CN107664631B - Device and method for detecting biological marker based on smart phone and preparation of sample thereof - Google Patents
Device and method for detecting biological marker based on smart phone and preparation of sample thereof Download PDFInfo
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- 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
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Abstract
The invention relates to a device for detecting a biological marker based on a smart phone, which mainly comprises a light source, a first optical filter, a glass slide, a second optical filter, a converging lens and the smart phone which are assembled in sequence; the first optical filter is arranged in an emergent light path of the light source; the side edge of the glass slide is arranged in an emergent light path of the first optical filter; the sample glass slide is loaded with a sample to be tested which generates excitation light after illumination; the second optical filter is arranged in an excitation light path of the sample to be detected; the converging lens is arranged in an emergent light path of the second optical filter; the camera of the smart phone is arranged in the refraction light path of the convergent lens. The device can detect samples outdoors at any time by utilizing the convenience function of the smart phone.
Description
Technical Field
The invention relates to a device and a method for identifying a biological marker and a method for preparing a sample before identification, belonging to the technical field of biological detection.
Background
In the context of global health, the supervision of food, environment, etc. is dependent on the traditional fluorescence microscope of a central laboratory, such as: inverted fluorescence microscope, confocal microscope, SIM microscope;
conventional fluorescence microscopes are expensive, bulky, heavy bench-top fluorescence microscopes. A bench top fluorescence microscope requires a complex and expensive microscope system, which includes: high numerical aperture objectives and other cumbersome optical elements, which limit the use of fluorescence microscopes in remote and resource-starved areas;
in contrast, relatively inexpensive smartphones have become popular. With the development of technology, the imaging system of the smart phone is advanced, and the fluorescent detection device for establishing the cheap and powerful biological marker by using the smart phone has great prospect.
Disclosure of Invention
The invention aims to solve the technical problem of providing a device for detecting a biological marker by using a smart phone, which can detect a sample outdoors at any time by utilizing the convenience function of the smart phone.
The invention solves the technical problems as follows:
the device for detecting the biological marker based on the smart phone mainly comprises a light source, a first optical filter, a sample slide, a second optical filter, a converging lens and the smart phone which are assembled in sequence; the first optical filter is arranged in an emergent light path of the light source; the side edge of the glass slide is arranged in an emergent light path of the first optical filter; the sample slide is loaded with a sample to be tested which generates excitation light after illumination; the second optical filter is arranged in an excitation light path of the sample to be detected; the converging lens is arranged in an emergent light path of the second optical filter; the camera of the smart phone is arranged in a refraction light path of the converging lens.
The technical scheme of the invention is further defined as follows:
furthermore, the light source is a semiconductor light source, and the light source can adopt an LED (light emitting diode) due to the advantages of low price and stable light emission of the LED; meanwhile, the LED can be replaced by a low-power LD (laser diode), and consistent results can be achieved.
For such LED light source specification requirements: since the light emission angle of the LED is large, an LED having a power of 1W or more is required to achieve sufficient excitation efficiency; the LED needs to be provided with a sufficient heat dissipation matching device; the light emitted by the LED is to have a waveness such as: the light emitted by the ultraviolet LED is mainly concentrated between 380 nm and 440 nm.
The first filter is a bandpass filter, and the common bandpass filter can meet the demands of people. The bandpass filter is to transmit light in a specific wavelength range and block other unwanted light (light in other wavelength bands emitted from the LED), so that the bandpass filter ensures that light emitted from the light source has very high transmittance in the bandpass region, but very low transmittance on both sides of the bandpass region.
The second optical filter is a long-pass optical filter, and the transmittance of the second optical filter in the transmission area is more than 90%; the long-pass filter is light emitted after being excited by fluorescent substances, and impurity light such as excitation light, autofluorescence, stray light and the like is filtered out, so that the signal-to-noise ratio is increased. The transmittance of the filter in the transmission region is required to be more than 90%.
