Inflammation marker and virus antibody synchronous detection chip based on thermal annealing gold nanoparticles and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to an inflammatory marker and virus antibody synchronous detection chip based on thermal annealing gold nanoparticles and application of the inflammatory marker and virus antibody synchronous detection chip. The invention provides a multiple detection chip for detecting CRP, SAA and tick-borne virus antibodies IgG / IgM in serum based on thermal annealing gold nanoparticles, the sample size demand is reduced in magnitude, the detection limitation of special crowds is broken through, the detection sensitivity is remarkably improved, and low-concentration target substances are accurately recognized. The method also has multiple detection capability, breaks through the limitation of single detection in the traditional technology, is excellent in clinical verification result, and ensures the detection reliability and practicability.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a chip for the simultaneous detection of inflammatory markers and viral antibodies based on thermally annealed gold nanoparticles and its application. Background Technology

[0002] Early symptoms of tick-borne virus (such as TBEV, SFTSV, ALSV, YEZV) infection are nonspecific. The core of their diagnosis revolves around the virus itself, viral antigens, and the body's immune response. The diagnosis techniques are mainly divided into three categories: etiological diagnosis, serological diagnosis, and molecular biological diagnosis.

[0003] Etiological diagnosis, which directly detects viral particles or their components, is one of the "gold standards" for confirming infection, especially suitable for the early stages of infection. Viral isolation and culture isolate live viruses from samples such as patient blood and cerebrospinal fluid, which can identify the virus species. However, this method is time-consuming (usually requiring several days to weeks) and has high requirements for laboratory biosafety levels (e.g., TBEV requires a BSL-3 laboratory), so it is rarely used routinely in clinical practice. Direct immunofluorescence (DFA) uses specific fluorescent antibodies to label viral antigens in samples, and the results are observed through a fluorescence microscope. This method is relatively simple and rapid (results within hours), but has low sensitivity and is mainly used for detecting viruses in acute-phase samples or ticks. Its disadvantages are that it is time-consuming, has low sensitivity, and is costly.

[0004] Serological diagnosis determines infection status by detecting antibodies (IgM and IgG) produced by the body against the virus, and is suitable for mid-to-late stages of infection or epidemiological investigations. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used serological method in clinical practice, detecting IgM and IgG antibodies separately. It is standardized, high-throughput, and relatively low-cost, making it suitable for large-scale sample screening. Western blotting, as a confirmatory assay for ELISA, detects specific viral proteins targeted by antibodies, exhibiting extremely high specificity. It is mainly used for further validation of ELISA-positive samples, ruling out false positives. Indirect immunofluorescence assay (IIFA) is similar in principle to DFA, but determines the result by detecting the binding of antibodies to viral antigens in the sample. This method has good specificity, but is relatively cumbersome to operate and relies on subjective interpretation, limiting its application compared to ELISA. However, traditional methods also have limitations, including the inability to diagnose early and the potential for cross-reactivity.

[0005] Molecular biological diagnostic testing of viral nucleic acids (DNA or RNA) is characterized by high sensitivity, strong specificity, and rapid detection speed, making it a core technology for the early diagnosis of tick-borne viruses. Reverse transcription polymerase chain reaction (RT-PCR) targets RNA viruses such as TBEV and SFTSV, first reversing RNA into cDNA, then amplifying and detecting it via PCR. This method can detect viral nucleic acids within 1-3 days after infection, enabling rapid diagnosis of acute-phase infection and making it the preferred choice for early clinical diagnosis. Real-time quantitative RT-PCR (qRT-PCR) adds fluorescent probes to RT-PCR, allowing real-time monitoring of the amplification process and quantification of viral load. It not only confirms infection but also assesses the severity of infection based on viral load, guiding clinical treatment. Multiplex PCR / RT-PCR can simultaneously detect the nucleic acids of multiple tick-borne viruses (such as TBEV, SFTSV, ALSV, and YEZV), suitable for patients with unexplained fever and a history of tick bites, significantly improving diagnostic efficiency and reducing missed and misdiagnoses. Next-generation sequencing (NGS) uses high-throughput sequencing to obtain all nucleic acid sequences in a sample, which are then compared with known virus databases to detect unknown or rare tick-borne viruses. This method has extremely high specificity and sensitivity, but it is expensive and data analysis is complex, so it is mainly used for scientific research or the diagnosis of difficult cases.

[0006] Current testing technologies have significant limitations, failing to simultaneously detect both inflammatory markers and viral antibodies in a single test. In clinical settings, physicians often need to simultaneously assess a patient's inflammatory response (e.g., using inflammatory markers like C-reactive protein and procalcitonin) and viral infection status (e.g., confirming infection or immune protection through antibody testing). Current technology requires two separate tests. This not only prolongs the diagnostic process, typically requiring 2-4 hours for results, but also increases the complexity of sample processing, potentially delaying treatment decisions, especially during public health emergencies or emergency care, and increasing the risk of missing optimal intervention opportunities.

[0007] Current detection technologies have significant limitations in sensitivity, making it difficult to accurately identify target substances in low-concentration samples. In the early stages of viral infection, the concentration of viral antibodies in patients is typically low (e.g., antibody titers may be below the detection limit within 1-3 days of infection). Furthermore, some individuals with weakened immune systems (such as those with chronic diseases or immunodeficiency) produce significantly fewer antibodies even after infection than the general population. In these situations, existing technologies are prone to false negatives, leading to missed diagnoses. For example, in the early stages of COVID-19 infection, approximately 15%-20% of low-concentration antibody samples were mistakenly identified as negative due to insufficient detection sensitivity. This could not only allow for the covert spread of the virus but also delay early isolation and treatment of patients.

