CN111537602A - QCM signal amplification method based on crystal growth - Google Patents
QCM signal amplification method based on crystal growth Download PDFInfo
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- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
- G01N29/02—Analysing fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2291/00—Indexing codes associated with group G01N29/00
- G01N2291/01—Indexing codes associated with the measuring variable
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Abstract
The invention discloses a QCM signal amplification method based on crystal growth, and belongs to the technical field of biological detection. According to the invention, functional groups with different proportions are modified on the surface of the crystal to prepare the self-assembled film with corresponding mixed functional groups, and the self-assembled film can quantitatively regulate and control the growth of the crystal on the surface of the quartz crystal microbalance wafer; the amount of surface crystals is related to-COOH/-N (CH)3)3The proportion presents a good linear relation; the crystallization induced by the oligonucleotide chain amplifies the resonance frequency change signal by nearly 1000 times.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a QCM signal amplification method based on crystal growth.
Background
The Quartz Crystal Microbalance (QCM) analysis method is a detection method that is easy to operate, has the advantages of no need of labeling and real-time detection of surface binding reactions, and has been widely used in biochemical analysis. The quartz crystal microbalance sensor has the characteristics of high precision, high sensitivity and high stability, and can detect the magnitude order below nanogram. The quartz crystal microbalance sensor can provide not only kinetic information about the adsorption process but also information about the surface coverage of the finally formed adsorption layer. The kinetic parameters can be obtained by a frequency-time graph, the wafer frequency will vary with the mass of the adsorbed species, and finally the mass of the adsorbed species can be calculated from the total frequency drop. There are two main methods of transporting the analyte into the sensor and then measuring the change in the resonant frequency of the wafer. One such method is known as the "dip and dry" method, which is a method of calculating the mass adsorbed by a wafer by measuring the resonance frequency of the wafer twice, before and after attachment of the target analyte to the probe-modified wafer, respectively, and by calculating the difference between the changes in the resonance frequency of the two times. Another method is a flow injection analysis system that delivers an analyte into a sensor and measures the change in its frequency. The small volume sample is transferred into the sensor by the flow injection analysis system, the oscillating circuit in the sensor is connected with the frequency converter, the change of the mass is converted into the change of the resonance frequency, and finally the frequency converter is connected with the computer, and the change of the resonance frequency is displayed on the computer in the form of data. The method can realize the on-line analysis of the data to obtain the real-time data, which is beneficial to the research of the combination degree of the bonding system from low to high. The quartz crystal microbalance can measure the data of the change of the resonance frequency and the energy loss of the bonding reaction.
However, the quartz crystal microbalance cannot obtain good signal response when detecting analytes with small molecular mass or analytes with low concentration and large molecular mass; to increase the sensitivity of the quartz crystal microbalance, it is necessary to expand the signal response.
Therefore, providing a QCM signal amplification method based on crystal growth is a problem that needs to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention provides a QCM signal amplification method based on crystal growth, which achieves the purpose of mass amplification by controlling the growth of crystalline substances on the surface of a crystal, and realizes microanalysis of a quartz crystal microbalance.
In order to achieve the purpose, the invention adopts the following technical scheme:
a QCM signal amplification method based on crystal growth comprises the following specific operation steps:
(1) cleaning a wafer;
(2) self-assembled film modification of wafers: spreading the cleaned wafer on the bottom of a small beaker, and adding-N (CH) with different ratios3)3Adding the mixed solution of-COOH into a small beaker, and keeping the temperature of the mixed solution in a gas bath constant temperature oscillator at 37 ℃ for reaction for 12 hours; after the reaction is finished, taking out the wafer, and washing away unreacted substances on the wafer by using absolute ethyl alcohol; drying the wafer in a nitrogen atmosphere to obtain a self-assembled film modified wafer;
(3) and (3) crystallization: taking out the self-assembled film modified wafer, installing the wafer in an open detection cell of a quartz crystal microbalance detection system, opening QCM software, preheating to 37 ℃, and starting to measure; when the resonance frequency stabilized within. + -. 2Hz in air, 500. mu.l of 6mM CaCl was added to the open cell2Solution, when the resonance frequency is stabilized within +/-2 Hz, adding excessive ammonium carbonate solid powder into the reaction space, placing the solution nearby calcium chloride solution by using a carrier to create a carbon dioxide atmosphere and provide reactant carbon dioxide for crystallization of calcium chloride; and recording the signal response of the quartz crystal microbalance wafer surface with different modifications in the crystallization process through the quartz crystal microbalance sensor, and storing data after the signal is stable.
