# US 9,040,240 B2 · Arsenic assay using ELISA

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- Number: US 9,040,240 B2
- Document ID: US9040240B2
- Status: Granted
- Kind: B2
- Application number: 14/001,648
- Filed: 2012-02-23
- Published: 2013-12-19
- Granted: 2015-05-26
- Earliest claimed: 2011-02-25
- Assignees: Universite De Lorraine
- Applicants: Pascal Poupin
- Inventors: Pascal Poupin
- Web page: https://patentgrep.com/patent/US9040240B2

## CPC

- C12Q 1/689: for bacteria
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/84: involving inorganic compounds or pH
- C12Q 1/68: involving nucleic acids
- 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
- 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 invention relates to a process involving analyzing the presence of arsenic in a sample, comprising the following steps: (a) contacting a sample suspected of containing arsenic on a solid support functionalized with a DNA fragment comprising an ArsR protein binding site, site on which an ArsR protein is attached, the presence arsenic causing separation of the ArsR protein from the DNA fragment; (b) incubation of the sample in contact with the said support; (c) elimination of ArsR protein separated from the DNA fragment by washing the support after the incubation carried out in step (b); and (d) analyzing by an ELISA technique to detect the presence or absence of ArsR protein remaining attached to the DNA fragment.

## Claims

### Claim 1

A process for analyzing arsenic in a sample, comprising the following steps:
(a) contacting a sample suspected of containing arsenic on a solid support functionalized with a DNA fragment comprising an ArsR protein binding site, site on which an ArsR protein is attached, the presence of arsenic causing separation of the ArsR protein from the DNA fragment;
(b) incubation of the sample in contact with the said support;
(c) elimination of ArsR protein separated from the DNA fragment by washing the support after the incubation carried out in step (b); and
(d) analyzing by an ELISA technique to detect the presence or absence of ArsR protein remaining attached to the DNA fragment.

### Claim 2 (depends on 1)

The process according to claim 1, said process further comprising a step (e) of comparison of the measurement made in step (d) with a control sample containing arsenic.

### Claim 3 (depends on 1)

The process according to claim 1, wherein the functionalization of the DNA fragment on the solid support is achieved by streptavidin-biotin bond.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the incubation step (b) is carried out for a time period ranging from 1 minute to 90 minutes.

### Claim 5 (depends on 1)

The process according to claim 1, wherein the incubation step (b) is carried out at a temperature ranging from 15° C. to 37° C.

### Claim 6 (depends on 1)

The process according to claim 1, wherein the contacting step (a) is carried out with a buffer solution whose pH level is comprised between 6 and 8.

### Claim 7 (depends on 1)

The process according to claim 1, wherein the DNA fragment containing an ArsR protein binding site is a promoter of the arsenic resistance gene of E. coli, or a part of the promoter of the arsenic resistance gene of E. coli enabling binding of an ArsR protein.

### Claim 8 (depends on 1)

The process according to claim 1, wherein the sequence of the DNA fragment containing an ArsR protein binding site is SEQ ID NO. 1, SEQ ID NO. 17, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 or a sequence having at least 80% of identity with SEQ ID NO. 1, SEQ ID NO. 17, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25 or SEQ ID NO. 26.

### Claim 9 (depends on 1)

The process according to claim 1, wherein the solid support is made of polystyrene, polyethylene or polypropylene.

### Claim 10 (depends on 1)

The process according to claim 1, wherein the ArsR protein is covalently bound to a known sequence of a recognition peptide, for the implementation of the ELISA test.

### Claim 11 (depends on 10)

The process according to claim 10, wherein the sequence of the recognition peptide is selected from among SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and a sequence having at least 80% of identity with these sequences.

### Claim 12

A kit intended for the detection of arsenic in a sample, said kit comprising:
a solid support functionalizable or functionalized with a DNA fragment comprising an ArsR protein binding site;
a DNA fragment containing an ArsR protein binding site;
an ArsR protein;
a means for detection by ELISA of the presence of the ArsR protein.

### Claim 13 (depends on 12)

The kit according to claim 12, wherein the means for detection by ELISA of the presence of the ArsR protein, is a detection means using colorimetry or luminescence.

### Claim 14 (depends on 12)

The kit according to claim 12, further comprising arsenic at a determined concentration.

### Claim 15 (depends on 12)

The kit according to claim 12, wherein the solid support is a plate or a microplate containing a plurality of wells.