QCM and LSPR dual-technology fusion biological detection system and method

Abstract

The invention discloses a QCM (Quartz Crystal Microbalance) and LSPR (Local Surface Plasmon Resonance) dual-technology fusion biological detection method, which comprises the following steps of: preparing a primer according to an object to be detected, fixing a nano composite probe on the surface of a piezoelectric quartz wafer, switching on a control circuit, controlling the reaction temperature through a temperature control optimization algorithm, and introducing a PBS (Phosphate Buffer Solution) until the output frequency of a sensing chip is stable; recording the frequency as a reference frequency f1 when the frequency is no-load, introducing a nucleic acid sample into a measuring cell, fully reacting the nucleic acid sample with a primer until a chip output frequency signal is stable, recording the frequency at the moment as f2, obtaining multiple parameters of a biological membrane according to two frequency changes (f1-f2) Hz in combination with a biological membrane multi-parameter fusion algorithm, and calculating a result according to the parameters of the biological membrane. According to the invention, high-precision, quick-response, high-integration and miniaturized intelligent sensing biological detection is realized, and the system can be applied to early diagnosis of serious diseases such as tumors and cardiovascular and cerebrovascular diseases, and has a wide prospect in the POCT (point-of-care testing) market.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a biological detection system and method integrating QCM and LSPR dual technologies. Background Art

[0002] At present, nucleic acid testing is a powerful means to detect viral activity. The main detection methods at this stage include antigen-antibody, sequencing, PCR, CRISPR and other detection technologies, which are made into portable detection kits. However, due to its cumbersome operation, high requirements for equipment and operators, long time consumption, expensive reagents and other limiting factors, it cannot fully meet the requirements of fast and accurate detection process and on-site screening.

[0003] Application No.: CN201811353335.6 proposes a constant temperature measurement cell for QCM and LSPR dual technology biomolecule detection to solve the above-mentioned problems existing in the existing biodetection technology. However, the device does not disclose how to integrate QCM and LSPR technologies to achieve rapid biodetection. Summary of the invention

[0004] In order to achieve rapid, portable and low-cost biological detection, the present invention proposes a biological detection method integrating QCM and LSPR technologies, which comprises the following steps: S1. Configure corresponding primers according to the analyte; S2. Fixing the nanocomposite probe on the surface of the piezoelectric quartz wafer of the measuring cell; S3. Turn on the control circuit to control the temperature control system of the measuring cell within the set temperature range for reaction; S4. PBS buffer is introduced into the measuring cell until the output frequency of the dual technology fusion chip of the measuring cell is stable, and this frequency is recorded as the reference frequency f1 when no load; S5. Pass the biological sample into the measurement pool, and let the biological sample and the primer react fully until the chip output frequency signal is stable, and record the frequency at this time as f2; S6. Based on the two frequency changes (f1-f2) Hz, combined with the frequency change curve and LSPR spectrum collected by QCM, various parameters of the biofilm are obtained. Finally, the sample properties are judged based on the calculation results of the biofilm parameters.

[0005] Preferably, in step S6, QCM can detect the total mass of the coupling including the solvent, and LSPR can measure the dry mass of the biomolecules. The two techniques of QCM and LSPR are combined to obtain the mass of the coupling solvent by subtracting the molecular dry mass from the total mass.

[0006] Preferably, the LSPR spectrum is collected and analyzed by the spectrum analysis software of the spectrophotometer; the measurement cell includes a QCM frequency signal acquisition system, which integrates an FPGA control module, receives the frequency of AD sampling measurement through the FPGA, and finally sends it to the host computer for processing through the serial port of the FPGA.

[0007] Preferably, in step S1, the primer length is 20-50 bp, the TM values ​​of the front and rear primers are close, and the configuration of the primers is performed by consulting literature or using the Primeblast function of NCBI to design primers for the conserved region of the virus.

[0008] Preferably, in step S6, the biofilm parameters include: dry mass of the biomolecular film, mass of the coupling solvent, film thickness, and density.

[0009] In addition, a QCM and LSPR dual-technique fusion biological detection system is proposed, including: Measurement pool: a container for nucleic acid reaction and placement of dual technology fusion chips; Dual technology fusion chip: converts biological signals into photoelectric signals; Biological probe: binds to nucleic acid molecules and causes changes in sensor signals; QCM driver module: drives the chip to work.

[0010] Preferably, the measuring cell comprises an upper cover, a base and a temperature control system, wherein the upper cover is provided with a cavity for accommodating a dual-technology fusion chip, the top electrode of the quartz wafer in the upper cover is a ring-shaped gold electrode, and the bottom electrode is a gold electrode.

[0011] Preferably, the temperature control system uses MSP430 as the main control chip and adopts an incremental PID temperature control algorithm to achieve rapid and accurate temperature control with an error of ±0.1°C.

