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WO2018131053A1 - Procédé de détection microbienne - Google Patents

Procédé de détection microbienne Download PDF

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Publication number
WO2018131053A1
WO2018131053A1 PCT/IN2018/050019 IN2018050019W WO2018131053A1 WO 2018131053 A1 WO2018131053 A1 WO 2018131053A1 IN 2018050019 W IN2018050019 W IN 2018050019W WO 2018131053 A1 WO2018131053 A1 WO 2018131053A1
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WO
WIPO (PCT)
Prior art keywords
phbv
pda
minutes
film
phb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2018/050019
Other languages
English (en)
Inventor
Sachin DUBEY
Usman Khan
Maya K V
Mohammad Saquib KHAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Module Innovations Pvt Ltd
Original Assignee
Module Innovations Pvt Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Module Innovations Pvt Ltd filed Critical Module Innovations Pvt Ltd
Publication of WO2018131053A1 publication Critical patent/WO2018131053A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/09—Carboxylic acids; Metal salts thereof; Anhydrides thereof
    • C08K5/092—Polycarboxylic acids

Definitions

  • the present invention relates to microbial detection method. More particularly the present invention relates to microbial detection methods using films of polymersomes.
  • the present invention further relates to color changing property of liposomes of polymers including natural, synthetic, Biodegradable and non biodegradable for the detection of biological entities including bacteria E.coli and ability to distinguish the detection of E.coli from other bacteria like S.Aureus, S.typhimurium, Klebsiella.
  • UTI Urinary Tract Infections
  • E.coli E.coli, S.Aureus, Klebsiella, Enterococcus etc. 50-55% UTI cases of the 150 million globally are due to E.coli. It becomes important to detect the presence of bacteria in urine to prescribe the right set of antibiotics.
  • Current methods of detection include nitrite and leucocyte detection strips, which give a yes/no result or performing time and resource intensive culture. Detection of bacteria is equally important in food and water contaminated with them to prevent people falling ill and other associated ailments .
  • Main objective of the present invention is to provide method of bacterial detection using polymeric structures such as films
  • Another objective of the present invention is to provide films of PHBV/PHB/PCDA polymersomes for colorimteric detection of bacteria.
  • Yet another objective of the present invention is to provide an instrument free method for detection of microbes.
  • the invention describes the method of making polymersomes of PolyHydroxybutyrate(PHB) and Polyhydroxy butyrate-co-valerate(PHBV).
  • the invention also describes polymersomes of PHB and PHBV containing liposomes of diacetylene compounds like 10, 12 Pentacosadiynoic acid(PCDA), TCDA without ligands for specific bacterial detection.
  • the invention further describes casted films of the above polymersomes comprised of but not limited to PHBV/PCDA.
  • the invention also describes E.coli detection by the said film indicated by blue to red color change upon subject to interaction with the said bacteria in but not limited to Nutrient Broth.
  • the present invention provides films of PHBV/PDA such as DMPC comprising 2-20 wt% of PHBV/PHB and PDA: Lipid ratio from 6:4 to 9: 1 wherein the said film causes color change upon bacterial detection.
  • step (d) Mixing PDA monomer obtained in step a with lipid DMPC in said ratios along with solution obtained in step c with total volume made up to 10ml.
  • step (d) Obtaining a thin film of the mixture in step (d) by removal of solvents using rotary evaporator at 40-45 ° C at vacuum of 100-180 mbar for 15-20 minutes and 60mbar for 5 minutes.
  • the present films provide a method of detecting bacteria E.coli
  • the stimulus is selected from Bacteria, heat, organic solvents
  • the detection is based on color change
  • the color change is permanent and irreversible
  • the present invention provides polymeric structure for the detection of microbes and a method of detection thereof.
  • One of the polymeric structures being thin films with size ranging from um in one dimension and cm in other dimension comprising of structural polymers including but not limited to Poly hydroxyButyrate (PHB) and PolyHydroxy butyrate-co-valerate (PHBV) and Polydiacetylene (PDA) including but not limited to 10, 12 Pentacosadiynoic acid (PCD A), 10,12 Tricosadiynoic acid (TCDA) and lipid including but not limited to DMPC, DOPE, cholesterol.
  • structural polymers including but not limited to Poly hydroxyButyrate (PHB) and PolyHydroxy butyrate-co-valerate (PHBV) and Polydiacetylene (PDA) including but not limited to 10, 12 Pentacosadiynoic acid (PCD A), 10,12 Tricosadiynoic acid (TCDA) and lipid including but
  • the films comprising polymersomes of the above materials can be used readily peeled from the glass beaker and is having enough rigidity and resilience for use as strip for bacterial detection.
  • the film comprised of liposomes of PDA/Lipid with membrane fluidity that could be perturbed by stimuli such as bacterial interaction, heat, high alkaline pH, organic solvents, pressure etc)
