FORM 2
THE PATENTS ACT-1970
(39 of 1970) &
The Patent Rules, 2006
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
1. TITLE OF THE INVENTION
Method for microbial detection
2. APPLICANT
a) Name Module Innovations Private Limited
b) Nationality Indian
c) Address 100, NCL Innovation Park
Dr Homi Bhabha Road, Pashan
Pune-411008, MH
India

Title
Method for Microbial Detection
Field of Invention
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.
Background of Invention
Bacteria and microbes are a looming threat that affects humans and animals alike. In humans, diseases like Urinary Tract Infections (UTI) are known to be caused by bacteria such as 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 .
In a scenario where resources are limited , there is a clear need of a method which can perform bacterial/microbial detection at the Point of Care (PoC), without electricity, trained manpower and sophisticated readout instruments. There have been previous attempts to make methods to detect bacteria using gold nanoparticles conjugated with antibodies, electronic devices, which can indicate biological species, but all the available instruments need power to provide the

readouts. It is also crucial to note that resource limited settings such as rural areas are extremely sensitive to price and a higher cost system would be an economical challenge for the rural strata
Thus there is an unmet need for a detection method that can detect microbes in clinical/non-clinical samples, without lab and or electric instruments. The features of the method should be such that it provides a visual and colorimetric detection.
Objects of the Invention
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.
Summary of the Invention
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.
In an embodiment present invention provides a process for making polymeric films of the polymersomes comprising the steps of:
a. obtaining the monomer of PDA by dissolving Polymer of PDA of a
predetermined quantity in 10 ml of solvent and then extruding it by PTFE
filter of the range 0.45-0.80um.
b. Removal of solvent by rotary evaporator of the above solution at vacuum
for 15-20 minutes
c. Solution of PHBV/PHB in dichlorobenzene by stirring 2-20 wt% for 2-4
hours.
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.
e. 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.
f. 10 ml of DI water/PBS buffer was added to the thin film to hydrate and
sonicated in bath sonicator at temperatures from 65◦-80◦C for 10-20
minutes.
g. The hydrated film in (f) was kept at +4◦ C for time ranging from 4-24
hours.
In yet another embodiment the present films provide a method of detecting bacteria E.coli
In another embodiment the stimulus is selected from Bacteria, heat, organic solvents
In yet another embodiment the detection is based on color change

In yet another embodiment the color change is permanent and irreversible
In another embodiment the invention provides specific bacterial detection without use of specific receptors
Detailed description of the invention:
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
(PCDA), 10,12 Tricosadiynoic acid (TCDA) and lipid including but not limited to
DMPC, DOPE, cholesterol.
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
a. obtaining the monomer of PDA here PCDA and TCDA by dissolving a
said quantity in 10 ml of chloroform and then extruding it by PTFE filter
of the range 0.45-0.80um
b. Removal of chloroform by rotary evaporator of the above solution at
vacuum for 15-20 minutes

c. Solution of PHBV/PHB in dichlorobenzene by stirring 2-20 wt% for 2-4
hours.
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.
e. 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.
f. 10 ml of DI water/PBS buffer was added to the thin film to hydrate and
sonicated in bath sonicator at temperatures from 65◦-80◦ C for 10-20
minutes.
g. The hydrated films in (f) was kept at +4 C for time ranging from 4-24
hours.
The invention also provides a method of detecting bacteria in contaminated water comprising the steps of:
a. Providing polymeric films of PHB/PHBV, 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,
b. Observing the color change in film after adding Urine containing bacteria
indicating contamination, where the bacteria can range from E.coli,
S.aureus, S.typhimurium, Klebsiella, enterococci,P.aeroginosa

Examples:
Following examples are given by way of illustration and should not be construed to limit the scope of the invention.
Example 1: Preparation of liposomes of Polyhydroxybutyrate(PHB), 10,12 Pentacosadiynoic acid(PCDA) and DMPC.
A. Making monomeric form of PDA-10,12 Pentacosadiynoic acid:
1 gm of PCDA obtained from Sigma Aldrich was dissolved in 10ml of analytical grade chloroform obtained from Rankem and was sonicated in bath sonicator for 15-20 minutes at RT. The solution was then extruded through a 0.45um PTFE syringe filter to obtain a clear solution. This solution was added to round bottom flask and rotary evaporated to obtain 80% yield. The monomer thus obtained was stored in dark to prevent photo crosslinking.
B. Making thin film of PHB, PCDA, 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.
C. Hydration of the film:
10 ml of DI water or PBS buffer was added to the RB flask containing thin film in step B. This was sonicated at temperatures in the range of 65-80C for 10-20 minutes. The hydrated film is kept at +4◦ C for 4-24 hours to allow the formation of liposomes.

