CN107664631A - The preparation of equipment, detection method and its sample based on smart mobile phone detection biological marker - Google Patents
The preparation of equipment, detection method and its sample based on smart mobile phone detection biological marker Download PDFInfo
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- CN107664631A CN107664631A CN201710612729.8A CN201710612729A CN107664631A CN 107664631 A CN107664631 A CN 107664631A CN 201710612729 A CN201710612729 A CN 201710612729A CN 107664631 A CN107664631 A CN 107664631A
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Classifications
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- 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
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- Biochemistry (AREA)
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- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention is a kind of equipment based on smart mobile phone detection biological marker, is mainly made up of the light source of assembled in sequence, the first optical filter, load sample slide, the second optical filter, plus lens and smart mobile phone；First optical filter is placed in the emitting light path of light source；The side of the load sample slide is placed in the emitting light path of the first optical filter；The testing sample that exciting light is produced after illumination is mounted with load sample slide；Second optical filter is placed in the excitation light path of testing sample；Plus lens is placed in the emitting light path of the second optical filter；The camera of smart mobile phone is placed in the refractive light paths of plus lens.The facilitating functions of the equipment utilization smart mobile phone can detect to sample at any time out of doors.
Description
Technical field
The present invention relates to a kind of identification equipment of biological marker, recognition methods, and the sample preparation methods before identification,
Belong to technical field of biological.
Background technology
Under the background of Global Health, supervision is carried out to food, environment etc. and all relies on the traditional glimmering of central laboratory
Light microscope, such as：Inverted fluorescence microscope, Laser Scanning Confocal Microscope, SIM microscopes；
Traditional fluorescence microscope is expensive, bulky, bulky desk-top fluorescence microscope.Desk-top fluorescence shows
Micro mirror needs complicated and expensive microscopic system, just includes among these：The object lens of high-NA and other bulky optics
Element, this limitation fluorescence microscope is in remote and scarcity of resources regional use；
On the contrary, relatively cheap smart mobile phone has been popularized.With the development of science and technology, the imaging system of smart mobile phone is
Highly advanced, using smart mobile phone, the fluorescence detection device for establishing cheap and powerful biological marker is with very big
Prospect.
The content of the invention
The technical problem to be solved by the invention is to provide a kind of facilitating functions using smart mobile phone can out of doors with
When sample is detected smart mobile phone detection biological marker equipment.
The present invention solves the technical scheme of above technical problem：
It is a kind of based on smart mobile phone detection biological marker equipment, mainly by the light source of assembled in sequence, the first optical filter,
Load sample slide, the second optical filter, plus lens and smart mobile phone composition；First optical filter is placed in the emitting light path of light source
In；The side of the load sample slide is placed in the emitting light path of the first optical filter；Produced after being mounted with illumination on the load sample slide
The testing sample of raw exciting light；Second optical filter is placed in the excitation light path of testing sample；The plus lens is placed in
In the emitting light path of two optical filters；The camera of the smart mobile phone is placed in the refractive light paths of plus lens.
The technical scheme that further limits of the present invention as：
Further, light source is semiconductor light source, and light source can use LED, is in order at the cheap, luminous steady of LED
The advantages of determining；LED can also be replaced with the LD (laser diode) of low-power simultaneously, it is possible to achieve consistent result.
For such a LED light source specification requirement：Because LED lighting angle is larger, therefore effect is excited in order to reach enough
Rate, it is necessary to power more than 1W LED；LED will have sufficiently radiating corollary apparatus；The light that LED is sent will have wave band, all
Such as：Ultraviolet LED, the light sent will be concentrated mainly between 380-440nm.
The needs of first optical filter is bandpass filter, and in general bandpass filter can meets us.The filter of band logical
Mating plate passes through the light of particular range of wavelengths, and stops other unwanted light (light for its all band that LED is sent), so
Bandpass filter will ensure there is very high transmitance in bandpass region by the light that light source is sent, but the two of bandpass region
While there is extremely low light transmittance.
