CN107421934A - A kind of real-time detection chip system of novel portable bacterium and detection method - Google Patents
A kind of real-time detection chip system of novel portable bacterium and detection method Download PDFInfo
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- CN107421934A CN107421934A CN201710653527.8A CN201710653527A CN107421934A CN 107421934 A CN107421934 A CN 107421934A CN 201710653527 A CN201710653527 A CN 201710653527A CN 107421934 A CN107421934 A CN 107421934A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
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Abstract
The invention belongs to Bacteria Detection technical field, more particularly to a kind of real-time detection chip system of novel portable bacterium and detection method, the detecting system integrates fluorescence detection function and dielectrophoresis enrichment function in one, it can either realize that object bacteria is highly enriched in microchip, improve detection sensitivity, reduce target bacteria detection limit, and can enough realizes the high precision test in situ of bacterium.Additionally it is possible to be done directly detection and analysis and data output inside detecting system, the portable detection in real time of bacterium is realized.The detection method high sensitivity, the degree of accuracy are good and simple to operate, are applicable bacterial concentration under a variety of varying environments and detect.
Description
Technical field
The invention belongs to Bacteria Detection technical field, and in particular to a kind of real-time detection chip system of novel portable bacterium
And detection method.
Background technology
The recognition detection of bacterium has in the field such as control and prevention of disease, clinical diagnosis and food sanitation safe and environmental monitoring
Important research is worth and Practical significance.The recognition detection method of bacterium has colony counting method, Immunological Method, flow cytometer at present
The Bacteria Culture of a variety of methods such as counting method, molecular biology method, fluorescence analysis detection method, wherein colony counting method as routine
Detection technique, the shortcomings of time-consuming, testing result inaccuracy be present, using limited；And Immunological Method, Flow cytometry
There is the defects of cumbersome or expensive in the methods of method, molecular biology method, in the quick knowledge for food-borne pathogens
Limitation and bottleneck are not still had and in terms of efficient detection；And bacterial fluorescence analyzing detecting method is because its effect is reliable, operation letter
It is single, have been to be concerned by more and more people.
The fluorescence detecting system of bacterial fluorescence analyzing detecting method, due to needing to add optically focused between light source and detector
Separate optical elements such as lens, and need external power supply, display system etc., so as to get the amplified filtering of electric signal after export to
Work station or recorder, integrated, miniaturization difficult, portable detection can not be both realized, also limit use environment, such as this problem
A kind of LED induction optical fiber type integrated PIN photo detector of the group disclosed in the A of patent document CN 101441177 it is miniature glimmering
Photodetector, external power supply is not only needed to realize to power elements such as excitation source, photodetectors, and need to be by detection gained
Output can not be completed to detect work, detector body self-contained portable to work station or recorder after the amplified filtering of signal arrived
Product is big, significantly limit its portability, real-time and working environment etc., and only rests on experimental stage, there is no method scale
Use.
At this stage, bacterium easy, integrated efficient detection technology are related real-time still in starting and experimental study stage
Site Detection instrument and method are still immature.
The content of the invention
It is an object of the present invention to provide a kind of real-time detection chip system of novel portable bacterium, the detecting system
Integrated fluorescence detection function and dielectrophoresis enrichment function can either realize effective richness of the object bacteria in microchip in one
Collection, improving detection sensitivity, reduce target bacteria detection limit, and can enough realizes the high precision test in situ of bacterium, moreover it is possible to
It is enough that detection and analysis and data output are done directly inside detecting system, realize the real-time detection of bacterium.
It is a further object to provide the application real-time detection chip system detectio of above-mentioned novel portable bacterium is thin
The method of bacterium, methods described combination magnetic effect and dielectrophoresis effect double mode separation and concentration, detection sensitivity is high, object bacteria inspection
Rising limit substantially reduces, and the degree of accuracy is good, and stability is good, simple to operate, is applicable the bacterial concentration detection of a variety of environment.