The converging lens is an aspheric condensing lens. Because it has a larger clear aperture and a higher NA than a spherical lens, these lenses are well suited for light from LEDs or similar light sources. The condensing lenses have a shorter focal length and so are more closely adjacent to each other or to other optical elements, which is a desirable choice for focusing light to a detector or other light collecting element, such as a cell phone camera.
Further, at the same time, for optimum performance, the flatter side of the aspherical condenser corresponds to the incident light direction.
Further, the distance from the aspherical condenser to the sample corresponds to the focal length of the focusing lens.
A method of detecting a biomarker, comprising the steps of:
(1) Obtaining a biological marker;
(2) The obtained biological marker is mixed with the probe 1, the probe 2, the SA-magnetic bead and the SA-quantum dot to react to form a sandwich structure of SA-magnetic bead, biotin-probe 1, biological marker, biotin-probe 2 and SA-quantum dot;
(3) And (3) sample injection detection: adding the liquid containing the sandwich structure of the biological marker on a glass slide, placing the glass slide in detection equipment, imaging fluorescent points, and shooting by a smart phone to obtain an imaging picture;
(4) The mobile phone app analyzes and processes the imaging picture.
The system uses magnetic beads as a solid phase carrier and uses Biotin-probe 1 as a capture probe to capture biological markers in pathogenic liquid such as serum; meanwhile, the SA-quantum dot and the Biotin-probe 2 are connected to form a signal probe; both signaling and capture probes can specifically bind to the biomarker without collision of binding sites, thus forming a sandwich structure.
This can interfere with the identification of the capture probe to the biomarker, considering the confounding of the medium components of the actual pathogenic liquid; while the system determines the minimum capture and signaling probe amounts according to the maximum amount of biomarker that the body can tolerate in order to avoid signal degradation (false negative) due to insufficient fluorescent probes.
The technical scheme of the invention is further defined as follows:
further, in the step (1), the biomarker is one of DNA, RNA, protein, biological small molecule, cell, bacterium, virus, parasite, veterinary drug residue, food additive, and heavy metal ion.
Further, the sample preparation process in the step (2) specifically comprises the following steps:
a) Adding a proper amount of PBS buffer solution after mixing the SA-magnetic beads uniformly, and magnetically centrifuging to remove supernatant to obtain a precipitate 1;
b) Adding a proper amount of Biotin-probe 1 and PBS reaction solution into the precipitate 1, continuously and uniformly mixing at room temperature to ensure that the magnetic beads are in a suspended state for 0.5-3 hours, and magnetically centrifuging to remove supernatant to obtain precipitate 2;
c) In the reaction period of the step B), adding a proper amount of SA-quantum dots in PBS reaction liquid into a new container, and vibrating and uniformly mixing to obtain a solution 1;
d) Adding a certain amount of Biotin-probe 2 into the solution 1 in the step C), continuously and uniformly mixing at room temperature to ensure that the magnetic beads are in a suspension state, stopping mixing after a certain time, adding all liquid into the precipitate 2, and simultaneously adding the biological marker to continuously and uniformly mix to obtain the solution 2;
e) And D), magnetically centrifuging the solution 2 in the step D) to remove supernatant, washing the precipitate with PBS buffer solution, re-suspending the precipitate in PBS reaction solution, and refrigerating and preserving to obtain a sample to be tested.
Further, the mobile phone app processing method in the step (5) specifically comprises the following steps: the method comprises the following steps of:
a) Acquiring operation information of a user on a function control in an app interface;
b) If the operation information meets the detection requirement, the app calls a mobile phone camera to shoot a specimen imaging picture and stores the picture in a local storage space;
c) The app performs comparison and analysis on the pictures according to a local database;
d) The comparison is successful, and the result is displayed on a mobile phone screen and is uploaded to a cloud server for storage;
e) And if the comparison is unsuccessful, uploading the picture to a cloud server for machine or manual analysis, and returning an analysis result to a mobile phone screen for display.
Further, in the step (2), the minimum amounts of the Biotin-probe 1 and the Biotin-probe 2 are determined according to the maximum amount of the biomarker which the body can tolerate.