[0012] This product combines QCM and LSPR, and can be used to obtain multiple parameters such as dry mass of biomolecular membranes, coupling solvent mass, membrane thickness, density, etc. This is not only the biggest technical advantage of this product, but also the difference from existing biochips. LSPR technology uses the characteristic that nanoparticles are smaller than the wavelength of the incident light. When the frequency of the incident light is consistent with the plasma oscillation frequency, resonance occurs to form an LSPR spectrum, which is sensitive to changes in the surrounding environment; QCM technology uses piezoelectric properties to sense tiny changes in the surface nanogram mass level. The combination of the two technologies can complement each other to form a high-precision, fast-response, highly integrated, and miniaturized intelligent sensing detection system, which can be used in the early diagnosis of serious diseases such as tumors, cardiovascular and cerebrovascular diseases in the future, and has broad prospects in the POCT market. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 It is a detection flow chart of the present invention. DETAILED DESCRIPTION

[0015] Embodiment 1, as Figure 1 The QCM and LSPR dual technology fusion biological detection method of the present invention comprises the following steps: S1. Configure corresponding primers according to the test object; for most RNA viruses, primers can be designed by consulting literature or using the Primeblast function of NCBI to design primers for the conserved regions of the virus. For viruses with fast transmission speed and strong mutation ability such as the new coronavirus, the genome mutation rate of different virus strains should be analyzed, and the most conservative region should be selected to design primers. The length of the primers is preferably 20-50bp, and the TM values ​​of the front and back primers should be close. After the primers are designed, they can be delivered to the company for synthesis. In this case, the primer concentration is configured to 20μmol / L; S2. Fixing the nanocomposite probe on the surface of the piezoelectric quartz wafer of the measuring cell; S3. Turn on the control circuit to control the temperature control system of the measuring cell within the set temperature range for reaction; S4. PBS buffer is introduced into the measuring cell until the output frequency of the dual technology fusion chip of the measuring cell is stable, and this frequency is recorded as the reference frequency f1 when no load; S5. Pass the biological sample into the measurement pool, and let the biological sample and the primer react fully until the chip output frequency signal is stable, and record the frequency at this time as f2; S6. According to the two frequency changes (f1-f2) Hz, combined with the frequency change curve and LSPR spectrum collected by QCM, various parameters of the biofilm are obtained. Finally, the sample properties are judged according to the calculation results of the biofilm parameters. In this step, the LSPR spectrum is collected and analyzed by the spectral analysis software of the spectrophotometer; the QCM frequency signal acquisition system integrates the FPGA control module, receives the frequency of AD sampling measurement through FPGA, and finally sends it to the host computer for processing through the serial port of FPGA, which makes the operation simpler.

[0016] As a more specific implementation, in step S6, QCM can detect the total mass of the coupling including the solvent, and LSPR can measure the dry mass of the biomolecule. The two technologies of QCM and LSPR are combined to obtain the mass of the coupling solvent by subtracting the molecular dry mass from the total mass.

[0017] As a more specific implementation, in step S6, the biofilm parameters include: dry mass of the biomolecular film, mass of the coupling solvent, film thickness, and density. By obtaining the above parameters, the type of the biological sample to be detected can be determined.

[0018] Example 2, the QCM and LSPR dual technology fusion biological detection system of the present invention comprises: Measuring cell: a container for nucleic acid reaction and placement of dual-technology fusion chip. The measuring cell used in this example refers to the constant temperature measuring cell for QCM and LSPR dual-technology biomolecule detection proposed in application number: CN201811353335.6; Dual technology fusion chip: converts biological signals into photoelectric signals; the dual technology fusion chip refers to the QCM and LSPR biosensors with transparent electrodes described in CN201811353324.8. The improvement of the biosensor chip in this case is to replace the top electrode of the quartz wafer with a ring-shaped gold electrode. This new structure allows the middle area of ​​the ring electrode to be transparent, forming a single transmission and double absorption phenomenon of nanoparticles, thereby enhancing the LSPR spectral intensity and optical sensitivity, making the measurement value more accurate; Biological probe: binds to nucleic acid molecules and causes changes in sensor signals; the equipment can replace detection probes according to the actual detection needs of customers, detect different types of viruses, and provide customized services; QCM driving module: drives the chip to work; see application number: CN201811353335.6 for the QCM detection system in a constant temperature measurement cell for QCM and LSPR dual technology biomolecule detection.

[0019] The present invention has the following innovative features: (1) The first QCM / LSPR dual technology fusion solution The first nucleic acid detection based on QCM and LSPR composite technology, with improved detection sensitivity, reduced false negative rate and simple operation. The equipment uses a combination of QCM and LSPR technologies. In liquid, QCM can detect the total mass of coupling including solvent, but cannot measure the "dry mass" of actual biological molecules. LSPR can measure the "dry mass" of biological molecules, but requires different refractive indices between the adsorption layer and the solvent. Combining QCM and LSPR technologies, we can obtain the mass of the coupling solvent by subtracting the molecular dry mass from the total mass.