For achieving the above object, the present invention provides following technical scheme：
A kind of real-time detection chip system of novel portable bacterium, including integral or integral type the detection module of assembling and processing
Module, light tight lid that the detection module includes from top to bottom setting gradually, chip mounting table, optical path unit and it is arranged at
DEP on chip mounting table excites unit, and the top of the processing module is integrated with circuit unit, the middle part of the processing module
Display typing unit is integrated with, the bottom of the processing module is integrated with separate power source element, it is characterised in that：
The light tight lid covers whole chip mounting table, can avoid the interference of veiling glare, improve accuracy of detection and data
Accurate reliability；
The middle part of the chip mounting table is provided with the chip rack of hollow out, and for placing chip to be measured, chip is in the chip
Trim locations can be moved left and right on rack, it is ensured that the hot spot of chip area to be detected alignment excitation light path, while hollow out is set
While meter can reduce the use of multiple beam optical fiber, avoid exciting light loss, improve detection sensitivity；
The optical path unit is made up of excitation light path and light path, and the light path is located at homonymy with excitation light path and is in
90 ° of angles, not only while miniaturization is advantageous to, the focus adjustment for meeting excitation light path collector lens can be taken into account apart from need
Will, and the stability of mounting structure can be significantly improved, it is ensured that the structure precision of the stationarity of signal output and whole instrument
Distribution, the excitation light path include excitation source, the first filter plate and collector lens, and the exciting light that excitation source is sent is through first
After filter plate filtration, the area to be detected of chip is focused on by collector lens, the light path includes the second filter plate and photoelectricity
Detector, the exciting light that excitation source is sent focus on chip area to be detected by collector lens after the filtration of the first filter plate, believed
Number intensity is good, no interference of stray light, excite caused by fluorescence after the filtration of the second filter plate, received by photodetector, and will
Optical signal is converted into electric signal, high sensitivity, it is good to repeat detection stability；
The DEP excites unit to include pumping signal generating unit, AD conversion unit and data processing unit, and the DEP is excited
The outside of unit is provided with outer cover metal shielding box, and the outer cover metal shielding box is described using the Faraday shield pattern being grounded
Pumping signal generating unit provides 0-10 Vpp excitation voltage and 1KHz -5MHz stimulating frequency for DEP chips, described
AD conversion unit realized to the AD conversion of electric signal, the data processing unit realize calculating to the data signal of AD conversion and
Processing, and be delivered to display typing unit and show that the DEP excites unit to be arranged on chip mounting table, while outside it
Outer cover metal shielding box is set, on the one hand can effectively avoid DEP from exciting high frequency ac signal caused by unit electric to other
Element produces electromagnetic interference, on the other hand may insure that DEP excites high frequency ac signal caused by unit to have stable amplitude
With frequency to ensure DEP concentration effects, excite unit to provide suitable and stable excitation voltage and stimulating frequency by DEP, make
Object bacteria while being enriched in region to be detected, removes Excess reagents, kept away under the positive dielectrophoresis force effect of micro- interdigital electrode
Exempt from the interference of background；
The circuit unit includes electric signal amplifying unit, filter unit, and the AD conversion unit of units shared is excited with DEP
And data processing unit, the electric signal amplifying unit realize the amplification to electric signal, the filter unit filters out High-frequency Interference
Signal, the circuit unit and DEP excite unit by multi-channel electronic switch realize signal switch, realize DEP arousal functions and
Integrating for fluorescence detection function, basis is provided for the real-time further miniature portable of detection chip system of novel portable bacterium,
The photodetector connection of the circuit unit and optical path unit, the signal of photodetector detection amplify through circuit unit, turned
Output extremely display typing unit after changing；
The display typing unit includes display screen, button, master control borad PCB, and the display typing unit is connected with circuit unit,
Realize that the acquisition, processing, result of data are shown and instruction input；
The separate power source element excites unit to be connected by switching with excitation source, photodetector, master control borad PCB and DEP,
Can be respectively and/or simultaneously excitation source by switching control, photodetector, master control borad PCB and DEP excite unit to supply
Electricity.
Further, the portable bacterium real-time detector is handheld structure, and described detection module is horizontally disposed with, institute
The processing module stated is vertically arranged, and the right flank of detection module is bonded or is integral type closely with the left surface of processing module.
Further, the supporting micro-fluidic chip for the portable bacterium real-time detector is made up of cover plate and egative film,
The cover plate is provided with least one pair of DEP enrichment detection zones, and the DEP enrichments detection zone is rectangle, each DEP enrichments detection
Area connects an injection port by sample channel, and each pair DEP enrichment detection zones are collected to same outlet by flowing out pipeline,
And the outlet is located on the center line of its corresponding a pair of DEP enrichments detection zone, can ensure that each pair DEP is enriched with detection zone
The flow velocity of interior detection sample is consistent, and 20-30 is integrated with to micro- interdigital electrode, institute on egative film corresponding to the DEP enrichments detection zone
State micro- interdigital electrode a length of 1500 μm -2000 μm, a width of 18 μm -23 μm, the micro- interdigital electrode of each pair at intervals of 18 μm of -23 μ
The paired design of m, DEP enrichment detection zone, can be detected to sample to be tested and reference substance by identical condition simultaneously, so as to
Testing result is corrected, improves the method degree of accuracy, or detects two samples simultaneously, improves detection efficiency.
The inventors discovered that being found when integrated DEP enrichment functions and fluorescence detection function are in one, DEP excites unit
Pumping signal generating unit caused by micro-fluidic chip need DEP high frequency ac signals, have to other electrical equipments very strong
Electromagnetic interference, influence the reliability of testing result；Meanwhile the DEP high frequency ac signals need stable amplitude and frequency, with
Ensure the DEP concentration effects of bacterium.The present inventor has found by substantial amounts of research, excites the outside of unit to set outer cover to DEP
Metal shielding box, and using the Faraday shield pattern of ground connection simultaneously, can admirably realize that DEP excites unit and circuit unit
Etc. being electrically isolated from one another for other parts, DEP can not only be effectively avoided to excite high frequency ac signal caused by unit to other
Electrical equipment produces electromagnetic interference, and be able to ensure that DEP excite high frequency ac signal caused by unit have stable amplitude and
Frequency, it is ensured that DEP concentration effects.