  • the invention discloses polymeric films which change color upon interaction with bacteria which can be readily observed by naked eye and quantified.
  • the invention provides a method of making polymer films of PHBV/PCDA/DMPC for bacterial detection purposes, which comprises
  • step d Mixing PDA monomer obtained in step a with lipid DMPC/DOPE in said ratios along with solution obtained in step c with total volume made up to 10ml.
  • step d Obtaining a thin film of the mixture in step d by removal of solvents using rotary evaporator at 40 ° -45 ° C at vacuum of 100-180 mbar for 15-20 minutes and 60mbar for 5 minutes.
  • the invention also provides a method of detecting bacteria in contaminated water comprising the steps of: a. Providing polymeric films of PHB/PHB V, PDA(PCDA/TCDA) and; b. Observing the color change in film after adding water containing bacteria indicating contamination, where the bacteria can range from E.coli, S. aureus, S.typhimurium, Klebsiella, enterococci, P.aeroginosa
  • the invention also provides a method of detecting urine contaminated with bacteria comprising the steps of: a. Providing polymeric films of PHB/PHBV, PDA(PCDA/TCDA), lipid(DMPC/DOPE/cholseterol) and,
  • Example 1 Preparation of liposomes of Polyhydroxybutyrate(PHB), 10,12 Pentacosadiynoic acid(PCDA) and DMPC.
  • PCDA and DMPC in the ratio ranging from 9: 1 to 6:4 was taken and dissolved in 5ml of Chloroform. Separately 5 wt% PHB was dissolved in 10ml dichrlomethane for 2 hours. All of them were mixed and rotary evaporated to obtain thin film on the round bottom flask.
  • Example 2 Preparation of liposomes of Polyhydroxy butyrate-co valerate(PHBV), 10,12 Pentacosadiynoic acid(PCDA) and DMPC.
  • PCDA and DMPC in the ratio ranging from 9: 1 to 6:4 was taken and dissolved in 5ml of Chloroform. Separately 5 wt% PHBV was dissolved in 10ml dichloromethane for 2 hours. All of them were mixed and rotary evaporated to obtain thin film on the round bottom flask.
  • Example 3 Preparation of liposomes of Polyhydroxybutyrate(PHBV), 10,12 Pentacosadiynoic acid(TCDA) and DMPC.
  • TCDA and DMPC in the ratio ranging from 9: 1 to 6:4 was taken and dissolved in 5ml of Chloroform. Separately 5 wt% PHBV was dissolved in 10ml dichrlomethane for 2 hours. All of them were mixed and rotary evaporated to obtain thin film on the round bottom flask.
  • Example 4 Color change by Short UV, indicating crosslinking
  • the polymeric film obtained changed from white to Blue upon exposure to UV radiation of 254nm, indication that the croslinking property of PDA remains intact even after self assembling in liposomal structure with another polymer
  • Example 5 Color change indicating bacterial detection
  • the polymeric films were cut in sizes of IcmXlcm and crosslinked by exposure to UV radiation of 254nm. The color of white films turned blue.
  • Figure 1 shows red content in Escherechia coli
  • Figure 2 explains red content in Staphylococcus aureus
  • Figure 3 explains blue content in Escherechia coli
  • Figure 4 shows blue content in Staphylococcus aureus
  • Figure 5 shows comparision of Red content in film of polymersomes of PHBV/PCDA/DMPC with Escherechia coli and Staphylococcus aureus
  • Figure 6 Shows the average size of the polymersome is around 300nm.
  • Figure 1 shows red content of the film upon interaction with E.coli bacteria from 0 hours, 6 hour and 24 hour.
  • the increase in the red component of RGB indicates that the film of PHBV/PCDA/DMPC is able to detect specifically the E.coli bacteria.
  • Red % increase in 6 hours is 126% and 24 hours is 273%.
  • Figure 2 shows red content of the film upon interaction with S.aureus.
  • the red values do not increase significantly to be noticed by naked eyes in the 0-24 hours time period.
  • Red %increase in 6 hours is 0% and 24 hours is 37%
  • Figure 3 shows the drop in blue content of the film of polymersomes of PHBV/PCDA/DMPC. There is a significant reduction in the Blue value over 0-24 hours period which corresponds to the increase in the red value in Figure 1.
  • Figure 4 indicates the drop in blue content of the film of polymersomes of PH BV/PCDA/DM PC upon interaction with Staphylococcus aureus. There is no significant drop noticable to the naked eye, which corresponds with the Figure 2
  • Figure 5 compares the red content of film of polymersomes of PHBV/PCDA/DM PC when it interacts with E.coli and S.aureus. The difference can easily be seen by the naked eye further supported by this graph, where dashed line represents Red content with E.coli interaction and straight line represents interaction with E.coli
  • Figure 6 is a SEM image of the polymersome showing the size of the polymersome in the polymeric film.
  • PCDA 10, 12 Pentacosadiynoic acid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des films polymères pour la détection microbienne basés sur des polymersomes de PHBV/PDA/lipide pour la détection colorimétrique et sans instrument d'entités biologiques comme des bactéries dans des échantillons cliniques et non cliniques 5 pour le diagnostic de maladies bactériennes. L'invention concerne en outre le procédé de préparation de ces structures polymères pour ladite application.
PCT/IN2018/050019 2017-01-14 2018-01-12 Procédé de détection microbienne Ceased WO2018131053A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201621031262 2017-01-14
IN201621031262 2017-01-14

