CN108717129A - A kind of SEM suitable for biological sample detects sample preparation reagents box and its application - Google Patents

Abstract

It includes porous plate to detect sample preparation reagents box and its application, the kit the invention discloses the SEM suitable for biological sample, and the silicon chip of polylysine modification, and the reagent etc. of processing sample are provided in the hole of porous plate.SEM by the kit for biological sample is detected, and mainly has following steps：Biological sample solution is added dropwise on the silicon chip of the polylysine modification in porous plate；Remove extra biological sample solution；Then it is incubated with buffer solution, then after being handled with phosphotungstic acid, and through the alcohol treatment drying containing hexamethyldisilazane, obtains SEM and detect sample, and carry out SEM detections.The SEM detections that the kit can be used for biological sample use, and have many advantages, such as that experimental result is reproducible, analysis result accuracy is high.

Description

A kind of SEM suitable for biological sample detects sample preparation reagents box and its application

Technical field

The invention belongs to detection kit fields, in particular to one kind being suitable for excretion body, cell, bacterium, virus or raw The biological samples such as object tissue carry out the detection sample preparation preparation box and its application of SEM (scanning electron microscope) detection.

Background technology

Scanning electron microscope (SEM) is a kind of microscopic appearance observation work between transmission electron microscope and light microscope Tool, mainly observes the configuration of surface of sample using secondary electron signal imaging, i.e., removes scanning sample with extremely narrow electron beam Product generate various effects, wherein the mainly effect of secondary electron emission of sample, base by the interaction of electron beam and sample The X rays topographs of sample surfaces amplification can be generated in secondary electron, this seems chronologically to be set up when sample is scanned , i.e., obtain intensified image using the method being imaged point by point.

In the prior art, many biological samples (such as excretion body, cell, bacterium, virus or biological tissue) cannot be fine Directly use scanning electron microscope or atomic force microscope observation, need to handle biological sample, allow biological sample energy SEM is enough matched, it could observation analysis.It is existing to utilize the means such as metal spraying, to biological sample surface metallization so that raw Object sample surface is conductive, preferably observes.However, metalling may lead to the loss of biological sample fine structure, Spraying plating is uneven may also to be caused to be experimental result poor repeatability, and deviation occurs in analysis result.It is therefore desirable to be changed to this Into.

Invention content

Technical problem to be solved of the embodiment of the present invention is, provides a kind of SEM samples suitable for detection of biological samples Product kit.Biological sample is carried out by the kit to be processed into detection sample, the original structure shape of biological sample can be kept State, and test experience result consistency is good.

To achieve the above object, it the technical scheme is that the kit includes porous plate, is set in the hole of porous plate It is equipped with the silicon chip and SEM detection reagents of polylysine modification.

SEM detection reagents include PBS buffer solution, glutaraldehyde, PFA paraformaldehydes, phosphotungstic acid aqueous solution, two silicon of hexamethyl Azane and ethyl alcohol.

The present invention provides a kind of SEM detection methods based on the SEM sample reagent boxes, include following steps：

1) silicon chip of polylysine modification is placed in a hole of porous plate；

2) biological sample solution to be measured is added dropwise on the silicon chip of the polylysine modification in porous plate, is incubated；

3) extra biological sample solution is removed；

4) 1X PBS buffer solution is added, and adds 2wt% glutaraldehydes, 4wt%PFA poly first in 1X PBS buffer solution Aldehyde, 4 DEG C of incubation 30min-16h；

5) it is cleaned with 1X PBS buffer solution；

6) processing of 0.1-1% phosphotungstic acids is added；

7) ultra-pure water cleans；

8) again successively with 30%, 50%, 70%, 90% alcohol treatment 5min；

9) 100% alcohol treatment 5min, is repeated 2 times；

10) contain the alcohol treatment 15-30s of 50% volume ratio hexamethyldisilazane；

11) 100% hexamethyldisilazane handles 15-30s；

12) hexamethyldisilazane is removed；

13) it is placed in vacuum desiccator dry；

14) and then the silicon chip through above-mentioned steps treated polylysine modification is placed on SEM and is detected.

