Dynamic-driven portable dual-mode immunoassay platform for marking core-shell nanoparticles

Abstract

The invention belongs to the field of early disease diagnosis, and particularly relates to a core-shell nanoparticle labeled portable dual-mode immunoassay platform based on dynamic driving, which comprises an antibody-gold nanoparticle biological coupling system, an amplification system, a color development system and a colorimetric-atomic emission spectrum detection system. The Au-coated Cu core-shell nano-particles are dynamically driven to realize signal amplification, colorimetric preliminary screening and atomic emission accurate quantification are combined, CEA (0.02 ng / mL), cTnI (0.35 fg / mL) and A beta (10.3 pg / mL) can be detected in an ultra-sensitive manner, and the Au-coated Cu core-shell nano-particles are suitable for bedside detection of early diseases.

Description

Technical Field

[0001] This invention belongs to the field of early disease diagnosis, specifically relating to a portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling. Background Technology

[0002] In the field of disease diagnosis, early intervention is the core prerequisite for reducing the incidence and mortality of severe illness. The key to early diagnosis lies in the accurate capture of low-concentration disease biomarkers (such as carcinoembryonic antigen CEA, cardiac troponin IcTnI, and β-amyloid Aβ) in biological samples. Clinical data show that the serum CEA concentration of early-stage cancer patients is mostly below 5 ng / mL, and the cTnI content is below pg within 1 hour of an acute myocardial infarction. Moreover, the dynamic changes are very important. Similarly, the Aβ concentration in the cerebrospinal fluid of patients in the early stage of Alzheimer's disease is only at the pg level. The detection of these trace biomarkers poses a severe challenge to existing technologies.

[0003] Current mainstream detection technologies can be divided into two categories, but both have limitations that are difficult to overcome: one category is laboratory-level precision detection technologies, such as inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectrometry (ICP-OES), and enzyme-linked immunosorbent assay (ELISA). Among them, although ICP-MS / OES can achieve detection limits from pg to fg, the equipment is bulky (requiring several square meters of laboratory space), weighs over 50kg, and relies on a stable 220V power supply and professional operators, making it unsuitable for on-site testing scenarios such as primary healthcare and field rescue; although ELISA is relatively simple to operate, it relies on enzyme-catalyzed linear signal amplification, and the detection limit is mostly in the ng level, which is difficult to meet the needs of early biomarker detection, and it is easily affected by matrix components such as albumin and glutathione in serum, with a false positive rate as high as 8%~15%. Another type is point-of-care testing (POCT) technology, such as traditional colorimetric methods and fluorescence immunochromatography. Although these technologies have the advantages of portability (equipment weight <1kg) and low operating threshold, their sensitivity is generally insufficient. Colorimetric methods rely on visual identification of color changes, and the detection limit is mostly above 10ng / mL. Moreover, "color drift" is prone to occur in complex biological matrices. Although fluorescence methods can reduce the detection limit to 1ng / mL, the fluorescence signal is easily affected by ambient light and sample autofluorescence, resulting in poor stability.

[0004] In recent years, nanoparticle labeling technology has become a research hotspot for improving detection sensitivity. Its core lies in utilizing the optical / electrical properties of nanoparticles such as gold and silver to amplify signals. However, existing research faces two major bottlenecks: First, the signal amplification mechanism is limited. Most techniques only focus on the static morphological control of nanoparticles (such as particle size and shell thickness), rarely exploring the dynamic laws of core-shell structure growth, resulting in linear signal amplification that struggles to break through the sensitivity limit. Second, there is the challenge of balancing performance—pursuing high sensitivity requires introducing complex signal amplification modules (such as multi-step enzymatic reactions), sacrificing portability and detection speed; simplifying the process to adapt to POCT scenarios leads to a significant decrease in sensitivity. This triangular contradiction of "sensitivity-portability-reliability" has long constrained the application of POCT platforms in early disease diagnosis, necessitating the development of a novel immunoassay technology that simultaneously achieves high-sensitivity signal amplification, rapid on-site operation, and accurate result verification.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling, comprising an antibody-gold nanoparticle bioconjugation system, an amplification system, a colorimetric system, and a colorimetric-atomic emission spectroscopy detection system; wherein: The colorimetric-atomic emission spectroscopy detection system amplifies the signal of core-shell nanoparticles through kinetic driving, and combines colorimetric screening with precise quantitative detection by atomic emission spectroscopy.

[0007] Preferably, the preparation method of the antibody-nanoparticle bioconjugate system includes the following steps: (I) Preparation of gold nanoparticles The gold source solution is heated, and then a reducing stabilizer is added to obtain gold nanoparticles. (II) Preparation of antibody-gold nanoparticle bioconjugate system (II-1) The secondary antibody was added to the gold nanoparticles and stirred to react; (II-2) Continue adding bovine serum albumin and stirring to react; (II-3) Centrifuge and wash to obtain the antibody-gold nanoparticle bioconjugate system.

[0008] Preferably, in step (I): The gold source solution is a chloroauric acid solution with a concentration of 10-12%. And / or, the reducing stabilizer is a sodium citrate solution with a concentration of 1-2%.

[0009] Preferably, in steps (II-1) and (II-2), the temperature of the stirring reaction is 20-30°C, and the stirring reaction time is 30-40 min.

