CN104034693B - A kind of method that porous silicon micro-cavity biosensor based on reflective light intensity detects biomolecule - Google Patents

Abstract

The present invention a kind of method that porous silicon micro-cavity biosensor based on reflective light intensity detects biomolecule is provided it is characterised in that：Main experimental apparatus are helium neon laser and luminous power detection meter；Laser instrument incides on the material with Porous Silicon Microcavity as substrate at an angle, receive meter with power and receive reflected light, find the superpower angle of the corresponding minimum light of micro-cavity structure, then add biological change porous silicon layer refractive index, detect the corresponding angle of minimum intensity of light power of reflected light again, by the change of front and rear angles, to detect interpolation biological concentration.

Description

A Porous Silicon Microcavity Biosensor Based on Reflected Light Intensity to Detect Biomolecules method

technical field

The invention relates to a method for detecting biomolecules, in particular to a method for detecting biomolecules based on a porous silicon microcavity biosensor based on reflected light intensity.

Background technique

Porous silicon biosensors have the characteristics of high sensitivity, fast response, good real-time performance, label-free, remote control, compact structure, no electromagnetic interference and high safety, and are now widely used to study the interaction between biomolecules , an important research tool for gene expression, new drug development, environmental testing, food safety and nuclear radiation monitoring.

There are two kinds of bio-optical sensing methods: one is to use the change principle of the refractive index of biomolecules, and its main feature is that biomolecules do not need to be labeled, and the sample preparation is simple; the other is to use the change principle of biomolecular fluorescent labels, the main features The detection is simple and highly sensitive. Among various biosensor materials, porous silicon is a good biosensor material with the following advantages:

1. Porous silicon biosensors have a very high internal surface area, which facilitates the combination of sensing molecules (bioactive probes) stationed in porous silicon and the measured molecules. Therefore, compared with planar biosensors, the unit of porous silicon The signal capacity of the area is significantly enhanced;

2. The honeycomb pore structure of porous silicon wraps biological molecules naturally, which prevents the leaching of biological components well, so the stability of the biosensor is strong;

3. The size of the pores of porous silicon can be controlled by current corrosion, so for biomolecules of different sizes, corresponding porous silicon sensors can be prepared correspondingly, which improves the anti-interference ability of the sensor;

4. The porous silicon biosensor is non-toxic and harmless, and has very good biocompatibility and adsorption;

5. Porous silicon can maintain biological activity. Cells such as neuron cells, rat liver epithelial parenchymal cells and human embryonic kidney cells have been grown on porous silicon. These biological cells grown on porous silicon can carry out normal metabolism, which is significantly better than those grown on glass;

6. Porous silicon can be integrated with other devices, and the preparation method of porous silicon is quite mature and stable, which can be used quickly and conveniently. Porous silicon materials can be widely used in the preparation of optical biosensors, and many biomolecules can be modified accordingly. Porous silicon bio-optical sensors can also be used in the research and development of MEMS devices, and are in the stage of research and development.

At present, there are a large number of biosensors published in academic journals at home and abroad, and more and more researches are moving from theory to practical application. For example, in the research of two-dimensional photonic crystal biosensors prepared on glass substrates and porous silicon biosensors with one-dimensional multilayer bandgap structures, the biological characteristics of nanoporous silicon and the bandgap structure of optical waveguides or photonic crystals The trend of combining sensors will greatly improve the performance of current biosensors.

Porous silicon has been widely used in experimental research and application as a substrate material for biological detection. Various porous silicon multilayer structures can be prepared by alternately using different currents during electrochemical corrosion of porous silicon. Electrochemical etching technology combined with photolithography and other technologies can also prepare various structures such as porous silicon waveguides and porous silicon gratings. Regardless of the type of porous silicon biosensor, the fundamental principle is that the biomolecules enter the porous structure of the porous silicon layer and increase the refractive index of the porous silicon layer. The increase in the refractive index is related to the amount of biomolecules entering. Therefore Using the change of the refractive index of the porous silicon layer, it is possible to add biological experiments through computer simulation.

There are many biosensors based on porous silicon microcavities reported so far, and the detection methods include: reflection spectrum detection and Raman and fluorescence spectrum detection.