[0020] (2) Independently developed a stepless dual technology control device ①LSPR spectrum is collected and analyzed by the spectrum analysis software of the spectrophotometer; the frequency signal acquisition system integrates the FPGA control module, receives the frequency of AD sampling measurement through FPGA, and finally sends it to the host computer for processing through the serial port of FPGA, which makes the operation simpler; ② Nanoparticles are prepared in the middle of the ring-shaped gold electrode of the chip. This new structure allows the middle area of ​​the ring electrode to transmit light, forming a one-time transmission and two-time absorption phenomenon of the nanoparticles, thereby enhancing the LSPR spectrum intensity and optical sensitivity, making the measurement value more accurate; ③ The LSPR technology in the dual-technology chip provides a label-free, highly sensitive detection method; ④ Integrate multiple structural units on the same substrate, and the liquid capacity of the non-polar sample measurement pool is 300μL, which has the technical advantages of saving sample liquid, miniaturization and facilitating biomedical testing.

[0021] The detection equipment of the present invention selects nucleic acid detection of Norovirus, hand, foot and mouth disease virus and other nucleic acid detection as verification carriers for independent development and design of dual technology fusion chips. The equipment uses QCM technology and LSPR technology to establish a method for rapid detection of various antibodies or viruses at the same time. Nanoparticles are prepared on a quartz wafer and the team's independently developed Wuji QCM / LSPR dual technology fusion chip is used for sensitivity detection and false result analysis. 120 clinical serum sample detection comparisons were conducted with an accuracy rate of 98%, solving the pain points of the times that the characteristics of complex biological molecules cannot be fully obtained due to the single detection technology, and the biological process cannot be dynamically synchronized in real time due to the influence of external factors. It provides a portable detection device with high sensitivity and low false probability for modern virus detection, effectively filling the technical gap of rapid diagnosis of modern RNA virus nucleic acid detection.

[0022] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A QCM and LSPR dual technology fusion biological detection method, characterized in that: The steps include: S1. Configure corresponding primers according to the analyte; S2. Fixing the nanocomposite probe on the surface of the piezoelectric quartz wafer of the measuring cell; S3. Turn on the control circuit to control the temperature control system of the measuring cell within the set temperature range for reaction; S4. PBS buffer is introduced into the measuring cell until the output frequency of the dual technology fusion chip of the measuring cell is stable, and this frequency is recorded as the reference frequency f1 when no load; S5. Pass the biological sample into the measurement pool, and let the biological sample and the primer react fully until the chip output frequency signal is stable, and record the frequency at this time as f2; S6. Based on the two frequency changes (f1-f2) Hz, combined with the frequency change curve and LSPR spectrum collected by QCM, various parameters of the biofilm are obtained. Finally, the sample properties are judged based on the calculation results of the biofilm parameters.

2. The QCM and LSPR dual technology fusion biological detection method according to claim 1, characterized in that: In step S6, QCM can detect the total mass of the coupling including the solvent, and LSPR can measure the dry mass of the biomolecule. The two technologies of QCM and LSPR are combined to obtain the mass of the coupling solvent by subtracting the molecular dry mass from the total mass.

3. The QCM and LSPR dual technology fusion biological detection method according to claim 2, characterized in that: The LSPR spectrum is collected and analyzed by the spectrum analysis software of the spectrophotometer; the measurement cell includes a QCM frequency signal acquisition system, which integrates an FPGA control module, receives the frequency of AD sampling measurement through the FPGA, and finally sends it to the host computer for processing through the serial port of the FPGA.

4. The QCM and LSPR dual technology fusion biological detection method according to claim 3, characterized in that: In step S1, the length of the primers is 20-50 bp, the TM values ​​of the front and rear primers are close, and the configuration of the primers is performed by consulting literature or using the Primeblast function of NCBI to design primers for the conserved region of the virus.

5. The QCM and LSPR dual technology fusion biological detection method according to claim 4, characterized in that: In step S6, the biofilm parameters include: dry mass of the biomolecular film, mass of the coupling solvent, film thickness, and density.

6. A QCM and LSPR dual technology fusion biological detection system, characterized by: include: Measurement pool: a container for nucleic acid reaction and placement of dual technology fusion chips; Dual technology fusion chip: converts biological signals into photoelectric signals; Biological probe: binds to nucleic acid molecules and causes changes in sensor signals; QCM driver module: drives the chip to work.

7. The QCM and LSPR dual technology fusion biological detection system according to claim 6, characterized in that: The measuring cell comprises an upper cover, a base and a temperature control system, wherein the upper cover is provided with a cavity for accommodating a dual-technology fusion chip, the top electrode of the quartz wafer in the upper cover is a ring-shaped gold electrode, and the bottom electrode is a gold electrode.

8. The QCM and LSPR dual technology fusion biological detection system according to claim 7, characterized in that: The temperature control system uses MSP430 as the main control chip and adopts an incremental PID temperature control algorithm to achieve rapid and accurate temperature control, with a temperature control error of ±0.1°C.

Images