Nanomaterial-based Bacterial Sensors

Abstract

This invention relates to a sensor that detects bacteria cells comprising (a) a primary negatively charged, nanoparticulate sensing material; (b) a secondary positively charged, fluorescent sensing material; (c) a housing; and (d) at least one illuminator; wherein said housing contains said primary negatively charged, nanoparticulate sensing material, said secondary positively charged fluorescent sensing material and a sample potentially comprising bacteria in aqueous medium, wherein said illuminator provides light of at least one pre-specified wavelength .sub.i to excite at least said secondary positively charged, fluorescent material, wherein said secondary positively charged, fluorescent material electrostatically attached to bacteria cells provides at least one fluorescent response at a second different wavelength .sub.n wherein both i and n are integers, wherein said negatively charged, nanoparticulate sensing material electrostatically attached to said fluorescent material suppresses fluorescing of said fluorescent material at said second wavelength .sub.n; and wherein said housing permits illumination of the contents of said housing by said illuminator and wherein said housing further permits the detection of a fluorescent response at a second wavelength .sub.n. The negatively charged material includes (dsDNA coated) spherical AuNPs and graphene oxide (GO). The positively charged fluorescent material includes water soluble cationic conjugated polyelectrolytes (COPE) or positively charged peptide/polymer labeled with fluorescence dye. The sensor makes use of the FRET phenomenon between the primary and secondary sensing materials. The sensor allows making a distinction between living and dead bacteria and can measure the total bacteria count. A method for detecting bacteria utilizing the sensor is another part of the invention.

Claims

1. A sensor that detects bacteria cells comprising: a primary negatively charged, nanoparticulate sensing material; a secondary positively charged, fluorescent sensing material; a housing; and at least one illuminator; wherein said housing contains said primary negatively charged, nanoparticulate sensing material, said secondary positively charged, fluorescent sensing material and a sample potentially comprising bacteria in aqueous medium, wherein said illuminator provides light of at least one pre-specified wavelength .sub.i to excite at least said secondary positively charged, fluorescent material, wherein said secondary positively charged, fluorescent material electrostatically attached to bacteria cells provides at least one fluorescent response at a second different wavelength .sub.n wherein both i and n are integers, wherein said negatively charged, nanoparticulate sensing material electrostatically attached to said fluorescent material suppresses fluorescing of said fluorescent material at said second wavelength .sub.n; and wherein said housing permits illumination of the contents of said housing by said illuminator and wherein said housing further permits the detection of a fluorescent response at said second wavelength

2. The sensor according to claim 1, wherein said primary negatively charged, nanoparticulate sensing material is selected from the group of metal nanoparticles and graphene oxide.

3. The sensor according to claim 2, wherein the metal of said metal nanoparticle is selected from the group of gold and silver.

4. The sensor according to claim 2 or 3 wherein said metal nanoparticle is a metal nanoparticle to which dsDNA is conjugated.

5. The sensor according to claim 1 wherein said secondary positively charged, fluorescent sensing material is selected from the group of cationic conjugated polyelectrolytes (COPE), peptides labeled with fluorescence dye and polymers labeled with fluorescence dye or being fluorescent.

6. The sensor of claim 5 wherein said cationic conjugated polyelectrolyte is selected from -conjugated polymers comprising side chains with cationic groups.

7. The sensor of claim 6 wherein said cationic groups comprise moieties selected from the group of quarternary ammonium and pyridinium.

8. The sensor of claim 6 or 7 wherein said -conjugated polymer is selected from the group of in each case cationic polythiophenes, poly(p-phenylene)s, poly(fluorene)s, poly(phenylene ethynylene)s, poly(fluorine ethynylenes)s, poly(phenylene vinylene)s, poly(naphthalene vinylene)s, poly(fluorine vinylene)s and copolymers comprising these polymers.

9. The sensor of claim 5 wherein said peptide comprises 5 to 100 amino acids and comprises amino acids selected from the group of histidine, lysine, arginine and mixtures thereof.

