DETAILED ACTION
This is the first action in response to US Patent Application No. 18/274,507, filed 27 July, 2023, as the National Stage Entry of International Application PCT/US2022/070512, and with priority to Provisional Application 63/145,391, filed 03 February, 2021. All claims 1-22 are pending and have been fully considered.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statement filed 27 July, 2023, has been fully considered.
With respect to the cited US Patent Application Publication US 2021/0018499 A1 (Mao et al.), it is noted that the cited reference has the same set of inventors (Mao and Liu) and the same applicant/assignee (University of Georgia Research Foundation) as the instant application. Also, US 2021/0018499 A1 was published (Jan 21, 2021) less than one year before the effective filing date (Feb 03, 2021, associated with parent provisional application 63/145,391) of the instant application. Accordingly, it is clear that the reference US 2021/0018499 A1 is not eligible for use in a rejection under 35 U.S.C. 102 or 103 because an exception under 35 U.S.C. 102(b)(1) applies (the reference was published by the instant inventors within the one year grace period prior to the effective filing date of the instant application) and an exception under 35 U.S.C. 102(b)(2) applies (the reference and application have a common assignee).
The information disclosure statement filed 27 July, 2023, further cites US 10,676,719 B2 and US 2020/0129981 A1; these publications are similarly excepted under 35 U.S.C. 102(b)(1&2). However, it is noted that the subject matter of these publications (US 10,676,719 B2 and US 2020/0129981 A1) was previously published in the documents US 2017/0029782 A1 and WO 2018/195451, respectively, which cannot be excepted under 35 U.S.C. 102(b)(1) because they were published more than one year before the effective filing date of the instant application.
Claim Objections
Claims 1 and 17 are objected to for the minor informalities indicated below:
-Claim 1, line 25 (first line of clause beginning “a magnetic source”), should be adjusted to read “configured to produce”;
-Claim 1, last line, “the sample fluid” should read “the fluid sample” to match the corresponding recitations of “the fluid sample” earlier in claim 1;
-Claim 17, line 3, “a sample fluid” should be adjusted to read “a fluid sample” to match the corresponding recitations of “the fluid sample” later in the claim; and
-Claim 17, line 3, the comma between “unlabeled” and “microparticles” should be omitted.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 8 and 19 rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 8 recites the limitations “each smaller micro-curve” (line 3) and “each larger micro-curve” (line 4) with insufficient antecedent basis. It is suggested that claim 8 be adjusted to depend from claim 7, which recites “alternating small and large micro-curves”, and the language of claim 8 be adjusted to refer to the micro-curves of claim 7. That is, the limitation at line 3 of claim 8 should be adjusted to read “each small
Claim 19 recites the limitation "the target cells” at line 1 of the claim. There is insufficient antecedent basis for this limitation in the claim. It is suggested that claim 19 be adjusted to depend from claim 18.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-7, 9, 17 and 19-22 are rejected under 35 U.S.C. 103 as being unpatentable over Spuhler et al. (US 2019/0264166 A1) in view of Lin et al. (US 2010/0044232 A1) and Stone et al. (US 2012/0080360 A1).
Regarding claim 1, Spuhler et al. (US 2019/0264166 A1) teaches a multiple-stage microfluidic device for separating cells/particles in a sample (microfluidic sorting device—title, abstract; generalized embodiment of Fig. 1 includes an inertial focusing region 104 and a magnetophoresis region 102—[0071]). An exemplary embodiment of the device of Spuhler is depicted in Fig. 4 below, with annotations added to identify key structures of the device.
