CN109813692A - A Capillary Analysis and Detection Method Based on Ultrasonic Aggregation - Google Patents
A Capillary Analysis and Detection Method Based on Ultrasonic Aggregation Download PDFInfo
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- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 claims abstract description 17
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- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims description 18
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 9
- 239000011787 zinc oxide Substances 0.000 claims description 9
- 239000010931 gold Substances 0.000 claims description 7
- 229910052737 gold Inorganic materials 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 6
- 239000000919 ceramic Substances 0.000 claims description 6
- 239000004793 Polystyrene Substances 0.000 claims description 5
- 229920002223 polystyrene Polymers 0.000 claims description 5
- 229910052709 silver Inorganic materials 0.000 claims description 5
- 239000004332 silver Substances 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 3
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- 238000004544 sputter deposition Methods 0.000 claims description 3
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- 102000053602 DNA Human genes 0.000 description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000004411 aluminium Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention provides a kind of capillary analysis detection method based on ultrasound aggregation, belongs to technical field of analytical chemistry.This method is by will be in the nano particle mixed injection capillary after trace object, signal mark molecule and special sex modification; assemble the nano particle after capturing marker by ultrasonic unit to realize the aggregation to ultra trace object, and Raman microscope or fluorescence microscope is combined to realize SERS or fluorescence detection to object.This method has the advantages such as detection speed is fast, detection limit is low, achievable automatic detection, is realizing that fast accurate is analyzed and ultra trace context of detection has important value.
Description
Technical field
The present invention relates to technical field of analytical chemistry, particularly relate to a kind of capillary analysis detection side based on ultrasound aggregation
Method.
Background technique
Accurate quick detection to trace determinand is the key that modern clinic medical treatment detection.Capillary is a kind of cost
Cheap and portable material, may be implemented the acquisition of micro-example, is widely used in acquiring sample in hospital, is clinical
Standing consumptive material, but the correlation technique that capillary is used to detect is actually rare.Be currently known capillary is used for detect
Method also needs to carry out capillary complicated pre-treatment mostly, is not easy to popularization and long-time storage on a large scale.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of capillary analysis detection method based on ultrasound aggregation, the party
Method has the advantages such as detection speed is fast, detection limit is low, achievable automatic detection, is realizing fast accurate analysis and ultra trace
Context of detection has important value, provides a kind of feasible scheme for the capillary analysis detection of trace object.
This method is by the nano particle mixed injection capillary after trace object, signal mark molecule and special sex modification
In, the nano particle after capturing marker is assembled to realize the aggregation to ultra trace object by ultrasonic unit, and combine
Raman microscope or fluorescence microscope realize SERS or fluorescence detection to object.
Specifically include that steps are as follows:
(1) signaling molecule is marked with label probe, obtains signal mark molecule；
(2) special sex modification is carried out to nano particle；
(3) mixed liquor is made in the nano particle after trace object, signal mark molecule and special sex modification；
(4) mixed liquor made from step (3) is automatically injected capillary through capillary action；
(5) mixed liquor in capillary is under ultrasonic unit driving, the nano particle that will specifically bind with object
Aggregation, obtains aggregation；
(6) SERS of object is carried out by Raman microscope or fluorescence microscope to aggregation obtained in step (5)
Or fluorescence detection.
Wherein, label probe can make trace object with SERS or fluorescence signal, label in conjunction with trace object
The signaling molecule of probe modification is fluorescent molecule or Raman molecular, such as R6G, Rox or TPE.
Modifier on nano particle is the molecule (DNA, RNA, aptamers etc.) for special marker, special marker
Molecule include DNA, RNA, aptamers；Trace object includes DNA, RNA.
Ultrasonic unit in step (5) includes signal generator, signal amplifier and acoustic transducer；Acoustic transducer
It is separately connected with signal generator and signal amplifier；Acoustic transducer is ultrasonic tabletting ceramics or interdigital transducer.
Interdigital transducer (IDT) is by one layer of zinc oxide film of electromagnetism sputtering sedimentation in FTO glass substrate, in zinc oxide
By one layer of aluminium film of chemical vapor deposition on layer, zinc oxide film central part is covered with Kapton adhesive tape in deposition of aluminum film layer
Lid, is routed to be formed on four sides of aluminium film by traditional photoetching process and lift-off technology.
When carrying out SERS detection, the nano particle includes gold nanorods, gold nanosphere, Silver nanorod, silver nanoparticle ball.
When carrying out fluorescence detection, the nano particle includes polystyrene nanoparticles, nano silicon particles.
Ultrasonic piezoelectric ceramics is the piezoelectric ceramics that homogeneous ultrasonic wave is generated under specific frequency.
Vltrasonic device is to generate ultrasonic wave by signal generator to pass to acoustic transducer through signal amplifier amplification, final to pass
Into capillary glass tube, special ultrasonic wave can make nanoparticle aggregate, to realize to the SERS detection of object or fluorescence
Detection.
More capillaries can also be lined up capillary array by above-mentioned apparatus, form high throughput analysis detection device.
