or assessing the likelihood of development of a disease associated with an abnormal level of a biomolecule in a subject.

## Claims

### Claim 1

An electrochemical biosensor for measuring concentration of an analyte in a fluid, the electrochemical biosensor comprising:
a) a sensing electrode having deposited onto its surface a polyaniline (PANI)-silver-silver oxide core-shell (Ag@AgO) nanocomposite that immobilizes a binding agent capable of specifically binding to the analyte to form a binding agent-analyte complex and wherein the binding of the analyte to the binding agent causes electron transfer resistance at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-analyte complexes,
b) a detection solution comprising a soluble redox probe, and
c) a test equipment capable of measuring the electrochemical response at the sensing electrode surface.

### Claim 2 (depends on 1)

The electrochemical biosensor of claim 1, wherein the sensing electrode is fabricated from a noble metal.

### Claim 3 (depends on 2)

The electrochemical biosensor of claim 2, wherein the noble metal is copper, silver, platinum, gold, bismuth, palladium, osmium, iridium, ruthenium, and rhodium.

### Claim 4 (depends on 1)

The electrochemical biosensor of claim 1, wherein the binding agent is a binding protein, an antibody, or an aptamer that specifically binds to the analyte and the analyte is a biomolecule.

### Claim 5 (depends on 4)

The electrochemical biosensor of claim 4, wherein the biomolecule is a hormone, a protein, a polysaccharide, a lipid, a polynucleotide, or a metabolite.

### Claim 6 (depends on 5)

The electrochemical biosensor of claim 5, wherein the hormone is a peptide hormone or a steroid hormone.

### Claim 7 (depends on 6)

The electrochemical biosensor of claim 6, wherein the peptide hormone is selected from Thyroid-stimulating hormone (TSH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Prolactin (PRL), Growth hormone (GH), Adrenocorticotropic hormone (ACTH), Vasopressin, Oxytocin, Thyrotropin-releasing hormone (TRH), Gonadotropin-releasing hormone (GnRH), Growth hormone-releasing hormone (GHRH), Corticotropin-releasing hormone (CRH), Somatostatin, Calcitonin, Parathyroid hormone (PTH), FGF-23 (phosphatonin), Osteocalcin, Erythropoietin (EPO), Human chorionic gonadotropin (HCG), Insulin, Glucagon, Amylin, Atrial-natriuretic peptide (ANP), Gastrin, Secretin, Cholecystokinin (CCK), Fibroblast Growth Factor 19 (FGF19), Neuropeptide Y, Ghrelin, PYY3-36, Insulin-like growth factor-1 (IGF-1), Angiotensinogen, Thrombopoietin, Hepcidin, Betatrophin, Leptin, Retinol Binding Protein 4, Adiponectin, and Irisin.

### Claim 8 (depends on 6)

The electrochemical biosensor of claim 6, wherein the steroid hormone is selected from progesterone, aldosterone, testosterone, estradiol, and cortisol.

### Claim 9 (depends on 1)

The electrochemical biosensor of claim 1, wherein the soluble redox probe is ferro-ferri cyanide.

### Claim 10

A method of measuring concentration of an analyte in a fluid, the method comprising:
a) providing an electrochemical biosensor comprising:
A) a sensing electrode having deposited onto its surface a polyaniline (PANI)-silver-silver oxide core-shell (Ag@AgO) nanocomposite that immobilizes a binding agent capable of specifically binding to the analyte form a binding agent-analyte complex and wherein the binding of the analyte to the binding agent causes electron transfer resistance at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-analyte complexes,
B) a detection solution comprising a soluble redox probe, and
C) a test equipment which measures the electrochemical response at the sensing electrode surface,
b) contacting the sensing electrode with the fluid under conditions that allow the formation of the binding agent-analyte complexes for a period of time sufficient to allow the formation of the binding agent-analyte complexes,
c) washing the sensing electrode with a washing solution to remove unbound and non-specifically bound chemicals from the sensing electrode,
d) introducing a detection solution over the sensing electrode,
e) measuring the electrochemical response at the sensing electrode surface, and
f) calculating the concentration of the analyte in the fluid based on the electrochemical response at the sensing electrode surface.

### Claim 11 (depends on 10)

The method of claim 10, wherein the binding agent is a binding protein, an antibody, or an aptamer that specifically binds to the analyte and the analyte is a biomolecule.

### Claim 12 (depends on 11)

The method of claim 11, wherein the biomolecule is a protein, a hormone, or a metabolite.

### Claim 13 (depends on 12)

The method of claim 12, wherein the hormone is a peptide hormone or a steroid hormone.

### Claim 14 (depends on 13)

The method of claim 13, wherein the peptide hormone is selected from TSH, FSH, LH, PRL, GH, ACTH, Vasopressin, Oxytocin, TRH, GnRH, GHRH, CRH, Somatostatin, Calcitonin, PTH, FGF-23, Osteocalcin, EPO, HCG, Insulin, Glucagon, Amylin, ANP, Gastrin, Secretin, CCK, FGF19, Neuropeptide Y, Ghrelin, PYY3-36, IGF-1, Angiotensinogen, Thrombopoietin, Hepcidin, Betatrophin, Leptin, Retinol Binding Protein 4, Adiponectin, and Irisin.

### Claim 15 (depends on 13)

The method of claim 13, wherein the steroid hormone is selected from progesterone, aldosterone, testosterone, estradiol, and cortisol.

### Claim 16 (depends on 10)

The method of claim 10, wherein the soluble redox probe is ferro-ferri cyanide.

### Claim 17

A method of detecting the presence of a cortisol-related chronic condition in a subject, the method comprising:
a) providing an electrochemical biosensor comprising:
A) a sensing electrode having attached to its surface a binding agent capable of specifically binding to cortisol to form a binding agent-cortisol complex and wherein the binding of cortisol to the binding agent causes electron transfer resistance at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-cortisol complexes, B) a detection solution comprising a soluble redox probe, and
C) a test equipment which measures the electrochemical response at the sensing electrode surface,
b) contacting the sensing electrode with the biofluid under conditions that allow the formation of the binding agent-cortisol complexes for a period of time sufficient to allow the formation of the binding agent-cortisol complexes,
c) washing the sensing electrode with a washing solution to remove unbound and non-specifically bound chemicals from the sensing electrode,
d) introducing a detection solution over the sensing electrode,
e) measuring the electrochemical response at the sensing electrode surface,
f) calculating the concentration of cortisol in the biofluid based on the electrochemical response at the sensing electrode surface, and
g) identifying the subject as having a cortisol-related chronic condition;
if the concentration of cortisol in the biofluid is higher or lower than the concentration of cortisol in the biofluid of a subject not having the cortisol-related chronic condition, wherein the biofluid of the subject and the subject not having the cortisol-related chronic condition are collected at similar times of day.

### Claim 18 (depends on 17)

The method of claim 17, wherein the binding agent is a binding protein, an antibody, or an aptamer that specifically binds to cortisol.

### Claim 19 (depends on 17)

The method of claim 17, wherein the soluble redox probe is ferro-ferri cyanide.