Triblock PolyA-Mediated Protein Biosensor Based on a Size-Matching Proximity Hybridization Analysis.

The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.

A multi-channel electrochemical biosensor based on polyadenine tetrahedra for the detection of multiple drug resistance genes.

Antimicrobial resistance poses a serious threat to human health due to the high morbidity and mortality caused by drug-resistant microbial infections. Therefore, the development of rapid, sensitive and selective identification methods is key to improving the survival rate of patients. In this paper, a sandwich-type electrochemical DNA biosensor based on a polyadenine-DNA tetrahedron probe was constructed. The key experimental conditions were optimized, including the length of polyadenine, the concentration of the polyadenine DNA tetrahedron, the concentration of the signal probe and the hybridization time. At the same time, poly-avidin-HRP80 was used to enhance the electrochemical detection signal. Finally, excellent biosensor performance was achieved, and the detection limit for the synthetic DNA target was as low as 1 fM. In addition, we verified the practicability of the system by analyzing E. coli with the MCR-1 plasmid and realized multi-channel detection of the drug resistance genes MCR-1, blaNDM, blaKPC and blaOXA. With the ideal electrochemical interface, the polyA-based biosensor exhibits excellent stability, which provides powerful technical support for the rapid detection of antibiotic-resistant strains in the field.

Development of a Single-Molecule Nanobalance Ratiometric Electrochemical DNA Biosensor Using a Triblock Poly-Adenine Probe.

The development of electrochemical DNA biosensors has been limited by their reliability and reproducibility due to many interfering factors such as electrode properties, DNA surface densities, and complex biological samples. In this work, we developed a nanobalance polyA hairpin probe (polyA-HP), which was effectively assembled onto the gold electrode surface through the affinity between the central polyA fragment and the Au surface. One flanking probe of the polyA-HP captured the target sequence together with a MB-labeled signal probe, and the other flanking probe captured a reference probe simultaneously. The MB signal related to the amount of target was normalized by the reference Fc signal; thus, the signal-to-noise (S/N) was as high as 2000, and the reproducibility was remarkably improved to 2.77%, even facing deliberately changed experiment conditions. By designing a hairpin structure at the terminal of the polyA-HP, the selectivity and specificity were dramatically improved for the analysis of mismatched sequences. The analysis performance of biological samples was dramatically improved after normalization, which is critical for its practicability. Our novel biosensor is a universal single-molecule platform for ratiometric biosensors with excellent performance in real samples, indicating great potential for next-generation high-precision electrochemical sensors.

Triblock probe-polyA-probe electrochemical interfacial engineering for the sensitive analysis of RNAi plants.

RNA interference (RNAi) is currently under fast development, which brings improved crop quality and new activity against pests in agriculture, by producing RNAs to specifically inhibit gene expression. This technology, in turn, creates a pressing need for sensitive and specific analytical methods of exogenous RNA molecules in genetically modified (GM) crops for safety assessment and regulation of RNAi plants and their products. In this work, we developed a novel RNA electrochemical biosensor for the analysis of GM maize samples based on a polyA-DNA capturing probe containing three DNA segments: the central polyA segment combined onto a gold electrode surface with adjustable configuration and density, and two flanking DNA probes simultaneously captured the RNA targets through hybridization. Both the assembling and hybridization capability of our probe were demonstrated, and we systematically optimized the analytical conditions. Finally, the ultrasensitive detection of 10 fM RNA was realized without any amplification processes, and the specificity was verified by analyzing non-target maize samples. Our electrochemical biosensor provided a reliable and convenient measurement strategy for RNAi safety and quality assessment, and more importantly, our PAP (probe-polyA-probe) capturing probe exhibited an innovative design for the detection of large RNA molecules with complex secondary structures.

Medium- to long-term outcomes of vaginally assisted laparoscopic sacrocolpopexy in the treatment of stage III-IV pelvic organ prolapse.

BACKGROUND: Vaginally assisted laparoscopic sacrocolpopexy (VALS) refers to the placement of synthetic meshes through the vagina in addition to traditional laparoscopic sacrocolpopexy. In this study, we aimed to investigate the medium- to long-term efficacy and safety of VALS for treating stage III-IV pelvic organ prolapse (POP). METHODS: The study was designed as a case series at a single center. Patients with stage III-IV POP in our hospital from January 2010 to December 2018 were included. Perioperative parameters, objective and subjective outcomes, and complications were assessed. RESULTS: A total of 106 patients completed the follow-up and were included in our study. Within a median follow-up duration of 35.4 months, the objective cure ratio of VALS reached 92.45% (98/106), and the subjective success rate was 99.06% (105/106). Patients reported significant improvements in subjective symptoms. In eight patients suffering anatomic prolapse recurrence, two posterior POP cases were treated by posterior pelvic reconstruction surgery, while six anterior POP cases did not need surgical therapies. The reoperation rate was 1.89% (2/106). No intraoperative complications occurred. Three patients (2.83%) had postoperative fever, and one (0.94%) had wound infection during hospitalization. Six patients (5.66%) had mesh exposure on the vaginal wall, and de novo urinary incontinence occurred in two patients (1.89%) during the follow-up period. CONCLUSION: VALS is an effective and safe surgical method for treating severe POP. Therefore, VALS should be considered in the treatment of severe POP due to its favorable subjective and objective outcomes, relatively low rate of infection and acceptable rate of mesh exposure.

