FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13) 1. Title of the invention: SPIN COLUMNS AND METHOD OF FABRICATION THEREOF
2. Applicant(s)

NAME NATIONALITY ADDRESS
WOBBLE BASE BIORESEARCH PRIVATE LIMITED Indian Wobble Base Bioresearch (P) Ltd., President Plaza, Wing-A, Floor 3, SN GeneLab Premises, Nanpura, Surat, Gujarat 395 001, India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.

TECHNICAL FIELD
[0001] The present subject matter relates in general to spin columns, and in particular, to spin columns and method of fabrication thereof.
BACKGROUND
[0002] Analysis of cellular components, such as quantifying proteins, nucleic acid, and the like, is a pivotal step in molecular biology and has been used, for example, in diagnostic kit. Typically, analysis of cellular components can be divided into four steps, namely, cell disruption, filtration, removal of unwanted biomolecules, and analyte concentration. Analyte concentration refers to extraction of the analyte to form a concentrated solution. Spin column-based analyte concentration is a solid phase extraction method to quickly purify nucleic acids when the sample is not available in bulk, for example, in case of extraction of ribonucleic acid (RNA) from mammalian blood.
[0003] Generally, spin column-based purification involves usage of two columns - a first column also called as shredder column for homogenization of cell suspension and to filter cellular debris from interfering with downstream nucleic acid purification process; and a second column also called purification column. The second column can hold membranes coated with chemicals that can selectively bind to the analyte at a first salt concentration and pH and can release it subsequently at a second salt concentration and pH.
BRIEF DESCRIPTION OF DRAWINGS
[0004] The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to reference like features and components.

[0005] Fig. 1 depicts an example hybrid spin column of the present subject matter, in
accordance with an implementation of the present subject matter.
[0006] Fig. 2 depicts a method of fabrication of a hybrid spin column, in accordance
with an implementation of the present subject matter.
[0007] Fig. 3 depicts an ancillary apparatus used in fabricating the hybrid spin column,
in accordance with an implementation of the present subject matter.
[0008] Fig. 4(a) depicts an example apparatus to perform a punching action, in
accordance with an implementation of the present subject matter.
[0009] Fig. 4(b) depicts a top perspective view of the example apparatus to perform
the punching actions, in accordance with an implementation of the present subject
matter.
[00010] Fig. 5 depicts an example apparatus to perform an affixing action, in
accordance with an implementation of the present subject matter.
DETAILED DESCRIPTION
[00011] The present subject matter provides hybrid spin columns and methods of fabrication of spin chambers and components ancillary to fabrication of the spin columns
[00012] Spin column-based purification is a technique for extraction and concentration of nucleic acid which typically involves sequential steps of lysis, binding, washing and elution. To lyse, cells of a sample are broken open with a lysis procedure which disrupts the cell membrane and the nucleus to release the analyte, for example, nucleic acid. The lysis procedure may include physical means, such as cavitation, sonication, grinding, and the like; and chemical means, such as, by using lytic enzymes. [00013] For binding, a buffer solution is then added to the sample comprising disrupted cells along with ethanol or isopropanol. This forms the binding solution. The binding solution is transferred to a spin column and the spin column is inserted in a centrifuge. The centrifuge forces the binding solution through a silica gel membrane that is inside

the spin column. If the pH and salt concentration of the binding solution are optimal, the nucleic acid will bind to the silica gel membrane as the solution passes through. [00014] To wash, a wash buffer is added to the spin column. The spin column is put in a centrifuge again, forcing the wash buffer through the silica gel membrane. This removes any remaining impurities from the silica gel membrane, leaving only the nucleic acid bound to the silica gel.
[00015] To elute, the wash buffer is removed and an elution buffer (or simply water) is added to the spin column. The spin column is put in the centrifuge again, forcing the elution buffer through the silica gel membrane. The elution buffer removes the nucleic acid from the membrane and the nucleic acid is collected from the bottom of the spin column.
[00016] Generally, spin column-based purification involves usage of two columns. A first column can be used in the lysing stage. The first column, also called as shredder column, can help in homogenization of cell suspension and can filter cellular debris to prevent interference with downstream nucleic acid purification process. Filtered sample from the first column can be transferred to a second column for the downstream nucleic acid purification process.
[00017] The second column, also called purification column, can hold the chemically coated membranes for selectively bind to the analytes at a first salt concentration and pH and can release it subsequently at a second salt concentration and pH at the time of elution.
[00018] This approach of using two different columns, namely, the first column and the second column, is time consuming and expensive. Further, the first column and the second column are fabricated from plastic and, therefore, add to volume of plastic disposable and thus are not environment friendly. Additionally, during transfer from the first column to the second column, nucleic acids may be retained in the first column due to non-specific binding. Further, there is also a chance of loss of sample and thereby the nucleic acids during transfer. This could be detrimental to sample analysis

