From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

Ultrasensitive Electrochemiluminescence Biosensor Based on DNA-Bio-Bar-Code and Hybridization Chain Reaction Dual Signal Amplification for Exosomes Detection.

Exosomes have received considerable attention as potent reference markers for the diagnosis of various neoplasms due to their close and direct relationship with the proliferation, adhesion, and migration of tumor. The ultrasensitive detection of cancer-derived low-abundance exosomes is imperative, but still a great challenge. Herein, we report an electrochemiluminescence (ECL) biosensor based on the DNA-bio-bar-code and hybridization chain reaction (HCR)-mediated dual signal amplification for the ultrasensitive detection of cancer-derived exosomes. In this system, two types of aptamers were modified on the magnetic nanoprobe (MNPs) and gold nanoparticles (AuNPs) with numerous bio-bar-code DNA, respectively, which formed "sandwich" structures in the presence of specific target exosomes. The "sandwich" structures were separated under magnetic field, and the numerous bio-bar-code DNA were released by dissolving AuNPs. The released bio-bar-code DNA triggered the HCR procedure to produce a good deal of long DNA duplex structure for embedding in hemin, which generated strong ECL signal in the presence of coreactors for ultrasensitive detection of exosomes. Under the optimal conditions, it exhibited a good linearly of exosomes ranging from 10 to 104 exosomes particle µL-1 with limit of detection down to 5.01 exosome particle µL-1. Furthermore, the high ratio of ECL signal and minor change of ECL intensity indicated the good specificity, stability, and repeatability of this ECL biosensor. Given the good performance for exosome analysis, this ultrasensitive ECL biosensor has a promising application in the clinical diagnosis of early cancers.

Integrated Mini-Pillar Platform for Wireless Real-Time Cell Monitoring.

Cell culture as the cornerstone of biotechnology remains a labor-intensive process requiring continuous manual oversight and substantial time investment. In this work, we propose an integrated mini-pillar platform for in situ monitoring of multiple cellular metabolism processes, which achieves media anchoring and cell culture through an arrayed mini-pillar chip. The assembly of polyaniline (PANI)/dendritic gold-modified microelectrode biosensors exhibits high sensitivity (63.55 mV/pH) and excellent interference resistance, enabling real-time acquisition of biosensing signals. We successfully employed such integrated devices to real-time measuring pH variations in multiple cells and real-time monitoring of cell metabolism under drug interventions and to facilitate in situ assisted cultivation of 3-dimensional (3D) cell spheroids. This mini-pillar array-based cell culture platform exhibits excellent biosensing sensitivity and real-time monitoring capability, offering considerable potential for the advancement of biotechnology and medical drug development.

Rapid fluorescent nucleic acid sensing with ultra-thin gold nanosheets.

Fluorescently labeled DNA oligonucleotides and gold nanospheres have been frequently utilized in biosensors, providing efficient nucleic acid detection. Nevertheless, the restricted loading capacity of gold nanospheres undermines overall sensitivity. In this study, we employed four-atom-thick ultrathin gold nanosheets (AuNSs), utilizing a "pre-mix model" for rapid target nucleic acid detection. In this approach, fluorescently labeled DNA probes were pre-incubated with the target nucleic acid, followed by the addition of AuNSs for probe adsorption and fluorescence quenching. With the developed method, we efficiently and rapidly detected the SARS-CoV-2 N gene sequence within 30 min, involving a brief 15-min target pre-incubation and a subsequent 15-min adsorption of free probes and fluorescence quenching by AuNSs. This method exhibited heightened sensitivity compared to gold nanospheres, boasting a limit of detection (LOD) of 0.808 nM. Furthermore, exceptional recovery was achieved in simulated biological samples. The study introduces an effective strategy for nucleic acid sensing characterized by rapidity, heightened sensitivity, ease of operation, and robustness. These findings encourage further development of rapid biomarker sensing methods employing 2D nanomaterials.

Ultrasensitive electrochemical microRNA-21 detection based on MXene and ATRP photocatalytic strategy.