Example 2: Preparation of liposomes of Polyhydroxy butyrate-co valerate(PHBV), 10,12 Pentacosadiynoic acid(PCDA) and DMPC.
A. Making monomeric form of PDA-10,12 Pentacosadiynoic acid
1 gm. of PCDA obtained from Sigma Aldrich was dissolved in 10ml of analytical grade chloroform obtained from Rankem and was sonicated in bath sonicator for 15-20 minutes at RT. The solution was then extruded through a 0.45um PTFE syringe filter to obtain a clear solution. This solution was added to round bottom flask and rotary evaporated to obtain 80% yield. The monomer thus obtained was stored in dark to prevent photo crosslinking.
B. Making thin film of PHBV, PCDA, 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.
C. Hydration of the film
10 ml of DI water or PBS buffer was added to the RB flask containing thin film in step B. This was sonicated at temperatures in the range of 65-80C for 10-20 minutes. The hydrated film is kept at +4◦ C for 4-24 hours to allow the formation of liposomes
Example 3: Preparation of liposomes of Polyhydroxybutyrate(PHBV), 10,12 Pentacosadiynoic acid(TCDA) and DMPC.
A. Making monomeric form of PDA-10,12 Tricosadiynoic acid
1 gm. of TCDA obtained from Sigma Aldrich was dissolved in 10ml of analytical grade chloroform obtained from Rankem and was sonicated in bath sonicator for 15-20 minutes at RT. The solution was then extruded

through a 0.45um PTFE syringe filter to obtain a clear solution. This solution was added to round bottom flask and rotary evaporated to obtain 80% yield. The monomer thus obtained was stored in dark to prevent photo crosslinking.
B. Making thin film of PHBV, TCDA, 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.
C. Hydration of the film
10 ml of DI water or PBS buffer was added to the RB flask containing thin film in step B. This was sonicated at temperatures in the range of 65-80C for 10-20 minutes. The hydrated film is kept at +4 C for 4-24 hours to allow the formation of liposomes
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
A. Crosslinking of polymer film
The polymeric films were cut in sizes of 1cmX1cm and crosslinked by exposure to UV radiation of 254nm. The color of white films turned blue.
B. Exposure to bacteria

The films were dipped in Nutrient broth in which was added 20-50ul of water and urine spiked with E.coli. The bacterial count was in the order of 102-108cfu/ml. Color changed from Blue to Red and bright red from 6hrs-24hrs time, indicating E.coli. Other bacteria such as S.aureus and S.typhimurium did not show color change within 24 hours indicating specificity of the system towards E.coli without any antibody or ligand.
Brief Description of drawings:
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.
Detailed description of the drawings:
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 PHBV/PCDA/DMPC 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/DMPC 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.
Abbreviations used:
PHB: PolyHydroxyButyrate
PHBV: PolyHydroxy Butyrate-co-Valerate
PCDA: 10,12 Pentacosadiynoic acid.
TCDA: 10,12 Tricosadiynoic acid
DMPC:1,2-dimyristoyl-sn-glycero-3-phosphocholine
DOPE :1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
Advantages:
• Point of Care detection of microbes
• No instrumentation
• Visual colorimetric readouts

• Does not require trained manpower
• Clinical and non-clinical samples can be tested
• Specificity without use of ligands such as antibodies.

I/We Claim,
1. A method for microbial detection in sample comprising the steps of:
a) providing polymeric films essentially consisting of PHB/PHBV, PDA
b) adding the sample containing bacteria indicating contamination
c) observing the visual color change
Wherein, method of preparation of polymeric films further comprises the steps of
a) obtaining the PDA monomer by dissolving 2gm in 10 ml of solvent and then extruding it by PTFE filter of the range 0.45-0.80um.
b) removal of solvent by rotary evaporator of the above solution at vacuum for 15-20 minutes to obtain the powder of PDA monomers
c) Solution of PHBV/PHB in dichlorobenzene by stirring 2-20 wt% for 2-4 hours.
d) Mixing PHBV/PHB solution obtained, with PDA monomer and lipid DMPC/DOPE in 6:4, 7:3 and 8:2 molar ratios and total volume made up to 10ml.
e) Obtaining a thin polymeric 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 50- 60mbar for 5 minutes.
f) 10 ml of Distilled Water was added to the polymeric thin film for hydration and sonicated in bath sonicator at temperatures from 65◦-80◦C for 10-20 minutes.
g) The hydrated film in (f) was kept at +4° C for time ranging from 4-24 hours to allow self assembly of the polymers and lipids to form polymersomes.
2. A method for microbial detection in sample as claimed in claim 1; wherein
the sample is infected with bacteria selected from the group of E.Coli,
S.Aureus, Klebsiella, Enterococcus, Pseudomonas and Proteus mirabilis.

3. Method of preparation of polymeric films as claimed in claim 1, the solvent is selected from Chloroform, Dichloromethane and Dichlorobenzene.
4. Method of preparation of polymeric films as claimed in claim 1; wherein the size of the polymersome is in the range of 300nm to 450 nm.
5. Method of preparation of polymeric films as claimed in claim 1; wherein the shape of the polymersomes is spherical bilayered.