CN105929149B - A kind of optical detector based on magnetic enrichment and total internal reflection - Google Patents

Abstract

The invention discloses an optical detector based on magnetic enrichment and total internal reflection, which is characterized in that it comprises: an electronically controlled dual magnetic field enrichment and separation system, an optical detection system and a constant temperature heating system, The collection and separation system is used to generate alternating magnetic fields to realize the two-way driving and enrichment of magnetic particles on the chip; the optical detection system uses total internal reflection detection technology to realize the detection and analysis of optical images; the constant temperature heating The system includes a temperature sensor and a heater chip to control the temperature of the detection environment. The detector of the invention adopts electronically controlled dual magnetic field technology, total internal reflection optical detection technology and image analysis software technology to realize automatic and rapid detection of low-concentration substances in samples. The instrument is portable, one-step operation, simple and fast.

Description

An Optical Detector Based on Magnetic Enrichment and Total Internal Reflection

technical field

The invention belongs to the technical field of biomedical detection, in particular to an optical detector based on magnetic enrichment and total internal reflection.

Background technique

For the detection of low-concentration substances in samples (especially in blood samples), immunoassay techniques are usually used. In order to reduce interference, it is first necessary to perform pretreatment steps such as centrifugation and removal of blood cells on the blood sample, which increases the detection time and detection steps. In order to increase the accuracy and sensitivity of detection, many immune reactions use magnetic particle separation technology to separate low-concentration substances to be detected from samples, and the magnetic particle complex also needs to be separated by centrifugation and washed. The detection sensitivity is high. But the steps are cumbersome. Moreover, the instruments used are large and expensive, complicated to operate, high in cost, and require professional inspectors to operate, which limits the inspection time and inspection site, and cannot be used for grassroots and on-site inspections.

The increase of urinary microalbumin (urinary microalbumin, English abbreviation: mAlb) is often seen in diabetic nephropathy, hypertension, and preeclampsia, and is an early sensitive indicator of kidney damage. If the microalbuminuria in the urine is found to be in the range of 20mg/L-200mg/L after the physical examination, it belongs to microalbuminuria. If the patient can repair the nephron and reverse the fibrosis treatment, the renal microalbuminia can be completely repaired. Balls to eliminate proteinuria. And when the microalbumin in urine exceeds 200mg/L, it proves that the patient with kidney disease has leaked a large amount of albumin, and the development of kidney disease is only one step away from the irreversible stage.

Contents of the invention

In view of this, the purpose of the present invention is to prepare an optical detector based on magnetic enrichment and total internal reflection. The detector of the present invention adopts electronically controlled dual magnetic field technology, total internal reflection optical detection technology and image analysis software technology to realize automatic and rapid detection of low-concentration substances in samples. The instrument is portable, one-step operation, simple and quick.

In order to achieve the above object, the present invention provides an optical detector based on magnetic enrichment and total internal reflection, which includes: an electronically controlled dual magnetic field enrichment and separation system, an optical detection system and a constant temperature heating system. The collection and separation system is used to generate alternating magnetic fields to realize bidirectional driving and enrichment of magnetic particles on the chip; the optical detection system uses total internal reflection detection technology to realize the detection and analysis of optical images; the constant temperature heating The system includes a temperature sensor and a heater chip to control the temperature of the detection environment.

The electronically controlled dual magnetic field enrichment and separation system uses (small reinforced) magnetic columns or magnetic sheets placed on the upper and lower sides of the detection chip to alternately power on/off to generate alternating magnetic fields to achieve two-way detection of magnetic particles on the chip. drive and enrichment. Preferably, the electronically controlled double magnetic field enrichment and separation system includes: a power supply, an electromagnetic control circuit program and two identical (small) magnetic columns.

Wherein, the electromagnetic control circuit program can respectively energize the two magnetic columns to generate magnetism and de-energize to eliminate the magnetism according to the set frequency, so as to realize the separation and enrichment of magnetic particles.

Preferably, the power-on time range is 0.1-30 seconds; the cycle is 0-100 cycles; the last residence time for magnetic particle separation is 0.5-100 s; the power-on intensity is 0.1-50V. More preferably, the power-on time range is 2-10 seconds; the cycle period is 5-20 cycles; the last residence time for magnetic particle separation is 5-20 s.