The beneficial effects of the invention are as follows:
(1) the invention mainly adopts a simple optical accessory and a smart phone to establish a field detection platform, and the detection platform can detect in the field.
(2) The mobile phone optical accessory adopts 3D printing, is low in price and light and portable.
(3) The smart phone is a basic computer, can perform digital image processing, save and communicate fluorescent images, can transmit on-site images to a remote central laboratory, and can accept analysis results of the central laboratory.
(4) Is suitable for being used in remote areas. Laboratory facilities are scarce but cell phone infrastructure is extensive; high power leds are inexpensive. In summary, our mobile microscope is field portable and can be used for microbial detection and fluorescence imaging in remote mountainous areas.
(5) The method is simple to operate without professional training, and can be used for rapidly, sensitively and selectively detecting the biological target identifier in clinic.
(6) The magnetic beads and the quantum dot system are established by using the biological markers, so that detection signals are amplified on one hand; in another aspect, the magnetism of the magnetic beads is utilized to separate unbound probes or biomarkers, thereby simplifying the procedure.
Drawings
FIG. 1 is a schematic diagram of an implementation structure of a single molecule detection apparatus based on a smart phone;
FIG. 2 is a handset app architecture;
FIG. 3 is a sandwich complex of biomarkers;
fig. 4 is a fluorescence imaging diagram.
Detailed Description
Example 1
Implementation case:
the LED is powered by a purple LED with luminous power of 5W and luminous wavelength of 380-440nm as a light source, and a 9-12V power adapter is equipped for driving the LED;
a 12V power adapter is adopted to drive a heat dissipation device comprising an electric fan, so that the temperature of an LED is prevented from being too high;
a rectangular sample bracket with the length of 4cm, the width of 3cm and the height of 0.5cm is adopted, and one end of the bracket is provided with a circular groove with the diameter of 25mm and the height of 200um for stably placing a slide;
a bandpass filter with a center wavelength of 400nm and a full width at half maximum of 40nm is adopted; the filter has an inner diameter of 21mm, an outer diameter of 25mm and a height of 6.3mm; the filter is placed in a special filter holder that can be easily inserted into a filter slot on the device; the filter disc groove is positioned between the LED and the sample support;
the sample is marked by CdSe/ZnS quantum dots, and the quantum dots can be excited by excitation light of 380-550nm and have highest emission efficiency at 625 nm.
A long-pass filter with a cut-off wavelength of 550nm is adopted to filter out the excitation light and the scattered light with the short wavelength below 550 nm; the outer diameter of the long-pass filter is 25-mm, the clear aperture is 21mm, and the height is 3.5mm; the long-pass filter is placed in a special filter groove;
an aspheric condensing lens is used for collecting light, and the lens has a large clear aperture and is very suitable for light emitted by an LED or similar light source; the planar base of the lens is a cylinder with a height of 2.4mm and a diameter of 15 mm;
the total height of the lens was 8.0mm, the focal length was 12mm, and the back focal length was 7mm.
To sum up: all optical elements (including the inner lens of the handset) are centered; switching on a power supply, and driving a heat dissipation device by an adapter to cool the LED; after the light emitted by the LED passes through the band-pass filter, the light is screened into ultraviolet light with a certain wave band (about 400nm with the strongest light); pumping the ultraviolet light to the edge of the slide to further excite the quantum dots on the biological marker; the quantum dots are excited by ultraviolet light and emit emitted light with the center of 625 nm; in the direction perpendicular to the excitation light, the emitted light passes through a 550nm long-pass filter, and some scattered light and excitation light are blocked; an aspherical condenser lens to collect the emitted light; the camera of the mobile phone automatically focuses on the lens and images; and (3) automatically analyzing the imaging picture by the APP customized on the mobile phone to give a diagnosis result.