CN101710118A discloses an optical immunodetection method based on a porous silicon ternary structure microcavity. The method adopts an electrochemical corrosion method based on computer precise control to prepare a porous silicon microcavity, wherein the upper and lower Bragg structures in the porous silicon microcavity are formed by Three current densities are alternately formed by electrochemical corrosion. It is characterized in that the coding of porous silicon microcavities prepared under different conditions can realize multiple detection. Combining in the holes of the microcavity, the type of antigen or antibody in multiple detection is marked by the coding of the porous silicon microcavity, and the concentration of the corresponding antigen or antibody in the sample is detected through the change of the spectral peak position before and after the biological reaction. The detection method Include the following steps:

1) The antigen or antibody is immobilized in the hole of the porous silicon microcavity. If it is a multiplex detection that requires coding, then a coded porous silicon microcavity corresponds to immobilizing a specific antigen or antibody of a biomolecule to be tested, and washes out the unbound molecules and seal the blank bonds of unbound biomolecules in the porous silicon microcavity, record and measure the spectrum of the porous silicon microcavity immobilized with antigen or antibody;

2) Different porous silicon microcavities react with different concentrations of solutions to be tested, so that the antigen-antibody can be specifically combined in the pores of the porous silicon microcavity, rinse after the reaction, record the spectrum and code of the porous silicon microcavity to determine the type and concentration.

This optical immunoassay method not only has many excellent performances of porous silicon and photonic bandgap structure sensors, but also has good structural stability, and multiple detections can be realized through coded detection technology. In addition, because the preparation method adopted is relatively simple and the price is relatively low, it has certain commercial application prospects.

CN1922486A discloses a method for analyzing contents in a biological sample, comprising: a) under suitable binding conditions, contacting the biological sample with a nanoporous semiconductor sensor, the nanoporous semiconductor sensor comprising a nanoporous semiconductor structure and one or more first probes connected to said porous semiconductor structure, said nanoporous semiconductor structure comprising a central layer disposed between an upper layer and a lower layer each comprising 5 to 20 alternating layers a porous layer; said one or more first probes specifically bind to at least one analyte in said sample, forming one or more binding complexes; b) under conditions suitable to promote their specific binding, contacting the one or more binding complexes with a Raman-active probe; c) illuminating the sensor to induce a fluorescent emission from the sensor, the emission producing a Raman spectrum from the binding complex; and d) detecting a Raman signal generated by said bound complex; wherein a Raman signal associated with said bound analyte indicates the presence and type of said analyte in said sample.

The method provides a method for analyzing content in a biological sample such as serum using cascaded Raman sensing. A fluorescent nanoporous biosensor having probes that specifically bind a known analyte is contacted with a biological sample to form one or more binding complexes attached to the porous semiconductor structure. Contacting the bound complex with a Raman-active probe, which specifically binds the bound complex, illuminates the biosensor, producing a fluorescent emission from the biosensor. The Raman signal generated by the bound complex is detected, and the Raman signal associated with the bound protein-containing analyte indicates the presence of the protein-containing compound in the sample.

The above-mentioned reflection spectrum, Raman spectrum, and fluorescence spectrum detection all have the disadvantage of requiring expensive and precise corresponding spectrum analyzers. Therefore, it is necessary to research and develop a detection technology that can detect organisms with simple experimental instruments.

Contents of the invention

The object of the present invention is to provide a porous silicon microcavity biosensor based on reflected light intensity detection, and a method for biological detection thereof.

The biggest difference between the present invention and the previous detection technology is that simple experimental instruments are used to detect organisms. The main experimental instruments are helium-neon lasers and optical power detectors (both common and cheap conventional optical configurations). The laser is incident on the material based on the porous silicon microcavity at a certain angle, and the reflected light is received by a power receiver, and the angle corresponding to the minimum light intensity power of the microcavity structure is found, and then biological substances are added to change the refractive index of the porous silicon layer, and then Detect the angle corresponding to the minimum light intensity power of the reflected light, and detect the added biological concentration through the change of the front and rear angles.

As shown in Figure 1, the reflection spectrum of this porous silicon microcavity is a spectrum with a defective peak. When the laser light with a fixed wavelength of 633nm is incident, the minimum value of reflected light intensity appears at 30°, as shown in Figure 1 The structure of this porous silicon microcavity is a multilayer porous silicon structure: (HL)6C(LH)6, H is a high refractive index layer (refractive index 1.52, physical thickness 104nm), L is a low refractive index layer (refractive index Ratio 1.21, physical thickness 131nm), C is the defect layer (refractive index 1.21, physical thickness 131nm), because by modulating the current, you can prepare multi-layer porous silicon with various refractive indices (between 1.2-2.0) and thickness, The distribution of structural parameters selected here corresponds to the corresponding galvanic corrosion parameters.

