Fast quantitative detection system and method for cfDNA

Abstract

The application discloses a cfDNA rapid quantitative detection system, which comprises a light source for providing incident light required for detection, a first polarizer through which light emitted by the light source passes, a birefringent crystal through which polarized light emitted by the first polarizer passes, and a prism having an incident surface, a detection surface and an emission surface, wherein the detection surface is provided with a nano-gold substrate for reaction with a cfDNA detection sample, and the emission surface is provided with a second polarizer, a filter and a detector.

Description

Technical Field

[0001] This invention belongs to the field of biomedical diagnostic technology, and specifically relates to a rapid quantitative detection system and method for cfDNA. Background Technology

[0002] In the field of biomedical diagnostics, cell-free cellular DNA (cfDNA) is a DNA fragment located in the peripheral blood circulation outside the cells. Its content is very rare, it is usually double-stranded, and the fragment is relatively short (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 the relevant characteristics of diseases and is one of the ideal biomarkers for "liquid biopsy".

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

[0004] Further analysis of plasma free metabolic kinetics in nasopharyngeal carcinoma patients during radiotherapy revealed that the biological half-life of cfDNA was 3.8 days (range 2.4–4.4 days). Tenna V et al.'s study on the plasma cfDNA metabolic kinetics of colorectal and bladder cancer patients found that the expression level in plasma decreased after 14 days of follow-up following complete surgical tumor resection.

[0005] Therefore, the rapid clearance of cfDNA can be used to assess the sensitivity, short-term efficacy, and prognosis of clinical radiotherapy and chemotherapy. Timely detection of changes in cfDNA levels enables rapid and convenient monitoring of disease progression. Currently, several methods and techniques (RT-PCR, NGS, etc.) for the rapid, economical, and non-invasive identification of 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 real-time PCR systems, the amplicons are 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 rapid quantitative detection system for plasmon resonance-based RCA-cfDNA (PRC-cfDNA) using photothermal methods. This detection system includes, according to the optical path direction,...

[0007] A broadband white LED light source is used to provide the incident light required for detection.

[0008] The first polarizer is through which the light emitted from the light source passes;

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

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

[0011] The detection surface is equipped with a gold nanoparticle substrate for reaction with the cfDNA detection sample.

[0012] The light emitted from the birefringent crystal is incident on the incident surface of the prism, refracted for the first time to the detection surface, acts on the gold nanoparticle substrate, refracted for the second time to the exit surface of the prism, and then refracted for the third time before exiting from the exit surface.

[0013] The second polarizer is used to receive light emitted from the exit surface of the prism.

[0014] The filter is the source of light emitted from the second polarizer.

[0015] The detector receives filtered light, which then performs calculations and analyses on the received detection signal. Attached Figure Description

[0016] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of the invention are illustrated in the drawings by way of example and not limitation, wherein:

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

[0018] Figure 2 A 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 A schematic diagram illustrating the modification effects and signals of each component according to one embodiment of the present invention.

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

[0021] Figure 5 A schematic diagram illustrating the direct detection of EGFR binding effect after blocking with different concentrations of MCH according to one embodiment of the present invention.

[0022] Figure 6A schematic diagram illustrating the direct detection of RCA-EGFR binding effect after blocking with different concentrations of MCH according to one embodiment of the present invention.

[0023] Figure 7 A schematic diagram illustrating the amplification effect of the LSPR system for direct detection of EGFR-RCA according to one embodiment of the present invention.

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

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

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

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

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

[0029] Figure 13 An RCA reaction sequence information table according to one embodiment of the present invention. Detailed Implementation

[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 further divided into temperature-dependent amplification technology (PCR)[3] and isothermal amplification technology (LAMP, RCA, etc.)[4,5] based on the temperature change requirements of nucleic acid amplification technology.

[0031] Although PCR-based amplification technology is currently the gold standard for nucleic acid detection, it still exhibits certain limitations for the detection of small nucleic acid fragments (cfDNA). For example, the short length of cfDNA fragments (160-180 bp) makes primer design very challenging; the temperature dependence of PCR detection places higher demands on the precise control of instrument temperature. Furthermore, PCR and sequencing methods require a large amount of advanced analytical equipment and a highly trained workforce, limiting their application in resource-constrained areas. Point-of-care testing (POCT) has advantages such as low cost, small size, and simple operation, and has broad application prospects in resource-limited environments. Next-generation sequencing (NGS)-related cfDNA detection also requires detection after nucleic acid amplification, and the detection takes 1-2 days to complete. Results require professional processing and interpretation; the high cost and massive data processing limit its clinical application. Therefore, there is an urgent need for a sensitive detection method to achieve quantitative detection of low-concentration cfDNA. Specifically, for the detection of small nucleic acids such as cfDNA, ctDNA, and miRNA in liquid biopsies, there is an urgent need to develop highly sensitive and accurate equipment to overcome the barriers of classic detection technologies. This disclosure utilizes the laser photothermal effect to control the nucleic acid amplification temperature while enhancing detection sensitivity, 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 in the control group. Studies have shown that ctDNA fragment lengths (60–145 bp) are shorter than those of cfDNA fragment lengths from non-tumor tissues (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 changes in plasma cfDNA HER2 dynamics are associated with reduced HER2 amplification detected in plasma after trastuzumab combined with chemotherapy and improved response and overall survival [8]. Although PCR-based detection methods have shown potential for quantitative detection of cfDNA, high-quality detection of cfDNA requires a sufficiently large number of clinical samples (5–10 ml plasma) to achieve sensitive and accurate cancer diagnosis based on cfDNA due to the low concentration of cfDNA (30–180 ng cfDNA / mL plasma).