in case of small sample sizes, for example, while analyzing nucleic acids obtained from mammalian blood.
[00019] The present subject matter addresses these and other problems and provides a hybrid spin column which provides an integrated functionality of filtering cell debris and purification in a single spin column. The hybrid spin column, also referred to hereinafter as the spin column, comprises a body portion having a first end and a second end provided opposite to the first end. The first end may be open to receive a suspended sample. The suspended sample can comprise a sample suspended in chemical reagents, for example, a buffer solution. The suspended sample can comprise an analyte, debris, and the chemical reagent.
[00020] A plurality of membrane layers may be disposed in the body portion substantially towards the second end of the body portion. The plurality of membrane layers comprises a filtration membrane layer to receive the suspended sample from the first end of the hybrid spin column. The filtration membrane layer can be selected from material such that debris from the suspended sample can be filtered and retained on the filtration membrane layer and only the analyte and chemical reagents can pass through to the binding membrane layer. The filtration membrane layer, thus, helps in filtration. [00021] A binding membrane layer can be provided below the filtration membrane layer. The binding membrane layer comprises the binding chemicals coated thereon for interacting and binding with the analyte. The binding membrane layer may interact with the analyte at a first salt concentration and pH. The binding membrane layer can also comprise other chemicals coated thereon, such as chaotropic agents, stabilizing agents, preservatives, and the like. Thus, the binding membrane layer provides the functionality of purification and concentration of the analyte.
[00022] The plurality of membrane layers may also comprise a support membrane layer provided below the binding membrane layer for providing mechanical support to the binding membrane layer and the filtration membrane layer. In one example, the support membrane layer can also comprise binding chemicals for binding to analyte,

for example, nucleic acids, to retain any analyte that may have passed through the binding membrane layer.
[00023] Therefore, by using the spin column of the present subject matter, dual functionality of filtering cell debris and purification and quantification of analyte can be achieved in a single spin column. This helps in reducing the number of plastic disposable by half and also reduces chances of wastage of sample that can otherwise occur during transfer of sample from one column to another.
[00024] In one example, to further improve analyte purification and concentration, a sealing member, for example, an O-ring, may be placed on top of the filtration membrane layer. The sealing member holds the plurality of membrane layers securely at the second end of the spin column. Further, the sealing member also seals the filtration membrane layer at peripheries of the spin column and prevents cellular debris from flowing towards the binding membrane layer via the peripheries when the spin column is being centrifuged for washing and elution of the analyte.
[00025] The present subject matter also provides a method for fabrication of the spin column. The method comprises obtaining punches from bulk materials of each layer of the plurality of membrane layers. As will be understood, the punches are pieces obtained from the bulk material by using a punching apparatus. The present subject matter also provides an apparatus to obtain the punches. The obtained punches may be placed towards the second end of the spin column. The sealing member may then be affixed on the filtration membrane layer of the plurality of membrane layers. The present subject matter also provides an apparatus for affixing the sealing member. [00026] The present subject matter, therefore, provides a hybrid spin column which integrates filtering of cell debris and purification and concentration of analyte from the sample. This reduces time consumed for analysis of the analyte. Further, number of plastic disposables used for a single analysis is reduced by half.
[00027] The above and other features, aspects, and advantages of the subject matter will be better explained with regard to the following description and accompanying