Second optical filter is long pass filter, and its transmitance in penetrating region is more than 90%；Long logical optical filter is will
The light emitted after being excited through fluorescent material, the impurity light such as exciting light, autofluorescence and veiling glare is filtered, so as to increase
Signal to noise ratio.It is required that this optical filter transmitance in penetrating region is more than 90%.
The plus lens is aspherical condenser.It is because it has bigger clear aperature and Geng Gao than spherical lens
NA, therefore these lens are especially suitable for the light that LED or similar light source are sent.Collector lens has shorter focal length, so more
It can each other be close to or closer to other optical elements, be to need to focus the light into detector or other light collection device (such as hands
Machine camera) ideal chose.
Further, meanwhile, in order to which in order to obtain optimum performance, the more flat side of aspherical condenser corresponds to incident light side
To.
Further, the distance of aspherical condenser to sample meets the focal length of condenser lens.
A kind of method for detecting biological marker, is carried out in accordance with the following steps：
(1) biological marker is obtained；
(2) biological marker obtained forms SA- magnetic with probe 1, probe 2, SA- magnetic beads, SA- quantum dot hybrid reactions
Interlayer structure as pearl+Biotin- probe 1+ biological marker+Biotin- probe 2+SA- quantum dots；
(3) sample detection：The liquid of the above-mentioned interlayer structure containing biological marker is added on slide, is placed in detection
In equipment, phosphor dot is imaged, smart mobile phone shooting obtains imaging picture；
(4) mobile phone app analyzes and processes to imaging picture.
Body series go to capture the diseases such as serum by the use of magnetic bead as solid phase carrier by the use of Biotin- probes 1 as capture probe
Biological marker in stoste；SA- quantum dots and the connection composition signal probe of Biotin- probes 2 simultaneously；Signal probe and capture
Probe can specific combination biological marker and binding site do not conflict, material is thus formed interlayer structure.
Mix in view of the middle composition of actual cause of disease liquid, this can identification of the noise capture probe to biological marker；Simultaneously
In order to avoid due to caused by fluorescence probe deficiency signal reduce (false negative), body series according to body it is patient maximum
The amount of biological marker determines the amount of minimum capture probe and signal probe.
The technical scheme that further limits of the present invention as：
Further, in step (1), biological marker DNA, RNA, protein, biological micromolecule, cell, bacterium, disease
One kind in poison, parasite, residue of veterinary drug, food additives, heavy metal ion.
Further, the process of sample preparation is specially in step (2)：
A) SA- magnetic beads add appropriate PBS after mixing, and magnetic centrifugal removes supernatant and obtains precipitation 1；
B appropriate Biotin- probes 1 and PBS reaction solutions) are added into precipitation 1, lasting mix is ensured at magnetic bead at room temperature
In suspended state 0.5-3 hours, magnetic centrifugal removes supernatant and obtains precipitation 2；
C) in step B) during reaction, add SA- quantum dots in appropriate PBS reaction solutions in new container, concussion is mixed
It is even to obtain solution 1；
D) in step C) solution 1 in add a certain amount of Biotin- probes 2, lasting mix is ensured at magnetic bead at room temperature
Stop mixing after suspended state, certain time, all liquid is added in precipitation 2, while add biological marker continuation
It is uniformly mixed so as to obtain solution 2；
E) by step D) in the magnetic centrifugal of solution 2 remove supernatant, after precipitation is washed with PBS, by precipitation weight
It is suspended from PBS reaction solutions, it is stored refrigerated to obtain sample to be tested.
Further, step (5) mobile phone app processing methods are specially：Carry out in accordance with the following steps：
A operation information of the user to functionality controls in app interfaces) is obtained；
B) if the operation information meets testing requirements, app calling mobile phones camera shooting sample imaging picture simultaneously will
Picture is stored in local storage space；
C) picture is compared according to local data base by App；
D) compare and analyze successfully, as a result display is uploaded to cloud server storage simultaneously on mobile phone screen；
E) comparison is unsuccessful, and picture is uploaded to cloud server progress machine or manual analysis, analysis result are back to hand
Machine screen display.
Further, in step (2), according to body patient maximum biological marker amount it is minimum to determine
The amount of Biotin- probes 1 and Biotin- probes 2.
