CN105738342B - It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver - Google Patents

It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver Download PDF

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CN105738342B
CN105738342B CN201610105916.2A CN201610105916A CN105738342B CN 105738342 B CN105738342 B CN 105738342B CN 201610105916 A CN201610105916 A CN 201610105916A CN 105738342 B CN105738342 B CN 105738342B
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aptamers
sers
pathogen
bacterium
nano silver
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CN105738342A (en
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高玮村
夏志平
李乾学
李志萍
曲晗
李博
王习文
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Institute of Military Veterinary Academy of Military Medical Sciences PLA
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Institute of Military Veterinary Academy of Military Medical Sciences PLA
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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
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

The invention discloses a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver, it is the method for reaching the enhancing of object bacteria SERS signal specificity in pathogen surface in situ synthesizing nano-silver shell using aptamers as holder, purpose is intended to the SERS signal of specificity enhancing aimed strain, simplify data analysis program, detected bacterial strain is intuitively identified by Raman spectrogram.Pathogenic bacteria are quickly detected based on SERS technologies and aptamers, can detect staphylococcus aureus and Listeria monocytogenes in 1h quantification.And accomplish in the short time of integration（2s is the 1/10 1/5 of normal integration time）It inside can also collect low noise and stablize spectrogram.SERS technologies are directly applied to by aptamers are label-free for the first time, for the first time in the case of no Raman labels object, bacterium is intuitively distinguished by spectrogram, to realize SERS technologies in Bacteria Detection field extensively using providing the foundation.