The reflectance spectrum shown in Figure 1 will shift to the right as a whole after adding biomolecules, that is, increasing the refractive index of each layer of the porous silicon microcavity, that is, the minimum value of reflected light intensity is no longer 30° As shown in Figure 2, the minimum value of reflected light intensity is close to 33° after the uniform increase of the refractive index of line A by 0.02, and the minimum value of reflected light intensity of line B is 0.04 evenly increased by the refractive index of 36°.

Preparation of porous silicon:

There are many explanations for the formation principle of porous silicon. For example, the preparation of porous silicon by electrochemical corrosion is to use single crystal silicon as an anode and pass a constant DC current in HF acid solution for anodic oxidation. Single crystal silicon in the HF acid solution due to different applied voltages or different constant currents, there will be two situations: when the current is greater than a certain critical value, the single crystal silicon will be peeled off. When the current is lower than this critical value, honeycomb porous silicon will appear on the surface of single crystal silicon.

Preparation of porous silicon by electrochemical anodic etching of p-type single crystal silicon.

Step 1. Use a P-type <100> single-sided polished single crystal silicon wafer with a resistivity of 4-8Ω-m and a thickness of 100±10μm. Silicon wafers were ultrasonically washed with acetone, absolute ethanol, and deionized water for 15 minutes before the experiment. Perform electrochemical etching on the cleaned single crystal silicon wafer. The corrosion solution is prepared by mixing 49% hydrofluoric acid and 95% ethanol according to the volume ratio HF:CH ₃ CH ₂ OH=1:1-3. The corrosion process was guided by constant current sources with current densities of 200-300mA/ ^cm2 and 600-800mA/ ^cm2 , respectively. First use a current density of 200-300mA/cm ² , corrosion time of 10-30s, pause for 5-10s, then use a current density of 600-800mA/cm ² , corrosion time of 10-30s, and pause for 5-10s to This alternate etching takes 2-4 cycles. Corrode the middle microcavity with 600-800mA/cm ² for 30-60s, pause for 10-20s in the middle, and finally use a current density of 600-800mA/cm ² , etch for 15-25s, pause for 5-10s, and then use a current density of 200 -300mA/cm ² , the corrosion time is 15-25s, and alternately corrodes 2-4 cycles. During the experiment, low temperature is required, and the corrosion tank is placed in an ultrasonic tank in a mixed solution of ice and water.

Step 2. Carrying out alkoxysilane modification to the obtained porous silicon.

Step 3. reacting the obtained alkoxysilane-modified porous silicon with 4-(diethylamino) salicylaldehyde to obtain porous silicon with aromatic tertiary amine groups.

Step 4. Soak the obtained porous silicon with tertiary aromatic amine groups in ion-exchanged water to remove residual organic matter.

Preferably, in the step 2, porous silicon is dispersed in an organic solvent at a mass ratio of 1:20-1:40, ultrasonically treated for 1-2h, alkoxysilane is added dropwise, and reacted at 90-100°C for 2-4h; After the completion, the solvent was removed by filtration, washed several times with absolute ethanol, and dried in vacuum to obtain alkoxysilane-modified porous silicon.

Further preferably, the organic solvent is toluene or xylene or THF or DMF, and the choice of solvent has little effect on the sensitivity of the porous silicon that is finally obtained; the alkoxysilane is preferably γ-aminopropyltrimethoxysilane or γ - Aminopropyltriethoxysilane, wherein the mass ratio of alkoxysilane to porous silicon is 1:0.1-1:0.5.

For step 3, disperse the alkoxysilane-modified porous silicon obtained in step 2 into absolute ethanol, sonicate for 10-20min, and add 4-(diethylamino)salicylaldehyde (CAS: 17754-90-4) , Stirred and refluxed for 6-8h, poured out the upper suspension, filtered to remove the solvent, washed several times with absolute ethanol, and dried in vacuum to obtain porous silicon with aromatic tertiary amine groups. The mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde is 1:5-1:10.

In the present invention, the porous silicon prepared by alternating current is modified to obtain modified porous silicon with simple preparation process and low price, and the detection sensitivity of some products can be as high as 8800±50nm/refractive index unit or more.

