CN108931510A - The detection method of porous silicon biological sensor - Google Patents
The detection method of porous silicon biological sensor Download PDFInfo
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- CN108931510A CN108931510A CN201810527584.6A CN201810527584A CN108931510A CN 108931510 A CN108931510 A CN 108931510A CN 201810527584 A CN201810527584 A CN 201810527584A CN 108931510 A CN108931510 A CN 108931510A
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- 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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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The present invention relates to a kind of detection methods of porous silicon biological sensor comprising：Target dna is modified into p type single crystal silicon, hybridization reaction is occurred into for CdSe/ZnS quantum dot-labeled DNA probe and the target dna, obtains porous silicon biological sensor；By excitation light source, the porous silicon biological sensor of the target dna of excitation setting concentration generates fluorescence, obtains fluorescent image by digital microscope, the relationship fit standard curve of target DNA concentration described in the sum of the grayscale values by fluorescent image；The gray value for measuring the fluorescent image of the porous silicon biological sensor of sample to be tested calculates the target DNA concentration of sample to be tested according to standard curve.The present invention is 88pM for detecting the detection limit of DNA, detection sensitivity is much higher than the porous silicon micro-cavity biosensor based on variations in refractive index, testing cost is low, digital microscope and general optical device need to be only used, and traditional method using Fluorescence Spectrometer detection is not used to the detection of biochip.
Description
Technical field
The present invention relates to a kind of field of biosensors, more particularly to a kind of detection of porous silicon biological sensor
Method.
Background technique
The advantages that optical biosensor is due to the easy, quick, accurate of it and strong selectivity is studied extensively by people.It is more
Hole silicon can be used for preparing the optical biosensor or biochip of each class formation as a kind of excellent biomaterial.
There are two main classes for the detection mechanism of porous silicon optical biosensor at present.The first kind is to caused by biological respinse
What variations in refractive index was detected, main method is reflection spectrometry or angle spectrometry based on Porous Silicon Microcavity.Second class is
Change in fluorescence caused by biological respinse is detected, main method is based on porous silicon Prague (Bragg) reflecting mirror or micro-
The fluorescence intensity measurements of chamber.In order to further increase the high detection sensitivity of porous silicon optical biosensor, people are introduced
Semiconductor Colloidal Quantum Dots.Semiconductor-quantum-point has wide excitation wavelength range and narrow launch wavelength range, and glimmering
Light spectrum is narrow and symmetrical, and photostability is strong, and fluorescence intensity is strong, and quantum yield is high, and size adjustable, the anti-light ability of drifting is strong, fluorescence
Service life is long, and water-soluble quantum dot (QDs) shows good bio-compatibility after functional modification, can specificity connection life
Object molecule can carry out biological living label and detection.In first kind method, by quantum dot-labeled probe biomolecule, put
Variations in refractive index caused by big biological respinse.In the second class method, using quantum dot as fluorescent label biology point
Sub- probe.
The method sensitivity of fluorescent marker specific refractivity variation at present is higher, but optical biosensor biological respinse draws
The change in fluorescence risen still detects it using Fluorescence Spectrometer, expensive.
Summary of the invention
It is a primary object of the present invention to provide a kind of detection method of novel porous silicon biological sensor, institute
Technical problems to be solved are to allow to carry out quick, parallel low cost detection, thus more suitable for practical.
The object of the invention to solve the technical problems adopts the following technical solutions to realize.It proposes according to the present invention
A kind of porous silicon biological sensor detection method comprising：
Target dna is modified into p type single crystal silicon, CdSe/ZnS quantum dot-labeled DNA probe and the target dna are sent out
Raw hybridization reaction, obtains porous silicon biological sensor；
By excitation light source, the porous silicon biological sensor of the target dna of excitation setting concentration generates fluorescence, by counting
Word microscope obtains fluorescent image, and the relationship fit standard of target DNA concentration described in the sum of the grayscale values by fluorescent image is bent
Line；
The gray value for measuring the fluorescent image of the porous silicon biological sensor of sample to be tested, calculates according to standard curve
The target DNA concentration of sample to be tested.
