CfDNA rapid quantitative detection system and method

Abstract

The invention discloses a cfDNA rapid quantitative detection system, which comprises, along a light path direction, a light source used for providing incident light required by detection; light emitted by the light source passes through the first polarizer; the polarized light emitted by the first polarizer enters the birefringent crystal; the shuttle mirror is provided with an incident surface, a detection surface and an emergent surface, the detection surface is provided with a nanogold substrate and is used for reacting with a cfDNA detection sample, emergent light of the birefringent crystal enters the incident surface of the prism, is refracted to the detection surface for the first time, acts on the nanogold substrate, is refracted to the emergent surface of the prism for the second time, is refracted for the third time and then exits from the emergent surface; a second polarizer to which light emitted from the prism emission surface enters; a filter into which the light emitted from the second polarizer is incident; and the light passing through the filter reaches the detector, and the detector calculates and analyzes the received detection signal.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical diagnosis, and in particular relates to a cfDNA rapid quantitative detection system and method. Background Art

[0002] In the field of biomedical diagnosis, cell-free DNA (cfDNA) is a type of DNA fragment located outside cells in the peripheral circulating blood. It is very rare, often double-stranded, and has a short fragment size (160-180 bp) [2]. cfDNA can be produced by cell necrosis, apoptosis, or self-release. Therefore, it carries cell-specific genetic information. cfDNA can reflect disease-related characteristics and is one of the ideal biomarkers for "liquid biopsy."

[0003] Studies have found that the total cfDNA concentration in whole blood is about 2.8-8.70ng / ml. During the occurrence and development of diseases such as tumors, cfDNA in plasma and serum may change throughout the tumor progression and is an ideal marker for real-time detection of disease status. Therefore, detecting biomarkers at multiple time points will provide important information for patient management. Studies have shown that the half-life of cfDNA is about 16.3 minutes (4-30 minutes).

[0004] Further analysis of the plasma free metabolomic characteristics of nasopharyngeal carcinoma patients during radiotherapy showed that the biological half-life of cfDNA was 3.8 days (2.4-4.4 days). Tenna V et al. studied the metabolomic characteristics of cfDNA in the plasma of patients with colorectal cancer and bladder cancer and found that the expression level in the plasma of patients after complete tumor removal decreased after 14 days of follow-up.

[0005] Therefore, the characteristics of rapid clearance of cfDNA can be used to determine the sensitivity, short-term efficacy and prognosis of clinical chemoradiotherapy. Timely detection of changes in the amount of cfDNA can achieve rapid and convenient monitoring of disease progression. At present, some methods and technologies (RT-PCR, NGS, etc.) that can quickly, economically and non-invasively identify biomarkers at different time points during the disease process are used to detect biomarkers in plasma and serum. The most commonly used nucleic acid amplification method is polymerase chain reaction (PCR). In the real-time PCR system, the amplicon is monitored in real time during the PCR reaction, and the time (or number of thermal cycles) required to reach the threshold concentration is highly correlated with the initial copy number of the amplified target nucleic acid. Summary of the invention

[0006] One embodiment of the present invention is a photothermal-based plasmon RCA-cfDNA rapid quantitative detection system. The detection system includes, according to the direction of the light path,

[0007] LED wide-spectrum white light source, used to provide the incident light required for detection;

[0008] a first polarizer, through which light emitted by the light source passes;

[0009] a birefringent crystal, into which the polarized light emitted by the first polarizer is incident;

[0010] The shuttle mirror has an incident surface, a detection surface and an exit surface, and is used to change the propagation direction of light.

[0011] The detection surface is provided with a nano-gold substrate for reacting with the cfDNA detection sample.

[0012] The outgoing light of the birefringent crystal is incident on the incident surface of the prism, is refracted to the detection surface for the first time, acts on the nano-gold substrate, is refracted to the exit surface of the prism for the second time, and is refracted for the third time before exiting from the exit surface;

[0013] A second polarizer, the light emitted from the prism output surface is incident on the second polarizer;

[0014] a filter, the light emitted from the second polarizer being incident on the filter;

[0015] Detector, the light passing through the filter reaches the detector, and the detector calculates and analyzes the received detection signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and other objects, features and advantages of the exemplary embodiments of the present invention will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present invention are shown in an exemplary and non-limiting manner, in which:

[0017] Figure 1 A flowchart of a photothermal-based RCA amplification reaction according to one embodiment of the present invention.

[0018] Figure 2 Schematic diagram of an optical path detection system based on plasmon photothermal and LSPR according to one embodiment of the present invention.

[0019] Figure 3 Schematic diagram of the modification effects and signals of each component according to one embodiment of the present invention.

[0020] Figure 4 A schematic diagram showing results of an optical system and a gel system according to one embodiment of the present invention.

[0021] Figure 5 Schematic diagram of directly detecting the EGFR binding effect after blocking with different concentrations of MCH according to one embodiment of the present invention.

[0022] Figure 6Schematic diagram of directly detecting the RCA-EGFR binding effect after blocking with different concentrations of MCH according to one embodiment of the present invention.

[0023] Figure 7 Schematic diagram of the amplification effect of directly detecting EGFR-RCA using an LSPR system according to one embodiment of the present invention.

[0024] Figure 8 Schematic diagram of laser power and RCA reaction effects at different temperatures according to one embodiment of the present invention.

[0025] Fig. 9 A schematic diagram of EGFR detection results according to one embodiment of the present invention.

[0026] Fig.10 A schematic diagram of PTEN detection results according to one embodiment of the present invention.

[0027] Fig.11 A schematic diagram of CDKN2A detection results according to one embodiment of the present invention.

[0028] Fig.12 A schematic diagram of TP53 detection results according to one embodiment of the present invention.

[0029] Fig.13 An RCA reaction sequence information table according to one embodiment of the present invention. DETAILED DESCRIPTION

[0030] Currently, quantitative detection methods for cfDNA are mainly divided into two categories: nucleic acid amplification technology and sequencing technology. Among them, nucleic acid amplification technology can be divided into variable temperature amplification technology (PCR)[3] and isothermal amplification technology (LAMP, RCA, etc.)[4,5] according to its requirement for temperature changes.