[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 low 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 detection platform depends on the availability of a particular 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, they may require significant time for validation, are less flexible, and are more expensive.

[0034] On the other hand, considering the advantages of photothermal plasmon resonance (SPR) detection platforms, the interaction between gold nanomaterials (approximately 1–100 nm in diameter) and light can generate localized surface plasmon resonance (LSPR). When light irradiates the surface of metal nanostructures (such as gold and silver), it excites collective oscillations of free electrons on the surface of these materials, forming localized surface plasmon resonance. This resonance effect leads to strong absorption and scattering of light on the nanostructure surface, thereby altering the optical properties of the material. LSPR technology has wide applications in chemical sensing, biosensing, photothermal therapy, and catalysis. LSPR-based sensors can perform label-free detection, real-time monitoring, and high-sensitivity detection of the interaction between metal surfaces and analyte molecules, such as on gold nanoparticle surfaces. The main method for microfluidic sensors using gold nanomaterials is to induce the LSPR effect on the surface of gold nanoparticle films using optical prism coupling.

[0035] LSPR sensors based on gold 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 to SPP-based sensors, LSPR sensors can resonate directly with light, eliminating the need for bulky optical coupling components and facilitating high integration. Furthermore, the wavelength and peak shape of the LSPR resonance peak can be adjusted by controlling parameters such as the size, shape, and spacing of the metal nanostructures, facilitating sensor optimization. Although the bulk refractive index sensitivity of LSPR sensors (~100-1000 nm RIU⁻¹) is typically much lower than that of SPP sensors (>3000 nm RIU⁻¹), the strong local electric field within a short distance on the metal surface still allows for high sensitivity to molecular bonding behavior near the surface.

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

[0037] Although the RCA reaction can be performed at room temperature, diurnal temperature variations can significantly impact quantitative detection results. Therefore, temperature-controlled uniformity is crucial for cfDNA amplification. Furthermore, due to the low cfDNA concentration, even with timely RCA amplification of the target sequence, the detection capability of conventional LSPR systems is limited by the efficiency constraints of linear amplification. Compared to conventional LSPR-RCA detection systems, the PT-RCA system is a temperature-controlled, highly sensitive detection system that integrates heating and LSPR.

[0038] If we review the existing detection methods, we can find that, since the content of cfDNA in real body fluids (blood, urine, etc.) is relatively low, 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 cycling, which usually takes 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 accomplished using quantitative polymerase chain reaction (qPCR), which requires highly trained laboratory personnel, numerous liquid handling steps, and expensive equipment such as thermal cyclers. Isothermal amplification offers an alternative to qPCR, allowing nucleic acid amplification at a single, constant temperature, eliminating the need for thermal cyclers. Common isothermal amplification methods include loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), rolling circle amplification (RCA), and nucleic acid sequence-based amplification (NASBA). In contrast, commonly used PCR methods are time-consuming, require specialized laboratory setups and expensive equipment, and necessitate dedicated rooms and personnel for nucleic acid amplification. Furthermore, digital PCR testing is costly. To overcome these drawbacks, various isothermal nucleic acid amplification methods, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), deoxyribonucleic acid (DNA) nanoscaffold-based hybridization chain reaction, and nucleic acid sequence-based amplification, have been developed to overcome the need for complex thermal cycling equipment associated with qRT-PCR.

[0040] Compared to quantitative polymerase chain reaction (qPCR), rolling circle amplification (RCA) is also a nucleic acid amplification technique used to amplify specific nucleic acid sequences for further detection and analysis. It is applied in fields such as pathogen detection, gene expression analysis, and genetic disease diagnosis. Unlike qPCR, RCA uses circular DNA as a template and undergoes isothermal amplification under the action of phi29 DNA polymerase, generating a large number of repetitive DNA sequences. RCA can be performed at a constant temperature, eliminating the need for temperature cycling and thus avoiding the need for expensive thermal cyclers. RCA amplification is rapid, yielding large amounts of DNA products in a short time, making it suitable for on-site testing and rapid screening, especially in situations lacking specialized equipment. Furthermore, RCA can be detected using various methods, including direct observation of the turbidity and fluorescence signal of the precipitate.