10. The sensor of claim 5 wherein said polymer comprises quaternary ammonium, biguanidine, phosphonium, guanidine, sulfonium, or pyridinium groups.

11. The sensor of claim 5 wherein said fluorescent dye is selected from the group of Abz (Anthranilyl, 2-Aminobenzoyl), N-Me-Abz (N-Methyl-anthranilyl, N-Methyl-2-Aminobenzoyl), FITC (Fluorescein isothiocyanate), 5-FAM (5-carboxyfluorescein), 6-FAM (6-Carboxyfluorescein), TAMRA (Carboxytetramethyl rhodamine), Mca (7-Methoxycoumarinyl-4-acetyl), AMCA/Amc (Aminomethylcoumarin Acetate), Dansyl (5-(Dimethylamino) naphthalene-1-sulfonyl), EDANS (5-[(2-Aminoethyl)amino]naphthalene-1-sulfonic acid), Atto (Atto465, Atto488, Atto495, Atto550, Atto647), Cy3 (1-(5-carboxypentyl)-3,3-dimethyl-2-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium chloride), Cy5 (1-(5-carboxypentyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dienyl)-3H-indolium chloride), Alexa Fluor (Alexa Fluor 647, Alexa488, Alexa532, Alexa546, Alexa594, Alexa633, Alexa647), Bodipy, Dylight (DyLight 488, DyLight 550), Trp (Tryptophan), Lucifer Yellow ((ethylene diamine) 6-Amino-2-(2-amino-ethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5,8-disulfonic acid) and mixtures thereof.

12. The sensor of claim 1 wherein the detection principle is based on the competitive electrostatic interaction or binding of the positively charged, fluorescent and nanoparticulate materials with either the bacteria cells or the negatively, charged nanoparticulate materials.

13. The sensor of claim 1 wherein said second wavelength is in the near infrared (NIR) region of the light.

14. A method for detecting bacteria comprising the steps of: (a) providing a system comprising: a. a primary negatively charged, nanoparticulate sensing material; b. a secondary positively charged, fluorescent sensing material that electrostatically attached to said primary negatively charged, nanoparticulate sensing material shows suppressed florescence; and c. at least one illuminator; (b) adding a sample comprising bacteria cells in aqueous medium to the system; (c) providing light of at least one pre-specified wavelength to excite at least said fluorescent sensing material; (d) detecting or measuring the fluorescence of said fluorescent sensing material at a second wavelength after electrostatically attachment of fluorescent sensing material to the bacteria cells in said sample.

15. The method of claim 14 wherein the electrostatic attachment of the positively charged, fluorescent materials to the negatively charged bacteria cells competes with the electrostatic attachment of the positively charged, fluorescent materials to the negatively, charged nanoparticulate materials for at least 2 minutes and up to 30 minutes before detecting or measuring the fluorescence.

16. The method of claim 14 wherein the system additionally comprises a light detector and an analysis unit that changes its status when the light detector signal exceeds a pre-set fluorescence value to indicate the presence of a certain concentration or total amount of total bacteria cells.

17. The method of claim 14 or 16 wherein the charge density of said secondary positively charged, fluorescent sensing material is adjusted by choice of said material to achieve a pre-set fluorescence value that is identical to a desired detection limit of bacteria cells.

18. The method of claim 14 wherein the method is used to detect whether said bacteria cells are alive or dead.

19. The method of claim 14 wherein the total bacteria count is detected.

20. The method of claim 14 wherein the bacteria cells comprise E. coli or S. aureus strains.

Description

DESCRIPTION OF DRAWINGS

[0085] The accompanying drawings illustrate a disclosed embodiment or reaction scheme and serve to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are designed for purposes of illustration of examples only, and not as a limitation of the invention.

[0086] FIG. 1 shows a schematic illustration of the bacteria cell detection in a fluorimetric competition assay by using positively charged luminescent materials and negatively charged dsDNA-AuNPs.