Description of drawings

Figure 1. Simulated reflectance spectrum of a porous silicon microcavity with a fixed incident wavelength of 633nm

Figure 2. The simulated reflection spectrum of a porous silicon microcavity with a fixed incident wavelength of 633nm (line A is a uniform increase in refractive index of 0.02, and line B is a uniform increase in refractive index of 0.04)

detailed description

Example 1:

(1) Prepare porous silicon by electrochemical etching method: use P-type <100> single-sided polished single-crystal silicon wafer, its resistivity is 4Ω-m, and the thickness is 100μm. Silicon wafers were ultrasonically washed with acetone, absolute ethanol, and deionized water for 15 minutes before the experiment. Perform electrochemical etching on the cleaned single crystal silicon wafer. The corrosion solution is prepared by mixing 49% hydrofluoric acid and 95% ethanol according to the volume ratio HF:CH ₃ CH ₂ OH=1:1. The corrosion process was guided by constant current sources with current densities of 200mA/ ^cm2 and 800mA/ ^cm2 , respectively. Firstly, the current density is 200mA/cm ² , the etching time is 10s, pause for 5s, and then the current density is 800mA/cm ² , the etching time is 10s, and then pause for 5s, so as to alternately corrode for 2 cycles. The middle microcavity is corroded with 800mA/cm ² for 30s and paused for 10s. Finally, the current density is 800mA/cm ² , the etching time is 15s, and the pause is 5s. Then the current density is 200mA/cm ² and the etching time is 15s. Corrosion for 2 cycles. During the experiment, low temperature is required, and the corrosion tank is placed in an ultrasonic tank in a mixed solution of ice and water;

(2) The porous silicon obtained in step (1) is modified with alkoxysilane: the porous silicon is dispersed in toluene at a mass ratio of 1:20, ultrasonically treated for 1 hour, γ-aminopropyltrimethoxysilane is added dropwise, and reacted at 100°C for 2 hours; After the reaction is completed, the solvent is removed by filtration, washed several times with absolute ethanol, and dried in vacuum to obtain alkoxysilane-modified porous silicon; the mass ratio of γ-aminopropyltrimethoxysilane to porous silicon is 1:0.1;

(3) react the alkoxysilane-modified porous silicon obtained in step (2) with 4-(diethylamino) salicylaldehyde to obtain porous silicon with aromatic tertiary amine groups: the alkoxysilane-modified silicon obtained in step (2) Disperse the porous silicon in absolute ethanol, ultrasonically treat for 10min, add 4-(diethylamino)salicylaldehyde (CAS: 17754-90-4), stir and reflux for 6h, pour out the upper layer suspension, filter to remove the solvent and use Washing with water and ethanol for several times, and vacuum drying to obtain porous silicon with aromatic tertiary amine groups; the mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde is 1:5;

(4) Soak the porous silicon with aromatic tertiary amine groups obtained in step (3) in ion-exchanged water to remove residual organic matter.

Stability testing of the prepared modified porous silicon: the modified porous silicon sample is placed in a flow cell made of polymethyl methacrylate, and phosphate buffer solutions with different pH values are sequentially pumped through the microflow Into the flow cell, wash the flow cell with pH 7.4, 0.01M phosphate buffer for 5 minutes before each buffer solution. Optical reflectance interference spectroscopy picks up optical signals through γ-type optical fibers, and monitors the change of optical thickness of the film in real time. It was found that the optical thickness of the modified porous silicon changes very stably in the phosphate buffer solution with a pH range of 2-12.

Sensitivity detection: drop 10uL of organic compounds with different refractive indices on the surface of the silicon wafer, detect the change of optical thickness, draw a linear relationship graph with the horizontal axis of refractive index and the vertical axis of optical thickness, and change the optical thickness of 1 unit with the refractive index The thickness variation is the sensitivity of the sample. The sensitivity of porous silicon in Example 1 of the present invention is 8850±50 nm/refractive index unit.

test biomolecules;

The main experimental instruments are helium-neon lasers and optical power detectors (both common and cheap conventional optical configurations). The laser is incident on the material based on the porous silicon microcavity at a certain angle, and the reflected light is received by a power receiver, and the angle corresponding to the minimum light intensity power of the microcavity structure is found, and then biological substances are added to change the refractive index of the porous silicon layer, and then Detect the angle corresponding to the minimum light intensity power of the reflected light, and detect the added biological concentration through the change of the front and rear angles.

In advance, a concentration-angle shift standard curve is prepared for defined concentrations of biomolecules. In order to obtain the concentration of the target molecule through the angular displacement value of the test result.

