CN117282129B - Serum bilirubin removing device and method - Google Patents
Serum bilirubin removing device and method Download PDFInfo
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- CN117282129B CN117282129B CN202311576323.0A CN202311576323A CN117282129B CN 117282129 B CN117282129 B CN 117282129B CN 202311576323 A CN202311576323 A CN 202311576323A CN 117282129 B CN117282129 B CN 117282129B
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- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 title claims abstract description 150
- 210000002966 serum Anatomy 0.000 title claims abstract description 144
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000000926 separation method Methods 0.000 claims abstract description 125
- 239000011324 bead Substances 0.000 claims abstract description 123
- 238000001914 filtration Methods 0.000 claims abstract description 77
- 239000007788 liquid Substances 0.000 claims abstract description 71
- 239000012528 membrane Substances 0.000 claims abstract description 53
- 230000009471 action Effects 0.000 claims abstract description 31
- 238000001179 sorption measurement Methods 0.000 claims abstract description 24
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 20
- 239000003292 glue Substances 0.000 claims description 18
- 238000007789 sealing Methods 0.000 claims description 16
- 239000002245 particle Substances 0.000 claims description 11
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 9
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- 239000004472 Lysine Substances 0.000 claims description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 8
- 239000011148 porous material Substances 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 5
- 239000012466 permeate Substances 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 4
- 238000007790 scraping Methods 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 230000004888 barrier function Effects 0.000 claims description 2
- 230000021523 carboxylation Effects 0.000 abstract description 3
- 238000006473 carboxylation reaction Methods 0.000 abstract description 3
- 239000000499 gel Substances 0.000 description 22
- 238000005119 centrifugation Methods 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 8
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- 239000002105 nanoparticle Substances 0.000 description 4
- 238000004544 sputter deposition Methods 0.000 description 4
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 2
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- 238000005538 encapsulation Methods 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- 230000036512 infertility Effects 0.000 description 1
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- 210000004185 liver Anatomy 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2676—Centrifugal separation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of serum bilirubin removal, in particular to a serum bilirubin removal device and a method, which are characterized in that an initial serum liquid is trapped at the middle section of a separation tube by using an auxiliary ball and an inert separation gel, meanwhile, nano-scale magnetic beads are subjected to carboxylation treatment, and then bilirubin corresponding adsorption components and carboxylated magnetic beads are subjected to coupling reaction to generate special magnetic beads carrying the adsorption components, the magnetic beads are attached to the tube wall of the separation tube under the action of a magnetic field, so that a filtering membrane at the bottom of the tube is opened for facilitating centrifugal separation, and trace residual serum liquid in the separation tube can be effectively filtered out under the multiple actions of centrifugal separation, magnetic field control and the auxiliary ball, so that all low bilirubin serum samples can be extracted.
Description
Technical Field
The invention belongs to the technical field of serum bilirubin removal, and particularly relates to a serum bilirubin removal device and a serum bilirubin removal method.
Background
Bilirubin is a major metabolite of ferriporphyrin compounds in the human body, and mainly completes the entire metabolic process in the liver. Bilirubin in the blood is greatly increased when a patient suffers from liver disease or under certain factors. Bilirubin has a certain antioxidant effect and is related to the regeneration function of human liver cells.
Bilirubin is a common interfering substance in clinical testing and scientific research testing. The too high bilirubin concentration in serum can cause interference to the detection of almost all serum test items, especially the interference of some important test items such as myocardial enzymes, myocardial infarction markers, blood sugar, electrolytes and the like is very large, thereby causing error of test results and further seriously affecting the accurate judgment of the clinician on the test results. In clinical examination, 3-5 ml of blood of a patient is generally extracted for detection of up to 30-50 examination items, but the current method for removing bilirubin in blood mainly adopts three methods of a microporous filtering membrane, a resin adsorbent and an agarose gel as a carrier and a polysaccharide type bilirubin adsorption material to remove bilirubin, and large-scale equipment is generally adopted for operation, so that the required blood volume is large, and bilirubin removal cannot be carried out on a trace (such as 500 microliters) blood sample required by clinical examination, so that the method cannot be suitable for clinical examination, the bilirubin removal operation of the current clinical examination operation is inconvenient, and the accuracy of a serum examination result is disturbed, and therefore, a bilirubin removal technology favorable for clinical examination needs to be developed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a serum bilirubin removing device and a serum bilirubin removing method, which are used for effectively removing bilirubin from trace blood required by clinical examination, so as to extract trace serum liquid, and the serum bilirubin removing device and the serum bilirubin removing method are suitable for clinical examination, improve the convenience of bilirubin removing operation in clinical examination and ensure the accuracy of serum examination results.
The invention is implemented by the following technical scheme:
the serum bilirubin removing device comprises a separation tube, wherein the separation tube is a tube body with two open ends, a sealing cover is arranged at the inlet end of the separation tube, a filtering end is arranged at the outlet end of the separation tube, and a filtering membrane is arranged in the filtering end;
an auxiliary ball is embedded in the inner cavity of the separation tube, a circle of inert separation glue is adhered to the inner wall of the separation tube, and the inert separation glue is positioned between the auxiliary ball and the inlet end of the separation tube; the end face of the inert separating glue, which faces the auxiliary ball, is provided with an arc concave surface, the arc concave surface of the inert separating glue is matched with the arc convex surface of the auxiliary ball, and the inert separating glue can fill or isolate an annular gap between the auxiliary ball and the inner wall of the separating tube;
the separating tube is used for being embedded in the collecting tube, a magnetic ring is arranged on the periphery of the collecting tube, and a magnetic field acting area of the magnetic ring is overlapped with an inner cavity area of the separating tube.
