Target-induced DNA nanomachine operation for the detection of proteins.

Accurate and sensitive detection of disease-associated proteins in early stage of patients plays an important role in timely treatment and successfully extending patients' lives. To meet this demand, we herein rationally designed a flexible target-induced DNA nanomachine operation (TIDNMO) sensor for the detection of proteins. The TIDNMO system was composed of DNA nanoswitch and DNA walker. Triplex DNA nanoswitch was triggered by specific target, followed by the release of the walking strand, which initiated the DNA walker amplification as signal output. In addition, the Exo III could drive walking strand autonomously move on gold nanoparticle surface to realize 2 orders of magnitude signal amplification. What's more, this sensor could transform its suitable functional recognition element of DNA nanoswitch to recognize other specific molecule and realize different targets sensing based on identical walking tracks. Considering the facile reporter elements and efficient amplification performance, the present DNA nanomachine as a sensor could achieve a detection limit of 68 pM for anti-Dig antibody, 0.95 pM for mucin-1 respectively, along with a superb specificity. Furthermore, the method reported here opened a new chapter in disease-related protein sensing for the development of clinical early diagnosis.

A simple, rapid and sensitive sandwich immunoassay based on poly(N-isopropylacrylamide) for the detection of alpha-fetoprotein.

Alpha-fetoprotein (AFP), as a tumor marker, plays a vital role in the diagnosis of liver cancer. In this work, a novel sandwich immunoassay based on a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), was developed for the detection of AFP. This immunoassay could realize one-step rapid reaction within 1 h, and facilitate the separation of the target molecules by incorporating PNIPAM. In this method, a conjugate of PNIPAM and capture antibody (Ab1) was successfully synthesized as a capture probe and the synthetic method of PNIPAM-Ab1 was simple, while the detection antibody (Ab2) was labeled with fluorescein isothiocyanate (FITC) to form a fluorescent detection probe. By employing a sandwich immunoassay, the method achieved quantitative determination of AFP, exhibiting a wide linear range from 5 ng/mL to 200 ng/mL and a low detection limit of 2.44 ng/mL. Furthermore, it was successfully applied to the analysis of spiked human serum samples and the screening of patients with hepatic diseases in clinical samples, indicating its potential application prospect in the diagnosis of liver cancer.

A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay.

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.

UV-induced oxidase activity of carbon dots in visible UVA dosage, Escherichia coli quantification and bacterial typing.

Ultraviolet (UV) light and foodborne pathogenic bacteriais are an important risk to the environment's safety. They endanger human health, and also lead to outbreaks of infectious disease, posing great threats to global public health security, national economy, and social stability. The appearance of carbon dot (CD) nanozymes offers a new perspective to solve the problems of detection of UV light and pathogenic bacteria in environment. This paper reports the preparation of CDs with dual enzyme-like activities (superoxide dismutase activity and UV-induced oxidase activity). The product can catalyze the oxidation of the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB) under UV light (365 nm) to achieve rapid color development. Based on the excellent fluorescence properties of CDs, the colorimetric-fluorescence dual-channel real-time detection of UVA dose was realized, the mechanism underlying the catalytic oxidation of TMB by UV-induced oxidase CDs was also investigated. Furthermore, a portable CDs-TMB-PA hydrogel was prepared which could realize the real-time monitoring of UV in outdoor environment with the assistance of smartphone. Based on the pH dependency of the CD nanozymes and specific glycolytic response of the pathogenic bacteria Escherichia coli (E. coli) O157:H7, the direct, simple, quick, and sensitive typing and detection have been realized. This research offers new perspectives for studying CD nanozymes and their applications in UV and bacterial detection, demonstrating the remarkable potential of CD nanozymes in detecting environmental hazards.

Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.

Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target a different site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing a fluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16 pM, which is a decrease of the LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.

Nucleic Acid Amplification-Free Digital Detection Method for SARS-CoV-2 RNA Based on Droplet Microfluidics and CRISPR-Cas13a.

