Electroluminescent enhanced biosensor and method of making the same

Abstract

The application provides an electroluminescence-enhanced biosensor and a preparation method thereof. The electroluminescence-enhanced biosensor comprises, from bottom to top, a flexible substrate, a coplanar electrode layer, a mixed luminescence layer, a functional modification layer and a biological recognition layer which are sequentially stacked; the coplanar electrode layer is formed by forming a pair of parallel electrodes on the flexible substrate with a conductive material; the mixed luminescence layer is formed by uniformly coating an alternating current electroluminescence material on the coplanar electrode layer; the functional modification layer comprises an adhesion layer arranged on the mixed luminescence layer and a material layer arranged on the adhesion layer; the adhesion layer is made of a positively charged biocompatible polymer, and the material layer is made of a two-dimensional material containing a phosphate group; the biological recognition layer is formed by fixing specific antibodies on the material layer and then sealing unreacted sites with a sealing material; after a sample to be measured is added to the biological recognition layer, an alternating current voltage is applied, and a detection signal is read.

Description

Technical Field

[0001] This invention relates to the field of sensor technology, specifically to an electroluminescence-enhanced biosensor and its fabrication method. Background Technology

[0002] Highly sensitive and reliable detection of biomarkers is crucial for early disease identification, accurate classification, and dynamic monitoring. Clinically, glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1), as specific indicators of central nervous system injury, are rapidly released into the bloodstream after traumatic brain injury or stroke. Human β-defensin 2 (HBD-2), interleukin-6 (IL-6), and C-reactive protein (CRP) are important response factors to infection and systemic inflammation, widely used for monitoring diseases such as sepsis and chronic inflammation. The concentrations of these biomarkers in human serum are typically at extremely low levels, ranging from picograms per milliliter (pg / mL) to nanograms per milliliter (ng / mL), placing extremely high demands on the sensitivity of the detection platform.

[0003] Currently, mainstream clinical testing methods such as enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR, while possessing high accuracy, generally suffer from problems such as expensive equipment, cumbersome operation procedures, and long testing cycles, making it difficult to meet the needs of point-of-care testing (POCT) in primary healthcare settings or resource-limited scenarios. Therefore, developing a low-cost, easy-to-operate, highly sensitive, and field-usable biosensor platform is of urgent practical significance.

[0004] In recent years, ACEL-based biosensors have gradually become a research hotspot in the POCT field due to their advantages such as fast response, no need for external light source, signal visualization, and simple device structure. Existing technologies achieve label-free detection by immobilizing antibodies or nucleic acid probes on the surface of ACEL devices and utilizing biorecognition events to regulate interfacial conductivity.

[0005] However, existing ACEL biosensors still face significant technical bottlenecks: First, the sensing interface lacks sufficient active sites, resulting in low antibody immobilization efficiency and limiting the ability to capture low-abundance targets; second, in complex biological samples (such as human serum), non-specific adsorption is severe, which easily leads to increased background noise and decreased signal-to-noise ratio; third, traditional devices rely on transparent conductive oxides (such as ITO) as top electrodes, which are not only costly but also difficult to be compatible with flexible substrates, restricting large-scale manufacturing and portable applications.

[0006] To improve the efficiency of interface functionalization, two-dimensional materials (such as graphene) have been introduced into the field of sensing due to their high specific surface area and excellent electrical properties. However, two-dimensional materials typically lack surface functional groups and cannot effectively bind to biomolecules, limiting their application in biosensing.

[0007] Therefore, there is an urgent need for a novel interface engineering strategy that can endow the sensing surface with high-density bio-coupling sites and good hydrophilicity, while being compatible with low-cost, flexible ACEL devices. This would enable highly sensitive, selective, label-free, and visual detection of a variety of low-concentration biomarkers, and support portable readout and modular multi-target adaptation. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an electroluminescence-enhanced biosensor, its fabrication method, and its applications.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides an electroluminescence-enhanced biosensor, comprising, from bottom to top, a flexible substrate, a coplanar electrode layer, a hybrid light-emitting layer, a functionalized modification layer, and a biorecognition layer. The coplanar electrode layer is formed by forming a pair of parallel electrodes on the flexible substrate using a conductive material; it is understood that this pair of parallel electrodes includes a positive electrode and a negative electrode. The hybrid light-emitting layer is formed by uniformly coating the coplanar electrode layer with an alternating current electroluminescent material. The functionalized modification layer includes an adhesion layer disposed on the hybrid light-emitting layer and a material layer disposed on the adhesion layer. The adhesion layer is made of a positively charged biocompatible polymer, and the material layer is made of a two-dimensional material containing phosphate groups. The biorecognition layer is formed by immobilizing a specific antibody on the material layer and then sealing unreacted sites with a sealing material. After a sample to be tested is dropped onto the biorecognition layer, the target protein in the sample binds to the specific antibody, forming an antigen-antibody complex; an alternating current voltage is applied, and a detection signal is read.