Further, the filtering membrane is pressed and fixed through the positioning ring, the outer edge of the positioning ring is fixed on the filtering end, and the positioning ring is matched with the separating tube.
Further, the positioning ring and the separating tube form interference fit; the outlet end of the filtering end is a conical outlet.
Further, the assembly clearance between the outer wall of the auxiliary ball and the inner wall of the separation tube is less than or equal to 0.5mm.
The invention also provides a serum bilirubin removing method, which adopts the serum bilirubin removing device and comprises the following steps:
s1: selecting a nanoscale magnetic bead material, and carboxylating the nanoscale magnetic bead material to fix carboxyl functional groups on the surfaces of the magnetic beads;
s2: coupling the conjugate with carboxylated nanoscale magnetic beads to obtain special magnetic beads carrying the conjugate, wherein the conjugate is one or more of lysine, aminoethanol and arginine;
s3: taking the separation tube, opening the outlet end of the separation tube upwards, attaching the auxiliary ball to the inert separation gel, adding serum liquid and special magnetic beads into the separation tube from the outlet end of the separation tube, taking the auxiliary ball as a carrier of the serum liquid, and taking the inert separation gel as a carrier of the auxiliary ball;
embedding a filtering end into an outlet end of a separating tube, covering a sealing cover at an inlet end of the separating tube, taking the sealing cover as a contact medium of mixing equipment, uniformly mixing substances in the separating tube through the mixing equipment, fully dispersing a plurality of special magnetic beads into serum liquid to form a mixed material, taking out the separating tube, standing for a preset time, and adsorbing bilirubin in serum by a conjugate on the surface of the special magnetic beads;
s4: embedding the separating tube into the collecting tube, ensuring that the inlet end of the separating tube and the inlet end of the collecting tube are both upward, enabling the filtering end to be connected with the inner cavity of the collecting tube, ensuring that the periphery of the separating tube is provided with a magnetic ring, enabling special magnetic beads deposited at the bottom of the separating tube and covering the filtering membrane to be adsorbed by the magnetic force of the magnetic ring, enabling the special magnetic beads to be attached and positioned at the inner wall of the separating tube under the action of the magnetic force, and enabling the surface of the covered filtering membrane to be opened;
s5: the collecting pipe connected with the separating pipe is arranged in a centrifugal device, the pipe orifice of the separating pipe is ensured to be upward, the centrifugal device is started to carry out centrifugal separation operation, so that serum liquid in the separating pipe permeates from the open surface of the filtering membrane and drops into the collecting pipe downward, the adsorption force of the magnetic ring on the special magnetic beads is kept, the separated special magnetic beads and bilirubin are obtained in the separating pipe through centrifugal operation, and a filtered serum sample is obtained in the collecting pipe.
In step S2, the conjugate and carboxylated magnetic beads are subjected to a coupling reaction, wherein the ratio of the conjugate to the carboxylated nanoscale magnetic beads is 1:100-1:200.
Further, in step S3, adding serum liquid and special magnetic beads into the separation tube from the outlet end of the separation tube, wherein the mass ratio of the serum liquid to the special magnetic beads is 10: 1-20:1.
Further, in step S3, the mixing device is a vortex mixer.
Further, in step S5, the centrifugal device is started to perform centrifugal separation, and the auxiliary ball generates a scraping effect on the serum liquid on the pipe wall under the action of centrifugal force, so as to promote the permeation of the serum liquid from the open surface of the filtering membrane.
Further, in step S5, the rotational speed of the centrifugal separation operation is 1000r/min to 2000r/min.
In step S5, the time for the centrifugal separation operation is 4 to 6 minutes.
Further, the average filter pore diameter of the filter membrane is smaller than the average particle diameter of the nanoscale magnetic beads.
Further, the average particle diameter of the nano-scale magnetic beads is 200 nm-400 nm, and the average filter pore diameter of the filter membrane is 30 nm-50 nm.
The beneficial effects of the invention are as follows: according to the invention, the initial serum liquid is trapped at the middle section of the separating tube by using the auxiliary ball and the inert separating gel, so that full separation of all serum samples is facilitated, meanwhile, a large number of carboxyl groups are fixed on the surfaces of the magnetic beads by carboxylation treatment of the nanoscale magnetic beads, then, the corresponding adsorption components of bilirubin are subjected to coupling reaction with the carboxylated magnetic beads, so that a large number of special magnetic beads carrying the adsorption components are finally generated, the coupled special magnetic beads can be used for adsorbing serum bilirubin, and the nanoscale size of the special magnetic beads can provide a larger adsorption contact area, so that the full bilirubin adsorption efficiency is ensured; and then the magnetic beads are attached to the wall of the separation tube under the action of a magnetic field, so that the filter membrane at the bottom of the tube is opened to facilitate centrifugal separation, and the special magnetic beads are not blocked by the filtering operation due to the position control action of the magnetic field.
