JP7284473B2 - Blood component separation method and anti-clogging agent for microfluidic device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a blood component separation method using a microfluidic device and an anti-clogging agent for the microfluidic device used in the same method.

Various techniques have been disclosed so far for methods of analyzing or separating biological components using microfluidic devices. In such microfluidic devices, clogging of channels is always a problem, and various attempts have been made to solve this problem.

For example, Patent Literature 1 below discloses that clogging is prevented by applying ultrasonic vibrations. Further, Patent Document 2 below discloses that an AC voltage is applied to prevent clogging.

In Non-Patent Document 1 below, by surface-modifying the silicon material of a microfluidic device with polyethylene glycol, zwitterionic sulfobetainesilane, zwitterionic polyethyleneglycol-sulfobetainesilane copolymer, etc., clogging, etc., can be prevented. is disclosed. In addition, Non-Patent Document 2 below discloses a method for preventing thrombus formation and the like in a medical device by dielectrophoresis.

The following non-patent document 3 discloses that surface modification of a silicon material of a microfluidic device with polyethylene glycol leads to extension of life. In addition, Non-Patent Document 4 below discloses that coating the silicon material of a microfluidic device with heparin reduces interaction with blood components. Furthermore, Non-Patent Document 5 below discloses that adsorption and sorption can be suppressed by surface-modifying a microfluidic device with a cyclic olefin copolymer.

In addition, Non-Patent Documents 6 and 7 below state that thrombin inhibitors are effective in preventing clogging of microfluidic devices. Specific examples of thrombin inhibitors include D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) in Non-Patent Document 6 and glyceroprotein IIb/IIIa in Non-Patent Document 7. It was tirofiban that inhibited

JP 2011-185839 A JP 2008-175812 A

T.J.Plegue, K.M.Kovach, A.J.Thompson & J.A.Potkay, "Stability of Polyethylene Glycol and Zwitterionic Surface Modifications in PDMS Microfluidic Flow Chambers." Langmuir, 2018, 34(1), p.492-502 S.Kinio & J.K.Mills, "Effects of dielectrophoresis on thrombogenesis in human whole blood." Electrophoresis, 2017, 38, p.1755-1763 K.M.Kovach, J.R.Capadona, A.Sen Gupta & J.A.Potkay, "The effects of PEG-based surface modification of PDMS microchannels on long-term hemocompatibility." 2014, DOI: 10.1002/jbm.a.35090 S.Thorslund, J.Sanchez, R.Larsson, F.Nikolajeff & J.Bergquist, "Bioactive heparin immobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface properties." 2005, DOI: 10.1016/j.colsurfb.2005.10 .009 R.K.Jena & C.Y.Yue, "Cyclic olefin copolymer based microfluidic devices for biochip applications: Ultraviolet surface grafting using 2-methacryloyloxyethyl phosphorylcholine." 2012, DOI: 10.1063/1.3682098 J.D'Silva, R.H.Austin & J.C.Sturm, "Inhibition of clot formation in deterministic lateral displacement arrays for processing large volumes of blood for rare cell capture." Lab Chip, 2015, 15, p.2240-2247 K.H.K.Wong, J.F.Edd, S.N.Tessier, W.D.Moyo, B.R.Mutlu, L.D.Bookstaver, K.L.Miller, S.Herrara, S.L.Stott & M. Toner, "Anti-thrombotic strategies for microfluidic blood processing." Lab Chip, 2018, 18, p.2146-2155

Clogging is always a problem when attempting to separate certain components, such as cells, that are suspended in blood using microfluidic devices. Platelets are assumed to be the cause of such clogging, but it is difficult to completely eliminate the clogging even if the surface of the microfluidic device is coated with, for example, an anticoagulant. is likely to be involved in clogging.

An object of the present invention is to reduce clogging in a blood component separation method using a microfluidic device, without subjecting the microfluidic device to special treatment or providing a special mechanism. do.

A first aspect of the present application has an inlet positioned upstream, an outlet positioned downstream, and a channel positioned between the inlet and the outlet, wherein the channel includes A blood component separation method for separating blood components by a microfluidic device in which a plurality of microposts are arranged at predetermined intervals, wherein a DNA aggregating agent is added as an anti-clogging agent to a blood sample flowing in from the inlet. It is

In a second aspect of the present application, in addition to the configuration of the first aspect, the DNA flocculating agent is polyamine or hexaamminecobalt(III) chloride.

In a third aspect of the present application, in addition to the configuration of the second aspect, the polyamine is linear.