In a preferred example, two small magnetic columns are located on both sides of the detection chip, and a suitable small coil needs to be matched with the optical detection cell. In order to achieve the smallest magnet to generate the largest magnetic field, the small magnetic columns can be powered to generate Magnetic fields and coils that eliminate magnetic fields and have magnetically enhanced iron cores or cobalt-nickel-iron cores.

The optical detection system adopts a total internal reflection detection technology, which includes two parts: an emission optical path and a detection optical path.

Wherein, the emitting light path is mainly a light source emitting module, which includes a light source control circuit board and a program, and an LED or laser parallel light source.

The light source control circuit board and program can set the intensity of the LED or laser, and the current adjustment range is preferably 0-1mA; the LED current value can be modified through key input or the upper computer through the RS485 interface, and then the brightness of the light source can be changed.

The LED or laser parallel light source includes components such as a beam expander and an aperture. The parameters such as the divergence angle and the light spot of the light source can be adjusted by means of collimation and aperture to form a beam of uniform parallel light.

Wherein, the laser parallel light source adopts a red parallel beam, and the wavelength is preferably 600-650 nm.

Wherein, the detection optical path includes an optical detection module, image recognition software, a microscope lens or a telecentric lens.

Wherein, the optical detection module can use CCD, CMOS, light-emitting diode or photomultiplier tube, etc. to perform optical image detection, wherein a CCD camera is preferred, and the pixel of the CCD camera is more preferably more than 1 million pixels; The heart lens is used to capture images. Optical acquisition rates and resolutions such as detectable micron-sized images are preferred for the present invention.

In a preferred example, the core acquisition part of the emission light path and the detection light path is sealed with a cassette, and the inner wall of the cassette is blackened to eliminate stray light interference. The incident angle and the angle of the receiving light path vary with the chip material. The material is determined and the angle is fixed.

Wherein, preferably, the image recognition software uses software such as MATLAB or C to realize image display, storage, grayscale recognition analysis comparison and scale analysis.

The constant temperature heating system includes a temperature sensor and a heating sheet. The constant temperature heating system controls the temperature of the detection environment through a temperature sensor and a heating plate, preferably controlled at a constant temperature of 37°C, so that the immune reaction can be carried out in a specific temperature environment, and the reaction difference error caused by temperature can be eliminated.

The temperature sensor obtains the temperature data of the reaction area in real time, and then uses the PID algorithm to calculate the output variation of the heating plate voltage, and the heating plate drive circuit outputs the adjusted heating voltage to realize the precise control of the area temperature.

In the detection of the present invention, the specific operation is preferably: add the chip, preheat the instrument, set the appropriate value of the magnetic field and light source, add the sample, the instrument automatically enters the detection mode, after the detection is completed, the image is taken, the software recognizes the image, and according to the pre-calibrated value, calculate the concentration of the analyte in the sample, and display the value.

In the present invention, the test sample can be whole blood, plasma, serum, urine, saliva, body fluid, etc. The invention adopts the principle of immunodetection, and is suitable for the substances to be detected with low content in samples. By immobilizing multiple antibodies on the chip, the detector can detect multiple indicators on one chip at a time.

The chip suitable for the detector of the present invention can be a highly transparent plastic material, glass or crystal material, etc.

The present invention has following beneficial effect:

(1) The present invention adopts an electronically controlled double magnetic field enrichment and separation mode, which effectively improves the reaction speed, simplifies the experimental steps, and realizes one-step detection.

(2) The total internal reflection optical detection technology adopted in the present invention is combined with the magnetic particle technology to realize the portable design of the instrument. The instrument is simple and effective, and is beneficial to on-site detection, so it has a very good application prospect.

Description of drawings

What Fig. 1 shows is the system block diagram of the optical detector of the present invention.

Fig. 2 shows the schematic diagram of magnetic enrichment and separation detection realized by the optical detector of the present invention during operation.

Fig. 3 shows the detection principle diagram of the optical detector of the present invention integrating magnetic separation and total internal reflection during operation.