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Accordingly, the device of Spuhler includes a first microfluidic channel having a first and second end; a first fluid inlet at the first end of the microfluidic channel and configured to receive a fluid sample comprising the sample combined with a ferrofluid (a fluid sample containing both non-magnetizable analytes and analytes bound to magnetic particles enters the system 400 at input port 402…the sample is then separated evenly into two parallel channels 404a and 404b…and enters inertial focusing regions 408a and 408b—[0107]; magnetic particles may include ferrite particles which are ferromagnetic—[0025], [0134]; viewing Fig. 4, it is evident that the input port 402 defines a first microfluidic channel with a first fluid inlet at a first end thereof, the inlet receiving a fluid including ferromagnetic particles);
an inertial focusing stage (408a, 408b) downstream of the second end of the first microfluidic channel (402), wherein the first microfluidic channel splits into two or more serpentine focusing channels (408a, 408b), each serpentine focusing channel having a plurality of alternating micro-curves (waves) configured to focus cells/particles within the sample into a narrow stream to produce a focused fluid sample stream and wherein the two or more serpentine focusing channels form a convergence at a second end of the inertial focusing stage (fluid from parallel channels 404a and 404b enters inertial focusing regions 408a and 408b—[0107]; inertial focusing regions are channels having the shape of a wave with large and small turns…common streamlines from each channel flow into a single channel 410 in a magnetophoresis region 412—[0108]; Fig. 1 shows related inertial focusing region 104, from which it is clear that the wave pattern includes a plurality of alternating micro-curves—see [0101]);
a magnetic separation stage (412) at the second end of the inertial focusing stage (408a-b), wherein the convergence of the serpentine focusing channels is located at a first end of the magnetic separation stage to form a magnetic separation channel configured such that the focused fluid sample stream exiting the convergence of the focusing channels enters the magnetic separation channel (410) in a central portion of the magnetic separation channel (streamlines from each focusing channel 408a and 408b flow into channel 410—[0108]; Fig. 4 depicts convergence of focusing channels 408a and 408b at the center of channel 410)
a magnetic source (see magnets 201 in Fig. 2E) in the magnetic separation stage configured to produce a substantially symmetric magnetic field having a field maximum along an inner longitudinal axis of the magnetic separation channel sufficient to cause cells/particles flowing in the ferrohydrodynamic separation channel to be deflected away from the center of the magnetic separation channel towards the sides of the magnetic separation channel as a function of the size of the cells/particles (magnetophoretic region 412 configured to deflect magnetizable particles within each flow stream as discussed in other embodiments—[0108]; Fig. 2E of Spuhler depicts an embodiment of a magnetophoresis region defined by a sextupole magnet configuration identical to the sextupole magnet configuration disclosed in the instant application at Fig. 2A, which the instant application at page 29, lines 17-28 indicates generates a maximum magnetic flux density at the center of the channel; accordingly, Spuhler teaches an embodiment of the device 400 with a magnetophoretic region 412 having magnets arranged in the sextuple configuration of Fig. 2E, which is structural identical to the disclosed magnetic arrangements and thus presumed to achieve the recited functions and magnetic field distribution);
three or more outlets (414a-b, 416a-b, 418) at a second end of the magnetic separation stage (412), each outlet positioned to receive cells/particles in fluid flowing along a different portion of the magnetic separation channel (410) from each of the other outlets such that cells/particles in the sample fluid are separated (channel 410 is fluidly coupled to outer channels 414a and 414b for receiving buffer fluid, channels 416a and 416 b for receiving analytes that are not bound to magnetic particles, and a central channel 418 for receiving analytes that are bound to magnetic particles—[0109])
Provided below is Fig. 2E of Spuhler, which depicts the most relevant embodiment of a magnetophoresis region (412), wherein the arrangement of the magnets (201) and channels (208) of Spuhler in Fig. 2E is identical to disclosed embodiments of the instant invention (instant Fig. 2A and page 29, lines 17-28 of the instant disclosure indicate that an identical sextuple arrangement induces maximum magnetic flux density at the center of the channel).
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With further respect to the first microfluidic channel splitting into the two or more serpentine focusing channels, it is acknowledged that the claim requires the split occurs at a second end of the first microfluidic channel and at a first end of the inertial focusing stage. This claim language appears to require the split in the first microfluidic lead directly into to the serpentine channels, whereas Fig. 4 of Spuhler as relied upon above depicts a pair of microchannels (404a-b) positioned between the split in the first microchannel (402) and the serpentine channels (408a-b). The examiner holds that the microchannels (404a-b) of Fig. 4 can reasonably considered part of the inertial focusing stage as they each lead to corresponding serpentine channels (408a-b) and fairly define an inlet part of the serpentine channel, such that the presence of the microchannels (404a-b) is not inconsistent with the claim.
Alternatively, it is noted that there is no evidence to suggest that the positioning of the split in the first microfluidic channel is critical as long as the first microfluidic channel is capable of delivering fluid from a common first inlet to a pair of serpentine channels. Therefore, it would be obvious to a person having ordinary skill in the art to rearrange the device of Spuhler such that the split in the first microchannel is positioned at the first end of the serpentine channels (408a-b) of the inertial focusing stage (i.e., arranging the device so microchannels 404a-b are merged into a single microchannel which forms an extension of the first microfluidic channel 402 identified above, wherein the extended first microfluidic channel splits directly into the serpentine channels 408a-b) because such rearrangement would not significantly alter function of the device and would advantageously maintain the sequence of filtering, inertial focusing, and magnetic separation of the sample fluid which enables the isolation and recovery of a target analyte (see Spuhler at Fig. 4, liquid flows from input/first microchannel 402 through filters 406, inertial focusing region 408a-b, and magnetic separation region 412; different types of analytes sorted into distinct outlets—see [0109]). Such modification may also reduce the number of distinct filter units (406) required by the device, advantageously simplifying the design of the device. See MPEP 2114.04(VI.)(C.) regarding the obviousness of the rearrangement of parts of the prior art when the rearrangement would not modify the operation of a device.