[0031] Although PCR-based amplification technology is currently the gold standard for nucleic acid detection, it still shows certain limitations for the detection of small fragments of nucleic acid (cfDNA). For example, due to the short size of cfDNA fragments (160-180bp), primer design is very challenging; the temperature dependence of PCR detection places higher requirements on the precise control of instrument temperature. In addition, methods such as PCR and sequencing require a large number of advanced analytical equipment and well-trained labor, which limits their application in resource-limited areas. POCT has the advantages of low cost, small size, and simple operation, and has broad application prospects in resource-limited environments. cfDNA detection related to second-generation sequencing (NGS) also needs to be tested on the basis of nucleic acid amplification, and the test takes 1-2 days to complete. The results need to be processed and interpreted by professionals; the huge cost and huge data processing limit clinical application to a certain extent. Therefore, there is an urgent need for a sensitive detection method to achieve quantitative detection of low-concentration cfDNA. That is, for the detection of small nucleic acids such as cfDNA, ctDNA, and miRNA in liquid biopsy, it is urgent to develop highly sensitive and high-precision equipment to surpass the barriers of classical detection technology. The present invention uses laser photothermal effect to control the temperature of nucleic acid amplification and enhance detection sensitivity at the same time, thereby achieving highly sensitive quantitative detection of low-concentration cfDNA.

[0032] In a study of prostate cancer patients with benign prostatic hyperplasia (BPH), cfDNA levels were reported to be significantly higher than those in the control group. Studies have shown that the fragment length of ctDNA (60-145 bp) is shorter than the fragment length of cfDNA from non-tumor tissue (165-180 bp). Polymerase chain reaction (PCR) methods and sequencing technologies have been used to detect and quantify specific cfDNA associated with target cancers [7]. Studies have shown that the dynamic changes of HER2 in plasma cfDNA and the reduction of HER2 amplification detected in plasma after trastuzumab combined with chemotherapy are associated with improved response and overall survival [8]. Although PCR-based detection methods have shown the potential for quantitative detection in cfDNA detection, due to the low concentration of cfDNA (30-180 ng cfDNA / mL plasma), in order to achieve sensitive and accurate cancer diagnosis based on cfDNA, a sufficiently large amount of clinical samples (5-10 ml plasma) is required to achieve high-quality detection of cfDNA.

[0033] In addition, in real-time PCR instruments, fluorescence detection systems are used to monitor the amplification process. However, for the application of real-time PCR in small point-of-care testing devices, fluorescence detection systems are relatively bulky and expensive. ddPCR can detect as little as 0.01-1.0% of genomic material and can be used to identify potential rare mutations and calculate copy number variations [5,6]. The choice of cfDNA testing platform depends on the availability of a specific center, cost, and whether multiplexing is required in tumors with multiple targets. Next-generation sequencing (NGS) platforms can provide a wider range of targets for testing, but on the other hand, it can take a lot of time to validate, has less flexibility, and is more expensive.

[0034] On the other hand, from the advantages of the photothermal plasmon detection platform, the interaction between gold nanomaterials (diameter is about 1 to 100 nm) and light can produce localized surface plasmon resonance LSPR (Localized Surface Plasmon Resonance). When light is irradiated to the surface of metal nanostructures (such as gold, silver, etc.), it will stimulate the collective oscillation of free electrons on the surface of these materials, forming a localized surface plasmon resonance. This resonance effect will cause light to be strongly absorbed and scattered on the surface of the nanostructure, thereby changing the optical properties of the material. LSPR technology has a wide range of applications in chemical sensing, biological detection, photothermal therapy, and catalysis. Sensors based on LSPR can detect the interaction between metal surfaces and molecules to be measured without labels, in real time, and with high sensitivity, such as nanogold surfaces. The main method of microfluidic sensors of gold nanomaterials is to use optical prism coupling to induce LSPR effect on the surface of gold nanoparticle films for detection.

[0035] Sensors based on LSPR nanostructures are increasingly being used. Changes in the refractive index of the environment can be detected in real time using the wavelength or intensity of the LSPR resonance peak. Compared with sensors based on SPP, LSPR sensors can resonate directly with light, eliminating bulky optical coupling components and facilitating high integration. In addition, the wavelength and peak shape of the LSPR resonance peak can be adjusted by controlling parameters such as the size, shape, and distance of the metal nanostructure, making it easier to optimize the sensor. Although the bulk refractive index sensitivity of LSPR sensors (~100-1000nm RIU-1) is usually much smaller than that of SPP sensors (>3000nm RIU-1), due to the strong local electric field within a short distance on the metal surface, it can still have a high sensitivity to the molecular binding behavior close to the surface.

[0036] On the other hand, from the advantages of PT-RCA in detecting cfDNA, due to the characteristics of hot carriers generated by LSPR nanomaterials decaying through electron-electron scattering and converting their energy into heat energy, they are also widely used in photothermal catalytic reactions. In the LSPR-enhanced photothermal catalytic system, LSPR excitation generates a high-energy local electric field, which can further enhance the light absorption around the nanostructure. Light can also be used to excite the generation of high-energy carriers and thermal energy, which can be used to drive or enhance surface chemical reactions, and can achieve new reaction pathways and product selectivity that break through the limitations of traditional thermal catalysis. Therefore, LSPR-enhanced photothermal catalysis comes from the synergistic combination of three properties: hot carrier generation, local heating effect, and optical near-field enhancement. Therefore, hot carriers and thermal effects synergistically enhance the overall reaction rate, and the two mechanisms work to varying degrees, which is specifically related to the catalytic reaction and the properties of the catalyst itself.

[0037] Although the RCA reaction can be performed at room temperature, the deviation of daytime temperature will also have a great impact on the quantitative detection results. Therefore, controlling the uniformity of temperature is extremely important for the amplification of cfDNA. In addition, due to the low content of cfDNA, the target sequence was amplified by RCA in time, but due to the efficiency limitation of linear amplification, the ability of ordinary LSPR systems to detect targets is limited. Compared with ordinary LSPR-RCA detection systems, the PT-RCA system is a temperature-controlled, high-sensitivity detection system that integrates heating and LSPR.