[0041] Rolling circle amplification (RCA) requires only one primer to amplify the target product. The reaction is triggered when both the primer and the circular template are present. Due to the powerful strand displacement function of the phi 29 polymerase, as the primer rotates around the circular template, it displaces the primer that initially annealed to the template, continuously generating dozens to hundreds of new tandem repeats of long ssDNA until the DNA polymerase activity is exhausted. The amplification efficiency of linear RCA is approximately 10-1. 5 Copying. The low requirements of RCA technology for instruments and testing conditions make it a promising technology for application in point-of-care testing (POCT) and rapid large-scale screening.

[0042] Among these methods, solid-phase RCA (Reactive Chemical Acquisition) reduces the washing step compared to traditional liquid-phase RCA, making it more advantageous for detecting low-concentration samples (such as cfDNA). Furthermore, liquid-phase RCA is often an endpoint detection method, requiring precise control of the detection time point, resulting in lower repeatability. Some liquid-phase RCA methods rely on colorimetric methods to read the detection signal, further increasing the error in the results. Solid-phase RCA, on the other hand, overcomes these drawbacks by reading the reaction signal in real time.

[0043] In liquid-phase detection systems, the gold nanoparticles used to generate photothermal energy need to maintain a relatively uniform density and distribution to ensure consistent temperature throughout the reaction system. However, the elongation and spontaneous folding of nucleic acid chains during nucleic acid reactions can affect temperature uniformity to some extent.

[11] Solid-phase nanochips can maintain a consistent temperature while preserving a uniform light spot.

[0044] When reading the results, in liquid phase detection, nanoparticles undergo changes at different scales, resulting in color changes. While this method allows for better naked-eye detection, it has limitations for more precise quantitative detection. Furthermore, liquid phase detection involves a washing process, which often introduces errors during liquid transfer. Solid-phase chip detection overcomes this problem by using continuous liquid flow for washing. Compared to traditional LSPR sensing technology, PT-LSPR detection has the advantage of improving the signal-to-noise ratio (sensitivity) of RCA amplified cfDNA detection through laser temperature control and LSPR technology. PT-LSPR detection, or Phase Thermal-Localized Surface Plasmon Resonance (LSPR) detection, is a biosensing technology based on the LSPR effect. The LSPR optical effect refers to the phenomenon where, when light shines on nanoparticles made of noble metals, if the frequency of the incident photons matches the surface plasmon resonance frequency of the metal nanoparticles, strong light absorption occurs, resulting in a significant resonance absorption peak in the spectrum. The LSPR effect is highly sensitive to changes in the refractive index of the surrounding medium, and therefore can be used as a highly sensitive chemical and biological sensor.

[0045] Furthermore, compared to liquid-phase AuNPs particles, solid-phase AuNPs chips exhibit more uniform temperature control.

[12] Gold nanoparticles possess high photoconversion efficiency, and the heat generated on the chip surface can largely maintain the rigid structure of long nucleic acid amplification chains, allowing them to participate more actively in the amplification reaction. The generation of LSPR signals on gold nanoparticle chips is based on electron-to-electric conversion, while photothermal LSPR is a higher-energy manifestation. Therefore, photothermal activity also plays a role in signal amplification. The localized surface plasmon resonance phenomenon generated when the gold nanostructures on these chips interact with incident light can be used to detect and analyze chemical or biological reactions occurring on or near the chip surface, as these reactions lead to changes in the LSPR resonance frequency.

[0046] Considering the photothermal conversion efficiency of different materials, PPy@IOs, noble metal particles, and gold nanoparticles (AuNPs) exhibit size- and shape-dependent optical properties.

[13] When the collective oscillation of free charges resonates with the electric field of incident light, strong light absorption is achieved in both SPR and local SPR modes. The absorbed photon energy can be effectively converted into heat and dissipated into the surrounding medium. Therefore, plasma devices can serve as optically driven heaters and can be remotely controlled.

[14] .

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

[0048] According to one or more embodiments, a rapid quantitative detection system for photothermal plasmon resonance-based RCA-cfDNA is disclosed. Figure 1 The diagram shows the basic flowchart of the detection method for photothermal cyclic recombination amplification (RCA) 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 role is to immobilize the target molecule, laying the foundation for the next step of detection.

[0050] S102. MCH blocks non-specific sites. MCH is used here as a blocking agent to block non-specific binding sites on the gold nanoparticle substrate that have not participated in the reaction. This reduces 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 being a DNA or RNA sequence, is detected that is associated with disease biomarkers or other biomolecules. The presence of the target molecule can be detected here using specific methods, such as fluorescent labeling or changes in optical signals.