The device of Spuhler clearly differs in structure from the device of the instant claim in that:
-Spuhler does not teach at least two sheathing fluid channels fluidly connected to one or more sheathing fluid inlets, the sheathing fluid channels configured such that the convergence of the serpentine focusing channels is between the at least two sheathing fluid channels; and the sheathing fluid channels being arranged so that the convergence of the serpentine focusing channels converges with the at least two sheathing fluid channels at a first end of the ferrohydrodynamic separation stage such that a sheathing ferrofluid exiting the sheathing fluid channels enters the ferrohydrodynamic separation channel on the periphery of the ferrohydrodynamic separation channel serving to further narrow the focused fluid sample stream and adjust its starting position in the ferrohydrodynamic separation channel.
With respect to the claimed sheathing fluid, Lin et al., in the analogous art of microfluidic devices for manipulating particles in a fluid (abstract), teaches a microfluidic device (100) which includes sheath fluid inlet channels (C,D; 118, 120) which are positioned at opposite sides of a sample inlet channel (A,B; 106) and which converge with the sample inlet channel at a first end of a main channel (104) (see Figs. 1A-B), the main channel being subjected to a magnetic field which separates different types of particles (microfluidic device 100 with main channel 104 having an inlet 106 and hydrodynamic focusing channels 118 and 120, which force the fluid from input 106 towards the center into a narrower sheath of fluid…magnetic field applied while microfluidic device 100 is in operation—Figs. 1A-B, [0034]; invention provides cell separation—[0033] Fig. 3 shows magnet positioned near main channel, and Fig. 1C shows magnetic field through main channel). See Figs. 1A-B of Lin below.
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Lin further indicates that the technique of hydrodynamic focusing with the sheath fluid (delivered by channels 118 and 120 from inlet sources C and D) focuses the sample flow into a fine central stream that allows adjustment of the position and width of the sample stream in the main channel ([0044]). Lin further suggest that introducing metal particles into the sheath (shear) streams can yield a stronger magnetic force acting on particles ([0061]), wherein iron particles with high magnetic susceptibility are suitable metal particles ([0060]).
Therefore, it would be obvious to a person having ordinary skill in the art to modify the device of Spuhler to include at least two sheathing fluid channels arranged to the sides of and converging with the sample inlet to the magnetic separation channel (the sample inlet to the magnetic separation channel 410 of Spuhler being the outlet of the convergence of the serpentine channels 408a-b, see Fig. 4 of Spuhler), as substantially seen in Lin, for the benefit of delivering a magnetically susceptible sheath fluid (sheath fluid including magnetically susceptible iron particles—see Lin at [0061], [0066]) into the magnetic separation channel so that the sheath fluid can hydrodynamically focuses the sample stream (Lin at [0034], [0044]), allow for adjustment of the starting position of the sample stream within the magnetic separation chamber (Lin at [0044]), and increase the strength of magnetic forces within the device (Lin at [0061]).
As modified above, the device of Spuhler is structurally indistinct from the device of claim 1. Nonetheless, it is acknowledged that the device of Spuhler is intended to operate by separating magnetic or magnetically bound particles/cells from the non-magnetic or non-magnetically bound particles/cells (Spuhler at [0109]), whereas the instant device employs a ferrohydrodynamic separation mechanism which is capable of separating cells/particles based on size as a result of a magnetic buoyancy effect (which is understood to involve magnetic particles of a ferrofluid concentrating in a portion of a fluid flow in response to a magnetic field and effectively pushing non-magnetic particles out of said portion of the fluid flow, wherein larger particles are deflected a greater distance by this effect relative to smaller particles). Although the specifics of this functional distinction are not explicitly recited in claim 1 in a manner that would necessarily yield a further structural distinction between the claimed device and the modified device of Spuhler, this functional difference has nonetheless been considered in the assessment of obviousness below to promote compact prosecution and to address associated limitations in other claims of the instant application in a consolidated manner.