figures. It should be noted that the description and figures merely illustrate the principles of the present subject matter along with examples described herein and, should not be construed as a limitation to the present subject matter. It is thus understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and examples thereof, are intended to encompass equivalents thereof. Further, for the sake of simplicity, and without limitation, the same numbers are used throughout the drawings to reference like features and components.
[00028] Fig. 1 depicts an example hybrid spin column 100, in accordance with an implementation of the present subject matter. The hybrid spin column 100, hereinafter also referred to as spin column 100, comprises a body portion 102. The body portion 102 may be a cylindrical hollow structure. The body portion 102 can comprise a first end 104 and a second end 106 provided opposite to the first end 104. A lid 108 may be provided on the first end 104. In one example, the lid 108 may be coupled to the first end 104 by a bendable flange. In other examples, the lid 108 may be a separate component which may be press-fitted at the first end 104 of the spin column 100. The lid 108 may be fabricated from polypropylene.
[00029] The second end 106 can comprise a tapering portion 110 having a first cross-section towards the body portion 102 and a second cross-section away from the body portion 102. Diameter of the first cross-section is the same as the diameter of the body portion 102 while diameter of the second cross-section is smaller than the diameter of the first cross-section. The second end 106 can also comprise a barrel portion 112 which extends from the second cross-section of the tapering portion 110 and has a diameter substantially the same as that of the second cross-section. The barrel portion 112 may be open at the free end to allow flow of fluid to the outside.
[00030] In one example, the body portion 102 may be fabricated from plastic, for example, from polypropylene. However, other materials may be used as well. In one

example, the spin column 100 may be a commercially available recharged spin column with a flat bottom and net structure at the bottom to support the plurality of membrane layers. In one example, the spin column 100 is a paper-based spin column prepared using 0.5 ml tubes fitted with the lid 108.
[00031] The spin column 100 can hold a plurality of membrane layers which provide the integrated functionality of filtering cellular debris from homogenized sample and purification of analyte from filtered sample. The plurality of membrane layers may be formed in the body portion 102 above the tapering portion 110.
[00032] The plurality of membrane layers can comprise a filtration membrane layer 114a. The filtration membrane layer 114a can receive a suspended sample. The suspended sample can be understood as a sample suspended in chemical reagents, such as a buffer. The sample can comprise an analyte. The filtration membrane layer 114a can receive the suspended sample from the first end 104 of the hybrid spin column 100. The filtration membrane layer 114a may be porous and may be made of material selected such that debris, for example, from lysed cells, in the sample can be filtered and retained on the filtration membrane layer 114a and only the analyte and the chemical reagents in the suspended sample can pass through to the binding membrane layer 114b. Porosity of the filtration membrane layer 114a may, thus, be based on the debris to be filtered.
[00033] The filtration membrane layer 114a may also have multiple sheets. In one example, each sheet of the filtration membrane layer 114a can be fabricated from Whatman grade 5. In other examples, other material may be used depending on the size of the debris to be filtered. Further, the filtration membrane layer 114a may be fabricated from multiple sheets of different material having different properties, such as, porosity. As will be understood, each membrane layer may be placed coaxially in the spin column 100.
[00034] The plurality of membrane layers can also comprise a binding membrane layer 114b. The binding membrane layer 114b can be provided below the filtration

membrane layer 114a. The binding membrane layer 114b can comprise the binding chemicals coated thereon for interacting and binding with the analyte at a first salt concentration and pH. The binding membrane layer 114b can also comprise other chemicals coated thereon, such as chaotropic agents, stabilizing agents, preservatives, and the like. The binding membrane layer 114b can be fabricated from multiple sheets. In one example, the binding membrane layer 114b is fabricated from microfibre coated cellulose sheets. In other examples, other material can be used. Further, each sheet forming the binding membrane layer 114b can be fabricated from materials having different properties, such as porosity. The binding membrane layer 114b, therefore, provides functionality of purification and concentration of analyte.
[00035] In one example, when the analyte to be purified and concentrated is nucleic acid, the binding membrane layer 114b may be coated with silica gel. The binding membrane layer 114b with silica gel may be prepared by suspending silica gel of pore size 60-A, 5 – 25 µm particle size in 7.5 molar guanidium thiocyanate or guanidium isothiocyanate solution. The binding membrane layer 114b may be soaked in the suspension and dried to obtain the coated binding membrane layer 114b.
[00036] The plurality of membrane layers can also comprise a support membrane layer 114c. The support membrane layer 114c is for providing mechanical support to the layers provided above the support membrane layer 114c, i.e., the filtration membrane layer 114a and the binding membrane layer 114b. In one example, the support membrane layer 114c can also comprise binding chemicals for binding to any analyte residual analyte which does not bind to the binding membrane layer 114b. For example, the support membrane layer 114c can comprise silica gel coated thereon for specific interaction of nucleic acids and thereby for further purification and concentration of nucleic acid.
[00037] In one example, the support membrane layer 114c is fabricated from set of 2-4 glass microfibre coated cellulose sheet of circular size and diameter 7.00 mm. Depending on experimental requirements, the number of sheets to form the support