[0087] FIG. 2 shows (a) a fluorescence spectrum (.sub.ex=334 nm) of CCPE before mixing with dsDNA-AuNPs and bacteria (initial, top line), after mixing with dsDNA-AuNPs without bacteria (F.sub.blank, bottom line), and after mixing with dsDNA-AuNPs with E. coli sample (F.sub.sample, middle line) and (b) the percentage of fluorescence increase from fluorimetric competition assay by using CCPE and dsDNA-AuNPs as a function of E. coli (square) and S. aureus concentration (circle).

[0088] FIG. 3 shows the percentage of fluorescence increase from the fluorimetric competition assay by using peptide-FITC and dsDNA-AuNPs as a function of live E. coli concentration.

[0089] FIG. 4 shows the percentage of fluorescence increase from the fluorimetric competition assay by using CCPE and dsDNA-AuNPs as a function of the concentration of live E. coli (dots) and deadautoclaved E. coli (squares). In FIG. 4, CTW means Cooling Tower Water (water collected from cooling tower in Singapore). Bacteria spiked in and autoclaved CTW (to kill any live bacteria) were tested. The CTW is different from DI water used for other bacteria sample preparation as the CTW contains other impurities from the cooling tower such as dust, fine particles, ion species, etc.

[0090] FIG. 5 shows a schematic illustration of the bacteria cell detection in a fluorimetric competition assay by using positively charged luminescent materials and negatively charged GO.

[0091] FIG. 6 shows (a) a fluorescence spectrum (.sub.ex=334 nm) of CCPE before mixing with GO and bacteria (initial, top line), after mixing with GO without bacteia (F.sub.blank,bottom line), and after mixing with dsDNA-AuNPs with E. coli sample (F.sub.sample, middle line); and (b) the percentage of fluorescence increase from the fluorimetric competition assay by using CCPE and GO as a function of E. coli (squares) and S. aureus concentration (circles).

[0092] FIG. 7 shows the percentage of fluorescence increase from the fluorimetric competition assay by using peptide-FITC and GO as a function of live E. coli concentration.

[0093] FIG. 8 shows the percentage of fluorescence increase from the fluorimetric competition assay by using CCPE and GO as a function of the concentration of live E. coli (dots) and dead-autoclaved E. coli (squares).

[0094] FIG. 9 shows the fluorescence spectra of PBS, pulpy orange juice, soy sauce, ketchup, and CCPE in these matrices. The auto-fluorescence background from juice, soy sauce, and ketchup mask the CCPE fluorescence intensity.

[0095] FIG. 10 shows the fluorescence spectra of 20 times diluted orange juice (bottom line), CCPE-410 in the diluted juice (top line), and CCPE-410 mixed with GO in diluted juice without (3.sup.rd from top line) and with E. coli sample in diluted juice (2.sup.nd from top line).

[0096] FIG. 11 shows the auto-fluorescence background intensity of PBS solution, pulpy orange juice, soy sauce, ketchup in various excitation/emission wavelength. Low fluorescence background intensity is observed in NIR region, where the excitation/emission conditions are at 590/630 nm.

INDUSTRIAL APPLICABILITY

[0097] According to the invention a sensor has been provided that can detect and quantify the total amount of bacteria in water. This sensor may have numerous applications in the analysis of the hygienic status of industrial water, public recreational water, marine ballast water discharge or drinking water. The sensor may be used in environmental monitoring and may be used to indicate the exceeding of pre-set bacteria levels in samples. Depending on the sensing materials used the sensor may also be used to indicate whether bacteria in a sample are alive or dead.

[0098] The sensor and the related method further have a potential use in hygienic beverage or food analysis as interferences from colorations may be overcome.

[0099] The sensor and the related method can further serve as a platform technology. Through further development (e.g. modifying the surface of the sensing materials with bio-affinity materials etc.) technologies to target a specific bacteria type may be delivered to meet specific bacteria detection requirements.

[0100] It will be apparent that various other modifications and adaptations of the invention are available to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.