Drawings
FIG. 1 is a process flow diagram of an embodiment of the present invention;
FIG. 2 is a schematic diagram showing a serum bilirubin removal apparatus according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing a loading state according to an embodiment of the present invention;
FIG. 4 is a schematic diagram illustrating a blending operation according to an embodiment of the present invention;
FIG. 5 is a diagram illustrating an embodiment of a separation structure according to the present invention;
FIG. 6 is a schematic diagram illustrating a separation state according to an embodiment of the present invention;
FIG. 7 is a schematic diagram illustrating a centrifugal operation according to an embodiment of the present invention.
In the figure: 10-separating tube, 10 a-sealing cover, 10 b-inert separating gel, 11-filtering end, 11 a-filtering membrane, 11 b-positioning ring, 12-special magnetic beads, 20-collecting tube, 21-mixed material, 22-serum liquid, 23-serum sample, 30-magnetic ring, 40-auxiliary ball, 50-vortex mixer, 60-centrifuge and 61-rotary disk.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples of the specification.
Example 1
As shown in fig. 2, a serum bilirubin removal apparatus comprises a separation tube 10, wherein the separation tube 10 is a tube body with two open ends, a sealing cover 10a is arranged at the inlet end of the separation tube 10, a filtering end 11 is arranged at the outlet end of the separation tube 10, and a filtering membrane 11a is arranged in the filtering end 11; therefore, the detachability of the filtering end 11 and the filtering membrane 11a is ensured, the inverted sample adding of the separating tube 10 is facilitated, the filtering membrane 11a is not affected in advance, and the quality of the subsequent separated serum is further ensured;
an auxiliary ball is embedded in the inner cavity of the separation tube 10, a circle of inert separation gel 10b is adhered to the inner wall of the separation tube 10, and the inert separation gel 10b is positioned between the auxiliary ball and the inlet end of the separation tube 10; the end face of the inert separating glue 10b facing the auxiliary ball is provided with an arc concave surface, the arc concave surface of the inert separating glue 10b is matched with the arc convex surface of the auxiliary ball, and the inert separating glue 10b can fill and separate an annular gap between the auxiliary ball and the inner wall of the separating tube 10; due to the structural design of the auxiliary ball embedded into the separating tube 10, when the separating tube 10 is inverted to hold serum liquid, most of serum liquid can be blocked by the auxiliary ball, the assembly gap between the auxiliary ball and the inner wall of the separating tube 10 is blocked by the inert separating glue 10b, so that the serum liquid is further ensured not to leak from the assembly gap at the edge of the auxiliary ball, the sealing cover 10a and the tube wall close to the inlet end are prevented from being adhered by the serum liquid, and all the serum liquid can be blocked at the middle section of the tube body of the separating tube 10 by the auxiliary ball, so that all serum samples are promoted to be in a separating action area, and the subsequent separating operation is facilitated, so that all the serum liquid can be fully separated;
meanwhile, the inert separation gel 10b is a semi-solidified substance special for serum separation operation in the industry, and the components of the inert separation gel have hydrophobicity, are not mixed with serum liquid and do not interfere with serum detection projects, so that the inert separation gel 10b is adhered and fixed in a concave area formed by an auxiliary ball and the separation tube 10 in a filling and smearing mode, thereby having good sealing and blocking effects and not affecting the subsequent serum bilirubin detection result;
the separating tube 10 is used for being embedded in the collecting tube, the periphery of the collecting tube is provided with a magnetic ring, and the magnetic field action area of the magnetic ring is overlapped with the inner cavity area of the separating tube 10, so that the magnetic ring can be ensured to effectively adsorb magnetic beads of the subsequent mixed material 21, the filtering membrane 11a is fully opened, and the smooth implementation of the centrifugation operation is ensured.
In this embodiment, the filtering membrane 11a is pressed and fixed by the positioning ring 11b, the outer edge of the positioning ring 11b is fixed on the filtering end 11, the positioning ring 11b is matched with the separating tube 10, the positioning ring 11b is used for fixing the physical form of the filtering membrane 11a, and meanwhile, the positioning ring 11b is used as an embedding body for butt-jointing the separating tube 10, so that the removability of the filtering end 11 is ensured.
In this embodiment, the positioning ring 11b forms an interference fit with the separating tube 10, so as to ensure the mechanical strength of the assembly of the filtering end 11 and the separating tube 10 (enough to withstand the subsequent centrifugal force);
preferably, adhesive glue can be arranged at the outer edge of the positioning ring 11b and the attaching area of the inner wall of the separating tube 10, the type of the adhesive glue is inert separating glue, and the mechanical strength and the tightness of the assembly of the separating tube 10 are further ensured by utilizing the viscosity of the inert separating glue.
In this embodiment, the outlet end of the filtering end 11 is a conical outlet, which is beneficial for centralized drainage of separated serum liquid and falls into the collection tube.
In this embodiment, the assembly gap between the outer wall of the auxiliary ball and the inner wall of the separation tube 10 is less than or equal to 0.5mm, so that the assembly gap between the outer edge of the auxiliary ball is ensured to be small enough, and a certain sliding property can be maintained.