[0008] Furthermore, current technologies require a relatively large sample volume, typically 5-10 mL of venous blood, making them unsuitable for groups with limited blood samples, such as children, the elderly, and critically ill patients. Children, especially infants, have inherently low blood volumes (e.g., newborns have a total blood volume of only about 300-500 mL), and drawing a large amount of blood at once can lead to risks such as anemia and shock. Clinically, multiple small blood draws are often required, increasing the suffering of the child and the difficulty for parents to cooperate. The elderly generally have poor vascular elasticity and weak coagulation function, making them prone to subcutaneous hematomas after large blood draws. Moreover, the procedures are complex and difficult to implement for rapid clinical screening.

[0009] Emerging and re-emerging tick-borne viruses pose a significant threat to global public health. Infections caused by these pathogens can lead to severe clinical manifestations such as hemorrhagic fever, neurological complications, and fatal thrombocytopenia. In recent years, more than ten pathogenic tick-borne viruses have been discovered in China, posing a major challenge to accurate disease diagnosis. Most of these viruses, such as tick-borne encephalitis virus (TBEV), are characterized by a short viral phase in infected individuals. Therefore, nucleic acid testing alone often fails to detect the pathogen. Thus, exploring host-specific biomarkers and developing virus-specific antibody detection methods are crucial to improving the accuracy and reliability of diagnosis. Summary of the Invention

[0010] In view of this, the purpose of this invention is to provide a chip for the simultaneous detection of inflammatory markers and viral antibodies based on thermally annealed gold nanoparticles and its application, which enables a reduction in sample volume requirements, breaks through the limitations of testing in special populations, significantly improves detection sensitivity, accurately identifies low-concentration target substances, and has multiple detection capabilities, breaking the limitations of traditional "single detection" technology, resulting in excellent clinical validation results and ensuring the reliability and practicality of the detection.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a detection chip based on thermally annealed gold nanoparticles, with inflammatory markers and antiviral neutralizing antibodies as detection targets, specifically comprising:

[0013] (i) Thermally annealed gold nanoparticle substrate; and

[0014] (ii) Capture antibodies against inflammatory markers; and

[0015] (iii) Viral antigens;

[0016] The capture antibody and the viral antigen are coated on the heat-annealed gold nanoparticle substrate;

[0017] The heat-annealed gold nanoparticle substrate includes a glass substrate and a gold nanoparticle monolayer film.

[0018] The inflammatory markers include C-reactive protein and / or serum amyloid A;

[0019] The viral antigens include at least one of the following: antigens derived from tick-borne encephalitis virus, antigens derived from Alonshan virus, antigens derived from fever with thrombocytopenia syndrome virus, and antigens derived from Yezo virus.

[0020] In some specific embodiments of the present invention, the method for preparing the above-mentioned detection chip includes:

[0021] Gold nanoparticle monolayers are formed through self-assembly at the oil-water interface.

[0022] The gold nanoparticle monolayer film was transferred to a glass slide and thermally annealed to form dense nano-interstic gaps, thus obtaining the thermally annealed gold nanoparticle substrate.

[0023] The detection chip is obtained by coating the capture antibody and viral antigen onto the heat-annealed gold nanoparticle substrate.

[0024] In some specific embodiments of the present invention, the thermal annealing conditions of the above-mentioned detection chip are as follows:

[0025] 100~500℃, which can be 100℃, 200℃, 300℃, 400℃ or 500℃;

[0026] 0.5 to 3 hours, which can be 30 minutes, 45 minutes, 50 minutes, 60 minutes, 120 minutes or 180 minutes;

[0027] The heating rate is 5~30℃ / min, which can be 6℃ / min, 7℃ / min, 8℃ / min, 9℃ / min, 10℃ / min, 11℃ / min, 12℃ / min, 13℃ / min, or 14℃ / min.

[0028] In some specific embodiments of the present invention, the formation of a gold nanoparticle monolayer film by self-assembly at the oil-water interface of the above-mentioned detection chip includes:

[0029] Chloroauric acid solution was mixed with deionized water and boiled, then mixed with sodium citrate dihydrate and kept boiling to obtain a colloidal solution of gold nanoparticles.

[0030] Hexane and a colloidal solution of gold nanoparticles were placed in the same container to form an oil-water interface. Ethanol was then added, and a dense monolayer of gold nanoparticles was formed at the interface.

[0031] In some specific embodiments of the present invention, the concentration of the chloroauric acid solution in the above-mentioned detection chip is 1% (1g / 100 mL).

[0032] In some specific embodiments of the present invention, the volume ratio of the chloroauric acid solution to the deionized water in the above-mentioned detection chip is 1:(295~301), which can be 1:296, 1:297, 1:298, 1:299 or 1:300.

[0033] In some specific embodiments of the present invention, the concentration of sodium citrate dihydrate in the above-mentioned detection chip is 2% (1g / 100 mL).

[0034] In some specific embodiments of the present invention, the volume ratio of sodium citrate dihydrate to deionized water in the above-mentioned detection chip is 1:(295~301), which can be 1:296, 1:297, 1:298, 1:299 or 1:300.

[0035] The present invention also provides the application of the above-mentioned detection chip in the preparation of products for diagnosing tick-borne virus infection, the products including reagent kits or systems.

[0036] In some specific embodiments of the present invention, the tick-borne viruses used in the above applications include at least one of tick-borne encephalitis virus, Arunsan virus, fever with thrombocytopenia syndrome virus, and Yezo virus.

[0037] The present invention also provides a detection kit, including the above-described detection chip.

[0038] In some specific embodiments of the present invention, the kit further includes labeled antibodies.

[0039] The present invention also provides a detection system, including the above-mentioned detection chip.

[0040] The present invention also provides a detection method, including detection based on the above-described detection chip, the above-described detection kit, or the above-described detection system.

[0041] In some specific embodiments of the present invention, the above detection method includes: collecting 1 μL of the sample to be tested and using the above detection chip, the above detection kit, or the above detection system to detect it and obtain the detection result.

[0042] The present invention also provides a diagnostic method, including detection based on the above-described detection chip, the above-described detection kit, or the above-described detection system.

