CN101514986A - Label-free biochemical detection method reinforced by utilizing local surface plasma - Google Patents
Label-free biochemical detection method reinforced by utilizing local surface plasma Download PDFInfo
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- CN101514986A CN101514986A CNA2009100781931A CN200910078193A CN101514986A CN 101514986 A CN101514986 A CN 101514986A CN A2009100781931 A CNA2009100781931 A CN A2009100781931A CN 200910078193 A CN200910078193 A CN 200910078193A CN 101514986 A CN101514986 A CN 101514986A
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Abstract
A label-free biochemical detection method reinforced by utilizing local surface plasma is characterized in that the label-free biochemical detection method comprises the steps as follows: (1) preparing an LSPR detection chip according to a detected object; (2) selecting a specific biological molecule to activate the chip according to the detected object, and testing the spectrum reference value of the chip by a spectrum test system; (3) leading in a to-be-detected sample for detection, and obtaining a spectrum curve by test; and (4) judging whether the to-be-detected sample contains the detected object by analysing the spectral peak movement state of the spectrum, so as to quickly detect the detected object with high sensitivity and without a label, meanwhile, the array chip is used for realising high-efficiency and multi-channel detection. The method of the invention does not need complex equipment and does not use radioactive isotope, enzyme or fluorescence as a label, has the remarkable characteristics of low cost and high sensitivity, and can realise array chip. The method provides a simple and practical new method for fast detection of biochemical molecule.
Description
Technical field
The invention belongs to biochemical field of detecting, relate to a kind of novel biochemical detection method, particularly a kind of high sensitivity biochemical detection method that utilizes the local surface plasma resonance enhancement techniques.
Background technology
Sensing technology is one of three big pillars of modern information technologies, is playing a significant role aspect national security, scientific experimentation, health care and the environmental monitoring.The biochemical sensitive technology is the important branch of sensing technology, is related to the fields such as closely bound up public safety, virus, Bacteria Detection, clinical medicine, environment measuring of living with people.Tradition biochemical sensitive technology needs mark usually, volume is big, sensitivity is low (usually in the nanomole magnitude), process is loaded down with trivial details, efficient is low.Wherein 90% workload is used in mark, main radioactive isotope, enzyme or the fluorescence etc. of using are as marker, poor stability and poor stability, can not satisfy the fast demand of (so that handling and control danger in real time), sensitive (so that surveying extremely toxic substance of trace), special efficacy (getting rid of the interference and the pollution of non-pathogenic composition), urgent wish development high sensitivity of new generation, efficiently, accurately, easily and fast, economic biochemical molecular detecting method and technology.In recent years, unmarked detection method is developed rapidly, elliptically polarized light, light-addressable potentiometric, ion sensing fet, surface acoustic wave and little day equality sensing technology of quartz crystal oscillator have occurred, has improved detection efficiency.Wherein mode of propagation surface plasma body resonant vibration (SPR) biology sensor is developed and practical application rapidly, and existing how tame unit has carried out nearly 20 years research, the commercialization of part achievement both at home and abroad.Yet SPR mode of propagation chip is difficult to array, integrated, chipization, and Detection Techniques remain further to be improved.
Along with development of technology, a kind of novel sensing technology---local surface plasma resonance strengthens the attention that (LSPR) is subjected to the various countries scientific research personnel day by day, be meant and utilize the electronics that has transmissibility in the nano particle, generate an electromagnetic field around nano particle, this has just determined the amount of the sensing on the responsive basis of refractive index.Because the conduction electron collective oscillation only occurs in special wavelength, the optionally photonic absorption that nano particle shows can be surveyed with ultraviolet-visible spectrometer easily.Multinomial characteristics such as, applied range convenient and swift, highly sensitive, monitoring in real time with it, be subjected to researchist's favor deeply, and walk in the forward position of sensor research, utilize this novel research means crucial meaning to be arranged for the aspects such as fundamental research, medical diagnosis and treatment of life science.