Present invention discover that the shape and size of DEP enrichment detection zones are very big on the influence of bacterium secondary concentration effect, DEP is rich
Collection detection zone is designed as rectangle, while integrated 20-30 is to a length of 1500 μm -2000 μm, a width of 18 μm -23 μm, the micro- fork of each pair
Refer to electrode at intervals of 18 μm -23 μm of micro- interdigital electrode, the radical length of enrichment detection zone and effectively rich can be dramatically increased
Collect detection zone area, improve capture rate and detection sensitivity.
The real-time detection chip system of novel portable bacterium provided by the invention, it is excellent by the distribution of rational space and structure
Change, shielding design etc., while system accuracy is ensured, by increasing capacitance it is possible to increase the utilization rate in space, length size are no more than
30 centimetres, weight is no more than 1kg, meets miniaturization and the requirement of portability.
Second aspect, the present invention provide the side of the application real-time detection chip system detectio bacterium of above-mentioned novel portable bacterium
Method, comprise the following steps：
Step 1 is once enriched with：The aptamers of selection energy specific recognition target bacteria, prepare nanometer magnetic bead-adaptor complex
（MNPs-Apt）, by gained nanometer magnetic bead-adaptor complex and object bacteria sample incubation, and it is marked with fluorometric reagent,
Nanometer magnetic bead-aptamers-object bacteria compound of fluorescence labeling is prepared（MNPs-Apt- object bacterias）, in the work of external magnetic field
With the once enrichment of lower completion object bacteria, the nanometer magnetic bead-adaptation for being specifically bound to obtain to object bacteria using aptamers
Body-object bacteria complex stabilities are high, and high specificity, concentration effect is good, and the object bacteria that can be significantly improved in sample to be tested is dense
Degree；
Two enrichments of step 2：The interdigital microelectrode of array will be integrated with（DEP）Micro-fluidic chip be placed in above-mentioned integrated dielectric electricity
On the chip mounting table of the portable bacterium real-time detector of swimming, by moving left and right trim locations on chip rack, make
DEP detection zones are corresponding with the hot spot of excitation light path, excite unit to be connected with DEP the interdigital microelectrode of chip array, by step 1
Gains injection micro-fluidic chip injection port is once enriched with, controlling switch makes separate power source element excite unit and master control for DEP
Plate PCB powers, and by showing that typing unit button provides suitable DEP stimulating frequencies and voltage for chip, completes object bacteria
Secondary enrichment, object bacteria sample is enriched in DEP detection zones under the effect of micro- interdigital electrode positive dielectrophoresis force, while more
Remaining nanometer magnetic bead and fluorescent labeling reagent flows out detection zone due to that can not be captured by DEP with fluid, can effectively avoid carrying on the back
Scape fluorescence disturbs, by secondary enrichment, it is possible to increase the sensitivity of Bacteria Detection simultaneously reduces detection limit；
Step 3 detects：Controlling switch makes separate power source element be obtained for excitation source, photodetector and master control borad PCB power supplies
Object bacteria sample fluorescence value testing result is obtained, the exciting light line focus lens focus that excitation source is sent is in chip DEP detection zones
Domain, induction mark produce fluorescence in the fluorometric reagent of captured bacterium surface, after testing the optical filter filtering of light path, photodetection
Device receives, and is delivered to circuit unit, and eventually passes through display typing unit and carry out data acquisition, processing, and output obtains fluorescent value
Testing result.
According to the method for detection bacterium of the present invention, fluorometric reagent marks in step 1 method be by organic fluorescence reagent with
Nanometer magnetic bead-adaptor complex, the sample containing object bacteria are incubated nanometer magnetic bead-adaptation that fluorescence labeling is prepared simultaneously
Body-object bacteria compound, or after fluorometric reagent first is prepared into fluorometric reagent-adaptor complex with suitable aptamers,
Fluorometric reagent-adaptor complex, nanometer magnetic bead-adaptor complex and object bacteria are incubated simultaneously fluorescence labeling is prepared
Nanometer magnetic bead-aptamers-object bacteria compound；Preferably, the method that fluorometric reagent marks in step 1 is that fluorometric reagent is first
After fluorometric reagent-adaptor complex being prepared into suitable aptamers, then by fluorometric reagent-adaptor complex, nano magnetic
Nanometer magnetic bead-aptamers-object bacteria that fluorescence labeling is prepared in pearl-adaptor complex and object bacteria incubation simultaneously is compound
Thing, the fluorescence interference of other miscellaneous bacterias can be removed, improve the sensitivity and the degree of accuracy of detection method.