[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 without the need for complex thermal cycling equipment. During RCA, a circular template and a specific DNA polymerase (such as...) are used... DNA polymerase is used to synthesize long single-stranded DNA, which is complementary to a circular template to form tandem repeat sequences, thereby enabling the mass replication of the target sequence.

[0053] Figure 1 In English, "plasma" refers to the liquid portion of blood, primarily composed of water, electrolytes, proteins, hormones, nutrients, and waste products. It is one of the main components of blood, with the remainder including red blood cells, white blood cells, and platelets. Alternatively, it can refer to blood-like fluids in certain organisms, such as hemolymph in some invertebrates.

[0054] Therefore, the embodiments of this disclosure Figure 1 The flowchart illustrates a highly integrated detection system that immobilizes target molecules using specific capture sequences, blocks non-specific sites using MCH to improve detection specificity, and then amplifies the target molecules using RCA technology to enhance the signal and achieve high-sensitivity detection. The solutions described in this disclosure are suitable for rapid, low-cost on-site detection, especially in resource-constrained environments.

[0055] exist Figure 1 The document also includes an optical path diagram for experimentally measuring the polarization absorption characteristics of gold nanoparticles in a photothermal plasmon-based RCA-cfDNA rapid quantitative detection device. The detection principle diagram of this optical path diagram is shown below. Figure 2 As shown, the English labels of the components are in Figure 2 The corresponding Chinese label is provided. This testing device consists of the following components:

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

[0057] The first polarizer is used to pass the light emitted by the LED light source. The function of the polarizer is to allow light waves vibrating in a specific direction to pass through, thereby producing polarized light.

[0058] A birefringent crystal is a crystal through which polarized light then enters. This type of crystal allows light to undergo birefringence, meaning that the speed of light travels at different speeds in two perpendicular directions, resulting in different refractive indices.

[0059] A prism, with an incident surface, a detection surface, and an exit surface, is used to change the direction of light propagation or focus the beam. A sample containing the cfDNA to be detected is placed on the detection surface of the prism. Incident light, after passing through a birefringent crystal, enters the incident surface of the prism, undergoes a first refraction to the detection surface, and acts on the sample, where the sample interacts with the light. After a second refraction to the exit surface of the prism, it exits after a third refraction. A gold nanoparticle microfluidic chip is disposed on the detection surface of the prism; this is essentially an AuNI chip with a microfluidic channel added, equivalent to a micro-reaction chamber, providing space for the analyte to react. LED light, after passing through a filter and a birefringent crystal, illuminates the gold nanoparticles placed on the prism, generating plasmon resonances (LSPR). LSPR occurs on the AuNI chip.

[0060] The second polarizer is used to receive light emitted from the exit surface of the prism.

[0061] A filter is a filter that receives light emitted from a second polarizer. The filter is used to filter or block light of a specific wavelength for analysis or detection.

[0062] Finally, the filtered light reaches the detector, which converts the optical signal into an electrical signal for further analysis and measurement.

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

[0064] Figure 2 This is a schematic diagram of the optical path for measuring the polarization absorption characteristics of gold nanoparticles in this experiment. Because gold nanoparticles have a broad optical absorption spectrum, an LED light source was chosen.

[0065] First, a polarizer modulates the circularly polarized light into linearly polarized light. Then, a birefringent crystal separates the light into incident light (p-polarized light) and reference light (s-polarized light). The incident light is incident on a prism at a certain angle. The light absorbed by the polarization coupling of the gold nanoparticles is primarily p-polarized, with minimal absorption of s-polarized light. A broadband white light source uses the birefringent crystal to adjust the optical path difference between the s-polarized and p-polarized states, constructing a phase-modulated common-path interference carrier signal. After refraction by a prism, this signal is obliquely incident on the microfluidic sensing interface located on the prism's detection surface.

[0066] By adjusting the ATR incident angle within the 600-800° incident angle range, the position of the LSPR resonance wavelength λLSPR in the white light spectrum can be adjusted. A broadband white light source uses a birefringent crystal to adjust the optical path difference between the s-polarization and p-polarization states, constructing a phase-modulated common-path interference carrier signal. This signal is then refracted by a prism and incident obliquely onto the microfluidic sensing interface. The refractive index of the sample is measured to calculate the refractive index resolution (RIR) at different ATR incident angles. ATR stands for Attenuated Total Reflection, an optical phenomenon where light undergoes total internal reflection at the interface when incident from a high-refractive-index medium to a low-refractive-index medium if the incident angle is greater than a critical angle. Under total internal reflection, an electromagnetic field called an "evanescent wave" or "attenuated wave" is formed near the interface, whose intensity rapidly decreases with increasing distance from the interface. In LSPR (Locally Surface Plasmon Resonance) sensors, ATR (Automatic Transmission Resonance) technology is used to enhance the LSPR signal because the evanescent waves generated by ATR can interact with metallic nanostructures at the nanoscale, thereby exciting LSPR modes. By precisely controlling the incident angle of the ATR, the intensity and distribution of the evanescent waves can be tuned, thus 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, thus affecting the sensor's sensitivity and detection limit.