With respect to this functional distinction, Stone et al. (US 2012/0080360 A1), in the analogous art of the manipulation of particles in channels (title), teaches an embodiment (Figs. 7A-B) of a particle sorting system wherein a fluid (710) carrying smaller particles (712), larger particles (714), and magnetic particles (as in a magnetic fluid, e.g., a ferrofluid) moves past magnets (716, 718, 724, 726) arranged about a fluid channel (720) ([0050]). The arrangement of magnets creates a magnetic field minimum (722, 728), wherein the larger particles (714) are transported toward the magnetic field minimum to a greater extent than the smaller particles (712), allowing non-magnetized particles of different size to be diverted to different outlets ([0050], Figs. 7A-B). Stone indicates that the device functions on an exclusion principle, wherein the movement of magnetic particles away from the magnetic field minimum causes movement of non-magnetic particles towards the magnetic field minimum, and the effect is stronger when the concentration of magnetic particles is higher ([0034]). Stone also indicates the strength of the effect (i.e., the displacement of non-magnetic particles by the exclusion from the net movement of magnetic particles) is stronger on non-magnetic particles with a larger drag coefficient, which correlates to lager diameter particles ([0050],[0052]). Also, it is noted Stone suggests that the sample (of different sized particles 712, 714, and magnetic particles) enters the device alongside a sheathing fluid (cladding fluid—[0050]) which may also contain magnetic particles ([0044]).
Therefore, it would be obvious to a person having ordinary skill in the art to combine the above teachings (of Spuhler, Lin, and Stone) and adapt the modified device of Spuhler for use with a sample which includes non-magnetic particles of different sizes and magnetic particles (e.g., a ferrofluid—see Stone at [0050]) and a sheath fluid also containing magnetic particles (see cladding fluid of Stone at [0044], [0055], and sheathing/shear fluid of Lin [0061], [0066]) for the benefit of driving the magnetic particles toward a center of the magnetic separation channel (Spuhler at [0077] indicates that the magnet arrangement drives magnetic particles toward a center of the channel) such that larger non-magnetic particles are strongly deflecting away from the center of the channel and smaller non-magnetic particles are less strongly deflected away from the center of the channel, allowing the collection of different sized particles at the device outlets (Stone at [0034], [0050], [0052], discusses how an exclusion effect caused by the net motion of magnetic particles within a magnetic field allows for non-magnetic particles to be separated by size). When adapted for such use, the magnetic separation stage of Spuhler clearly defines a ferrohydrodynamic separation stage consistent with the instant disclosure and achieves all functions recited in claim 1.
Regarding claim 2, the combination of Spuhler, Lin, and Stone, teaches the multi-stage microfluidic device of claim 1. As discussed with respect to claim 2 above, Spuhler teaches an embodiment of wherein the magnetic separation stage (412, Fig. 4) employs the magnet arrangement of Fig. 2E, wherein the magnets (201) are arranged about the magnetic separation channel (channel 410 in Fig. 4, corresponding to channels 208 in Fig. 2E) such that the magnetic source comprises an array of magnets comprising a top array and bottom array, wherein the magnetic separation stage (channel 208 in Fig. 2E corresponds to an embodiment of channel 410 of magnetic separation stage 412 in Fig. 4) is sandwiched between and substantially centrally aligned between the top magnet array and the bottom magnet array, wherein the magnets in the top array are oriented to repel the magnets in the bottom array (see Fig. 2E provided with respect to claim 1 above, which clearly meets the limitation so claim 2; also, as modified with respect to claim 1 above, the magnetic separation stage of Spuhler is configured as a ferrohydrodynamic separation stage).
Regarding claim 3, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 2, and Spuhler further teaches the array of magnets comprises six magnets arranged in a sextupole configuration (see Spuhler at Fig. 2E, which is identical to the sextupole configuration disclosed in instant Fig. 2A).
Regarding claim 4, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. Spuhler further teaches one or more filters between the inlet and the serpentine channels configured to separate debris from the fluid sample (each channel 404a-b may include one or more inlet filters 406 to remove or break up large debris in the fluid sample—[0107]). Additionally, as discussed with respect to claim 1 above, it would be obvious to a person having ordinary skill in the art to rearrange the device of Spuhler such that the microchannels (404a-b) are combined into a single microchannel including filters (406), the microchannel forming an extension of the inlet port (402) and branching at a second end into the serpentine channels (408a-b; see Fig. 4 of Spuhler) because such modification would not significantly alter the operation of the device and advantageously may simplify the design of the device (see rejection of claim 1 above). In such a modified embodiment, the filters are positioned along the first microfluidic channel (single microchannel formed by merging microchannels 404a and 404b) between the inlet (402) and the second end (the second end corresponding to a position just upstream of inertial focusing phase 408a-b where the modified first microchannel splits).