Above-mentioned apparatus can also connect micro fluidic device, form micro-fluidic automatic fluid injection detection device.
The advantageous effects of the above technical solutions of the present invention are as follows:
(1) the capillary analysis detection method of the invention based on ultrasound aggregation is fast with detection speed, detection limit is low,
The advantages such as automatic detection can be achieved, realizing that fast accurate analysis and ultra trace context of detection are trace with important value
The capillary analysis detection of object provides a kind of feasible scheme.
(2) ultrasonic aggregation apparatus of the invention generates ultrasonic wave, and the nano particle after aggregation capture marker is realized pair
The aggregation of ultra trace object, and Raman microscope or fluorescence microscope is combined to realize SERS or fluorescence detection to object,
Realize enriched with trace detection.
Detailed description of the invention
Fig. 1 is Vltrasonic device structural schematic diagram used in the capillary analysis detection method of the invention based on ultrasound aggregation；
The signal of comparison is dispersed in capillary and assembled to nanometer rods when Fig. 2 is ultrasound opening and closing in the embodiment of the present invention
Figure, wherein (a) is ultrasonic closed state, is (b) ultrasonic opening state；
Fig. 3 is the process schematic that DNA modification gold nano grain and marker combine in the embodiment of the present invention；
Fig. 4 is the Raman map of marker ultrasound aggregation detection in capillary in the embodiment of the present invention；
Nano particle when Fig. 5 is ultrasound opening and closing in the embodiment of the present invention disperses in capillary and assembles the signal of comparison
Figure, wherein (a) is ultrasonic closed state, it is (b) ultrasonic opening state；
Fig. 6 is in the embodiment of the present invention for detecting the combination schematic diagram of object.
Specific embodiment
To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with attached drawing and tool
Body embodiment is described in detail.
The present invention provides a kind of capillary analysis detection method based on ultrasound aggregation.
This method is by the nano particle mixed injection capillary after trace object, signal mark molecule and special sex modification
In, the nano particle after capturing marker is assembled to realize the aggregation to ultra trace object by ultrasonic unit, and combine
Raman microscope or fluorescence microscope realize SERS or fluorescence detection to object.
As shown in Figure 1, ultrasonic unit used in this method includes signal generator, signal amplifier and acoustic transducer；
Acoustic transducer is separately connected with signal generator and signal amplifier；Acoustic transducer is ultrasonic tabletting ceramics or interdigital transducing
Device.
It is explained combined with specific embodiments below.
Embodiment 1
The present embodiment is related to a kind of SERS detection verifying of capillary analysis detection device based on ultrasound aggregation, used to receive
Rice grain is gold nanorods, and the specific method is as follows for the verifying:
Step 1, micro-fluidic automatic detection Vltrasonic device based on capillary is built:
By one layer of zinc oxide film of electromagnetism sputtering sedimentation in FTO glass substrate, pass through chemical gaseous phase on zinc oxide film
One layer of aluminium film is deposited, is covered zinc oxide film central part with Kapton adhesive tape in deposition of aluminum film layer.Pass through traditional photoetching
Method and lift-off technology are routed to be formed interdigital transducer (IDT) on four sides of aluminium film, and IDT and signal generator and signal amplifier lead to
Cross conducting wire connection.
Signal generator is divided by two conducting wires by acoustic signals input signal amplifier, then by the positive and negative anodes of signal amplifier
It Lian Jie not interdigital transducer.Signal amplifier is used to the acoustic signals that amplified signal generator generates, and is amplified to IDT needs
Voltage amplitude.
Capillary is put on a pair of of interdigital transducer, capillary is coupled by couplant with interdigital transducer center and connect
Touching.
Step 2, the DNA modification on gold nanorods:
0.1M~0.15M dithiothreitol (DTT) and 0.15M~0.25M phosphate buffer (PBS) are added into DNA chain,
Disulfide bond functional group is handled under conditions of pH=8.0 1~2 hour, with NAP-5 column purification.By treated, DNA adds to Jenner
In rice stick (1~2OD/1mL), room under conditions of PBS is lauryl sodium sulfate (SDS) of 0.01M~0.02M and 0.01%
Temperature is incubated for 20~25 minutes.It keeps increasing sodium chloride concentration while SDS concentration, increases 0.05M, ultrasonic treatment 10~20 every time
Second, it is incubated for 20~25 minutes at room temperature, until sodium chloride concentration reaches 0.8~1.0M.It is incubated overnight at room temperature.Repeatedly washing gold
Gold nanorods after modification are finally resuspended in 0.01%SDS by nanometer rods.
Step 3, the detection of object:
Gold nanorods mixed liquor after trace object and modification is automatically injected in capillary through capillary action, is stood
Make the modifier and the automatic base pairing of target to be measured on gold nanorods, gold nanorod aggregation is made by ultrasonic unit, institute
It states gold nanorod aggregation object and serves as coarse noble metal substrate, enhancing SERS detects signal；It is adsorbed between the gold nano grain of aggregation
The trace object to be measured for having modified signal, carries out SERS detection to accumulation point with micro- Confocal laser-scanning microscopy instrument at this time, most
The detection to trace object to be measured in capillary is realized eventually.