Summary of the invention
The problem to be solved in the present invention is: shortcoming such as overcome existing biochemical sensitive Technology Need specific installation, cost height, the cycle is long, sensitivity is low, utilize the specific reaction between LSRP (local surface plasma technology) and biochemical molecular, move the high sensitivity that realizes specific molecular by observing spectrum, exempt from mark, multichannel high-efficient is surveyed; Chip by the later stage dissociate handle after, reusable.
The technical solution adopted for the present invention to solve the technical problems is: a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen is characterized in that: comprise that step is as follows:
(1) prepares the detection chip of substrate surface band metal micro-nanostructure according to detected object;
(2) select for use specific biomolecule that chip is activated according to detected object,, obtain the chip reference value by the spectrum of this chip of test system and test;
(3) introduce testing sample and detect, obtain the curve of spectrum by test;
(4) judge whether contain detected object in the testing sample by analyzing the spectrum moving state.
Detected object in the described step (1) is bacterium or toxin or protein.
Detection chip prepares by following steps in the described step (1): (a) select chip base for use, clean and do hydrophilic treatment; (b) produce nanostructured at substrate surface by nanosphere self assembly or photoetching or nano impression; (c), obtain to have the chip of nanometer metal structure at substrate surface with nanostructured metalization.
The material of the chip base in the above-mentioned described step (a) is glass or quartzy or silicon or germanium.
Detection chip in the described step (1) is the array chip, the array number is from 1 * 1～150 * 150, the subelement size is 5 μ m * 5 μ m～10mm * 10mm, can activate different subelements at multiple test substance by the point sample mode, realizes that efficient, multi-channel parallel detects.
Metal micro-nanostructure on the detection chip in the described step (1) is triangle or rhombus or pentalpha or cylindrical or two-layer composite, described metal material is gold or silver or the compound gold of silver surface, and the nanostructured characteristic dimension is from 30nm～1000nm.
Detection in the described step (3) can be online detection or the reacted detection of chip, and the described determinand reaction time is 1 minute to 20 hours.
Detection in the described step (3), its detection mode is tested for utilizing intermolecular specific reaction, binding antibody on chip earlier, by antibody test antigen or on chip conjugated antigen be used to detect antibody.
The environment that detects in the described step (3) is under vacuum or nitrogen environment or the atmospheric environment; Simultaneous temperature is at-50 ℃～80 ℃.
Analysis spectrum moving state in the described step (4) is meant that spectrum spectrum peak position amount of movement is greater than 10nm.
The advantage that the present invention is compared with prior art had is: this method has does not need complex apparatus, do not need to use radioactive isotope, enzyme or fluorescence etc. as marker, have that cost is low, highly sensitive, applied range, safety, stability are high, have spatial resolution height (reaching single nano particle level), have good bioaffinity, etc. distinguishing feature, and can realize the array chip, for the fast detecting biochemical molecular provides a kind of simple and practical new method.
Description of drawings
Fig. 1 is that the present invention realizes the schematic flow sheet that label-free biochemical is surveyed;
Fig. 2 is the activation method of the specific detection of employing in the embodiment of the invention 1, and conjugated antigen on chip judges whether contain test substance in the measured object by detecting antibody earlier;
Fig. 3 adopts the reference value and the figure as a result that surveys after thing moves of spectrometer test gained for the present invention;
Among the figure: 1, metal construction, 2, substrate, 3, activated molecule, 4, antibody, 5, antigen, 6, chip reference measurement curve, 7, in conjunction with the test curve behind the measured object.
Embodiment
Introduce the present invention in detail below in conjunction with the drawings and the specific embodiments.
The label-free biochemical detection method that utilizes local surface plasma to strengthen of the present invention, its particular flow sheet comprises following step as shown in Figure 1:
(1) prepares the detection chip of substrate surface band metal micro-nanostructure according to detected object;
(2) select for use specific biomolecule that chip is activated according to detected object,, obtain the chip reference value by the spectrum of this chip of test system and test;
(3) introduce testing sample and detect, obtain the curve of spectrum by test;
(4) judge whether contain detected object in the testing sample by analyzing the spectrum moving state.