Development of pattern recognition based on nanosheet-DNA probes and an extendable DNA library.

Pattern recognition, also called "array sensing," is a recognition strategy with a wide and expandable analysis range, based on high-throughput analysis data. In this work, we constructed a sensor array for the identification of targets including bacterial pathogens and proteins by using FAM-labeled DNA probes and 2D nanosheet materials. We designed an ordered and extendible DNA library for the collection of recognition probes. Unlike traditional DNA probes with random and massive sequences, our DNA library was constructed following a 5-digit binary number (00000-11111, 0 = CCC, and 1 = TTT), and especially, 8 special symmetry sequences were chosen from the library. Two different nanosheet materials were used as the quencher. When targets were added, the interaction between DNA and the nanosheets was competitively affected, and as a result, the fluorescence signal changed accordingly. Finally, by using our fluorescent sensor array, 17 bacteria and 8 proteins were precisely recognized. We believe that our work has provided a simple and valuable strategy for the improvement of the recognition range and discrimination precision for the development of pattern recognition.

A polyA DNA probe-based ultra-sensitive and structure-distinguishable electrochemical biosensor for the analysis of RNAi transgenic maize.

Since the application of RNA interference (RNAi) is rapidly developing in GMO technology, accurate and sensitive detection of functional RNA molecules was urgently needed, for the safety and functional assessment of RNAi crops. In this work, we developed an electrochemical biosensor for transgene-derived long RNA based on a poly-adenine (polyA) DNA capture probe. The polyA self-assembling monolayer (SAM) provided enhanced interface stability and optimized surface density for the subsequent hybridization of the long RNA molecule. A multiple reporter probe system (MRP) containing 12 reporter probes (RPs) and 2 spacers was applied to open the complex molecular secondary structure and hybridize with the long RNA, with the critical assistance of dimethyl sulfoxide (DMSO). By using 3 addressable RPs, structural recognition was performed among long stem-loop RNA, long dsRNA (no loop), and siRNA. Excellent selectivity was achieved when the extracted total RNA samples were directly analyzed. When reverse transcription recombinase polymerase amplification (RT-RPA) technology was combined, the sensitivity was improved to 10 aM. To the best of our knowledge, this is the first electrochemical biosensor with the excellent capability of quantification and structural analysis of the long RNA of the RNAi GMO. Our work shows great potential in a wide range of RNAi GMO samples.

DNA Framework-Mediated Electrochemical Biosensing Platform for Amplification-Free MicroRNA Analysis.

MicroRNAs (miRNAs) have been explored as biomarkers for early diagnosis of diseases like cancers. However, it remains challenging to detect low-level miRNAs in the total RNA from real samples in a facile approach. In this work, we report a two-phase miRNA biosensing strategy based on a modular framework nucleic acid (FNA) platform, which combines the high efficiency of homogeneous reaction and the convenience of heterogeneous biosensing. In the first phase, free DNA probes bind target miRNAs in a homogeneous solution, forming a DNA-RNA complex with high base stacking energy. Then, at the second phase, the universal FNA interface on the electrode selectively mediated the transition of the complex from the solution onto the interface for electrochemical signal generating and transduction. By applying this method, we detected as few as 1 aM of miR-141, a cancer marker miRNA, without the need for nucleic acid amplification. The dynamic range spans 10 orders of magnitude. We demonstrate multiplex miRNA detection and discrimination of highly homologous miRNAs with mismatches as few as a single base. We also show that this system can detect miR-141 in only 50 ng of total RNA samples from real cells, which allows discrimination of prostate cancer cells with normal cells. We envision this platform may satisfy the need for facile and high-throughput screening of early cancer markers.

Ultrasensitive Electrochemical Biosensor of Bacterial 16S rRNA Gene Based on polyA DNA Probes.