[0010] Preferably, in step (II-3), the centrifugal washing method is as follows: (1) Centrifuge at 2000-2500g for 20-30 minutes and retain the supernatant; (2) Centrifuge again at 8000-9000g for 30-40 minutes and retain the precipitate; (3) Finally, centrifuge and wash at 6000-7000g for 30-40 minutes. After resuspending the precipitate, the antibody-gold nanoparticle bioconjugate system is obtained.

[0011] Preferably, the amplification system comprises a copper sulfate solution and an L-ascorbic acid solution; wherein: The concentration of the copper sulfate solution is 80-120 mM; The concentration of the L-ascorbic acid solution is 20-30 mM.

[0012] Preferably, the colorimetric system comprises ferric chloride solution and potassium ferricyanide solution; wherein: The concentration of the ferric chloride solution is 2-4 mM; The concentration of the potassium ferricyanide solution is 2-4 mM.

[0013] Preferably, the colorimetric-atomic emission spectroscopy detection system includes an adjustable discharge system, a solid-structure microplasma discharge chamber, and wherein: The adjustable discharge system is used to adjust the height difference between the capillary inlet and the discharge end in order to achieve precise adjustment and control of the injection volume. The solid structure microplasma discharge chamber is used to generate a closed inert atmosphere to improve stability.

[0014] The beneficial effects of this invention are as follows: The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling provided by this invention fundamentally solves the core contradictions of "insufficient sensitivity, poor portability, and low reliability of results" in the background technology through innovative "kinetic signal amplification + dual-mode detection integration + miniaturized device design". It realizes ultra-sensitive on-site detection of early disease biomarkers and provides an efficient solution for early diagnosis in scenarios such as primary healthcare and field rescue.

[0015] 1. Nonlinear signal amplification, breaking through the sensitivity limit: To address the limitation of linear amplification in traditional nanoparticle labeling technology, this invention uses Au@Cu core-shell nanoparticles as signal markers. The growth of the copper shell follows a kinetically controlled, self-limiting growth pattern, enabling nonlinear signal amplification—compared to the traditional Au@Ag linear amplification system (CEA detection limit 0.9 ng / mL). [1] The detection limit of this platform for CEA has been reduced to 0.02 ng / mL, the detection limit for cTnI has reached 0.35 fg / mL, and the detection limit for Aβ has reached 10.3 pg / mL, with a significant improvement in sensitivity, fully meeting the trace detection needs of early disease biomarkers.

[0016] 2. Dual-mode detection synergy enhances result reliability: Addressing the shortcomings of single detection methods, this platform integrates dual-mode detection of "colorimetric initial screening + atomic emission precise quantification": The colorimetric mode achieves visually visualized semi-quantification through the Prussian blue colorimetric reaction (color gradient change from yellow to blue), and can complete on-site initial screening within 5 minutes; the atomic emission mode (based on miniaturized CLEGD-OES), with its closed inert atmosphere design and precise sample introduction control, significantly improves its resistance to matrix interference, and can perform laboratory-level precise quantification of positive samples in the initial screening. Cross-validation of the two modes significantly reduces the false positive rate, solving the problem of low reliability of traditional POCT results.

[0017] 3. Miniaturized design for adaptable field testing scenarios: Addressing the issue of poor portability of large precision instruments, this platform features core optimizations to the testing system: The adjustable discharge system achieves precise control of the sample injection volume by controlling the height difference of the capillary; the volume of the solid structure micro-plasma discharge chamber is reduced while maintaining a closed inert atmosphere; the overall equipment is lightweight and supports external lithium battery power supply, enabling use in fields and primary healthcare facilities without a stable power grid, filling the gap in the application of high-sensitivity detection technology in field scenarios.

[0018] 4. Stable and versatile process, lowering the application threshold: Addressing the issues of poor stability and narrow applicability of nanoparticle conjugation systems, this invention clarifies the key parameters for antibody-gold nanoparticle conjugation (such as stirring at 20~30℃ for 30~40 min and three-step gradient centrifugation). The conjugation system can be stably stored at 4℃ for more than 15 days. At the same time, the platform can detect multiple biomarkers such as CEA, cTnI, and Aβ by changing different antibodies. Serum samples only require simple ultrafiltration treatment (centrifugation at 8000 rpm for 15 min) before being used, without the need for professional pretreatment equipment, which greatly lowers the threshold for clinical application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of a portable colorimetric / capillary-microplasma spectroscopy system.

[0021] Figure 2 This is a schematic diagram of a sandwich immunoassay method based on signal amplification induced by one-step copper deposition.

[0022] Figure 3 (A) is a schematic diagram of Au@Cu synthesis; Figure 3 (B) is the UV-Vis absorption spectrum: a is labeled gold nanoparticles with antibodies + copper deposition reagent; b is labeled gold nanoparticles with antibodies + deionized water; c is carbonate buffer solution + copper deposition reagent; Figure 3 (C) shows the UV-Vis spectra of gold nanoparticles and Au@Cu nanolabels; Figure 3 (D) is a transmission electron microscope and mapping image of gold nanoparticles and Au@Cu nanolabels; Figure 3 (E) is the in-situ time-resolved UV-Vis absorption spectrum used to monitor the formation of Au@Cu.