As shown in fig. 1, this embodiment also provides a serum bilirubin removal method, which adopts the serum bilirubin removal apparatus described above, and includes the following steps:
s1: selecting nanoscale magnetic bead materials, wherein the average particle size of the nanoscale magnetic beads is 400nm, and carboxylating the nanoscale magnetic bead materials to fix carboxyl functional groups on the surfaces of the magnetic beads so as to obtain a base material required by coupling, and meanwhile, the nanoscale particle size can be beneficial to subsequent full adsorption operation;
s2: the arginine and carboxylated nanoscale magnetic beads are subjected to coupling reaction, the mass ratio of the arginine to the nanoscale magnetic beads is 1:100, and the special magnetic beads 12 carrying the arginine are obtained, and the arginine can be used for adsorbing serum bilirubin, and meanwhile, the coupling carrying effect of the magnetic beads is utilized, so that the arginine carried by the magnetic beads and the bilirubin adsorbed by the arginine can be subjected to magnetic field-assisted separation operation in the follow-up process;
s3: as shown in fig. 3, the separation tube 10 is taken, the outlet end of the separation tube is opened upwards, the auxiliary ball 40 falls under the action of the dead weight of the separation tube and is attached to the arc concave surface of the inert separation gel 10b, and meanwhile, the inert separation gel 10b is embedded into a groove gap formed by the auxiliary ball 40 and the separation tube 10 to form a sealing barrier by utilizing the semi-solidification property of the inert separation gel 10 b;
serum liquid and special magnetic beads 12 are added into the separation tube 10 from the outlet end (as above in fig. 3) of the separation tube 10, and the mass ratio of the serum liquid to the special magnetic beads 12 is 10:1, at this time, the auxiliary ball 40 is used as a carrier of serum liquid, and the inert separating gel 10b is used as a carrier of auxiliary ball, so that the serum liquid can be trapped in the middle section of the separating tube 10, and the wall of the inlet end of the separating tube 10 and the sealing cover 10a can not be adhered by serum, so that all the serum liquid is ensured to be in a separating action area;
preferably, a certain distance is reserved between the liquid surface of the serum liquid 22 and the port of the outlet end of the separation tube 10, so that the serum liquid is prevented from adhering to the upper part in advance, and the quality of the subsequent separation operation is further ensured;
as shown in fig. 4, the filtering end 11 is embedded into the outlet end of the separating tube 10, the sealing cover 10a is ensured to cover the inlet end of the separating tube 10, the sealing cover 10a is used as a contact medium of a mixing device, the mixing operation is carried out on the mixed material 21 in the separating tube 10 through the vortex mixer 50 (the bottom sealing cover 10a is used as a force transmission medium of the mixing operation), a plurality of special magnetic beads 12 are fully dispersed in serum liquid 22, the separating tube 10 is taken out and kept stand for a preset time, and the conjugate on the surface of the special magnetic beads 12 is used for adsorbing bilirubin in serum; the nanoscale size of the magnetic beads can provide a larger adsorption contact area through the adsorption, encapsulation and uniform mixing contact action of arginine, so that the sufficient bilirubin adsorption efficiency is ensured;
in other embodiments, the separating tube 10 may also adopt an inclined posture for mixing, and the mixing operation can be normally performed only by ensuring that the sealing cover 10a contacts with the end face of the vortex mixer 50 and the separating tube 10 is in an inverted state with the outlet end facing upwards;
meanwhile, the vibration frequency of the vortex mixer 50 is controlled to be less than or equal to 120n/min, preferably 120n/min, so that the mixed material 21 is prevented from sputtering under the low-speed mixing effect, the filtering membrane 11a is prevented from being influenced by sputtering, and meanwhile, the sufficient mixing of serum liquid and the special magnetic beads 12 can be ensured under the frequency effect.
S4: as shown in fig. 5, the separating tube 10 is embedded in the collecting tube 20, the inlet end of the separating tube 10 and the inlet end of the collecting tube 20 are both upward, the filtering end 11 is connected with the inner cavity of the collecting tube 20, and the magnetic ring 30 is arranged on the periphery of the separating tube 10; specifically, in order to ensure that the magnetic ring 30 is not polluted by sputtering, the magnetic ring 30 is arranged on the outer wall surface of the collecting pipe 20 to form a sleeving structure of the magnetic ring 30, the collecting pipe 20 and the separating pipe 10, the special magnetic beads 12 deposited at the bottom of the separating pipe 10 and covering the filtering membrane 11a are adsorbed by the magnetic force of the magnetic ring 30, the special magnetic beads 12 are attached and positioned at the inner wall of the separating pipe 10 under the action of the magnetic force, the surface of the covered filtering membrane 11a is promoted to be opened, so that the opened filtering membrane 11a fully performs the centrifugation operation, and the magnetic beads are adsorbed on the side wall of the pipe body due to the position control action of the magnetic field, thus the blocking of the filtering operation is not caused, and the centrifugal filtration in the follow-up centrifugation operation is facilitated to extract trace and residual serum liquid 22;
preferably, in this step, the type of the filtering membrane 11a is a nano-sized polycarbonate filtering membrane, which ensures the sterility of the centrifugal filtering environment; the average filter pore diameter of the filter membrane 11a is smaller than the average particle diameter of the magnetic beads, specifically, the average filter pore diameter of the filter membrane 11a is 50nm, so that 400nm magnetic bead particles are effectively intercepted, and a smooth serum filter channel is provided;
s5: as shown in fig. 7, the collecting tube 20, which is provided with the separating tube 10, is placed in the rotary table 61 of the centrifuge 60, the mouth of the separating tube 10 and the cover 10a are kept upward, a balance tube body of the same weight as that used for balancing the rotary table 61 is mounted in the opposite side direction of the separating tube 10, the centrifuge 60 is started to rotate the rotary table 61, the centrifugal separation operation is performed, the rotary table 61 for the centrifugal separation operation rotates at 1000r/min for 4min, the serum liquid 22 in the separating tube 10 permeates through the open surface of the filtering membrane 11a and drops down into the collecting tube 20, the adsorption force of the magnetic ring 30 to the special magnetic beads 12 is maintained at the inner wall of the separating tube 10, the special magnetic beads 12 and bilirubin adsorbed by the special magnetic beads 12 are obtained in the separating tube 10 by the centrifugal operation, and the filtered serum sample 23 is obtained in the collecting tube 20 in the state as shown in fig. 6.