[0010] Taking the detection signal as the intensity of the luminescence signal as an example, after the sample to be tested is dropped onto the biorecognition layer, the target protein in the sample binds to the specific antibody, forming an antigen-antibody complex. Based on the electron charge conduction mechanism induced by the protein structure, the antigen-antibody complex synergistically enhances the interfacial electron conduction. When an AC voltage is applied, the current density in the low-resistance region increases, and the exciton recombination efficiency improves, resulting in an increase in electroluminescence intensity with increasing target concentration. The light signal can be objectively and rapidly interpreted using a portable spectrometer (peak 470 nm) or illuminance quantification. The intensity of the luminescence signal is positively correlated with the target concentration. It should be noted that the AC voltage is specifically 10 kHz and 200 V; the detection signal also includes parameters such as luminescence frequency, impedance change, and RGB component signals.

[0011] Further, the flexible substrate comprises one of the following flexible materials: polyethylene terephthalate (PET), paper-based, polyimide (PI), and thermoplastic polyurethane elastomer (TPU). Preferably, the thickness of the flexible substrate is 110-125 μm. The conductive material used for the coplanar electrode layer includes conductive silver paste (such as Elec-X130), carbon paste, gold paste, or conductive polymer (such as PEDOT:PSS). Preferably, the thickness of the electrode is 4-7 μm. The AC electroluminescent material used for the hybrid light-emitting layer includes one or more of copper-doped zinc sulfide (ZnS:Cu), manganese-doped zinc sulfide (ZnS:Mn), strontium aluminate-doped europium, and dysprosium (SrAl2O4:Eu,Dy). Preferably, the thickness of the hybrid light-emitting layer is 30-32 μm.

[0012] Furthermore, the biometric layer includes multiple sub-sensing regions for immobilizing various specific antibodies, with each sub-sensing region immobilizing one specific antibody. By modifying each region with different antibodies, the detection signal can be distinguished by the difference in the illuminated color of the luminescent material in each region, enabling simultaneous detection of multiple targets.

[0013] Furthermore, the electroluminescence-enhanced biosensor includes a detection area comprising the flexible substrate, a coplanar electrode layer, a hybrid light-emitting layer, a functionalized modification layer, and a biometric layer superimposed on it; wherein the pair of parallel electrodes extend outward from the detection area while maintaining a preset distance. This configuration results in a clearly defined detection area for the electroluminescence-enhanced biosensor, facilitating the addition of the sample and enabling connection to a built-in AC inverter to apply voltage.

[0014] Furthermore, the positively charged biocompatible polymer includes one or more of chitosan, gelatin, polylysine, and polyethyleneimine; the phosphate-containing two-dimensional material includes one or more of phosphoric graphene, phosphorylated carbon nanotubes, and phosphorylated Mxene.

[0015] Furthermore, the specific antibody includes one or more of anti-GFAP, anti-CRP, glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1, human β-defensin 2, interleukin-6, and C-reactive protein; the blocking material includes one of synthetic polymeric blocking agents such as 1% bovine serum albumin (BSA), skim milk powder, casein, polyvinyl alcohol (PVA), and polyethylene glycol (PEG).

[0016] This invention also provides a method for fabricating the above-mentioned electroluminescence-enhanced biosensor, the method comprising: (1) Prepare a flexible substrate; (2) A pair of parallel electrodes are formed on the flexible substrate with conductive material, and after curing, a coplanar electrode layer is formed to obtain a device with a coplanar electrode layer superimposed. (3) A uniformly coated AC electroluminescent material is applied to the coplanar electrode layer and dried to form a mixed light-emitting layer, thereby obtaining a device with a superimposed mixed light-emitting layer. It should be noted that when printing AC electroluminescent material, two layers are generally printed to ensure that the AC electroluminescent material on the resulting mixed light-emitting layer is uniformly distributed.

[0017] (4) After activation, the device with the superimposed mixed light-emitting layer is immersed in a positively charged biocompatible polymer solution overnight to form an adhesion layer, thus obtaining a device with an adhesion layer; after reactivation, a two-dimensional material solution containing phosphate groups is dropped onto the adhesion layer and dried at room temperature to form a material layer, thus obtaining a device with a material layer; it should be noted that in some embodiments, in order to form an adhesion layer on the device, the entire device is immersed in a positively charged biocompatible polymer solution, but in subsequent processing, the biometric layer is only superimposed on the adhesion layer on the side away from the flexible substrate. That is, with the flexible substrate as the bottom layer, the biometric layer is superimposed on the top adhesion layer to form a sensing area.