Regarding claim 5, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. As discussed with respect to claims 1 and 4 above, it would be obvious to a person having ordinary skill in the art to merge the microchannels 404a-b or Spuhler into a single channel forming part of the first microfluidic channel (see rejections of claims 1 and 4 above). Spuhler depicts the microchannel (404) bending toward an inertial focusing stage (see Fig. 4, channel 404a bends toward inertial focusing stage 408a). Spuhler also suggests forming the device as a single integrated device ([0107]). A person having ordinary skill in the art would recognize that curves in the first microchannel, such as the curve in the microchannel (404a) of Spuhler (Fig. 4), can advantageously provide a device with a more compact form factor. Therefore, when modifying the microchannels (404a-b) of Spuhler to form the first microchannel of claim 1 (see rejections of claims 1 and 4 above), it would be obvious to a person having ordinary skill in the art to include at least one or more bends at the second end of the first microfluidic channel before the inertial focusing stage for the benefit of achieving a microfluidic channel having a more compact form factor.
Regarding claim 6, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. Spuhler indicates that the two or more serpentine channels each comprise a plurality of alternating micro-curves (inertial focusing region channel has the shape of a wave having large and small turns, wherein a radius of curvature can change after each inflection point of the wave—see [0101], Fig. 1, [0108], Fig. 4). Although Fig. 4 of Spuhler does appear to depict tens of waves (i.e., alternating micro-curves) along the inertial focusing region (408a-b), Spuhler does not explicitly suggest about 30-50 alternating micro-curves. However, Spuhler does discuss how the serpentine channels are configured to precisely align particles along a lateral position within a microfluidic channel (e.g., along a common streamline) ([0098]), wherein the effectiveness of the inertial focusing is dependent on properties of the particles to be separated, the geometry of the microchannel, and the flow conditions in the microchannel ([0100], [0102]). Additionally, it is fairly implied the focusing effect is narrowed by each successive curve (consider Fig. 1 of Spuhler showing particles becoming increasingly focused along the length of inertial focusing region). Accordingly, it would be obvious to a person having ordinary skill in the art to arrive at a number of alternating micro-curves within the claimed range of 30 to 50 by way of routine optimization of the serpentine channel geometry to achieve an improved focusing effect on the targeted types of particles at desired flow conditions.
Regarding claim 7, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. Spuhler further teaches the serpentine focusing channels comprise alternating small and large micro-curves (inertial focusing region channel has the shape of a wave having large and small turns, wherein a radius of curvature can change after each inflection point of the wave—see [0101], [0108], Fig. 4; Fig. 1 shows wave pattern having alternating small and large curves at inertial focusing stage 104).
Regarding claim 9, , the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. Although Fig. 4 of Spuhler depicts the ferrohydrodynamic separation channel (410) as a straight channel, Spuhler indicates the ferrohydrodynamic region (412) can be configured according to any of the disclosed embodiments ([0108]), which includes an embodiment (Fig. 2E) wherein the channel (410) includes two channels (208a, 208b) ([0096]) which may be coupled together in series ([0095]). Thus, Spuhler teaches the ferrohydrodynamic separation channel (410) comprises a first section (208a) and a second section (208b) such that the ferrohydrodynamic separation channel passes through the magnetic field twice to increase separation of the particles. Spuhler does not explicitly indicate that the first and second sections are connected by a substantially u-shaped curve, but it would be obvious to a person having ordinary skill in the art when connecting the first and second sections in series (as suggested by Spuhler at [0095]) to use a u-shaped curve for the evident benefit of achieving a spatially compact connection (as opposed to the connection looping around back to an upstream side of the magnet array) which can maintain laminar flow conditions with focused particle streams (as opposed to a non-curved connection which could be more likely to disrupt flow streams).
Regarding claim 17, the claim is a method of using the device of claim 1. Spuhler in view of Lin and Stone teaches the device of claim 1. Use of the device of Spuhler as modified with respect to claim 1 defines a method of enriching and/or separating unlabeled microparticles in a sample comprising a plurality of components, the method comprising:
introducing a sample fluid comprising the sample with the unlabeled, microparticles and a first ferrofluid into the first fluid inlet of a multi-stage microfluidic device according to claim 1 at a first flow rate (Spuhler at [0107] indicates that a fluid sample containing both non-magnetizable analytes and analytes bound to magnetic particles enters the system 400, and indicates that the magnetic particles may include ferrite particles which are ferromagnetic—[0025], [0134]; the device of Spuhler is modified with respect to claim 1 in view of Stone to instead receive a flow of non-magnetic particles of different sizes combined with a ferrofluid, see Stone at [0050] and the rejection of claim 1 above);
flowing the fluid sample through the inertial focusing stage and focusing the microparticles in the fluid sample into a focused fluid sample stream (Spuhler at [0108] indicates that the channels of inertial focusing 408a and 408b focus analytes into a common streamline);
combining the focused fluid sample stream from the inertial focusing stage with a sheathing ferrofluid at the first end of the ferrohydrodynamic separation stage such that the sheathing ferrofluid serves to further narrow the focused fluid sample stream of microparticles in the fluid sample (the device of Spuhler as modified with respect to claim 1 in view of Lin includes sheathing ferrofluid channels which deliver a ferrofluid for narrowing the focused fluid sample stream—see hydrodynamic focusing channels 118 and 120 of Lin at [0034], and suggestion of Lin to include metal particles in the focusing channels at [0061]).