What Fig. 4 shows is the system hardware block diagram of the optical detector of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

Example 1

The optical detector based on magnetic enrichment and total internal reflection of the present invention includes: an electronically controlled dual magnetic field enrichment and separation system, an optical detection system and a constant temperature heating system, and the electronically controlled dual magnetic field enrichment and separation system is used to generate alternating magnetic fields , to achieve bidirectional drive and enrichment of magnetic particles on the chip; the optical detection system adopts total internal reflection detection technology to realize the detection and analysis of optical images; the constant temperature heating system includes temperature sensors and heating plates to control the detection environment temperature.

The electronically controlled double magnetic field enrichment and separation system includes: power supply, electromagnetic control circuit program, and two identical small magnetic columns, which generate alternating magnetic fields through alternate power on/off to realize bidirectional drive and enrichment of magnetic particles on the chip .

Among them, the electromagnetic control circuit program can respectively energize the two magnetic columns to generate magnetism and de-energize to eliminate magnetism according to the set frequency, so as to realize the separation and enrichment of magnetic particles.

The optical detection system adopts total internal reflection detection technology, which includes two parts: emission light path and detection light path. The emission light path is mainly a light source emission module, which includes a light source control circuit board and program, and an LED or laser parallel light source. Among them, the light source control circuit board and the program can set the intensity of the LED or laser; modify the LED current value through the key input or the upper computer through the RS485 interface, and then change the brightness of the light source.

LED or laser parallel light source includes components such as beam expander and diaphragm. The parameters such as divergence angle and light spot of the light source can be adjusted by collimation and diaphragm to form a uniform beam of parallel light.

The laser parallel light source adopts red parallel beams. The detection optical path includes an optical detection module, image recognition software, and a microscope lens. Among them, the optical detection module uses a CCD camera with a pixel of more than 1 million pixels; at the same time, it needs to cooperate with a microscope lens to shoot images. Image recognition software uses software such as MATLAB or C to realize image display, storage, grayscale recognition, analysis, comparison and scale analysis.

The constant temperature heating system controls the temperature of the detection environment at a constant temperature of 37°C through a temperature sensor and a heating plate, so that the immune reaction can be carried out in a specific temperature environment, and the reaction difference error caused by temperature can be eliminated.

The temperature sensor obtains the temperature data of the reaction area in real time, and then uses the PID algorithm to calculate the output variation of the heating plate voltage, and the heating plate drive circuit outputs the adjusted heating voltage to realize the precise control of the area temperature.

During the detection, the specific operation is: add the chip, preheat the instrument, set the appropriate value of the magnetic field and light source, add the sample, the instrument automatically enters the detection mode, after the detection is completed, the image is taken, the software recognizes the image, and calculates according to the pre-calibrated value The concentration of the analyte in the sample is displayed and the value is displayed.

1 and 4 for details, FIG. 1 is a system block diagram of the optical detector of the present invention, and FIG. 4 is a hardware block diagram. When working, the optical detector is mainly composed of a detection chip 1, a control circuit 2, a light source 3, electromagnetic coils 4-1 and 4-2, a detection camera 5, and a host computer 6.

After starting up, preheating and self-starting completes the self-inspection of the instrument, including the electromagnetic drive circuit 2-1 driving the electromagnetic coils 4-1 and 4-1 to perform magnetic field drive scanning, and the light source drive circuit 2-2 to perform brightness calibration and temperature control of the light source 3 The circuit 2-3 performs regional constant temperature self-inspection, the indicator light 2-4 prompts, and the control circuit 2 completes parameter setting with the upper computer 6 and the camera 5 through the communication interface 7 .

When performing detection, the detection chip 1 is placed on the instrument chip bracket, the regional constant temperature control circuit 2-3 drives the heating plate to raise the temperature of the detection chip 1 to 37°C, and 1 drop of the sample (containing mAlb, urine microalbumin) is added to the In the sample pool 1-2 of the detection chip 1, the sample enters the reaction pool 1-1 through the channel 1-3 under the action of siphon to realize automatic sampling.

There are optical detectors next to the positions 1-3 of the chip channel, which capture the change of the light signal of the sample passing through, and trigger the detection, so as to realize the self-starting of the instrument detection.