The object of the invention to solve the technical problems also can be used following technical measures and further realize.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein the porous silicon biological
The preparation method of sensor includes：
Porous silicon Bragg mirror is made in p type single crystal silicon surface, linking objective DNA obtains porous silicon sample；
CdSe/ZnS quantum dot is activated, is coupled with amido modified DNA probe, obtains quantum dot probe；
The quantum dot probe is penetrated into the porous silicon sample, obtains porous silicon biological sensor.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein described porous silicon Prague is anti-
Mirror is penetrated to be alternately stacked by the first current sheet and the second current sheet；
The optical thickness of first current sheet and the second current sheet meets formula (1)：
n1d1=n2d2=λ/4 (1)
Wherein, n1And d1The refractive index and thickness of respectively the first current sheet；
n2And d2The refractive index and thickness of respectively the first current sheet；
λ is the central wavelength of porous silicon Bragg mirror；
And first current sheet refractive index be greater than the second current sheet refractive index.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein described porous silicon Prague is anti-
The central wavelength for penetrating mirror is 600-620nm.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein described porous silicon Prague is anti-
Mirror is penetrated to be prepared by electrochemical erosion method；
The electrolytic etching liquid of electrochemical erosion method, with volume percentage comprising：
Hydrofluoric acid：40-60%
Ethyl alcohol：40-60%.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein described porous silicon Prague is anti-
The Porous Silicon area of mirror is penetrated as circle, diameter 0.5-1cm.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein the crystal orientation of the p type single crystal silicon
For<100>, resistivity is 0.01-0.06 Ω cm, with a thickness of 300-500 μm.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein the excitation light source be argon from
Sub- laser, the wavelength of the argon ion laser are 460-500nm.
Preferably, the detection method of porous silicon biological sensor above-mentioned, wherein the standard curve is：
Wherein, x is the target DNA concentration of porous silicon biological sensor, unit nmol/L；
Y is the average gray value that porous silicon biological sensor generates fluorescent image.
By above-mentioned technical proposal, the detection method of porous silicon biological sensor of the present invention at least has following excellent
Point：
The detection method of porous silicon biological sensor of the present invention is examined by calculating the average gray value variation of image
The concentration of DNA is surveyed, this method is 88pM for detecting the detection limit of DNA.Detection sensitivity is much higher than based on variations in refractive index
Porous Silicon Microcavity (PSM) biosensor, although it is slightly below traditional using Fluorescence Spectrometer detect method, detection at
This is low, need to only use digital microscope and general optical device, and traditional method using Fluorescence Spectrometer detection without
Method is used for the detection of biochip.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is the light path schematic diagram of porous silicon biological sensor fluorescent image of the present invention；
Fig. 2 is embodiment porous silicon Bragg mirror surface SEM figure；
Fig. 3 is embodiment porous silicon Bragg mirror section SEM figure；
Fig. 4 is the fluorogram of embodiment quantum dot and DNA probe coupling front and back；
Fig. 5 is the reflectance spectrum figure of embodiment porous silicon sample and porous silicon biological sensor；
Fig. 6 is the porous silicon biological sensor fluorogram of embodiment setting target DNA concentration；
Fig. 7 is the fitted figure of embodiment setting target DNA concentration and gray value；
Fig. 8 is the fitted figure of comparative example setting target dna and fluorescence spectrum.
Specific embodiment
It is of the invention to reach the technical means and efficacy that predetermined goal of the invention is taken further to illustrate, below in conjunction with
Attached drawing and preferred embodiment, to its specific embodiment party of the detection method of porous silicon biological sensor proposed according to the present invention
Formula, structure, feature and its effect, detailed description is as follows.In the following description, different " embodiment " or " embodiment " refers to
It is not necessarily the same embodiment.In addition, the special characteristic, structure or feature in one or more embodiments can be by any suitable shape
Formula combination.