In a fourth aspect of the present application, the linear polyamine has a molecular weight of 88 or more and 3000 or less.

In a fifth aspect of the present application, in addition to the configuration of the fourth aspect, the polyamine is either one or both of spermidine and spermine.

In a sixth aspect of the present application, in addition to the configuration of the fifth aspect, the polyamine is spermidine, and the spermidine concentration in the blood sample is 50 μM or more and 1 mM or less.

In a seventh aspect of the present application, in addition to the configuration of any one of the second to sixth aspects, a DNA degrading enzyme is further added to the blood sample as an anti-clogging agent.

In an eighth aspect of the present application, the DNase is DNase I in addition to the configuration of the seventh aspect.

In a ninth aspect of the present application, in addition to the configuration of the eighth aspect, the DNase I concentration in the blood sample is 0.0002 mg/ml or more and 2 mg/ml or less.

The microfluidic device clogging preventive agent of the tenth aspect of the present application contains a DNA flocculating agent in a buffer solution as a solvent.

In the eleventh aspect of the present application, in addition to the configuration of the tenth aspect, the DNA flocculating agent is polyamine or hexaamminecobalt(III) chloride.

In a twelfth aspect of the present application, in addition to the configuration of the eleventh aspect, the polyamine is linear.

In a thirteenth aspect of the present application, in addition to the configuration of the twelfth aspect, the linear polyamine has a molecular weight of 88 or more and 3000 or less.

In a fourteenth aspect of the present application, in addition to the configuration of the thirteenth aspect, the polyamine is spermidine.

The fifteenth aspect of the present application further contains a DNase in addition to the configuration of any one of the eleventh to fourteenth aspects.

In the sixteenth aspect of the present application, in addition to the configuration of the fifteenth aspect, the DNase is DNase I.

Since the present invention is configured as described above, in the blood component separation method using a microfluidic device, without subjecting the microfluidic device to special treatment or providing a special mechanism, It is possible to reduce clogging.

1 is a schematic diagram showing a microfluidic device according to an embodiment of the present disclosure in plan view; FIG. 2 is an enlarged schematic diagram of the front half of the channel of the microfluidic device of FIG. 1. FIG. 1 is a schematic diagram showing a microfluidic device of an example in a plan view; FIG. FIG. 4 is an enlarged schematic diagram of the channel of the microfluidic device of FIG. 3 ; 4 is a micrograph showing the state of the channel after the blood sample has flowed through the microfluidic device. 4 is an electron micrograph showing the state around the microposts in the channel after the blood sample has flowed through the microfluidic device. 4 is an electron micrograph showing the state around the microposts in the channel after the blood sample has flowed through the microfluidic device. It is a microphotograph obtained by DAPI-staining a channel after a blood sample has flowed through the microfluidic device and performing fluorescence observation of DNA. It is a micrograph obtained by performing fluorescence observation of platelets by staining with an anti-CD61 antibody in a channel after a blood sample has flowed through the microfluidic device. Fig. 10 is a micrograph showing the state of the channel after a blood sample to which spermidine is not added has flowed through the microfluidic device. 2 is a micrograph showing the state of a channel after a blood sample to which 50 μM of spermidine has been added is passed through a microfluidic device. 2 is a micrograph showing the state of a channel after a blood sample to which 500 μM of spermidine has been added is passed through a microfluidic device. 2 is a microphotograph showing the state of a channel after a blood sample to which 1 mM spermidine was added was passed through a microfluidic device. 2 is a micrograph showing the state of a channel after a blood sample added with 5 mM spermidine is passed through a microfluidic device. 2 is a micrograph showing the state of a channel after a blood sample added with 500 μM of spermidine and 0.2 mg/ml of DNase I was allowed to flow through a microfluidic device. 1 is a micrograph showing the state of a channel after a blood sample to which 0.2 mg/ml of DNase I has been added is passed through a microfluidic device.

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic plan view showing a microfluidic device 10 used in the blood component separation method of the present embodiment. The microfluidic device 10 has an inlet 11 located upstream and an outlet 12 located downstream, and a channel 13 is interposed between the inlet 11 and the outlet 12 . The inflow port 11 is composed of a specimen inflow port 11A into which the blood specimen flows and a medium inflow port 11B into which a fluid medium for flowing the blood specimen in the downstream direction flows. A fluid medium is, for example, a suitable buffer. The flow path 13 is composed of a front half portion 13A having a relatively wide width and a rear half portion 13B having a width half that of the front half portion 13A. The total length and width of the flow path 13 can be set variously depending on the object to be measured. In this embodiment, the total length of the flow path 13 is about 11 mm, and the width of the front half 13A of the flow path 13 is about 1 mm. The outflow port 12 includes a first outflow port 12A branched from the terminal end of the front half portion 13A of the flow channel 13, and an outer second outflow port 12B and an inner second outflow port 12B branched and extended from the rear end of the rear half portion 13B. 3 outlets 12C.