[0038] If we review the existing detection methods, we can find that due to the low content of cfDNA in real body fluids (blood, urine, etc.), its quantitative detection is currently mainly achieved by methods based on digital droplet PCR or next-generation sequencing (NGS). However, methods such as NGS sequencing rely on repeated thermal cycles and usually require more than 2-14 days to read. The instrument-intensive and time-consuming process hinders the application of point-of-care testing (POC) [1].

[0039] Currently, the diagnosis of many diseases is done using quantitative polymerase chain reaction (qPCR), which requires highly trained laboratory personnel, many liquid handling steps, and expensive equipment such as thermal cyclers. Isothermal amplification offers an alternative to qPCR, allowing nucleic acid amplification to be performed at a single, constant temperature, eliminating the need for a thermal cycler. Common isothermal amplification methods include loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), and nucleic acid sequence-based amplification (NASBA). The currently used PCR method is time-consuming, requires a dedicated laboratory setup and expensive instrumentation, and requires dedicated rooms and professionals to perform nucleic acid amplification. In addition, digital PCR testing is expensive. To overcome these shortcomings, various isothermal nucleic acid amplification methods, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), hybridization chain reaction based on deoxyribonucleic acid (DNA) nanoscaffolds, and nucleic acid sequence-based amplification, have been developed to overcome the need for complex thermal cycling equipment associated with qRT-PCR.

[0040] Compared with quantitative polymerase chain reaction (qPCR), rolling circle amplification (RCA) is also a nucleic acid amplification technology used to amplify specific nucleic acid sequences for further detection and analysis. It is used in pathogen detection, gene expression analysis, genetic disease diagnosis and other fields. Unlike qPCR, RCA uses circular DNA as a template and performs isothermal amplification under the action of phi29 DNA polymerase to generate a large number of repeated DNA sequences. RCA can be performed at a constant temperature and does not require temperature cycling, so expensive thermal cyclers are not required. RCA has a fast amplification speed and can obtain a large amount of DNA products in a shorter period of time. It is suitable for scenarios such as on-site detection and rapid screening, especially in the absence of professional equipment. In addition, RCA can be detected by a variety of methods, including direct observation of the turbidity of the precipitate, fluorescence signals, etc.

[0041] RCA (rolling circle amplification) only needs one primer to amplify the target product. The reaction can be triggered when the primer and the circular template are present at the same time. Since phi 29 polymerase has a strong chain displacement function, when the primer rotates around the circular template, it will replace the primer that initially annealed to the circular template, and continuously generate dozens to hundreds of new tandem repeats of long ssDNA around the circular template until the activity of the DNA polymerase is exhausted. The amplification efficiency of linear RCA is about 10 5 The RCA technology has low requirements for instruments and testing conditions, which makes it have great application potential in POCT and rapid large-scale screening.

[0042] Compared with the traditional liquid-phase RCA detection method, solid-phase RCA reduces the washing steps and has more advantages in detecting low-concentration samples (such as cfDNA). In addition, liquid-phase RCA is mostly an endpoint detection method, which requires precise control of the detection time point, resulting in low detection repeatability. Some liquid-phase RCAs read the detection signal through colorimetry, which increases the error of the detection results. Solid-phase RCA can overcome these shortcomings by reading the reaction signal in real time.

[0043] In the liquid phase detection system, the gold nanoparticles used to generate photothermal energy need to maintain a relatively uniform density and distribution to maintain the temperature consistency of the entire reaction system. However, since the nucleic acid reaction process will produce the extension and spontaneous folding of the nucleic acid chain, it will have a certain degree of influence on the temperature uniformity.

[11] The solid-phase nanochip can maintain a consistent temperature while maintaining a uniform light spot.

[0044] When reading the results, for liquid phase detection, the nanoparticles will undergo changes in different scales, resulting in changes in color. Although this method can better achieve naked eye detection, it has limitations for more accurate quantitative detection. At the same time, there will be a washing process in liquid phase detection, which often produces errors in the liquid transfer process. Solid phase chip detection overcomes this problem by washing through continuous liquid flow. Compared with traditional LSPR sensing technology, the advantage of PT-LSPR detection is that the signal-to-noise ratio (sensitivity) of RCA amplification detection of cfDNA is improved through laser temperature control and detection of LSPR technology. PT-LSPR detection, namely Phase Thermal-Localized Surface Plasmon Resonance detection, is a biosensor technology based on the localized surface plasmon resonance (LSPR) effect. The LSPR optical effect refers to when light is irradiated on nanoparticles composed of precious metals, if the frequency of the incident photon matches the surface plasmon resonance frequency of the metal nanoparticles, strong light absorption will occur, resulting in a significant resonance absorption peak in the spectrum. The LSPR effect is very sensitive to changes in the refractive index of the surrounding medium and can therefore be used as a highly sensitive chemical and biological sensor.

[0045] In addition, compared with liquid-phase AuNPs particles, solid-phase AuNPs chips can regulate temperature more evenly.

[12] . Nanogold has a high light-to-energy conversion efficiency and the heat generated on the chip surface can largely maintain the rigid structure of the longer nucleic acid amplification chain and participate in the amplification reaction to a greater extent. The generation of LSPR signals on the nanogold chip is based on electron-to-electric conversion, and the LSPR generated by photothermal is a higher energy form. Therefore, photothermal also plays a role in signal amplification to a certain extent. When the gold nanostructure on this nanogold chip interacts with the incident light, the localized surface plasmon resonance phenomenon is generated, and the generated signal can be used to detect and analyze chemical or biological reactions occurring on the chip surface or in the nearby medium, because these reactions will cause changes in the LSPR resonance frequency.

[0046] If the photothermal conversion efficiency of different materials is considered, PPy@IOs, noble metal particles, and gold nanoparticles AuNPs have size- and shape-dependent optical properties.

[13] . SPR and localized SPR modes achieve strong light absorption when the collective oscillation of free charges resonates with the electric field of incident light. The absorbed photon energy can be efficiently converted into heat and dissipated into the surrounding medium. Therefore, plasmonic devices can act as optically driven heaters that can be remotely controlled.

[14] .

[0047] Localized surface plasmon resonance (LSPR) biosensing systems are suitable for different types of clinical analytes. Due to the enhanced plasmon field near the nanostructure, the LSPR sensing system shows high sensitivity to local changes, including refractive index changes and molecular binding. By analyzing local changes, real-time and label-free detection of micro-nanoscale analytes can be achieved.