Further, the cleaning step of the wafer in the step (1) is as follows: placing the wafer in piranha rinse (H)2SO4：H2O23: 1 by volume) for 10 minutes, and then according to H2O：H2O2：NH3·H2Preparing alkali liquor according to the volume ratio of O to 1:1, heating and boiling the prepared alkali liquor on an electric heating sleeve, and soaking the wafer in the boiled alkali liquor for 15 minutes; the alkali liquor is kept in a boiling state all the time in the soaking process. Washing with ultrapure water, soaking in ultrapure water for 5 minutes, washing with absolute ethyl alcohol, and soaking in absolute ethyl alcohol for 5 minutes; blowing dry with nitrogen gas to dry the waferThe mixture was placed on a clean bench and irradiated with ultraviolet rays for 20 minutes to remove organic substances from the surface.
Further, the different proportions of-N (CH) in the step (2)3)3The preparation method of the mixed solution of-COOH was as follows: weighing-N (CH)3)3and-COOH, respectively adding absolute ethyl alcohol to dissolve them to obtain solutions, mixing them according to a certain proportion to make-N (CH)3)3and-COOH at a final concentration of 10 mM.
The invention selects-N (CH)3)3(i.e., -NMe)3) and-COOH as a terminal functional group. -N (CH)3)3The functional group can completely inhibit the crystallization process of the surface calcium carbonate crystal, and the capability can effectively prevent the non-specific mineral from precipitating and crystallizing; the-COOH functional groups are capable of inducing a process of promoting the crystallization of the surface calcium carbonate crystals, which promotes the generation of solid crystals of sufficient quality for signal amplification. Theoretically, when calcium chloride and carbon dioxide are supplied simultaneously, as the carboxyl terminal functional group increases on the sensor surface, the amount of calcium carbonate crystals formed on the sensor surface should increase, and the detected frequency change signal response should also gradually increase.
According to the technical scheme, compared with the prior art, the invention discloses a QCM signal amplification method based on crystal growth, and the self-assembled film of the corresponding mixed functional group is prepared by modifying functional groups with different proportions on the surface of the crystal, and can quantitatively regulate and control the crystal growth on the surface of the quartz crystal microbalance wafer; the amount of surface crystals is related to-COOH/-N (CH)3)3The proportion presents a good linear relation; the crystallization induced by the oligonucleotide chain amplifies the resonance frequency change signal by nearly 1000 times.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the present invention modified by only-N (CH)3)3The wafer surface and the bare gold wafer surface and the wafer surface modified only with-COOH;
FIG. 2 is a schematic view showing a scanning electron microscope of the present invention modified with only-N (CH)3)3The surface of the wafer, the surface of the bare gold wafer, and the surface of the wafer modified with-COOH only;
wherein A is modified by-N (CH)3)3The wafer surface crystallization condition of (1); b is the surface crystallization condition of the bare gold wafer; c is the crystallization condition of the surface of the wafer only modified with-COOH;
FIG. 3 is a drawing showing the modification of the present invention with-N (CH) in different proportions3)3Frequency change during crystallization of the COOH wafer surface; from bottom to top, 0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1,10: 0;
FIG. 4 is a graph showing the frequency variation of the quartz crystal microbalance of the present invention with respect to different-COOH/-N (CH)3)3A proportional linear operating curve;
FIG. 5 is a graph showing the change of resonance frequency caused by 50mM DNA of the present invention and its crystallization process.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Nucleotides used in the present invention were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and have the sequence 5' -COOH-ATG TCC CTC AGA CCC TTT- (CH)2)6-SH-3’。HS-C11-NMe3Cl and HS-C11-COOH were purchased from Prochimia. For detection of quartz crystal microbalanceThe wafer of (a) was purchased from Biolin Scientific and the gold plate for Raman detection was purchased from BioNavis Ltd. CaCl2，(NH4)2CO3And Tris (2-carboxyethyl) phosphinihydrochlorides (tcep) from alatin reagent, Tris (hydroxymethyl) aminomethane (Tris) from SangonBiotech co., Ltd. (shanghai china), disodium Ethylenediaminetetraacetate (EDTA) from iron tower reagent, and sodium chloride (NaCl) from national drug group chemicals, Ltd., all of which were analytically pure. The pH of the immobilization buffer (10mM Tris, 1mM EDTA, 1M NaCl, 1mM TCEP, pH 7.4) was adjusted with 0.1M HCl or 0.1M NaOH. Hydrogen peroxide (H)2O2) Concentrated sulfuric acid (H)2SO4) Ammonia (NH)3·H2O), absolute ethanol (C)2H5OH) and other chemical reagents used in the method are analytically pure and are not specially indicated, and the water used in the experimental process is ultrapure water.