Description

It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver
Technical field
The present invention relates to a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver, is using aptamers as holder Reach the method for object bacteria SERS signal specificity enhancing in pathogen surface in situ synthesizing nano-silver shell, belongs to food security inspection Survey field.
Background technology
Early in 1989, Holt et al. collected photosynthetic bacteria cell wall SERS spectra, to pull-up SERS detections The prelude of microorganism.SERS technologies were more ripe in the detection of bacterium in recent years, but due to the knot of bacterium and nano-particle Close is difficult regulation and control, obtained SERS spectrogram poor repeatabilities.Also, the SERS spectrogram difference of pathogen is very faint, some be difficult according to It is directly distinguished according to SERS collection of illustrative plates, needs to be classified with chemometrics method, increase the difficulty of Site Detection.For this Series of problems, we, which search out, a kind of can have specificity and the oligonucleotide sequence of high-affinity prop up target pathogens Frame, in one layer of nano silver shell of bacterium surface fabricated in situ, to achieve the purpose that specificity obtains SERS signal.This oligonucleotides Sequence specific recognition bacterium surface site and combines after folding, since it is in rugosity, makes to be adsorbed on the small of its surface and receive It is a big nano-particle that rice corpuscles, which is reunited, and this nanoparticle surface agglomerated into is coarse, and hot spot-effect becomes apparent from, and is increased Potent fruit is more ideal.And since binding site is fixed, so that hotspot's distribution is fixed, obtains high duplication, the SERS of homogeneity believes Number.Also, this method can only achieve the effect that aimed strain SERS enhances, and can intuitively be identified and be detected carefully by spectrogram Bacterium eliminates cumbersome data analysis step.
Invention content
The invention discloses a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver, be with aptamers be branch Frame reaches the method for object bacteria SERS signal specificity enhancing in pathogen surface in situ synthesizing nano-silver shell, and purpose is intended to specifically Property enhancing aimed strain SERS signal, simplify data analysis program, detected bacterial strain is intuitively identified by Raman spectrogram.
It is disclosed by the invention a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver, using following technical side Case：
Aptamers immerse in silver nitrate solution, sodium borohydride then is added quickly will be silver-colored with after pathogen specific binding Ion reduction reaches SERS signal specificity enhancing effect at nano silver shell.Referring to Fig. 1.
It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver described in the present invention, include the following steps：
1）In LB culture mediums 200rpm, 37 DEG C under the conditions of shake culture freezing pathogen 11 hours；It takes 1ml107Cfu/ml bacteriums MilliQ H2O is washed twice；Then bacterium is stored in 4 DEG C of refrigerators for use；
2）It is denaturalized two minutes for 95 DEG C using thermal cycler, 2 DEG C of drop is gradually cooled to 37 DEG C within every 40 seconds, the aptamers folded It is spare to be stored in 4 DEG C of refrigerators；
3）Aptamers capture pathogen：The aptamers that 300nM is folded respectively with specified pathogen bacterium and non-specific Opportunistic pathogen is incubated 20min, washs 2 times with buffer solution to remove unbonded aptamers, 4 DEG C save backup；
4）By step 3）The pathogen of middle aptamers capture is incubated 5min with 10mM silver nitrates, and it is molten that 10mM sodium borohydrides are added Supernatant is abandoned in liquid, centrifugation, and 1ml MilliQ H2O are resuspended, and 4 DEG C save backup；
5）By step 4）Middle sample carries out Raman scanning.
The good effect of invention is：Pathogenic bacteria are quickly detected based on SERS technologies and aptamers, it can be in 1h quantification Detect staphylococcus aureus and Listeria monocytogenes.And accomplish in the short time of integration（2s is normal integration time 1/10-1/5）It inside can also collect low noise and stablize spectrogram.SERS technologies are directly applied to by aptamers are label-free for the first time, For the first time in the case of no Raman labels object, bacterium is intuitively distinguished by spectrogram, to realize SERS technologies in Bacteria Detection Field extensive use provides the foundation.
Description of the drawings
Fig. 1 is principle of the invention figure；
Fig. 2 is 1 staphylococcus aureus aptamers in-situ reducing nano silver SERS figures of experimental example；
Fig. 3 is 2 Listeria monocytogenes aptamers in-situ reducing nano silver SERS figures of experimental example；
Fig. 4 is that staphylococcus aureus aptamers in-situ reducing nano silver SERS mixes golden yellow grape with conventional nano silver Coccus SERS compares figure；
Fig. 5 is that staphylococcus aureus aptamers in-situ reducing nano silver SERS quantitatively detects figure（105cfu/ml- 10cfu/ml）.
Specific implementation mode
By following embodiment further illustrate description the present invention, do not limit the invention in any way, without departing substantially from Under the premise of technical solution of the invention, easy to implement any of those of ordinary skill in the art made for the present invention changes Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1
1）Shake culture staphylococcus aureus and Listeria monocytogenes are distinguished in LB culture mediums（Condition：200rpm、37 DEG C culture 11 hours）.1ml a concentration of 10 is taken respectively7Cfu/ml staphylococcus aureuses and Listeria monocytogenes MilliQ H2O is washed twice.Then the bacterium washed is stored in 4 DEG C of refrigerators for further using；
2）In library, U.S. biological Co., Ltd synthesizes staphylococcus aureus aptamers （TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC）, use thermal cycler 95 DEG C are denaturalized two minutes, and 2 DEG C of drop is gradually cooled to 37 DEG C within every 40 seconds, and it is spare that the aptamers folded are stored in 4 DEG C of refrigerators；
3）The staphylococcus aureus aptamers that 300nM is folded increase Liszt with staphylococcus aureus and list respectively Bacterium is incubated 20min, washs 2 times with phosphate buffer to remove unbonded aptamers, 4 DEG C save backup；
4）By step 3）The staphylococcus aureus of middle aptamers capture and Listeria monocytogenes respectively with silver nitrate （10mM）It is incubated 5min, sodium borohydride is added（10mM）Solution, centrifugation, abandons supernatant, 1ml MilliQ H2O is resuspended, 4 DEG C of preservations It is spare；
5）It will（4）Middle sample carries out Raman scanning.
Embodiment 2
1）Shake culture staphylococcus aureus and Listeria monocytogenes are distinguished in LB culture mediums（Condition： 200rpm、 37 DEG C are cultivated 11 hours）.1ml a concentration of 10 is taken respectively7Cfu/ml staphylococcus aureuses and Listeria monocytogenes are used MilliQ H2O is washed twice.Then the bacterium washed is stored in 4 DEG C of refrigerators for further using；
2）In library, U.S. biological Co., Ltd synthesizes Listeria monocytogenes aptamers （TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG）, it is denaturalized two minutes, every 40 seconds for 95 DEG C using thermal cycler 2 DEG C of drop is gradually cooled to 37 DEG C, and it is spare that the aptamers folded are stored in 4 DEG C of refrigerators；
3）The Listeria monocytogenes aptamers that 300nM is folded respectively with staphylococcus aureus and Listeria monocytogenes It is incubated 20min, washs 2 times with phosphate buffer to remove unbonded aptamers, 4 DEG C save backup；
4）By step 3）The Listeria monocytogenes of middle aptamers capture and staphylococcus aureus respectively with silver nitrate （10mM）It is incubated 5min, sodium borohydride is added（10mM）Solution, centrifugation, abandons supernatant, 1ml MilliQ H2O is resuspended, 4 DEG C of preservations It is spare；
5）By step 4）Middle sample carries out Raman scanning.
Test example 1
Using embodiment 1 prepare sample, staphylococcus aureus SERS signal obtain high intensity enhancing, and with condition at The Listeria monocytogenes SERS signal enhancing of reason is faint, and Staphylococcus aureus can be directly identified without complicated data processing Bacterium.Referring to Fig. 2.
Test example 2
The sample prepared using embodiment 2, Listeria monocytogenes SERS signal obtain high intensity enhancing, and with condition processing The enhancing of staphylococcus aureus SERS signal it is faint, can directly be identified without complicated data processing and single increase Liszt Bacterium.Referring to Fig. 3.
It is received using aptamers in-situ reducing referring to spectral strength at 735cm-1 in Fig. 4 staphylococcus aureus SERS spectrograms Rice human lymph node method will be significantly larger than common nano silver mixing enhancing method.
Referring to Fig. 5 when being adapted to bulk concentration saturation, staphylococcus aureus aptamers in-situ reducing nanometer human lymph node method exists Spectral strength is linearly related with staphylococcus aureus concentration at 735cm-1（R2=0.97897）.