[0043] In some specific embodiments of the present invention, the above-mentioned diagnostic method includes: collecting 1 μL of the sample to be tested and using the above-mentioned detection chip, the above-mentioned detection kit, or the above-mentioned detection system to obtain the detection result.

[0044] The present invention has the following beneficial effects:

[0045] I. Sample size requirements have been reduced by a significant margin, overcoming testing limitations for special populations.

[0046] Compared to existing mainstream detection technologies, this invention achieves a revolutionary breakthrough in sample volume, requiring only 1 μL of serum to complete the entire testing process. This volume is far lower than that of the clinically commonly used enzyme-linked immunosorbent assay (ELISA), which typically requires 50-100 μL of serum sample. The sample requirement of this invention is only 1 / 50 to 1 / 100 of that of ELISA. From a clinical application perspective, this advantage is significant for special groups with limited blood samples: for newborns (total blood volume approximately 300-500 mL), 1 μL of sample represents only 0.0002% to 0.0003% of their total blood volume, eliminating concerns about anemia or physiological stress caused by blood collection; for elderly and frail patients or patients in the intensive care unit (ICU) requiring frequent monitoring, micro-volume blood collection reduces the number of vascular punctures, lowering the risk of infection and physical burden. In contrast, the 50-100 μL sample volume required for ELISA often necessitates a balance between the necessity of blood collection and patient tolerance in such populations, and may even prevent timely testing due to insufficient sample. This invention completely solves this clinical pain point.

[0047] II. Detection sensitivity has been significantly enhanced, enabling precise identification of low-concentration target substances.

[0048] In terms of detection sensitivity, this invention represents a qualitative leap compared to existing technologies, achieving a CRP detection limit at the fM (femtomolar) level and an antibody detection limit at the ng / mL (nanogram / mL) level. In comparison, traditional ELISA typically has a CRP detection limit at the nM (nanomolar) level, while this invention improves sensitivity by more than 100 times, accurately capturing the signal even when the CRP concentration in the sample is only 0.1 fM (approximately 0.01 ng / mL). For viral antibody detection, ELISA detection limits are mostly at the μg / mL (microgram / mL) level, while this invention raises the sensitivity to the ng / mL level, equivalent to identifying only 1 ng of antibody in 1 mL of serum—a level that covers the low antibody expression stage in the early stages of viral infection (within 1-2 days of infection), effectively avoiding false negatives caused by insufficient sensitivity in traditional technologies. For example, in the early stages of COVID-19 infection, the concentration of IgM antibodies in patients may only be 2-5 ng / mL, making ELISA prone to detection blind spots, while this invention can accurately detect them, buying crucial time for early isolation and treatment.

[0049] III. Powerful multi-detection capabilities, breaking through the limitations of traditional "single detection" technologies.

[0050] In existing technologies, commonly used methods such as ELISA are mostly limited to "single indicator detection." If it is necessary to detect inflammatory markers and viral antibodies simultaneously, multiple tests must be performed separately, which is not only time-consuming and labor-intensive, but also increases sample volume and testing costs. However, this invention, with its high-throughput detection technology of TA-GNP, can simultaneously detect inflammatory markers (such as CRP and SAA) and multiple viral antibodies (such as ALSV, YEZV, TBEV, and SFTSV antibodies) in a single test, greatly simplifying the testing process and reducing clinical workload. It is especially suitable for emergency or fever clinic scenarios where it is necessary to quickly determine the type of infection and the degree of inflammation.

[0051] IV. Excellent clinical validation results ensure the reliability and practicality of the test.

[0052] This invention is not limited to the laboratory stage but has undergone rigorous clinical sample validation. CRP testing results from 66 clinical samples showed that both sensitivity and specificity reached 100%. Details of the clinical validation show that the 66 samples covered patients of different ages (newborns to the elderly), different types of infection (bacterial and viral), and different stages of the disease (early, middle, and recovery phases). The test results were in complete agreement with the clinical diagnostic gold standard. This means that in practical clinical applications, this invention can accurately identify positive patients (no missed diagnoses) and precisely exclude negative individuals (no misdiagnoses), providing doctors with a reliable basis for developing treatment plans while avoiding the risks of overtreatment or missed treatment due to misdiagnosis.

[0053] V. Integrated rapid screening model, adapted to emergency needs of sudden outbreaks.

[0054] In the face of public health emergencies (such as influenza pandemics), the existing "stepwise testing" model (screening for inflammation first, then virus typing) is insufficient to meet the demands of large-scale rapid testing—traditional procedures take more than 3 hours per sample, limiting the daily testing capacity. The integrated "screening-typing" diagnostic model constructed in this invention can simultaneously complete "inflammatory status assessment + virus typing confirmation," significantly improving testing efficiency. Furthermore, its minimal sample requirement (1 μL) supports finger-prick blood collection, reducing sample collection time and lowering the risk of cross-infection. In contrast, ELISA requires specialized laboratory operations, takes 1-2 hours per sample, and has a daily testing capacity of only a few hundred samples, making it unsuitable for the emergency testing needs of sudden outbreaks. Therefore, this invention can rapidly construct an "early detection, early isolation, and early treatment" prevention and control system during public health emergencies, providing crucial technical support for epidemic prevention and control. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0056] Figure 1 Demonstrate the preparation process;

[0057] Figure 2 This diagram illustrates the simultaneous detection of CRP and SAA on a TA-GNP substrate. a) Digital photograph of the TA-GNP chip (scale bar: 0.5 cm); b) Printed layout for CRP / SAA detection; c) Schematic diagram of the sandwich immunoassay structure for CRP / SAA detection; d) Scanning electron microscope (SEM) image of the TA-GNP chip (scale bar: 500 nm); e) Fluorescence images of CRP / SAA detection performed on glass (left) and TA-GNP (right); f) Fluorescence enhancement performance of TA-GNP in CRP / SAA detection; g) Thermal map of the specificity of CRP and SAA detection on the TA-GNP chip.