Q-Sense E1 Quartz Crystal microbalance (Biolin Scientific);
hitachi 3400 scanning electron microscope imager (Hitachi corporation, japan);
laser confocal microscope raman spectrometer (Renisaw, uk);
an electronic balance (Shanghai Jingke Tianmei scientific instruments, Ltd.) can be accurate to 0.0001 g;
the electronic balance (Beijing Saedodus scientific instruments, Inc.) can be accurate to 0.00001 g;
pHS-3D type acidimeters (Shanghai Lei magnetic Instrument works);
model 16R centrifuge (zhhai black horse medical instruments ltd);
DHT type magnetic stirring temperature controlled electric heating jacket (Shandong Juancheng Hualu electric heating apparatus Co., Ltd.);
gas bath constant temperature oscillator (Tan City Tan Ji manufacturer);
clean bench (Suzhou Bolelel clean Equipment Co., Ltd.).
(1) Cleaning a wafer: the wafer (quartz crystal microbalance sensor wafer, Biolin sci, Sweden)Made by the company entific, having a diameter of 14mm and a fundamental frequency of 5MHz in piranha wash (H)2SO4：H2O23: 1 by volume) for 10 minutes, and then according to H2O：H2O2：NH3·H2Preparing alkali liquor according to the volume ratio of O to 1:1, heating and boiling the prepared alkali liquor on an electric heating sleeve, and soaking the wafer in the boiled alkali liquor for 15 minutes; the alkali liquor is kept in a boiling state all the time in the soaking process. Washing with ultrapure water, soaking in ultrapure water for 5 minutes, washing with absolute ethyl alcohol, and soaking in absolute ethyl alcohol for 5 minutes; blowing the wafer to dry by nitrogen, and placing the dried wafer in a purification workbench to irradiate for 20 minutes under ultraviolet rays to remove organic matters on the surface of the wafer;
(2) self-assembled film modification of wafers: spreading the cleaned wafers on the bottom of small beaker, and spreading-N (CH) on the cleaned wafers3)3Adding the-COOH solution into a small beaker, and keeping the temperature of the small beaker in a gas bath constant temperature oscillator to react for 12 hours at 37 ℃; after the reaction is finished, taking out the wafer, and washing away unreacted substances on the wafer by using absolute ethyl alcohol; drying the wafer in a nitrogen atmosphere to obtain a self-assembled film modified wafer;
(3) and (3) crystallization: taking out the self-assembled film modified wafer, installing the wafer in an open detection cell of a quartz crystal microbalance detection system, opening QCM software, preheating to 37 ℃, and starting to measure; when the resonance frequency stabilized within. + -. 2Hz in air, 500. mu.l of 6mM CaCl was added to the open cell2Solution, when the resonance frequency is stabilized within +/-2 Hz, adding excessive ammonium carbonate solid powder into the reaction space, placing the solution nearby calcium chloride solution by using a carrier to create a carbon dioxide atmosphere and provide reactant carbon dioxide for crystallization of calcium chloride; and recording the signal response of the quartz crystal microbalance wafer surface with different modifications in the crystallization process through the quartz crystal microbalance sensor, and storing data after the signal is stable.
Modified with only-N (CH)3)3The crystallization experiment was performed on the wafer surface of (1), the wafer surface modified with only-COOH, and the bare gold wafer surface. Sensing by quartz crystal microbalanceThe surface crystallization process is monitored in real time by the instrument, and the signal response result is shown in figure 1.