[0018] (5) After activation treatment, specific antibodies are dropped onto the material layer, and after incubation and fixation treatment, unreacted sites are blocked with a blocking material to form a biorecognition layer, thereby obtaining a device with a superimposed biorecognition layer, namely the electroluminescence enhanced biosensor (ACEL sensor device). The activation treatment involves activating with argon plasma for 5-15 minutes. The purpose of sealing unreacted sites is to prevent non-specific adsorption.

[0019] Furthermore, the preparation method specifically includes: (1) Prepare a flexible substrate; (2) A pair of parallel electrodes are formed on the flexible substrate by screen printing conductive silver paste, and then cured by baking at 120-125℃ for 20-30 minutes to form a coplanar electrode layer, thereby obtaining a device with a coplanar electrode layer superimposed. (3) Mix copper-doped zinc sulfide luminescent powder, polydimethylsiloxane (PDMS), EL adhesive and curing agent in a mass ratio of 7:7:5:1, and then set them on the top of the coplanar electrode layer by screen printing. Dry at 125°C for 30 minutes to form a mixed luminescent layer, and obtain a device with a superimposed mixed luminescent layer. (4) After activating with argon plasma for 5-15 min, the device with the superimposed mixed light-emitting layer is immersed in 0.05% w / v chitosan solution overnight to form an adhesion layer, thus obtaining a device with the superimposed adhesion layer; after activating with argon plasma for 5-15 min, 3 μL of 0.1 mg / mL phosphoric acid graphene aqueous solution is added to the adhesion layer and dried at room temperature for 3 hours to form a material layer, thus obtaining a device with the superimposed material layer; after step (4), the nitrated graphene material has a lateral dimension of 50-100 nm, a thickness of 1-4 nm, and a surface rich in phosphate groups, providing a large number of bio-coupling sites; (5) After activation by argon plasma for 5-15 min, 50 μg / mL of specific antibody (such as anti-GFAP, anti-CRP, etc.) is added, incubated at room temperature for 30 min, and fixed overnight at 4℃; then, the unreacted sites are blocked with 1% bovine serum albumin (BSA) to form a biorecognition layer, and the electroluminescent enhanced biosensor is obtained.

[0020] It should be noted that after obtaining the electroluminescence-enhanced biosensor, it can be washed three times with PBST buffer (containing 0.5% Tween 20) and stored at 4°C for later use.

[0021] The present invention also provides a detection method for the electroluminescence-enhanced biosensor as described above; the detection method includes: Add ≥1.5 μL of the test sample to the biorecognition layer, incubate at room temperature for 30 min, and wash three times with PBST buffer to remove unbound targets; Add 5-10 μL of ion-conducting mediator; ultrapure water can be used as the ion-conducting mediator. The purpose of adding the ion-conducting mediator is to bridge the electrodes to form a complete circuit.

[0022] When an alternating voltage is applied, the electroluminescent enhanced biosensor is illuminated, and the detection signal is read to obtain the target concentration of the sample to be tested.

[0023] Specifically, a pair of parallel electrodes of the electroluminescence-enhanced biosensor were connected to a built-in AC inverter (output: ~200 V, ~10 kHz); the device was illuminated, and electroluminescence was emitted from the top; the detection signal was acquired using a portable spectrometer and a portable illuminometer. The luminescence intensity spectrum (luminescence intensity, au) was obtained using the portable spectrometer; the luminescence luminance (cd / m²) was also obtained using the portable illuminometer. 2 ).

[0024] This invention further provides the aforementioned electroluminescence-enhanced biosensor for the rapid detection of five biomarkers associated with infection and inflammation. Specifically, these include GFAP, UCH-L1, HBD-2, IL-6, and CRP.

[0025] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages: This invention provides an electroluminescence-enhanced biosensor and its preparation method. 1) Screen-printed ACEL devices with coplanar electrode structure: No transparent conductive layer is required, which greatly reduces material and process costs and is suitable for flexible substrates; 2) Synergistic modification strategy of positively charged biocompatible polymers (chitosan as an example) and phosphate-containing two-dimensional materials (graphene phosphate as an example): Chitosan provides the adhesion basis, and graphene phosphate provides high-density active sites. The combination of the two achieves "low background + high signal + high signal-to-noise ratio". 3) Plasma surface modification technology: Ar plasma treatment for 5-10 minutes is required before the modification of the adhesion layer, the material layer, and the antibody.