[0068] LSPR sensors typically utilize the localized enhancement effect of incident light by metallic 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 also changes; this change can be observed by measuring the extinction spectrum of the nanostructure. Therefore, the resonant frequency (RIR) is generally related to the spectral resolution of the LSPR sensor; higher spectral resolution results in a higher RIR, meaning the sensor can detect smaller changes in refractive index.

[0069] PT-LSPR detection, in addition to utilizing the LSPR principle, also incorporates the phase thermo-optic effect. PT stands for Photothermal, meaning that when a metallic nanostructure absorbs light energy and converts it into heat energy, the resulting thermal expansion or refractive index change affects the phase of light propagation. PT-LSPR detection measures this phase change, which typically requires an interferometer or other instruments capable of detecting phase changes, such as a Mach-Zehnder interferometer or a fiber optic interferometer, to accurately measure the phase shift.

[0070] In the PT-LSPR-RCA detection system, a homogenized 532nm laser beam is incident normally onto the LSPR microfluidic chip and interacts with gold nanoparticles (AuNI) through the PDMS chamber, generating a localized photothermal effect. This effect is achieved by using 0-5W / cm². 2 The energy density of the homogenized laser spot is adjusted within a certain range to achieve temperature regulation of plasmon photothermal effects; the signal-to-noise ratio of the LSPR sensing signal is optimized, and the rules and characteristics of reducing dual-path optical signal interference are analyzed to improve the stability and signal-to-noise ratio of the signal in photothermal biosensing. Specifically, in this embodiment, the PT-LSPR-RCA abbreviation combination, proposed for the first time, refers to an RCA detection method and system based on photothermal mediated LSPR sensing, integrating photothermal PT, localized surface plasmon resonance sensing LSPR, and rolling ring amplification RCA—these three technologies form the core technical components of this embodiment. Here, gold nanosheets and PDSM are bonded together via Plasma / ionization bonding, forming a closed microfluidic chamber composed of the upper and lower layers bonded together.

[0071] For quantitative detection, system stability and the determination of the limit of detection (LOD) are crucial. To accurately control temperature and improve detection sensitivity, this disclosure establishes a photothermal RCA amplification system for detecting trace amounts of cfDNA in bodily fluids. Traditional gold nanoparticle (AuNP)-based chips exhibit 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 fluorescent labeling and other methods to amplify signals and achieve the detection of trace nucleic acids. Compared to these labeled detection technologies, the detection system of this disclosure achieves highly sensitive quantitative detection without the introduction of additional labels.

[0072] According to one or more embodiments, a rapid quantitative detection method for plasmon resonance-based RCA-cfDNA is provided. The first step involves preparing the experimental materials, including: cDNA (capture, 100 μM), TCEP, MCH, 75% ethanol, EGFR single-stranded template (10 nM), RCA circular template, dNTPs, PHI29 DNA polymerase, PHI29 DNA polymerase buffer, albumin, ddH2O, PBS, and a gold nanoparticle substrate.

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

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

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

[0076] ③ Prepare EGFR template single strands of various concentrations or extract 50 μL of patient cfDNA, heat in a 90℃ metal bath for 3 minutes, then immediately place in an ice bath to maintain the rigid structure of the nucleic acid strand to be tested.

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

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

[0079] S201: Install the gold nanoparticle substrate, adjust the optical path, and collect data for approximately 30 minutes (adjust the time according to the spectral stability). Process the data to check if the baseline has stabilized. After reaching steady state, collect data again (50 μL / min). Wait for a baseline period, then pour the prepared capture from step ① above (10 μL / min—300 μL) into the container and allow it to react with the gold nanoparticle substrate for approximately 30 minutes. Rinse the reaction chamber with PBS (approximately 30 minutes). After the experiment, process the data to check if the capture has bonded to the gold nanoparticle substrate and if the baselines before and after the experiment have stabilized.

[0080] S202, after the baseline stabilizes, open a new file and begin the MCH blocking experiment. Adjust the optical path to a slightly less sensitive state, and first collect baseline data for a period of time (50 μL / min), then add the working solution prepared in step ② (20 μL / min). React for 30 minutes, then rinse the reaction chamber with PBS. Process the data after the experiment.

