CN117147511A - Time-resolved fluorescence immune flow type optical detection device and method based on magnetic particles - Google Patents
Time-resolved fluorescence immune flow type optical detection device and method based on magnetic particles Download PDFInfo
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- CN117147511A CN117147511A CN202311025204.6A CN202311025204A CN117147511A CN 117147511 A CN117147511 A CN 117147511A CN 202311025204 A CN202311025204 A CN 202311025204A CN 117147511 A CN117147511 A CN 117147511A
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- 239000006249 magnetic particle Substances 0.000 title claims abstract description 70
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 230000003287 optical effect Effects 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 8
- 230000005284 excitation Effects 0.000 claims abstract description 21
- 239000012530 fluid Substances 0.000 claims abstract description 13
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 102000036639 antigens Human genes 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 10
- 229910052747 lanthanoid Inorganic materials 0.000 claims abstract description 9
- 150000002602 lanthanoids Chemical class 0.000 claims abstract description 9
- 238000003018 immunoassay Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 3
- 238000007689 inspection Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000001914 filtration Methods 0.000 abstract description 2
- 239000011324 bead Substances 0.000 description 18
- 239000004005 microsphere Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 201000009310 astigmatism Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The invention provides a time-resolved fluorescence immune flow type optical detection device and a method based on magnetic particles, wherein the time-resolved fluorescence immune flow type optical detection device based on the magnetic particles comprises a capillary, a conveying unit and a detection unit, the conveying unit is used for conveying the magnetic particles and non-Newtonian fluid into the capillary, the detection unit comprises a light source, a first focusing lens and a detector which are sequentially arranged, excitation light emitted by the light source passes through the first focusing lens and then is focused in the capillary, and the detector receives optical signals; further comprises: fluorescent light emitted by magnetic particles positioned at the downstream of the focusing point of the excitation light in the capillary tube sequentially passes through a second converging lens and a light filtering device and is received by a detector; the lanthanide element marks the antibody or antigen on the magnetic particles; the processor is used for sending a signal acquisition instruction to the detector after the detector receives the optical signal and the interval time delta t. The invention has the advantages of high sensitivity and the like.
Description
Technical Field
The invention relates to single molecule detection, in particular to a time-resolved fluorescence immune flow type optical detection device and method based on magnetic particles.
Background
Compared with the traditional chemiluminescence method, the flow cytometry detection technology has the greatest advantages that multi-parameter high-low flux analysis can be carried out on tens of thousands of cell particulate matters in one second, and the flow cytometry detection technology has the advantages of being high in speed, high in precision and good in accuracy, and is one of the most advanced cell quantitative analysis technologies in the current generation. At present, the flow cell detection fluorescent signal has a plurality of influencing factors, is interfered by nonspecific fluorescence, has high background value, low detection sensitivity and low repeatability. In practical immunoflow detection, the magnetic bead microsphere is used as a solid phase carrier, the antibody and the antigen are specifically combined on the magnetic bead to form an antigen-antibody complex, and the luminescent substance is specifically combined with the immunomagnetic bead to form a fluorescent marker. Meanwhile, the magnetic bead microsphere of the capture antibody has a non-negligible autofluorescence signal, so that the sensitivity of fluorescence detection is seriously reduced.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a time-resolved fluorescence immune flow type optical detection device based on magnetic particles.
The invention aims at realizing the following technical scheme:
the time-resolved fluorescence immune flow type optical detection device based on the magnetic particles comprises a capillary, a conveying unit and a detection unit, wherein the conveying unit is used for conveying the magnetic particles and non-Newtonian fluid into the capillary, the detection unit comprises a light source, a first convergent lens and a detector which are sequentially arranged, excitation light emitted by the light source passes through the first convergent lens and then is focused in the capillary, and the detector receives an optical signal transmitted through the capillary; the time-resolved fluorescence immune flow optical detection device based on the magnetic particles further comprises:
the fluorescent light emitted by the magnetic particles positioned at the downstream of the focusing point of the excitation light in the capillary tube sequentially passes through the second converging lens and the optical filter device and is received by the detector;
a lanthanide element that labels antibodies or antigens on the magnetic particles;
and the processor is used for sending an instruction for acquiring a fluorescent signal after the interval time delta t to the detector after the detector receives the optical signal.