After the detection is started, the sample analyte entering the reaction cell 1-1 undergoes a binding reaction with the anti-mAlb-nano-magnetic particles in the reaction cell to form a mAlb-anti-mAlb-nano-magnetic particle complex. The electromagnetic coil drive circuit 2-1 in the control circuit 2 turns on the electronically controlled dual magnetic field enrichment and separation mode, alternately powers on the upper electromagnetic coil 4-1 and the lower electromagnetic coil 4-2, and forms a magnetic field whose direction alternately changes up and down. Under the action of the changing magnetic field, the anti-mAlb-nano-magnetic particles move up and down in the reaction cell 1-1, which increases the contact with the mAlb in the sample, increases the reaction binding speed, and shortens the reaction time.

The mAlb-anti-mAlb-nanomagnetic particle complex is subjected to an alternating magnetic field, and will collide with the bottom of the reaction cell 1-1, and will combine with the paired antibody firmly fixed at the bottom of the reaction cell 1-1 to form antibody 2-mAlb-antibody mAlb-nanomagnetic particle composite, so that the magnetic particles are fixed on the surface at the bottom of the reaction cell 1-1.

After three minutes of reaction, the mAlb-anti-mAlb-nanomagnetic particle complexes are fixed on the bottom of the reaction cell, and the quantity tends to be stable. Stop changing the direction of the magnetic field, keep the upper electromagnetic coil 4-1 powered on, and maintain the direction of the magnetic field upward. Under the action of the upward magnetic force, the redundant unfixed magnetic particles will be attracted to the upper side of the reaction cell 1-1.

The mAlb-anti-mAlb-nanomagnetic particle complex combined with the analyte is fixed on the lower side of the reaction cell 1-1 through double-antibody binding, and its density is proportional to the concentration of the analyte. Since the magnetic particles are fixed at the bottom of the reaction cell 1-1, the total internal reflection interface at the bottom of the reaction cell 1-1 will be destroyed. The emitted light beam from the original light source 3 is totally reflected at the interface at the bottom of the reaction cell 1-1. At this time, due to the existence of fixed magnetons, the reflected light will be weakened, and the reflected light intensity is inversely proportional to the number of fixed magnetic particles.

After acting on the upward magnetic field for 10 seconds, the unfixed magnetic particles are basically attracted to the upper layer. Turn on the image acquisition by the camera 5, capture and shoot the total internal reflection image of the magnetic particles at the bottom of the reaction cell 1-1, and send it to the host computer 6 for software identification and analysis, obtain the reflected light intensity information, and further calculate the concentration of mAlb in the sample , and display the test results.

Fig. 2 and Fig. 3 are schematic diagrams of magnetic enrichment and separation detection realized by the optical detector of the present invention during operation. First, the two reactants (for example, antibody 1 combined with magnetic particles to form composite magnetic particles fixed on the top of the chip; antibody 2 fixed on the bottom of the chip) were respectively fixed on the inner surface film of the chip, and then the sample to be tested was added to the sample. The combined reaction between the antigen and the composite magnetic particle on the chip occurs; the upper and lower magnetic coils are respectively energized according to a certain frequency through the electronically controlled double magnetic field to generate a magnetic field that changes up and down. The bottom of the chip moves and binds to Antibody 2 to form a firm bond that attaches to the bottom of the chip. When the magnetic field acts, the unbound composite magnetic particles are attracted to the top of the chip. The upper and lower magnetic fields are applied repeatedly to accelerate the combination and separation of the antigen-composite magnetic particles and the antibody below. The antibody-magnetic particles move up and down in the reaction cell, which increases the contact with the substance to be tested in the sample and improves the reaction combination speed. Reduced reaction time. After the reaction is sufficient, a magnetic field is applied to attract the unbound redundant composite magnetic particles to the top of the chip, and the reacted antigen-composite magnetic particles remain below the chip through the binding of antibody 2 to form magnetic particle aggregation. , the gray value of the aggregation area formed by the magnetic particles will change accordingly, and the concentration of the analyte is directly proportional to the density of the nano-magnetic particles finally fixed on the bottom side of the reaction tank. After the converging parallel light emitted by the light source enters the chip through the optical angle surface of the chip, the collected magnetic particles cause the change of the refractive index of the surface film. After total reflection, the light is irradiated on the CCD area array or other optical element receivers, and the whole reaction is recorded in real time. The signal changes in the process can be processed by using the collected CCD images or other optical images of different gray levels, using corresponding software, and the modulus is converted into a light intensity data curve. After calibration, the concentration of the measured object can be obtained. parameter.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention. For example: appearance types of magnetic coils, types of optical devices, different optical path preparation methods, etc.

