Amplification Strategy Research Articles

Metal Ion-Based Chemical Coordination Amplification: A Chromophore In Situ Deposition Strategy for Visual Sensitivity-Enhanced Lateral Flow Immunochromatography Assays.

Traditional lateral flow immunochromatography assays (LFIAs) have faced low sensitivity for trace detection due to the lack of colorimetric brightness. The current strategies to improve sensitivity commonly have the disadvantages of an uncontrollable enhancement process or high background interference, leading to huge obstacles for signal readout. Herein, an in situ metal ion-based chemical coordination amplification (MICCA) strategy has been reported. Metal ion clusters on metal-organic frameworks could coordinate with chromophores to produce colored complexes for visual signal enhancement. A Zr-based metal AIEgen framework (MAF) loaded with Prussian blue was chosen as the dual-mode signal tag for colorimetric and fluorescent readout. MAF could be employed as a grafting substrate to in situ deposit chromophores through the coordination with Zr4+ clusters and arsenazo III. The process of MICCA was in situ, controllable, and free of background interference. For target cancer biomarker alpha-fetoprotein (AFP), the limit of detection (LOD) by the naked eye was 25 ng/mL, and the LODs of MICCA and fluorescence were 5 ng/mL, which was 5-fold decreased. Significantly, MICCA-LFIA could effectively differentiate between AFP-positive and AFP-negative clinical serum samples. The quantitative results were highly consistent with clinical results (R2 = 0.9927). This work explored the application of metal ion-based chemical coordination reactions in signal amplification strategies and provided ideas for high-sensitivity LFIA development.

Lens-Free Holographic Imaging-Based Immunosensor Using Unpaired Data Set Signal-to-Noise Ratio-Enhanced Modal Transformation for Biosensing.

Conventional microscopes have limited capacities to reconcile the trade-off between the lens and field of view (FOV). Thus, the imaging field and accuracy of immunosensors remain restricted. In this study, a holographic deep learning unpaired modal transformation-assisted immunosensor is presented, combining a portable lens-free holographic imaging device with a CuO2@SiO2 nanoparticle-based click reaction signal amplification strategy for accurate antibiotic detection. The immunosensor achieves both large FOV imaging (10.3-fold improvement over the microscope) and signal-to-noise ratio-enhanced holographic reconstruction (signal-to-noise ratio of 32.65 dB, structural similarity index of 0.83) by constructing a modal transformation model with unpaired data sets, thus resolving the complexity of one-to-one matching of data sets required by conventional methods. The immunosensor detects chloramphenicol with high sensitivity and a wide linear range (limit of detection = 3.54 pg/mL, dynamic range of 10 pg/mL to 50 ng/mL) within 40 min. As a portable detection device, it demonstrates potential as a sensitive and on-site detection platform for food safety inspection and clinical diagnosis.

Enhanced Photoelectrochemical Sensing of T-2 Toxin via Photoelectron Transfer Mediated by SrTiO3-CdIn2S4/LaNiO3 Composite and Mn2+-Dependent DNAzyme Amplification Strategy

Enhanced Photoelectrochemical Sensing of T-2 Toxin via Photoelectron Transfer Mediated by SrTiO3-CdIn2S4/LaNiO3 Composite and Mn2+-Dependent DNAzyme Amplification Strategy

Applying hollow octahedron PtNPs/Pd-Cu2O nanozyme and highly conductive AuPtNPs/Ni-Co NCs to colorimetric -electrochemical dual-mode aptasensor for AFB1 detection.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. This toxin is highly carcinogenic and toxic, posing a serious threat to human and animal health. AFB1 primarily enters the human body through contaminated food, particularly peanuts, corn, nuts, and wheat. These foods are easily contaminated with AFB1 during planting, harvesting, storage, and processing. Therefore, the establishment of an accurate and sensitive method for the detection of AFB1 is essential to safeguard food safety and human health. This study applied catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), hollow octahedron PtNPs/Pd-Cu2O bifunctional nanozyme, and AuPtNPs/Ni-Co NCs to develop a colorimetric-electrochemical dual-mode aptasensor for AFB1 detection. Here, Pd-Cu2O corner-etched octahedra with cavities and oxygen vacancy defects were first designed, followed by the synthesis of PtNPs/Pd-Cu2O through the loading of PtNPs to enhance the peroxidase-like activity of Pd-Cu2O. PtNPs/Pd-Cu2O effectively catalyzed the generation of blue oxidation products from 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). It also enhances the redox reaction of methylene blue (MB) at the electrode and improves the electrochemical signal. AuPtNPs/Ni-Co NCs possess a large specific surface area and excellent conductivity, enhanced the redox signal of MB. Additionally, the aptasensor combined the enzyme-free amplification strategies of CHA and HCR to improve sensitivity. The aptasensor reached limit of detection (LOD) 0.76pg/mL in electrochemical mode and 0.49pg/mL in colorimetric mode. The designed colorimetric-electrochemical dual-mode aptasensor exhibits high selectivity, specificity, stability, and reliability. The dual-mode aptasensor had been successfully applied to the spiked analysis of edible oils, peanuts, and cornmeal, demonstrating excellent recovery rates. Therefore, the dual-mode aptasensor had a wide application prospect in the field of quality supervision and food safety.