Beneficial effects of the present invention are：
1. invention is main to establish a Site Detection platform, the detection using simple an optical accessory and smart mobile phone
Platform can be detected at the scene；
2. the mobile phone optical accessory uses 3D printing, cheap, light and handy and can carry with；
3. smart mobile phone is a basic computer, Digital Image Processing can be carried out, and fluoroscopic image preservation and
Communication, the image at scene can be delivered to far central laboratory, and the analysis result of central laboratory can be received；
4. it is adapted to be used in some remote areas.The facility in laboratory is rare but mobile phone infrastructure is extensive；
Powerful light emitting diode is cheap.In summary, our Cellscope is countryside portable, can be remote
Mountain area carry out microorganism detection and fluorescence imaging.
5. needing not move through the training of specialty, simple to operate, clinic uses, it is possible to achieve quick, sensitive, selective detection
The other thing of biological label.
6. establishing magnetic bead and quantum dot system using biological marker, detection signal is on the one hand exaggerated；Still further aspect
Uncombined probe or biological marker are separated using the magnetic of magnetic bead, simplifies operation.
Brief description of the drawings
Fig. 1 is the implementation structural representation of the Single Molecule Detection device based on smart mobile phone；
Fig. 2 is mobile phone app frameworks；
Fig. 3 is the sandwich complex of biological marker；
Fig. 4 is fluorescence imaging figure.
Embodiment
Embodiment 1
Case study on implementation：
We use luminous power as 5W, and the purple LED that emission wavelength is 380-440nm is light source, while is equipped with 9-12V
Power supply adaptor be used for drive LED；
One heat abstractor for including electric fan is driven using 12V power supply adaptor, avoids LED temperature mistake
It is high；
Using a long 4cm, wide 3cm, the highly rectangular sample holder for 0.5cm, one end of support has one directly
Footpath is 25mm, highly the circular groove for 200um, for stable placement slide；
It for 400nm, full width at half maximum (FWHM) is 40nm bandpass filters to use a centre wavelength；The interior diameter of the optical filter is
21mm, overall diameter 25mm, it is highly 6.3mm；The optical filter is placed in a special filter disc support, and the support can be with
Filter disc groove on easy insertion apparatus；The position of the filter disc groove is between LED and sample holder；
Sample is marked using CdSe/ZnS quantum dots, the quantum dot can be by 380-550nm excitation, simultaneously
There is highest emission effciency in 625nm.
A cutoff wavelength is used as the long pass filters of 550nm, for filter the exciting light of below 550nm short wavelength and
Scatter light；Long pass filter overall diameter 25mm, the clear aperature 21mm, are highly 3.5mm；The long pass filter is placed on specially
Optical filtering film trap in；
Light is collected using a spherical projection lens, the lens possess very big clear aperature, are especially suitable for LED
Or the light that similar light source is sent；The planar substrates of the lens are that a height is 2.4mm, a diameter of 15mm cylinder；
The total height of the lens is 8.0mm, focal length 12mm, back focal length 7mm.
In summary：All optical element (interior lens for including mobile phone) all centers are to straight；Switch on power, adapter
Driving heat abstractor cools to LED；The LED light sent has been screened into certain wave band after through bandpass filter
The ultraviolet light of (400nm or so light is most strong)；Ultraviolet light side pump is further excited on the other thing of biological label to the edge of slide
Quantum dot；Quantum dot receives ultraviolet excitation, launches the transmitting light that center is 625nm；In the direction vertical with exciting light,
Launch the long pass filter that light passes through 550nm, some scattering light and exciting light are blocked；Spherical projection lens are launched to collect
Light；The camera of mobile phone is focused on lens automatically, and is imaged；The APP customized on mobile phone carries out automatic to imaging picture
Analysis, provides diagnostic result.