According to the method for detection bacterium of the present invention, further comprise the steps：By the standard liquid of object bacteria according to step
Rapid 3 method measures fluorescence difference, using concentration as abscissa, using the fluorescent value measured as ordinate, establishes canonical plotting, will
The sample to be tested of two enrichments of step 2 measures fluorescence difference by the method for step 3, substitutes into canonical plotting and obtains sample to be tested
Middle object bacteria concentration value.
The dielectric of the high magnetic of method combination nanometer magnetic bead provided by the invention, the specificity of aptamers and bacterium is special
Property, integrated magnetic effect and dielectrophoresis effect are efficiently separated enrichment, can greatly promote the separation and concentration of target bacteria twice
Efficiency, Bacteria Detection sensitivity is improved, the original position of object bacteria is realized, quickly and efficiently detects；In addition, method provided by the invention
Herein in connection with the portable bacterium real-time detector of integrated dielectrophoresis provided by the invention, object bacteria in complex samples can be realized
Real-time detection, can promote fast-bacteria-detection technical applicationization develop.
Brief description of the drawings
Fig. 1 is the structural representation of the real-time detection chip system of novel portable bacterium of the present invention；
Fig. 2 is the detection module structural representation of the real-time detection chip system of novel portable bacterium of the present invention；
Fig. 3 is the processing module structural representation of the real-time detection chip system of novel portable bacterium of the present invention；
Fig. 4 is the supporting microfluidic chip structure schematic diagram for the real-time detection chip system of novel portable bacterium of the present invention；
Fig. 5 is the schematic diagram of the real-time detection chip system of novel portable bacterium of the present invention；
Fig. 6 is the fluorescence signal intensity difference of embodiment 2 and salmonella concentration ATCC14028 Log value linear relationship charts；
In figure,
1- detection modules, the light tight lids of 11-, 12- chip mounting tables, 121- chip racks, 13- optical path units,
131- excitation light paths, 132- light paths, 14-DEP excite unit；
2- processing modules, 21- circuit units, 22- show typing unit, 23- separate power source elements；
90-DEP is enriched with detection zone, 91- injection ports, 92- outlets, the micro- interdigital electrodes of 93-.
Specific embodiment
Embodiment 1 integrates the portable bacterium real-time detector of dielectrophoresis
The real-time detection chip system of novel portable bacterium as shown in Figure 1-Figure 3, including integral or integral type the inspection of assembling
Module 1 and processing module 2 are surveyed, the detection module 1 includes light tight lid 11, the chip mounting table from top to bottom set gradually
12nd, optical path unit 13 and the DEP being arranged on chip mounting table 12 excite unit 14, and the top of the processing module 2 integrates
There is circuit unit 21, the middle part of the processing module 2 is integrated with display typing unit 22, and the bottom of the processing module 2 integrates
There is separate power source element 23, the light tight lid 11 covers whole chip mounting table 12, can avoid the interference of veiling glare, carry
The accurate reliability of high measurement accuracy and data；
The middle part of the chip mounting table 12 is provided with the chip rack 121 of hollow out, chip energy on the chip rack 121
Enough move left and right trim locations, it is ensured that the hot spot of chip area to be detected alignment excitation light path, while hollow out is designed to reduce
While multiple beam optical fiber uses, avoid exciting light loss, improve detection sensitivity；
The optical path unit 13 is made up of excitation light path 131 and light path 132, the light path 132 and excitation light path 131
Positioned at homonymy and it is in 90 ° of angles, not only while miniaturization is advantageous to, can takes into account and meet excitation light path collector lens
Focus adjustment distance needs, and can significantly improve the stability of mounting structure, it is ensured that the stationarity of signal output and whole
The structure precision distribution of instrument, the excitation light path 131 include excitation source, the first filter plate and collector lens, the detection
Light path 132 includes the second filter plate and photodetector, and the exciting light that excitation source is sent is after the filtration of the first filter plate, by gathering
Optical lens focuses on chip area to be detected, and signal intensity is good, no interference of stray light, excites caused fluorescence to be filtered through the second filter plate
Later, received by photodetector, and convert optical signals into electric signal, high sensitivity, it is good to repeat detection stability；
The DEP excites unit 14 to include pumping signal generating unit, AD conversion unit and data processing unit, and the DEP swashs
The outside of bill member 14 is provided with outer cover metal shielding box, and the outer cover metal shielding box uses the Faraday shield pattern of ground connection,
The pumping signal generating unit provides 0-10 Vpp excitation voltage and 1KHz -5MHz stimulating frequency for DEP chips,
The AD conversion unit realizes the AD conversion to electric signal, and the data processing unit realizes the meter to the data signal of AD conversion
Calculate and handle, and be delivered to typing display unit 22 and show, the DEP excites unit 14 to be arranged on chip mounting table 12, together
Outer cover metal shielding box is set outside Shi Qi, on the one hand can effectively avoid DEP from exciting high-frequency ac caused by unit 14 to believe
Number to other electrical equipments produce electromagnetic interference, on the other hand may insure that DEP excites high frequency ac signal caused by unit 14
With the amplitude and frequency stablized to ensure DEP concentration effects, exciting unit 14 to provide by DEP, suitable and that stablizes excites electricity
Pressure and stimulating frequency, make object bacteria under the positive dielectrophoresis force effect of micro- interdigital electrode, while being enriched in region to be detected,
Excess reagents are removed, avoid the interference of background；
The circuit unit 21 includes electric signal amplifying unit, filter unit, and excites the shared AD conversion of unit 14 with DEP
Unit and data processing unit, the electric signal amplifying unit realize the amplification to electric signal, and the filter unit filters out high frequency
Interference signal, the circuit unit 21 and DEP excite unit to realize that signal switches by multi-channel electronic switch, realize that DEP is excited
Function and fluorescence detection function it is integrated, provided for the real-time further miniature portable of detection chip system of novel portable bacterium
Basis, the circuit unit 21 are connected with the photodetector of optical path unit 13, and the signal of photodetector detection is through circuit list
Output extremely display typing unit 22 after the amplification of member 21, conversion；The display typing unit 22 includes display screen, button, master control borad
PCB, it is connected with circuit unit 21, realizes that result is shown and instruction input；
The separate power source element 23 excites unit to connect by switching with excitation source, photodetector, master control borad PCB and DEP
Connect, can be respectively and/or simultaneously excitation source by switching control, photodetector, master control borad PCB and DEP excite unit
Power supply.