First, test the reflectance spectrum of the porous silicon microcavity when no biological small molecules are added, and record the angle at the minimum value of light intensity;

Second, test the reflectance spectrum after adding small biomolecules, and record the angle at the minimum value of light intensity;

Then, compare the redshift of the angle corresponding to the minimum value of the light intensity, and calculate the angular difference of the movement. Correspond the angular displacement value with the standard curve, and read the concentration value of the biological small molecule.

Example 2:

The corrosion process was guided by constant current sources with current densities of 300mA/ ^cm2 and 600mA/ ^cm2 , respectively. Others are the same as in Embodiment 1, and the sensitivity of porous silicon is 8800±50 nm/refractive index unit.

Example 3:

The organic solvent in step (2) is THF, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8850±50nm/refractive index unit.

Example 4:

The organic solvent in step (2) is DMF, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8850±50nm/refractive index unit.

Example 5:

The organic solvent in step (2) is xylene, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8850±50nm/refractive index unit.

Embodiment 6:

The mass ratio of γ-aminopropyltrimethoxysilane to porous silicon in step (2) is 1:0.5, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8900±50 nm/refractive index unit.

Embodiment 7:

The mass ratio of γ-aminopropyltrimethoxysilane to porous silicon in step (2) is 1:1, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8830±50 nm/refractive index unit.

Embodiment 8:

The mass ratio of γ-aminopropyltrimethoxysilane to porous silicon in step (2) is 1:2, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8810±50 nm/refractive index unit.

Embodiment 9:

The mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde in step (3) is 1:10, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8870 ± 50nm/refractive index unit .

Example 10:

The mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde in step (3) is 1:2, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8330±50nm/refractive index unit .

Example 11:

The mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde in step (3) is 1:15, and the others are the same as in Example 1. The sensitivity of the obtained porous silicon reaches 8410 ± 50nm/refractive index unit .

Example 12:

The corrosion process was guided by constant current sources with current densities of 20mA/ ^cm2 and 80mA/ ^cm2 , respectively. Others are the same as in Embodiment 1, and the sensitivity of porous silicon is 8300±50 nm/refractive index unit.

Example 13:

The corrosion process was guided by constant current sources with current densities of 30mA/ ^cm2 and 60mA/ ^cm2 , respectively. Others are the same as in Embodiment 1, and the sensitivity of porous silicon is 8200±50 nm/refractive index unit.

Comparative example 1:

Step (1) is to prepare porous silicon by electrochemical etching, the etching solution is hydrofluoric acid: absolute ethanol volume ratio is 3:1; the current density is 600mA/cm ² , the etching time is 20 seconds; others are the same as in Example 1 . The sensitivity of porous silicon in Comparative Example 1 of the present invention is 8600±100 nm/refractive index unit.

Comparative example 2:

The current density is 200mA/cm ² , and the etching time is 20 seconds; others are the same as in Comparative Example 1. The sensitivity of porous silicon in Comparative Example 2 of the present invention is 8100±100 nm/refractive index unit.

Comparative example 3:

The treatment in step (3) was not carried out, and the others were the same as in Example 1. It was found that the stability of porous silicon was poor, and the optical thickness varied greatly in the phosphate buffer solution with a pH range of 2-12. The sensitivity is less than 8000±100nm/refractive index unit.

Comparative example 4:

Step (2) was not performed, and the others were the same as in Example 1. It was found that the stability of porous silicon was poor, and the optical thickness varied greatly in a phosphate buffer solution with a pH range of 2-12.

Comparative example 5:

N-type <100> single crystal silicon is used, and the others are the same as in Example 1. The sensitivity of porous silicon is 8500±100 nm/refractive index unit.

Through the comparison of Example 1 and Comparative Examples 3-4, it can be seen that the treatment of steps (2) and (3) of the present invention have an important impact on the stability and sensitivity of the final porous silicon.

Through the comparison of Example 1 and Comparative Examples 1-2, it can be seen that the sensitivity of porous silicon obtained by alternating current etching in step (1) is better than that obtained by single current intensity etching.

The comparison between Example 1 and Comparative Example 5 shows that the selection of single crystal silicon has an influence on the sensitivity of the final porous silicon. P-type single crystal silicon is more suitable for the present invention.

A comparison between Examples 1 and 2 shows that the selection of alternating currents of 200-300mA/cm ² and 600-800mA/cm ² has certain influence on the sensitivity of the final porous silicon. However, it is more sensitive than the products obtained by selecting alternate currents of 20-30mA/cm ² and 60-80mA/cm ² respectively.

The comparison between Example 1 and 3-5 shows that the choice of solvent in step (2) has no effect on the sensitivity of the final porous silicon.