[0023] Figure 4 (A) is 0–1.5 mM Fe 2+ Color photographs of solutions; Figure 4 (B) is Fe after adding 3mM K3[Fe(CN)6] 2+ Color photographs; Figure 4 (C) represents Fe at different concentrations 2+ The UV-Vis absorption spectrum of 3mM K3[Fe(CN)6] in the (0-1.5mM) system. Figure 4 (D) is the emission line of Cu atoms (324.8 / 327.4 nm); Figure 4 (E) is the curve of Cu atom emission intensity changing with time.

[0024] Figure 5 The calibration curves for Cu element are (A) ICP-OES and (B) portable CLEGD-OES system.

[0025] Figure 6 The optimal conditions for the immune response are: (A) immune response time; (B) coating antibody concentration; and (C) labeled antibody concentration.

[0026] Figure 7The optimization of copper deposition conditions includes: (A) Copper deposition solution concentration: a) CuSO4; b) AA; (B) Copper amplification time; and (C) Copper deposition solution concentration.

[0027] Figure 8 This is a dual-mode detection scheme for multiple biomarkers. The inset of the calibration curves for CEA (A), cTnI (C), and Aβ (E) in the contrast colorimetric detection mode is a corresponding photograph under natural light; the calibration curves for CEA (B), cTnI (D), and Aβ (F) in the portable CLEGD-OES detection mode; (G) Flowchart for on-site detection of cTnI in serum using portable atomic spectroscopy. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example

[0029] (a) The main reagents, experimental equipment and characterization equipment required for this embodiment are shown in Table 1, Table 2 and Table 3 respectively.

[0030] Table 1 Main Reagents

[0031] Table 2 Experimental Equipment

[0032] Table 3 Characterization equipment

[0033] (II) Experimental Methods 2.1 Solution Preparation (i) Coating buffer, 0.05M sodium carbonate / sodium bicarbonate buffer (pH=9.6, 0.26 g Na2CO3 and 0.34 g NaHCO3 dissolved in 100 mL deionized water); (ii) Phosphate-buffered saline (PBS), 0.01 M, pH 7.4 (1.095 g of Na2HPO4•2H2O, 0.60 g of NaH2PO4•2H2O and 8.76 g of NaCl were dissolved in 1 L of water). (iii) Blocking buffer / detection buffer: Dissolve 0.10 g of BSA in 10 mL of 0.01 M PBS and store at 4 °C. (iv) Washing buffer: 50 μL of Tween20 dissolved in 100 mL of 0.01 M PBS; (v) Copper amplification solutions, solution A (100 mM CuSO4) and solution B (25 mM AA), are prepared fresh and used immediately; (vi) Colorimetric solutions: colorimetric solution A (3mM FeCl3) and colorimetric solution B (3mM K3Fe(CN)6), prepared fresh and used immediately; (vii) Serum samples were first centrifuged in a 100kDa ultrafiltration tube (8000 rpm, 15 min) to remove interfering substances (such as GSH, HSA, etc.), and then the filtrate was diluted 100 times and sandwich immunoassay was performed.

[0034] 2.2 Preparation of gold nanoparticles (AuNPs) First, 50 μL of a 10% chloroauric acid (HAuCl4) solution was added to the flask and heated to boiling. Then, 4 mL of a 1% sodium citrate solution was rapidly added while stirring continuously. The solution changed from colorless to wine red within minutes, and was kept at boiling for 30 minutes with continuous stirring. The resulting gold nanoparticles were cooled to room temperature and stored at 4°C.

[0035] 2.3 Preparation of antibody-AuNPs (Ab2-AuNPs) bioconjugates Adjust the pH of 50 mL of the above gold nanoparticle system to 8.0 using 0.2 mol / mL Na₂CO₃. Dissolve 500 μg of secondary antibody in 5 mL of PBS and adjust the pH to 7.4. Then, add the pH-adjusted secondary antibody to the gold nanoparticle system and stir at room temperature for 30 min to couple AuNPs with the secondary antibody. Next, add 5 mL of 10% BSA and stir at room temperature for 30 min. After coupling, centrifuge and wash the mixture. First, centrifuge at 2000 g for 20 min and retain the supernatant; second, centrifuge at 8000 g for 30 min and retain the precipitate; third, centrifuge at 6000 g for 30 min and repeat three times. The precipitate is resuspended in PBS for subsequent use.

[0036] 2.4 Construction of a portable colorimetric-atomic emission spectroscopy (CLEGD-OES) detection system Figure 1A detailed schematic diagram of a portable colorimetric / capillary liquid electrode glow discharge-atomic emission spectroscopy (CLEGD-OES) system is shown. To further improve the stability and analytical performance of the entire device, the following modifications were made: First, the discharge system was changed from a fixed to an adjustable type. This allows for precise adjustment and control of the sample injection volume by adjusting the height difference between the capillary inlet and discharge ends. Excessive sample injection volume per unit time results in excess moisture remaining in the discharge chamber, creating water mist that interferes with spectral detection; insufficient injection volume per unit time disrupts the continuity of microplasma excitation and may even extinguish the microplasma. Adjusting the height difference between the capillary inlet and discharge ends improves the sensitivity and stability of the microplasma. Second, except for the visible area of ​​the discharge section, the microplasma discharge chamber was changed from a hollow structure to a solid structure. The advantages of this design include generating a closed, inert atmosphere, especially at low flow rates, reducing the possibility of microplasma contact with the external atmosphere, improving equipment safety, and reducing the impact of air fluctuations on microplasma stability. Third, an integrated colorimetric system enables qualitative identification of the target analyte. The fourth modification is that the HNO3 in the buffer system, originally at pH=3, has been replaced with HNO3 at a volume concentration of 5%. + The concentration of HNO3 is crucial for maintaining the stability of plasma discharge. In addition, 5% HNO3 was introduced into the fused silica capillary using a 5mL syringe for cleaning purposes.