The portable bacterium real-time detector is handheld structure, and described detection module 1 is horizontally disposed, described place
Reason module 2 is vertically arranged, and the right flank of detection module 1 is bonded or is integral type closely with the left surface of processing module 2.
As shown in figure 4, the supporting micro-fluidic chip for the portable bacterium real-time detector is by cover plate and egative film structure
Into, the long wide 2.5cm of 3cm, material is advisable with PDMS, is made using thermoplastic method, and cover plate is provided with two DEP and is enriched with detection zone 90,
Two DEP enrichment detection zones 90 are rectangle, and length is 3000 μm, and wide is 2000 μm, in two DEP enrichment detection zones 90
In the heart away from for 10mm, each DEP enrichments detection zone 90 passes through sample intake passage and connects an injection port 91, a width of 500 μ of sample intake passage
M, a height of 50 μm, the distance of the corresponding DEP enrichment detection zones of injection port 91 is 5000 μm, and two DEP are enriched with detection zone
（90）Same outlet 92 is pooled to, outlet 92 is on the center line of two detection zones, away from detection zone air line distance
7mm, 25 pairs of micro- interdigital electrodes 93 are integrated with egative film corresponding to the DEP enrichments detection zone 90, each micro- interdigital electrode is a length of
1900 μm, a width of 20 μm, the micro- interdigital electrode of each pair at intervals of 20 μm.
Upright emulsion tube and sample introduction liquid storage tank are installed in micro-fluidic chip inlet and outlet position and go out sample negative pressure pump company using preceding
Connect, be placed in the chip rack of the portable bacterium real-time detector of integrated dielectrophoresis, and be connected with DEP excitation modules.
The salmonella typhimurium ATCC14028 of embodiment 2 is detected
The foundation of 2.1 standard curves
Using salmonella typhimurium ATCC14028 as target bacteria, from the excellent quantum dot of photoluminescent property（QDs）As fluorescence
Labelled reagent.Coupling reagent is separately added into 200 μ L nanometer magnetic beads solution and 100 μ L QDs（EDC（60mM）And NHS
（30mM）Solution）, lucifuge concussion reaction 30min.The amido modified object bacteria specific recognition aptamers of 1OD are then added, instead
After answering 1h, Magneto separate simultaneously cleans 3 times, and decibel obtains nanometer magnetic bead-adaptor complex（MNPs-Apt）With quantum dot-aptamers
Compound（QDs-Apt）；
100 μ L salmonella typhimuriums ATCC14028 are inoculated in 300-600mL LB culture mediums, 37 DEG C be incubated overnight after put down
Plate is counted and draws bacterial concentration, and gained object bacteria is diluted into 50,10 by gradient concentration with PBS2、103、104、105、106 cfu/
The standard items liquid of 6 concentration of mL, experimental comparison group is used as by the use of same amount of PBS；
Nanometer magnetic bead-adaptor complex is added into above-mentioned object bacteria standard items liquid and comparison liquid respectively（MNPs-Apt）And amount
Sub- point-adaptor complex（QDs-Apt）, after being incubated 30min, separation and concentration is carried out to sample using external magnetic field and is resuspended in
In 100 μ L deionized waters；
The object bacteria standard items liquid and comparison liquid that pass through Magneto separate and be resuspended in deionized water are respectively placed in micro-fluidic DEP cores
Corresponding to a pair of DEP enrichment detection zones of piece in two injection port liquid storage tanks, using embodiment 1 integrated dielectrophoresis it is portable
Formula bacterium real-time detector applies DEP excitation signals, DEP capture enrichment object bacterias to the interdigital microelectrode of array of two detection zones
10min, open excitation source and carry out fluoroscopic examination, testing result is recorded, with object bacteria sample and check sample fluorescence intensity
Difference is ordinate, using the concentration of object bacteria standard items liquid as abscissa, establishes standard curve, sees Fig. 6, detection range 102
-105Cfu/mL, detection are limited to 50 cfu/mL.
