CN111579522A - Terahertz imaging-based relative quantitative detection method for biomacromolecule content - Google Patents
Terahertz imaging-based relative quantitative detection method for biomacromolecule content Download PDFInfo
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Abstract
The invention relates to a relative quantitative detection method for biomacromolecule content based on terahertz imaging, and belongs to the field of biomacromolecule detection. The method comprises the following steps: s1: obtaining a nitrocellulose membrane or polyvinylidene fluoride membrane containing biological macromolecules; s2: placing the film to be detected on a terahertz time-domain spectrometer for scanning imaging to obtain a time-domain spectrum of a point; s3: fourier transform and smoothing the time domain spectrum data to obtain frequency domain spectrum data; s4: taking N points on each film to be measured, measuring the thickness of each point, and taking the average value as the thickness of the film to be measured; s5: and calculating, analyzing and recombining the frequency domain spectral data of each pixel point of the film to be measured, and converting the frequency domain spectral data into the gray value of the corresponding pixel point, thereby forming a visual image reflecting the spatial distribution condition of the content of the biomacromolecule on the film. The invention does not need antibody, has simple and convenient operation, high detection speed and accuracy, saves time and labor cost and realizes label-free detection at the same time.
Description
Technical Field
The invention belongs to the field of biomacromolecule detection, and relates to a terahertz imaging-based relative quantitative detection method for biomacromolecule content.
Background
The currently used biomolecule detection technique is immunostaining (Western Blot), which is a commonly used experimental method in molecular biology, biochemistry and immunogenetics. The basic principle is to stain a gel electrophoresis treated cell or biological tissue sample with a specific antibody. Information on the expression of a specific protein in the analyzed cell or tissue is obtained by analyzing the position and depth of staining.
Antigen-antibody based reactions lead to better specificity but also to more complex operations with consequent material, time and labor costs. Moreover, for some proteins that cannot obtain high quality antibodies, the accuracy of the analysis cannot be guaranteed, or even the experiment cannot be performed at all. The development of label-free detection technology can greatly simplify the detection operation steps, does not need antibodies, labels and the like, and avoids the situation that the target object cannot be detected due to the lack of antibodies. Therefore, the development of a feasible label-free detection method can greatly improve the detection efficiency and save the cost.
Disclosure of Invention
In view of the above, the present invention aims to provide a relative quantitative detection method for biomacromolecule content based on terahertz imaging, which solves the problems of material, time and labor cost caused by the need of antibodies (high antibody titer, high sensitivity is a decisive factor for successful experiment), complex operation, etc., in the existing method for quantitative and qualitative analysis of proteins based on immunology technology. The method does not need an antibody, is simple and convenient to operate, has high detection speed and accuracy, saves time and labor cost, and simultaneously realizes label-free detection.
In order to achieve the purpose, the invention provides the following technical scheme:
a relative quantitative detection method for biomacromolecule content based on terahertz imaging specifically comprises the following steps:
s1: obtaining a nitrocellulose membrane (NC) or a polyvinylidene fluoride membrane (PVDP) containing biological macromolecules such as proteins or DNA;
s2: placing a film to be detected on a two-dimensional translation table utilizing a terahertz time-domain spectrometer, and performing scanning imaging to obtain time-domain spectrums of all pixel points;
s3: fourier transform and smoothing are carried out on the obtained time domain spectrum data set to obtain a frequency domain spectrum data set of the film to be detected; meanwhile, terahertz spectrum data of air needs to be collected, and the terahertz spectrum data is used as a reference signal after Fourier transformation and smoothing;
s4: taking N points on each film to be measured, measuring the thickness of each point, and taking the average value as the thickness of the film to be measured;
s5: and calculating, analyzing and recombining the frequency domain spectral data of each pixel point of the film to be measured, and converting the frequency domain spectral data into the gray value of the corresponding pixel point, thereby forming a visual image reflecting the spatial distribution condition of the content of the biomacromolecule on the film.
Further, the step S2 specifically includes: fixing a film to be measured on a two-dimensional translation table through a clamping device of a terahertz time-domain spectrometer, placing the film to be measured in a measuring light path of a terahertz spectrum system, moving the two-dimensional translation table, realizing scanning of a film to be measured, and recording a time-domain spectrum of each pixel point; wherein, the scanning step is 0.25mm, and the scanning speed is 50 mm/s.