In addition, according to the semi-solidification property of the inert separation gel 10b, the inert separation gel 10b can not generate obvious flow phenomenon at the centrifugal speed of 3000r/min, so that the inert separation gel 10b can basically keep still or generate slight sliding displacement under the low-speed centrifugal action of the embodiment;
in addition, in other embodiments, the centrifuge 60 adopts the ultracentrifugation operation, at this time, the inert separating gel 10b has fluidity, and since the auxiliary ball 40 is blocked between the inert separating gel 10b and the serum liquid 22 and the inert separating gel 10b itself has viscosity, the inert separating gel 10b is difficult to penetrate from the assembling gap of less than 0.5mm at the edge of the auxiliary ball 40, so even in the ultracentrifugation state, the fluidity of the inert separating gel 10b will not affect the centrifugation operation of the serum liquid under the blocking action of the auxiliary ball 40.
Meanwhile, in the present embodiment, under the action of centrifugal force, the auxiliary ball 40 moves downward together with the serum liquid 22 under the action of centrifugal force, and during the downward movement of the auxiliary ball 40, the side wall of the ball can scrape the residual serum liquid 22 adhered to the inner wall of the separation tube 10, so that the residual serum liquid 22 is scraped to the bottom of the separation tube 10; in addition, the special magnetic beads 12 adsorbed on the inner wall of the separation tube 10 can be extruded into and occupy the corner space of the tube bottom (the position shown in fig. 6) under the double blocking action of the auxiliary ball 40 and the filtering membrane 11a, so as to further promote the residual serum liquid 22 on the tube wall and the tube bottom to be extruded out of the filtering membrane 11a, finally promote the residual serum liquid 22 to be completely filtered out of the membrane holes of the filtering membrane 11a under the action of centrifugal force, and obtain the filtered whole serum sample 23 in the collecting tube 20. The mode can further ensure that a trace amount of serum liquid is fully separated and extracted through the action of the auxiliary ball 40, so that bilirubin removal operation is effectively performed on trace amount of blood required by clinical examination, the convenience of bilirubin removal operation in clinical examination is improved, and the accuracy of a serum examination result is ensured.
In this embodiment, the separating tube 10, the collecting tube 20 and the auxiliary ball 40 are made of medical plastic materials, so as to ensure that the subsequent serum test items are not interfered by impurities.
In this embodiment, the TBIL index detection is performed on the serum sample 23 after separation and filtration, and at the same time, since the serum sample 23 is derived from the serum liquid of the hepatitis patient, and the serum liquid (not subjected to separation operation) of the same batch of hepatitis patients is subjected to comparative detection, the total bilirubin of the serum liquid not subjected to separation operation is 232.1 μmol/L, and the total bilirubin of the serum sample 23 after filtration is 19.3 μmol/L, so that it is confirmed that the serum bilirubin removal method provided in this embodiment has an obvious bilirubin separation effect.
In this embodiment, a precision electronic scale is used to measure the total mass of the collection tube 20 and the collected serum sample in this embodiment, record the weight data m1= 3.4843g, and subtract the original weight of the collection tube 20 (m0= 3.0829) to obtain the mass m1= 0.4014g of the collected serum sample, which is used as a reference standard for the subsequent comparative analysis;
in the centrifugation process, the magnetic beads are fixed on the side wall by the magnetic field, so that the centrifugation process is not blocked by the magnetic beads, and residual serum can be smoothly filtered out, the magnetic beads and bilirubin components coupled and adsorbed by the magnetic beads are fixed on the side wall of the tube body by the magnetic ring 30, separation of bilirubin is further promoted, trace residual serum liquid 22 in the separation tube 10 can be effectively filtered out under the multiple actions of centrifugation, magnetic field control and auxiliary balls, and then all low bilirubin serum samples 23 are extracted.