Sample to be tested salmonella typhimurium ATCC14028 Concentration Testing
Nanometer magnetic bead-adaptor complex is prepared with reference to the method for step 2.1（MNPs-Apt）With quantum dot-adaptor complex
（QDs-Apt）, by nanometer magnetic bead-adaptor complex（MNPs-pt）With quantum dot-adaptor complex（QDs-Apt）Add
In sample to be tested and check sample, it is respectively 150 μ g/mL and 200 μ g/mL to make its final concentration, is placed in 37 in shaking tableoC 150
Rpm shakings are incubated 30 min, and reaction terminates rear Magneto separate frame separation, removes supernatant, PBS 2 times, be resuspended in 100 μ L
In PBS, completion is once enriched with；
Sample to be tested and check sample are placed in two injection ports corresponding to a pair of DEP enrichment detection zones of micro-fluidic DEP chips
In liquid storage tank, the array of two detection zones is pitched using the portable bacterium real-time detector of the integrated dielectrophoresis of embodiment 1
Refer to microelectrode and apply DEP excitation signals, DEP capture enrichment object bacteria 10min, open excitation source and carry out fluoroscopic examination, remember
Testing result is recorded, the difference for measuring concentration samples and check sample fluorescence intensity is 0.352, and the standard for bringing step 2.1 foundation into is bent
In line, the content for trying to achieve salmonella typhimurium ATCC14028 in sample to be tested is 3.291 × 103cfu/mL。
Claims (6)
1. a kind of real-time detection chip system of novel portable bacterium, including integral or integral type the detection module (1) of assembling
With processing module (2), the detection module (1) includes light tight lid (11), the chip mounting table from top to bottom set gradually
(12), optical path unit (13) and the DEP being arranged on chip mounting table (12) excite unit (14), the processing module (2)
Top be integrated with circuit unit (21), be integrated with the middle part of the processing module (2) display typing unit (22), the processing
The bottom of module (2) is integrated with separate power source element (23), it is characterised in that：
The light tight lid (11) covers whole chip mounting table (12)；
The middle part of the chip mounting table (12) is provided with the chip rack (121) of hollow out, for placing chip to be measured；
The optical path unit (13) is made up of excitation light path (131) and light path (132), and the light path (132) is with swashing
Luminous road (131) is located at homonymy and is in 90 ° of angles, and the excitation light path (131) includes excitation source, the first filter plate and gathered
Optical lens, the exciting light that excitation source is sent are focused on the area to be detected of chip by collector lens after the filtration of the first filter plate,
The light path (132) includes the second filter plate and photodetector, excites caused fluorescence after the filtration of the second filter plate,
Received by photodetector, and convert optical signals into electric signal；
The DEP excites unit (14) to include pumping signal generating unit, AD conversion unit and data processing unit, the DEP
The outside of unit (14) is excited to be provided with outer cover metal shielding box, the outer cover metal shielding box is using the Faraday shield mould being grounded
Formula, the pumping signal generating unit provide 0-10Vpp excitation voltage and 1KHz-5MHz stimulating frequency, institute for DEP chips
AD conversion of the AD conversion unit realization to electric signal is stated, the data processing unit realizes the meter to the data signal of AD conversion
Calculate, processing, and be delivered to display typing unit (22) display；
The circuit unit (21) includes electric signal amplifying unit, filter unit, and excites the shared AD of unit (14) with DEP
Converting unit and data processing unit, the electric signal amplifying unit realize the amplification to electric signal, and the filter unit filters out
High-frequency interferencing signal, the circuit unit (21) and DEP excite unit to realize that signal switches by multi-channel electronic switch, realize
Integrated, the circuit unit (21) and the photodetector company of optical path unit (13) of DEP arousal functions and fluorescence detection function
Connect；
The display typing unit (22) includes display screen, button, master control borad PCB, is connected with circuit unit (21), realizes data
Result show and instruction input；
The separate power source element (23) excites unit by switch with excitation source, photodetector, master control borad PCB and DEP
Connection.
2. portable bacterium real-time detector as claimed in claim 1, it is characterised in that：The portable bacterium detects in real time
Device is handheld structure, and described detection module (1) is horizontally disposed, and described processing module (2) is vertically arranged, detection module
(1) right flank is bonded or is integral type closely with the left surface of processing module (2).