[0058] Figure 3 Analysis of inflammatory markers in emerging viral infections is shown. Image a shows a schematic diagram of CRP and SAA detection in the serum of the emerging viral infection group (ALSV, YEZV, TBEV, and SFTSV PCR positive) and the control group; images b and c show the fluorescence images of CRP and SAA detection in the emerging viral infection group (b) and the control group (c); and image d shows the corresponding fluorescence signals of CRP and SAA detection in patients with emerging viral infection (n=19) and the control group (n=19).

[0059] Figure 4 This paper presents enhanced CRP quantification analysis in clinical serum using the TA-GNP platform. A) shows a schematic diagram of the detection workflow and fluorescence images of positive samples (#32, #35, #26), negative samples (#2, #47), and blank controls. B and C) show fluorescence images (b) and calibration curve (c) of CRP detection using TA-GNP. The limit of detection (LOD) is defined as the blank mean plus three standard deviations. D) shows the CRP level determination results of TA-GNP (blue) and conventional clinical methods (orange) in 66 clinical serum samples, with the dashed line representing the cutoff value. E) shows that the specificity and sensitivity of TA-GNP for CRP detection in the 66 clinical samples were both 100%. F) shows the correlation between TA-GNP and clinical methods in CRP detection results in the 66 samples. G) shows a box plot of CRP levels in the 66 clinical serum samples detected by TA-GNP, compared with the results of the clinical method.

[0060] Figure 5This invention illustrates the clinical testing of the detection chip, where a shows a schematic diagram of CRP and SAA detection in the serum of newly infected virus groups (ALSV, YEZV, TBEV, and SFTSV PCR positive) and control groups; b and c show the fluorescence images of CRP and SAA detected in the newly infected virus groups and control groups, respectively; d shows the corresponding fluorescence signals of CRP and SAA detected in newly infected virus patients (n=19) and control groups (n=19).

[0061] Figure 6 The changes in systemic inflammatory markers in emerging viral diseases are shown. a) Box plots of CRP levels in serum of ALSV-, YEZV-, TBEV-, and SFTSV PCR-positive samples compared to control serum; b) Box plots comparing CRP levels in the emerging virus infection group and control group; c) Box plots of SAA levels in serum of ALSV-, YEZV-, TBEV-, and SFTSV PCR-positive samples compared to control serum; d) Box plots comparing SAA levels in the emerging virus infection group and control group. *P < 0.05, **P < 0.01, ****P < 0.0001, determined by the Wilcoxon–Mann–Whitney test.

[0062] Figure 7 The images show the expression of recombinant ALS virus capsid protein VP2 in *E. coli* for immunoassay. In lane a, the purity of C-His-tagged ALSV-VP2 (lane 2) and bovine serum albumin (lane 1) was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In lane b, Western blot analysis of ALSV-VP2 using anti-His antibody (lane 2) was performed, with lane M representing the protein molecular weight marker.

[0063] Figure 8 As shown in the diagram, recombinant nucleoprotein (NP) of YEZV is expressed in Escherichia coli for immunoassay. In the diagram, a shows the purity of C-His-tagged YEZV-NP (lane 2) and bovine serum albumin (lane 1) assessed using SDS-PAGE; b shows Western blot analysis of YEZV-NP using anti-His antibody (lane 2), with lane M representing the protein molecular weight marker.

[0064] Figure 9 The image shows the expression of the recombinant envelope protein (EP) of tick-borne encephalitis virus (TBEV) in Escherichia coli for immunoassay. In image a, the purity of C-His-tagged TBEV-EP (lane 2) and bovine serum albumin (lane 1) was assessed using SDS-PAGE; in image b, Western blot analysis of TBEV-EP using anti-His antibody (lane 2), with lane M representing the protein molecular weight marker.

[0065] Figure 10 To facilitate immunoassay, recombinant nucleoprotein (NP) of SFTSV was expressed in Escherichia coli. In example, (a) SDS-PAGE was used to assess the purity of C-His-tagged SFTSV-NP (lane 2) and bovine serum albumin (lane 1); (b) Western blot analysis of SFTSV-NP using anti-His antibody (lane 2), with lane M representing the protein molecular weight marker. Detailed Implementation

[0066] This invention discloses a chip for the simultaneous detection of inflammatory markers and viral antibodies based on thermally annealed gold nanoparticles and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0067] Acute-phase proteins, such as C-reactive protein (CRP) and serum amyloid A (SAA), are key biomarkers for systemic inflammation induced by viral infection. Meanwhile, serotyping of virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) is crucial for differentiating between the acute and convalescent phases. Current serological methods, such as enzyme-linked immunosorbent assays (ELISA), often lack sufficient early detection sensitivity, require large sample volumes, and hinder multiplex analysis of host inflammatory responses and pathogen-specific antibodies. Recent advances in plasma-enhanced fluorescence substrates have enabled highly sensitive multiplexed detection.

[0068] This invention develops a substrate based on heat-annealed gold nanoparticles (TA-GNP) for the simultaneous quantification of acute-phase proteins (CRP / SAA) and detection of antibodies against pathogenic tick-borne viruses. Four representative viruses were selected in the examples: TBEV, Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), Alonshan Virus (ALSV), and Yezo Virus (YEZV). TBEV and SFTSV are considered the most pathogenic and clinically significant viruses in China, while ALSV and YEZV are classified as emerging pathogens with epidemic potential. The TA-GNP platform utilizes dense gold nanopores to achieve 25.3-fold and 37.6-fold fluorescence enhancement for CRP and SAA, respectively. Ultra-low concentrations (43.375 fM) of CRP can be detected using only 1 μL of human serum. Clinical validation of 66 serum samples showed that the sensitivity and specificity for CRP were both 100%, exhibiting strong correlation with clinically available methods (R0.05). 2 =0.874). This platform also amplified IgG and IgM antibody signals by 26.4-fold and 27.7-fold, respectively, with a detection limit of 0.625 ng / mL for rabbit-derived antibodies. -1 In a clinical cohort study, the TA-GNP biosensor differentiated 23 patients infected with tick-borne viruses from healthy controls by elevating CRP / SAA levels, demonstrating its application in inflammation profile analysis and early infection screening. By seamlessly integrating acute-phase protein quantification and viral detection into a single “screening and typing” paradigm, this technology provides a transformative diagnostic tool for rapid clinical decision-making during emerging viral outbreaks.