Claims (14)

1. a kind of optical detector based on magnetic enrichment and total internal reflection, which is characterized in that it includes：Automatically controlled double magnetic field enrichments point From system, Systems for optical inspection and constant-temperature heating system, automatically controlled double magnetic field concentration and separation systems are for generating alternate magnetic , to realize bi-directional drive and the enrichment to magnetic particle on chip；The Systems for optical inspection uses total internal reflection detection technique, To realize the detection and analysis processing of optical imagery comprising transmitting light path and light path two parts, the light path packet Optical detecting module and telecentric lens are included, the optical detecting module uses CCD, CMOS, light emitting diode or photomultiplier To carry out optical imagery detection；The constant-temperature heating system includes temperature sensor and heating plate, for controlling detection environment Temperature；Wherein, automatically controlled double magnetic field concentration and separation systems are the magnetic pole or magnetic by being placed on detection chip or more both sides Piece, alternating power up/down generate alternate magnetic field, to realize bi-directional drive and the enrichment to magnetic particle on chip；

Wherein, the core collecting part of the transmitting light path and light path is closed using magazine, and magazine carries out inner wall hair Interference of stray light is eliminated in black processing, and incident angle and the angle for receiving light path are different with chip material variation, and chip material is true Fixed, incident angle and the angle for receiving light path are fixed.

2. the optical detector according to claim 1 based on magnetic enrichment and total internal reflection, which is characterized in that described automatically controlled Double magnetic field concentration and separation systems include power supply, electromagnetic control circuit and two identical magnetic poles.

3. the optical detector according to claim 2 based on magnetic enrichment and total internal reflection, which is characterized in that wherein, institute It states electromagnetic control circuit and realizes that the frequency according to setting powers up generation magnetism to two magnetic poles respectively and goes electricity elimination magnetism, thus Realize the separation and enrichment of magnetic particle.

4. the optical detector according to claim 3 based on magnetic enrichment and total internal reflection, which is characterized in that wherein, institute The time range for stating power-up is 0.1~30 second；Cycle period is 0~100 period；The last stop for magnetic particle separation Time is 0.5~100s；The intensity of power-up is 0.1~50V.

5. the optical detector according to claim 3 based on magnetic enrichment and total internal reflection, which is characterized in that wherein, institute The time range for stating power-up is 2~10 seconds；Cycle period is 5~20 periods；When being stopped for magnetic particle separation last time Between be 5~20s.

6. the optical detector according to claim 1 based on magnetic enrichment and total internal reflection, which is characterized in that wherein, institute It is light source emitting module to state transmitting light path, and it includes control circuit for light source plate, LED or parallel laser light sources.

7. the optical detector according to claim 6 based on magnetic enrichment and total internal reflection, which is characterized in that the light source Control circuit board is for setting the intensity of LED or laser.

8. the optical detector according to claim 7 based on magnetic enrichment and total internal reflection, which is characterized in that wherein electric current Adjusting range is 0~1mA.

9. the optical detector according to claim 6 based on magnetic enrichment and total internal reflection, which is characterized in that the laser Source of parallel light is using red collimated light beam.

10. the optical detector according to claim 9 based on magnetic enrichment and total internal reflection, which is characterized in that red is flat The wavelength of row light beam is 600~650nm.

11. the optical detector according to claim 1 based on magnetic enrichment and total internal reflection, which is characterized in that the figure As identification software divides the comparison of the display of image, storage and gray scale discriminance analysis and scale using MATLAB or C software realizations Analysis.

12. the optical detector according to claim 1 based on magnetic enrichment and total internal reflection, which is characterized in that the CCD The pixel of camera is more than 1,000,000 pixels.

13. the optical detector according to claim 1 based on magnetic enrichment and total internal reflection, which is characterized in that the perseverance The temperature that warm heating system passes through temperature sensor and heating plate control detection environment.

14. the optical detector according to claim 13 based on magnetic enrichment and total internal reflection, which is characterized in that the control The temperature of system detection environment is in 37 DEG C of temperature constant states.