Further, the step S5 specifically includes the following steps:
s51: according to a corresponding formula, calculating terahertz spectrum data of corresponding pixel points, and performing image reconstruction according to terahertz spectrum parameters, wherein the terahertz spectrum parameters comprise time domain spectrum information: peak-to-peak and unilateral peak height, etc.; frequency domain spectral information: the amplitude, phase, extinction coefficient, absorption and refractive index of the transfer function, etc. (which may be the value of a certain frequency, or the average of a certain range of frequencies);
s52: counting a numerical value corresponding to a certain parameter at a certain point of the film to be tested to obtain a highest value and a lowest value, and dividing the difference between the highest value and the lowest value into M parts corresponding to different gray values;
s53: converting the corresponding parameter values of all the points of the film to be detected into gray values, corresponding to a space coordinate system, and reconstructing an image;
s54: repeating the above operations on different parameters to obtain images reconstructed by different parameters;
s55: and screening the clearest image for display.
Further, in the step S51, the refractive index n (ω), the extinction coefficient k (ω), the absorption coefficient α (ω), and the complex dielectric constant in the frequency domain spectrum information
The calculation formulas of (A) and (B) are respectively as follows:
wherein A (ω) and
the amplitude and the phase of the transfer function are respectively, and the value of the transfer function is equal to the ratio of the amplitudes of the sample frequency domain signal and the reference frequency domain signal; omega is angular frequency, d is the thickness of the sample to be measured, c is the propagation speed of the terahertz wave in the air,1and2corresponding to the real and imaginary parts of the complex permittivity, respectively.
Further, in step S51, the calculation formula of the terahertz energy loss is:
wherein, PreIs the relative energy of a single transmission spectrum, EsAnd ErThe frequency dependent amplitudes of the fourier transforms of the sample signal and the reference signal, respectively.
The invention has the beneficial effects that: the method of the invention does not need an antibody, is simple and convenient to operate, has high detection speed and accuracy, saves time and labor cost, and simultaneously realizes label-free detection. Compared with the traditional marking method, the method has better economy, applicability and simplicity.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a comparison chart of the flow of the terahertz spectral imaging method of the present invention and the conventional western blotting method;
FIG. 2 is a diagram of a terahertz spectral imaging system;
FIG. 3 is a schematic diagram of protein sample detection;
FIG. 4 is a terahertz time-domain spectrogram and a frequency-domain spectrogram of a single pixel point;
FIG. 5 is a diagram of imaging effects of different parameters;
FIG. 6 is a schematic diagram of the terahertz imaging result of a protein sample.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention in a schematic way, and the features in the following embodiments and examples may be combined with each other without conflict.
Wherein the showings are for the purpose of illustrating the invention only and not for the purpose of limiting the same, and in which there is shown by way of illustration only and not in the drawings in which there is no intention to limit the invention thereto; to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
The same or similar reference numerals in the drawings of the embodiments of the present invention correspond to the same or similar components; in the description of the present invention, it should be understood that if there is an orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "front", "rear", etc., based on the orientation or positional relationship shown in the drawings, it is only for convenience of description and simplification of description, but it is not an indication or suggestion that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes, and are not to be construed as limiting the present invention, and the specific meaning of the terms may be understood by those skilled in the art according to specific situations.
Referring to fig. 1 to 6, fig. 1 shows a relative quantitative detection method for biomacromolecule (such as protein) content based on terahertz imaging, which specifically includes the following steps:
step 1: preparing a nitrocellulose membrane (NC) or a polyvinylidene fluoride membrane (PVDP) containing protein and other biological macromolecules by using a traditional method:
step 2: total protein extraction, such as:
1) extraction of total protein from adherent monolayer cells:
(1) the medium is poured off and the bottle is inverted over absorbent paper to allow the absorbent paper to suck the medium dry (or the bottle is placed upright for a while to allow the residual medium to flow to the bottom of the bottle and then removed by pipetting).
(2) 3ml of 4 ℃ pre-cooled PBS (0.01M pH 7.2-7.3) was added to each flask of cells. The cells were washed by gentle shaking for 1min and then the wash solution was discarded. The above operation was repeated twice, and the cells were co-washed three times to wash out the culture solution. After discarding the PBS, the flasks were placed on ice.