A fluorescent aptasensor for kanamycin detection in milk, seafood and water samples using DNA-AgNCs and exonuclease I-assisted recycling amplification strategy

A fluorescent aptasensor for kanamycin detection in milk, seafood and water samples using DNA-AgNCs and exonuclease I-assisted recycling amplification strategy

Cocatalysis of catalytic hairpin assembly and ternary heterojunction Bi2S3@MoS2@Bi2MoO6 promotes ultra-sensitive electrochemical of detection MiRNA-21.

Ternary heterojunction Bi2S3/MoS2/Bi2MoO6 was designed as a signal probe to develop a dual signal amplification strategy empowered electrochemical biosensor for sensitive miRNA-21 detection by combining with catalytic hairpin assembly (CHA). The combination of the Bi2S3/MoS2/Bi2MoO6 heterojunction as a tracer indication probe and the CHA amplification strategy not only took fully use of the highly dense nanowire interwoven structure and superior active region of the probe, but also endowed the ability to improve the molecular hybridization efficiency by collision, which significantly avoided the cumbersome chain design and greatly simplified the step-by-step construction of the electrode surface. Hairpin H1 was first added dropwise to the gold nanoparticle-decorated electrode surface, and then opened by the introduced miRNA-21 to initiate the specific hybridization. Once the H2-MB/Bi2S3/MoS2/Bi2MoO6 heterojunction was added, more sensitive and rapid detection of miRNA-21 would be achieved through the introduction of methylene blue since the cocatalysis of hairpin assembly and ternary heterojunction Bi2S3@MoS2@Bi2MoO6. Finally, the constructed biosensor demonstrated effective performance in the detection of miRNA-21 within the linear range of 1 fM to 100pM with a detection limit of 0.31 fM. The satisfactory analysis of human blood samples further validated the preferable reliability and practicability of this new strategy for the ultrasensitive detection of miRNA.

Multi-site enzymatic repairing amplification (MSERA) enables ultrasensitive detection of terminal deoxynucleotidyl transferase activity.

A novel multi-site enzymatic repairing amplification strategy is developed for high-sensitive terminal deoxynucleotidyl transferase quantification through combining enzymatic repairing amplification and lesion base-involved terminal extension. This method may provide a sensitive and flexible tool for molecular diagnosis and drug discovery.

Dual recycling signal amplification strategy based on autocatalytic entropy-driven circuit and DNAzyme for colorimetric detection of platelet-derived growth factor-BB.

Platelet-derived growth factor-BB (PDGF-BB), an important protein biomarker, is closely associated with tumorigenesis. Therefore, it is important to develop a simple and sensitive method to detect PDGF-BB. Herein, we developed a dual recycling signal amplification strategy for colorimetric and sensitive detection of PDGF-BB using a PDGF-BB specific aptamer. In the presence of PDGF-BB, the first entropy-driven circuit (EDC) reaction cycle was triggered for colorimetric signal amplification. Meanwhile, a catalytic Mg2+-dependent DNAzyme was formed on one side of the EDC product, which could cleave its substrate and generate numerous "mimic trigger" DNA products. Then, an autocatalytic EDC (AEDC) reaction was triggered by the "mimic trigger" DNA, and the signal amplification efficiency of this colorimetric assay was significantly improved. This AEDC- and DNAzyme-based colorimetric assay showed a good linear range from 5.0 to 100 nM for PDGF-BB and the limit of detection was calculated to be 2.2 nM. In addition, PDGF-BB has been quantitatively detected in human serum samples. This colorimetric assay can be easily extended to detect different protein biomarkers by using other recognition elements (aptamers) and shows great potential for clinical diagnosis and prognosis.