[0037] The capillary tube is used for sample introduction. Microplasma in the discharge chamber excites characteristic elements to emit corresponding atomic emission spectra, which are then detected by a charge-coupled device (CCD) for quantitative analysis. A booster controls the electrode voltage, and a battery controls the power supply for the entire device. The lifting platform and discharge chamber are adjustable, allowing for precise adjustment and control of the sample introduction volume by adjusting the height difference between the capillary's sample introduction and discharge ends. Excessive sample introduction per unit time results in excess moisture remaining in the discharge chamber, creating water mist that interferes with spectral detection; insufficient introduction per unit time disrupts the continuity of microplasma excitation, potentially extinguishing the microplasma. Furthermore, different metals require different amounts of energy to be excited; adjusting the height of the two electrodes in the lifting platform and discharge chamber allows for the generation of different energies. The portable power supply only controls the power to the immunoassay platform, which contains a 96-well plate for sample incubation and the immunoassay reaction. A temperature controller manages the temperature of the immunoassay platform.

[0038] The colorimetric platform is located at the back of the device and is equipped with a colorimetric system for signal comparison to achieve semi-quantitative analysis of the target analyte. The sample cell contains a 96-well plate with metal particles introduced into it to provide the sample.

[0039] 2.5 Study on the growth kinetics of Au@Cu core-shell structure To investigate the growth kinetics of copper shells on gold nanoparticles, 1 mL of gold nanoparticle solution and 500 μL of copper sulfate solution were vortexed in a glass bottle. An equal volume of ascorbic acid solution was then added and thoroughly mixed. The reaction mixture was immediately transferred to a microcuvette, and the growth kinetics were monitored using in-situ time-resolved UV-Vis absorption spectroscopy.

[0040] 2.6 Immunoassay Protocol Figure 2 This study demonstrates the principle of immunoassay based on signal amplification induced by Au@Cu core-shell nanostructures. A signal amplification strategy for metal core-shell nanostructures suitable for colorimetric / capacitively coupled liquid electrode glow discharge-emission spectroscopy (CLEGD-OES) platforms was developed, and a microplate-based sandwich immunoassay was used to detect carcinoembryonic antigen (CEA), cardiac troponin I (cTnI), and β-amyloid 42 (Aβ-42).

[0041] Step 1 (Antibody Immobilization and Blocking): The captured antibody was immobilized on a 96-well plate by physical adsorption. The plate was then triple-washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20.

[0042] Step 2: Add 380 μL of blocking buffer to each well to saturate unbound active sites.

[0043] Step 3 (Antigen Binding and Incubation): Add 50 μL of standard solution or sample and 100 μL of antibody-gold nanoparticle bioconjugate (Ab(2)-AuNPs) to the designated wells and incubate at 37°C for 2 hours to promote antigen-antibody binding. After incubation, wash three times with washing buffer, then wash with ultrapure water, and then soak in ultrapure water for 1 hour to completely remove non-specifically bound antibodies.

[0044] Step 4 (Copper-enhanced signal amplification): Add 50 μL of copper enhancement solution A and solution B, and incubate at 37°C for 8 minutes to allow copper to catalytically deposit on the gold nanoparticle label. After amplification, terminate the reaction and remove residual reagents by rigorous washing. Dual-mode detection: For colorimetric detection, add 50 μL of colorimetric solution A (3 mM FeCl3) and colorimetric solution B (3 mM K3[Fe(CN)6]) to each well sequentially to form a gradient color development for visual reading. The formation of Prussian blue enables semi-quantitative analysis of the target concentration. For atomic spectroscopy detection, digest with 50% concentrated nitric acid for 10 minutes, and then record the atomic emission spectrum signal through capillary injection.

[0045] (III) Results and Discussion 3.1 Preparation and Characterization of Au@Cu Nanolabels Figure 3A demonstrates the process of forming gold-core copper-shell (Au@Cu) nanoparticles via an adsorption-reduction step. The successful formation of the core-shell structure is confirmed by UV-Vis absorption spectroscopy: a characteristic absorption peak of copper appears at 710 nm. Figure 3 B), the characteristic absorption peak of gold nanoparticles at 520 nm disappears ( Figure 3 C), the solution color changed significantly from wine red to gray, indicating that the outer copper shell effectively masked the gold core. Subsequent studies showed that gold nanoparticles, acting as a catalyst, significantly accelerated the reduction and deposition of copper, thereby enhancing the macroscopic colorimetric effect and strongly validating the feasibility of this signal enhancement strategy. The morphology of the synthesized gold nanoparticles and the resulting Au@Cu composite was characterized by transmission electron microscopy. Figure 3 D). The original gold nanoparticles are spherical with a diameter between 21-23 nm. Kinetic-driven reduction reactions promote Cu... 2+ Specific adsorption occurs on the surface of gold nanoparticles. Subsequently, a uniform copper shell is formed through ascorbic acid (AA)-mediated reduction and in-situ spontaneous growth, resulting in a slight increase in particle size. This phenomenon is consistent with dynamic light scattering (DLS) results.