[0069] Specifically, the present invention provides a multiplex detection chip for inflammatory markers (CRP, SAA) and tick-borne virus antibodies (IgG / IgM) in serum, comprising: a substrate based on thermally annealed gold nanoparticles (TA-GNP), an inflammatory marker detection method, a tick-borne virus antibody detection method, and a multiplex detection chip.

[0070] In one example, the preparation method of the TA-GNP substrate is as follows:

[0071] Glass slides were treated with piranha solution (H2SO4:H2O2 = 3:1) and heated at 120°C for 1 hour. They were then immersed in a 35 nm gold nanoparticle (GNP) suspension, and a GNP monolayer was formed through self-assembly at the water / oil interface. The membrane was then transferred to a glass slide and thermally annealed at 300°C for 1 hour (heating rate 10°C / min) to form dense nano-interstic gaps. The advantages of the TA-GNP substrate prepared by this method include: the thermal annealing process forms dense nano-interstic gaps, significantly enhancing the fluorescence signal; multiplex detection array design: capable of simultaneously printing inflammatory marker antibodies and viral antigens; micro-sample detection capability: requiring only 1 μL of serum, suitable for limited blood sample populations; high sensitivity and specificity: fluorescence enhancement of 25–37 times, demonstrating excellent clinical validation performance.

[0072] In one example, the antibody / antigen microarray printing method is as follows:

[0073] Using a Nanoplotter 2.1 printer, spotting volume was 5 nL; CRP (50 µg / mL) and SAA (100 µg / mL) capture antibodies were printed; ALSV, YEZV, TBEV, and SFTSV viral antigens (100 µg / mL) were printed.

[0074] In one example, the detection process is as follows:

[0075] Add 1 μL of serum sample to the chip subunit; incubate for 30 minutes, wash; add IR800-labeled detection antibody (CRP / SAA) or secondary antibody (viral antibody); incubate again, wash, and dry with nitrogen; acquire fluorescence images using a MidaScan scanner (excitation wavelength 670 / 785 nm); analyze the fluorescence signal using software, plot the calibration curve, and calculate the LOD.

[0076] In the example, the detection performance is:

[0077] CRP detection limit: 43.375 fM; SAA detection limit: value not specified, but fluorescence enhancement was 37.6-fold; Viral antibody detection limit: 0.625 ng / mL (ALSV); Clinical sample validation: CRP detection sensitivity / specificity was 100% in 66 serum samples.

[0078] In practical implementation, the present invention has the following advantages:

[0079] I. TA-GNP substrate preparation method: thermal annealing process to construct fluorescence-enhancing core

[0080] The TA-GNP substrate is prepared using an innovative thermal annealing process, which can form a dense nano-gap structure, providing key support for fluorescence signal enhancement.

[0081] II. Multi-detection array design: Precisely prints multiple types of probes to achieve simultaneous detection of "inflammation + virus".

[0082] Leveraging the superior performance of the TA-GNP substrate, this invention designs a microarray chip capable of simultaneously detecting CRP, SAA, and multiple viral antibodies. The detection array is constructed on the surface of the TA-GNP substrate using inkjet printing technology, dividing the detection area into multiple independent units, and precisely printing different probes according to the detection target.

[0083] The core advantages of this array design are: first, it achieves "one chip, multiple indicators" detection through modular layout, simultaneously capturing CRP, SAA, and multiple viral antibodies without the need to replace the chip or add samples multiple times; second, each detection unit is independently separated, avoiding cross-contamination between probes and ensuring detection specificity. Compared with traditional single-detection chips (such as ELISA chips that can only detect CRP), the microarray chip of this invention significantly improves detection efficiency and can flexibly adjust the types of viral antigens according to clinical needs, adapting to the detection requirements of different infection types. The probe combination (inflammatory antibody + viral antigen), printing layout, and adaptation method with the TA-GNP substrate of this microarray chip are all unique.

[0084] III. Microsample Detection Capabilities and Methods: High-efficiency detection with 1 μL of serum, preserving the complete detection process.

[0085] This invention enables the detection to be completed with only 1 μL of serum. The innovation and protective value of this detection method are reflected in the establishment of a standardized process of "micro-sample dilution-rapid binding-efficient washing", which ensures that only 1 μL of serum sample is needed to meet the detection requirements and is suitable for groups with limited blood samples, such as newborns and the elderly. From a clinical application perspective, this method can reduce the physiological impact of blood collection on patients while improving detection efficiency. Its complete operation process and parameter settings have clear technical barriers.

[0086] IV. High Sensitivity and Specificity: Breakthrough in Fluorescence Enhancement Performance, Focusing on Early Diagnostic Applications of Tick-borne Viruses

[0087] Leveraging the fluorescence enhancement effect (25-37 times) of the TA-GNP substrate and precise array design, this invention achieves high sensitivity and specificity in detection, with its application in the early diagnosis of tick-borne virus infection being a key area of ​​focus. Performance validation data shows that the chip has a detection limit of 40 fM for CRP and 0.625 ng / mL for tick-borne virus antibodies. In clinical validation, the CRP detection results of 66 clinical cases showed that both sensitivity and specificity reached 100%, with no false negatives or false positives. Its application value and protective significance in the early diagnosis of tick-borne virus infection lies in the fact that patients with early-stage tick-borne virus infection (1-3 days after infection) often present with atypical symptoms and low antibody concentrations, making them prone to missed diagnoses using traditional techniques. This invention, however, can capture low-concentration antibody signals with high sensitivity, enabling early diagnosis. Simultaneously, the chip can detect CRP, SAA (to assess the degree of inflammation), and tick-borne virus antibodies (to clarify the type of infection), providing a basis for clinical development of combined "antiviral therapy + anti-inflammatory intervention" regimens. From a technical protection perspective, the application scenarios and performance advantages of this chip in the diagnosis of specific diseases (tick-borne virus infection) are key features that distinguish it from general detection technologies.