The invention also aims at providing a time-resolved fluorescence immune flow type optical detection method based on magnetic particles, which is realized by the following technical scheme:
the time-resolved fluorescence immunoassay method based on the magnetic particles comprises the following steps:
(A1) The conveying unit conveys magnetic particles and non-Newtonian fluid into the capillary tube, and the magnetic particles are focused in the central axis direction of the capillary tube; the lanthanide element marks the antibody or antigen on the magnetic particles;
(A2) The excitation light emitted by the light source passes through the first focusing lens and then is focused in the capillary, the magnetic particles pass through the focusing point, the detector receives the optical signal transmitted through the capillary, and the output electrical signal is sent to the processor;
(A3) The processor sends an instruction for collecting fluorescent signals after the interval time delta t to the detector after the detector detects the optical signals;
(A4) The magnetic particles flowing downstream of the focal point of the excitation light within the capillary emit fluorescence that in turn passes through a second converging lens and a filter device to be received by the detector.
Compared with the prior art, the invention has the following beneficial effects:
1. the sensitivity is high;
the detection range is staggered by laser light spots, namely, the influence of superposition of signals of magnetic particles (coded magnetic beads) and fluorescent signals of marks is staggered, so that the influence of short-service-life fluorescent signals of the magnetic particles (coded magnetic beads) on detection is effectively eliminated, only lower background signals are left, the sensitivity is improved, and qualitative and quantitative analysis of proteins is realized;
the free sample signal and the marked sample signal can be effectively distinguished by the double-channel measurement of forward dispersion (detector receiving) and fluorescence (detector receiving);
the setting of the interval delta t range enables the collection of the marked fluorescent signals to be completely staggered with the short-service-life fluorescent signals of the magnetic particles, and the marked fluorescent signals are imaged at the center of the detection surface of the detector, so that the detection sensitivity is further improved;
2. the specificity combination of the magnetic bead coding microsphere improves the accuracy and the linear range of detection, the sample consumption is small, the blood drawing amount required by a subject is small, the whole time required by detection can be improved, and the accuracy, the cost, the timeliness and the labor are greatly improved.
3. The detection volume is reduced, the capillary light window interface is eliminated, the Raman signal interference of the solution is reduced, and the background is effectively reduced;
4. a continuous laser can be used for time-resolved fluorescence microsphere testing, reducing costs relative to high pulsed lasers.
Drawings
The present disclosure will become more readily understood with reference to the accompanying drawings. As will be readily appreciated by those skilled in the art: the drawings are only for illustrating the technical scheme of the present invention and are not intended to limit the scope of the present invention. In the figure:
fig. 1 is a schematic structural diagram of a time-resolved fluorescence immunoflow optical detection apparatus based on magnetic particles according to an embodiment of the present invention.
Detailed Description
Fig. 1 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and reproduce the invention. For the purpose of explaining the technical solution of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art will appreciate variations or alternatives derived from these specific embodiments that fall within the scope of the invention. Those skilled in the art will appreciate that the features described below can be combined in various ways to form multiple variations of the invention. Thus, the invention is not limited to the following alternative embodiments, but only by the claims and their equivalents.