A kind of detection method for porous silicon biological sensor that one embodiment of the present of invention proposes comprising：
Prepare porous silicon biological sensor：The surface of p type single crystal silicon is made by porous silicon using electrochemical erosion method
Bragg mirror, linking objective DNA obtain porous silicon sample；EDC and sulfo-NHS is added to click through CdSe/ZnS quantum
Row activation, and be coupled with amido modified DNA probe, obtain quantum dot probe；Quantum dot probe is penetrated into described porous
Silicon sample obtains porous silicon biological sensor.
Determine standard curve：As shown in Figure 1, using argon ion laser 1 as excitation light source, exciting light passes sequentially through diaphragm
2, lens 3, lens 4, laser got on porous silicon biological sensor 7 by reflecting mirror 6, inspires porous silicon by diaphragm 5
The fluorescence of biological sensor enters digital microscope 9 by long pass filter piece 8, shows fluorescent image by computer 10
Gray scale.Lens 3 and lens, 4 constitute collimating and beam expanding system, and long pass filter piece 8 is 600nm long pass filter piece., excitation sets dense
The porous silicon biological sensor of the target dna of degree generates fluorescence, obtains fluorescent image by digital microscope, passes through fluorogram
The relationship fit standard curve of target DNA concentration described in the sum of the grayscale values of picture, standard curve are as follows：
Wherein, x is the target DNA concentration of porous silicon biological sensor, unit nmol/L；
Y is the average gray value that porous silicon biological sensor generates fluorescent image.
Measure the concentration of sample to be tested：Measure the gray scale of the fluorescent image of the porous silicon biological sensor of sample to be tested
Value calculates the target DNA concentration of sample to be tested according to the formula (2) of standard curve.
Preferably, porous silicon Bragg mirror is alternately stacked by the first current sheet and the second current sheet；First
The optical thickness of current sheet and the second current sheet meets formula (1)：
n1d1=n2d2=λ/4 (1)
Wherein, n1And d1The refractive index and thickness of respectively the first current sheet；
n2And d2The refractive index and thickness of respectively the first current sheet；
λ is the central wavelength of porous silicon Bragg mirror；
And first current sheet refractive index be greater than the second current sheet refractive index.
Preferably, the central wavelength of porous silicon Bragg mirror is 600-620nm.
The refractive index of first current sheet is 1.00-1.10, with a thickness of 130-150nm；
The refractive index of second current sheet is 1.45-1.55, with a thickness of 90-110nm.
Preferably, porous silicon Bragg mirror is prepared by electrochemical erosion method；
The electrolytic etching liquid of electrochemical erosion method, with volume percentage comprising：
Hydrofluoric acid：40-60%
Ethyl alcohol：40-60%.
Preferably, the crystal orientation of p type single crystal silicon is<100>, resistivity is 0.01-0.06 Ω cm, with a thickness of 300-500 μ
m。
Preferably, the Porous Silicon area of porous silicon Bragg mirror is circle, diameter 0.5-1cm.
Preferably, the wavelength of argon ion laser is 460-500nm.
Embodiment
One embodiment of the present of invention proposes a kind of detection method of porous silicon biological sensor comprising：
Prepare porous silicon biological sensor：Selection resistivity is 0.01-0.06 Ω cm, and crystal orientation is<100>, thickness
For 400 ± 10 μm of p type single crystal silicon, circle, diameter 0.8cm is in standby Porous Silicon area of drawing up.Pass through single slot anode electrification
It learns etch and prepares porous silicon Bragg mirror, it is 1 that electrolytic etching liquid, which is by volume ratio,：1 hydrofluoric acid (concentration 40%)
It is configured with dehydrated alcohol.Current density when electrochemical corrosion porous silicon Bragg mirror is 110mA/cm2、60mA/cm2It is right
The etching time answered is 1s, 1.2s.Porous silicon Bragg mirror is alternately stacked by the first current sheet and the second current sheet
At, the first current sheet with a thickness of 140nm, refractive index 1.08, the second current sheet with a thickness of 100nm, refractive index 1.52.