[0026] 4) Blocking of unmodified sites: After antibody modification is completed, blocking materials are used to block unmodified sites to reduce non-specific adsorption; 5) Modular antibody replacement design: Different biomarkers can be detected by simply changing the terminal antibody on the same device platform, which has high versatility; 6) Multi-target detection design: The biometric layer can be divided into multiple blocks and distinguished by different luminescent material coatings. Each region is modified with different antibodies, and the detection signal can be distinguished by the difference in the illumination color of the luminescent material in each region, so as to realize the simultaneous detection of multiple targets.

[0027] Compatible with portable optical readout devices: supports spectrometers or low-cost illuminometers, enabling true point-of-care testing (POCT) applications. Attached Figure Description

[0028] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments, and unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0029] Figure 1 The following are schematic diagrams of the ACEL sensor device provided in this embodiment of the invention: (a) structural diagram, (b) physical diagram, (c) schematic diagram of portable spectrometer detection, and (d) schematic diagram of portable lumen illuminance meter detection.

[0030] Figure 2 The water contact angles of each layer in the ACEL sensor construction process provided in this embodiment of the invention are characterized as follows: (a) luminescent layer 93.85°; (b) chitosan layer 87.98°; (c) chitosan layer after plasma modification treatment 43.08°; (d) two-dimensional material layer 93.41°; (e) two-dimensional material after plasma treatment 55.18°.

[0031] Figure 3 The material optimization of the biofunctionalized layer of the ACEL sensor provided in this embodiment of the invention is as follows: (a) prepared by two-dimensional material or chitosan solution alone; (b) the preparation concentration result of the superimposed two-dimensional material layer with a fixed chitosan concentration of 0.05%; (c) the preparation concentration result of the substrate chitosan layer with a fixed two-dimensional material concentration of 0.1 mg / mL.

[0032] Figure 4 This is a resistance characterization of the ACEL sensor device construction process provided in the embodiments of the present invention.

[0033] Figure 5 The following are the linearity results (UCH-L1) detected by the ACEL sensor device (a) using a portable spectrometer provided in the embodiments of the present invention, with the inset showing the colorimetric results obtained; and (b) the linearity results (GFAP) detected by the ACEL sensor device using a portable lumen illuminometer. Detailed Implementation

[0034] The inventors found that the main drawbacks of existing technologies include: the sensing interface lacks efficient bioconjugation sites; traditional ACEL surfaces have few functional groups, resulting in low antibody immobilization; poor material hydrophilicity makes it difficult for biomolecules to be uniformly adsorbed, thus affecting reaction efficiency; severe non-specific adsorption leads to high background noise and low signal-to-noise ratio in complex samples such as serum; device fabrication relies on expensive transparent electrodes (such as ITO), which increases costs and limits flexibility and wearable applications; the detection system relies on large optical equipment, making true portability impossible; in addition, the lack of modular design means that changing the detection target requires redesigning the entire device, resulting in a long development cycle.

[0035] Based on this, the present invention provides an electroluminescence-enhanced biosensor, its preparation method, and its application, aiming to solve the following main technical problems: 1) Insufficient detection sensitivity: Although existing mainstream methods (such as enzyme-linked immunosorbent assay and real-time quantitative PCR) have a certain degree of accuracy, they are difficult to achieve high-sensitivity detection of biomarkers (such as GFAP, UCH-L1, IL-6, etc.) at concentrations as low as picograms per milliliter (pg / mL). 2) High dependence on equipment and high cost: Existing technologies generally rely on large and precision instruments (such as fluorescence plate readers and PCR instruments), resulting in high testing costs and complex operations, making them difficult to apply to primary healthcare or resource-limited scenarios; 3) The detection process is time-consuming: Traditional immunoassays usually require multiple steps of incubation, washing and signal amplification, and the overall detection time can take several hours, which cannot meet the needs of POCT for rapid response; 4) Low efficiency of sensor interface functionalization: Existing electroluminescent sensors are limited by the low density of active sites on the sensor surface and poor hydrophilicity, resulting in low antibody immobilization efficiency, which affects the capture ability and signal-to-noise ratio. 5) Weak anti-interference ability: In complex biological samples (such as human serum), non-specific adsorption is severe, which can easily cause false positives or signal drift. 6) Complex device structure and high manufacturing cost: Traditional ACEL devices often require transparent conductive oxides (such as indium tin oxide, ITO) as the top electrode, which are expensive and have complex processes, making them unsuitable for large-scale flexible device manufacturing.