flowing the focused fluid sample stream of microparticles in the ferrohydrodynamic separation channel such that the substantially symmetric magnetic field produced by the magnetic force hydrodynamically causes cells/particles flowing in the channel to be focused away from the center of the channel towards the sides of the channel as a function of the size of the particles, such that larger particles move further toward the sides of the channel than smaller particles (Spuhler teaches fluid from the inertial focusing stage 408a-b flowing into the channel 410 of the magnetized region 412—[0108]—wherein the magnets are arranged in a manner consistent with the instant disclosure [compare Fig. 2E of Spuhler to Fig. 2A of the instant disclosure] which drives magnetic particles toward the center of the channel—see Spuhler at [0077], [0093], [0096]; Stone fairly suggests that the movement of magnetic particles toward the center of the channel would have the effect of deflecting non-magnetic particle to the periphery of the channel, with larger particles being pushed further—see Stone at [0050], [0052]).
collecting separated particles from the at least 3 outlets (Spuhler indicates outlets 414, 416, and 418 collect different types of particles).
Regarding claim 19, the combination of Spuhler, Lin, and Stone teaches method of claim 17. Spuhler further teaches the target cells are circulating tumor cells (the targeted and detected cells can be cancer cells, including circulating tumor cells—[0147]).
Regarding claim 20, the combination of Spuhler, Lin, and Stone teaches method of claim 17. The microparticles of interest within the device of Spuhler include white blood cells and circulating tumor cells (see e.g., Figs. 5A-B, [0110]). Based on at least the instant specification at page 32, lines 15-16 and 17-18, circulating tumor cells have a diameter within the range of 15 μm-25 μm, and white blood cells have a diameter in the range of 8-14 um in diameter. Thus, the microparticles of Spuhler have varying physical diameters in a range of about 4-40 μm (the microparticles of Spuhler are white blood cells and circulating tumor cells which having varying diameters laying within the claimed range as evidenced by the instant specification).
Regarding claim 21, the combination of Spuhler, Lin, and Stone teaches method of claim 17. Spuhler, Lin, and Stone, do not explicitly teach a focused fluid sample stream entering the ferrohydrodynamic separation channel having a width of 4-100 μm. However, Spuhler indicates that the focused fluid sample has narrow stream lines when entering the magnetic separation stage (see Fig. 5A, [00110]), wherein the total width of the channel of the magnetic separation stage is between 100 μm and 5mm ([0089]), and wherein providing the fluid sample in inertially focused streams allows highly sensitize magnetic sorting of cell populations ([0104]). Accordingly, it would be obvious to a person having ordinary skill in the art to configure the device of modified Spuhler to provide a focused sample stream into the first end of the ferrohydrodynamic separation channel having a width within the claimed range of 4-100 μm by routine optimization directed toward narrowing (i.e., focusing) the steam so that the particles therein are advantageously more susceptible to magnetic sorting effects (consider Spuhler at [0104]), without narrowing the stream to a point where it is too small for the target cells or unable to support a suitable sample flow rate.
Regarding claim 22, , the combination of Spuhler, Lin, and Stone teaches method of claim 17. Spuhler further indicates that the fluid sample is processed in the device at a flow rate of about 200-1400 L/min (analytes to be separated/isolated at high flow rates, e.g., at least approximately 50 μL/min, at least approximately 100 μL/min, at least approximately 150 μL/min, at least approximately 300 μL/min, at least approximately 500 μL/min, or at least approximately 1000 μL/min—[0083]).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Spuhler et al. (US 2019/0264166 A1) in view of Lin et al. (US 2010/0044232 A1) and Stone et al. (US 20120080360 A1), as applied to claim 1 above, and further evidenced by Toner et al. (US 9,196,913 B2).
Regarding claim 8, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. As discussed with respect to claims 6-7 above, Spuhler teaches that the serpentine channel include smaller and larger micro-curves (wave having large and small turns—[0101], [0108], Figs. 1 and 4). As depicted in Fig. 1 of Spuhler, it is evident the width of the serpentine channel is narrower at the smaller-micro curves and the serpentine channel width is greater at the larger micro-curves (see inertial focusing region 104 in Fig. 1). Nonetheless, Spuhler does not particularly teach that the interior width of the serpentine channel is about 50-200 μm at a crest portion of each smaller micro-curve and about 100-400 μm at a crest portion of each larger micro-curve.