The central wavelength of porous Bragg mirror is 610nm；Fig. 2 and Fig. 3 is respectively that the scanning electron of porous Bragg mirror is aobvious
Micro mirror surfaces pattern and Cross Section Morphology figure.
Target dna is diluted to nine concentration with PBS, modifies Prague transmitting mirror with the target dna of 9 kinds of various concentrations,
Obtain porous silicon sample；
EDC and sulfo-NHS is added to activate CdSe/ZnS quantum dot, and is coupled with amido modified DNA probe,
Obtain quantum dot probe；As shown in figure 4, curve 11 is the fluorogram of the CdSe/ZnS quantum dot of activation, curve 12 is quantum dot
The fluorogram of probe；After DNA probe coupling, the fluorescence peak of quantum dot solution is moved to 627nm from 625nm, shows CdSe/
ZnS quantum dot pass flag DNA probe；
Quantum dot probe is penetrated into the porous silicon sample, obtains porous silicon biological sensor.Such as Fig. 5 institute
Show, curve 13 is the reflectance spectrum figure of porous silicon sample, and curve 14 is the reflectance spectrum figure of porous silicon biological sensor；It is more
Hole silicon sample high reflectance zone central wavelength is located at 610nm or so, and the wave-length coverage of high reflectance zone is more between 560nm to 660nm
The high reflectance zone central wavelength of hole luminescence from silicon biosensor is located at 625nm, the wave-length coverage of high reflectance zone from 575nm to
Between 675nm, the reflectance spectrum red shift of porous silicon biological sensor indicates DNA molecular successful cross.
Determine standard curve：It is 488nm argon ion laser as excitation light source using wavelength, exciting light passes sequentially through light
Laser is got on porous silicon biological sensor sample by reflecting mirror, inspires porous silicon by door screen, lens, lens, diaphragm
The fluorescence of biological sensor enters digital microscope by the long pass filter piece of 600nm, shows fluorogram by computer
The gray scale of picture.As shown in fig. 6, curve 15,16,17,18 and 19 be respectively 0.1nmol/L, 0.25nmol/L, 0.5nmol/L,
The porous silicon biological sensor of the target dna of 1.0nmol/L and 2.5nmol/L generates fluorescence, is obtained by digital microscope
Fluorescent image chooses the more uniform region of one piece of gray scale to calculate average gray value.Pass through the sum of the grayscale values institute of fluorescent image
The relationship fit standard curve for the target DNA concentration stated, as shown in fig. 7, standard curve is as follows：
Wherein, x is the target DNA concentration of porous silicon biological sensor, unit nmol/L；
Y is the average gray value that porous silicon biological sensor generates fluorescent image；
Fitting coefficient is 0.998.
Measure the concentration of sample to be tested：Measure the gray scale of the fluorescent image of the porous silicon biological sensor of sample to be tested
Value calculates the target DNA concentration of sample to be tested according to the formula (2) of standard curve.Since the gray value minimum of digital picture becomes
Changing unit is 1, therefore limits about 88pM by the detection that formula 2 calculates target DNA concentration.
Comparative example
A comparative example of the invention proposes a kind of detection method of porous silicon biological sensor, and it is glimmering to prepare porous silicon
The method of optical biosensor is identical as embodiment, is detected with traditional spectral fluorometer to fluorescence intensity, strong by fluorescence
The relationship fit standard curve of degree and target DNA concentration, as shown in figure 8, standard curve is as follows：
Y=185.16+201.99C (3)
Wherein, C is the target DNA concentration of porous silicon biological sensor, unit nmol/L；
Y is the fluorescence intensity of porous silicon biological sensor；
Fitting coefficient is 0.990.