membrane layer 114c and diameter and size of each sheet in the support membrane layer 114c and the material used may vary. Further, each sheet forming the support membrane layer 114c can be fabricated from material having different properties, such as porosity.
[00038] A sealing member 116, for example, an O-ring, may be placed on top of the filtration membrane layer 114a. The sealing member 116 holds the plurality of membrane layers securely in the body portion 102. Further, the sealing member 116 also seals the filtration membrane layer 114a at peripheries of the spin column 100 and prevents cellular debris from flowing towards the binding membrane layer 114b via peripheries of the spin column 100 when the spin column 100 is centrifuged for washing and elution of the analyte.
[00039] In operation, a sample comprising cells is subjected to cell disruption outside the spin column 100. The cell disruption may be performed by physical or chemical means. The physical means may include grinding, sonication, cavitation, and the like. The chemical means may include subjecting to the sample to lytic enzymes, chaotropic agents, detergents, and the like. The sample comprising the disrupted cells may be suspended in a buffer solution to maintain a particular salt concentration and pH. The suspended sample is then introduced into the spin column 100. The spin column 100 is placed in a first centrifuge tube and subjected to centrifugation at 10000 rpm for 1-2 minutes.
[00040] During centrifugation, the filtration membrane layer 114a filters cellular debris, such as, disrupted cell wall components. The filtration membrane layer 114a allows analyte and chemical reagents to pass through to the binding membrane layer 114b. Further, the filtration membrane layer 114a may also allow unspecified proteins and unfiltered cellular components to pass through depending on their size. [00041] The analyte can bind to the binding membrane layer 114b at the salt concentration and pH of the suspended sample. However, similar to the filtration membrane layer 114a, the binding membrane layer 114b may allow unspecified

proteins and unfiltered cellular components to pass through. Since the barrel portion 112 is open, the filtrate comprising the unspecified proteins, unfiltered cellular components, and chemical reagents, can pass from the spin column 100 into the first centrifuge tube. The spin column 100 may then be subjected to repeated centrifugation in the presence of a wash solution to release any non-specifically bound components of the sample, such as lipids, proteins, and the like. The wash solution may have the same salt concentration and pH as the suspended sample to ensure that the analyte remains bound to the binding membrane layer 114b.
[00042] The hybrid spin column 100 may then be separated from first centrifuge tube. The hybrid spin column 100 may comprise the analyte bound to the binding membrane layer 114b and the debris on the filtration membrane layer 114a. The hybrid spin column 100 may then be placed in a second centrifuge tube. An elution solution may be introduced into the spin column 100 to alter the salt concentration and pH for release of the analyte from the binding membrane layer 114b. The second centrifuge tube may be subjected to centrifugation. The analyte released by the binding membrane layer 114b can pass through the hybrid spin column 100 to the second centrifuge tube. Thus, the second centrifuge tube can be used for collecting the analyte along with the elution solution. Therefore, the present subject matter provides for filtration and purification of analyte in a single spin column 100.
[00043] Fig. 2 depicts a method 200 of fabrication of a hybrid spin column, in accordance with an implementation of the present subject matter. The hybrid spin column fabricated may be the hybrid spin column 100 as shown in Fig. 1. At block 202, the method 200 comprises obtaining punches from bulk materials of each layer of the plurality of membrane layers. As will be understood, the punches are residual pieces obtained from the bulk material by using an apparatus that can be used for punching. The plurality of membrane layers may be the filtration membrane layer 114a and the binding membrane layer 114b.