(3) Add 10. mu.l PMSF (100mM) to 1ml of lysate and shake well on ice. (PMSF could be mixed with the lysate until no crystals were present after shaking.)
(4) 400 μ l of lysis solution containing PMSF was added to each flask of cells and lysed on ice for 30min, and the flask was shaken back and forth often to fully lyse the cells.
(5) After lysis, the cells were scraped to one side of the flask using a clean scraper (action is faster) and then the cell debris and lysate were transferred to a 1.5ml centrifuge tube using a gun (the whole procedure was performed on ice as much as possible).
(6) Centrifuge at 12000rpm for 5min at 4 deg.C (pre-centrifuge pre-cool).
(7) The centrifuged supernatant was transferred to a 0.5ml centrifuge tube and stored at-20 ℃.
2) Extraction of total protein in tissue:
(1) placing a small amount of tissue blocks in a spherical part in a homogenizer of 1-2 ml, and shearing the tissue blocks as much as possible by using clean scissors.
(2) Add 400. mu.L of single detergent lysate (containing PMSF) to the homogenizer for homogenization. And then placed on ice.
(3) After a few minutes, the tissue is rolled for a while and then placed on ice, and the rolling is repeated several times to grind the tissue as much as possible.
(4) After the lysis is carried out for 30min, the lysate can be transferred to a 1.5ml centrifuge tube by a pipettor, then the lysate is centrifuged for 5min at 12000rpm at 4 ℃, and the supernatant is taken and subpackaged in a 0.5ml centrifuge tube and is placed at-20 ℃ for preservation.
3) Extraction of total protein from adherent cells treated with drug:
since some cells are exfoliated by the influence of the drug, the cells in the culture solution should be collected in addition to the operation according to 1). The following is the extraction of total cellular protein from the culture broth:
(1) the culture was poured into 15ml centrifuge tubes and centrifuged at 2500rpm for 5 min.
(2) The supernatant was discarded, 4ml of PBS was added and washed with a gentle blow with a gun, and then centrifuged at 2500rpm for 5 min. The supernatant was discarded and washed with PBS once more.
(3) After washing the supernatant with a gun, 100. mu.L of lysis buffer (containing PMSF) was added and lysed for 30min on ice, and one bomb was frequently used during lysis to lyse the cells sufficiently.
(4) Mixing the lysate with the lysate in the culture flask, centrifuging at 12000rpm for 5min at 4 deg.C, collecting supernatant, packaging in 0.5ml centrifuge tube, and storing at-20 deg.C.
And step 3: SDS-PAGE electrophoretically separates protein samples and transfers them to a solid support. The method specifically comprises the following steps: western blot experiment: the Western blot immune analysis process includes separating protein via gel electrophoresis, transferring the protein band on the gel onto nitrocellulose membrane under the action of electric field, blocking, combining with the antibody as probe to the protein to be detected, washing, combining the filter membrane with secondary reagent-radioactive label or horseradish peroxidase or alkaline phosphatase coupled anti-immunoglobulin antibody, further washing, and determining the position and abundance of the antigen-antibody complex on the filter membrane via autoradiography or in-situ enzyme reaction.
Reagents required by Western blot experiment
(1) And (3) an IgG standard product.
(2) Goat anti-human horseradish peroxidase (HRP) labeled IgG antibody.
(3) Transferring buffer, adding 3.03g of Tris, 14.4g of Gly14 and 200ml of methanol, adding triple distilled water to 1000ml of solution, and storing in a refrigerator at 4 ℃.
(4) Tris Buffer (TBS): tris 2.42g, NaCl 29.2g, dissolved in 600ml of triple distilled water, adjusted to pH7.5 with 1N HCl, and then supplemented with triple distilled water to 1000 ml.
(5) Rinse liquid (TTBS): 500ml of TBS solution was added Tween 20250 ul.
(6) Sealing liquid: 5% skimmed milk powder.
(7) Antibody buffer 1.5g BSA was dissolved in 50ml TTBS.
(8) Preparing a color development liquid DAB (3.3-diaminobenzidine ): 5mg DAB was dissolved in 10ml citric acid buffer (2.6 ml 0.01mol/L citric acid, 0.02mol/L Na2HPO417.39ml) and 30% H2O 210. mu.l was added (prepared immediately).