The flow path 13 is formed in a flat plate shape having a flow direction as a longitudinal direction, and a micropost array in which a plurality of microposts 20 are arranged in a forest at predetermined intervals as shown in FIG. formed. The micropost 20 has a substantially circular shape in plan view. The size of the microposts 20 can be appropriately set according to the size of the microfluidic device 10 and the object to be measured. In this embodiment, the diameter of the microposts 20 is about 20 μm.

In the front half portion 13A of the channel 13, as shown in FIG. 2, the rows of the microposts 20 in the longitudinal direction are arranged so as to incline rightward in the direction of flow. As a result, the components of the blood specimen that are larger than the predetermined size flow while shifting to the right in the flow direction, and flow out from the first outlet 12A in the middle of the channel 13 .

On the other hand, in the rear half portion 13B of the channel 13, the rows of the microposts 20 in the longitudinal direction are arranged so as to be inclined in the opposite direction to the front half portion 13A. As a result, among the remaining components in the blood sample, relatively large components flow while shifting leftward in the direction of flow, and finally flow out from the second outlet 12B. A relatively small component flows through the center without deviating from the flow direction, and finally flows out from the third outlet 12C.

As described above, the largest components in the blood sample are recovered from the first outlet 12A, the medium ones are recovered from the second outlet 12B, and the smallest ones are recovered according to their sizes. By recovering from the third outlet 12C, it is possible to separate the blood components according to the size of the components.

A DNA aggregating agent is added to the blood sample as an anti-clogging agent that prevents clogging between the microposts 20 . The DNA agglutinating agent as an anti-clogging agent may be added to the blood sample in advance before flowing from the inlet 11, or may be added to the fluid medium. A DNA flocculating agent refers to a compound having the effect of flocculating DNA, and examples thereof include polyamines and hexaamminecobalt (III) chloride. In particular, polyamine has the effect of bending and condensing the DNA strands in the blood sample, so it has the effect of preventing clogging between the microposts 20 caused by DNA in the blood sample. Hexamminecobalt (III) chloride exists as hexaamminecobalt (III) ions in blood samples, and these ions have the effect of aggregating DNA strands.

This polyamine is preferably linear and has a molecular weight of 88 or more and 3000 or less, and more preferably is one or both of spermidine and spermine. Moreover, when this polyamine is spermidine, the concentration of spermidine in the blood sample is desirably 50 μM or more and 1 mM or less. Here, the concentration of spermidine in the blood sample means the final concentration at the stage of flowing through the channel 13 . Concerning the concentration of spermidine, if it is less than 50 μM, the anti-clogging effect is weak, and if it exceeds 1 mM, clogging occurs again. Spermidine has three amino groups, whereas spermine has four amino groups, so that the effect of aggregating DNA is higher.

In addition, it is desirable that a DNA degrading enzyme is further added to the blood sample as the anti-clogging agent. The DNase as an anti-clogging agent may be added to the blood sample in advance before it flows from the inlet 11, or may be added to the fluid medium. By degrading DNA in the blood sample, the DNase has the effect of preventing clogging between the microposts 20 caused by the DNA in the blood sample in combination with the effect of the polyamine described above. This DNase is preferably DNase I, and its concentration in the blood sample is preferably 0.0002 mg/ml or more and 2 mg/ml or less. Here, the concentration of DNase I in the blood sample means the final concentration at the stage of flowing through the channel 13 . When the concentration of DNase I is less than 0.0002 mg/ml, the effect of improving the anti-clogging effect of spermidine is small. On the other hand, the upper limit was set to 2 mg/ml, considering that the anti-clogging effect reaches a plateau even if it exceeds 2 mg/ml.

As the anti-clogging agent for the microfluidic device 10 described above, a buffer containing a DNA-aggregating agent, particularly polyamine, can be used as a solvent. Here, the type of the buffer is not particularly limited as long as it does not affect the components of the blood sample. For example, it is desirable to use phosphate buffered saline (PBS). . As the polyamine, spermidine is desirable as described above. Here, the concentration of spermidine in the antiloading agent may be adjusted to a predetermined concentration (for example, 50 μM or more and 1 mM or less) when the antiloading agent is added to the blood sample. For example, when the antiloading agent is added to the blood sample at a ratio of 1:1, the concentration of spermidine in the antiloading agent is twice the predetermined concentration described above (e.g., 100 μM or more and 2 mM below).