[00044] At block 204, the obtained punches are then placed in the spin column. In one example, the obtained punches may be placed towards the second end 106 of the spin column 100. In one example, the punches may be placed in the spin column 100 using tweezers. The filtration membrane layer 114a may be placed on the binding membrane layer 114b coaxially.
[00045] At block 206, the sealing member 116 can be affixed on the plurality of membrane layers. The sealing member 116 can hold the plurality of membrane layers securely at the second end 106 of the spin column 100 to obtain the hybrid spin column 100.
[00046] In one example, wherein the hybrid spin column 100 comprises the support membrane layer 114c, the obtained punches of the support membrane layer 114c may be placed below the binding membrane layer 114b at block 204. Thus, in this example, the support membrane layer 114c may be placed in the body portion 102 followed by the binding membrane layer 114b and the filtration membrane layer 114a prior to affixing the sealing member 116. The sealing member 116 may then be affixed on the filtration membrane layer 114a at block 206.
[00047] The present subject matter also provides ancillary apparatuses to obtain the punches of bulk materials and affix the sealing member over the plurality of membrane layers. An example ancillary apparatus is explained with reference to Fig. 3. [00048] Fig. 3 depicts an ancillary apparatus 300 used in fabricating the hybrid spin column, in accordance with an implementation of the present subject matter. The apparatus 300 may be used for obtaining the punches of bulk materials or affixing the sealing member over the plurality of membrane layers in the hybrid spin column 100. [00049] The apparatus 300 can comprise a stand 302 and a rod assembly 304. The stand 302 can comprise a first platform 306 having a holder 308. The holder 308 may be an aperture drilled into the first platform 306. In one example, the holder 308 may be used to hold a conduit, as explained with reference to Figs. 4(a) and 4(b). In another

example, the holder 308 may be used to hold a spin column, as explained with reference to Fig. 5. The spin column may be the spin column 100.
[00050] The stand 302 can also comprise a second platform 310 having a rod holder 312. The rod holder 312 can be used to hold a rod 314. In one example, the rod is a punching rod, as explained later with reference to Figs. 4(a) and 4(b). In another example, the rod is a stamping rod, as explained with reference to Fig. 5. The second platform 310 can be placed above and substantially parallel to the first platform 306, such that the holder 308 and the rod holder 312 are substantially coaxial. A support element 316 may support the second platform 310 above the first platform 306 and can spatially separate the first platform 306 from the second platform 310. The support element 316 provides mechanical strength to the apparatus 300.
[00051] The rod assembly 304 can be arranged on the second platform 310. The rod assembly 304 can comprise the rod 314. The rod 314 can be receivable in the rod holder 312 of the second platform 310 of the stand 302. An actuation arrangement 318 can be coupled to the rod 314. The actuation arrangement 318 can be used to cause movement of the rod 314 relative to the holder 308 to perform one of: a punching action and an affixing action.
[00052] Fig. 4(a) depicts an example apparatus 400 to perform a punching action, in accordance with an implementation of the present subject matter. The apparatus 400 is also hereinafter referred to as punching device 400. The punching device 400 can comprise the stand 302. In one implementation, the stand 302 is fabricated from steel. It is to be understood that the stand 302 may be fabricated from any other metal as well. The stand 302, as illustrated in Fig. 4(a), includes the first platform 306 and the second platform 310 substantially parallel to each other.
[00053] The stand 302 also comprises the support element 316 which couples the first platform 306 to the second platform 310. In one implementation, the support element 316 is substantially perpendicular to the first platform 306 and the second platform 310 and separates the first platform 306 and the second platform 310 by a distance. The

separation provides clearance between the first platform 306 and second platform 310 and may be used to place a conduit 402 into the holder 308 of the first platform 306 with ease. In one implementation, the support element 316 is welded to an upper side of the first platform 306 and an underside of the second platform 310. In other implementations, the support element 316 may be fastened to the first platform 306 and the second platform 310 by any method known in the art, for example by using fasteners.
[00054] The stand 302, as illustrated in Fig. 4(a), can also comprises legs 404. The legs 404 can support the first platform 306 and provide clearance space below the first platform 306 to accommodate the conduit 402 and the storage bag 406. In one implementation, the legs 404 may be welded to the underside of the first platform 306. In another implementation, the legs 404 may be fastened to the first platform 306 by any method known in the art.
[00055] The first platform 306 includes the holder 308 to hold the conduit 402. The conduit 402 may be a hollow cylindrical plastic tube. The holder 308 can hold the conduit 402 for passage of punches from the bulk membranes into a storage bag 406. The storage bag 406 may be coupled to a first end of the conduit 402 to receive punches while a second end of the conduit 402 may be coupled at the holder 308. The second platform 310 includes the rod holder 312 to hold the rod structure. The rod structure may be a punching rod 408.
[00056] In one example, the holder 308 and rod holder 312 may be apertures drilled into the first platform 306 and second platform 310, respectively. The diameter and cross-section of the holder 308 and rod holder 312 depend on the diameter and cross-section of the conduit 402 and the punching rod 408. The holder 308 and rod holder 312 are substantially co-axial. The diameter of the rod holder 312 is fabricated such that it provides tolerance for free movement of the punching rod 408 through it. The diameter of the holder 308 is fabricated such that the conduit form-fits in the holder