(9) Decoloring liquid: 250ml of methanol, 100ml of glacial acetic acid and distilled water to 1000 ml.
(10) Amino black staining solution (0.1% amino black-10B): dissolving 0.2g of amino black-10B in 200ml of destaining solution, fully stirring and dissolving, and filtering by using filter paper.
Western blot experiment procedure:
1) SDS-polyacrylamide gel electrophoresis of samples
The method is carried out according to the four experimental operation steps. During sample application, repeated spotting is carried out on the same gel in sequence, so that when electrophoresis is finished, one is used for immune identification, and the other is used for protein staining and banding, so as to facilitate mutual comparison and analysis of experimental results.
2) Transfer blot
(1) Preparation before transfer: cutting filter paper and nitrocellulose membrane (NC) into the same size as the glue, soaking the NC membrane in distilled water for 10-20min, and soaking in transfer buffer for balancing for 30 min.
(2) Gel balance: placing the SDS-PAGE gel plate after electrophoresis in a transfer buffer for balancing for 30-60min
(3) The layers are paved layer by layer without air bubbles and wrinkles between the layers.
(4) Starting transferring, connecting the positive electrode and the negative electrode, covering a cover, connecting a power supply, carrying out constant current of 0.8mA/cm, transferring for 1h at room temperature, dyeing the transferred gel with amino black 10B dyeing solution for 20min, and then carrying out decoloration and detection on the transferring effect.
3) Immunostaining
(1) The transferred NC membrane was blocked in 5% skim milk powder overnight at 4 ℃.
(2) TBS washing is carried out for 1-2 times and 10 min/time.
(3) Adding HRP labeled antibody, and keeping the temperature for 1 h.
(4) TBS was washed 3 times for 10 min.
(5) And transferring the NC membrane into DAB color development liquid, reacting in a dark place, and immediately washing with triple distilled water to terminate the reaction when the color development reaction reaches the optimal degree.
And 4, step 4: the standby film will air, utilize terahertz time domain spectrometer (as shown in fig. 2), scan the formation of image: the film to be measured is fixed on the two-dimensional translation table through the clamping device, the film to be measured is placed in a measuring light path of the terahertz spectrum system, and the two-dimensional translation table is moved so as to scan the blade to be measured and record the time domain spectrum of each point. Wherein, the scanning step is 0.25mm, and the scanning speed is 50 mm/s; performing Fourier transform and smoothing on the obtained spectrum data set to obtain a terahertz frequency domain spectrum data set of the film to be detected (the terahertz time domain spectrum data and the frequency domain spectrum data of a certain point of a sample to be detected are shown in figure 4); meanwhile, terahertz spectrum data of air needs to be collected, and the terahertz spectrum data is used as a reference signal after Fourier transformation and smoothing processing.
And 5: the thickness of each film was measured at 15 points, and the average value was taken as thickness information of the film.
Step 6: calculating, analyzing and recombining the spectral information of each pixel point of the film to be detected to obtain a visual image reflecting the spatial distribution condition of the substance content on the film, wherein the gray value of each point in the image represents the substance content of the point. The relative content of the proteins from different sources is reflected by the gray value deduced by the terahertz characteristic spectrum values at different positions on the film. The step 4 specifically comprises the following steps:
s51: according to a corresponding formula, calculating terahertz spectrum data of corresponding pixel points, and performing image reconstruction according to terahertz spectrum parameters, wherein the terahertz spectrum parameters comprise time domain spectrum information: peak-to-peak and unilateral peak height, etc.; frequency domain spectral information: the amplitude, phase, extinction coefficient, absorption and refractive index of the transfer function, etc. (which may be the value of a certain frequency, or the average of a certain range of frequencies);
refractive index n (omega), extinction coefficient k (omega), absorption coefficient α (omega), complex dielectric constant in frequency domain spectral information
The calculation formulas of (A) and (B) are respectively as follows:
wherein A (ω) and
the amplitude and the phase of the transfer function are respectively, and the value of the transfer function is equal to the ratio of the amplitudes of the sample frequency domain signal and the reference frequency domain signal; omega is angular frequency, d is the thickness of the sample to be measured, c is the propagation speed of the terahertz wave in the air,1and2corresponding to the real and imaginary parts of the complex permittivity, respectively.
