# US 2019/0376972 A1 · Amplification Technology Using Dual Enzyme Cascade Detection

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- Number: US 2019/0376972 A1
- Document ID: US20190376972A1
- Status: Published application
- Kind: A1
- Application number: 16/541,111
- Filed: 2019-08-14
- Published: 2019-12-12
- Earliest claimed: 2017-02-20
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Niren Murthy; Lee W. Riley; Tara Renee BeBoer
- Web page: https://patentgrep.com/patent/US20190376972A1

## CPC

- G01N 33/573: for enzymes or isoenzymes
- C12Q 1/26: involving oxidoreductase
- C12Q 1/34: involving hydrolase
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Diagnostic assays and methods employ a dual-enzyme cascade system comprised of two enzymatic amplifiers and two molecular probes, by (a) incubating a target enzyme with a target enzyme substrate to liberate a thiol; (b) incubating the thiol with a disulfide inactivated amplification enzyme to activate the amplification enzyme in an interchange reaction of the thiol and the disulfide; and (c) incubating the activated amplification enzyme with an amplification enzyme substrate to generate an amplified signal.

## Claims

### Claim 1

An amplification assay method, comprising:
(a) incubating a target enzyme with a target enzyme substrate to liberate a thiol;
(b) incubating the thiol with a disulfide inactivated amplification enzyme to activate the amplification enzyme in an interchange reaction of the thiol and the disulfide; and
(c) incubating the activated amplification enzyme with an amplification enzyme substrate to generate an amplified signal.

### Claim 2 (depends on 1)

The method of claim 1 wherein the amplification enzyme is a cysteine protease or a protease having cysteine protease activity.

### Claim 3 (depends on 1)

The method of claim 1 wherein the amplification enzyme is a cysteine protease selected from papain, bromelain, cathepsin K, and calpain, caspase-1 and separase, adenain, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus-type nsP2 peptidase, dipeptidyl-peptidase VI, deSI-1 peptidase, TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase, hedgehog protein, and dmpA aminopeptidase.

### Claim 4 (depends on 1)

The method of claim 1 wherein the target enzyme is a diagnostic enzyme selected from neuroaminidase, aldolase, creatine phosphokinase, gamma-glutamyl transpeptidase, lactic dehydrogenase, lipase, transaminases like glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase, acid phosphatase, alanine aminotransferase, alkaline phosphatase, amylase, angiotensin converting enzyme, aspartate aminotransferase, cholinesterase, lactate and renin.

### Claim 5 (depends on 1)

The method of claim 1 wherein the target enzyme is a microbial enzyme selected from β-lactamases, glycosidases, esterases and lipases, DNases, peptidases and proteases and coagulase.

### Claim 6 (depends on 1)

The method of claim 1 wherein the target enzyme substrate comprises a sulfenyl moiety, that is cleaved by the target enzyme to liberate a corresponding aromatic or alkyl thiol via an elimination mechanism.

### Claim 7 (depends on 1)

The method of claim 1 wherein the target enzyme substrate has a structure selected from:

### Claim 8 (depends on 1)

The method of claim 1 wherein the amplification enzyme substrate generates a colored or fluorescent product.

### Claim 9 (depends on 1)

The method of claim 1 wherein the amplification enzyme substrate generates an autocatalytic secondary amplifier.

### Claim 10 (depends on 1)

The method of claim 1 wherein the amplification enzyme substrate generates an autocatalytic secondary amplifier, that is a peptide, which liberates a self-immolative chemical moiety upon hydrolytic cleavage of the backbone peptide, to undergo intramolecular cyclization or elimination mechanisms and evolve additional thiol species to trigger further cysteine protease molecules.

### Claim 11 (depends on 1)

The method of claim 1 wherein the amplification enzyme is papain, and the amplification enzyme substrate is a papain probe having a structure selected from:

### Claim 12 (depends on 1)

The method of claim 1 wherein the target enzyme is β-lactamase and the target enzyme substrate is a thiophenol-β-lac having a structure selected from:

### Claim 13 (depends on 1)

The method of claim 1 wherein:
the amplification enzyme is papain; and
the target enzyme is β-lactamase.

### Claim 14 (depends on 1)

The method of claim 1 wherein:
the amplification enzyme is papain, and the amplification enzyme substrate is a papain probe having a structure selected from:
and
the target enzyme is β-lactamase and the target enzyme substrate is a thiophenol-β-lac having a structure selected from:

### Claim 15 (depends on 1)

The method of claim 1 wherein the method assays an unprocessed urine sample for target enzyme activity.

### Claim 16 (depends on 7)

The method of claim 7 wherein the method assays an unprocessed urine sample for target enzyme activity.

### Claim 17 (depends on 11)

The method of claim 11 wherein the method assays an unprocessed urine sample for target enzyme activity.

### Claim 18 (depends on 12)

The method of claim 12 wherein the method assays an unprocessed urine sample for target enzyme activity.

### Claim 19 (depends on 13)

The method of claim 13 wherein the method assays an unprocessed urine sample for target enzyme activity.

### Claim 20 (depends on 14)

The method of claim 14 wherein the method assays an unprocessed urine sample for target enzyme activity.