[0036] The electroluminescence-enhanced biosensor provided by this invention is a high-performance, low-cost, portable AC electroluminescence biomarker sensor based on a two-dimensional material modified with phosphate groups, enabling label-free, highly sensitive, visualized, and rapid detection of five inflammatory and neurological damage biomarkers.

[0037] The present invention will now be described in detail with reference to specific embodiments.

[0038] Example This invention provides an electroluminescence-enhanced biosensor, its fabrication method, and its application. The electroluminescence-enhanced biosensor is described in detail below.

[0039] 1) Detection Principle: This sensor is based on a conductivity-regulated electroluminescence mechanism: when the sample is added to the biorecognition layer (i.e., the sensing area), the target protein specifically binds to the surface antibody, forming an antigen-antibody complex. Based on the electron charge conduction mechanism induced by the protein structure, the antigen-antibody complex synergistically enhances interfacial electron conduction. When a 10 kHz, 200 V AC voltage is applied, the current density in the low-resistance region increases, and the exciton recombination efficiency improves, leading to an increase in electroluminescence intensity with increasing target concentration. The light signal can be objectively and rapidly interpreted using a portable spectrometer (peak 470 nm) or illuminance quantification. The luminescence signal intensity is positively correlated with the target concentration.

[0040] 2) such as Figure 1 As shown, the structure and fabrication steps of the ACEL sensor device (device structure as shown) Figure 1 a): - Flexible substrate: Made of polyethylene terephthalate (PET) film with a thickness of about 110-125 μm, it has good flexibility and mechanical stability; - Coplanar structure silver electrode layer: A pair of parallel electrodes are formed by screen printing conductive silver paste (such as Elec-X130), and cured by baking at 120-125℃ for 20-30 minutes. The electrode thickness is about 4-7 μm. - Hybrid light-emitting layer: The mixture of copper-doped zinc sulfide (ZnS:Cu) light-emitting powder, polydimethylsiloxane (PDMS), EL adhesive and curing agent in a mass ratio of 7:7:5:1 is screen-printed on the electrode in two layers and dried at 125℃ for 30 minutes, with a total thickness of about 30-32 μm. - Functionalized modification layer [plasma interface modification technology; chitosan layer + phosphorylated graphene nanomaterial layer]: Chitosan bottom layer: After argon plasma activation for 5-15 min, the device is immersed in 0.05% (w / v) chitosan solution overnight to form a hydrophilic adhesion layer; Phosphated graphene nanosheets: After argon plasma activation for 5-15 min, 0.1 mg / mL phosphated graphene aqueous solution (3 μL) was added dropwise and dried at room temperature for 3 hours; the material has a lateral dimension of 50-100 nm, a thickness of 1-4 nm, and a surface rich in phosphate groups, providing a large number of bioconjugation sites. - Biorecognition layer: After activation with argon plasma for 5-15 min, add 50 μg / mL of specific antibody (such as anti-GFAP, anti-CRP, etc.), incubate at room temperature for 30 min, and fix overnight at 4℃; then block unreacted sites with 1% bovine serum albumin (BSA) to prevent nonspecific adsorption. Finally, wash three times with PBST buffer (containing 0.5% Tween 20) and store at 4℃ for later use.

[0041] 3) Testing steps: - Sample loading: ≥1.5 μL of the sample to be tested, added dropwise to the detection area ( Figure 1 b) Incubate at room temperature for 30 min, then wash three times with PBST buffer to remove unbound targets.

[0042] - Loading medium: Add 5 μL of ultrapure water as an ion-conducting medium to bridge the bottom silver electrode and form a complete circuit.

[0043] - Apply AC voltage and read the signal: Connect the device to the built-in AC inverter (output: ~200 V, ~10 kHz); the device is lit up, and electroluminescence is emitted from the top; acquire the signal using a portable spectrometer and a portable illuminometer.

[0044] - Signal quantization: using a portable spectrometer ( Figure 1 c) Obtain the luminescence intensity spectrum, which shows a distinct broad peak at 470 nm (luminescence intensity, au); and a portable illuminance meter ( Figure 1 d) Obtain luminous intensity (cd / m²) 2 ).

[0045] The main differences of this invention include: ① Plasma Modification Technology: Plasma interface modification technology is added, eliminating the need for conventional activation steps (using glutaraldehyde to activate surface groups is complex and the reagents are toxic); Main plasma treatment parameters: Argon (Ar) treatment for 5-10 min. Through simple, rapid, and safe plasma modification, hydrophilic groups on the material surface are re-exposed, improving the hydrophilicity of the device surface and providing an excellent environment for device surface functionalization. Experimental results also confirm that... Figure 2 As shown, the water contact angle of each layer during the construction of the ACEL sensor is characterized. Figure 2 a) Emissive layer, 93.85 °; Figure 2 b) Chitosan layer 87.98 ° ( Figure 2 c) The chitosan layer was plasma-modified at 43.08 °; Figure 2 d) Two-dimensional material layer 93.41 °; Figure 2 e) Two-dimensional materials were plasma-treated at 55.18°.