[0081] S203. After the baseline stabilizes, open a new file and start EGFR detection. Once steady state is reached, reacquire data (50 μL / min), then add the prepared working solution (10 μL / min - 300 μL) from step ③ to allow EGFR to react with the capture for 30 minutes. Rinse the reaction chamber with PBS (approximately 30 minutes). After the experiment, process the data to check if EGFR has reached a stable state.

[0082] S204, turn on the laser that vertically illuminates the AuNI chip, with an optical power density of 174mW / cm². 2 Wait for the baseline to stabilize. To check for stability, you can collect and process the data first, or observe if the spectral changes are minimal. Once steady state is reached, collect data again (50 μL / min), and then flow the sample into the working solution prepared in step ④. After the working solution sample flows in, stop the working solution delivery pump, clamp the pipe, and allow the RCA reaction to proceed for 1.5 hours. This process is used to form a stable self-assembled monolayer on the metal surface to improve the sample's adsorption properties or to perform specific surface modifications.

[0083] The entire detection process is performed on a solid-state microfluidic chip, including the capture of cDNA sequences and binding via Au-S bonds (the chip is an AuNI solid-state chip), the binding of cDNA and cfDNA, and the subsequent RCA amplification of cfDNA.

[0084] In the above detection process, the detection signal at each step can be monitored in real time using Avantes software via a spectrometer. Data is recorded every 2 seconds, and each detected signal is saved to a folder. The signals can be processed using Matlab. Real-time signal reading is employed in the experiment to effectively sense the binding behavior of biomolecules in the photothermal field. The interference signal is calculated using the windowed Fourier transform (WFT) method to obtain the phase change information of plasmons at the sensing interface, achieving highly sensitive sensing of refractive index and biomolecule affinity. In the analysis and calculation, the data with the strongest reflection from the processed spectral signal are selected for analysis. For example, the following data and results can be obtained.

[0085] ① Nucleic acid sequence of RCA reaction.

[0086] Table 1 shows the RCA reaction sequence information. Figure 13 As shown. The terms used in Table 1 include:

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

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

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

[0090] Detection Target Sequence (EGFR): A template for the nucleic acid sequence to be tested.

[0091] In the Function description section, "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"; and "Target sequence cyclization helper" refers to "Target sequence cyclization helper".

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

[0093] ③ Testing the binding effect of each component.

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

[0095] 1. Capture sequence modification. The figure shows the modification level changing over time, from 500 seconds to 3000 seconds, with the modification level varying between 2.5 and 0.2.

[0096] 2. MCH modification: The figure shows the measured MCH modification levels at different time points, from 500 seconds to 3000 seconds, with the modification level varying between 2.6 and 0.2.

[0097] 3. EGFR capture: The figure shows the variation of EGFR capture level over time, from 500 seconds to 3000 seconds, with the capture level varying between 0.6 and 0.05.

[0098] 4. RCA amplification: The figure shows the change in RCA amplification level over time, from 500 seconds to 3000 seconds, with the amplification level varying between 2.0 and 0.05.

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

[0100] Figure 4 The results from the optical and gel systems in this embodiment show that the reaction can only be initiated when all RCA components are present. In the cfDNA-EGFR detection process, changes in the detection signal indicate successful binding of the analyte.

[0101] ④ This disclosure explores the blocking effect of different concentrations of MCH in its embodiments. For example... Figure 5 A schematic diagram showing the direct detection of EGFR binding after blocking with different concentrations of MCH. Figure 6 This diagram illustrates the direct detection of RCA-EGFR binding efficacy after blocking with different concentrations of MCH. The results show that adding MCH to the reaction system and diluting it at a ratio of 1:1000 resulted in the best blocking effect, and this concentration can be selected for subsequent experiments.

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

[0103] In this disclosure, when the LSPR system was used to directly detect the amplification effect of EGFR-RCA, the detection signal was found to be low. Further attempts were made to utilize laser irradiation to generate localized photothermal effects, resulting in a significant increase in the detection signal. Subsequent experimental testing requires further refinement of the EGFR-RCA detection signal to achieve the detection of low concentrations of EGFR-cfDNA in plasma.

[0104] ⑥ The effects of laser power and RCA reaction at different temperatures in the embodiments of this disclosure. For example... Figure 8 As shown, the photothermal response effect of LSPR-PT was verified. By exploring the different effects of EGFR-RCA at different photothermal temperatures, a light power density of 174 mW / cm² was selected here. 2 Experiments were conducted using lasers.

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

[0106] Epidermal growth factor receptor (EGFR) is an important cell surface receptor, and its mutations are closely related to the development and progression of various solid tumors, including non-small cell lung cancer (NSCLC). EGFR testing is often used to guide the use of targeted therapies, such as EGFR tyrosine kinase inhibitors.

