DESC:
[0022] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
[0023] The present invention discloses a miniature handheld device driven by in situ generated oxyhydrogen which is used to obtain high transformation efficiencies in bacterial strains of Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycobacterium tuberculosis and Helicobacter pylori. The proposed device demonstrates potential for delivery of exogenous DNA through cell wall of simple as well as complex bacterial strains wherein the bacterial cell wall has a complex structure which prevents the bacteria from environmental challenges and helps maintain its internal homeostasis.
[0024] Shock waves have been extensively used in various interdisciplinary arenas of research and their applications in biomedical engineering have been gaining prominence lately. Bacterial transformation is a technique of prime importance in field of genetic engineering where it finds applications from construction of genomic libraries to production of recombinant insulin for treatment of diabetes. The method of introducing a foreign DNA in a host cell using common techniques like electroporation and heat-shock method, is associated with a number of limitations like low transformation efficiency, tedious competent cell preparation and requirement of an extensively salt free medium.
[0025] Using shockwaves to perform bacterial transformation has attracted the interest of researchers around the globe because shockwaves not only help to improve the transformation efficiencies as compared to conventional methods but also demonstrates the potential to transform some complex bacterial species. Mycobacteria have a thick waxy cell wall due to the presence of mycolic acid which makes it one of the most difficult micro-organisms to transform. In this study, we report a new shockwave-assisted technique for high efficiency transformation of a range of bacterial species which include E.coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Helicobacter pylori as well as Mycobacteria.
[0026] The shockwave-assisted technique for high efficiency transformation of a range of bacterial species also known by the name “SuperPorator” comprises of two main components. The first component is an oxyhydrogen generator station. This is a table top system that generates the oxyhydrogen miniature in situ. The second component is a miniature handheld device which is used to administer the drug to the patient. Through alkaline electrolysis, the oxyhydrogen generator produces the required pressure of oxyhydrogen mixture during each operation of the device. The schematic of the oxyhydrogen generator station is shown in the figure 1. The oxyhydrogen mixture is tapped from the outlet as shown in the figure 1.
[0027] The fill pressure of oxyhydrogen mixture required to transform the different bacteria is different. This is due to the different thickness of the cell wall in the bacteria. The oxyhydrogen fill pressure for the different bacteria are listed below:
• E.coli–Oxyhydrogen fill pressure required is 3 bar
• Pseudomonas aeruginosa–Oxyhydrogen fill pressure required is 3.5 bar
• Salmonella Typhimurium–Oxyhydrogen fill pressure required is 4 bar
• Helicobacter pylori –Oxyhydrogen fill pressure required is 4 bar
• Mycobacteria–Oxyhydrogen fill pressure required is 10 bar

[0028] The miniature handheld device is a shock tube with an internal diameter 6 mm and comprises of two sections – a driver section of length at least 100 mm and driven section of length of at least 170 mm. Tracing paper of at least 95 GSM is used as diaphragm to separate the driver section and driven section of the shock tube. The driver section of the shock tube is filled to the required pressure of oxyhydrogen mixture and the mixture is detonated. This ruptures the diaphragm and a strong shockwave is created in the driven section of the shock tube.
[0029] The bacterial sample is accommodated in a stainless steel sterile cavity of diameter 6 mm and depth 5 mm. The direct impact of the shockwave followed by the products of detonation on the bacterial sample leads to issues of contamination. To avoid this, a suitable barrier is chosen for good energy transfer and to prevent the detonation products from impacting the bacteria. A silicone rubber membrane has been used between the shock tube and the bacteria.
[0030] FIG.1 illustrates the schematic view of the oxyhydrogen generator according to an embodiment of the present invention. According to the embodiment, the oxyhydrogen generator comprises of the electrolysis unit 101, the buffer chamber 102 and the recirculation pipes 103. The electrolysis unit comprises of at least four neutral plates which are stacked between two sets of electrode plate. This set of 6 plates constitutes one electrolysis cell. Similarly, 6 cells are stacked and connected in series as shown in the figure 1. Electrode plates and neutral plates are made out of stainless steel plates. An equal spacing of ~2 mmis maintained between the electrodes and the neutral plates by using electrical insulators. About 7M potassium hydroxide solution (400 g of KOH in 1 l of distilled water) is used as electrolyte. The chemical reactions at the anode and the cathode of the electrolysis unit during electrolysis enable to generate the hydrogen and the oxygen gases at the cathode and anode points. Since both these electrodes are confined to a single chamber, the hydrogen and oxygen gases get collected above the electrolyte solution in the electrolysis unit.