A Ti3C2TxMXene-based biosensor has been developed and the photocatalytic atom transfer radical polymerization (photo ATRP) amplification strategy applied to detect target miRNA-21 (tRNA). Initially, Ti3C2TxMXene nanosheets were synthesized from the Ti3AlC2 MAX precursor via selective aluminum etching. Then, functionalization of Ti3C2TxMXene nanosheets with 3-aminopropyl triethoxysilane (APTES) via silylation reactions to facilitate covalent bonding with hairpin DNA biomolecules specifically designed for tRNA detection. Upon binding with the tRNA, the hairpin DNA liberated the azide (N3) group, initiating a click reaction to affix to the photo ATRP initiator. Through the ATRP photoreaction, facilitated by an organic photoredox catalyst and light, a significant amount of ferrocenyl methyl methacrylate (FMMA) monomer was immobilized on the electrode. Therefore, the electrochemical signal is amplified. The electrochemical efficacy of the biosensor was assessed using square wave voltammetry (SWV). Under optimized conditions, the biosensor demonstrated remarkable sensitivity in detecting tRNA, with a linear detection range from 0.01 fM to 10 pM and a detection limit of 2.81 aM. The findings elucidate that the developed biosensor, in conjunction with the photo ATRP strategy, offers reproducibility, stability, and increased sensitivity, underscoring its potential applications within the experimental medical sector of the biomolecular industry.

Riboflavin-induced photo-ATRP electrochemical strategy for detection of biomarker trypsin.

The detection of trypsin and its inhibitors is important for both clinical diagnosis and disease treatment. Abnormal trypsin activity affects pancreatic function and leads to corresponding pathological changes in the body. Therefore, the study presented a riboflavin-induced photo-ATRP electrochemical assay of trypsin activity and its inhibitor, including detection of trypsin activity in real urine samples. Experiments were performed on indium tin oxide (ITO) electrodes modified with sulfhydryl groups of 3-mercaptopropionic acid, and target trypsin-specific cleavage of BSA-Au nanocluster (BSA-Au NCs) was followed by the modification of Au NCs to the electrodes using Au-S. The Au NCs immobilized monodeoxy-monomercapto-ß-cyclodextrin@adamantan-2-amine (SH-ß-CD@2-NH2-Ada) host-guest inclusion complexes to the electrode surfaces via Au-S. In a two-component photo-initiator system consisting of riboflavin as an initiator and ascorbic acid (AA) as a mild reducing agent under mild blue light radiation, a large number of electroactive substances were grafted onto the electrode surface to generate electrochemical signals. In addition, we have successfully realized the detection of clinical drug inhibitors of trypsin. The detection limit of the system is as low as 0.0024 ng/mL, which much littler than the average standard of trypsin in the patient's urine or serum. It's worth noting that this work will provide researchers with a different route to design electrochemical sensors based on non-covalent recognition strategies.

From synthesis to applications of biomolecule-protected luminescent gold nanoclusters.

Gold nanoclusters (AuNCs) are a class of novel luminescent nanomaterials that exhibit unique properties of ultra-small size, featuring strong anti-photo-bleaching ability, substantial Stokes shift, good biocompatibility, and low toxicity. Various biomolecules have been developed as templates or ligands to protect AuNCs with enhanced stability and luminescent properties for biomedical applications. In this review, the synthesis of AuNCs based on biomolecules including amino acids, peptides, proteins and DNA are summarized. Owing to the advantages of biomolecule-protected AuNCs, they have been employed extensively for diverse applications. The biological applications, particularly in bioimaging, biosensing, disease therapy and biocatalysis have been described in detail herein. Finally, current challenges and future potential prospects of bio-templated AuNCs in biological research are briefly discussed.

Double-Enhanced Photothermal Lateral Flow Biosensor Based on Dual Gold Nanoparticle Conjugates.

Bacterial toxins emerge as the primary triggers of foodborne illnesses, posing a significant threat to human health. To ensure food safety, it is imperative to implement point-of-care testing methods. Lateral flow biosensors (LFBs) based on gold nanoparticles (GNPs) have been commonly used for rapid detection, but their applicationis limited by low sensitivity. Based on the localized surface plasmon resonance and photothermal effect of dual gold nanoparticle conjugates (DGNPs), we developed a smartphone-integrated photothermal LFB (PLFB) with double-enhanced colorimetric and photothermal sensitivity. Through numerical simulations, we verified that DGNPs have significantly enhanced photothermal performance compared to single 15 nm GNPs (SGNPs), and applied DGNPs in PLFB for the detection of staphylococcus enterotoxin A (SEA). The colorimetric and photothermal limits of detection of DGNPs-based PLFB for SEA were 0.091 and 0.0038 ng mL-1, respectively. Compared with the colorimetric detection of the SGNPs-based LFB, the colorimetric detection sensitivity of the DGNPs-based PLFB was increased by 10.7 times, and the photothermal detection sensitivity was further improved by 255.3 times. Moreover, the PLFB exhibits robust reproducibility and exceptional specificity and is applicable for detecting SEA in milk samples. This smartphone-integrated PLFB based on DGNPs allows users to detect toxins simply, conveniently, and quickly and has huge application potential in the field of food safety.