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

[0108] The CDKN2A gene encodes the p16 protein, a cell cycle repressor protein whose loss-of-function mutations are associated with various cancers. CDKN2A testing helps identify high-risk tumor types, and immunohistochemical detection 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 mutations are among the most common gene mutations in cancer and are associated with various biological behaviors of tumors. TP53 testing helps assess tumor malignancy and prognosis.

[0110] The LSPR-PT system of this disclosure can enhance the signal of specific biomolecules to improve the detection performance of various biomolecules, including EGFR, PTEN, CDKN2A, and TP53. To verify its effectiveness, the following detection experiments were conducted in this disclosure embodiment.

[0111] Figure 9 The results are based on EGFR detection according to embodiments of this disclosure. The detection method used an LSPR-PT system to detect EGFR-cfDNA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, and 10 pM, respectively. The limit of quantification was 12.87 pM, and the lowest detection limit reached 45.86 pM. A standard curve for EGFR-cfDNA detection was plotted by fitting the EGFR-cfDNA concentration with the LSPR-PT system detection results.

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

[0113] Figure 10 The results are based on PTEN detection according to embodiments of this disclosure. The detection method used an LSPR-PT system to detect PTEN-cfDNA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, and 10 pM, respectively. The limit of quantification was 5.02 pM, and the lowest detection limit reached 13.92 pM. A standard curve for PTEN-cfDNA detection was plotted by fitting the PTEN-cfDNA concentration with the LSPR-PT system detection results.

[0114] PTEN-RCA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, 10 pM, 1 pM, and 500 fM were detected using the LSPR-PT system, and standard curves for PTEN-RCA detection were plotted. The PTEN-PT system significantly enhanced the ability to detect PTEN-RCA, with a limit of quantitation of 0.739 pM and a minimum detection limit of 0.044 pM.

[0115] Figure 11This is a schematic diagram of CDKN2A detection results according to an embodiment of the present disclosure. The detection method used an LSPR-PT system to detect CDKN2A-cfDNA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, and 10 pM, respectively. The limit of quantification was 1.73 pM, and the lowest detection limit reached 6.08 pM. A standard curve for CDKN2A-cfDNA detection was plotted by fitting the CDKN2A-cfDNA concentration with the LSPR-PT system detection results.

[0116] CDKN2A-RCA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, 10 pM, 1 pM, and 500 fM were detected using the LSPR-PT system, and a standard curve for CDKN2A-RCA detection was plotted. The CDKN2A-PT system showed significantly enhanced detection capability for CDKN2A-RCA, with a limit of quantitation of 0.145 pM and a minimum detection limit of 0.006 pM.

[0117] Figure 12 This is a schematic diagram of TP53 detection results according to an embodiment of the present disclosure. The detection was performed using an LSPR-PT system to detect TP53-cfDNA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, and 10 pM, respectively. The limit of quantification was 11.54 pM, and the lowest detection limit reached 6.04 pM. A standard curve for TP53-cfDNA detection was plotted by fitting the TP53-cfDNA concentration with the LSPR-PT system detection results.

[0118] TP53-RCA concentrations of 10 nM, 1 nM, 500 pM, 100 pM, 50 pM, 10 pM, 1 pM, and 500 fM were detected using the LSPR-PT system, and a standard curve for TP53-RCA detection was plotted. It can be seen that the TP53-PT system significantly enhances the ability to detect TP53-RCA, with a limit of quantitation of 0.298 pM and a minimum detection limit of 0.036 pM.

[0119] In summary, this disclosure addresses the current limitations of photothermal-regulated nucleic acid detection, which is limited to the in situ cellular level (and cannot perform quantitative detection, only qualitative analysis). It presents a rapid quantitative RCA method and system for specimens such as body fluids, which is a rapid amplification detection system that integrates detection and temperature control.

[0120] This method is simple and rapid, better meeting the requirements of POCT and reducing the laboratory environment requirements for testing. POCT is an abbreviation for "Point of Care Testing," meaning "bedside testing" or "on-site rapid testing." It allows rapid diagnostic tests to be performed at patient care points (such as at a patient's bedside, emergency room, doctor's office, or community health center) without having to send samples to a central laboratory for analysis.

[0121] The cfDNA microfluidic detection system in this embodiment integrates photothermal PT, localized surface plasmon resonance (LSPR) sensing, and rolling circle amplification (RCA). Amplification at the solid-phase interface is achieved through RCA amplification on a solid-phase chip. This solid-phase interface simultaneously serves as a photothermally mediated optical temperature control interface and a real-time detection interface, resulting in a significant improvement in detection sensitivity. Figure 7 As shown. Meanwhile,

[0122] Because detection is performed on a solid-phase chip, the transfer processes such as washing are reduced, enabling better detection of low-concentration biomarkers. Simultaneously, RCA amplification can amplify the detection signal, eliminating the need for multiple primers or reactants, resulting in a simpler reaction system and reducing the design process for identifying detection targets.