[0048] According to one or more embodiments, a photothermal-based plasmon RCA-cfDNA rapid quantitative detection system is provided. Figure 1 As shown, the basic process of the detection method based on photothermal cyclic recombination amplification RCA amplification reaction. Specifically, it includes the following steps:

[0049] S101. Capture sequence binding. In this step, a specific capture sequence is used to bind to the target molecule. The capture sequence is a single-stranded DNA or RNA that is partially complementary to the target nucleic acid sequence. Its function is to fix the target molecule and lay the foundation for the next step of detection.

[0050] S102. MCH blocks non-specific sites. MCH is used as a blocking reagent to block non-specific binding sites on the gold nanoparticle substrate that are not involved in the reaction. This can reduce the occurrence of non-specific binding in subsequent reactions, thereby improving the specificity and accuracy of the detection.

[0051] S103. Target molecule detection. In this step, the target nucleic acid sequence (target molecule), here the DNA or RNA sequence, is detected, which is related to the disease marker or other biological molecule. Here, the presence of the target molecule can be detected by specific methods, such as fluorescent labeling or optical signal changes.

[0052] S104. RCA amplification. Once the target molecule is detected, the signal is amplified using RCA technology. RCA is a highly efficient nucleic acid amplification method that can be performed at a constant temperature and does not require complex thermal cycling equipment. During the RCA process, a circular template and a specific DNA polymerase (such as DNA polymerase) is used to synthesize long single-stranded DNAs that are complementary to the circular template to form tandem repeat sequences, thereby achieving massive replication of the target sequence.

[0053] Figure 1 In the context of plasma, "plasma" refers to the liquid part of the blood, which is mainly composed of water, electrolytes, proteins, hormones, nutrients, and waste products. It is one of the main components of blood, the rest of which include red blood cells, white blood cells, and platelets. Alternatively, it can refer to the blood-like fluid in some organisms, such as hemolymph in some invertebrates.

[0054] Therefore, the embodiments of the present disclosure Figure 1 The flowchart in shows a highly integrated detection system, which fixes the target molecule through a specific capture sequence, uses MCH to block nonspecific sites to improve the specificity of detection, and then amplifies the target molecule through RCA technology to enhance the signal and achieve high-sensitivity detection. The scheme of the disclosed embodiment is suitable for rapid and low-cost on-site detection, especially in resource-limited environments.

[0055] exist Figure 1 The article also includes the optical path diagram of the experimental measurement of the polarization absorption characteristics of gold nanoparticles in the photothermal-based plasmon RCA-cfDNA rapid quantitative detection device. The detection principle diagram of the detection optical path diagram is shown in the figure below. Figure 2 As shown, the English labels of the parts are Figure 2 There are corresponding Chinese labels in the . The detection device consists of the following components:

[0056] LED light source, used to provide incident light required for detection;

[0057] A first polarizer, through which the light emitted by the LED light source passes. The function of the polarizer is to allow only light waves vibrating in a specific direction to pass through, thereby generating polarized light;

[0058] A birefringent crystal, into which the polarized light then enters. This crystal can cause the light passing through it to undergo birefringence, i.e., the light has different propagation speeds in two perpendicular directions, resulting in different refractive indices of the light;

[0059] The prism has an incident surface, a detection surface and an exit surface, which are used to change the propagation direction of light or focus the light beam. The test sample containing the cfDNA to be detected is placed on the detection surface of the prism. The incident light passing through the birefringent crystal is incident on the incident surface of the prism, and after the first refraction to the detection surface, it acts on the test sample, and the test sample interacts with the light. After the second refraction to the exit surface of the prism, it is refracted for the third time and then emitted from the exit surface. A gold nano-microfluidic chip is provided on the detection surface of the prism, that is, a microfluidic flow channel is added to the AuNI chip, which is equivalent to a micro-reaction chamber, providing reaction space for the substance to be tested. After passing through the filter and the birefringent crystal, the LED light is irradiated onto the gold nanosheet placed on the prism, and plasmons occur. LSPR occurs on the AuNI sheet;

[0060] a second polarizer, on which the outgoing light emitted from the prism exit surface is incident;

[0061] A filter, into which the light emitted from the second polarizer is incident, and the filter is used to screen or block light of a specific wavelength for analysis or detection;

[0062] Detector,Finally, the light that passes through the filter reaches the detector, which converts the optical signal into an electrical signal for further analysis and measurement.

[0063] In the disclosed example, prism coupling, i.e., attenuated total internal reflection (ATR) method, is used to excite surface plasmons. This excitation method is composed of a high refractive index prism device, a metal film / nanoparticles, and a low refractive index dielectric layer.

[0064] Figure 2 This is the principle diagram of the optical path for measuring the polarization absorption characteristics of gold nanoparticles in this experiment. Since the light absorption spectrum of gold nanoparticles is wide, LED light source is selected here.

[0065] First, the polarizer adjusts the circularly polarized light to linearly polarized light, and then the birefringent crystal divides the light into incident light (p-polarized light) and reference light (s-polarized light). The incident light is incident on the prism at a certain angle. The light absorbed by the polarization coupling of the gold nanoparticles is basically p-polarized light, and s-polarized light is basically not absorbed. The wide-spectrum white light source adjusts the optical path difference between the s-polarization state and the p-polarization state through the birefringent crystal, constructs a phase-modulated common optical path interference carrier signal, and after being refracted by the prism, it is obliquely incident on the microfluidic sensing interface located on the detection surface of the prism.

[0066] The ATR incident angle is adjusted within the range of 600-800 incident angles to achieve the position adjustment of the LSPR resonance wavelength λLSPR in the white light spectrum. The wide-spectrum white light source adjusts the optical path difference between the s-polarization state and the p-polarization state through a birefringent crystal, constructs a phase-modulated common optical path interference carrier signal, and obliquely incidents to the microfluidic sensing interface after being refracted by a prism. The refractive index resolution (RIR) of different ATR incident angles is calculated by detecting the refractive index of the sample with detection. ATR is the abbreviation of "Attenuated Total Reflection", which is an optical phenomenon. When light is incident from a high refractive index medium to a low refractive index medium, if the incident angle is greater than the critical angle, the light will be totally reflected at the interface. Under the condition of total reflection, the light wave will form an electromagnetic field called "attenuated wave" or "evanescent wave" near the interface, and its intensity decays rapidly with the increase of the distance from the interface. In LSPR (localized surface plasmon resonance) sensors, ATR technology is used to enhance the LSPR signal because the evanescent wave generated by ATR can interact with metal nanostructures at the nanoscale, thereby exciting the LSPR mode. By precisely controlling the incident angle of ATR, the intensity and distribution of the evanescent wave can be adjusted, thereby optimizing the performance of the LSPR sensor.