Study of V2CTx-MXene Based Immunosensor for Sensitive Label-Free Impedimetric Detection of SARS-CoV-2 Spike Protein.

Rapid and reliable immunosensing is undoubtedly one of the priorities in the efficient management and combat against a pandemic, as society has experienced with the SARS-CoV-2 outbreak; simple and cost-effective sensing strategies are at the forefront of these efforts. In this regard, 2D-layered MXenes hold great potential for electrochemical biosensing due to their attractive physicochemical properties. Herein, we present a V2CTx MXene-based sensing layer as an integral part of a label-free immunosensor for sensitive and selective detection of the SARS-CoV-2 spike protein. The sensor was fabricated on a supporting screen-printed carbon electrode using Nafion as an immobilizing agent for MXene and glutaraldehyde, the latter enabling effective binding of protein A for further site-oriented immobilization of anti-SARS-CoV-2 antibodies. A thorough structural analysis of the sensor architecture was carried out, and several key parameters affecting the fabrication and analytical performance of the immunosensor were investigated and optimized. The immunosensor showed excellent electroanalytical performance in combination with an impedimetric approach and exhibited a low detection limit of only 45 fM SARS-CoV-2 spike protein. Its practical applicability was successfully demonstrated by measuring the spike protein in a spiked artificial nasopharyngeal fluid sample.

Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

Wearable Vertical Graphene-Based Microneedle Biosensor for Real-Time Ketogenic Diet Management.

Ketogenic diets have attracted substantial interest in the treatment of chronic diseases, but there are health risks with long-term regimes. Despite the advancements in diagnostic and therapeutic methods in modern medicine, there is a huge gap in personalized health management of this dietary strategy. Hence, we present a wearable microneedle biosensor for real-time ketone and glucose monitoring. The microneedle array possesses excellent mechanical properties, allowing for consistent sampling of interstitial biomarkers while reducing the pain associated with skin puncture. Vertical graphene with outstanding electrical conductivity provides the resulting sensor with a high sensitivity of 234.18 µA mM-1 cm-2 and a low limit detection of 1.21 µM. When this fully integrated biosensor was used in human volunteers, it displayed an attractive analytical capability for tracking the dynamic metabolite levels. Moreover, the results of the on-body evaluation established a significant correlation with commercial blood measurements. Overall, this cost-effective and efficient sensing platform can accelerate the application of a ketogenic diet in personal nutrition and wellness management.

An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization.

BACKGROUND: Thrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. RESULTS: We designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au-S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate -COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL âˆ¼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SIGNIFICANCE: The biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

Bimetallic Coordination Polymers: Synthesis and Applications in Biosensing and Biomedicine.

Bimetallic coordination polymers (CPs) have two different metal ions as connecting nodes in their polymer structure. The synthesis methods of bimetallic CPs are mainly categorized into the one-pot method and post-synthesis modifications according to various needs. Compared with monometallic CPs, bimetallic CPs have synergistic effects and excellent properties, such as higher gas adsorption rate, more efficient catalytic properties, stronger luminescent properties, and more stable loading platforms, which have been widely applied in the fields of gas adsorption, catalysis, energy storage as well as conversion, and biosensing. In recent years, the study of bimetallic CPs synergized with cancer drugs and functional nanomaterials for the therapy of cancer has increasingly attracted the attention of scientists. This review presents the research progress of bimetallic CPs in biosensing and biomedicine in the last five years and provides a perspective for their future development.

Multiscale Heterogeneities-Based Piezoresistive Interfaces with Ultralow Detection Limitation and Adaptively Switchable Pressure Detectability.

Mechanical compliance and electrical enhancement are crucial for pressure sensors to promote performances when perceiving external stimuli. Here we propose a bioinspired multiscale heterogeneity-based interface to adaptively regulate its structure layout and switch to desirable piezoresistive behaviors with ultralow detection limitation. In such a multiscale heterogeneities system, the micro-/nanoscale spiny Ag-MnO2 heterostructure contributes to an ultralow detection limitation of 0.008 Pa and can perceive minor pressure increments under preloads with high resolution (0.0083%). The macroscale heterogeneous orientation of the cellular backbone enables anisotropic deformation, allowing the sensor to switch to rational sensitivity and working range (e.g., 580 kPa-1 for 0-20 kPa/54 kPa-1 for 60-140 kPa) as required. The sensor's stepwise activation progresses from the micro-/nanoscale heterostructure to the macroscale heterogeneous orientation, which can adaptively match diverse sensing tasks in complex applications scenarios. This multiscale heterogeneous and switchable design holds immense potential in the development of intelligent electromechanical devices, including wearable sensors, soft robotics, and smart actuators.