[0088] In summary, the TA-GNP-based multiplex detection chip, through the unique optical properties of gold nanoparticles and targeted modification technology, constructs an ultrasensitive detection system capable of simultaneously and accurately detecting two inflammatory markers, CRP (C-reactive protein) and SAA (serum amyloid A), as well as viral antibodies. Its core advantage lies in the efficient coupling of specific antibodies by the thioglycolic acid groups on the TA-GNP surface, forming a "gold nanoparticle-antibody" composite probe. When the probe binds to the target substance in the sample, it triggers the aggregation effect of the gold nanoparticles, producing a significant change in optical signal. Compared to traditional detection technologies, this chip improves the marker recognition capability by 1-2 orders of magnitude, accurately capturing signals even in the early stages of viral infection (antibody titers as low as 10 IU / mL), effectively avoiding false negatives due to low concentrations and providing a reliable basis for early clinical diagnosis.

[0089] The detection chip of this invention requires only 1 μL of serum sample to complete the entire detection process, achieving an "order-of-magnitude reduction" in sample volume compared to the 5-10 mL required by traditional technologies. In clinical applications, for special groups with limited blood samples (such as newborns, premature infants, elderly and frail patients, and patients in intensive care units), samples can be obtained simply by finger-prick blood collection or micro-venipuncture, avoiding the risks of anemia and vascular damage that may be caused by large-volume blood collection. Taking neonatal infection detection as an example, traditional technologies require drawing 2-3 mL of venous blood (approximately 0.5%-1% of the total blood volume of a newborn), while the TA-GNP detection chip only requires 1 μL of serum, greatly reducing the impact of blood collection on the physiological state of the newborn, while improving the convenience of clinical testing and patient compliance.

[0090] The TA-GNP detection chip achieves a 25-37 fold increase in fluorescence signal through the localized surface plasmon resonance (LSPR) effect of gold nanoparticles, effectively solving the problems of weak signals and susceptibility to background interference in low-concentration samples in traditional detection techniques. During the detection process, when the "gold nanoparticle-antibody" probe binds to the target biomarker, the gold nanoparticles amplify the emission signal of the fluorescent molecules through the LSPR effect, while suppressing non-specific background fluorescence, resulting in a signal-to-noise ratio improvement of more than 30 times.

[0091] This detection chip, through its multi-channel detection area design, constructs an integrated "screening-typing" diagnostic mode. It can simultaneously complete inflammation screening and viral infection typing in a single test, completely changing the traditional technology that requires multiple tests and cumbersome procedures. For example, in the diagnosis of respiratory infection patients, the traditional process requires first performing inflammatory marker detection (about 1 hour) and then viral antibody typing detection (about 2 hours), with a total time exceeding 3 hours. The TA-GNP detection chip, however, completes both tests simultaneously, directly outputting a comprehensive diagnostic report of "CRP concentration 20 mg / L (indicating moderate inflammation) + viral IgM positive (indicating recent infection)". This helps doctors quickly determine the patient's infection type and inflammation level, and rapidly formulate a treatment plan. Especially in public health emergencies or emergency treatment scenarios, it can significantly improve diagnostic efficiency and secure the best treatment opportunity for patients.

[0092] Terms and abbreviations involved: TA-GNP: Thermally Annealed Gold Nanoparticles; CRP: C-Reactive Protein; SAA: Serum Amyloid A; IgG / IgM: Immunoglobulin G / M; LOD: Limit of Detection.

[0093] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0094] The present invention will be further illustrated below with reference to the embodiments.

[0095] Example 1: Fabrication of a patterned protein detection chip

[0096] Preparation technology route such as Figure 1 As shown.

[0097] Add 2 mL of 1% (w / v, 1 g / 100 mL) chloroauric acid solution to 198 mL of deionized water and boil. Then add 1 mL of 2% (w / v, 2 g / 100 mL) sodium citrate dihydrate and maintain boiling for 20 min to obtain a 40 nm gold nanoparticle colloidal solution. Place a glass substrate (2.5 × 7.5 cm) on the substrate. 2 The gold nanoparticles were placed in a 90 mL colloidal solution, and then 20 mL of n-hexane was added to form an oil-water interface. Ethanol was then continuously injected, and a dense gold nanoparticle monolayer film was formed at the interface. The solution was then discharged from the bottom of the container, and the gold nanoparticle monolayer film was transferred onto a glass substrate. Finally, the substrate was placed in a tube furnace and annealed in an argon atmosphere for 1 hour at a heating rate of 10 °C / min to an annealing temperature of 300 °C, yielding the TA-GNP substrate.

[0098] Example 2: Detection of CRP and SAA levels on TA-GNP substrates

[0099] Using a Nanoplotter 2.1 printer, CRP (Abcam, 50 µg mL) was printed. -1 ) and SAA (Origene, 100 µgmL -1 Capture antibody microarrays were printed on TA-GNP substrates. After drying at ambient temperature for 2 hours, each substrate was divided into 16 subunits using a FRAME device (Nirmidas). Subunits were then blocked with 80 µL of 5% BSA (Sigma) for 30 minutes, followed by washing. Then, 60 µL of standard solution or serum sample was added and incubated for 30 minutes. After washing, 60 µL of IR800-labeled CRP (Abcam, 1.5 µg / mL) was added. -1 ) and SAA (Origene, 1.5 µg mL -1 The antibody mixture was incubated for 30 minutes. Finally, the substrate was washed, dried under a nitrogen stream, and imaged.