Sample treatment case: detection of antibodies against porcine circovirus type 2 Cap protein in porcine serum:
(1) after mixing 10mg/mL of streptavidin-coated magnetic beads (SA-magnetic beads), 20ul of the mixture was put into a 1.5mL centrifuge tube, 400ul of PBS buffer (0.01M, pH 7.4) was added, and magnetic separation was performed by using a 1.5mL magnetic centrifuge tube rack to remove the supernatant;
(2) 400ul of PBS buffer solution is added into the 1.5mL centrifuge tube, and the supernatant is removed by magnetic separation through a 1.5mL magnetic centrifuge tube rack; repeatedly washing twice, and washing the magnetic bead storage liquid;
(3) removing supernatant by magnetic separation, adding 40ul of bio-Cap (20 ug/ml), adding 400ul of PBS reaction solution (0.01M PBS PH 7.4, 1% BSA) and gently mixing, wherein the reaction of streptavidin and biotin is carried out for 30-60min at room temperature; during the reaction, placing the centrifuge tube on a rotary mixing instrument, and keeping the magnetic beads in a suspension state through upside-down rotation (20-30 r/min);
(4) during the reaction of (3), 5ul (1 umol/L) of streptavidin-shaped quantum dots (SA-QDs) are added into 400ul PBS reaction liquid, and the mixture is uniformly mixed by shaking on a vortex oscillator;
(5) adding 10ul of bio-protein L (0.1 ug/ul) into the solution of the step (4) and gently mixing, wherein the step is to react streptavidin and biotin, and the reaction is to be carried out for 30-60min at room temperature; during the reaction, placing the centrifuge tube on a rotary mixing instrument, and keeping the magnetic beads in a suspension state through upside-down rotation (20-30 r/min);
(6) after the reaction of (3) for 30-60min, performing magnetic separation by using a 1.5mL magnetic centrifuge tube rack to remove supernatant;
(7) after 30-60min of reaction in (5), centrifuge for 3min with a small-sized common centrifuge, and then add all liquid to the centrifuge tube in (6) with a pipette. Meanwhile, adding serum containing Cap protein antibody, mixing the centrifuge tube gently, and placing the centrifuge tube on a rotary mixer for mixing for 1-2h;
(8) after the centrifuge tube in the step (7) reacts for 1-2 hours, carrying out magnetic separation by using a 1.5mL magnetic centrifuge tube rack so as to remove supernatant; after three subsequent washes with multiplexed PBS, the pellet was resuspended in 50ul of PBS and stored at 4 ℃.
(9) 10ul of prepared samples can be added to a circle center slide with the diameter of 25mm, and then shooting and diagnosis can be carried out by using a matched fluorescence detection device based on a mobile phone.
Note that: in the system established by us: we determine the theoretical maximum in the amount of capture probes and signaling probes based on the amount of antibodies to CAP protein that results in the critical point of the body being just diseased.
The spherical CdSe/ZnS quantum dots are nanoparticles with stable diameters of 2-20 nm. But note that: the quantum dots are used because the quantum dots have the common characteristics of good light stability, wide excitation spectrum, narrow emission spectrum, good biocompatibility, long fluorescence life, larger Stokes shift and the like, that is, the material and the size of the quantum dots are not key factors influencing the experimental results of the quantum dots, so that all the quantum dots conforming to the common characteristics of the quantum dots can be used.
The solid phase carrier used by us is SA-magnetic beads, which are used for grabbing and enriching the probes. But note that: we can also use COOH-beads, NH 2-beads, azide beads to capture and enrich the probe; we can also use other solid phase substrates such as slides to capture probes: the substrate is formed from materials such as silica, polydimethylsiloxane, polymethyl methacrylate, polystyrene, polycarbonate, cyclic olefin copolymer, polyamide, polyethylene, polypropylene, polyphenylene oxide, polyoxymethylene, polyetheretherketone, polytetrafluoroethylene, polyvinylchloride, polyvinylidene fluoride, polybutylene terephthalate, fluorinated ethylene propylene, or perfluoroalkoxyalkane.