Example two
This example provides embodiments of different conjugate components comprising the steps of:
s1: selecting a nanoscale magnetic bead material, wherein the average particle size of the nanoscale magnetic beads is 200nm, and providing more reaction area for a conjugate (amino ethanol) with smaller molecular weight; carboxylation treatment is carried out on the nanoscale magnetic bead material, so that carboxyl functional groups are fixed on the surface of the magnetic bead, thus obtaining a base material required by coupling, and meanwhile, the nanoscale particle size can be beneficial to the subsequent full adsorption operation;
s2: coupling reaction is carried out on the amino ethanol and carboxylated magnetic beads, the mass ratio of the amino ethanol to the nanoscale magnetic beads is 1:200, and the special magnetic beads 12 carrying the amino ethanol are obtained, and the amino ethanol can be used for adsorbing serum bilirubin, and meanwhile, the coupling carrying effect of the magnetic beads is utilized, so that the amino ethanol carried by the magnetic beads and the bilirubin adsorbed by the amino ethanol can be subjected to magnetic field auxiliary separation operation in the follow-up process;
s3: taking a separation tube 10, opening the outlet end of the separation tube upwards, and adding serum liquid and special magnetic beads 12 into the separation tube 10 from the outlet end of the separation tube 10, wherein the mass ratio of the serum liquid to the special magnetic beads 12 is 20:1, uniformly mixing substances in a separation tube 10 by a vortex mixer to ensure that a plurality of special magnetic beads 12 are fully dispersed in serum liquid 22, taking out the separation tube 10, standing for a preset time, and adsorbing bilirubin in serum by conjugates on the surfaces of the special magnetic beads 12; the nanoscale size of the magnetic beads can provide a larger adsorption contact area through the adsorption, encapsulation and uniform mixing contact action of the amino alcohol, so that the sufficient bilirubin adsorption efficiency is ensured;
s4: the separation tube 10 is embedded into the collecting tube 20, the inlet end of the separation tube 10 and the inlet end of the collecting tube 20 are both upward, the filtering end 11 is connected with the inner cavity of the collecting tube 20, the magnetic ring 30 is arranged on the outer wall surface of the collecting tube 20 to form a sleeve structure of the magnetic ring 30-the collecting tube 20-the separation tube 10, the special magnetic beads 12 which are deposited at the bottom of the separation tube 10 and cover the filtering membrane 11a are adsorbed by the magnetic force of the magnetic ring 30, the special magnetic beads 12 are attached and positioned at the inner wall of the separation tube 10 under the action of the magnetic force, the surface of the covered filtering membrane 11a is made to be open, so that the opened filtering membrane 11a can fully implement the centrifugation operation, and the magnetic beads are adsorbed on the side wall of the tube body due to the position control action of the magnetic field, so that the blocking of the filtering operation cannot be caused, and the centrifugal filtering operation is facilitated to extract trace and residual serum liquid 22;
preferably, the average filter pore diameter of the filter membrane 11a is 30nm, so that the magnetic bead particles of 200nm are effectively intercepted;
s5: the collecting tube 20 connected with the separating tube 10 is put into a centrifugal device, the mouth of the separating tube 10 is ensured to be upward, the centrifugal device is started to perform centrifugal separation, and as the magnetic bead particle size is smaller in the embodiment, more sufficient centrifugal effect is required, the rotating speed of the centrifugal separation is set to 2000r/min, the centrifugal time is set to 6min, the serum liquid 22 in the separating tube 10 permeates from the open surface of the filtering membrane 11a and drops into the collecting tube 20 downward, the adsorption force of the magnetic ring 30 on the special magnetic bead 12 is maintained, the special magnetic bead 12 and bilirubin adsorbed by the special magnetic bead 12 are maintained at the inner wall of the separating tube 10, the separated special magnetic bead 12 and bilirubin are obtained in the separating tube 10 through the centrifugal operation, and the filtered serum sample 23 is obtained in the collecting tube 20.