However, Spuhler incorporates by reference Toner (US 8,186,913 B2), wherein Toner teaches inertial focusing channels having a width within the range of 10 to 1000 μm (column 3, lines 39-43), wherein embodiments of the focusing channels of Toner have an asymmetrically curving geometry wherein the radius of curvature and the width of the channel changes in a periodic manner to create a single focused particle stream (column 11, lines 36-40; column 16, lines 48-54). Toner further teaches an example of such an asymmetrically curving channel wherein the width of the channel is 100 μm at the smaller curve and 160 μm at the larger curve—which lays within the claimed ranges—said exemplary channel of Toner being effective for focusing a 10 μm particle into a single stream at a Reynolds number of 12 (Example 24—Fig. 41, column 47, lines 30-40). Therefore, it would be obvious to a person having ordinary skill in the art to configure the device of modified Spuhler such that the width at the crest of the smaller micro-curve is 100 μm and the width at the crest of the larger micro-curve is 160 μm, as seen in Toner, for the benefit of focusing particles into a single stream (see Toner at Fig. 41, column 47, lines 30-40).
Claims 10-11 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Spuhler et al. (US 2019/0264166 A1) in view of Lin et al. (US 2010/0044232 A1) and Stone et al. (US 20120080360 A1), as applied to claim 1 above, and further evidenced Nagrath et al. (US 20150293010 A1)
Regarding claim 10, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. Initially, it is noted that claim 10 limits an apparatus (multi-stage microfluidic device) based on the material worked upon (lysed blood sample) by the apparatus; accordingly, claim 10 does not impose a further structural limitation on the claimed device as per MPE 2115, which indicates that a recitation of a material worked upon does not limit an apparatus claim. Nonetheless, to promote compact prosecution it is noted that Spuhler does teach that the sample is a blood sample and that the cells/particles include white blood cells and target cells (whole blood is filtered in region 106 to remove red blood cells and platelets, while white blood cells and circulating tumor cells continue to the inertial focusing region—[0071]-[0072]; Figs. 5a-B show the magnetic separation stage 412 separating white blood cells from circulating tumor cells—[0110]). While Spuhler does not indicate that the blood sample is lysed, it would be obvious to a person having ordinary skill in the art to use a lysed blood sample for the benefit of providing an alternative technique for removing red blood cells from the sample, as evidenced by Nagrath (Nagrath, in the analogous art of microfluidic devices for detecting rare cells—title, abstract—indicates that red blood cell lysis is a commonly used technique to remove red blood cells from whole blood when red blood cells are not the target cell of a separation technique—[0111])
Regarding claim 11, the combination of Spuhler, Lin, Stone, and Nagrath teaches the multi-stage microfluidic device of claim 10. Spuhler further teaches the target cells are circulating tumor cells (the targeted and detected cells can be cancer cells, including circulating tumor cells—[0147]).
Regarding claim 18, the combination of Spuhler, Lin, and Stone teaches the method of claim 17. Spuhler further teaches the sample is a blood sample, the microparticles are cells, and the cells include white blood cells and target cells (whole blood is filtered in region 106 to remove red blood cells and platelets, while white blood cells and circulating tumor cells continue to the inertial focusing region—[0071]-[0072]; Figs. 5a-B show the magnetic separation stage 412 separating white blood cells from circulating tumor cells—[0110]). Spuhler, Lin, and Stone do not explicitly suggest that the blood sample is lysed. However, as disused with respect to claim 10 above, Nagrath, in the analogous art of microfluidic devices for detecting rare cells (title, abstract), indicates that red blood cell lysis is a commonly used technique to remove red blood cells from whole blood when the red blood cells are not the target cell of a separation technique ([0111]). Therefore, it would be obvious to a person having ordinary skill in the art to use a lysed blood sample for the benefit of providing an alternative technique of removing red blood cells from the sample, as evidenced by Nagrath (see Nagrath at [0111]).
Claims 12-16 are rejected under 35 U.S.C. 103 as being unpatentable over Spuhler et al. (US 2019/0264166 A1) in view of Lin et al. (US 2010/0044232 A1) and Stone et al. (US 20120080360 A1), as applied to claim 1 above, and further in view of Koser (US 20120237997 A1).