[0067] Refractive Index Resolution (RIR) refers to the ability of a measuring device or method to distinguish the smallest difference between two materials with different refractive indices. In sensors based on localized surface plasmon resonance (LSPR), RIR is an important parameter that determines the smallest refractive index change that the sensor can detect, which in turn affects the sensitivity and detection limit of the sensor.

[0068] LSPR sensors typically use the local enhancement effect of incident light on metal nanostructures (such as gold or silver nanoparticles) to detect changes in refractive index. When the refractive index of the medium surrounding the nanostructure changes, the resonant frequency of the LSPR will also change, and this change can be observed by measuring the extinction spectrum of the nanostructure. Therefore, RIR is usually related to the spectral resolution of the LSPR sensor. The higher the spectral resolution, the higher the RIR, which means that the sensor can detect smaller refractive index changes.

[0069] In addition to using the LSPR principle, PT-LSPR detection also combines the phase thermo-optic effect. PT refers to Photothermal, which means that the metal nanostructure absorbs light energy and converts it into heat energy, causing thermal expansion or refractive index changes, which in turn affects the propagation phase of light. PT-LSPR detection measures the phase change of light, which usually requires an interferometer or other instrument that can detect phase changes, such as a Mach-Zehnder interferometer or a fiber interferometer, to accurately measure the phase change.

[0070] In the PT-LSPR-RCA detection system, the 532nm laser beam after spot homogenization is incident on the LSPR microfluidic chip in a normal direction and interacts with the gold nanoisland (AuNI) nanoparticles through the PDMS chamber to generate localized photothermal effects. 2 The energy density of the homogenized spot laser is adjusted within a range to achieve temperature regulation of the plasmon photothermal effect; and the signal-to-noise ratio of the LSPR sensing signal is optimized, the rules and characteristics of reducing the interference of dual-path optical signals are analyzed, and the stability and signal-to-noise ratio of the signal in photothermal biosensing are improved. That is, in the disclosed embodiment, the PT-LSPR-RCA abbreviation combination proposed for the first time refers to the RCA detection method and system based on photothermal-mediated LSPR sensing, which integrates the three technologies of photothermal PT, localized surface plasmon resonance sensing LSPR, and rolling ring amplification RCA, and becomes the technical core component of the disclosed embodiment. Here, the gold nanosheets and PDSM are bonded together by Plasma / ionization bonding, that is, a closed microfluidic chamber formed by bonding the upper and lower layers.

[0071] For quantitative detection, the stability of the system and the determination of the minimum detection limit are extremely important for the detection system. In order to accurately control the temperature and improve the detection sensitivity, the present invention establishes a photothermal-based RCA amplification system to detect trace cfDNA contained in body fluids. Traditional gold nanoparticle (AuNPs) chips have relatively low sensitivity (~nM) for nucleic acid detection, and most isothermal amplification analyses require fluorescent well labeling and expensive reagents. For example, some studies have used methods such as fluorescent labeling to amplify signals to achieve detection of trace nucleic acids. Compared with these labeled detection techniques, the detection system disclosed in the present invention can achieve highly sensitive quantitative detection without the introduction of other markers.

[0072] According to one or more embodiments, a photothermal-based plasmon RCA-cfDNA rapid quantitative detection method is firstly prepared for experimental materials, including: cDNA (capture, 100 μM), TCEP, MCH, 75% ethanol, EGFR template single strand (10 nM), RCA-circular template (circular template), dNTPs, phi29 DNA polymerase, phi29 DNA polymerase buffer, albumin, ddH2O, PBS, nanogold substrate.

[0073] Secondly, configure the experimental materials, including the following steps:

[0074] ① Take 10μL capture (PBS dilution, 1:9), 5μL TCEP and 90μL PBS into a 1.5ml EP tube and react at room temperature for at least 30 minutes.

[0075] ② Take 990μL 75% ethanol into a 1.5ml EP tube and add 10μL MCH to mix.

[0076] ③ Heat the prepared EGFR template single-strand of each concentration or 50 μL of extracted patient cfDNA in a 90°C metal bath for 3 minutes, and then immediately place in an ice bath to maintain the rigid structure of the nucleic acid chain to be detected.

[0077] ④ Add 20μL RCA-circular template, 5μL dNTPs, 15μL ddH2O, and 5μL phi29 DNA polymerase buffer into a 1.5ml EP tube, heat in a 90℃ metal bath for 3 minutes and immediately put in an ice bath. After cooling, add 2.5μL phi29 DNA polymerase and 1μL albumin.

[0078] Then comes the measurement operation process, including:

[0079] S201, install the nanogold substrate, adjust the optical path, collect data for about 30 minutes (adjust the time according to the spectral stability), and process the data to see if the baseline is stable. After reaching steady state, re-collect data (50μL / min), wait for a period of baseline data, and then flow in the capture processed in step ① above (10μL / min—300ul) to let the capture react with the nanogold substrate for about 30 minutes, and rinse the reaction chamber with PBS (about 30min). After the experiment, process the data to see whether the capture has been combined with the nanogold substrate and whether the baselines before and after are stable.

[0080] S202, after the baseline is stable, reopen a file and start the MCH blocking experiment. Adjust the light path to a slightly insensitive state, collect baseline data for a period of time (50μL / min), and flow the working solution configured in step ② (20μL / min). React for 30 minutes and rinse the reaction chamber with PBS. Process the data after the experiment.

[0081] S203, after the baseline is stable, reopen a file and start EGFR detection. After reaching steady state, re-collect data (50μL / min), then flow the working solution treated in step ③ (10μL / min-300ul) to let EGFR react with capture for 30 minutes, and rinse the reaction chamber with PBS (about 30min). After the test, process the data to see if EGFR has been rinsed to a stable state.