[0100] Experimental results and analysis:

[0101] Figure 2 Figures a to d in the diagram illustrate the principle and microscopic structure of the detection chip of this invention. To verify the serum detection capability of the TA-GNP platform, a specific detection protocol for C-reactive protein (CRP) was established and validated using clinical serum samples. Figure 4 (a) First, the sensitivity of the TA-GNP platform for CRP detection was evaluated by serially diluting CRP and measuring the resulting fluorescence signals. The results showed that the TA-GNP platform had a detection limit (LOD) as low as 43.375 fM for CRP, indicating its potential for high-sensitivity detection. Figure 4(b, c in the text).

[0102] Subsequently, the established detection protocol was applied to a cohort of 66 clinical serum samples, including 56 positive and 8 negative samples. Notably, the TA-GNP platform demonstrated 100% specificity and sensitivity in CRP detection, showing a significant correlation with the conventional clinical method (immunoturbidimetry) used for comparison, with a correlation coefficient of 0.874, confirming the reliability of the method. Figure 4 (d to f in the middle).

[0103] To achieve simultaneous detection of CRP and SAA from a single serum sample, the specificity of these two biomarkers was validated. Standard solutions of CRP and SAA were introduced into each subunit, followed by incubation and washing to remove unbound components. A mixed detection antibody (targeting both biomarkers and conjugated with IR800) was then applied for fluorescent labeling. Subsequent fluorescence signal analysis showed that the TA-GNP substrate produced significantly brighter fluorescence compared to the glass substrate, with a 25.3-fold increase in CRP signal and a 37.6-fold increase in SAA signal. Figure 2 (e and f in the original text). Furthermore, TA-GNP exhibits excellent specificity in the detection of CRP and SAA, demonstrating its robust performance as a biosensing platform. Figure 2 These results highlight the potential of TA-GNP substrates for ultrasensitive quantification of proteins in the acute phase, paving the way for their application in rapid clinical diagnosis.

[0104] This study focused on a group of 19 patients who were confirmed by PCR to be infected with novel viruses (including ALSV, YEZV, TBEV, and SFTSV), all of whom were diagnosed after tick bites. These results were compared with a control group of 19 healthy individuals. Figure 5 'a' in the diagram is a schematic representation.

[0105] Fluorescence imaging analysis results as follows Figure 5 As shown in b to d, in patient samples diagnosed with viral infection (labeled P1-P19), significantly elevated fluorescence signals corresponding to CRP and SAA were observed, indicating that the viral pathogen triggered a strong inflammatory response. In contrast, the fluorescence signals of these inflammatory markers were weaker in the serum of healthy controls, highlighting the specificity and sensitivity of the detection method of this invention.

[0106] Example 3: Detection of newly emerging viral antibody standards on TA-GNP substrate

[0107] On TA-GNP substrate (100 µg mL) -1Antigen microarrays of ALSV, YEZV, TBEV, and SFTSV were printed on the substrate. After drying for 2 hours, the substrate was divided into 16 subunits using a FRAME device. Subunits were blocked with 80 µL of 5% BSA for 30 minutes and then washed. 60 µL of rabbit antibody standard was then incubated in each subunit for 30 minutes. After washing, anti-rabbit IgG (Licor, 1 µg / mL) bound to IR800 was... -1 Incubate for 30 minutes and detect the bound antibodies. Finally, wash the substrate, dry it with nitrogen, and image it.

[0108] Experimental results and analysis:

[0109] The printed antigen array was incubated with rabbit-derived specific antibodies against ALSV, YEZV, TBEV, and SFTSV. After thorough washing, fluorescence detection was performed using secondary antibodies labeled with IR800. Figure 3 'a' in the diagram is a schematic representation. The resulting fluorescence images and thermograms reveal the excellent specificity of the TA-GNP platform in detecting antibodies against these emerging viruses. Figure 3 (d in the text)

[0110] To further validate the platform's performance, the rabbit-derived antibody was serially diluted. Notably, the limit of detection (LOD) for TA-GNP against ALSV antibody was as low as 0.625 ng / mL. Figure 3 (e). High sensitivity was also observed in the detection of YEZV, TBEV, and SFTSV antibodies. Figure 3 (f to h in the original text). This high-sensitivity combination platform's ability to distinguish different stages of infection highlights the potential of the multiplex antibody detection system of this invention. Overall, the integration of this technology provides an efficient and effective method for early diagnosis and accurate virus detection, enhancing the ability to respond rapidly to emerging viral threats.

[0111] Example 4: Detection of anti-IgG and anti-IgM on TA-GNP substrate

[0112] Human IgG (Thermo, 50 µg mL) -1 ) and IgM (Yarewell, 50 µg mL -1 The microarray was printed onto a TA-GNP substrate. After drying for 2 hours, the substrate was divided into 16 subunits using a FRAME device. The subunits were blocked with 80 µL of 5% BSA for 30 minutes and then washed. Then, IR800-labeled anti-human IgG (Licor) and Cy5-labeled anti-human IgM (Vector Laboratories) were coated and incubated for 30 minutes. The substrate was then washed, dried under nitrogen, and imaged.

[0113] Experimental results and analysis:

[0114] In addition to facilitating rapid screening, the ability to accurately detect viruses is crucial for managing emerging viral infections. The presence of specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies can provide key insights into the stages of viral infection, thus distinguishing between the acute and recovery phases of the disease. To this end, an antigen array containing antigens from ALSV, YEZV, TBEV, and SFTSV was designed and printed on the TA-GNP platform. This innovative approach enables the simultaneous detection of IgM and IgG antibodies against these four viruses.

[0115] The fluorescence signals generated by using anti-human IgG coupled with IR800 to detect IgG and anti-human IgM coupled with Cy5 to detect IgM on a glass substrate and a TA-GNP platform were compared and analyzed. The results showed that on the TA-GNP platform, the fluorescence enhancement of anti-human IgG-IR800 reached 26.4 times, and the fluorescence enhancement of anti-human IgM-Cy5 reached 27.7 times, highlighting the platform's excellent sensitivity and specificity. Figure 3 (b, c in the text).