[0046] 3.2 Signal Amplification Effect of the Dynamic Growth Model The signal amplification characteristics of the kinetic growth model were quantitatively evaluated by comparing the nonlinear Au@Cu system with traditional linear amplification systems such as Au@Ag. The Au@Cu system achieved a detection limit of 0.02 ng / mL for CEA, more than 40 times lower than the Au@Ag system (0.9 ng / mL). This enhancement stems from the nonlinear growth kinetics of diffusion-controlled copper deposition, which differs from the linear response of traditional systems. Theoretically, Cu shell growth follows a kinetically controlled, self-limiting process, enabling ultra-trace detection of cTnI down to 0.35 fg / mL. This well-defined kinetic behavior supports the construction of a highly sensitive dual-mode detection platform combining visual semi-quantitative analysis and atomic emission spectroscopy.

[0047] 3. Principle and Feasibility of Dual-Mode Biodetection The colorimetric detection mode in this study is essentially based on the coupling of copper signal amplification and the classic Prussian blue reaction, which can convert the concentration of the target biomarker into a visually perceptible color gradient. Following the sandwich immunoassay and copper signal amplification steps detailed in the experimental section, the amount of copper deposited on the gold nanoparticle label is directly proportional to the target concentration (e.g., CEA, cTnI, or Aβ). The colorimetric principle involves two consecutive reactions: 1. In-situ generation of Cu 2+ and Fe 2+ Substances similar to: metallic copper (Cu) deposited on nanoparticle markers 0 Fe in the colorimetric solution 3+Oxidation with (3mM FeCl3) results in in-situ formation of Cu. 2+ and Fe 2+ : Cu 0 +2Fe 3+ →Cu 2+ +2Fe 2+ ; 2. Formation of indigo: The generated Fe... 2+ The ions immediately react with ferric tricyanide (III) ions (K3[Fe(CN)6]) in the solution to form insoluble indigo (Fe4[Fe(CN)6]3): 4Fe 2+ +3[Fe(CN)6] 3− →Fe4[Fe(CN)6]3↓ like Figure 4 A, Figure 4 As shown in B, Fe was subjected to different concentrations... 2+ Adding K3[Fe(CN)6] to the solution produces a concentration-dependent color gradient: from yellow (lower [Fe] ) to ... 2+ The color transitions from green to blue (high [Fe]) and eventually turns blue. 2+ This indicates that the catalytic formation process of Prussian blue (Fe4[Fe(CN)6]3) can be read visually and spectrophotometrically. Ultraviolet-Visible spectroscopy (UV-Vis) Figure 4 C) Confirmation: With Fe 2+ With increasing concentration, the absorbance at 710 nm increases sharply—a characteristic of Prussian blue formation—while the absorbance of K3[Fe(CN)6] at 415 nm decreases only slightly. The absorbance at 710 nm and Fe... 2+ A strong linear correlation was observed between concentrations, indicating that the intensity of the generated blue color is directly related to the amount of deposited copper. This copper amount is non-linearly amplified through a kinetically regulated growth process and is quantitatively correlated with the initial target concentration. This dual-reaction mechanism firmly links the amplified copper signal to the target, providing a reliable basis for semi-quantitative on-site screening.

[0048] For atomic emission spectrometry detection, a microcapillary liquid electrode glow discharge photoemission spectroscopy (CLEGD-OES) system was used to achieve highly sensitive quantitative detection. This method utilizes a low-power (≤30W), low-voltage (~500V) micro-plasma generated between the liquid electrode and the ground electrode to effectively excite copper atoms released from the amplified immune complex. Quantitative analysis was achieved by monitoring the characteristic atomic emission spectrum of copper (wavelength 327.7nm). For atomic emission spectrometry detection, a dual-mode immunoassay device was used for quantitative detection. This device recorded the characteristic atomic emission spectrum of copper at 324.8 / 327.4nm. Figure 4D), and the spectral time curve of copper atom emission at a wavelength of 324.8 nm ( Figure 4 E).

[0049] 3.5 Copper calibration curves for ICP-OES and CLEGD-OES Under optimal conditions, ICP-OES and CLEGD-OES calibration curves for copper were constructed using a series of elemental standard solutions. Figure 5 The linear range exceeds 5 orders of magnitude, R 2 >0.999.

[0050] 3.6 Optimization of Immunoassay Parameters Immunoassay conditions included the concentrations of coating and labeled antibodies, and the incubation time. All conditions were optimized for best analytical performance. In this study, the detection of β-amyloid protein used antibodies and antigens from a commercial kit; therefore, antibody concentration optimization was not involved. Consequently, the optimization of immunoassay parameters was based on CEA as a reference.