[0082] S204, turn on the laser that irradiates the AuNI chip vertically, with an optical power density of 174 mW / cm 2 ), wait for the baseline to be stable. If it is stable, you can collect data first and process it or see if the spectrum changes little. After reaching a steady state, re-collect data (50μL / min), and then flow in the working solution treated in step ④. After the working solution sample flows in, stop the working solution delivery pump, clamp the pipeline with a clamp, and perform RCA reaction for 1.5 hours to form a stable self-assembled monolayer on the metal surface to improve the adsorption performance of the sample or perform specific surface modification.

[0083] The entire detection process is carried out on a solid-state microfluidic chip, including the capture of cDNA sequences through Au-S bond binding (the chip is a solid-state chip of AuNI), the binding of cDNA to cfDNA, and the subsequent RCA amplification of cfDNA.

[0084] In the above detection process, through the spectrometer, each step of the detection signal can be monitored in real time by avantes software. One data is recorded every 2 seconds, and the signal of each detection is saved to a folder, and the signal can be processed by Matlab. In the experiment, a real-time signal reading method is used to effectively sense the binding behavior of biomolecules in the photothermal field. The interference signal is calculated by the window Fourier transform method (WFT) to obtain the phase change information of the plasmon at the sensing interface, and realize high-sensitivity refractive index and biomolecule affinity sensing. In the analysis and calculation, the data with the strongest reflection in the processed spectral signal is selected for analysis. For example, the following data and results can be obtained.

[0085] ①Nucleic acid sequence of RCA reaction.

[0086] Table 1 RCA reaction sequence information Fig.13 The terms involved in Table 1 include:

[0087] Linear target sequence (Padlock): A long chain involved in the synthesis of the RCA circular template. After combining with PLP to form a double strand, it is linked by T4 DNA ligase to form a circular structure.

[0088] Patch Probe (PLP): Patch chain that links Padlock and is complementary to the Padlock chain.

[0089] Capture sequence: A capture sequence that is partially complementary to EGFR.

[0090] Detection target sequence (EGFR): Template of the nucleic acid sequence to be detected.

[0091] In the function description column, "Padlock and Target sequence amplification template" refers to "padlock and target sequence amplification template"; "Capture" refers to "capture"; "Quantitative determination" refers to "quantitative analysis"; "Target sequence amplification template" refers to "target sequence amplification template"; "Target sequence cyclization helper" refers to "target sequence cyclization helper".

[0092] ②Verification of the amplification effect of RCA reaction.

[0093] ③Detection of the combination effect of each component.

[0094] like Figure 3 As shown, the modification effects and signals of each component demonstrate the changes in time series of different modification and amplification technologies, which are used to evaluate the efficiency and stability of different modification or amplification technologies over time.

[0095] 1. Capturing sequence modifications. The figure shows how the modification level changes over time. From 500 seconds to 3000 seconds, the modification level changes between 2.5 and 0.2.

[0096] 2. MCH modification. The figure shows the measured values ​​of MCH modification levels at different time points. From 500 seconds to 3000 seconds, the modification levels vary between 2.6 and 0.2.

[0097] 3. EGFR capture. The figure shows the change of EGFR capture level over time. From 500 seconds to 3000 seconds, the capture level varies between 0.6 and 0.05.

[0098] 4. RCA amplification. The figure shows the change of RCA amplification level over time. From 500 seconds to 3000 seconds, the amplification level varies between 2.0 and 0.05.

[0099] Figure 3 The ordinate may represent the relative level of modification or amplification, while the abscissa represents time in seconds.

[0100] Figure 4 The results of the optical system and gel system in the disclosed embodiment show that the reaction can only be initiated when all RCA components are present. In the cfDNA-EGFR detection process, the change in the detection signal indicates that the object to be detected has successfully bound.

[0101] ④ The disclosed embodiments explore the blocking effect of MCH at different concentrations. Figure 5 Schematic diagram of directly detecting the EGFR binding effect after blocking with different concentrations of MCH. Figure 6 This is a schematic diagram of directly detecting the RCA-EGFR binding effect after blocking with different concentrations of MCH. The results show that the best blocking effect is achieved when MCH is added to the reaction system and diluted 1:1000. This concentration can be selected for subsequent experiments.

[0102] ⑤ The EGFR-RCA detection effects without photothermal and with photothermal in the disclosed embodiments are as follows: Figure 7 shown.

[0103] In the present disclosure, when the LSPR system was tried to directly detect the amplification effect of EGFR-RCA, it was found that the detection signal was low, so further attempts were made to use laser irradiation to generate local photothermal effects, which greatly improved the detection signal. Subsequent experimental tests need to further analyze the detection signal of EGFR-RCA to achieve the detection of low-concentration EGFR-cfDNA in plasma.

[0104] ⑥ The RCA reaction effect of laser power and different temperatures in the embodiments of the present disclosure. Figure 8 As shown in the figure, the photothermal reaction effect of LSPR-PT was verified by exploring the different effects of EGFR-RCA at different photothermal temperatures. Here, the light power density was selected to be 174mW / cm 2 Experiment with laser.

[0105] ⑦ The EGFR / TP53 / CDKN2A / PTEN direct detection results and RCA detection results (detection concentration curve and standard quantitative curve) of the disclosed embodiment are as follows. Clinically, EGFR detection, PTEN detection, CDKN2A detection, and TP53 detection are all tumor-related biomarker detection.

[0106] Epidermal Growth Factor Receptor (EGFR) is an important cell surface receptor whose mutation is closely related to the occurrence and development of various solid tumors such as non-small cell lung cancer (NSCLC). EGFR detection is often used to guide the use of targeted therapeutic drugs, such as EGFR tyrosine kinase inhibitors.

[0107] PTEN is a tumor suppressor protein whose loss-of-function mutations are commonly found in a variety of cancers, including breast cancer, thyroid cancer, etc. PTEN testing helps understand the molecular characteristics of the tumor and may indicate the use of specific targeted therapies.