The above examples only represent smartphone-based implementations, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that the smart phone may be replaced with a portable electronic product such as a tablet computer, a digital camera, a video camera, a mini-scanner, an imager, etc.; the system can also be replaced by a series of small mobile terminals such as an image collector, an electronic sensor, an electronic chip, an image sensor and the like which are designed by oneself, and data transmission and image processing are carried out based on wifi and Bluetooth; of course, the system can also be replaced by a large mobile terminal similar to a notebook computer, a desktop computer, a projector and the like, and can upload data and feed back results through the Internet.
Compared with the prior art, the design has the remarkable progress as follows:
1.1 ultra high signal to noise ratio: fluorescent imaging of nanoscale objects is a challenging task, the most important of which is the signal-to-noise ratio. One of the reasons for the poor signal-to-noise ratio is stray light. The invention uses lateral excitation light, long pass filters and bandpass filters to create a very efficient background subtraction mechanism necessary to resolve the extremely weak fluorescent signals generated by the nano-scale biomarkers.
1.2 a lighter design achieves powerful functions:
(1) the invention adopts the existing hot technology-3D printing, and the optical accessory required by printing by using the thermoplastic material ABS is lighter;
(2) the invention adopts the lens with low numerical aperture to reduce the sensitivity of the depth of field, so that the lens can focus on the surface of a sample on the premise of avoiding a complex focusing process;
(3) the invention adopts a sample chip, and a plurality of miniature sample adding holes are arranged on the chip, so that the detection of a plurality of biological markers can be realized at one time, and the invention has subverted significance for the identification of a plurality of biological tables needing large-flux detection.
1.3 powerful apps:
(1) the android custom app which is designed by the user can perform preliminary analysis on fluorescent images shot by the mobile phone;
(2) meanwhile, the preliminary analysis result and the fluorescence image can be sent to a central laboratory to carry out systematic diagnosis;
(3) the handset app can accept diagnostic results from a central laboratory. This allows one to diagnose some infectious diseases in field conditions and to obtain detailed diagnostic results within a few hours. For some acute animal diseases, the earlier the disease is found, the greater the loss caused by the spread of the disease can be avoided.
(4) The mobile phone app is used for establishing a data network, so that the time-space evolution of different species, diseases or infectious diseases can be dynamically tracked, and the causal relationship of the time-space modes can be better investigated and identified in a larger range, thereby providing an important tool for epidemiology.
1.4, the operation is simple, and the usability is strong:
(1) after the guidance of half an hour, the common personnel can operate by themselves;
(2) the invention has good stability, and the diagnosis result can not be overturned along with the proficiency of operators, so the usability is strong.
(3) Good repeatability and high sensitivity.
1.5 wide application range:
(1) the invention is suitable for animal protection mechanisms of various large farms and basic layers: the personnel on the base layer can use the portable single-molecule detection device to carry out high-flux disease screening; in resource-starved areas, the animal farm is followed and evaluated for on-site vaccination campaigns.
(2) The invention can be used as a tool for biomedical research and field infectious disease diagnosis.
(3) The invention applies a single-molecule technology to the detection field, and can realize fluorescence imaging of biological markers such as DNA, RNA, protein, biological small molecules, cells, bacteria, viruses, parasites, veterinary drug residues, food additives, heavy metal ions and the like.
In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.