[0051] Incubation time is crucial for achieving high analytical performance. Firstly, the immunoreaction time was optimized (including 20, 40, 60, 80, and 100 min). For example... Figure 6 As shown in Figure A, the signal intensity increases significantly with increasing incubation time, but the upward trend becomes relatively mild when the incubation time exceeds 60 minutes. Insufficient incubation time may result in a signal intensity lower than normal; excessive incubation time increases the background signal intensity. Therefore, 60 minutes was chosen as the incubation time. Coating is the first step in the immunoassay. Different concentrations (5, 10, 15, 20, 25, 30, 35, and 40 μg / mL) of coated antibody were tested at the highest concentration (1 μg / mL) of labeled antibody (35 μg / mL) and standard CEA. The strongest signal value was observed when the coated antibody concentration was 5 μg / mL. Figure 6 B). For other concentrations of coated antibodies, the signal values ​​of the immune response remained largely consistent; therefore, 5 μg / mL was selected as the optimal concentration of CEA-coated antibody in this embodiment. The labeled antibody in the immune response acts as a signal tag and captures the target analyte. The effect of colloidal gold-labeled antibody concentration in the range of 5 μg / mL to 40 μg / mL was investigated (coated antibody concentration of 5 μg / mL, CEA antigen at the highest detection concentration of 1 μg / mL). Figure 6 As shown in Figure C, the signal intensity increased rapidly as the antibody concentration increased from 5 μg / mL to 35 μg / mL, and remained almost constant at higher concentrations. Therefore, a colloidal gold-labeled CEA antibody at a concentration of 35 μg / mL was selected for the immunoassay.

[0052] 3.7 Optimization of Colorimetric Mode Conditions The AuNPs-mediated copper deposition process is constrained by various experimental parameters, among which different deposition solution concentrations, deposition times, and chromogenic reagent concentrations all affect the final results. Optimization results for these factors are presented in [the table / document / etc.]. Figure 7 In order to obtain the best signal amplification results, the deposition solution concentrations were selected as 6.25 mM, 12.5 mM, 25 mM, 50 mM, 75 mM and 100 mM. Figure 7 A), the deposition times were selected as 4 min, 6 min, 7 min, 8 min, 9 min, and 10 min ( Figure 7 B), select colorimetric reagent concentrations of 1mM, 2mM, 3mM, 4mM, and 5mM ( Figure 7 C) Optimization tests were conducted. When the deposition time was 4 min and 6 min, most 96-well plates were yellow or yellowish-green, indicating that the copper deposition thickness on the AuNPs surface was thin due to the short deposition time, making it difficult to distinguish analytes of different concentrations based on color differences. When the deposition time was increased to 9 min, green and blue appeared in the 96-well plates, but the color difference was not significant enough for practical analysis. When the deposition time was 8 min, a clear color gradient (yellow, green, blue) was shown, which corresponded well to the analyte concentration gradient (from low to high). In this case, semi-quantitative analysis could be easily achieved visually, therefore, a deposition time of 8 min was determined to be the optimal experimental condition. The chromogenic reagent can control the degree of colorimetric reaction; the amount of chromogenic reagent should be appropriately increased. However, excessive concentration of chromogenic reagent can also produce background interference; therefore, a concentration of 3 mM was selected as the optimal chromogenic reagent concentration.

[0053] 3.8 Detection performance of the dual-mode immunoassay This portable analytical system allows for simple semi-quantitative analysis via visual colorimetry and quantification via the CLEDD-OES system. In the 96-well microplate, the colorimetric reaction induced by copper deposition exhibits a clear gradient with increasing analyte concentration. After adding the developing solution, the amount of Prussian blue produced is directly proportional to the target concentration. As the target analyte concentration increases, the color changes from yellow to yellow-green, then to green, and finally to blue. In the low concentration range, the color is yellow to yellow-green; in the medium concentration range, the color is green; and in the high concentration range, green-blue and blue are produced. Therefore, due to the concentration-dependent multicolor transition, different levels of analytes can be easily distinguished visually. Taking carcinoembryonic antigen (CEA) as an example, this portable dual-mode immunoassay platform demonstrated excellent detection performance. In colorimetric mode, as the CEA concentration increased, the solution exhibited a characteristic color gradient from yellow, yellow-green, green to blue, enabling rapid semi-quantitative screening by visual inspection. The detection limit for this mode was validated by measuring the absorbance of Prussian blue at 710 nm using a UV-Vis spectrophotometer, and the limit of detection was 0.1 ng / mL. Figure 8 A). In quantitative analysis mode, the portable capillary glow discharge atomic spectroscopy system (CLEGD-OES) exhibited good logarithmic correlation (calibration curve: y = 0.021log[CEA] + 0.037, R² = 0.991) in the concentration range of 0.1 to 1000 ng / mL, with a detection limit as low as 0.02 ng / mL. Figure 8 B). Performance comparisons show that the sensitivity of this portable CLEGD-OES system is comparable to that of large laboratory-grade instruments: the detection limit of commercial ICP-OES is 0.01 ng / mL (calibration curve: y = 954261log[CEA] + 507724, R² = 0.999). The superior sensitivity exhibited by CLEGD-OES is mainly attributed to the efficient signal amplification effect achieved by Au@Cu core-shell nanoparticles through kinetically driven nonlinear copper deposition. In summary, this platform successfully integrates the rapid screening capability of colorimetry with the precise quantitative capability of atomic spectroscopy.