308 without undergoing deformation or passing through the holder 308 on application of pressure for punching.
[00057] For punching, the punching rod 408 may be placed in the rod holder 312. The punching rod 408 can comprise a first portion 410 and a second portion 412. The first portion 410 of the punching rod 408 may be below the second platform 310 and the second portion 412 may be above the second platform 310. A lower circumference of the punching rod 408 can be made sharp such that it pierces membrane layers to create punches from bulk membranes to obtain punches of the bulk membranes. The second portion 412 of the punching rod 408 can comprise a spring member 414 coupled to it. The spring member 414 may be coaxial with the second portion 412 of the punching rod 408.
[00058] The actuation arrangement 318 of the punching device 400 can comprise a lever 416 which can be coupled to the second portion 412 of the punching rod 408 and a vertical part 418 to support the lever 416. The lever 416 can comprise a free end 420, a pivoted end 422, and a mounting point 424. The free end 420 may be used for actuation of the punching device or apparatus 400 to cause movement of the punching rod 408 relative to the holder 308 to perform the punching action. The mounting point 424 can be a point between the free end 420 and the pivoted end 422 where the second portion 412 of the punching rod 408 is coupled to the lever 416. For example, as shown in Fig. 4(a), the second portion 412 may have a slit through which the lever 416 can pass and be coupled to the punching rod 408. The lever 416 may be movable in the slit. The lever 416 can be pivotable about the pivoted end 422.
[00059] The vertical part 418 can be coupled to the lever 416 at the pivoted end 422. The vertical part 418 may be disposed on the second platform 310. The vertical part 418 can be spaced apart from the second portion 412 of the punching rod 408. As the operation of punching device 400 is spring-loaded, to restrict movement of the punching rod 408, a stopper 426 may be provided. The stopper 426 may be provided on the first portion 410 of the punching rod 408.

[00060] Fig. 4(b) depicts a top perspective view of the punching device 400, in accordance with an implementation of the present subject matter. Fig. 4(b) depicts bulk membrane provided below the punching rod 408 of the punching device 400. [00061] In operation, with reference to both Fig. 4(a) and 4(b), the conduit 402 can be placed in the holder 308 and the storage bag 406 can be coupled to the conduit 402 under the first platform 306. The punching rod 408, coupled to the actuation arrangement 318, can be placed in the rod holder 312. Bulk membrane layer, for example, the circular membrane layer may be placed on the first platform 306 below the punching rod 408. For punching, the lever 416 may be pushed downwards towards the second platform 310. The sharp edges of the punching rod 408 cuts through the membrane layer and the punches pass through the conduit 402 into the storage bag 406. When the lever 416 is released, the spring member 414 causes the punching rod 408 to retract in the rod holder 312. The stopper 426 restricts the punching rod 408. [00062] While Figs. 4(a) and 4(b) depict the example apparatus 400 which can be used for obtaining punches from bulk membranes, other examples are possible. Fig. 5 depicts yet another example apparatus 500, in accordance with an implementation of the present subject matter. The apparatus 500 may be used for affixing the sealing member 116 (refer Fig. 1) on the plurality of membrane layers.
[00063] Fig. 5 depicts sealing of the sealing member 116, in accordance with an implementation of the present subject matter. In said example, the sealing member 116 can be placed over the plurality of membrane layers in the spin column 100. The spin column 100 can then placed in the holder 308.
[00064] Similar to the apparatus 400, the apparatus 500 can comprise the stand 302 including the first platform 306 and the second platform 310 substantially parallel to each other. The support element 316 is substantially perpendicular to the first platform 306 and the second platform 310 and separates the first platform 306 and the second platform 310 by a distance.