[0046] ②

Chitosan layer + phosphographene nanomaterial layer

[0047] First, such as Figure 3 As shown in figure a, the detection performance of ACEL devices constructed using chitosan and phosphorylated graphene were compared. The results indicate that when using phosphorylated graphene alone, the detection signal difference (Δ) was the largest and the brightness was the highest at a concentration of 0.1 mg / mL; while when chitosan alone was used, a concentration of 0.05% showed the best performance, but its Δ was only half that of the graphene group. This demonstrates the superior performance of two-dimensional materials in ACEL sensor detection.

[0048] Next, to balance low background and high signal, the two were combined for modification (0.05% chitosan and 0.1 mg / mL graphene phosphate). The results showed that when the chitosan content was fixed at 0.05% ( Figure 3 (b) Reducing the graphene concentration to 0.1 mg / mL increased Δ by 27 times compared to the 10 mg / mL group, achieving a significant signal enhancement; conversely, when the graphene concentration was fixed at 0.1 mg / mL ( Figure 3 c) Chitosan at a concentration of 0.05% yielded the largest Δ, approximately 2.5 times that of 0.01% chitosan, resulting in a significant signal enhancement.

[0049] Comparative Example Its functionalized modification layer uses only 0.01% chitosan for modification and does not involve the modification of graphene phosphate materials. Otherwise, the settings are the same as those in the embodiments of the present invention.

[0050] It is also worth noting that the Δ( ) in the combined modification of the present invention Figure 3 b) and the comparative Δ ( Figure 3 a) In comparison, the Δ of the present invention significantly enhances the signal compared to the Δ of the comparative example. Simultaneously, the present invention also exhibits lower resistivity ( Figure 4 In this invention, the resistivity decreased by 50.0% after adding the target, while in the chitosan-modified group, the resistivity decreased by 37.5%. Secondly, the detection performance of the ACEL sensor for multiple targets was verified, which was not performed using similar methods. Under these conditions, this invention detected five targets (GFAP, UCH-L1, HumanBD-2, IL-6, and CRP), and all achieved good low detection limits. In particular, the detection limit for CRP (0.34 pg / mL) was an order of magnitude lower than that of the comparative example (0.33 μg / mL). It also exhibited a high signal-to-noise ratio. The detection results for the above five targets are as follows: When using a portable spectrometer, the results showed that when the GFAP concentration was 0-300 pg / mL, the spectral intensity had a linear relationship with the GFAP concentration, expressed as y = 0.1002x + 23.17, R... 2=0.9963, LOD=4.13 pg / mL (SNR=3); meanwhile, the linear relationship between spectral intensity and UCH-L1 concentration ranged from 70 to 1600 pg / mL, with the equation y=0.04059x+9.728, R 2 = 0.9577, LOD=5.17 (SNR=3); the linear relationship between spectral intensity and HumanBD-2 concentration in the concentration range of 30-800 pg / mL is y=0.04997x+25.95, R 2 =0.9985, LOD=1.24 pg / mL (SNR=3), and the linear concentration range of IL-6 spectral intensity is 20-300 pg / mL, the relationship is y=0.1256x+19.04, R 2 =0.9989, LOD=1.47 pg / mL (SNR=3); spectral intensity ( Figure 5 a) When the linear concentration range of CRP is 4-256 pg / mL, the relationship is y=0.08193x+26.50, R 2 =0.9826, LOD=0.34 pg / mL (SNR=3). When measured using a portable illuminometer, the results show that the luminance (LOD) at GFAP concentrations of 0-300 pg / mL is... Figure 5 b) A linear relationship exists between y and GFAP concentration, expressed as y = 0.0476x + 13.12, R0 2 =0.9392, LOD=43.5 pg / mL (SNR=3); meanwhile, the linear concentration range of lumen output and UCH-L1 concentration was 70-1600 pg / mL, with the relationship y=0.01615x+7.328, R 2 = 0.9577, LOD=43.10 (SNR=3); the linear relationship between lumen output and HumanBD-2 concentration in the concentration range of 30-800 pg / mL is y=0.01391x+13.80, R 2 =0.9801, LOD=7.18 pg / mL (SNR=3), and the linear concentration range of IL-6 with lumen luminance of 20-300 pg / mL, the relationship is y=0.04624x+9.542, R 2 =0.9880, LOD=7.33 pg / mL (SNR=3); when the linear concentration range of CRP for lumen luminance is 4-256 pg / mL, the relationship is y=0.04283x+9.258, R 2 =0.9823, LOD=4.41 pg / mL (SNR=3). Furthermore, the detection time of this invention is shorter, requiring only 30 min, compared to similar methods.[1] The reaction needs to be completed in 1 hour.