In addition, the anticlogging agent of the microfluidic device 10 preferably further contains a DNase, and the DNase is preferably DNase I as described above. Here, the concentration of DNase I in the anti-clogging agent is set to a predetermined concentration (for example, 0.0002 mg/ml or more and 2 mg/ml or less) when the anti-clogging agent is added to the blood sample. It should be adjusted. For example, if the antiloading agent is added to the blood sample at a 1:1 ratio, the concentration of DNase I in the antiloading agent will be twice the concentration given above (e.g., 0.0004 mg /ml or more and 4 mg/ml or less).

Hereinafter, embodiments of the present disclosure will be described with reference to the drawings.

(1) Appearance of Microfluidic Device FIG. 3 is a schematic plan view showing the microfluidic device 10 used in the examples. A blood sample flows in from the inlet 11 , flows through the channel 13 , and flows out from the outlet 12 . The distance between inlet 11 and outlet 12 is 20 mm, and the width of channel 13 is 2 mm.

In the channel 13, as shown in the enlarged view of FIG. 4, micropost arrays in which microposts 20 having a substantially half-moon shape in plan view and having a maximum diameter of about 20 μm are arranged at intervals of about 20 μm in the width direction. is formed.

(2) Method for Producing Microfluidic Device Two types of the microfluidic device 10 were prepared, one formed based on a silicon substrate and the other formed based on a glass substrate.

(2-1) Silicon substrate A silicon substrate having an oxide film formed on its surface was subjected to patterning by photolithography and wet etching so that the oxide film became a desired micropost array. Using this oxide film as a mask, the silicon layer is etched to a thickness of 50 μm by silicon deep plasma etching (DeepRIE) to form a micropost array as shown in FIG. got

(2-2) Glass substrate A film resist (HM-4056, Hitachi Chemical Co., Ltd.) is laminated on the surface of the glass substrate to a film thickness of 56 μm, exposed to form a desired micropost array, and then developed to form a film resist resin. After forming a micropost array as shown in FIG. 4, the microfluidic device 10 was obtained by covering with a cover glass.

(3) Verification of Clogging Factor Heparinized blood was diluted 1:1 with PBS containing DNase I at a concentration of 0.4 mg/ml to obtain a blood sample. As a result, the final concentration of DNase I in the blood sample was 0.2 mg/ml. 4 ml of this blood sample was fed by a syringe pump at a flow rate of 200 μl/min from the inlet 11 of the microfluidic device 10 made of the silicon substrate, and clogging in the channel 13 was observed. That is, after the blood sample was delivered, only PBS was delivered to wash away unadsorbed blood components. In this state, as shown in the microphotograph of FIG. 5, clogging between the microposts 20 was observed.

The cover glass was removed from the microfluidic device 10 in this state, the micropost array was exposed, a glutaraldehyde solution was dropped, and the device was stored under cold temperature for 3 hours to fix the biological material. Then, after washing off excess glutaraldehyde solution with PBS, freeze-drying was performed by drawing a vacuum while freezing with liquid nitrogen. The dried samples were coated with gold by sputtering and observed with an electron microscope.

As a result, as shown in FIGS. 6 and 7, it was found that a fibrous substance was entangled around the microposts 20, and this substance was found to be the cause of the clogging.

Next, since DNA and platelets were suspected as candidates for this substance, 4',6-diamidino-2-phenylindole (DAPI) and a red fluorescent dye-labeled anti-CD61 antibody were simultaneously administered as shown in FIG. It was applied to the micropost array in the state, and fluorescence observation was performed with a microscope. DAPI has the property of binding to DNA and exhibiting blue fluorescence. Anti-CD61 antibody is an antibody that specifically recognizes CD61, which is a platelet surface antigen.

As a result, first, as shown in FIG. 8, it was recognized that blue fluorescence was abundant in the portions in contact with the microposts 20 . On the other hand, it was found that the red fluorescence shown in FIG. As a result, it was inferred that the DNA was deposited around the micropost 20 before the platelets, and the platelets were adsorbed to the deposited DNA. As a result, it was suggested that the clogging of the microfluidic device 10 is caused by DNA becoming fibrous and entangling with the microposts 20 .