Label-Free and Sequence-Independent Isothermal Amplification Strategy for the Simultaneous Detection of Genomic 5-Methylcytosine and 5-Hydroxymethylcytosine.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are crucial epigenetic modifications in eukaryotic genomic DNA that regulate gene expression and are associated with the occurrence of various cancers. Here, we combined bisulfite conversion with 4-acetamido-2,2,6,6-tetramethyl-1-oxopiperridinium tetrafluoroborate (ACT+BF4-, TCI) oxidation to develop a label-free and sequence-independent isothermal amplification (BTIA) assay for a genome-wide 5mC and 5hmC analysis. The BTIA strategy can distinguish 5mC and 5hmC signatures from other bases with high sensitivity and good specificity, avoiding sophisticated chemical modifications and expensive protein labeling. Moreover, the utilization of terminal deoxynucleotidyl transferase (TdT) enables the proposed strategy to detect global 5mC and 5hmC without sequence dependence. With only 78 ng of input of genomic DNA, global 5mC and 5hmC levels were accurately quantified in cells (including cancer cells of A549, T47D, and K562 and normal cells of HEK-293T, CHO, and NRK-52E) and clinical whole blood samples (including healthy control, precancerous cervical cancer, and confirmed cervical cancer) within 18 h. The detection results suggested that 5mC was highly expressed in cancer cells. More importantly, a significant increase in 5mC was observed in precancerous cervical cancer and further upregulation in confirmed cervical cancer, suggesting a correlation between 5mC and cancer occurrence and development. However, 5hmC showed the reverse result in these tested cells and clinical samples. Collectively, the BTIA strategy can be easily performed on the ordinary heating apparatus in almost all research and medical laboratories, showing a significant application in the early screening of cervical cancer in the clinic.

Microfluidic-Enabled Self-Directed Hydrogel Microspheres for Multiplexed MicroRNA Assays.

Multiplexed microRNA (miRNA) detection has proven valuable in disease diagnosis; yet, the development of advanced tools for their analysis remains a subject of broad interest. Here, we propose a novel single-particle method for multiplexed miRNA detection using self-directed hydrogel microspheres, which feature supersegmented compartments for loading analyte probes and an air-encapsulated region that grants the microsphere a unique preferred posture in aqueous solutions. By exploiting microfluidic technology, we can widely adjust the size of the microspheres and the number of compartments can be widely adjusted. The air-encapsulated region is located at the edge of the microsphere's symmetry axis, resulting in a spontaneous orientation that facilitates efficient multitarget signal readout in a standard manner without requiring active energy input. The microspheres exhibit excellent temperature stability, ensuring consistent performance under varying thermal conditions. Additionally, signal amplification strategies, such as hybridization chain reaction, are compatible with microspheres, enabling sensitive miRNA quantification. As a concept of proof, precise miRNA detection was demonstrated using these features. We hope that the proposed biocompatible microsphere tools will find broader application prospects in various clinical diagnostic settings.

Multiple-Signal Amplification Strategy to Fabricate an Ultrasensitive Electrochemiluminescence Magnetic Immunosensor for Detecting Biomarkers of Alzheimer's Disease via Iridium-Based Self-Enhancing Nanoemitters.

Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive decline, significantly impairing the daily life of elderly individuals. The low abundance of blood-based biomarkers in AD necessitates higher analytical technique requirements. Herein, one novel iridium-based ECL self-enhanced nanoemitter (TPrA@Ir-SiO2) was unprecedentedly reported, and it was further used to construct an ultrasensitive ECL magnetic immunosensor by a multiple-signal amplification strategy to unequally sensitively and accurately detect the AD blood-based biomarker (P-tau181) in this work. The initial signal amplification was accomplished via incorporating a new efficient iridium-based luminophore named Ir(mdq)2(acac) and a corresponding coreactant into silica nanoparticles to successfully obtain TPrA@Ir-SiO2. In addition, the specific and high-affinity interactions between streptavidin and biotin were subsequently employed to further facilitate signal amplification. Based on the advantages of the luminophore itself and the high-affinity interactions between biotin and streptavidin, the corresponding ECL immunosensor proposed in this work exhibited remarkable sensitivity, covering a wide linear range from 0.1 pg/mL to 0.1 μg/mL, and achieved an ultralow limit of detection of 68.58 fg/mL (S/N = 3), and it also exhibited outstanding recovery (98-104%) and RSD (1.92-4.86%) in the detection of serum samples by the spiking method. These remarkable results undoubtedly demonstrate the potential of self-enhanced ECL nanoemitters combined with a synergistic signal amplification strategy bearing streptavidin-biotin in detecting AD blood-based biomarkers, providing accurate and reliable solutions for early diagnosis and monitoring of AD, which would open a new avenue to effectively reduce the burden on AD patients' families and society in the future.

PCR-Free Self-Calibrated Ratiometric Electrochemical Genosensor Utilizing a Dual-Signal Amplification Approach for Genomic Detection of Mycobacterium tuberculosis

Abstract Tuberculosis (TB) remains a critical global health challenge. While many electrochemical (EC) biosensors have been developed for Mycobacterium detection, most relied on PCR amplification strategies. This study introduced a PCR-free, self-calibrated ratiometric electrochemical (REC) genosensor leveraging dual-signal amplification for the detection of Mycobacterium tuberculosis (MTB). Electroactive silver nanoparticles (AgNPs) were modified onto screen-printed gold electrodes, followed by chemisorption of a methylene blue (MB)-tagged peptide nucleic acid (PNA) probe targeting MTB genomic DNA. MTB presence triggered structural changes in the probe, reducing the MB redox current (IMB) as the detection signal, while the Ag current (IAg) diminished as an internal calibration marker. This ratiometric mechanism minimized system errors, enhancing detection reliability. The genosensor achieved a broad detection range (3.5 fM–35 nM) with a detection limit of 1.59 fM for non-amplified MTB DNA, showing high specificity, reproducibility, and accuracy comparable to standard diagnostic methods. It effectively detected MTB in simulated sputum samples, demonstrating its potential for reliable, rapid, and amplification-free TB diagnostics.

Simultaneous or separate detection of heavy metal ions Hg2+ and Ag+ based on lateral flow assays.

Alateral flow assay (LFA) was developed for the simultaneous or separate detection of mercury ion and silver ion based on isothermal nucleic acid amplification. T-Hg2+-T and C-Ag+-C were utilized in the isothermal nucleic acid amplification strategy to form specific complementary base pairs. Under the action of KF polymerase and endonuclease Nt.BbvCl, trace amounts of Hg2+ and Ag+ were converted to Product-Hg2+ and Product-Ag+ as bridges. Biotin-labeled capture strands (Biotin-DNA1, Biotin-DNA1, and Biotin-DNA3) immobilized on the test strips could capture the Au NPs-DNA nanoprobes by hybridization with the generated bridge products for monitoring of two heavy metal ions simultaneously or separately. Theassembly method of DNAs on the nanoprobes was explored, and the DNA sequences on the nanoprobes were designed so that only one kind of DNA strand was used to bind to all three capture DNA strands on the C, T1, and T2 bands. Under optimal detection conditions, the limits of detection for Hg2+ and Ag+ were 2.19 and 5.41pM, respectively, with desired selectivity and reproducibility.

Ultrasensitive detection of microRNAs based on cascade amplification strategy of RCA-PER and Cas12a.

Since microRNAs (miRNAs) serve as markers for early cancer diagnosis, it is crucial to develop a novel biosensor to detect miRNAs quickly, sensitively and selectively. Hence, we developed a fluorescence biosensor based on target miRNA-initiated rolling circle amplification (RCA) to generate RCA products with multiple tandem catalytic hairpin DNA templates that trigger primer exchange reactions (PER) which extend short single-strand DNA (ssDNA) primers into long ssDNA. Subsequently, the long ssDNA activates the trans-cleavage activity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to cleave a fluorescent reporter chain, enabling ultrasensitive detection of miRNAs through the output fluorescence signal. The biosensor could quantify miRNA-141 concentrations from 100 to 105 pM, with a detection limit of 94 fM. Therefore, the biosensing strategy proposed in this study offers a robust technique for the clinical diagnosis of miRNA-141.