Example 1
Fig. 1 schematically shows a block diagram of a time-resolved fluorescence immunoassay optical detection assembly based on magnetic particles according to an embodiment of the present invention, as shown in fig. 1, the time-resolved fluorescence immunoassay optical detection assembly based on magnetic particles includes:
a capillary 4 and a delivery unit for delivering magnetic particles and a non-newtonian fluid into the capillary 4, the magnetic particles being focused in the direction of the central axis of the capillary 4;
the detection unit comprises a light source 1, a first focusing lens 3 and a detector 6 which are sequentially arranged, wherein excitation light emitted by the light source 1 passes through the first focusing lens 3 and then is focused in the capillary 4, flowing magnetic particles pass through the focusing point of the excitation light one by one, and the detector 6 receives a front scattered signal;
a second converging lens 8, a filter 9 and a detector 10, wherein fluorescence emitted by the magnetic particles in the capillary 4 downstream of the focusing point (i.e. fluorescence receiving point is on the magnetic particles downstream of the focusing point) sequentially passes through the second converging lens 8 and the filter 9 and is received by the detector 10;
a lanthanide element that labels antibodies or antigens on the magnetic particles;
a processor 11, wherein the processor 11 is configured to send an instruction to the detector 10 to collect a fluorescence signal after an interval Δt after the detector 6 receives the optical signal.
In order to accurately receive specific fluorescence on the magnetic particles downstream of the focus point, further, the detection device further comprises:
a collimator lens 7, through which the fluorescence passes in sequence through the collimator lens 7, a second converging lens 8 and a filter device 9;
a is the minor axis of the focusing spot of the excitation light in the capillary, b is the radius of the magnetic particles, f 1 Is the focal length of the collimating lens, f 2 Is the focal length of the second converging lens, L is the detection surface diameter of the detector, V is the volumetric flow rate of the capillary non-Newtonian fluid, and D is the cross-sectional inner diameter of the capillary perpendicular to its central axis.
In order to reduce costs, further, the light source 1 is a continuous laser.
In order to reduce the light interference, further, the angle between the central axis of the second converging lens 8 and the central axis of the capillary 4 and the central axis of the first converging lens 3 is a right angle, and the angle between the central axis of the capillary 4 and the central axis of the first converging lens 3 is a right angle.
To reduce the volume of the device, the time-resolved fluorescence immunoflow optical detection device based on magnetic particles further comprises:
a dichroic mirror 2, and reflected light of the excitation light on the dichroic mirror 2 sequentially passes through the first condensing lens 3 and the capillary 4.
The time-resolved fluorescence immunoflow optical detection method based on the magnetic particles comprises the following steps of:
(A1) The conveying unit conveys magnetic particles and non-Newtonian fluid into the capillary tube 4, and the magnetic particles are focused in the central axis direction of the capillary tube 4; the lanthanide element marks the antibody or antigen on the magnetic particles;
(A2) The excitation light emitted by the light source 1 passes through the first focusing lens 3 and then is focused in the capillary 4, flowing magnetic particles pass through the focusing point one by one, the detector 6 receives a front astigmatism signal transmitted through the capillary 4, and an output electric signal is transmitted to the processor 11;
(A3) After the detector 6 detects the optical signal, the processor 11 sends an instruction to the detector 10 to collect the fluorescent signal after an interval Δt;
(A4) The magnetic particles flowing downstream of the focal point of the excitation light in the capillary 4 emit fluorescence that passes through the second converging lens 8 and the filter device 9 in turn, and is received by the detector 10.
In order to accurately receive the specific fluorescence on the magnetic particles downstream of the focusing point, further, the fluorescence passes through the collimating lens, the second converging lens and the light filtering device in order;
a is the focal spot short half axis length, b is the radius of the magnetic particles, f 1 Is the focal length of the collimating lens, f 2 Is the focal length of the second converging lens, L is the detection surface diameter of the detector, V is the volumetric flow rate of the capillary non-Newtonian fluid, and D is the cross-sectional inner diameter of the capillary perpendicular to its central axis.
In order to reduce costs, further, the light source 1 is a continuous laser.
In order to reduce the light interference, further, the angle between the central axis of the second converging lens 8 and the central axis of the capillary 4 and the central axis of the first converging lens 3 is a right angle, and the angle between the central axis of the capillary 4 and the central axis of the first converging lens 3 is a right angle.
To reduce the volume of the device, the time-resolved fluorescence immunoflow optical detection device based on magnetic particles further comprises:
a dichroic mirror 2, and reflected light of the excitation light on the dichroic mirror 2 sequentially passes through the first condensing lens 3 and the capillary 4.