When the quartz crystal microbalance detects the three kinds of wafers on line in real time, crystallization signals are generated. In the modification of only-N (CH)3)3The resonance frequency of the wafer is only reduced by about 20Hz, as shown in the curve I in FIG. 1, which shows that no solid crystal is generated on the surface of the quartz crystal microbalance wafer. For bare gold wafers (FIG. 1, curve II) and wafers modified with only-COOH (FIG. 1, curve III), a significant drop in resonance frequency was produced, which is a response manifestation of typical crystallization signals. The apparent change in resonance frequency indicates that there is a calcium carbonate crystallization reaction occurring on the wafer surface modified with only-COOH as well as on the bare gold wafer surface. The degree of the decrease in the resonance frequency reflects the difference in the amount of crystals grown on the wafer surface for the wafer modified with only-COOH and the bare gold wafer. The degree of the decrease in the resonance frequency of the wafer modified with only-COOH was greater, indicating that the surface of the wafer modified with only-COOH produced more crystals than the surface of the bare gold wafer.
And scanning the picture by using a scanning electron microscope to characterize the crystallization condition of the crystal surface. The scanning electron microscope picture is shown in FIG. 2, which is modified with only-N (CH)3)3The presence of calcium carbonate crystals was not observed on the wafer surface consistent with the absence of signal response from the quartz crystal microbalance sensor. The presence of calcium carbonate crystals was found on both the wafer surface modified with only-COOH as well as on the bare gold wafer surface. On the surface of the bare gold wafer, the amount of calcium carbonate crystals was small and irregular in shape. On the wafer surface modified with only-COOH, a large number of calcium carbonate crystals were observed, and these calcium carbonate crystals had a substantially uniform size and morphology. The signal response of the quartz crystal microbalance sensor and the result of the scanning electron microscope picture characterization jointly confirm-N (CH)3)3The functional group can effectively inhibit the formation of calcium carbonate crystals on the surface of the wafer. And a great response signal of the resonance frequency change of the quartz crystal microbalance sensor can be generated on the surface of the wafer of-COOH and a large amount of calcium carbonate crystals are generated as shown in a scanning electron microscope picture,both results simultaneously indicate that the-COOH functional groups are effective in promoting the formation of calcium carbonate crystals on the wafer surface. This phenomenon indicates that the calcium carbonate crystals are randomly adsorbed on the surface of the bare gold wafer and may be inadvertently and accidentally deposited; and the calcium carbonate crystal is induced by functional groups on the surface of the wafer modified by-COOH, and the property is more stable. The results provide a theoretical basis for feasibility by quantitatively regulating and controlling the amount of the calcium carbonate crystals growing on the surface of the wafer.
Example 2
When modification is carried out, the proportion of the functional groups modified on the surface of the wafer is regulated and controlled by adjusting the dosage proportion of two thiol substances with different terminal functional groups.
weighing-N (CH)3)3and-COOH, respectively adding absolute ethyl alcohol to dissolve them to obtain solutions, mixing them according to a certain proportion to make-N (CH)3)3and-COOH at a final concentration of 10mM, was subjected to self-assembled film modification of the wafer, followed by crystallization.
Modified with different proportions of-N (CH)3)3The change in resonance frequency during the detection of crystallization for the quartz crystal microbalance sensor wafer of-COOH is shown in FIG. 3. The results show that the reaction proceeds with-COOH/-N (CH)3)3The change in the resonant frequency gradually increases with increasing proportion. reacting-COOH/-N (CH)3)3The experiment was repeated three times for each ratio and the frequency change of the quartz crystal microbalance was plotted against the different-COOH/-N (CH)3)3The results are shown in fig. 4 for a proportional linear operating curve. The linear regression satisfies the equation y-10.8-1158.4R with the correlation coefficient R2Is 0.996, wherein y is the frequency change of the quartz crystal microbalance sensor during surface crystallization, r is the-COOH functional group and-N (CH)3)3Ratio of functional groups. According to Shaoerlubi equation, the change of the surface quality of the wafer and the change of the resonant frequency present a certain proportional relation; the linear relationship thus indicates that the amount of crystal grown on the wafer surface can be effectively controlled by the surface functional groups.
Further displaying by scanning electron microscope-COOH/-N(CH3)3Different proportions of the surface of the wafer are crystallized. When the proportion of-COOH was 10%, the amount of crystals induced was small, and it was again confirmed that-COOH was effective in inducing the generation of calcium carbonate crystals. It can be clearly seen from the pictures that the amount of crystallization on the wafer surface is dependent on-COOH/-N (CH)3)3The ratio increases. By modifying the wafer surface with different ratios of-COOH and-N (CH)3)3The self-assembled film of the functional group can effectively control the amount of crystal growing on the surface of the quartz crystal microbalance sensor.