Sample treatment case：The detection of the antibody of Swine serum moderate resistance carrying Cap gene of porcine circovirus type 2：
After 1. the magnetic bead (SA- magnetic beads) of 10mg/mL streptavidin mixes, take in 20ul to 1.5mL centrifuge tubes, add
Enter 400ul PBS (0.01M, PH 7.4), carry out Magneto separate using 1.5mL magnetic centrifugals pipe support so as to remove supernatant
Liquid；
2. adding 400ul PBS in above-mentioned 1.5mL centrifuge tubes, carried out using 1.5mL magnetic centrifugals pipe support
Magneto separate removes supernatant；Repetition is washed twice, wash clean magnetic bead storage liquid；
3. Magneto separate removes supernatant, 40ul bio-Cap (20ug/ml) is added, while adds 400ul PBS reactions
Liquid (0.01M PBS PH 7.4,1%BSA) simultaneously gently mixes, and this step is the reaction of Streptavidin and biotin, be
30-60min is reacted at room temperature；During reaction, centrifuge tube is placed on rotation blending instrument, by the rotation turned upside down (20~
Magnetic bead 30r/min) is set to keep suspended state；
4. during 3. reacting, the quantum dot (SA-QDs) of 5ul (1umol/L) streptavidin is taken to be added to
In 400ulPBS reaction solutions, vibrate and mix in vortex concussion instrument；
5. 10ul bio-protein L (0.1ug/ul) and soft mixing, this step are added in solution 4. is
The reaction of Streptavidin and biotin, 30-60min is reacted at room temperature；During reaction, centrifuge tube is placed on rotation and mixed
On instrument, magnetic bead is set to keep suspended state by the rotation (20~30r/min) turned upside down；
6. after 3. 30-60min has been reacted, Magneto separate is carried out using 1.5mL magnetic centrifugals pipe support so as to remove supernatant；
7. after 5. 30-60min has been reacted, with small-sized generic centrifuge centrifuge 3min, then with liquid-transfering gun by whole
Liquid is added in centrifuge tube 6..Meanwhile the serum of the antibody containing Cap protein is added, after the soft mixing of centrifuge tube, put
1-2h is mixed on instrument in rotation mixed give；
8. after the centrifugation tube reaction 1-2h in 7., using 1.5mL magnetic centrifugals pipe support carry out Magneto separate so as to remove on
Clear liquid；Then repeat after washing three times with PBS, be resuspended precipitating in the PBS with 50ul, 4 DEG C of preservations.
9. the ready-made samples of 10ul can will be taken to be added on a diameter of 25mm center of circle slide, then with supporting based on hand
The fluorescence detection device of machine is shot and diagnosed.
Note：In the system that we establish：We are according to the antibody for causing the body just CAP albumen of ill critical point
Amount is used as foundation, to determine the maximum in the theoretical value of the amount of capture probe and signal probe.
We for spherical CdSe/ZnS quantum dots, be nano-particle of the stable diameter in 2~20nm.But to note
Meaning：We use quantum dot, are because quantum dot has good photostability, wide PLE and narrow emission spectra, biology
Compatibility is good, fluorescence lifetime is long and the material with universal characteristics, in other words quantum dot such as larger Stokes shifts
It is not to influence the key factor of our experimental results with size, therefore meets all of the universal feature of foregoing quantum dot
Quantum dot we can use.
Our solid phase carrier is SA- magnetic beads, is for capturing and being enriched with probe.It should be noted that：We can be with
Probe is captured and is enriched with using COOH- magnetic beads, NH2- magnetic beads, Azide magnetic bead；We can also use slide etc. other solid
Phase substrate captures probe：Substrate is by silica, dimethyl silicone polymer, polymethyl methacrylate, polystyrene, poly- carbon
It is acid esters, cyclic olefin copolymer, polyamide, polyethylene, polypropylene, polyphenylene oxide, polyformaldehyde, polyether-ether-ketone, polytetrafluoroethylene (PTFE), poly-
The material shape such as vinyl chloride, Kynoar, polybutylene terephthalate (PBT), PEP or PFA
Into.