Example 2
Application example of magnetic particle-based time-resolved fluorescence immunoflow optical detection apparatus and method according to embodiment 1 of the present invention.
In this application example, as shown in fig. 1, the light source 1 adopts a continuous laser with a wavelength of 3mW and 356-360nm, and passes through the first focusing lens 3 (achromatic biconic lens) after being reflected by the light reflector 2 (dichroic mirror), so as to form a laser beam with a beam waist diameter of 10 μm (i.e. a minor axis a=5 μm of a focusing spot), and the laser beam is perpendicular to the central axis of the capillary 4; the laser beam passes through the capillary 4, passes through the third converging lens 5 and is received by the detector 6, the focal length of the third converging lens 5 is 16mm, the optical trap is arranged on the third converging lens 5, the detector 6 is a PD detector, the inner diameter D of the capillary is=75 mu m, and the volume flow rate V in the capillary is=5 mu L/min.
The collimating lens 7, the second converging lens 8, the optical filter 9 and the detector 10 (APD is adopted) are sequentially arranged, and the central axes of the collimating lens 7 and the second converging lens 8 and the central axis of the capillary 4 as well as the central axis of the first converging lens 3 are all right angles; focal length f of collimator lens 7 1 =3.1 mm, focal length f of second converging lens 8 2 The detection surface diameter l=180 μm of the detector 10=38.1 mm.
Feeding a non-newtonian fluid and magnetic beads into the quartz capillary 4, the lanthanide labeling antibodies or antigens on the magnetic beads; the number of the magnetic beads is 200W/mL, the magnetic beads are basically separated from each other in the sample liquid flow, and the magnetic beads are 113 mu m in distance and flow on the same line; magnetic bead radius b=1.5 μm.
The processor sends out a signal of light to the detector 10 at intervals after the detector 6 receives the signal of lightInstructions to collect fluorescence signals after = 1.0774ms, such that specific fluorescence on the magnetic beads is received by the detection face center of the detector 10 after an interval Δt.
The time-resolved fluorescence immune flow type optical detection method based on the magnetic particles, which is the working process of the optical detection device of the embodiment, comprises the following steps:
(A1) The conveying unit conveys the magnetic beads and the non-Newtonian fluid into the capillary tube, and the magnetic particles are focused in the central axis direction of the capillary tube; the lanthanide element marks the antibody or antigen on the magnetic particles;
(A2) Excitation light emitted by the light source 1 is reflected by the dichroic mirror 2, passes through the first focusing lens 3 and then is focused on the magnetic beads in the capillary tube 4, the detector 6 receives light signals, and the output electric signals are sent to the processor 11;
(A3) The processor 11 sends out an interval time to the detector 10 after the detector 6 detects the pre-astigmatism signalInstructions to collect fluorescence signals after = 1.0774 ms;
(A4) The magnetic beads flowing to the downstream of the focusing point of the excitation light in the capillary 4 emit fluorescence, the fluorescence passes through the collimating lens 7, the second converging lens 8 and the optical filter 9 in turn, and the detector 10 collects fluorescence signals according to the time interval Δt, so that the fluorescence is received by the center of the detection surface of the detector 10;
and (3) carrying out qualitative and/or quantitative analysis on the marked sample on the magnetic beads according to the forward scattered signal output by the detector 6 and the fluorescent signal output by the detector 10.
Claims (10)
1. The time-resolved fluorescence immune flow type optical detection device based on the magnetic particles comprises a capillary, a conveying unit and a detection unit, wherein the conveying unit is used for conveying the magnetic particles and non-Newtonian fluid into the capillary, the detection unit comprises a light source, a first convergent lens and a detector which are sequentially arranged, excitation light emitted by the light source passes through the first convergent lens and then is focused in the capillary, and the detector receives an optical signal transmitted through the capillary; the time-resolved fluorescence immune flow type optical detection device based on the magnetic particles is characterized by further comprising:
the fluorescent light emitted by the magnetic particles positioned at the downstream of the focusing point of the excitation light in the capillary tube sequentially passes through the second converging lens and the optical filter device and is received by the detector;
a lanthanide element that labels antibodies or antigens on the magnetic particles;
and the processor is used for sending an instruction for acquiring a fluorescent signal after the interval time delta t to the detector after the detector receives the optical signal.