The crystallization mechanism of the calcium carbonate crystals on the surface of the QCM wafer is as follows: adsorbing CaCl in reaction solution by-COOH group carried in probe sequence2Ca in (1)2+Ion, external world (NH)4)2CO3Continuous supply of CO2，CO2And Ca2+Ions combine at the target binding site to form CaCO3Seeding and continuously growing.
Example 3 verification of Signal amplification Capacity
In order to evaluate the ability of-COOH to induce signal amplification in the crystallization mechanism, the present invention selects an oligonucleotide chain to verify: 5'-ATG TCC CTC AGA CCC TTT-3', respectively; SEQ ID No. 1;
wherein the 5 'end is modified with COOH-, and the 3' end is modified with- (CH)2)6-SH。
As a result, as shown in FIG. 5, when the concentration of the oligonucleotide chain (50mM) was high, the change in the resonance frequency was only 10Hz, which was difficult to detect. When crystallization occurs, the resonance frequency drops by approximately 10000Hz and the signal expands by a factor of approximately 1000. The result shows that the quantitative control mechanism of the calcium carbonate surface crystallization process provides a wide prospect for signal amplification of the quartz crystal microbalance.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Linyi university
<120> QCM signal amplification method based on crystal growth
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>DNA
<213>Artificial Sequence
<400>1
atgtccctca gacccttt 18
Claims (3)
1. A QCM signal amplification method based on crystal growth is characterized by comprising the following specific operation steps:
(1) cleaning a wafer;
(2) self-assembled film modification of wafers: spreading the cleaned wafer on the bottom of a small beaker, and adding-N (CH) with different ratios3)3Adding the mixed solution of-COOH into a small beaker, and keeping the temperature of the mixed solution in a gas bath constant temperature oscillator at 37 ℃ for reaction for 12 hours; after the reaction is finished, taking out the wafer, and washing away unreacted substances on the wafer by using absolute ethyl alcohol; drying the wafer in a nitrogen atmosphere to obtain a self-assembled film modified wafer;
(3) and (3) crystallization: taking out the self-assembled film modified wafer, installing the wafer in an open detection cell of a quartz crystal microbalance detection system, opening QCM software, preheating to 37 ℃, and starting to measure; when the resonance frequency stabilized within. + -. 2Hz in air, 500. mu.l of 6mM CaCl was added to the open cell2Adding excessive ammonium carbonate solid powder into a reaction space after the resonance frequency is stabilized within +/-2 Hz; recording the signal response of the quartz crystal microbalance wafer surface with different modifications in the crystallization process through a quartz crystal microbalance sensor, and storing data after the signal is stable。
2. A QCM signal amplifying method according to claim 1, wherein the wafer cleaning step of step (1) is as follows: soaking the wafer in piranha lotion for 10 min, and treating with the following method2O：H2O2：NH3·H2Preparing alkali liquor according to the volume ratio of O to 1:1, heating and boiling the prepared alkali liquor on an electric heating sleeve, and soaking the wafer in the boiled alkali liquor for 15 minutes; washing with ultrapure water, soaking in ultrapure water for 5 minutes, washing with absolute ethyl alcohol, and soaking in absolute ethyl alcohol for 5 minutes; blow-drying with nitrogen, and placing the dried wafer in a clean bench to irradiate under ultraviolet rays for 20 minutes.
3. A QCM signal amplifying method according to claim 1, wherein the different ratios of-N (CH) in step (2)3)3The preparation method of the mixed solution of-COOH was as follows: weighing-N (CH)3)3and-COOH, respectively adding absolute ethyl alcohol to dissolve them to obtain solutions, mixing them according to a certain proportion to make-N (CH)3)3and-COOH at a final concentration of 10 mM.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022035457A1 (en) * | 2020-08-10 | 2022-02-17 | Saudi Arabian Oil Company | Methods for growing crystals on qcm sensors |
| CN114166681A (en) * | 2021-11-12 | 2022-03-11 | 常州大学 | QCM humidity sensor based on high polymer/inorganic composite sensitive material and preparation method thereof |
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| Title |
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| 吴从从: "基于结晶质量增大效应的石英晶体微天平检测技术研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022035457A1 (en) * | 2020-08-10 | 2022-02-17 | Saudi Arabian Oil Company | Methods for growing crystals on qcm sensors |
| CN114166681A (en) * | 2021-11-12 | 2022-03-11 | 常州大学 | QCM humidity sensor based on high polymer/inorganic composite sensitive material and preparation method thereof |
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