3. portable bacterium real-time detector as claimed in claim 1, it is characterised in that：It is supporting to be used for the portable bacterium
The micro-fluidic chip of real-time detector is made up of cover plate and egative film, and the cover plate is provided with least one pair of DEP enrichment detection zones
(90), the DEP enrichment detection zones (90) are rectangle, and each DEP enrichment detection zones (90) connect one by sample channel
Injection port (91), each pair DEP enrichment detection zones (90) are collected to same outlet (92) by flowing out pipeline, and it is described go out sample
Mouth (92) is located on the center line of its corresponding a pair of DEP enrichments detection zone, egative film corresponding to the DEP enrichment detection zones (90)
On be integrated with 20-30 to micro- interdigital electrode (93), a length of 1500 μm -2000 μm of micro- interdigital electrode, a width of 18 μm of -23 μ
M, the micro- interdigital electrode of each pair at intervals of 18 μm -23 μm.
4. the method for the portable bacterium real-time detector detection bacterium described in claim any one of 1-3, comprises the following steps：
Step 1 is once enriched with：The aptamers of selection energy specific recognition target bacteria, prepare nanometer magnetic bead-adaptor complex
(MNPs-Apt), by gained nanometer magnetic bead-adaptor complex and object bacteria sample incubation, and it is marked with fluorometric reagent,
Nanometer magnetic bead-aptamers-object bacteria compound (MNPs-Apt- object bacterias) of fluorescence labeling is prepared, in the work of external magnetic field
With the once enrichment of lower completion object bacteria；
Two enrichments of step 2：The micro-fluidic chip for being integrated with the interdigital microelectrode of array (DEP) is placed in collection described in claim 1
Into on the chip mounting table (12) of the portable bacterium real-time detector of dielectrophoresis, by left on chip rack (121)
Move right trim locations, make DEP detection zones corresponding with the hot spot of excitation light path (131), by the interdigital microelectrode of chip array with
DEP excites unit to connect, and step 1 is once enriched with to gains injection micro-fluidic chip injection port, and controlling switch makes independent current source
Unit (23) is that DEP excites unit and master control borad PCB to power, and by showing that typing unit (22) button provides properly for chip
DEP stimulating frequencies and voltage, complete the secondary enrichment of object bacteria；
Step 3 detects：Controlling switch makes separate power source element (23) for excitation source, photodetector and master control borad PCB power supplies,
Obtain object bacteria sample fluorescence value testing result.
5. detection method as claimed in claim 4, it is characterised in that：The method that fluorometric reagent marks in step 1 is will be organic
Fluorometric reagent is incubated and receiving for fluorescence labeling is prepared simultaneously with nanometer magnetic bead-adaptor complex, the sample containing object bacteria
Rice magnetic bead-aptamers-object bacteria compound, or fluorometric reagent is first prepared into fluorometric reagent-be adapted to suitable aptamers
After nanocrystal composition, fluorometric reagent-adaptor complex, nanometer magnetic bead-adaptor complex and object bacteria are incubated preparation simultaneously
Obtain nanometer magnetic bead-aptamers-object bacteria compound of fluorescence labeling；Preferably, the method for fluorometric reagent mark is in step 1
After fluorometric reagent first is prepared into fluorometric reagent-adaptor complex with suitable aptamers, then fluorometric reagent-aptamers are multiple
Compound, nanometer magnetic bead-adaptor complex and object bacteria be incubated simultaneously be prepared nanometer magnetic bead-aptamers of fluorescence labeling-
Object bacteria compound.
6. the detection method as described in claim 4 or 5, it is characterised in that：Still further comprise following steps：By object bacteria
Standard liquid measures fluorescence difference according to the method for step 3, using concentration as abscissa, using the fluorescent value measured as ordinate, builds
Vertical canonical plotting, the sample to be tested of two enrichments of step 2 is measured into fluorescence difference by the method for step 3, substitutes into standard curve
Target bacteria concentration value in sample to be tested is obtained in figure.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001269A (en) * | 2018-09-19 | 2018-12-14 | 重庆大学 | Integrate the bacterium chip and its detection method of DEP separation, magnetic microsphere selective enrichment and EIS in situ detection |
| CN110174382A (en) * | 2019-05-23 | 2019-08-27 | 安徽华米生物科技有限公司 | It is a kind of for detecting the device of sewage bacterial concentration |
| CN110907418A (en) * | 2019-11-27 | 2020-03-24 | 中国科学院上海光学精密机械研究所 | Device for detecting optical characteristics of liquid sample in pipette head |
| CN113176118A (en) * | 2021-04-29 | 2021-07-27 | 江苏大学 | Portable gas fax bacterium real-time acquisition and detection device and method |