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

[0124] 1.Wang Y,Kong SL,Su XD.A centrifugation-assisted visual detection ofSNP in circulating tumor DNA using gold nanoparticles coupled with isothermalamplification.RSC Adv 2020;10(3):1476-148310.1039 / c9ra09029k.

[0125] 2. Keller L, Belloum Y, Wikman H, Pantel K. Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond. Br J Cancer 2021; 124(2):345-35810.1038 / s41416-020-01047-5.

[0126] 3.Hauser S,Zahalka T,Fechner G,Muller SC,Ellinger J.Serum DNAhypermethylation in patients with kidney cancer:results of a prospectivestudy.Anticancer Res 2013;33(10):4651-4656

[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 spirit and principles of this invention have been described with reference to several specific embodiments, it should be understood that this invention is not limited to the disclosed specific embodiments, and the division of aspects does not imply that the features in these aspects cannot be combined; such division is merely for the convenience of description. This invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. A rapid quantitative detection system for cfDNA, characterized in that, The system includes, depending on the optical path direction, A light source, used to provide the incident light required for detection; The first polarizer is through which the light emitted from the light source passes; A birefringent crystal, into which polarized light emitted from the first polarizer is incident; A prism, having an incident surface, a detection surface, and an exit surface, is used to change the direction of light propagation. The detection surface is equipped with a gold nanoparticle substrate for reaction with the cfDNA detection sample. The light emitted from the birefringent crystal is incident on the incident surface of the prism, refracted for the first time to the detection surface, acts on the gold nanoparticle substrate, refracted for the second time to the exit surface of the prism, and then refracted for the third time before exiting from the exit surface. The second polarizer is the source of light emitted from the exit surface of the prism. The filter is the source of light emitted from the second polarizer. The detector receives light that has passed through a filter, and then performs calculations and analysis on the received detection signal. The prism detection surface is equipped with a nano-gold microfluidic chip, which is a solid-phase microfluidic chip, and the nano-gold substrate of the microfluidic chip is AuNI; A first laser is incident along the normal direction onto the microfluidic AuNI sensor chip, and interacts with the AuNI nanoparticles on the gold nanoparticle substrate through the PDMS chamber of the microfluidic chip to generate a local photothermal effect. The detection system performs the detection of the sample on the solid-phase microfluidic chip, and the specific steps include: capturing the sequence and binding it to the gold nanoparticle substrate to fix the target molecule; using MCH to block non-specific sites to improve the specificity of detection; amplifying the target molecule through RCA; and detecting the target molecule to enhance the signal and achieve high-sensitivity detection.

2. The detection system according to claim 1, characterized in that, The light source is a broadband white LED light source.

3. The detection system according to claim 1, characterized in that, The birefringent crystal splits the incident light into p-polarized light, which serves as the incident light from the prism, and s-polarized light, which serves as the reference light.

4. The detection system according to claim 3, characterized in that, The light source adjusts the optical path difference between the polarization states of s-polarized light and p-polarized light through the birefringent crystal to construct a phase-modulated common-path interference carrier signal, which is then refracted by the prism and incident on the microfluidic chip sensing interface located on the prism detection surface.

5. The detection system according to claim 1, characterized in that, The first laser is a 532nm laser after the spot homogenization process. It is incident on the microfluidic chip in the normal direction and interacts with the gold nano island AuNI nanoparticles through the PDMS chamber to generate local photothermal effect.

6. The detection system according to claim 5, characterized in that, Through 0-5W / cm 2 The energy density of the homogenized laser spot is adjusted within a certain range to achieve temperature regulation of plasmonic photothermal effect; and the signal-to-noise ratio of the LSPR sensing signal is optimized.

7. The detection system according to claim 1, characterized in that, The amplification of the target molecule via RCA includes: turning on a laser that vertically irradiates the AuNI chip at a power density of 174 mW / cm². 2 The solution flows into the RCA reaction working solution, and the RCA reaction proceeds for 1.5 hours.

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

9. A rapid quantitative detection method for cfDNA, characterized in that, The detection method is based on the detection system as described in any one of claims 1 to 8, and the detection method includes, A nano-gold substrate is mounted on the prism detection surface of the detection system, and the optical path is adjusted. The sample solution is poured in and reacts with the gold nanoparticle substrate. The reaction chamber is then rinsed with PBS. Obtain test data; The entire detection process is performed on a solid-state microfluidic chip, including cDNA capture sequence binding via Au-S bonds, cDNA binding to cfDNA, and subsequent cfDNA RCA amplification. The nucleic acid amplification temperature is controlled by laser photothermal effect to enhance detection sensitivity.

Images