Enhanced Interstitial Fluid Extraction and Rapid Analysis via Vacuum Tube-Integrated Microneedle Array Device.

Advancing the development of point-of-care testing (POCT) sensors that utilize interstitial fluid (ISF) presents considerable obstacles in terms of rapid sampling and analysis. Herein, an innovative strategy is introduced that involves the use of a 3D-printed, hollow microneedle array patch (MAP), in tandem with a vacuum tube (VT) connected through a hose, to improve ISF extraction efficiency and facilitate expedited analysis. The employment of negative pressure by the VT allows the MAP device to effectively gather ≈18 µL of ISF from the dermis of a live rabbit ear within a concise period of 5 min. This methodology enables the immediate and minimally invasive measurement of glucose levels within the body, employing personal healthcare meters for quantification. The fusion of the VT and MAP technologies provides for their effortless integration into a comprehensive and mobile system for ISF analysis, accomplished by preloading the hose with custom sensing papers designed to detect specific analytes. Moreover, the design and functionality of this integrated VT-MAP system are intuitively user-friendly, eliminating the requirement for specialized medical expertise. This feature enhances its potential to make a significant impact on the field of decentralized personal healthcare.

l-Cysteine-Tuned the Hierarchical Structure Based on Benzimidazole: Synthesis, Characterization, and Application in Ratiometric Electrochemiluminescence Immunoassay.

Efficient and robust electrochemiluminescence (ECL) emitters are crucial for enhancing the ECL immunosensor sensitivity. This study introduces a novel ECL emitter, CoBIM/Cys, featuring a hierarchical core-shell structure. The core of the structure is created through the swift coordination between the sulfhydryl and carboxyl groups of l-cysteine (l-Cys) and cobalt ions (Co2+), while the shell is constructed by sequentially coordinating benzimidazole (BIM) with Co2+. This design yields a greater specific surface area and a more intricate porous structure compared to CoBIM, markedly enhancing mass transfer and luminophore accessibility. Moreover, the l-Cys and Co2+ core introduces Co-S and Co-O catalytic sites, which improve the catalytic decomposition of H2O2, leading to an increased production of hydroperoxyl radicals (OOH•). This mechanism substantially amplifies the ECL performance. Leveraging the competitive interaction between isoluminol and BIM for OOH• during ECL emission, we developed a ratiometric immunosensor for cardiac troponin I (cTnI) detection. This immunosensor demonstrates a remarkably broad detection range (1 pg mL-1 to 10 ng mL-1), a low detection limit (0.4 pg mL-1), and exceptional reproducibility and specificity.

ASD-Net: a novel U-Net based asymmetric spatial-channel convolution network for precise kidney and kidney tumor image segmentation.

Early intervention in tumors can greatly improve human survival rates. With the development of deep learning technology, automatic image segmentation has taken a prominent role in the field of medical image analysis. Manually segmenting kidneys on CT images is a tedious task, and due to the diversity of these images and varying technical skills of professionals, segmentation results can be inconsistent. To address this problem, a novel ASD-Net network is proposed in this paper for kidney and kidney tumor segmentation tasks. First, the proposed network employs newly designed Adaptive Spatial-channel Convolution Optimization (ASCO) blocks to capture anisotropic information in the images. Then, other newly designed blocks, i.e., Dense Dilated Enhancement Convolution (DDEC) blocks, are utilized to enhance feature propagation and reuse it across the network, thereby improving its segmentation accuracy. To allow the network to segment complex and small kidney tumors more effectively, the Atrous Spatial Pyramid Pooling (ASPP) module is incorporated in its middle layer. With its generalized pyramid feature, this module enables the network to better capture and understand context information at various scales within the images. In addition to this, the concurrent spatial and channel squeeze & excitation (scSE) attention mechanism is adopted to better comprehend and manage context information in the images. Additional encoding layers are also added to the base (U-Net) and connected to the original encoding layer through skip connections. The resultant enhanced U-Net structure allows for better extraction and merging of high-level and low-level features, further boosting the network's ability to restore segmentation details. In addition, the combined Binary Cross Entropy (BCE)-Dice loss is utilized as the network's loss function. Experiments, conducted on the KiTS19 dataset, demonstrate that the proposed ASD-Net network outperforms the existing segmentation networks according to all evaluation metrics used, except for recall in the case of kidney tumor segmentation, where it takes the second place after Attention-UNet.