[0108] The CDKN2A gene encodes p16 protein, a cell cycle inhibitory protein whose loss-of-function mutations are associated with a variety of cancers. CDKN2A testing helps identify high-risk types of tumors, and immunohistochemical testing of p16 can serve as a surrogate marker for HPV infection.

[0109] The TP53 gene encodes the p53 protein, a key tumor suppressor protein. TP53 mutation is one of the most common gene mutations in cancer and is associated with a variety of biological behaviors of tumors. TP53 testing helps assess the malignancy and prognosis of tumors.

[0110] The LSPR-PT system of the disclosed embodiment can enhance the signal of specific biological molecules to improve the detection performance of various biological molecules including EGFR, PTEN, CDKN2A, and TP53. To verify its effect, the disclosed embodiment conducts the following detection experiments.

[0111] Fig. 9 This is the EGFR detection result according to the embodiment of the present disclosure. The test detected EGFR-cfDNA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, and 10pM respectively through the LSPR-PT system, with a quantitative detection limit of 12.87pM and a minimum detection limit of 45.86pM. By fitting the concentration of EGFR-cfDNA with the detection results of the LSPR-PT system, a standard curve for EGFR-cfDNA detection was drawn.

[0112] The LSPR-PT system was used to detect EGFR-RCA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, 10pM, 1pM and 500fM, and a standard curve for EGFR-RCA detection was drawn. The ability of the LSPR-PT system to detect EGFR-RCA was significantly enhanced, with a quantitative detection limit of 0.05pM and a minimum detection limit of 0.024pM. Compared with direct binding detection, the detection sensitivity of the detection system with added RCA amplification was greatly improved, and the LOD and LOQ were increased by 3 orders of magnitude.

[0113] Fig.10 This is the PTEN detection result according to the embodiment of the present disclosure. The detection detected PTEN-cfDNA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, and 10pM respectively through the LSPR-PT system, with a quantitative detection limit of 5.02pM and a minimum detection limit of 13.92pM. By fitting the concentration of PTEN-cfDNA with the detection results of the LSPR-PT system, a standard curve for PTEN-cfDNA detection was drawn.

[0114] The LSPR-PT system was used to detect PTEN-RCA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, 10pM, 1pM and 500fM, and a standard curve for PTEN-RCA detection was drawn. The ability of the PTEN-PT system to detect PTEN-RCA was significantly enhanced, with a quantitative detection limit of 0.739pM and a minimum detection limit of 0.044pM.

[0115] Fig.11Schematic diagram of the CDKN2A detection results according to the embodiment of the present disclosure. The detection detected CDKN2A-cfDNA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, and 10pM respectively through the LSPR-PT system, with a quantitative detection limit of 1.73pM and a minimum detection limit of 6.08pM. The standard curve of CDKN2A-cfDNA detection was drawn by fitting the concentration of CDKN2A-cfDNA with the detection results of the LSPR-PT system.

[0116] The LSPR-PT system was used to detect CDKN2A-RCA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, 10pM, 1pM and 500fM, and a standard curve for CDKN2A-RCA detection was drawn. The ability of the CDKN2A-PT system to detect CDKN2A-RCA was significantly enhanced, with a quantitative detection limit of 0.145pM and a minimum detection limit of 0.006pM.

[0117] Fig.12 Schematic diagram of TP53 detection results according to the embodiment of the present disclosure. The detection was performed by the LSPR-PT system to detect TP53-cfDNA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, and 10pM, respectively. The quantitative detection limit was 11.54pM, and the minimum detection limit could reach 6.04pM. The standard curve of TP53-cfDNA detection was drawn by fitting the concentration of TP53-cfDNA with the detection results of the LSPR-PT system.

[0118] The LSPR-PT system was used to detect TP53-RCA at concentrations of 10nM, 1nM, 500pM, 100pM, 50pM, 10pM, 1pM and 500fM, and a standard curve for TP53-RCA detection was drawn. It can be seen that the ability of the TP53-PT system to detect TP53-RCA is significantly enhanced, with a quantitative detection limit of 0.298pM and a minimum detection limit of 0.036pM.

[0119] In summary, the present invention aims at the current photothermal-regulated nucleic acid detection which only focuses on the in situ cellular level (and cannot perform quantitative detection, only qualitative detection), and realizes a rapid quantitative detection RCA method and system for specimens such as body fluids. It is a rapid amplification detection system that integrates detection and temperature control.

[0120] This method is simple and fast, which can better meet the requirements of POCT and reduce the laboratory environment requirements for testing. POCT is the abbreviation of "Point of Care Testing", which means "bedside testing" or "on-site rapid testing". It allows rapid diagnostic testing at the patient's care point (such as the patient's bedside, emergency site, doctor's office or community health center) without the need to send the sample to a central laboratory for analysis.

[0121] The cfDNA microfluidic detection system in the disclosed embodiment integrates photothermal PT, localized surface plasmon resonance sensing LSPR, and rolling circle amplification RCA, and realizes amplification at the solid phase interface through RCA amplification of the solid phase chip. The solid phase interface is also a photothermal-mediated optical temperature control interface and a real-time detection interface, thereby greatly improving the detection sensitivity. Figure 7 At the same time,

[0122] Since the detection is performed on a solid-phase chip, the washing and other transfer processes are reduced, and low-concentration detection markers can be better detected. At the same time, the RCA amplification reaction can amplify the detection reaction signal, without the need for multiple primers or reactants to react, the reaction system is more concise, and the design process of designing the detection target is reduced.

[0123] The public documents involved in this disclosure are as follows.

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[0127] 4.Huang T,Gibbons C,Rashid S,Priston MK,Bedford HM,Mak-Tam E,MeschinoWS.Prenatal screening for trisomy 21:a comparative performance and costanalysis of different screening strategies.BMC Pregnancy Childbirth 2020;20(1):71310.1186 / s12884-020-03394-w.

[0128] 5.Hu F,Li J,Zhang Z,Li M,Zhao S,Li Z,Peng N.Smartphone-Based DropletDigital LAMP Device with Rapid Nucleic Acid Isolation for Highly SensitivePoint-of-Care Detection.Anal Chem 2020;92(2):2258-226510.1021 / acs.analchem.9b04967.