[00065] The first platform 306 can comprise the holder 308. The holder 308 may be used to hold the spin column 100 comprising the plurality of membrane layers placed therein. The stand 302 can also comprises legs 404. The legs 404 can support the first platform 306 and provide clearance space below the first platform 306 to accommodate the spin column 100.
[00066] The second platform 310 can comprise the rod holder 312. For affixing the sealing member 116 on the plurality of membrane layers, the rod may be a stamping rod 502. The stamping rod 502 can be coupled in the rod holder 312. The stamping rod 502 can comprise a first portion 504 and a second portion 506. The first portion 504 can be below the second platform 310. A lower circumference of the first portion 504 can be blunt to enable affixing of the sealing member 116 on the plurality of membrane layers in the spin column 100.
[00067] The second portion 506 of the stamping rod 502 can be above the second platform 310. Threads may be provided on the second portion 506. The threads of the second portion 506 can mate with threads of threads provided on an inner surface of the rod holder 312.
[00068] To cause movement of the stamping rod 502 relative to the holder 308 to perform the affixing action, the actuation arrangement can comprise a knob 508. The knob 508 can be coupled to the second portion 506 of the stamping rod 502. The knob 508 can be coupled to the stamping rod 502 away from the second platform 310. Rotation of the knob 508 can cause the stamping rod 502 to move towards and away from the first platform 306.
[00069] Unlike the punching rod 408, lower circumference of the stamping rod 502 formed in first portion 504 below the second platform 310 is blunt. Thus, in operation, on rotation of the knob, the stamping rod 502 can moves downwards into the spin column 100 and seal the sealing member 116 on the plurality of membrane layers forming the hybrid spin column 100. On counter rotation, the stamping rod 502 can be retracted and the hybrid spin column 100 can be detached or removed from the holder

308. To restrict movement of the stamping rod 502, a top stopper 510a may be provided in the second portion 506 of the stamping rod 502 and a bottom stopper 510b may be provided in the first portion 504 of the stamping rod 502.
[00070] The present subject matter, therefore, provides a hybrid spin column 100 which integrates filtering of cell debris and purification and concentration of analyte from the sample is provided by the present subject matter. The hybrid spin column 100 reduces time consumed for analysis of the analyte. Further, number of plastic disposables used for a single analysis is reduced by half. The present subject matter also provides a method for preparing the spin column 100 and apparatuses that is ancillary to the preparation. Further, the hybrid spin column 100 provides enhanced quality and quantity of extracted analyte as an additional step of transfer from shredder column to purification column is mitigated.
[00071] While the present subject matter has been explained with reference to nucleic acid purification, the hybrid spin column 100 can be modified for various other purposes as will be understood. For example, the binding membrane layer 114b may be coated with unrelated nucleic acid which can be used as a template for internal control during replication process. The hybrid spin column 100 can be suitably modified for purification of amplicons of varying sizes generated by replication processes for downstream processes, such as nucleotide sequencing and gene cloning. The hybrid spin column can also be suitably modified for protein purification as well. [00072] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible. As such, the scope of the present subject matter should not be limited to the description of the preferred examples and implementations contained therein.

I/We Claim:
1. A hybrid spin column (100) comprising:
a body portion (102) comprising:
a first end (104) to receive a suspended sample, wherein the suspended sample comprises an analyte and debris suspended in a chemical reagent;
a second end (106) provided opposite to the first end (104); and a plurality of membrane layers disposed in the body portion (102) substantially towards the second end (106), wherein the plurality of membrane layers comprises:
a filtration membrane layer (114a) to receive suspended sample from the first end (104) of the hybrid spin column (100), to retain debris from the suspended sample, and to allow the analyte and the chemical reagent to pass through; and
a binding membrane layer (114b) provided below the filtration membrane layer (114a) which comprises binding chemicals coated thereon for interacting and binding with the analyte.
2. The hybrid spin column (100) as claimed in claim 1, wherein the plurality of membrane layers comprises a support membrane layer (114c) provided below the binding membrane layer (114b) to support the plurality of membrane layers.
3. The hybrid spin column (100) as claimed in claim 2, wherein the support membrane layer (114c) comprises binding chemicals coated thereon for interacting and binding with the analyte.