At first adopt glass to select chip base for use, clean and do hydrophilic treatment as substrate; Produce the about 5 μ m of area * 5 μ m at substrate surface by the nanosphere self assembly, the array number is the rhombus Nano Silver structure that 1 * 1 quadrilateral is arranged, and the nanostructured characteristic dimension is 300nm; With nanostructured metalization, obtain to have the chip of Nano Silver structure by silver-plated at substrate surface.Next make the fixing reactive group (shown among Fig. 2 3) of going up in metal Nano structure surface, will be fixed on the reactive group with the antibody (shown among Fig. 2 4) of detected target bacteria molecule by zero-distance coupling reagent then.By above program, finish the activation chip processes.In the reference value of utilizing spectrum test testing of equipment chip in the vacuum environment shown among Fig. 36.Introduce thing to be detected by the mode that drips at last, target molecule is by specific reaction and corresponding antibodies.Treat fully reaction back (reaction time is about 2 hours), in vacuum environment, in 40 ℃～50 ℃ the temperature range, test spectral distributes, and obtains the curve shown in 7 among Fig. 3.6,7 curves in the comparison diagram 3 find that spectrum peak position takes place obviously to move (amount of movement is about 50nm), then judge to contain the bacterium composition of surveying to some extent in the testing sample; This detection chip by the later stage dissociate handle after, reusable.
Present embodiment is substrate to melt quartz, and the triangle metal gold structure that adopts hexagon to arrange is a detection chip; The label-free biochemical detection method test toxin that utilizes local surface plasma of the present invention to strengthen, the specific reaction process of employing is a conjugated antigen on chip.
At first adopt and melt quartz, clean and do hydrophilic treatment as substrate; Producing characteristic dimension at substrate surface by the method for nano-photoetching is 50nm, the triangle nanostructured that hexagon is arranged; With nanostructured metalization, obtain to have the chip of nm of gold structure by gold-plated at substrate surface.Next make the fixing reactive group of going up in metal Nano structure surface, will be fixed on the reactive group with the antigen of detected target bacteria molecule by zero-distance coupling reagent then.By above program, finish the chip activation process.(temperature is the normal temperature state) utilizes the reference value of the spectrum test system testing chip of building on experiment table in atmospheric environment, introduce thing to be detected to chip surface by fluid channel at last, if contain corresponding antibody in the measured object, promptly can combine with corresponding antigen by specific reaction.On-line testing spectral distribution in this atmospheric environment, under the normal temperature state, whether the analytical spectra peak position (amount of movement is greater than 10nm) takes place obviously to move, in order to judge whether contain the bacterium composition of surveying to some extent in the testing sample.
Embodiment 3
Present embodiment is substrate with silicon, method by photoetching is produced foraminous die plate, the chip array number is 150 * 150, by integrated 150 * 150 kinds of proteantigens of the method for point sample, whether the label-free biochemical detection method test measured object that utilizes local surface plasma of the present invention to strengthen contains various types of protein.
At first adopt silicon as substrate, clean and do hydrophilic treatment; Producing number of arrays at substrate surface by nano impression is 150 * 150 nano-structure array, and each subelement size is 1000 μ m * 1000 μ m; With nanostructured metalization, obtain to have the chip of nanometer metal structure at substrate surface.Structure in each subelement is according to the determinand different characteristic, can adopt the structure that has nothing in common with each other, the individual layer pentalpha is arranged, also by the silver of the double-layer nanometer up and down structure of forming by surface structure and lower surface configuration, composite nanostructure is also arranged, in the surface attachment of silver-colored structure the layer of metal gold to play the effect that prevents the chip oxidation deactivation.The nanostructured characteristic dimension is from 30nm～1000nm.Utilize biological point sample instrument that chip is carried out point sample, in conjunction with the antibody (or antigen) of different proteins.Under nitrogen environment, utilize the reference value of integrated testing of equipment chip, temperature is at-50 ℃～80 ℃, at last again by point sample in each subregion in conjunction with on testing sample, treat that fully reaction back (about 30min) is in nitrogen environment build-in test all subregion spectral distribution, temperature is at-50 ℃～80 ℃.Whether the analytical spectra peak position (amount of movement is greater than 10nm) takes place obviously to move, in order to judge whether contain the protein component of surveying to some extent in the testing sample.