The calculation formula of the terahertz energy loss is as follows:
wherein, PreIs the relative energy of a single transmission spectrum, EsAnd ErThe frequency dependent amplitudes of the fourier transforms of the sample signal and the reference signal, respectively.
S52: counting a numerical value corresponding to a certain parameter at a certain point of the film to be tested to obtain a highest value and a lowest value, and dividing the difference between the highest value and the lowest value into 256 parts corresponding to different gray values;
s53: converting the corresponding parameter values of all the points of the film to be measured into gray values, corresponding to the space coordinate system, and reconstructing an image, as shown in fig. 6;
s54: repeating the above operations on different parameters to obtain images reconstructed by different parameters;
s55: the clearest image was screened for display as shown in fig. 5.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (5)
1. A relative quantitative detection method for biomacromolecule content based on terahertz imaging is characterized by comprising the following steps:
s1: obtaining a nitrocellulose membrane or polyvinylidene fluoride membrane containing biological macromolecules;
s2: placing a film to be detected on a two-dimensional translation table utilizing a terahertz time-domain spectrometer, and performing scanning imaging to obtain time-domain spectrums of all pixel points;
s3: fourier transform and smoothing are carried out on the obtained time domain spectrum data set to obtain a frequency domain spectrum data set of the film to be detected; meanwhile, terahertz spectrum data of air needs to be collected, and the terahertz spectrum data is used as a reference signal after Fourier transformation and smoothing;
s4: taking N points on each film to be measured, measuring the thickness of each point, and taking the average value as the thickness of the film to be measured;
s5: and calculating, analyzing and recombining the frequency domain spectral data of each pixel point of the film to be measured, and converting the frequency domain spectral data into the gray value of the corresponding pixel point, thereby forming a visual image reflecting the spatial distribution condition of the content of the biomacromolecule on the film.
2. The relative quantitative detection method for biomacromolecule content based on terahertz imaging according to claim 1, wherein the step S2 specifically comprises: fixing a film to be measured on a two-dimensional translation table through a clamping device of a terahertz time-domain spectrometer, placing the film to be measured in a measuring light path of a terahertz spectrum system, moving the two-dimensional translation table, realizing scanning of a film to be measured, and recording a time-domain spectrum of each pixel point; wherein, the scanning step is 0.25mm, and the scanning speed is 50 mm/s.
3. The relative quantitative detection method for biomacromolecule content based on terahertz imaging according to claim 1, wherein the step S5 specifically comprises the following steps:
s51: according to a corresponding formula, calculating terahertz spectrum data of corresponding pixel points, and performing image reconstruction according to terahertz spectrum parameters, wherein the terahertz spectrum parameters comprise time domain spectrum information: peak-to-peak and unilateral peak height; frequency domain spectral information: the amplitude, phase, extinction coefficient, absorption and refractive index of the transfer function;
s52: counting a numerical value corresponding to a certain parameter at a certain point of the film to be tested to obtain a highest value and a lowest value, and dividing the difference between the highest value and the lowest value into M parts corresponding to different gray values;
s53: converting the corresponding parameter values of all the points of the film to be detected into gray values, corresponding to a space coordinate system, and reconstructing an image;
s54: repeating the above operations on different parameters to obtain images reconstructed by different parameters;
s55: and screening the clearest image for display.
4. The relative quantitative detection method for biomacromolecule content based on terahertz imaging according to claim 3, wherein in step S51, the calculation formula of the refractive index n (ω) and the extinction coefficient k (ω) in the frequency domain spectral information is as follows:
wherein A (ω) and
the amplitude and the phase of the transfer function are respectively, and the value of the transfer function is equal to the ratio of the amplitudes of the sample frequency domain signal and the reference frequency domain signal; ω is the angular frequency, d is the thickness of the sample to be measured, and c is the propagation speed of the terahertz wave in air.
5. The relative quantitative detection method for biomacromolecule content based on terahertz imaging according to claim 3, wherein in step S51, the calculation formula of terahertz energy loss is as follows:
wherein, PreIs the relative energy of a single transmission spectrum, EsAnd ErThe frequency dependent amplitudes of the fourier transforms of the sample signal and the reference signal, respectively.
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