4. The hybrid spin column (100) as claimed in claim 1, wherein a sealing member (116) is placed on the filtration membrane layer (114a) to hold the plurality of membrane layers securely at the second end (106) of the hybrid spin column (100).
5. The hybrid spin column (100) as claimed in claim 1, wherein the filtration membrane layer (114a) is porous, wherein a porosity of the filtration membrane layer (114a) is based on the debris to be filtered.
6. The hybrid spin column (100) as claimed in claim 1, wherein the binding membrane layer (114b) is a microfibre coated cellulose sheet comprising binding chemicals coated thereon.
7. A method of fabrication of a hybrid spin column (100), the method comprising:
obtaining punches from bulk materials of each layer of a plurality of membrane layers, wherein the plurality of membrane layers comprises:
a filtration membrane layer (114a) to receive a suspended
sample, to retain debris from the suspended sample, and to allow an
analyte in the suspended sample and chemical reagents in the suspended
sample to pass through; and
a binding membrane layer (114b) which comprises binding
chemicals coated thereon for interacting and binding with the analyte;
placing the obtained punches towards a second end (106) of a body portion (102) of a spin column (1000, wherein the filtration membrane layer (114a) is placed on the binding membrane layer (114b); and
affixing a sealing member (116) on the filtration membrane layer (114a) to hold the plurality of membrane layers securely at the second end of the spin column (100) to obtain the hybrid spin column (100).

8. The method as claimed in claim 7, wherein the plurality of membrane layers
comprises a support membrane layer (114c), wherein the method comprises:
placing the obtained punches of the support membrane layer (114c) below the binding membrane layer (114b) prior to affixing the sealing member (116).
9. An apparatus (300, 400, 500) comprising:
a stand (302) comprising:
a first platform (306) having a holder (308), wherein the holder (308) is to hold one of: a conduit (402) and a spin column (100);
a second platform (310) having a rod holder (312) to hold a rod (314), wherein the second platform (310) is placed above and substantially parallel to the first platform (306) and separated from the first platform (306); and
a support element (316) to support the second platform (310) above the first platform (306), wherein the holder (308) and the rod holder (312) are substantially coaxial; and
a rod assembly (304) arranged on the second platform (310), wherein the rod assembly (304) comprises:
the rod (314) which is receivable in the rod holder (312) of the second platform (310) of the stand (302); and
an actuation arrangement (318) coupled to the rod (314), wherein the actuation arrangement (318) is to cause movement of the rod (314) relative to the holder (308) to perform one of: a punching action and an affixing action.
10. The apparatus (400) as claimed in claim 9, wherein the rod (314) is a punching
rod (408) comprising:

a first portion (410) below the second platform (310), wherein a lower circumference of the first portion (410) is sharp to create punches from bulk membranes to obtain punches of the bulk membranes; and
a second portion (412) above the second platform (310), wherein the second portion (412) comprises a spring member (414).
11. The apparatus (400) as claimed in claim 10, wherein the actuation arrangement
(318) comprises:
a lever (416) coupled to the second portion (412) of the punching rod
(408) away from the second platform (310), wherein the lever (416) comprises:
a free end (420) for actuation of the apparatus (400) to cause
movement of the punching rod (408) relative to the holder (308) to
perform the punching action;
a pivoted end (422), wherein the lever (416) is pivotable about the pivoted end (422); and
a mounting point (424) between the free end (420) and the
pivoted end (422), wherein the second portion (412) of the punching rod
(408) is coupled to the lever (416) at the mounting point (424); and
a vertical part (418) disposed on the second platform (310) spaced apart
from the second portion (412) of the punching rod (408), wherein the vertical
part (418) is coupled to the lever (416) at the pivoted end (422).
12. The apparatus (500) as claimed in claim 9, wherein the rod (314) is a stamping
rod (502) comprising:
a first portion (504) below the second platform (310), wherein a lower circumference of the first portion (504) is blunt for affixing of a sealing member (116) on a plurality of membrane layers in a hybrid spin column (100); and

a second portion (506) above the second platform (310), wherein the second portion (506) comprises threads, wherein the threads of the second portion (506) mate with threads provided in the rod holder (312).
13. The apparatus (500) as claimed in claim 12, wherein the actuation arrangement
(318) comprises a knob (508) coupled to the second portion (506) of the stamping rod (502) away from the second platform (310) of the stand (302), wherein rotation of the knob (508) causes movement of the stamping rod relative to the holder (308).