| CN113252632A (en) * | 2021-06-25 | 2021-08-13 | 成都瀚辰光翼生物工程有限公司 | Sample concentration processing method and device, sample processing equipment and readable storage medium |
| CN113671172A (en) * | 2021-08-11 | 2021-11-19 | 中国科学院微电子研究所 | Microfluidic detection device |
| CN114705665A (en) * | 2022-06-02 | 2022-07-05 | 圣湘生物科技股份有限公司 | Fluorescence detection device and fluorescence detection method |
| GB2605697A (en) * | 2021-04-29 | 2022-10-12 | Univ Jiangsu | Not published |
| CN116337728A (en) * | 2023-05-30 | 2023-06-27 | 天津大学 | Fluorescence detection device of monolithic integrated micro-flow cytometer |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060219939A1 (en) * | 2004-12-03 | 2006-10-05 | Nano Science Diagnostic, Inc. | Method and apparatus for low quantity detection of bioparticles in small sample volumes |
| CN101693875A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on columnar microelectrode array and electrofusion method |
| CN101738418A (en) * | 2010-01-21 | 2010-06-16 | 重庆大学 | Microfluidic chip system integrating cell operation and detection |
| CN102507706A (en) * | 2011-10-11 | 2012-06-20 | 重庆大学 | Microfluidic chip analysis microsystem for detecting bacterium dielectric electrophoresis impedance |
| US8398839B1 (en) * | 2009-06-05 | 2013-03-19 | Sandia Corporation | System for particle concentration and detection |
| CN103529006A (en) * | 2013-10-18 | 2014-01-22 | 大连海事大学 | Portable fluorescence detection device based on micro-fluidic chip and detection method thereof |
-
2017
- 2017-08-02 CN CN201710653527.8A patent/CN107421934B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060219939A1 (en) * | 2004-12-03 | 2006-10-05 | Nano Science Diagnostic, Inc. | Method and apparatus for low quantity detection of bioparticles in small sample volumes |
| US8398839B1 (en) * | 2009-06-05 | 2013-03-19 | Sandia Corporation | System for particle concentration and detection |
| CN101693875A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on columnar microelectrode array and electrofusion method |
| CN101738418A (en) * | 2010-01-21 | 2010-06-16 | 重庆大学 | Microfluidic chip system integrating cell operation and detection |
| CN102507706A (en) * | 2011-10-11 | 2012-06-20 | 重庆大学 | Microfluidic chip analysis microsystem for detecting bacterium dielectric electrophoresis impedance |
| CN103529006A (en) * | 2013-10-18 | 2014-01-22 | 大连海事大学 | Portable fluorescence detection device based on micro-fluidic chip and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| HE XIAOXIAO ET AL.: "Rapid and ultrasensitive Salmonella Typhimurium quantification using positive dielectrophoresis driven on-line enrichment and fluorescent nanoparticles label", 《BIOSENSORS AND BIOELECTRONICS》 * |
| WANG RENJIE ET AL.: "Rapid and sensitive detection of Salmonella typhimurium using aptamer-conjugated carbon dots as fluorescence probe", 《ANALYTICAL METHODS》 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001269A (en) * | 2018-09-19 | 2018-12-14 | 重庆大学 | Integrate the bacterium chip and its detection method of DEP separation, magnetic microsphere selective enrichment and EIS in situ detection |
| CN109001269B (en) * | 2018-09-19 | 2021-03-02 | 重庆大学 | Bacterial chip integrating DEP separation, magnetic microsphere selective enrichment and EIS in-situ detection and detection method thereof |
| CN110174382A (en) * | 2019-05-23 | 2019-08-27 | 安徽华米生物科技有限公司 | It is a kind of for detecting the device of sewage bacterial concentration |
| CN110174382B (en) * | 2019-05-23 | 2020-11-20 | 安徽维嵩生产力促进有限公司 | Device for detecting sewage bacteria concentration |
| CN110907418A (en) * | 2019-11-27 | 2020-03-24 | 中国科学院上海光学精密机械研究所 | Device for detecting optical characteristics of liquid sample in pipette head |
| GB2605697B (en) * | 2021-04-29 | 2023-04-19 | Univ Jiangsu | Portable real-time airborne fungi acquiring and detecting equipment and method |
| GB2605697A (en) * | 2021-04-29 | 2022-10-12 | Univ Jiangsu | Not published |
| WO2022227395A1 (en) * | 2021-04-29 | 2022-11-03 | 江苏大学 | Portable airborne fungus real-time collection and detection device and method |
| US11566980B2 (en) | 2021-04-29 | 2023-01-31 | Jiangsu University | Portable real-time airborne fungi acquiring and detecting equipment and method |
| CN113176118A (en) * | 2021-04-29 | 2021-07-27 | 江苏大学 | Portable gas fax bacterium real-time acquisition and detection device and method |
| CN113252632A (en) * | 2021-06-25 | 2021-08-13 | 成都瀚辰光翼生物工程有限公司 | Sample concentration processing method and device, sample processing equipment and readable storage medium |
| CN113671172A (en) * | 2021-08-11 | 2021-11-19 | 中国科学院微电子研究所 | Microfluidic detection device |
| CN113671172B (en) * | 2021-08-11 | 2024-07-02 | 中国科学院微电子研究所 | Microfluidic detection device |
| CN114705665A (en) * | 2022-06-02 | 2022-07-05 | 圣湘生物科技股份有限公司 | Fluorescence detection device and fluorescence detection method |
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| CN116337728B (en) * | 2023-05-30 | 2023-08-18 | 天津大学 | Fluorescence detection device of monolithic integrated micro-flow cytometer |
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