[0051] In summary, this invention provides a highly sensitive, label-free, and visualized ACEL sensor for biomarker detection. This ACEL sensor utilizes phosphorylated graphene nanosheets as an interface engineering material, significantly improving antibody immobilization efficiency and hydrophilicity. After antibody modification, bovine serum albumin can be used to block unmodified sites, preventing non-specific adsorption and achieving an ultra-low detection limit at the pg / mL level, meeting the needs of early clinical screening. Furthermore, this ACEL sensor employs a coplanar electrode screen printing process, eliminating the need for a transparent conductive layer, enabling low-cost, scalable, and flexible device manufacturing. Its modular architecture of "immobilized device + replaceable antibody" facilitates rapid adaptation to different biomarkers. It offers advantages such as visualized detection results, compatibility with portable spectrometers or illuminometers, and support for rapid on-site detection.

[0052] It is understood that the specific choices in the embodiments are merely examples. In fact, there are multiple options for each component in this invention, all of which can achieve the purpose of the invention. Specifically: 1) Substrate replacement: PET can be replaced by flexible materials such as paper base, PI (polyimide), and TPU.

[0053] 2) Electrode material replacement: Silver paste can be replaced with carbon paste, gold paste or conductive polymer (such as PEDOT:PSS), as long as it can form a coplanar electrode; 3) Replacement of the light-emitting layer: ZnS:Cu can be replaced with other AC electroluminescent materials, such as ZnS:Mn, SrAl2O4:Eu, Dy, etc.; it can be printed in sections to achieve multi-target detection and differentiation.

[0054] 4) Adhesion layer replacement: Chitosan can be replaced with positively charged biocompatible polymers such as gelatin, polylysine, and polyethyleneimine; 5) Two-dimensional material alternatives: In addition to phosphorylated graphene, two-dimensional materials containing phosphate groups, such as phosphorylated carbon nanotubes and phosphorylated Mxene, can also be used; 6) Functionalization alternatives: Antibody fixation can be achieved using covalent / non-covalent strategies such as EDC / NHS chemical conjugation and streptavidin-biotin system; 7) Biofunctional layer substitution: Antibodies other than GFAP, UCH-L1, HumanBD-2, IL-6, and CRP can also be modified to achieve specific detection of different corresponding target proteins; 8) Alternative blocking materials: In addition to BSA, skim milk powder, casein, and synthetic polymer blocking agents such as PVA and PEG can also be used to block modified sites.

[0055] 9) Readout method alternative: In addition to light intensity, parameters such as emission frequency, impedance change and RGB component signal can also be monitored as detection signals.

[0056] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. An electroluminescence-enhanced biosensor, characterized in that, It includes a flexible substrate, a coplanar electrode layer, a hybrid light-emitting layer, a functionalized modification layer, and a biometric layer, which are stacked sequentially from bottom to top. The coplanar electrode layer is formed by forming a pair of parallel electrodes on a flexible substrate using conductive material. The hybrid light-emitting layer is formed by uniformly coating an alternating current electroluminescent material onto the coplanar electrode layer; The functionalized modification layer includes an adhesion layer disposed on the hybrid light-emitting layer and a material layer disposed on the adhesion layer; the adhesion layer is made of a positively charged biocompatible polymer, and the material layer is made of a two-dimensional material containing phosphate groups; The biorecognition layer is formed by fixing specific antibodies onto the material layer and then sealing unreacted sites with a sealing material. After the sample to be tested is dropped onto the biorecognition layer, the target protein in the sample binds to the specific antibody to form an antigen-antibody complex; an AC voltage is applied and the detection signal is read.

2. The electroluminescence-enhanced biosensor according to claim 1, characterized in that, The flexible substrate includes one of the following flexible materials: polyethylene terephthalate, paper-based, polyimide, and thermoplastic polyurethane elastomer; The conductive material used in the coplanar electrode layer includes conductive silver paste, carbon paste, gold paste, or conductive polymer. The AC electroluminescent material used in the hybrid light-emitting layer includes one or more of copper-doped zinc sulfide, manganese-doped zinc sulfide, strontium aluminate-doped europium, and dysprosium.

3. The electroluminescence-enhanced biosensor according to claim 1, characterized in that, The biometric layer includes multiple sub-sensing regions for immobilizing various specific antibodies, with each sub-sensing region immobilizing one specific antibody.