Aptamer-Proximity Ligation Coupled with Rolling Circle Amplification Strategy for an Ultrasensitive Analysis of Tumor-Derived Extracellular Vesicles PD-L1.

Tumor-derived extracellular vesicles (T-EVs) PD-L1 are an important biomarker for predicting immunotherapy response and can help us understand the mechanism of resistance to immunotherapy. However, this is due to the interference from a large proportion of nontumor-derived EVs. It is still challenging to accurately analyze T-EVs PD-L1 in complex human fluids. Herein, a simple and ultrasensitive method based on the dual-aptamer-proximity ligation assay (PLA)-guided rolling circle amplification (RCA) for the analysis of T-EVs PD-L1 was developed. First, dual aptamers with strong binding affinity were utilized for the recognition of EpCAM and PD-L1 on EVs, and then the aptamer-based PLA occurred. With the aid of the high signal amplification ability of RCA guided by the dual-aptamer-based PLA and efficient magnetic separation, the biosensor could realize highly sensitive quantification of EpCAM and PD-L1 dual-positive EVs with a low detection limit of 7.5 particles/μL. In addition, this method based on the aptamer-PLA-guided RCA was used to discriminate cancer patients from healthy donors with 100% accuracy without additional purification. Overall, this strategy might provide a practical tool for the analysis of multiple proteins on EVs, exhibiting great potential in early cancer diagnosis and treatment.

Concentration-Bias-Free Discrimination of Single Nucleotide Variants Using Isothermal Nucleic Acid Amplification and Mismatch-Guided DNA Assembly.

Isothermal nucleic acid amplification techniques are promising alternatives to polymerase chain reaction (PCR) for amplifying and detecting nucleic acids under resource-limited conditions. While many isothermal amplification strategies, such as recombinase polymerase amplification (RPA), offer comparable sensitivity to PCR, they often lack the specificity and robustness for discriminating single nucleotide variants (SNVs), mainly due to the uncontrolled production of massive amplicons. Herein, we introduce a mismatch-guided DNA assembly (MGDA) approach capable of discriminating SNVs in the presence of high concentrations of wild-type (WT) interferences. We show that an optimal MGDA design can effectively suppress interfering signals from WT while maintaining high detection signals for the targeted SNV. A further introduction of a competitive sink probe allowed the detection of challenging SNVs, such as those containing G-T wobbles, with high sensitivity and specificity. Because it is highly specific and tolerant to massively produced interfering amplicons during isothermal nucleic acid amplification, we integrated MGDA with RPA for discriminating clinically relevant SNVs in point-of-care settings. We demonstrate that our RPA-MGDA is highly sensitive and specific, allowing the detection of as low as 1 aM SNVs with an allele frequency of 0.5%. We also evaluated the clinical potential of RPA-MGDA by analyzing epidermal growth factor receptor L858R mutations in tumor tissue samples collected from non-small-cell lung cancer patients (n = 44). A multiplexed RPA-MGDA assay was also developed for the simultaneous detection of pharmacogenetic mutations in buccal swab samples (n = 30).

Localized Bicirculating DNAzyme Self-Feedback Amplification Strategy for Ultra-Sensitive Fluorescence Biosensing of MicroRNA.

DNAzyme-based cascade networks are effective tools to achieve ultrasensitive detection of low-abundance miRNAs. However, their designs are complicated and costly, and the operation is time-consuming. Herein, a novel simple noncascade DNAzyme network is designed and its amplification effect is comparable to or even better than many cascading ones. It is a nonenzymatic, isothermal, bicirculating amplification network consisting of two toehold-mediated strand-displacement reactions and a localized DNAzyme amplification strategy. Taking microRNA-122 as a target model, this ultrasensitive fluorescence biosensor has a detection limit of 84 zmol L-1, which is 8-orders of magnitude lower than that of the nonamplification one. The ultrasensitivity mainly benefits from the exclusive design and positive self-feedback mechanism of the ingenious bicirculating DNAzyme amplification network. In addition, the utilization of superparamagnetic Fe3O4@SiO2 particles not only helps for the localization of DNAzymes but also facilitates the rapid separation of signal probes (output DNA-CdTe QDs). This fluorescent biosensor also has the advantages of specificity, speed, thermal stability, and low cost. This novel design paves a new way to simple and effective bioamplification strategy, which may be very attractive for biosensors, DNA logic gates, and DNA computers.