Example III
This example provides embodiments of different conjugate components comprising the steps of:
s1: selecting a nanoscale magnetic bead material, wherein the average particle size of the nanoscale magnetic beads is 300nm, and carboxylating the nanoscale magnetic bead material to fix carboxyl functional groups on the surfaces of the magnetic beads so as to obtain a base material required by coupling, and meanwhile, the nanoscale particle size of the base material can be beneficial to subsequent full adsorption operation;
s2: coupling reaction is carried out on lysine and carboxylated nanoscale magnetic beads, the mass ratio of the lysine to the nanoscale magnetic beads is 1:90, and the special magnetic beads 12 carrying the lysine are obtained, and the lysine can be used for adsorbing serum bilirubin, and meanwhile, the coupling carrying effect of the magnetic beads is utilized, so that the subsequent magnetic field assisted separation operation of the lysine carried by the magnetic beads and the bilirubin adsorbed by the magnetic beads can be facilitated;
s3: taking a separation tube 10, opening the outlet end of the separation tube upwards, adding serum liquid and special magnetic beads 12 into the separation tube 10 from the outlet end of the separation tube 10, wherein the mass ratio of the serum liquid to the special magnetic beads 12 is 9:1, uniformly mixing substances in a separation tube 10 by a vortex mixer to ensure that a plurality of special magnetic beads 12 are fully dispersed in serum liquid 22, taking out the separation tube 10, standing for a preset time, and adsorbing bilirubin in serum by conjugates on the surfaces of the special magnetic beads 12; the nanoscale size of the magnetic beads can provide a larger adsorption contact area through lysine adsorption wrapping and uniform mixing contact action, so that the sufficient bilirubin adsorption efficiency is ensured;
s4: as shown in fig. 2, the separating tube 10 is embedded in the collecting tube 20, the inlet end of the separating tube 10 and the inlet end of the collecting tube 20 are both upward, the filtering end 11 is connected with the inner cavity of the collecting tube 20, and the magnetic ring 30 is arranged on the periphery of the separating tube 10; specifically, in order to ensure that the magnetic ring 30 is not polluted by sputtering, the magnetic ring 30 is arranged on the outer wall surface of the collecting pipe 20 to form a sleeving structure of the magnetic ring 30, the collecting pipe 20 and the separating pipe 10, the special magnetic beads 12 deposited at the bottom of the separating pipe 10 and covering the filtering membrane 11a are adsorbed by the magnetic force of the magnetic ring 30, the special magnetic beads 12 are attached and positioned at the inner wall of the separating pipe 10 under the action of the magnetic force, the surface of the covered filtering membrane 11a is promoted to be opened, so that the opened filtering membrane 11a fully performs the centrifugation operation, and the magnetic beads are adsorbed on the side wall of the pipe body due to the position control action of the magnetic field, thus the blocking of the filtering operation is not caused, and the centrifugal filtration in the follow-up centrifugation operation is facilitated to extract trace and residual serum liquid 22;
preferably, the average filter pore diameter of the filter membrane 11a is 40nm, so that magnetic bead particles of 300nm are effectively intercepted;
s5: embedding an auxiliary ball 40 in the inner cavity of the separation tube 10, so that the auxiliary ball 40 is positioned above the liquid level of the serum liquid 22, and the assembly gap between the outer wall of the auxiliary ball 40 and the inner wall of the separation tube 10 is less than or equal to 0.5mm, thereby keeping the slidability of the auxiliary ball 40, and ensuring that the auxiliary ball 40 is a hollow plastic ball and the property of a specimen is not influenced; the collecting tube 20 connected with the separating tube 10 is put into a centrifugal device, the mouth of the separating tube 10 is ensured to be upward, the centrifugal device is started to perform centrifugal separation operation, the rotating speed of the centrifugal separation operation is set to be 1500r/min, the centrifugal time is set to be 5min, serum liquid 22 in the separating tube 10 permeates from the open surface of the filtering membrane 11a and drops into the collecting tube 20 downwards, the adsorption force of the magnetic ring 30 on the special magnetic beads 12 is maintained, the separated special magnetic beads 12 and bilirubin are obtained in the separating tube 10 through the centrifugal operation, the state shown in fig. 6 is formed, and a filtered serum sample 23 is obtained in the collecting tube 20.
Comparative example one
Performing a comparison experiment according to the same experimental data of the first embodiment, omitting the auxiliary ball 40 and the inert separation gel 10b in the first embodiment, putting the separation tube 10 and the collection tube 20 without the auxiliary ball 40 into a centrifugal device for centrifugation, and collecting a comparison serum sample 23;
measuring the total mass of the collecting pipe and the collected serum sample in the first comparative example by using a precise electronic scale, recording weight data M2= 3.3969g, and subtracting the original weight (M0= 3.0829) of the collecting pipe to obtain the mass m2=0.314 g of the collected serum sample, wherein the mass m2=0.314 g is used as a reference standard for comparative analysis;
comparison of the serum weight data m2 in this comparative example with the serum weight data m1 of example one gave the following comparative analysis table:
from the above comparative analysis table, it can be confirmed that the amount of the serum sample 23 collected in the first embodiment using the auxiliary ball 40 is significantly larger than that in the first embodiment without using the auxiliary ball 40, and the auxiliary ball 40 has a scraping guiding function for the residual adhering serum liquid, so that the collection rate of the serum sample 23 can be further improved, and the guiding function of the auxiliary ball can further ensure that the trace serum sample 23 is sufficiently separated and extracted.
The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any way, and it should be understood that the embodiments may be modified, altered, and substituted in other equivalent ways within the scope of the features defined in the claims, and all such modifications are intended to be included in the scope of the present invention.