Claims (8)
1. A method of detecting a biomarker, comprising the steps of:
(1) Obtaining a biological marker;
(2) Uniformly mixing the obtained biomarker with the probe 1, the probe 2, the SA-magnetic beads and the SA-quantum dots to form a sandwich structure of SA-magnetic beads, biotin-probe 1, the biomarker, biotin-probe 2 and SA-quantum dots;
(3) And (3) sample injection detection: adding the liquid containing the sandwich structure prepared in the step (2) on a glass slide, placing the glass slide in a device for detecting the biological marker based on a smart phone, imaging a fluorescent point, and shooting by the smart phone to obtain an imaging picture;
(4) The mobile phone app analyzes and processes the imaging picture;
the equipment for detecting the biological marker by the smart phone mainly comprises a light source, a first optical filter, a sample slide, a second optical filter, a converging lens and the smart phone which are assembled in sequence; the first optical filter is arranged in an emergent light path of the light source; the side edge of the glass slide is arranged in an emergent light path of the first optical filter; the sample slide is loaded with a sample to be tested which generates excitation light after illumination; the second optical filter is arranged in an excitation light path of the sample to be detected; the converging lens is arranged in an emergent light path of the second optical filter; the camera of the smart phone is arranged in a refraction light path of the converging lens;
the preparation process of the sample in the step (2) specifically comprises the following steps:
step A), adding a proper amount of PBS buffer solution after the SA-magnetic beads are uniformly mixed, and magnetically centrifuging to remove supernatant to obtain a precipitate 1;
step B), adding a proper amount of Biotin-probe 1 and PBS reaction solution into the precipitate 1, continuously and uniformly mixing at room temperature to ensure that the magnetic beads are in a suspended state for 0.5-3 hours, and magnetically centrifuging to remove supernatant to obtain precipitate 2;
step C), during the reaction of the step B), adding a proper amount of PBS (phosphate buffered saline) reaction solution and SA-quantum dots into a new container, and vibrating and uniformly mixing to obtain a solution 1;
step D), adding a proper amount of Biotin-probe 2 into the solution 1 in the step C), continuously and uniformly mixing at room temperature to ensure that the magnetic beads are in a suspended state for 0.5-3 hours, centrifuging, adding all liquid into the precipitate 2, and simultaneously adding a biological marker and continuously and uniformly mixing to obtain a solution 2;
and E) magnetically centrifuging the solution 2 in the step D) to remove supernatant, washing the precipitate with PBS buffer solution, re-suspending the precipitate in PBS reaction solution, and refrigerating and preserving to obtain a sample to be tested.
2. The method of detecting a biomarker according to claim 1, wherein:
in the step (1), the biological marker is one of DNA, RNA, protein, biological small molecules, cells, bacteria, viruses, parasites, veterinary drug residues, food additives and heavy metal ions.
3. The method of detecting a biomarker according to claim 1, wherein:
the mobile phone app processing method in the step (5) specifically comprises the following steps: the method comprises the following steps of:
acquiring operation information of a user on a function control in an app interface;
if the operation information meets the detection requirement, the app calls a mobile phone camera to shoot a specimen imaging picture and stores the picture in a local storage space;
the app performs comparison and analysis on the pictures according to a local database;
the comparison is successful, and the result is displayed on a mobile phone screen and is uploaded to a cloud server for storage;
and if the comparison is unsuccessful, uploading the picture to a cloud server for machine or manual analysis, and transmitting an analysis result back to a mobile phone screen for display.
4. The method of detecting a biomarker according to claim 1, wherein: in step (2), the minimum amounts of Biotin-probe 1 and Biotin-probe 2 are determined according to the maximum amount of biomarker that the body can tolerate.
5. The method of detecting a biomarker according to claim 1, wherein: the light source is a semiconductor light source; the first optical filter is a bandpass optical filter; the second optical filter is a long-pass optical filter, and the transmittance of the second optical filter in the transmission area is more than 90%; the converging lens is an aspheric condensing lens.
6. The method of detecting a biomarker according to claim 1, wherein: the flatter side of the aspheric condenser corresponds to the incident light direction.
7. The method of detecting a biomarker according to claim 1, wherein: the distance from the aspherical condenser to the sample corresponds to the focal length of the focusing lens.
8. The method of detecting a biomarker according to claim 1, wherein: the smart phone can be replaced by a portable electronic product, wherein the portable electronic product is one of a tablet personal computer, a digital camera, a video camera, a small scanner and an imager; or the small mobile terminal is replaced by a small mobile terminal, and the small mobile terminal is one of an image collector, an electronic sensor, an electronic chip and an image sensor and performs data transmission and image processing based on wifi and Bluetooth; or the large mobile terminal is replaced by the large mobile terminal, and the large mobile terminal is one of a notebook computer, a desktop computer and a projector and is used for uploading data and feeding back results through the Internet.
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