Modifier on the gold nanorods can be combined with object base pairing, and object capture is adsorbed on Jenner
Rice stick surface.
The detection device that the present embodiment is built using above-mentioned steps has carried out confirmatory gather to the single stranded DNA that ROX is modified
Collection and detection, as shown in figures 1-4.
Single-stranded DNA sequence to be measured after ROX is modified is 5'-TGA GGT AGT AGG TTG TAT AGT T-ROX-
3'；
DNA sequence dna in the gold nano grain of DNA modification are as follows: 5'-SH-A ACT ATA CAA CCT ACT ACC TCA-
3'。
The capillary analysis detection method based on ultrasound aggregation of the present embodiment is fast with detection speed, detection limit is low,
The advantages such as automatic detection can be achieved, realizing that fast accurate analysis and ultra trace context of detection are trace with important value
The capillary analysis detection of object provides a kind of feasible scheme.
Embodiment 2
The present embodiment is related to a kind of fluorescence detection verifying of capillary analysis detection device based on ultrasound aggregation, described glimmering
The step of light detection specific steps are with embodiment 1 is essentially identical, only unlike:
The nano particle is replaced with polystyrene nanoparticles.
The step 2, which replaces with, carries out DNA modification on polystyrene nanoparticles surface, specifically: by the surface 3~8mg
Carboxylic polystyrene microsphere is added to 5~10min of ultrasound in 0.5mL~1.5mL Tris-HCl buffer buffer solution, then
1mL~2mL EDC (5mmol/L) is added and 0.25mL~0.5mLNHS (2mmol/L) activates 10~20min, 50 are added later
~100 μ L DNA solutions (50 μ g/mL) are centrifugated under room temperature after oscillating reactions 45min~60min, are buffered with Tris-HCl
Liquid is cleaned multiple times and is centrifuged, and last dispersing and dissolving is in Tris-HCl buffer.
In the detecting step of the object, it is gathered in the fluorescent molecule captured on nano particle by ultrasonic unit
Together, so that fluorescent molecule is realized aggregation-induced emission, fluorescence detection is carried out to accumulation point with fluorescence microscope at this time, it is final to realize
Fluorescence detection to trace object to be measured in capillary.
The aggregation schematic diagram of the present embodiment is as shown in Figure 5.
The sequence of the single stranded DNA to be measured is 5'-TGA GGT AGT AGG TTG TAT AGT T-TPE-3'；
DNA sequence dna in the gold nano grain of the DNA modification are as follows: 5'-NH2-A ACT ATA CAA CCT ACT ACC
TCA-3'。
Embodiment 3
The present embodiment and the SERS of the capillary analysis detection device based on ultrasound aggregation of embodiment 1 detect basic phase
Together, unlike only:
In the DNA modification step of the gold nanorods, DNA sequence dna used are as follows: 5'-TGC ATC CAG GTC ATG-SH-
3'；
The single-stranded DNA sequence to be measured are as follows: 5'-AGA AGA TAT TTG GAA TAA CAT GAC CTG GAT
GCA-3'；
In the object detection process, the gold nanorods after signaling molecule chain and object, modification are mixed together note
Enter in capillary, wherein signaling molecule sequence are as follows: 5'-ROX-TTA TTC CAA ATA TCT TCT-3'；
The signaling molecule chain can make on object in conjunction with the trace object base pairing with SERS signal point
Son；
The detection schematic diagram of the present embodiment is as shown in Figure 6.
Embodiment 4
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 2
Together, unlike only:
The nano particle is replaced with nano silicon particles.
Embodiment 5
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 3
Together, unlike only:
The gold nanorods of the DNA modification are replaced with the gold nanorods that RNA is modified.
Embodiment 6
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 3
Together, unlike only:
The gold nanorods are replaced with gold nano grain.
Embodiment 7
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 3
Together, unlike only:
The gold nanorods are replaced with Silver nanorod.
Embodiment 8
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 3
Together, unlike only:
The gold nanorods are replaced with silver nano-grain.
Embodiment 9
The basic phase of detection method of the present embodiment and the capillary analysis detection device based on ultrasound aggregation of embodiment 1
Together, unlike only:
The acoustic transducer is replaced with ultrasonic piezoelectric ceramics.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, several improvements and modifications can also be made, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (8)
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| CN111122537A (en) * | 2019-12-23 | 2020-05-08 | 中国科学院合肥物质科学研究院 | A surface-enhanced Raman spectroscopy substrate based on sidewall opening transmission capillary, preparation method and application |
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| CN111122537A (en) * | 2019-12-23 | 2020-05-08 | 中国科学院合肥物质科学研究院 | A surface-enhanced Raman spectroscopy substrate based on sidewall opening transmission capillary, preparation method and application |
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Application publication date: 20190528 |