Publications (1)

Publication Number Publication Date
WO2018131053A1 true WO2018131053A1 (fr) 2018-07-19

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PCT/IN2018/050019 Ceased WO2018131053A1 (fr) 2017-01-14 2018-01-12 Procédé de détection microbienne

Country Status (1)

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WO (1) WO2018131053A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114099439A (zh) * 2021-11-19 2022-03-01 四川大学 一种性能优良的聚丁二炔囊泡的制备方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014063097A1 (fr) * 2012-10-19 2014-04-24 Danisco Us Inc. Stabilisation de membranes biomimétiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014063097A1 (fr) * 2012-10-19 2014-04-24 Danisco Us Inc. Stabilisation de membranes biomimétiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO, C.X. ET AL.: "SIZE EFFECT OF POLYDIACETYLENE VESICLES FUNCTIONALIZED WITH GLYCOLIPIDS ON THEIR COLORIMETRIC DETECTION ABILITY", THE JOURNAL OF PHYSICAL CHEMISTRY B, vol. 109, no. 40, 13 October 2005 (2005-10-13), pages 18765 - 18771, XP055511284 *
MEN, Y. ET AL.: "METHODS FOR PRODUCTION OF UNIFORM SMALL-SIZED POLYMERSOME WITH RIGID MEMBRANE", POLYMER CHEMISTRY, vol. 7, no. 24, 8 May 2016 (2016-05-08), pages 3977 - 3982, XP055511292 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114099439A (zh) * 2021-11-19 2022-03-01 四川大学 一种性能优良的聚丁二炔囊泡的制备方法

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