4. The electroluminescence-enhanced biosensor according to claim 1, characterized in that, The electroluminescent enhanced biosensor includes a detection area in which the flexible substrate, coplanar electrode layer, hybrid light-emitting layer, functionalized modification layer and biometric layer are superimposed; wherein, the pair of parallel electrodes extend outward from the detection area and maintain a preset distance.

5. The electroluminescence-enhanced biosensor according to claim 1, characterized in that, The positively charged biocompatible polymers include one or more of chitosan, gelatin, polylysine, and polyethyleneimine; The two-dimensional materials containing phosphate groups include one or more of phosphorylated graphene, phosphorylated carbon nanotubes, and phosphorylated Mxene.

6. The electroluminescence-enhanced biosensor according to claim 1, characterized in that, The specific antibodies include one or more of the following: anti-GFAP, anti-CRP, glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1, human β-defensin 2, interleukin-6, and C-reactive protein. The sealing material includes one of the following synthetic polymer sealing agents: 1% bovine serum albumin, skim milk powder, casein, polyvinyl alcohol, and polyethylene glycol.

7. A method for preparing an electroluminescence-enhanced biosensor according to any one of claims 1 to 6, characterized in that, The preparation method includes: (1) Prepare a flexible substrate; (2) A pair of parallel electrodes are formed on the flexible substrate with conductive material, and after curing, a coplanar electrode layer is formed to obtain a device with a coplanar electrode layer superimposed. (3) A mixed light-emitting layer is formed by uniformly coating an AC electroluminescent material on the coplanar electrode layer and drying it to obtain a device with a superimposed mixed light-emitting layer. (4) After activation treatment, the device with the superimposed mixed light-emitting layer is immersed in a positively charged biocompatible polymer solution overnight to form an adhesion layer, and the device with the superimposed adhesion layer is obtained; after activation treatment again, a two-dimensional material solution containing phosphate groups is dropped onto the adhesion layer and dried at room temperature to form a material layer, and the device with the superimposed material layer is obtained. (5) After activation treatment, specific antibodies are dropped onto the material layer, and after incubation and fixation treatment, unreacted sites are blocked with a blocking material to form a biorecognition layer, thereby obtaining a device with a superimposed biorecognition layer, namely the electroluminescence enhanced biosensor. The activation process involves activating with argon plasma for 5-15 minutes.

8. The method for fabricating the electroluminescence-enhanced biosensor according to claim 7, characterized in that, The preparation method specifically includes: (1) Prepare a flexible substrate; (2) A pair of parallel electrodes are formed on the flexible substrate by screen printing conductive silver paste, and then cured by baking at 120-125℃ for 20-30 minutes to form a coplanar electrode layer, thereby obtaining a device with a coplanar electrode layer superimposed. (3) Mix copper-doped zinc sulfide luminescent powder, polydimethylsiloxane, EL glue and curing agent in a mass ratio of 7:7:5:1, and then set them on the top of the coplanar structure electrode layer by screen printing. Dry at 125°C for 30 minutes to form a mixed luminescent layer, and obtain a device with a superimposed mixed luminescent layer. (4) After activating with argon plasma for 5-15 min, the device with the superimposed mixed light-emitting layer is immersed in 0.05% w / v chitosan solution overnight to form an adhesion layer, thus obtaining a device with the superimposed adhesion layer; after activating with argon plasma for 5-15 min, 3 μL of 0.1 mg / mL phosphorylated graphene aqueous solution is added to the adhesion layer, and the device is dried at room temperature for 3 hours to form a material layer, thus obtaining a device with the superimposed material layer. (5) After activation by argon plasma for 5-15 min, 50 μg / mL of specific antibody is added, incubated at room temperature for 30 min, and fixed overnight at 4℃; then, the unreacted sites are blocked with 1% bovine serum albumin to form a biorecognition layer, thus obtaining the electroluminescent enhanced biosensor.

9. A detection method, characterized in that, The detection method is used in the electroluminescence-enhanced biosensor as described in any one of claims 1 to 6; The detection method includes: Add ≥1.5 μL of the test sample to the biorecognition layer, incubate at room temperature for 30 min, and wash three times with PBST buffer to remove unbound targets; Add another 5-10 μL of ion-conducting mediator; When an alternating voltage is applied, the electroluminescent enhanced biosensor is illuminated, and the detection signal is read to obtain the target concentration of the sample to be tested.

10. An electroluminescent enhanced biosensor according to any one of claims 1 to 6 for the rapid detection of five biomarkers associated with infection and inflammation.

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