It is containing excretion body, cell, bacterium, virus or biological group that further setting, which is the biological sample solution to be measured, The physiological buffered solution knitted.

It is an advantage of the invention that：

(1) silicon chip of poly-D-lysine is provided through the invention, and there is very outstanding hydrophily, be conducive to biological sample This is adsorbed in silicon chip surface, improves sample preparation success rate, convenient for observation；

(2) detecting step through the invention, is handled using phosphotungstic acid so that biofilm structure has preferably conductive Property, be conducive to SEM observations.

(3) the hexamethyldisilazane disjunction of various concentration is used to handle, hmds is rapid after contacting with the air Hydrolysis, the hexamethyldisiloxane and trimethylsilane alcohol of a variety of organic solvents can be dissolved in by generating, this property determine its can rapidly by Dehydrating agent in sample and residual moisture content are replaced, quickly fully drying sample, the damage from atmospheric pressure to tissue, So that the 3 D stereo fine structure of tissue is stored in condition of living organism；

(4) present invention can effectively keep the stability of biological sample pattern to be measured, so that SEM test experience results Reproducible, analysis result accuracy is high.

Description of the drawings

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, according to These attached drawings obtain other attached drawings and still fall within scope of the invention.

The contact angle test chart of the original Si of Fig. 1；

Fig. 2 by present invention process treated Si contact angle test chart；

The SEM detection figures (being from left to right arranged in order) of Fig. 3 three embodiments of the present invention.

Specific implementation mode

To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing Step ground detailed description.

Prepare embodiment

1. the SEM of excretion body is detected：

1) Poly L-Lysine- silicon chips are placed in a hole of orifice plate

2) physiological buffer of 10 μ L bodies containing excretion is added dropwise in the coated Si on pieces of PLL, 4 DEG C are incubated an evening

3) physiological buffer of the extra body containing excretion is removed

4) 1X PBS (containing 2% glutaraldehyde, 4%PFA paraformaldehydes), 4 DEG C of incubation 30min is added

5) it is cleaned 2 times with 1X PBS

6) phosphotungstic acid processing is added

7) ultra-pure water cleans 3 times

8) the alcohol treatment 5min of 30%-50%-70%-90% is used respectively

9) 100% alcohol treatment 5min, is repeated 2 times

10) 15~30s of alcohol treatment containing 50%HMDS

11) 100%HMDS handles 15~30s

12) HMDS is removed

13) it is placed in vacuum desiccator and is dried overnight

14) SEM is tested, and SEM detects the picture of excretion body, referring to shown in Fig. 1 (left side).

The cell observation that embodiment 2 is cultivated

1) when cell coverage rate reaches 70-80% in culture dish, culture medium in culture dish is removed；

2) 2% glutaraldehyde, 4% paraformaldehyde is added) (1XPBS configurations), 4 DEG C, overnight；

3) fixer is removed, with 1xPBS Rapid Cleanings 2 times

4) plus 1% phosphotungstic acid (being configured with ultra-pure water), 4 DEG C, 4h

5) ultra-pure water Rapid Cleaning is used 3 times

6) 30%-50%-70%-90% Gradient elution using ethanols, 4 DEG C, 15min

7) absolute ethyl alcohol, room temperature, 2 each 15min

8) ethanol solution of 50%HMDS, 100%HMDS is used to handle 2min successively

9) removal HMDS is placed in vacuum drying chamber

10) laser cutting ware bottom obtains 8 × 8mm polystyrene sheet (parameters of laser cutting：Maximum/minimum power：30%, Speed：10mm/s)

11) it upward by the one side for having cell, is fixed on metab, is imaged with SEM.

3 tissue samples SEM imagings of embodiment

1) mouse anesthesia is put to death, takes out eyeball and fixes 5-10min in fixer is pre-chilled；

2) under Stereo microscope, trabecular meshwork is found, removes crystalline lens and vitreum.In materials it is noted that avoiding Crush injury avoids damage and destruction of the external force to viewing surface.