Measure the concentration of sample to be tested：The fluorescence for measuring the porous silicon biological sensor of sample to be tested, according to standard
The formula (3) of curve calculates the target DNA concentration of sample to be tested.The sensitivity of sensor is exactly the slope of formula (3), thus may be used
To obtain the sensitivity of sensor for 201.99/nM.There are random noises for Fluorescence Spectrometer fluorescence intensity used in experiment, glimmering
The minimum resolution of luminous intensity is about 5.According to the detectable limit formula of sensor：Detectable limit=instrument minimum resolution/biography
The sensitivity of sensor, the detectable limit of available sensor are about 25pM.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, according to
According to technical spirit any simple modification, equivalent change and modification to the above embodiments of the invention, this hair is still fallen within
In the range of bright technical solution.
Claims (9)
1. a kind of detection method of porous silicon biological sensor, which is characterized in that it includes：
Target dna is modified into p type single crystal silicon, CdSe/ZnS quantum dot-labeled DNA probe and the target dna generation is miscellaneous
Reaction is handed over, porous silicon biological sensor is obtained；
By excitation light source, the porous silicon biological sensor of the target dna of excitation setting concentration generates fluorescence, aobvious by number
Micro mirror obtains fluorescent image, the relationship fit standard curve of target DNA concentration described in the sum of the grayscale values by fluorescent image；
The gray value for measuring the fluorescent image of the porous silicon biological sensor of sample to be tested calculates to be measured according to standard curve
The target DNA concentration of sample.
2. the detection method of porous silicon biological sensor according to claim 1, which is characterized in that described is porous
The preparation method of luminescence from silicon biosensor includes：
Porous silicon Bragg mirror is made in p type single crystal silicon surface, linking objective DNA obtains porous silicon sample；
CdSe/ZnS quantum dot is activated, is coupled with amido modified DNA probe, obtains quantum dot probe；
The quantum dot probe is penetrated into the porous silicon sample, obtains porous silicon biological sensor.
3. the detection method of porous silicon biological sensor according to claim 2, which is characterized in that described is porous
Silicon Bragg mirror is alternately stacked by the first current sheet and the second current sheet；
The optical thickness of first current sheet and the second current sheet meets formula (1)：
n1d1=n2d2=λ/4 (1)
Wherein, n1And d1The refractive index and thickness of respectively the first current sheet；
n2And d2The refractive index and thickness of respectively the first current sheet；
λ is the central wavelength of porous silicon Bragg mirror；
And first current sheet refractive index be greater than the second current sheet refractive index.
4. the detection method of porous silicon biological sensor according to claim 3, which is characterized in that described is porous
The central wavelength of silicon Bragg mirror is 600-620nm.
5. the detection method of porous silicon biological sensor according to claim 2, which is characterized in that described is porous
Silicon Bragg mirror is prepared by electrochemical erosion method；
The electrolytic etching liquid of electrochemical erosion method, with volume percentage comprising：
Hydrofluoric acid：40-60%
Ethyl alcohol：40-60%.
6. the detection method of porous silicon biological sensor according to claim 2, which is characterized in that described is porous
The Porous Silicon area of silicon Bragg mirror is circle, diameter 0.5-1cm.
7. the detection method of porous silicon biological sensor according to claim 1, which is characterized in that the p-type
The crystal orientation of monocrystalline silicon is<100>, resistivity is 0.01-0.06 Ω cm, with a thickness of 300-500 μm.
8. the detection method of porous silicon biological sensor according to claim 1, which is characterized in that the excitation
Light source is argon ion laser, and the wavelength of the argon ion laser is 460-500nm.
9. the detection method of porous silicon biological sensor according to claim 1, which is characterized in that the standard
Curve is：
Wherein, x is the target DNA concentration of porous silicon biological sensor, unit nmol/L；
Y is the average gray value that porous silicon biological sensor generates fluorescent image.
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