(4) Anti-clogging test Spermidine was added to PBS to prepare an anti-clogging agent so that each final concentration as described later was obtained, and blood was diluted 1:1 with this agent. From the inlet 11 of the microfluidic device 10, the liquid is sent at an average flow rate of 270 mm / second in the channel for 15 minutes, and then only PBS is sent to wash away the unadsorbed blood components. The degree of clogging was observed with an optical microscope.

First, when the final concentration of spermidine in the blood sample was 0 μM, that is, when spermidine was not added to the blood sample, as shown in FIG. 10, severe clogging occurred in the micropost array.

On the other hand, in the blood sample with a final spermidine concentration of 50 μM, as shown in FIG. 11, clogging in the micropost array was remarkably improved, but a large amount of residue was still observed.

Furthermore, in the blood sample with a final spermidine concentration of 500 μM, as shown in FIG. 12, the clogging in the micropost array was further improved and the residue was reduced.

However, as shown in FIG. 13, in the blood sample with a final concentration of spermidine of 1 mM, clogging increased again, although the condition was improved compared to the state in FIG. 10 without spermidine added. was taken.

As shown in FIG. 14, in the blood sample with a final concentration of spermidine of 5 mM, clogging was as severe as in the state of FIG. 10 in which no spermidine was added.

From the above results, clogging-improving effects were observed when the final concentration of spermidine in the blood sample was at least in the range of 50 μM to 1 mM.

Next, in the case of spermidine at a final concentration of 500 μM, which showed the best clogging-improving effect among the above, when DNase I was further added to the blood sample at a final concentration of 0.2 mg/ml, the results shown in FIG. As shown, there was even less clogging. Therefore, it was found that it is more desirable to use spermidine and DNase I together as an antiloading agent.

In addition, when spermidine was not added and only DNase I was added to the blood sample to a final concentration of 0.2 mg/ml, as shown in FIG. Although clogging was improved compared to the state, it was not as good as when only spermidine was added at a final concentration of 500 μM (see FIG. 12).

(5) Conclusion As a result of the above, when the final concentration of spermidine in the blood sample is at least in the range of 50 μM to 1 mM, an improvement effect on clogging is observed, and the improvement effect is further enhanced by further adding DNase I to this. found to do. It was also found that although DNase I alone was effective in improving clogging, it was not as good as spermidine alone at a final concentration of 500 μM.

10 microfluidic device 11 inlet 11A sample inlet 11B medium inlet 12 outlet 12A first outlet 12B second outlet 12C third outlet 13 channel 13A front half 13B rear half 20 micropost

Claims (16)

An inlet located upstream, an outlet located downstream, and a channel located between the inlet and the outlet, wherein a plurality of microposts are arranged at predetermined intervals in the channel. A blood component separation method for separating blood components by an array of microfluidic devices, comprising:
A method for separating blood components, wherein a DNA agglutinating agent is added as an anti-clogging agent to the blood specimen flowing in from the inlet. 2. The method for separating blood components according to claim 1, wherein the DNA agglutinating agent is polyamine or hexaamminecobalt(III) chloride. 3. The method for separating blood components according to claim 2, wherein said polyamine is linear. 4. The method for separating blood components according to claim 3, wherein the linear polyamine has a molecular weight of 88 or more and 3000 or less. 5. The blood component separation method according to claim 4, wherein the polyamine is either one or both of spermidine and spermine. 6. The blood component separation method according to claim 5, wherein the polyamine is spermidine, and the spermidine concentration in the blood sample is 50 μM or more and 1 mM or less. 7. The method for separating blood components according to any one of claims 2 to 6, wherein the blood sample further contains a DNase as an anti-clogging agent. 8. The method for separating blood components according to claim 7, wherein said DNase is DNase I. 9. The blood component separation method according to claim 8, wherein the concentration of said DNase I in said blood sample is 0.0002 mg/ml or more and 2 mg/ml or less. An anti-clogging agent for microfluidic devices, comprising a DNA-aggregating agent contained in a buffer solution as a solvent. 11. The antiloading agent for microfluidic devices according to claim 10, wherein the DNA flocculating agent is polyamine or hexaamminecobalt(III) chloride. 12. The microfluidic device antiloading agent of claim 11, wherein the polyamine is linear. 13. The antiloading agent for microfluidic devices according to claim 12, wherein the linear polyamine has a molecular weight of 88 or more and 3000 or less. 14. The microfluidic device antiloading agent of claim 13, wherein the polyamine is spermidine. 15. The anticlogging agent for microfluidic devices according to any one of claims 11 to 14, further comprising a DNase. 16. The microfluidic device anti-clogging agent of claim 15, wherein the DNase is DNase I.

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