Most of the methods currently developed for RNA detection based on CRISPR were combined with nucleic acid amplification. As a result, such methods inevitably led to certain disadvantages such as multiple operations, expensive reagents, and amplification bias. To solve the above problems, we developed a highly sensitive and specific nucleic acid amplification-free digital detection method for SARS-CoV-2 RNA based on droplet microfluidics and CRISPR-Cas13a. In this assay, thousands of monodisperse droplets with a size of 30 µm were generated within 2 min by a negative pressure-driven microfluidic chip. By confining a single target RNA recognition event to an independent droplet, the collateral cleavage products of activated Cas13a could be accumulated in one droplet. By combining the droplet microfluidics and CRISPR-Cas13a, SARS-CoV-2 RNA could be easily detected within 30 min with a detection limit of 470 aM. The performance of this assay was verified by specificity experiments and spiking and recovery experiments with human saliva. Compared with many developed methods for SARS-CoV-2 RNA detection, our method is time- and reagent-saving and easy to operate. Taken together, this digital detection method based on droplet microfluidics and CRISPR-Cas13a provides a promising approach for RNA detection in clinical diagnostics.

A functional nanoflower based lateral flow immunoassay for the rapid and robust detection of pathogens.

In the face of complex public health emergencies and various social medical needs in new situations, it is urgent to establish rapid detection technology for the early detection of pathogens to control their spread and minimize the resultant health and societal impact. Point-of-care testing (POCT) that allows rapid, on-site, and affordable detection and monitoring of health conditions at home or away from clinical labs has received increasing attention in modern medicine. In this work, we have synthesized multifunctional magainin I-human chorionic gonadotropin (hCG)-Cu3(PO4)2 nanoflowers and demonstrated a new strategy for the fast diagnosis of pathogenic microorganisms by combining functional nanoflowers with a lateral flow immunoassay device. The prepared multifunctional nanoflowers immobilized many signal molecules, which solves the poor sensitivity of traditional lateral flow strips and realizes the highly sensitive detection of pathogenic microorganisms ("accurate detection"). Besides, this method can complete the rapid transformation of commercial-off-the-shelf lateral flow strips and realize the fast diagnosis of target analytes in case of an outbreak ("fast detection"). Therefore, the established rapid and highly sensitive lateral flow immunoassay for the detection of pathogenic microorganisms will effectively improve the early diagnosis efficiency of infectious diseases caused by pathogenic microorganisms and shorten the diagnosis time of diseases.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Ratiometric fluorescent Si-FITC nanoprobe for immunoassay of SARS-CoV-2 nucleocapsid protein.

Coronavirus disease 2019 (COVID-19) highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission. Here, we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles (NPs) for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N protein). The synthesis of Si-FITC NPs did not need post-modification, which addressed the issue of quantum yield reduction during the coupling reaction. Si-FITC NPs showed two distinct peaks, Si fluorescence at λ em = 385 nm and FITC fluorescence at λ em = 490 nm. In the presence of KMnO4, Si fluorescence was decreased and FITC fluorescence was enhanced. Briefly, in the presence of N protein, catalase (CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates. Subsequently, hydrogen peroxide (H2O2) and Si-FITC NPs/KMnO4 were injected into the microplate together. The decomposition of H2O2 by CAT resulted in remaining of KMnO4, which changed the fluorescence intensity ratio of Si-FITC NPs. The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL, and the detection limit was 0.003 ng/mL, which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL. The N protein concentration can be accurately determined in human serum. Furthermore, the COVID-19 and non-COVID-19 patients were distinguishable by this method. Therefore, the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR, HRXPS); stability investigation of Si-FITC NPs (photostability, pH stability, anti-interference ability); stability investigation of free FITC (pH value, KMnO4); quenching mechanism of KMnO4 (UV-vis absorption spectra, fluorescence lifetime decay curves); reaction condition optimization of biotin-CAT with H2O2 (pH value, temperature, time); detection of N protein using commercial ELISA Kit; selectivity investigation of assays for SARS-CoV-2 N protein detection; determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-5005-z.

Dual-Color Fluorescent Hydrogel Microspheres Combined with Smartphones for Visual Detection of Lactate.