Embodiment described above only expresses the embodiment based on smart mobile phone, and its description is more specific and detailed, but
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that smart mobile phone could alternatively be flat board
The portable type electronic products such as computer, digital camera, video camera, small scanner, imager；It may be replaced by designed, designed
Image acquisition device, electronic sensor, electronic chip, a series of small mobile terminals such as imaging sensor, based on Wiff and
Bluetooth carries out data transmission and image procossing；It is, of course, also possible to replace with similar to notebook computer, desktop computer, projecting apparatus
The Large-scale Mobile terminal of equivalent type, data can be carried out by internet and uploaded and result feedback.
The design has significant progress as follows relative to prior art：
The signal to noise ratio of 1.1 superelevation：Fluorescence imaging nanoscopic objects are a challenging tasks, most important among these
The reason for be exactly signal to noise ratio.One of the reason for signal to noise ratio is bad is exactly veiling glare.This invention is using broadside light, long logical optical filtering
Piece and bandpass filter create a very effective background subtraction mechanism, and this is for differentiating the biological marker by Nano grade
Caused extremely weak fluorescence signal is necessary.
Powerful function is realized in 1.2 lighter and handier designs：
1. it is attached to print required optics using thermoplastic ABS using hot technology -3D printing instantly for this invention
Part is lighter；
2. this invents the susceptibility that the depth of field is reduced using the lens of low numerical aperture, can thus complexity be being avoided
Focussing process on the premise of, make lens focus to sample surfaces；
3. this invention uses sample chip, there are multiple miniature wells on chip, so can disposably realize multiple lifes
The detection of thing marker, this has subversive meaning for the biological table identification of the big flux detection of some needs.
1.3 powerful APP：
1. the Android customization APP of our designed, designeds, preliminary analysis can be carried out to the fluoroscopic image that mobile phone is shot；
2. meanwhile preliminary analysis result and fluoroscopic image can be sent to central laboratory, carry out the diagnosis of system；
3. cell phone application can receive the diagnostic result from central laboratory.Under the conditions of this just allows us in the wild
Some infectious diseases are diagnosed, and detailed diagnostic result can be obtained within several hours.It is acute dynamic for some
Thing epidemic disease, massive losses caused by more early discovery disease then can avoid the diffusion of disease.
4. establishing data network using cell phone application, different plant species can be dynamically tracked, the space-time of disease or outbreak of communicable diseases is drilled
Become,；The causality of these spatiotemporal modes in the larger context, preferably can be investigated and identified, is provided for epidemiology
Important tool.
1.4 is simple to operate, and usability is strong:
1. ordinary person is after the guidance by half an hour, it is possible to Self-operating；
2. invention stability is fine, with the qualification of operating personnel subversive change will not occur for diagnostic result,
Therefore usability is strong.
3. reproducible, high sensitivity.
1.5 applications are wide：
1. this invention is suitable for major plant and the animal protection mechanism of basic unit：The personnel of basic unit can utilize this just
The Single Molecule Detection device for taking formula carries out high-throughout disorder in screening；In the area of scarcity of resources, scene is carried out to animal farm
The tracking and evaluation of vaccine inoculation campaign.
2. this invention can turn into biomedical research and the instrument of field Diagnosis of Infectious Diseases.
3. single molecule techniques are applied to detection field by this invention, it is possible to achieve to DNA, RNA, protein, small point of biology
The fluorescence of the biological markers such as son, cell, bacterium, virus, parasite, residue of veterinary drug, food additives, heavy metal ion into
Picture.
In addition to the implementation, the present invention can also have other embodiment.It is all to use equivalent substitution or equivalent transformation shape
Into technical scheme, all fall within the protection domains of application claims.
Claims (10)
- A kind of 1. equipment based on smart mobile phone detection biological marker, it is characterised in that：Mainly by the light source of assembled in sequence, One optical filter, load sample slide, the second optical filter, plus lens and smart mobile phone composition；First optical filter is placed in light source In emitting light path；The side of the load sample slide is placed in the emitting light path of the first optical filter；It is mounted with the load sample slide The testing sample of exciting light is produced after illumination；Second optical filter is placed in the excitation light path of testing sample；The convergence is saturating Mirror is placed in the emitting light path of the second optical filter；The camera of the smart mobile phone is placed in the refractive light paths of plus lens.