[0129] 6.Oellerich M,Schutz E,Beck J,Kanzow P,Plowman PN,Weiss GJ,WalsonPD.Using circulating cell-free DNA to monitor personalized cancertherapy.Crit Rev Clin Lab Sci 2017;54(3):205-21810.1080 / 10408363.2017.1299683.

[0130] 7.Ponti G,Maccaferri M,Percesepe A,Tomasi A,Ozben T.Liquid biopsywith cell free DNA:new horizons for prostate cancer.Crit Rev Clin Lab Sci2021;58(1):60-7610.1080 / 10408363.2020.1803789.

[0131] 8.Shoda K,Masuda K,Ichikawa D,Arita T,Miyakami Y,Watanabe M,KonishiH,Imoto I,Otsuji E.HER2 amplification detected in the circulating DNA ofpatients with gastric cancer:a retrospective pilot study.Gastric Cancer 2015;18(4):698-71010.1007 / s10120-014-0432-5.

[0132] 9.Lapin M,Oltedal S,Tjensvoll K,Buhl T,Smaaland R,Garresori H,JavleM,Glenjen NI,Abelseth BK,Gilje B,Nordgard O.Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreaticcancer.J Transl Med 2018;16(1):30010.1186 / s12967-018-1677-2.

[0133] 10.Ahmed HB,El-Hawary NS,Emam HE.Self-assembled AuNPs for ingrainpigmentation of silk fabrics with antibacterial potency.Int J Biol Macromol2017;105(Pt 1):720-72910.1016 / j.ijbiomac.2017.07.096.

[0134] 11.Moitra P,Alafeef M,Dighe K,Frieman MB,Pan D.Selective Naked-EyeDetection of SARS-CoV-2 Mediated by N Gene Targeted Antisense OligonucleotideCapped Plasmonic Nanoparticles.ACS Nano 2020;14(6):7617-762710.1021 / acsnano.0c03822.12.Chen CY,Yang HW,Hsieh PH,Hsieh CH,Wu MH.Development of aphotothermal bead-based nucleic acid amplification test(pbbNAAT)technique fora high-performance loop-mediated isothermal amplification(LAMP)-based point-of-care test (POCT).Biosens Bioelectron 2022;215:11457410.1016 / j.bios.2022.114574.

[0135] 13.Yang Y,Zheng X,Chen L,Gong X,Yang H,Duan X,Zhu Y.MultifunctionalGold Nanoparticles in Cancer Diagnosis and Treatment.Int J Nanomedicine 2022;17:2041-206710.2147 / IJN.S355142.

[0136] 14.Monshat H,Wu Z,Pang J,Zhang Q,Lu M.Integration of plasmonicheating and on-chip temperature sensor for nucleic acid amplificationassays.J Biophotonics 2020;13(7):e20200006010.1002 / jbio.202000060.

[0137] 15. Cakir O, Bakhshpour M, Yilmaz F, Baysal Z. Novel QCM and SPR sensors based on molecular imprinting for highly sensitive and selective detection of 2,4-dichlorophenoxyacetic acid in apple samples. Mater Sci Eng C Mater BiolAppl 2019;102:483-49110.1016 / j.msec.2019.04.056.

[0138] It is worth noting that, although the foregoing content has described the spirit and principle of the invention with reference to several specific embodiments, it should be understood that the invention is not limited to the disclosed specific embodiments, and the division of various aspects does not mean that the features in these aspects cannot be combined, and such division is only for the convenience of expression. The invention is intended to cover various modifications and equivalent arrangements included in the spirit and scope of the appended claims.

Claims

1. A cfDNA rapid quantitative detection system, characterized in that: The system includes, according to the direction of the optical path, A light source, used to provide incident light required for detection; a first polarizer, through which light emitted by the light source passes; a birefringent crystal, into which the polarized light emitted by the first polarizer is incident; The shuttle mirror has an incident surface, a detection surface and an exit surface, and is used to change the propagation direction of light. The detection surface is provided with a nano-gold substrate for reacting with the cfDNA detection sample. The outgoing light of the birefringent crystal is incident on the incident surface of the prism, is refracted to the detection surface for the first time, acts on the nano-gold substrate, is refracted to the exit surface of the prism for the second time, and is refracted for the third time before exiting from the exit surface; A second polarizer, the light emitted from the prism output surface is incident on the second polarizer; a filter, the light emitted from the second polarizer being incident on the filter; Detector, the light passing through the filter reaches the detector, and the detector calculates and analyzes the received detection signal.

2. The detection system according to claim 1, characterized in that: The prism detection surface is provided with a nano-gold microfluidic chip.

3. The detection system according to claim 1, characterized in that: The light source is an LED broad-spectrum white light source.

4. The detection system according to claim 1, characterized in that: The birefringent crystal separates incident light into p-polarized light as prism incident light and s-polarized light as reference light.

5. The detection system according to claim 4, characterized in that: The light source adjusts the optical path difference of the polarization state of the s-polarized light and the polarization state of the p-polarized light through the birefringent crystal to construct a phase-modulated common optical path interference carrier signal, which is refracted by the prism and incident on the microfluidic chip sensing interface located on the prism detection surface.

6. The detection system according to claim 5, characterized in that: The microfluidic chip is a solid phase microfluidic chip.

7. The detection system according to claim 5, characterized in that: The nano-gold substrate of the microfluidic chip is AuNI.

8. The detection system according to claim 7, characterized in that: The first laser is incident on the microfluidic AuNI sensor chip in a normal direction, and interacts with the AuNI nanoparticles on the nanogold substrate through the PDMS chamber of the microfluidic chip to generate a localized photothermal effect.

9. The detection system according to claim 1, characterized in that: The detection steps of the detection system for the detection sample include: capturing sequence binding to fix the target molecule, using MCH to block non-specific sites to improve the specificity of the detection, detecting the target molecule, and amplifying the target molecule through RCA to enhance the signal and achieve high-sensitivity detection.

10. The detection system according to claim 1, characterized in that: The detector includes spectral analysis electronics.

11. A method for rapid quantitative detection of cfDNA, characterized in that: The detection method is based on any detection system according to claims 1 to 10, and the detection method comprises: Install the nano-gold substrate on the prism detection surface of the detection system and adjust the optical path. The test sample solution flows in to react with the nano-gold substrate, and then the reaction chamber is rinsed with PBS to obtain the test data.

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