Sensitive Electrochemical Immunosensor for Procymidone Detection Based on a Supramolecular Amplification Strategy.

A sensitive electrochemical immunosensor for procymidone detection was developed based on a supramolecular amplification strategy. β-Cyclodextrin (β-CD)-based nanomaterials were employed to immobilize ferrocene derivative (FC)-functionalized antibodies/antigens through host-guest interactions. With the presence of procymidone, the formed β-CD-labeled bioconjugates were immobilized on the antibody-modified electrode after the immunoreaction, indicating fabrication of the immunosensor. The FC/β-CD complexes were with multiplex electroactive species and provided more sites for recognition groups, resulting in signal amplification of the sensor. Monitored with differential pulse voltammetry, the proposed immunosensor exhibited a wide linear range from 5 pM to 0.1 μM with a low detection limit (LOD) of 1.67 pM. The as-prepared immunosensor possessed high sensitivity, specificity, and stability and showed great potential for monitoring procymidone in the field of food safety.

Co-assemblies of Silver Nanoclusters and Fullerenols With Enhanced Third-Order Nonlinear Optical Response.

Exploring potential third-order nonlinear optical (NLO) materials attracts ever-increasing attention. Given that the atomically precise and rich adjustable structural features of silver nanoclusters (Ag NCs), as well as the unique π-electron conjugated system of carbon-based nanomaterials, a supramolecular co-assembly amplification strategy to enhance the luminescent intensity and NLO performance of the hybrids of the two components, are constructed and the relationship between structures and optical properties are investigated. By combining water soluble Ag NCs [(NH4)6[Ag6(mna)6] (H2mna = 2-mercaptonicotinic acid, abbreviated to Ag6─NCs hereafter) containing uncoordinated carboxyl groups with water-soluble fullerene derivatives modified with multiple hydroxyl groups (fullerenols, C60─OH), the π-electron delocalization is expanded owing to non-covalent hydrogen bonding effect between Ag6─NCs and C60─OH, which provides a feasible basis for realizing the NLO response. Then, the co-assemblies are doped into a PMMA matrix to prepare composite film and its NLO properties are evaluated by Z-scan technique. Remarkably, the effective nonlinear absorption coefficients β is of two orders magnitude higher than those of the Ag6─NCs assemblies at the absence of C60─OH. This work showcases a new approach for amplifying NLO responses which greatly facilitates the development of integrated photonic devices.

Wavelength-Resolved Magnetic Multiplex Biosensor for Simultaneous and Ultrasensitive Detection of Pneumonia Pathogens via Catalytic Hairpin Assembly Strategy with Luminescent Iridium Complexes.

Pneumonia is a prevalent acute respiratory infection and a major cause of mortality and hospitalization, and the urgent demand for a rapid, direct, and highly accurate diagnostic method capable of detecting both Streptococcus pneumoniae (S. pneumoniae) and Klebsiella pneumoniae (K. pneumoniae) arises from their prominent roles as the primary pathogens responsible for pneumonia. Herein, two luminescent iridium complexes with nonoverlapping photoluminescence spectra, iridium(III)-bis [4,6-(difluorophenyl)-pyridinato-N,C2'] picolinate (abbreviated as Ir-B) and bis (2-(3,5- dimethylphenyl) quinoline-C2,N') (acetylacetonato) iridium(III)) (abbreviated as Ir-R), were unprecedently proposed to construct a novel wavelength-resolved magnetic multiplex biosensor for simultaneous detection of S. pneumoniae and K. pneumoniae based on catalytic hairpin assembly (CHA) signal amplification strategy combined with dye-doped silica nanoparticles. Notably, the proposed wavelength-resolved multiplex biosensor not only exhibits a broad linear range from 50 pM to 10 nM but also demonstrates excellent recovery rates for S. pneumoniae (96.1-99.3%) and K. pneumoniae (94.8-101.5%) in real clinical samples, with corresponding relative standard deviation (RSD) values ranging from 2.57 to 3.15% for S. pneumoniae and 1.45 to 3.17% for K. pneumoniae. These favorable experimental outcomes undoubtedly offer a promising approach for the simultaneous detection of multiple pathogens in the future.