- 2. the equipment according to claim 1 based on smart mobile phone detection biological marker, it is characterised in that：The light source For semiconductor light source；First optical filter is bandpass filter；Second optical filter is long pass filter, and it is passed through in penetrating region Rate is more than 90%；The plus lens is aspherical condenser.
- 3. the equipment according to claim 2 based on smart mobile phone detection biological marker, it is characterised in that：It is aspherical poly- The more flat side of light microscopic corresponds to incident light direction.
- 4. the equipment according to claim 3 based on smart mobile phone detection biological marker, it is characterised in that：It is aspherical poly- The distance of light microscopic to sample meets the focal length of condenser lens.
- 5. a kind of method for detecting biological marker, is carried out in accordance with the following steps：（1）Obtain biological marker；（2）The biological marker of acquisition and probe 1, probe 2, SA- magnetic beads, SA- quantum dot hybrid reactions form SA- magnetic beads+ Interlayer structure as Biotin- probe 1+ biological marker+Biotin- probe 2+SA- quantum dots；（3）Sample detection：The liquid of the above-mentioned interlayer structure containing biological marker is added on slide, is placed in detection device In, phosphor dot is imaged, smart mobile phone shooting obtains imaging picture；（4）Mobile phone app analyzes and processes to imaging picture.
- 6. the method for detection biological marker according to claim 5, it is characterised in that：Step（1）In, described biological marker is DNA, RNA, protein, biological micromolecule, cell, bacterium, virus, parasitism One kind in worm, residue of veterinary drug, food additives, heavy metal ion.
- 7. the method for detection biological marker according to claim 5, it is characterised in that：Step（2）The preparation of sample Journey is specially：SA- magnetic beads add appropriate PBS after mixing, magnetic centrifugal removes supernatant and obtains precipitation 1；Appropriate Biotin- probes 1 and PBS reaction solutions are added into precipitation 1, lasting mix ensures that magnetic bead is in suspension at room temperature State 0.5-3 hours, magnetic centrifugal remove supernatant and obtain precipitation 2；In step B) reaction during, appropriate PBS reaction solutions are added in new container and SA- quantum dots, concussion are uniformly mixed so as to obtain Solution 1；In step C) solution 1 in add a certain amount of Biotin- probes 2, lasting mix ensures that magnetic bead is in and suspended at room temperature State 0.5-3 hours, centrifuge and all liq is added in precipitation 2, while add biological marker and continue to be uniformly mixed so as to obtain solution 2；By step D) in the magnetic centrifugal of solution 2 remove supernatant, after precipitation is washed with PBS, precipitation is resuspended in PBS It is stored refrigerated to obtain sample to be tested in reaction solution.
- 8. the method for detection biological marker according to claim 5, it is characterised in that：Step（5）Mobile phone app processing methods are specially：Carry out in accordance with the following steps：Obtain operation information of the user to functionality controls in app interfaces；If the operation information meets testing requirements, app calling mobile phones camera shooting sample imaging picture simultaneously deposits picture It is placed in local storage space；Picture is compared according to local data base by App；Comparison is analyzed successfully, and as a result display is uploaded to cloud server storage simultaneously on mobile phone screen；Compare unsuccessful, picture, which is uploaded to cloud server, to carry out machine or manual analysis, analysis result and be back to mobile phone screen Display.
- 9. the method for detection biological marker according to claim 5, it is characterised in that：Step（2）In, according to body institute The amount of patient maximum biological marker determines the amount of minimum Biotin- probes 1 and Biotin- probes 2.
- 10. the equipment according to claim 1 based on smart mobile phone detection biological marker, it is characterised in that：Intelligent hand Machine could alternatively be the portable type electronic products such as tablet personal computer, digital camera, video camera, small scanner, imager；May be used also To replace with a series of small-sized movable such as the image acquisition device of designed, designed, electronic sensor, electronic chip, imaging sensor Terminal, is carried out data transmission and image procossing based on Wiff and bluetooth；It is, of course, also possible to replace with similar to notebook computer, The Large-scale Mobile terminal of desktop computer, projecting apparatus equivalent type, data are carried out by internet and uploaded and result feedback.
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