[0031] FIG.2 illustrates the schematic view of the miniature handheld device according to an embodiment of the present invention. According to the embodiment, the miniature handheld device is a shock tube with an internal diameter of at least 6 mm and comprises of two sections – a driver section 201 of length at least 100 mm and driven section 202 of length of at least 170 mm. Tracing paper of at least 95 GSM is used as diaphragm in the diaphragm station 205 to separate the driver section 201 and driven section 202 of the shock tube. The driver section 201 of the shock tube is filled to the required pressure of oxyhydrogen mixture and the mixture is detonated using a battery 203 and detonation initiator 204. This ruptures the diaphragm and a strong shockwave is created in the driven section 202 of the shock tube.
[0032] The bacterial sample is accommodated in a stainless steel sterile cavity 207 of diameter of at least6 mm and depth of at least 5mm. The direct impact of the shockwave followed by the products of detonation on the bacterial sample leads to issues of contamination. To avoid this, a suitable barrier is chosen for good energy transfer and to prevent the detonation products from impacting the bacteria. A silicone rubber membrane 206 has been used between the shock tube and the bacteria.
[0033] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. ,CLAIMS:WE CLAIM:
1. A shockwave-assisted device that is capable of generating shockwaves for high efficiency in-vitro transformation of bacterial cells using a miniature detonation-driven shock tube device, the device comprising:
a in situ oxyhydrogen generator apparatus,
a driver section,
a driven section,
a diaphragm station;
a battery unit;
a detonation initiator;
a silicon rubber membrane attached over the driven section; and
a cavity in the silicon rubber membrane to house the bacteria sample.
wherein, the generated oxyhydrogen mixture from the in situ oxyhydrogen apparatus is tapped from the outlet situated on the oxyhydrogen generator and fed into the miniature handheld shockwave-assisted device in which the driver section of the shock tube is filled with at least 2.5 bar of oxyhydrogen mixture and the mixture is detonated using a battery and a detonation initiator which then ruptures the diaphragm and a strong shockwave is created in the driven section of the shock tube, the shock waves energy which then is transferred to the silicone rubber membrane provided over the handheld shockwave-assisted device and the resultant shockwave is further provided to the cavity in the silicon rubber membrane which holds the bacteria sample that is to be administered to the target.

2. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the miniature handheld device is driven by the in-situ generated oxyhydrogen which is used to obtain high transformation efficiencies in bacterial strains of Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycobacterium tuberculosis and Helicobacter pylori.
3. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the said miniature handheld shockwave device driven by in situ generated oxyhydrogen delivers exogenous DNA through cell wall of simple as well as complex bacterial strains wherein the bacterial cell wall has a complex structure which prevents the bacteria from environmental challenges and helps maintain its internal homeostasis.
4. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the wherein the shockwave-assisted device can generate high velocity jets through the detonation of in situ generated oxyhydrogen mixture (stoichiometric mixture of hydrogen and oxygen gases in the ratio 2:1).
5. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the miniature handheld device is a shock tube with an internal diameter 6 mm and comprises of two sections – a driver section of length at least 100 mm and driven section of length of at least 170 mm.
6. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein a tracing paper of at least 95 GSM is used as diaphragm to separate the driver section and driven section of the shock tube.
7. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the driver section of the shock tube is filled to the required pressure of oxyhydrogen mixture and the mixture is detonated and this action ruptures the diaphragm and a strong shockwave is created in the driven section of the shock tube.
8. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the bacterial sample is accommodated in a stainless steel sterile cavity of diameter 6 mm and depth 5 mm.
9. The shockwave-assisted in-vitro transformation of bacterial device according to claim 1, wherein the silicone rubber membrane has been used between the shock tube and the bacteria which acts as a suitable barrier for good energy transfer and to prevent the detonation of products from impacting the bacteria.