It can be seen from the comparison of Examples 1, 6-8 that the mass ratio of γ-aminopropyltrimethoxysilane to porous silicon in step (2) has an important influence on the sensitivity of the final porous silicon.

It can be seen from the comparison of Examples 1, 9-11 that the mass ratio of alkoxysilane-modified porous silicon to 4-(diethylamino) salicylaldehyde in step (3) has an important influence on the sensitivity of the final porous silicon.

Claims (7)

1. a kind of porous silicon micro-cavity biosensor based on reflective light intensity detect biomolecule method it is characterised in that：Make Capital equipment is helium neon laser and luminous power detection meter；Laser instrument incides at an angle On the material of substrate, receive meter with the power in light power detection meter and receive reflected light, find the corresponding minimum light of micro-cavity structure Superpower angle, then adds biomolecule and changes porous silicon layer refractive index, then the minimum intensity of light power pair detecting reflected light The angle answered, by the change of front and rear angles, to detect interpolation biological concentration；The preparation method of porous silicon is (1) to pass through electrochemistry Lithographic method prepares porous silicon, wherein adopts p type single crystal silicon as the raw material preparing described porous silicon；(2) (1) step is obtained Porous silicon carries out alkoxy silane modification；(3) the porous silicon of the alkoxy silane modification (2) step being obtained and 4-（Lignocaine） Bigcatkin willow aldehyde reaction, obtains the porous silicon with aryl tertiary amine group；(4) the porous silicon with aryl tertiary amine group (3) step being obtained It is immersed in ion exchange water, remove the Organic substance of remaining；

Described step (2) middle porous silicon with mass ratio for 1:20-1:40 are scattered in organic solvent, supersound process 1-2h, Deca alkane TMOS, 90-100^oC reacts 2-4h；Reaction is filtered to remove solvent after terminating, with absolute ethanol washing for several times, vacuum drying, Obtain final product the porous silicon of alkoxy silane modification；

For step (3), the porous silicon alkoxy silane that (2) step obtains modified is distributed in dehydrated alcohol, supersound process 10-20min, adds 4-（Lignocaine）Salicylide（CAS:17754-90-4）, pour out upper strata suspension after being stirred at reflux 6-8h, It is filtered to remove after solvent with absolute ethanol washing for several times, vacuum drying, obtain the porous silicon with aryl tertiary amine group.

2. method according to claim 1 it is characterised in that：

(1) step adopts P type<100>Single-sided polishing monocrystalline silicon piece, its resistivity is 4-8-m, and thickness is 100 ± 10 μ m；With acetone, dehydrated alcohol, deionized water, silicon chip is carried out respectively before preparation ultrasonic washing 15 minutes；To the list after over cleaning Crystal silicon chip carries out electrochemical corrosion；Corrosive liquid be by 49% Fluohydric acid. and 95% ethanol according to volume ratio HF:CH₃CH₂OH= 1:1-3 ratio mixes；Corrosion process is 200-300mA/cm by electric current density respectively²And 600-800mA/cm²Constant current Source guides；It is 200-300mA/cm first with electric current density², etching time is 10-30s, suspends 5-10s, closeer with electric current Spend for 600-800 mA/cm², etching time is 10-30 s, then suspends 5-10 s, with the 2-4 cycle of this alternating corrosion； Middle microcavity 600-800mA/cm²Corrosion 30-60s intermediate suspension 10-20s, is finally 600- with electric current density 800mA/cm², etching time is 15-25s, suspends 5-10s, then is 200-300mA/cm with electric current density², etching time is 15-25s, with the 2-4 cycle of this alternating corrosion；In preparation process, need low temperature, etching tank is put in frozen water mixed solution In ultrasonic bath.

3. method according to claim 1 it is characterised in that：Described organic solvent is THF or DMF.

4. method according to claim 1 it is characterised in that：Alkoxy silane is 1 with the mass ratio of porous silicon:0.1-1: 0.5.

5. method according to claim 1 it is characterised in that：Alkoxy silane be γ-aminopropyltrimethoxysilane or Gamma-aminopropyl-triethoxy-silane.

6. method according to claim 1 it is characterised in that：Porous silicon and 4- that alkoxy silane is modified（Lignocaine） The mass ratio of salicylide is 1:5-1:10.

7. method according to claim 1 it is characterised in that：It is non-that described porous silicon is used for optics as biosensor Labelling biological detection, wherein testing sample are serum or tissue fluid.