3) after the trabecular meshwork detached is first rinsed with precooling 0.1M PBS, it is put into 0.1M PH7.4PBS at once and (contains 2% + 4% paraformaldehyde of glutaraldehyde) in, 4 DEG C of fixed 12h.

4) smooth trabecular network truncation surface is cut out with knife after fixing.

5) it after fixed, is cleaned 3 times with 0.1M PBS (PH 7.4), 4 DEG C of each 10min.

6) it is stayed overnight again with 1% phosphotungstic acid, 4 DEG C of processing.

7) it is cleaned 3 times with 0.1M PBS (PH 7.4), 4 DEG C of each 10min.

8) 30%-50%-70%-90% Gradient elution using ethanols, be 10min, 4 DEG C.

9) 100% ethanol dehydration twice, be 10min, 4 DEG C.

10) again with the ethyl alcohol containing 50%HMDS, then 100%HMDS processing, difference 2min, room temperature.

11) after removing HMDS, dry 1 evening, rear kept dry are for use in vacuum desiccator freeze-dryer.

12) tissue is fixed on metab with conducting resinl, it is cross-section face-up, it is imaged using SEM.

The kit of the present invention can also be respectively used to SEM detection cells, bacterium, the biological sample such as virus or biological tissue Product.

The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.

Claims (5)

1. a kind of SEM sample reagent boxes suitable for detection of biological samples, it is characterised in that：The kit includes porous plate, The silicon chip of polylysine modification and the reagent of SEM detections are provided in the hole of porous plate.

2. a kind of SEM sample reagent boxes suitable for detection of biological samples according to claim 1, it is characterised in that：Institute The silicon chip for the polylysine modification stated is prepared by following steps：

(1) it is cut into the silicon chip of uniform sizes specification；

(2) silicon chip is cleaned；

(3) hydrophilic treated is carried out to silicon chip, heating water bath handles 10min in sulfuric acid, ammonia aqueous solution respectively；

(4) silicon chip obtained by (3) is dried up with nitrogen；

(5) dry silicon chip is placed in culture plate, and 0.1-1% poly-D-lysines is added, be incubated；

(6) silicon chip obtained by (5) is cleaned with 1X PBS buffer solution, obtains the silicon chip of polylysine modification.

3. the SEM sample reagent boxes according to claim 1 suitable for detection of biological samples, it is characterised in that：Described Step (2) is to be cleaned by ultrasonic 15min successively in dish detergent, methanol, acetone, deionized water respectively.

4. a kind of SEM detection methods based on SEM sample reagents box described in claim 1, it is characterised in that include following Step：

1) silicon chip of polylysine modification is placed in a hole of porous plate；

2) biological sample solution to be measured is added dropwise on the silicon chip of the polylysine modification in porous plate, is incubated；

3) extra biological sample solution is removed；

4) 1X PBS buffer solution is added, and adds 2wt% glutaraldehydes, 4wt%PFA paraformaldehydes in 1X PBS buffer solution, 4 DEG C It is incubated 30min-16h；

5) it is cleaned with 1X PBS buffer solution；

6) processing of 1% phosphotungstic acid is added；

7) ultra-pure water cleans；

8) again successively with 30%, 50%, 70%, 90% alcohol treatment 5min；

9) 100% alcohol treatment 5min, is repeated 2 times；

10) contain the alcohol treatment 15-30s of 50% volume ratio hexamethyldisilazane；

11) 100% hexamethyldisilazane handles 15-30s；

12) hexamethyldisilazane is removed；

13) it is placed in vacuum desiccator dry；

14) and then the silicon chip through above-mentioned steps treated polylysine modification is placed on SEM and is detected.

5. the SEM detection methods according to right 4, it is characterised in that：The biological sample to be measured be excretion body, cell, Bacterium, virus or biological tissue.