[0054] Disease biomarkers are highly diverse. This amplification method can detect not only antigen-based biomarkers but also, through the design principle of metal-based core-shell nanostructures, can be extended to the detection of other types of biomarkers. To evaluate the system's universal detection capability for multiple biomarkers, the system was further validated by detecting cardiac troponin I (cTnI) and β-amyloid (Aβ), which play key roles in myocardial infarction (MI) and Alzheimer's disease (AD). In the cTnI analysis ( Figure 8 C Figure 8 D), the colorimetric mode enables visual semi-quantitative detection with a detection limit of 0.1 ng / mL ( Figure 8 C); while CLEGD-OES-based quantification of cTnI showed a log-linear relationship from 0.01 to 100 pg / mL, as expressed by the equation y = 977.042log[cTnI] + 4698.299 (R 2 The detection limit is described as being as low as 0.35 fg / mL (=0.999). Figure 8 D) This system is also applicable to Aβ analysis ( Figure 8 E, Figure 8F), the signal showed a good logarithmic linear relationship with concentration in the concentration range of 15-240 pg / mL. The detection limit of the colorimetric mode reached 15 pg / mL (F). Figure 8 E), while the CLEGD-OES model exhibits a log-linear range of 15 to 240 pg / mL, conforming to y = 0.073log[Aβ] + 0.069 (R). 2 =0.993), with a detection limit of 10.3 pg / mL—comparable to the detection limit of 6.4 pg / mL for commercial ICP-OES.

[0055] These results validate the visualization and quantitative analysis capabilities of the portable dual-mode system over a wide dynamic range and confirm its effectiveness and versatility in detecting various disease biomarkers, such as CEA, cTnI, and Aβ. Overall, this portable device exhibits unique advantages such as good selectivity, high sensitivity, and a wide dynamic range, making it particularly suitable for the analysis of proteins in complex biological samples. Its on-site detection flowchart is shown below. Figure 8 As shown in G.

[0056] 3.9 Actual Sample Testing and Verification The complexity of the human body fluid matrix often leads to matrix interference, causing signal enhancement or attenuation in immunoassays and affecting detection accuracy. To evaluate the reliability of the one-step copper deposition-induced signal amplification technology and the portable colorimetric / CLEGD-OES dual-mode system in real sample detection, this embodiment analyzed real human serum samples provided by West China Hospital, with known concentrations of CEA, cTnI, and Aβ standards added (Table 4). In clinical samples, cerebrospinal fluid (CSF) is often considered the gold standard for detecting Alzheimer's disease markers such as Aβ due to its moderate biomarker concentration and low interference. However, CSF collection requires invasive procedures, limiting the sample volume and patient acceptance. In contrast, blood samples are more favored in clinical applications due to their non-invasive collection method. However, the concentration of target markers in plasma is usually low, requiring the detection system to have higher sensitivity. Relying on excellent signal amplification strategies and plasma excitation efficiency, this system successfully achieved multi-target simultaneous detection capability in spiked serum samples. The integrated colorimetric / CLEGD-OES system exhibits complementary functions: the colorimetric system enables rapid semi-quantitative screening, while the portable CLEGD-OES system provides laboratory-grade precise quantitative analysis. Therefore, this developed system can rapidly identify disease biomarkers, creating the possibility for timely therapeutic intervention.

[0057] Table 4. Analysis results of CEA, cTnI and Aβ in human serum

[0058] It has been reported that fluorescence assays, paper-based fluxoimmunoassays, chemiluminescence, and electrochemical assays can all be used to measure Aβ. Tables 5 and 6 detail the comparative analysis results of this invention with previous literature. Specifically, the device developed in this invention has certain advantages in terms of detection range and detection limit. In this study, AuNPs were introduced as signal tags, and a copper deposition strategy was used to further amplify the signal. Furthermore, in terms of sensitivity, operational portability, and instant detection efficiency, it is more competitive than other previously reported detection techniques and traditional ELISA methods, opening up new avenues for the rapid and efficient detection of Alzheimer's disease biomarkers.

[0059] Table 5 Comparative Analysis of Developed Point-of-Care Detection Technologies for Aβ

[0060] Table 6 Comparative Analysis of Developed cTnI Point-of-Care Detection Technologies

[0061] References: [1] He, J.; Hu, M.; Yang, L.; Yin, J.; Zhang, Z.; Tu, L.; Heng, Y.;Tang, L.; He, J.; Hou, X.; Jiang, 2024, 96 (49), 19196-19201. [2] Tang, M.; Pi, J.; Long, Y.; Huang, N.; Cheng, Y.; Zheng, H. Quantum dots-based sandwich immunoassay for sensitive detection of Alzheimer's disease-related Aβ1–42. Spectrochim Acta A. 2018, 201, 82-87. [3] Zhang, L.; Black, X.; Su, Y.; Niu, S.; Li, Y.; Liang, X.; Luo,H.Quantitative assessment of AD markers using naked eyes: point-of-caretesting with paper-based lateral flow immunoassay. J Nanobiotechnol. 2021, 19(1), 366. [4] Sheng, M.; Yu, L.; Peng, Y.; Wang, Q.; Huang, J.; Yang,X.Combination of Ternary Electrochemiluminescence System of BNQDs / AgMOG-K2S2O8 and Electrochemiluminescence Resonance Energy Transfer Strategy forUltrasensitive Immunoassay of Amyloid-β Protein.Anal. Chem. 2024, 96 (1), 41-48. [5] Gordon Pidal, JM; Moreno-Guzmán, M.; Montero-Calle, A.;Valverde, A.; Pingarrón, JM; Campuzano, S.; Calero, M.; Barderas, R.; Lopez, M. A.; Escarpa, A.Micromotor-based electrochemical immunoassays forreliable determination of amyloid-β (1–42) in Alzheimer's diagnosed clinicalsamples.Biosens. Bioelectron. 2024, 249, 115988. [6] Gagni, P.; Sola, L.; Cretich, M.; Chiari, M.Development of ahigh-sensitivity immunoassay for amyloid-beta 1–42 using a silicon microarrayplatform.Biosens. Bioelectron. 2013, 47, 490-495. [7] Pi, J.; Long, Y.; Huang, N.; Cheng, Y.; Zheng, H.A sandwichimmunoassay for detection of Aβ1-42 based on quantum dots.Talanta 2016, 146,10-15. [8] Zeng, L.; Lin, C.; Liu, P.; Sun, D.; Lu, J.Anisotropic aptamer-modified DNA tetrahedra / MOF nanoprobes for enhanced colorimetric aptasensingof cardiac troponin I.Chem. Eng. J. 2023, 474, 145525. [9] Çimen, D.; Bereli, N.; Günaydın, S.; Denizli, A.Detection ofcardiac troponin-I by optic biosensors with immobilized anti-cardiactroponin-I monoclonal antibody.Talanta 2020, 219, 121259.