Regarding claim 12, the combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. As similarly indicated with respect to claim 10, the limitations of claim 12 refer to a material worked on by the claim device and do not pose a clear structural limitation on the claimed device in view of MPEP 2115. Nonetheless, it is noted that the device of Spuhler as modified with respect to claim 1 includes a ferrofluid and a sheathing ferrofluid each comprise a plurality of magnetic nanoparticles, and a carrier fluid (Spuhler suggests the sample including ferromagnetic beads having diameters around 100 nm—[0025], [0134]; Stone similarly discusses forming a ferrofluid with iron or iron oxide particles—[0039], [0050]; Lin suggests iron nanoparticles—[0060]—introduced through sheathing fluid inlets—[0061]). The cited references do not explicitly discuss the ferrofluid including a surfactant. However, Koser—in the analogous art of label-free cell manipulation and sorting (title)—indicates that a ferrofluid is a colloidal mixture of nanometer sized magnetic particles covered by a surfactant and suspended in a carrier medium ([0029]), wherein the surfactant can be selected to be biocompatible and serves to stabilize the ferrofluid ([0033], [0069], [0076]), i.e., avoid particle aggregation. Therefore, it would be obvious to a person having ordinary skill in the art to select a ferrofluid for use with the modified device of Spuhler wherein the ferrofluid comprises magnetic nanoparticles, a surfactant, and a carrier fluid, as taught by Koser ([0029]), for the benefit of providing a ferrofluid which is stable (i.e., resistant to particle aggregation—see Koser at [0033], [0069], [0076]).
Regarding claim 13, the claimed kit incorporates the device of claim 1 combined with a superparamagnetic composition and carrier fluid which form a superparamagnetic fluid. The combination of Spuhler, Lin, and Stone teaches the multi-stage microfluidic device of claim 1. As substantially discussed with respect to claim 12 above, it would be obvious in view of Koser to provide the modified device of Spuhler with a superparamagnetic composition comprising a plurality of magnetic nanoparticles and a surfactant which is combined with a carrier fluid to form a biocompatible ferrofluid (see Koser at [0029], [0033], [0069], [0076]). Said ferrofluid defines a superparamagnetic fluid suitable for use as the ferrofluid and/or sheathing ferrofluid of claim 1.
Regarding claim 14, the combination of Spuhler, Lin, Stone, and Koser teaches the kit of claim 13. Claim 14 recites instructions for combining the magnetic nanoparticles, surfactant, and carrier fluid to make the superparamagnetic fluid and instructions for using the superparamagnetic fluid and the multi-stage microfluidic device to separate cells/particles in a fluid sample. MPEP 2112.01(III.) indicates that, in view of, e.g., In re Ngai, 367 F.3d 1336, 1339, 70 USPQ2d 1862, 1864 (Fed. Cir. 2004), printed instructions do not distinguish a claimed product from otherwise identical prior art product. Therefore, it would be obvious to a person having ordinary skill in the art to provide the kit of modified Spuhler with a set of instructions for the benefit of guiding a user in proper use of the kit.
Regarding claim 15, the combination of Spuhler, Lin, Stone, and Koser teaches the kit of claim 14. The modified kit of Spuhler includes a ferrofluid including a surfactant taught by Koser (see rejections of claims 12 and 13 above), wherein the surfactant of Koser is biocompatible (Koser: citrate is an effective surfactant in ferrofluids and is mostly biocompatible in cell cultures—[0033]).
Regarding claim 16, the combination of Spuhler, Lin, Stone, and Koser teaches the kit of claim 13. The cited references do not explicitly indicate that the ferrofluid and the sheathing ferrofluid have a concentration of magnetic nanoparticles of about 0.001- 1% (v/v). However, Stone recognized that the concentration of magnetic particles affects the strength of the exclusion effect which causes movement of non-magnetic particles when the fluid sample is subjected to a magnetic field ([0034]; [0082]-[0084]). Also, a person of ordinary skill in the art would recognize that excessively high nanoparticle concentrations could present biocompatibility issues and damage cells (consider Lin at [0061] indicating that metal particles can contaminate a sample and pose biocompatibility issues). Therefore, it would be obvious to a person having ordinary skill in the art to arrive at a magnetic particle concentration within the claimed range of 0.001-1% (v/v) by way of routine optimization of the modified kit of Spuhler, the optimization directed at achieving the benefit of a sufficiently strong exclusion force on non-magnetic particles within the stream (consider Stone at [0034] and [0082]-[0084]) without excessively contaminating or damaging biological samples (consider Lin at [0061]).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
US 2017/0029782 A1 and WO 2018/195451 are disclosures by the instant inventors that were published over one year before the effective filing date of the instant application, the publications disclosing particle separation devices and methods analogous to the claimed device.
Smith et al. (US 2015/0336096 A1) teaches microfluidic devices for sorting particles using high gradient magnetic fields, including embodiments (Fig. 9A) including an inertial focusing stage (904) and a magnetic deflection channel (906) ([0132]-[0133]).
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/BRADY C PILSBURY/Examiner, Art Unit 1799
/JENNIFER WECKER/Primary Examiner, Art Unit 1797