Fine tuning of porphyrin based-paddlewheel framework by imidazole derivative to boost electrochemiluminescence performance.

Precise tuning the structure of catalytic center is of great importance for the construction of enhanced electrochemiluminescence (ECL) emitters and the development of ECL amplification strategies, which is a key factor in improving the sensitivity of biosensors. In this work, we report the enhanced ECL emitters based on the porphyrin-based paddlewheel framework (PPF) with axial coordinated imidazole-like ligands (PPF/X, X = 2-methylimidazole (MeIm), imidazole (Im), benzimidazole (BIM)). In this system, the electron-donating ability of the axial ligands is positively correlated to its coordination ability to the paddlewheel units and the catalytic ability of the axially coordinated paddlewheel units. In addition, the electrochemical and ECL behavior of PPF/X (X = MeIm, Im, BIM) with different axial coordinated ligands are explored.

Design and application of dual-emission metal-organic framework-based ratiometric fluorescence sensors.

Metal-organic frameworks (MOFs) are novel inorganic-organic hybridized crystals with a wide range of applications. In the last twenty years, fluorescence sensing based on MOFs has attracted much attention. MOFs can exhibit luminescence from metal nodes, ligands or introduced guests, which provides an excellent fluorescence response in sensing. However, single-signal emitting MOFs are susceptible to interference from concentration, environment, and excitation intensity, resulting in poor accuracy. To overcome the shortcomings, dual-emission MOF-based ratiometric fluorescence sensors have been proposed and rapidly developed. In this review, we first introduce the luminescence mechanisms, synthetic methods, and detection mechanisms of dual-emission MOFs, highlight the strategies for constructing ratiometric fluorescence sensors based on dual-emission MOFs, and classify them into three categories: intrinsic dual-emission and single-emission MOFs with luminescent guests, and non-emission MOFs with other luminescent materials. Then, we summarize the recent advances in dual-emission MOF-based ratiometric fluorescence sensors in various analytical industries. Finally, we discuss the current challenges and prospects for the future development of these sensors.

Zinc porphyrin/MXene hybrids with phosphate-induced stimuli-responsive behavior for dual-mode fluorescent/electrochemiluminescent ratiometric biosensing.

Highly sensitive ratiometric biosensors have attracted much attention in biomarker detection, but most rely on single-mode signals, which can affect accuracy. The development of new principles and methods for dual-mode ratiometric sensing can enhance detection accuracy. Herein, the zinc(II) meso-tetra(4-carboxyphenyl) porphyrin/MXene (ZnTCPP/Ti3C2Tx) hybrids with phosphate-induced stimuli-responsive behavior are used to develop a novel dual-mode fluorescent/electrochemiluminescent (FL/ECL) ratiometric biosensor. The composites exhibit FL quenching and enhanced ECL behavior involving dissolved O2. The FL quenching of ZnTCPP/Ti3C2Tx is caused by energy transfer (EnT) and photo-induced electron transfer (PET) from ZnTCPP to Ti3C2Tx. While the introduction of MXene compensates for the inadequate conductivity of ZnTCPP, facilitating electron transfer, which further makes the surface ZnTCPP more capable of activating O2 to produce singlet oxygen (1O2), thereby generating enhanced cathodic ECL. Furthermore, phosphate ions (PO43-) can interact with the Ti sites of ZnTCPP/Ti3C2Tx, leading to competition for coordination with ZnTCPP, which in turn detaches ZnTCPP, resulting in enhanced FL and reduced ECL. On the basis of the phosphate-induced stimuli-responsive behavior, the dual-mode FL/ECL ratiometric biosensing of alkaline phosphatase (ALP) is achieved through ALP-catalyzed production of PO43- cascade effect with ZnTCPP/Ti3C2Tx. The linear detection range for ALP is 0.1-50 mU/mL, with a detection limit as low as 0.0083 mU/mL. This proposed ZnTCPP/Ti3C2Tx composites with stimuli-responsive behavior is expected to provide new ideas for the development of high-sensitivity dual-mode ratiometric biosensors with promising applications in the precise detection of important biomarkers.