[10] Wang, Y.; Yang, Y.; Chen, C.; Wang, S.; Wang, H.; Jing, W.;Tao, N.One-Step Digital Immunoassay for Rapid and Sensitive Detection ofCardiac Troponin I.ACS Sensors. 2020, 5 (4), 1126-1131.

[11] Kang, J.; Yeom, G.; Jang, H.; Oh, J.; Park, C.-J.; Kim, M.-G.

[12] Liao, M.; Hu, Z.; Chen, X.; Chen, M.; Yang, Z.; Pan, J.; (iv) Summary This study developed a portable dual-mode immunoassay platform that synergistically integrates kinetically driven Au@Cu core-shell nanolabels with colorimetric and atomic spectroscopy detection technologies. The system utilizes kinetically controlled, self-limiting growth of Au@Cu tags to amplify signals, enabling ultrasensitive on-site detection of multiple clinically relevant biomarkers, including carcinoembryonic antigen (CEA), cardiac troponin I (cTnI), and β-amyloid (Aβ), achieving atomic spectroscopy detection limits of 0.02 ng / mL, 0.35 fg / mL, and 10.3 pg / mL, respectively. Crucially, by combining user-friendly and rapid colorimetric screening with highly specific and accurate atomic spectroscopy detection, the system achieves cross-validation between the two detection modes, significantly improving reliability and reducing the risk of false positives. The platform requires minimal sample pretreatment, consumes little power, and has a compact structure, making it particularly suitable for field operations. With its modular design and adaptability to various antibodies, this technology holds great potential for early, accurate, and reliable screening of various diseases in resource-scarce environments.

[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling, characterized in that, This includes an antibody-gold nanoparticle bioconjugation system, an amplification system, a colorimetric system, and a colorimetric-atomic emission spectroscopy detection system; among which: The colorimetric-atomic emission spectroscopy detection system amplifies the signal of core-shell nanoparticles through kinetic driving, and combines colorimetric screening with precise quantitative detection by atomic emission spectroscopy.

2. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 1, characterized in that, The preparation method of the antibody-nanoparticle bioconjugate system includes the following steps: (I) Preparation of gold nanoparticles The gold source solution is heated, and then a reducing stabilizer is added to obtain gold nanoparticles. (II) Preparation of antibody-gold nanoparticle bioconjugate system (II-1) The secondary antibody was added to the gold nanoparticles and stirred to react; (II-2) Continue adding bovine serum albumin and stirring to react; (II-3) Centrifuge and wash to obtain the antibody-gold nanoparticle bioconjugate system.

3. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 2, characterized in that, In step (I): The gold source solution is a chloroauric acid solution with a concentration of 10-12%. And / or, the reducing stabilizer is a sodium citrate solution with a concentration of 1-2%.

4. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 2, characterized in that, In steps (II-1) and (II-2), the temperature of the stirring reaction is 20-30℃ and the stirring reaction time is 30-40 min.

5. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 2, characterized in that, In step (II-3), the centrifugal washing method is as follows: (1) Centrifuge at 2000-2500g for 20-30 minutes and retain the supernatant; (2) Centrifuge again at 8000-9000g for 30-40 minutes and retain the precipitate; (3) Finally, centrifuge and wash at 6000-7000g for 30-40 minutes. After resuspending the precipitate, the antibody-gold nanoparticle bioconjugate system is obtained.

6. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 1, characterized in that, The amplification system includes copper sulfate solution and L-ascorbic acid solution; wherein: The concentration of the copper sulfate solution is 80-120 mM; The concentration of the L-ascorbic acid solution is 20-30 mM.

7. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 1, characterized in that, The colorimetric system comprises ferric chloride solution and potassium ferricyanide solution; wherein: The concentration of the ferric chloride solution is 2-4 mM; The concentration of the potassium ferricyanide solution is 2-4 mM.

8. The portable dual-mode immunoassay platform based on kinetic-driven core-shell nanoparticle labeling according to claim 1, characterized in that, The colorimetric-atomic emission spectroscopy detection system includes an adjustable discharge system and a solid-structure microplasma discharge chamber; wherein: The adjustable discharge system is used to adjust the height difference between the capillary inlet and the discharge end in order to achieve precise adjustment and control of the injection volume. The solid structure microplasma discharge chamber is used to generate a closed inert atmosphere to improve stability.

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