Since it is difficult for human eyes to distinguish between two identical colors with only <15% variation in brightness, mono-color fluorescent hydrogel microspheres have some limitations in the detection of lactate. Herein, we prepared novel dual-color fluorescent hydrogel microspheres, which can achieve hue transformation. Microspheres were prepared by introducing a fluorescent nanoparticle as the reference signal while CdTe QDs were used as the response signal. We used smartphones with image processing software to collect and analyze data. In this way, the signal of lactate was converted to RGB (red, green, and blue) values, which can be quantitatively read. Within 10 to 1500 µM, the R/G values of the microspheres had a linear relationship with the logarithm of the lactate concentration. Moreover, color cards for lactate detection were prepared, from which the color change and concentration of lactate could be easily read by the naked eye. It is worth mentioning that this method was successfully applied to screen patients with hyperlactatemia.

Synthesis of bio-templated clickable quantum dots and a dual-emitting organic/inorganic complex for ratiometric fluorescence visual assay of blood glucose.

With the prevalence of diabetes, rapid and simple blood glucose monitoring has become more and more important. Here, we report the synthesis of bio-templated N3-CdZnTeS quantum dots (QDs), which are great fluorescent biological labels and were used for the fabrication of dual-emissive dye@protein-QD conjugates via copper-free click chemistry, such as the 5(6)-carboxyfluorescein@glucose oxidase-quantum dot (FAM@GOx-QDs) complex. When adding glucose, the red fluorescence of the CdZnTeS QDs sharply decreased, while the green fluorescence of FAM was invariable. A good linear relationship ranging from 0.3 to 30 µM was obtained for glucose detection, with the limit of detection as low as 0.035 µM. Notably, the DNA-bridging FAM@GOx-QDs complex exhibited enhanced enzyme activity and stability, and was applied for the differentiation of diabetic and healthy people by the naked eye.

Metabolic labeling of enterovirus 71 with quantum dots for the study of virus receptor usage.

Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.

Glow-type chemiluminescent hydrogels for point-of-care testing (POCT) of cholesterol.

Cholesterol is an essential compound for human health, and a high or low concentration of cholesterol is closely related to various diseases. Thus, developing a simple method for POCT of cholesterol has great significance in clinical diagnosis. In this work, alginate (Alg) hydrogels with glow-type chemiluminescence (CL) were prepared and applied for rapid and quantitative cholesterol detection via a smartphone. The glow-type CL hydrogels (HRP/COD/luminol/Alg hydrogels) contained luminol as a chemiluminescent reagent, horseradish peroxidase (HRP) and cholesterol oxidase (COD) for enzymatic cascade reactions. The HRP/COD/luminol/Alg hydrogels exhibited outstanding stability, which effectively avoided the enzyme inactivation during long-term storage. Furthermore, the HRP/COD/luminol/Alg hydrogels exhibited longer and more stable glow-type CL. With the help of COD catalytic specificity for cholesterol and bi-enzymatic cascade reactions, the glow-type CL hydrogels realized the specific and sensitive detection of cholesterol. The smartphone was used as a detector instead of a special large instrument for responding to the glow-type CL emission, and a LOD of 7.2 µM was obtained. Therefore, the proposed sensor expands the application of the glow-type CL in POCT and provides an alternative way for cholesterol detection in clinical diagnosis.

A fluorescence color card for point-of-care testing (POCT) and its application in simultaneous detection.

Diabetes mellitus has received much attention because its complications include liver, kidney, eye, heart and cerebrovascular diseases. Thus, it would be highly significant to develop a rapid and efficient method for glucose detection in biological samples. In this work, a point-of-care testing (POCT) method of glucose detection was proposed using a standard colorimetric card for semi-quantitative determination patterns. In the prepared fluorescence color card for glucose, a good linear relationship was acquired by plotting the ratio of the grayscale value (I/I0) versus the logarithm of glucose concentration within 100.0 to 1000.0 µmol L-1, and the LOD of glucose detection was 1.1 µmol L-1. A large number of actual samples (30 serum and 7 urine) were analyzed and the results demonstrated that this method had good potential to be applied in the primary screening of diabetic patients. In addition, this method is universal and can be applied in the simultaneous detection of multiple small molecules. It provides a new strategy for the primary screening of multiple diseases simultaneously, which presents excellent application potential.

DNAzyme Sensor Uses Chemiluminescence Resonance Energy Transfer for Rapid, Portable, and Ratiometric Detection of Metal Ions.

DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.

Investigating the effect of 6-mercaptohexanol on the performance of a biosensor based on nanosurface energy transfer between gold nanoparticles and quantum dots.