Claims (1)

1. it is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver, include the following steps：
1）In LB culture mediums 200rpm, 37 DEG C under the conditions of shake culture freezing pathogen 11 hours；Take 1ml107Cfu/ml is thin Bacterium is washed twice with MilliQ H2O；Then bacterium is stored in 4 DEG C of refrigerators for use；
2）It is denaturalized two minutes for 95 DEG C using thermal cycler, 2 DEG C of drop is gradually cooled to 37 DEG C within every 40 seconds, the aptamers storage folded It is spare in 4 DEG C of refrigerators；
3）Aptamers capture pathogen：The aptamers that 300nM is folded respectively with specified pathogen bacterium and non-specific opportunistic pathogen It is incubated 20min, washs 2 times with buffer solution to remove unbonded aptamers, 4 DEG C save backup；
4）By step 3）The pathogen of middle aptamers capture is incubated 5min with 10mM silver nitrates, and 10mM sodium borohydride solutions are added, Supernatant is abandoned in centrifugation, and 1ml MilliQ H2O are resuspended, and 4 DEG C save backup；
5）By step 4）Middle sample carries out Raman scanning.
CN201610105916.2A 2016-02-26 2016-02-26 It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver Expired - Fee Related CN105738342B (en)

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CN107228819B (en) * 2017-06-04 2019-12-13 胥振国 Flow cytometry detection method for staphylococcus aureus
CN110567936B (en) * 2019-09-05 2021-07-20 上海应用技术大学 Method for detecting cyromazine in milk based on nucleic acid aptamer
CN112098389B (en) * 2020-08-31 2022-04-22 华南理工大学 Detection method of Listeria monocytogenes
CN114199850B (en) * 2021-11-11 2024-05-14 江苏大学 Method for detecting three food-borne pathogenic bacteria based on Au@Ag NPs filter paper substrate

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CN104458704A (en) * 2014-12-24 2015-03-25 中国科学院合肥物质科学研究院 Method for detecting low-concentration mercury ions based on DNA modified SERS substrate
CN104597027A (en) * 2015-01-09 2015-05-06 江南大学 Raman multiple detection method based on silver nanoparticles tetrahedron
CN104964960A (en) * 2015-06-08 2015-10-07 江南大学 Ultra-sensitive method for detecting vascular endothelial growth factor (VEGF) based on tetrahedral silver-inlaid structure

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WO2005090948A2 (en) * 2003-12-29 2005-09-29 Intel Corporation Composite organic-inorganic nanoclusters
CN101923088A (en) * 2010-07-05 2010-12-22 中国人民解放军军事医学科学院野战输血研究所 Gold nanorod immunoprobe, and preparation method and application thereof
CN103954607A (en) * 2014-05-14 2014-07-30 江南大学 Construction method of ultra-sensitive surface-enhanced Raman spectrum (SERS) sensor for measuring Hg<2+>
CN104198464A (en) * 2014-09-23 2014-12-10 南京农业大学 Method for building surface enhanced Raman scattering detection system
CN104458704A (en) * 2014-12-24 2015-03-25 中国科学院合肥物质科学研究院 Method for detecting low-concentration mercury ions based on DNA modified SERS substrate
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CN104964960A (en) * 2015-06-08 2015-10-07 江南大学 Ultra-sensitive method for detecting vascular endothelial growth factor (VEGF) based on tetrahedral silver-inlaid structure

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