[0116] In the above embodiments, the ALSV antigen is the ALSV-VP2 protein, and its amino acid sequence is as follows:

[0117] MRTLIYILAVAALTMGKPNGAPDWGKIELLGQQLQSGIAKKNAEKTRYYAELIIKELPTQEVTYYAGLRTGEEIALTAGAAKAIIGEAVNLRVNQWTRAVGEYLTIDRHIPGLTLLITGEDVLNDLMEFGW SEQ ID NO: 1).

[0118] In the above embodiments, the YEZV antigen is the YEZV-NP protein, and its amino acid sequence is as follows:

[0119] MARLIEAKTREELDKWIHNLDTTHFLGLNTKYTKSLALQPCEPGSCPEAYISFDGKKSDELDAILSGALVDSLKFAAPIPECAWNLTNKIFTEGMAWFNANKDQKCMAWDKKYDTLRSTLPSPEDVAQYQIAARKWRKDIGYECSPNNGIMRGEVTKVYIAPKKYVNDMILMVTDMRSKRRHRLGITEEQEQEALSRKGAEHAPWVEDWVKGGEDRIFSLPSWGGWNKKTKKGLLLGGTAVAHLVQKKVITLKIFRSKVEKAGELLNNQQKMDELGIDVSQARLMHAHMEGCLKEAEGLIKDSVTSAYVQQGSALDTAFSTYYWMWKADVTLANFGALNEMAFLYGQKPVGQKKLLDTLKGTAYKWGSTLANMCATGDFNGERVHMHPGVFTPHRMSEMTAAIGAFPLSNPVKIKEGSASYRYLTNLKTSESNPAAKVVCELFNTHRRAHSDWNSPTSIVPPEHYFHQSLLERLGPFCHVSLVRGNALRVNIVEEMGAGGTHHHHHH (SEQ ID NO: 2);

[0120] In the above embodiments, the TBEV antigen is the TBEV-EP protein, and its amino acid sequence is:

[0121] MSRCTHLENRDFITGTQGTTRVTLVLELGGCVTITAEGKPSMDVWLDSIYQENPAKTREYCLHAKLSDTKVVARCPTMGPATLAEEHQSGTVCKRDQSDRGWGNHCGLFGKGSIVTCVKASCEAKKKATGHVYDANKIVYTVKVEPHTGDYVAANETHSGRKTASFTVSSEKTILTMGDYGDVSLLCRVASGVDLAQTVILELDKTSEHLPTAWQVHRDWFNDLALPWKHEGAQNWNNAERLVEFGAPHAVKMDVYNLGDQTGVLLKSLAGVPVAHIDGTKYHLKSGHVTCEVGLEKLKMKGLTYTMCDKTKFTWKRIPTDSGHDTVVMEVAFSGTKPCRIPVRAVAHGSPDVNVAMLITPNPTIETNGGGFIEMQLPPGDNIIYVGELSHQWFQKGSSIGRVFQKTRKGIERLTVIGEHAWDFGSTGGFLTSVGKALHTVLGGAFNHHHHHH (SEQ ID NO: 3).

[0122] In the above embodiments, the SFTSV antigen is the SFTSV-NP protein, and its amino acid sequence is:

[0123] MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVRDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVEKAETRLSITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNLHHHHHH (SEQ ID NO: 4).

[0124] Figures 7-10 Shows the expression of the ALSV, YEZV, TBEV and SFTSV antigen proteins of the present invention.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detection chip based on thermally annealed gold nanoparticles, characterized in that, The detection targets are inflammatory markers and antiviral neutralizing antibodies, specifically including: (i) Thermally annealed gold nanoparticle substrate; and (ii) Capture antibodies against inflammatory markers; and (iii) Viral antigens; The capture antibody and the viral antigen are coated on the heat-annealed gold nanoparticle substrate; The heat-annealed gold nanoparticle substrate includes a glass substrate and a gold nanoparticle monolayer film. The inflammatory markers include C-reactive protein and / or serum amyloid A; The viral antigens include at least one of the following: antigens derived from tick-borne encephalitis virus, antigens derived from Alonshan virus, antigens derived from fever with thrombocytopenia syndrome virus, and antigens derived from Yezo virus.

2. The detection chip as described in claim 1, characterized in that, Its preparation methods include: Gold nanoparticle monolayers are formed through self-assembly at the oil-water interface. The gold nanoparticle monolayer film was transferred to a glass slide and thermally annealed to form dense nano-interstic gaps, thus obtaining the thermally annealed gold nanoparticle substrate. The detection chip is obtained by coating the capture antibody and viral antigen onto the heat-annealed gold nanoparticle substrate.

3. The detection chip as described in claim 2, characterized in that, The conditions for heat annealing are 300°C, 1 hour, and a heating rate of 10°C / min.

4. The detection chip as described in claim 2, characterized in that, The formation of a gold nanoparticle monolayer film through self-assembly at the oil-water interface includes: Chloroauric acid solution was mixed with deionized water and boiled, then mixed with sodium citrate dihydrate and kept boiling to obtain a colloidal solution of gold nanoparticles. Hexane and a colloidal solution of gold nanoparticles were placed in the same container to form an oil-water interface. Ethanol was then added, and a dense monolayer of gold nanoparticles was formed at the interface.

5. The detection chip as described in claim 4, characterized in that, The concentration of the chloroauric acid solution is 1%.

6. The detection chip as described in claim 4, characterized in that, The concentration of the sodium citrate dihydrate is 2%.

7. The use of the detection chip as described in any one of claims 1 to 6 in the preparation of products for diagnosing tick-borne virus infection.

8. The application as described in claim 7, characterized in that, The tick-borne viruses include at least one of tick-borne encephalitis virus, Arunshan virus, fever with thrombocytopenia syndrome virus, and Yezo virus.

9. A test kit, characterized in that, Includes the detection chip as described in any one of claims 1 to 6.

10. A detection system, characterized in that, Includes the detection chip as described in any one of claims 1 to 6.

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