# US 9,435,805 B2 · Ultrasensitive detection of beta hemolytic Streptococcus

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- Number: US 9,435,805 B2
- Document ID: US9435805B2
- Status: Granted
- Kind: B2
- Application number: 13/825,076
- Filed: 2011-09-22
- Published: 2014-08-07
- Granted: 2016-09-06
- Earliest claimed: 2010-09-23
- Assignees: Woundchek Laboratories (US), Inc.
- Applicants: Mitchell C. Sanders; Dale Macy; Andrei Rakitin; Courtney Mankus
- Inventors: Mitchell C. Sanders; Dale Macy; Andrei Rakitin; Courtney Mankus
- Web page: https://patentgrep.com/patent/US9435805B2

## CPC

- G01N 33/56944: {Streptococcus}
- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- C12Q 1/37: involving peptidase or proteinase
- 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

The present invention is directed to a method for the ultrasensitive detection of beta hemolytic Streptococcus, a bacterium implicated in strep throat, using a specific protease marker. Also disclosed is a device as well as a biosensor, both of which are useful for the detection of beta hemolytic Streptococcus. The biosensor and the device can be used in conjunction with other reagents as part of a kit for detecting strep throat.

## Claims

### Claim 1

A device for detecting the presence or absence of a bacterium in a sample, comprising:
(a) a reaction chamber configured to hold the sample and comprising a first amplifier configured to release a substrate in response to an enzyme produced and/or secreted by the bacterium;
(b) a membrane;
(c) a second amplifier in fluid communication with the first amplifier, configured to bind to the released substrate and to produce a signal in the presence of the bacterium; and
(d) a lateral flow cassette comprising a conjugate pad in fluid communication with the membrane, a lateral flow strip, and a wicking pad;
wherein the membrane is configured to allow the sample to flow to the conjugate pad and lateral flow strip under conditions that allow the released substrate to bind to the second amplifier, wherein the membrane or conjugate pad filters the first amplifier away from the released substrate such that the first amplifier is prevented from entering the lateral flow strip, and wherein the lateral flow strip is configured to indicate the signal in the presence of the bacterium.

### Claim 2 (depends on 1)

The device of claim 1, wherein the signal on the lateral flow strip comprises a pattern.

### Claim 3 (depends on 2)

The device of claim 2, wherein the pattern is selected from the group consisting of a line, a cross and a circle.

### Claim 4 (depends on 1)

The device of claim 1, wherein the membrane is a size exclusion membrane.

### Claim 5 (depends on 4)

The device of claim 4, wherein the size exclusion membrane excludes particles of greater than about 1 micron.

### Claim 6 (depends on 4)

The device of claim 4, wherein the size exclusion membrane comprises an absorbent material, a non-absorbent material, an opaque material, a translucent material, or a hollow fiber.

### Claim 7 (depends on 1)

The device of claim 1, wherein the device comprises a solid support selected from a toothbrush, a wound dressing, a container for holding body fluids, a disk, a scope, a filter, a lens, a foam, a cloth, a paper, a suture, a dipstick and a swab.

### Claim 8 (depends on 1)

The device of claim 1, wherein the bacterium is Streptococcus pyogenes.

### Claim 9 (depends on 1)

The device of claim 1, wherein the first amplifier comprises a peptide substrate covalently attached to a bead.

### Claim 10 (depends on 9)

The device of claim 9, wherein the bead is about 1 to about 100 microns in diameter.

### Claim 11 (depends on 1)

The device of claim 1, wherein the substrate comprises an amino acid sequence selected from the group consisting of FLVMFLSG (SEQ. ID. NO.: 1), ILFTLTGCVG (SEQ. ID. NO.: 2) and GSNMYVYNIS (SEQ. ID. NO.: 3).

### Claim 12 (depends on 1)

The device of claim 1, wherein the first amplifier further includes one affinity label or at least two different affinity labels.

### Claim 13 (depends on 1)

The device of claim 1, wherein the substrate is covalently labeled with a label selected from the group consisting of: affinity tags, spin labels, antigen tags, epitope tags, haptens, enzyme labels, prosthetic groups, fluorescent materials, pH-sensitive materials, chemiluminescent materials, colorimetric components, bioluminescent materials, and radioactive materials.

### Claim 14 (depends on 1)

The device of claim 1, wherein the second amplifier is less than about 1 micron in diameter.

### Claim 15 (depends on 1)

The device of claim 1, wherein the second amplifier is labeled with an affinity tag.

### Claim 16 (depends on 1)

The device of claim 1, wherein the reaction chamber is removable.

### Claim 17 (depends on 1)

The device of claim 1, wherein the membrane or conjugate pad includes the second amplifier.

### Claim 18 (depends on 1)

The device of claim 1, wherein the membrane further includes the first amplifier.

### Claim 19 (depends on 1)

The device of claim 1, further comprising one or more reagents for detecting the enzyme produced and/or secreted by Streptococcus pyogenes.

### Claim 20 (depends on 1)

The device of claim 1, wherein the sample is incubated with the first amplifier; and wherein the released substrate is incubated with the second amplifier.

### Claim 21 (depends on 20)

The device of claim 20, wherein the sample is on a solid support.

### Claim 22 (depends on 21)

The device of claim 21, wherein the solid support is a selected from the group consisting of a wound dressing, a container for holding body fluids, a disk, a scope, a filter, a lens, a foam, a cloth, a paper, a suture, a dipstick, a toothbrush, a bead, and a swab.

### Claim 23

A method for detecting the presence or absence of a bacterium in a sample using a lateral flow device comprising: an enzyme reaction chamber having a membrane, wherein the membrane of the enzyme reaction chamber filters released substrate away from any unreleased substrate; and a lateral flow cassette comprising a conjugate pad in fluid communication with the membrane, a lateral flow strip, a wicking pad, and a flow strip chamber; the method comprising:
a) contacting the sample with a first amplifier in the enzyme reaction chamber under conditions that result in release of a substrate from the first amplifier in response to an enzyme produced and/or secreted by the bacterium;
b) collecting the released substrate onto a second amplifier in the enzyme reaction chamber under conditions that allow the released substrate to bind to the second amplifier;
c) allowing the sample to flow from the enzyme reaction chamber onto the conjugate pad, and
d) detecting the presence of a signal on the flow strip, wherein the presence of the signal indicates the presence of the bacterium in the sample and absence of a signal on the flow strip indicates the absence of the bacterium in the sample.

### Claim 24 (depends on 20)

The device of claim 20, wherein the bacterium is Streptococcus pyogenes.

### Claim 25 (depends on 20)

The device of claim 20, wherein the substrate comprises an amino acid sequence selected from the group consisting of FLVMFLSG (SEQ. ID. NO.: 1), ILFTLTGCVG (SEQ. ID. NO.: 2) and GSNMYVYNIS (SEQ. ID. NO.: 3).

### Claim 26 (depends on 1)

The device of claim 1, wherein the first amplifier is a conjugate capable of being modified by the enzyme which is produced and/or secreted by the bacterium.

### Claim 27 (depends on 1)

The device of claim 1, wherein the second amplifier comprises an affinity tag and a color-producing label.

### Claim 28 (depends on 1)

The device of claim 1, wherein the second amplifier comprises an NTA-gold labeled particle.

### Claim 29 (depends on 1)

The device of claim 1, wherein the lateral flow strip further comprises a charged surface or a binding partner configured to bind specifically to the released peptide.

### Claim 30 (depends on 1)

A method for detecting the presence or absence of a bacterium in a sample comprising contacting the sample with the device of claim 1, and detecting the presence or absence of a signal, wherein the presence or absence of the bacterium is detected.