Claims (10)
1, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen is characterized in that: comprise that step is as follows:
(1) prepares the detection chip of substrate surface band metal micro-nanostructure according to detected object;
(2) select for use specific biomolecule that chip is activated according to detected object,, obtain the chip reference value by the spectrum of this chip of test system and test;
(3) introduce testing sample and detect, obtain the curve of spectrum by test;
(4) judge whether contain detected object in the testing sample by analyzing the spectrum moving state.
2, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the detected object in the described step (1) is bacterium or toxin or protein.
3, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: detection chip prepares by following steps in the described step (1): (a) select chip base for use, clean and do hydrophilic treatment; (b) produce nanostructured at substrate surface by nanosphere self assembly or photoetching or nano impression; (c), obtain to have the chip of nanometer metal structure at substrate surface with nanostructured metalization.
4, the preparation of detection chip according to claim 3 is characterized in that: the material of the chip base in the described step (a) is glass or quartzy or silicon or germanium.
5, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the detection chip in the described step (1) is the array chip, the array number is from 1 * 1～150 * 150, the subelement size is 5 μ m * 5 μ m～10mm * 10mm, can activate different subelements at multiple test substance by the point sample mode, realize that efficient, multi-channel parallel detects.
6, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the metal micro-nanostructure on the detection chip in the described step (1) is triangle or rhombus or pentalpha or cylindrical or two-layer composite, described metal material is gold or silver or the compound gold of silver surface, and the nanostructured characteristic dimension is from 30nm～1000nm.
7, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the detection in the described step (3) can be online detection or the reacted detection of chip, and the described determinand reaction time is 1 minute to 20 hours.
8, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the detection in the described step (3), its detection mode is tested for utilizing intermolecular specific reaction, binding antibody on chip earlier, by antibody test antigen or on chip conjugated antigen be used to detect antibody.
9, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the environment that detects in the described step (3) is under vacuum or nitrogen environment or the atmospheric environment; Simultaneous temperature is at-50 ℃～80 ℃.
10, a kind of label-free biochemical detection method that utilizes local surface plasma to strengthen according to claim 1, it is characterized in that: the analysis spectrum moving state in the described step (4) is meant that spectrum spectrum peak position amount of movement is greater than 10nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792368A (en) * | 2014-01-27 | 2014-05-14 | 暨南大学 | Surface plasma resonance immunosense chip as well as preparation method and application thereof |
| CN104819954A (en) * | 2015-04-21 | 2015-08-05 | 曾安 | Method for detecting biological substance content in sample by near-infrared without markers |
| CN108106994A (en) * | 2017-12-15 | 2018-06-01 | 中国科学院光电技术研究所 | A kind of scan-type local enhances biochemical sensitive device |
| CN109668957A (en) * | 2018-11-14 | 2019-04-23 | 江苏科技大学 | QCM and LSPR biosensor with transparent electrode |
-
2009
- 2009-02-26 CN CNA2009100781931A patent/CN101514986A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792368A (en) * | 2014-01-27 | 2014-05-14 | 暨南大学 | Surface plasma resonance immunosense chip as well as preparation method and application thereof |
| CN103792368B (en) * | 2014-01-27 | 2015-10-07 | 暨南大学 | A kind of surface plasma body resonant vibration immune sensing chip and preparation method thereof and application |
| CN104819954A (en) * | 2015-04-21 | 2015-08-05 | 曾安 | Method for detecting biological substance content in sample by near-infrared without markers |
| CN104819954B (en) * | 2015-04-21 | 2018-04-17 | 曾安 | The method of biological substance content in label-free thing near infrared detection sample |
| CN108106994A (en) * | 2017-12-15 | 2018-06-01 | 中国科学院光电技术研究所 | A kind of scan-type local enhances biochemical sensitive device |
| CN109668957A (en) * | 2018-11-14 | 2019-04-23 | 江苏科技大学 | QCM and LSPR biosensor with transparent electrode |
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Application publication date: 20090826 |