2. The magnetic particle-based time-resolved fluoroimmunoassay optical detector of claim 1, further comprising:
the fluorescence sequentially passes through the collimating lens, the second converging lens and the optical filter device;
a is the minor axis of the focusing spot of the excitation light in the capillary, b is the radius of the magnetic particles, f 1 Is the focal length of the collimating lens, f 2 Is the focal length of the second converging lens, L is the detection surface diameter of the detector, V is the volumetric flow rate of the capillary non-Newtonian fluid, and D is the cross-sectional inner diameter of the capillary perpendicular to its central axis.
3. The magnetic particle-based time-resolved fluoroimmunoassay optical detector of claim 1, wherein the light source is a continuous laser.
4. The magnetic particle-based time-resolved fluoroimmunoassay optical instrument of claim 2, wherein the central axis of the second convergent lens is at right angles to the central axis of the capillary and the central axis of the first convergent lens, and the central axis of the capillary and the central axis of the first convergent lens are at right angles.
5. The magnetic particle-based time-resolved fluoroimmunoassay optical instrument of claim 1, further comprising:
a dichroic mirror, and reflected light of the excitation light on the dichroic mirror sequentially passes through the first condensing lens and the capillary tube.
6. The time-resolved fluorescence immunoassay method based on the magnetic particles comprises the following steps:
(A1) The conveying unit conveys magnetic particles and non-Newtonian fluid into the capillary tube, and the magnetic particles are focused in the central axis direction of the capillary tube; the lanthanide element marks the antibody or antigen on the magnetic particles;
(A2) The excitation light emitted by the light source passes through the first focusing lens and then is focused in the capillary, the magnetic particles pass through the focusing point, the detector receives the optical signal transmitted through the capillary, and the output electrical signal is sent to the processor;
(A3) The processor sends an instruction for collecting fluorescent signals after the interval time delta t to the detector after the detector detects the optical signals;
(A4) The magnetic particles flowing downstream of the focal point in the capillary tube fluoresce, which in turn passes through a second converging lens and a filter device to be received by the detector.
7. The magnetic particle-based time-resolved fluorescence immunoflow optical assay of claim 6, wherein the fluorescence passes through a collimating lens, a second converging lens, and a filter in that order;
a is the focal spot light spot short half shaftLength b is the radius of the magnetic particle, f 1 Is the focal length of the collimating lens, f 2 Is the focal length of the second converging lens, L is the detection surface diameter of the detector, V is the volumetric flow rate of the capillary non-Newtonian fluid, and D is the cross-sectional inner diameter of the capillary perpendicular to its central axis.
8. The magnetic particle-based time-resolved fluoroimmunoassay optical assay of claim 6, wherein the light source is a continuous laser.
9. The magnetic particle-based time-resolved fluoroimmunoassay optical inspection method of claim 6, wherein the angle between the central axis of the second convergent lens and the central axis of the capillary and the central axis of the first convergent lens is a right angle, and the angle between the central axis of the capillary and the central axis of the first convergent lens is a right angle.
10. The magnetic particle-based time-resolved fluoroimmunoassay optical assay of claim 6, wherein the reflected light of the excitation light on the dichroic mirror passes through the first focusing lens and the capillary tube in sequence.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310010497 | 2023-01-04 | ||
| CN2023100104974 | 2023-01-04 |
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| Publication Number | Publication Date |
|---|---|
| CN117147511A true CN117147511A (en) | 2023-12-01 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202311025204.6A Pending CN117147511A (en) | 2023-01-04 | 2023-08-15 | Time-resolved fluorescence immune flow type optical detection device and method based on magnetic particles |
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| CN (1) | CN117147511A (en) |
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- 2023-08-15 CN CN202311025204.6A patent/CN117147511A/en active Pending
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