Nanosurface energy transfer (NSET)-based sensors have been widely developed using various pairs of nanomaterials including gold nanoparticles (AuNPs) and quantum dots (QDs). However, a low signal to background ratio is one of the most important problems that researchers are continually trying to solve. Herein, we present a 6-mercaptohexanol (MCH) modified MCH/DNA-Au-QD sensor for the detection of nucleic acids and MUC1. Interestingly, an unexpected effect of MCH was found in enhancing the fluorescence recovery ratio, therefore yielding a higher signal to background ratio. Through further investigation, we perceive the enhancement as a result of lowering of the NSET efficiency between free DNA-AuNPs and free DNA-QDs, which arises from the stretching of adsorbed DNA on the surface of AuNPs. The employment of MCH endowed the sensor with a wider linear range from 5 nM to 120 nM and a relatively lower LOD of 1.19 nM in nucleic acid detection, outperforming the original DNA-Au-QD sensor. Furthermore, the application of the sensor can be further extended to MUC1 detection. This study offers a better understanding of the NSET process between QDs and AuNPs and also initiates a new approach for the performance optimization of analogous NSET-based sensors.

DNAzyme Walker for Homogeneous Detection of Enterovirus EV71 and CVB3.

When dealing with infectious pathogens, the risk of contamination or infection in the process of detecting them is nonnegligible. Separation-free detection will be beneficial in operation and safety. In this work, we proposed a DNAzyme walker for homogeneous and isothermal detection of enterovirus. The DNAzyme is divided into two inactivate subunits. When the subunit-conjugated antibody binds to the target virus, the activity of the DNAzyme recovers as a result of spatial proximity. The walker propels, and the fluorescence recovers. The final fluorescence intensity of the reaction mixture is related to the concentration of the target virus. The detection limit of this proposed method is 6.6 × 104 copies/mL for EV71 and 4.3 × 104 copies/mL for CVB3, respectively. Besides, this method was applied in detection of EV71 in clinical samples with a satisfactory result. The entire experiment is easy to operate, and the proposed method has great potential for practical use.

Point-of-care testing (POCT) of patients with a high concentration of uric acid by using alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs).

High concentration of uric acid is usually related to cardiovascular and cerebrovascular diseases. Developing a simple method for the rapid and efficient detection of uric acid has a great significance in clinical diagnosis. In this work, alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs) were prepared for the first time, and further used for point-of-care testing (POCT) of patients with a high concentration of uric acid. This strategy is mainly based on visual detection of H2O2, the product of uric acid after an enzymatic reaction. The proposed sensor (Alg@QDs-UOx MSs) has several advantages. First, it can reduce the interference of the proteins to the fluorescence of QDs. Second, Alg@QDs-UOx MSs help improve the stability of the CdZnTeS QDs as well as the activity of urate oxidase during storage. Third, it is easy to use, has fast response speed, and is of low cost. Therefore, the proposed sensor shows good application prospects. Simply through the built-in camera of a smartphone, we can visualize the urine samples from patients with a high concentration of uric acid within 10 minutes, and the accuracy rates were 100%. In the range of 100.0 µM to 900.0 µM, the I/I0 values and uric acid concentrations are in a great linear relationship (R2 = 0.9973), indicating that this method can be employed for quantitative analysis of uric acid in human urine (<10 mM). The limit of detection (LOD) is 20.3 µM.

Novel Method of Clickable Quantum Dot Construction for Bioorthogonal Labeling.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

Magnetic bead-enzyme assemble for triple-parameter telomerase detection at single-cell level.

In this work, we developed a triple-parameter strategy for the detection of telomerase activity from cancer cells and urine samples. This strategy was developed based on magnetic bead-enzyme hybrids combined with fluorescence analysis, colorimetric assay, or adenosine triphosphate (ATP) meter as readout. The application of magnetic bead-enzyme hybrids has the advantages of magnetic separation and signal amplification. These detection methods can be used individually or in combination to achieve the optimal sensing performance and make the results more convincing. Among them, the ATP meter with portable size had easy operation and low cost, and this response strategy provided a higher sensitivity at the single-cell level. The designed strategy was suitable as naked-eye sensor and point-of-care testing (POCT) for rapid assaying of telomerase activity. Graphical abstract Magnetic bead-enzyme assemble for triple-parameter telomerase detection.