Claims (9)
1. A serum bilirubin removing method adopts a serum bilirubin removing device, wherein the serum bilirubin removing device comprises a separating tube, and is characterized in that: the separation pipe is a pipe body with two open ends, the inlet end of the separation pipe is provided with a sealing cover, the outlet end of the separation pipe is provided with a filtering end, and a filtering membrane is arranged in the filtering end;
an auxiliary ball is embedded in the inner cavity of the separation tube, a circle of inert separation glue is adhered to the inner wall of the separation tube, and the inert separation glue is positioned between the auxiliary ball and the inlet end of the separation tube; the end face of the inert separating glue, which faces the auxiliary ball, is provided with an arc concave surface, the arc concave surface of the inert separating glue is matched with the arc convex surface of the auxiliary ball, and the inert separating glue can fill or isolate an annular gap between the auxiliary ball and the inner wall of the separating tube;
the separating tube is used for being embedded in the collecting tube, a magnetic ring is arranged on the periphery of the collecting tube, and a magnetic field acting area of the magnetic ring is overlapped with an inner cavity area of the separating tube;
under the action of centrifugal force, the auxiliary ball moves downwards together with the serum liquid under the action of centrifugal force, and in the downward moving process of the auxiliary ball, the side wall of the ball body can scratch the residual serum liquid adhered to the inner wall of the separation tube, so that the residual serum liquid is scraped to the bottom of the separation tube; the special magnetic beads adsorbed on the inner wall of the separation tube are extruded into and occupy the corner space of the tube bottom under the double barrier action of the auxiliary ball and the filtering membrane, so that the residual serum liquid on the tube wall and the tube bottom is promoted to be extruded to the transparent filtering membrane, and finally the residual serum liquid is promoted to be completely filtered out of the membrane holes of the filtering membrane under the centrifugal force action, and a filtered whole serum sample is obtained in the collecting tube;
the method comprises the following steps:
s1: selecting a nanoscale magnetic bead material, and carboxylating the nanoscale magnetic bead material to fix carboxyl functional groups on the surfaces of the magnetic beads;
s2: coupling the conjugate with carboxylated nanoscale magnetic beads to obtain special magnetic beads carrying the conjugate, wherein the conjugate is one or more of lysine, aminoethanol and arginine;
s3: taking the separation tube, opening the outlet end of the separation tube upwards, attaching the auxiliary ball to the inert separation gel, adding serum liquid and special magnetic beads into the separation tube from the outlet end of the separation tube, taking the auxiliary ball as a carrier of the serum liquid, and taking the inert separation gel as a carrier of the auxiliary ball;
embedding a filtering end into an outlet end of a separating tube, covering a sealing cover at an inlet end of the separating tube, taking the sealing cover as a contact medium of mixing equipment, uniformly mixing substances in the separating tube through the mixing equipment, fully dispersing a plurality of special magnetic beads into serum liquid to form a mixed material, taking out the separating tube, standing for a preset time, and adsorbing bilirubin in serum by a conjugate on the surface of the special magnetic beads;
s4: embedding the separating tube into the collecting tube, ensuring that the inlet end of the separating tube and the inlet end of the collecting tube are both upward, enabling the filtering end to be connected with the inner cavity of the collecting tube, ensuring that the periphery of the separating tube is provided with a magnetic ring, enabling special magnetic beads deposited at the bottom of the separating tube and covering the filtering membrane to be adsorbed by the magnetic force of the magnetic ring, enabling the special magnetic beads to be attached and positioned at the inner wall of the separating tube under the action of the magnetic force, and enabling the surface of the covered filtering membrane to be opened;
s5: the collecting pipe connected with the separating pipe is arranged in a centrifugal device, the pipe orifice of the separating pipe is ensured to be upward, the centrifugal device is started to carry out centrifugal separation operation, so that serum liquid in the separating pipe permeates from the open surface of the filtering membrane and drops into the collecting pipe downward, the adsorption force of the magnetic ring on the special magnetic beads is kept, the separated special magnetic beads and bilirubin are obtained in the separating pipe through centrifugal operation, and a filtered serum sample is obtained in the collecting pipe.
2. The method for removing serum bilirubin as claimed in claim 1, wherein: the filtering membrane is fixed by pressing through a positioning ring, the outer edge of the positioning ring is fixed on the filtering end, and the positioning ring is matched with the separating tube.
3. The method for removing serum bilirubin as claimed in claim 2, wherein: the positioning ring and the separating tube form interference fit; the outlet end of the filtering end is a conical outlet.
4. The method for removing serum bilirubin as claimed in claim 1, wherein: the assembly clearance between the outer wall of the auxiliary ball and the inner wall of the separation tube is less than or equal to 0.5mm.
5. The method for removing serum bilirubin as claimed in claim 1, wherein: in the step S2, the conjugate and carboxylated nanoscale magnetic beads are subjected to a coupling reaction, wherein the ratio of the conjugate to the carboxylated nanoscale magnetic beads is 1:100-1:200.
6. The method for removing serum bilirubin as claimed in claim 1, wherein: in the step S3, adding serum liquid and special magnetic beads into the separation tube from the outlet end of the separation tube, wherein the mass ratio of the serum liquid to the special magnetic beads is 10: 1-20:1.
7. The method for removing serum bilirubin as claimed in claim 1, wherein: in step S5, the centrifugal device is started to perform centrifugal separation operation, and the auxiliary ball generates a scraping effect on the serum liquid on the pipe wall under the action of centrifugal force, so that the permeation of the serum liquid from the open surface of the filtering membrane is promoted.
8. The method for removing serum bilirubin as claimed in claim 1, wherein: in the step S5, the rotation speed of centrifugal separation operation is 1000 r/min-2000 r/min; the time of the centrifugal separation operation is 4 min-6 min.
9. The method for removing serum bilirubin as claimed in claim 1, wherein: the average pore diameter of the filtering membrane is smaller than the average particle diameter of the nanoscale magnetic beads;
the average grain diameter of the nano-scale magnetic beads is 200 nm-400 nm, and the average filter pore diameter of the filter membrane is 30 nm-60 nm.
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