Traditional microbiology analysis is usually hindered by the long time-cost and lack of portability in many urgent situations. In this work, we developed a novel electrochemical DNA biosensor (E-biosensor) for sensitive analysis of the 16S rRNA gene of five bacteria, using a consecutive adenine (polyA) probe. The polyA probe consists of a polyA tail and a recognition part. The polyA tail can combine onto the gold surface with improved controllability of the surface density, by conveniently changing the length of polyA. The recognition part of the capture probe together with two biotin-labeled reporter probes hybridize with the target DNA and form a stable DNA-tetramer sandwich structure, and then avidin-HRP enzyme was added to produce a redox current signal for the following electrochemical detection. Finally, we realized sensitive quantification of artificial target DNA with a limit of detection (LOD) of 10 fM, and excellent selectivity and reusability were also demonstrated. Importantly, the detection capability was equally good when facing bacterial genomic DNA, due to the base-stacking force of our multireporter-probe system, which can help to break the second structure and stabilize the probe-target complexes. Our biosensor was constructed on a 16-channel electrode chip without any polymerase chain reaction (PCR) process needed, which took a significant step toward a portable bacteria biosensor.

Ultrasensitive Electrochemical DNA Biosensor Based on a Label-Free Assembling Strategy Using a Triblock polyA DNA Probe.

Multiblock DNA probe attracted a large amount of scientific attention, for the development of multitarget biosensor and improved specificity/sensitivity. However, the development of multiblock DNA probes highly relied on the chemical synthesis of organic linkers or nanomaterials, which limited their practicability and biological compatibility. In this work, we developed a label-free assembling strategy using a triblock DNA capture probe, which connects two DNA probes with its intrinsic polyA fragment (probe-PolyA-probe, PAP). The middle polyA segment has a high affinity to the gold electrode surface, leading to excellent reproducibility, stability, and regeneration of our biosensor. Two flanking capture probes were tandemly co-assembled on the electrode surface with consistent spatial relationship and exactly the same amount. When combined with the target DNA, the hybridization stability was improved, because of the strong base stacking effect of two capture probes. The sensitivity of our biosensor was proved to be 10 fM, with a wide analysis range between 10 fM to 1 nM. Our PAP-based biosensor showed excellent specificity when facing mismatched DNA sequences. Even single nucleotide polymorphisms can be distinguished by each probe. The excellent practicability of our biosensor was demonstrated by analyzing genomic DNA both with and without PCR amplification.

Fluorometric determination of HIV DNA using molybdenum disulfide nanosheets and exonuclease III-assisted amplification.

A convenient and ultrasensitive fluorometric method is described for the determination of HIV DNA. It exploits the strong difference in the affinities of MoS2 nanosheets for long ssDNA versus short oligonucleotide fragments. In addition, efficient signal amplification is accomplished by exonuclease III-assisted target recycling. When absorbed on the MoS2 nanosheets, the fluorescence of the FAM-labeled ssDNA probe (FP) is quenched. However, in the presence of HIV DNA, the FP hybridizes with target to form a duplex. As a result, the FP in the duplex will be stepwise hydrolyzed into short fragments by Exo III, and the fluorescence signal thus is retained because short fragments have low affinity for the MoS2 nanosheets. By using the Exo III-assisted target recycling amplification, the detection sensitivity is strongly improved. The sensor can detect DNA in a concentration as low as 5.3 pM (at an S/N ratio of 3), and the analytical range extends from 0.01 nM to 10 nM. The assay is simple, sensitive and specific, and conceivably represents a valuable tool in clinical studies related to the HIV. Graphical abstract Schematic presentation of fluorometric determination of HIV DNA based on molybdenum disulfide nanosheets and Exo III. When the fluorescence-tagged ssDNA probe hybridized with target to form a duplex, the Exo III-assisted target recycling amplification is generated. The method can detect as low as 5.3 pM HIV DNA.

Multiplexed aptasensing of food contaminants by using terminal deoxynucleotidyl transferase-produced primer-triggered rolling circle amplification: application to the colorimetric determination of enrofloxacin, lead (II), Escherichia coli O157:H7 and tropomyosin.

A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3'-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H2O2-mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL-1 with a linear range that extends from 1 pg mL-1 to 1 µg·mL-1. The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%. Graphical abstractSchematic representation of a TdT-RCA based aptasensor for multiple analytes related to food safety. It makes use of sandwich aptasensors and TdT-produced universal primer-triggered RCA reaction. dATP: deoxyadenosine triphosphate, TdT: Terminal Deoxynucleotidyl Transferase, RCA: rolling circle amplification, TMB: 3,3',5,5'-Tetramethylbenzidine.

Electrochemical detection of PCR amplicons of Escherichia coli genome based on DNA nanostructural probes and polyHRP enzyme.

Fast, portable and sensitive analysis of E. coli is becoming an important challenge in many critical fields (e.g., food safety, environmental monitoring and clinical diagnosis). Thus, electrochemical biosensing of PCR amplicons from the bacterial genome has attracted reasonable research attention. In this work, we utilized a 3D DNA tetrahedral probe to establish a "sandwich-type" electrochemical DNA biosensor for sensitive and specific analysis of a 250 bp unpurified PCR amplicon from the uidA gene of the E. coli genome. Asymmetric PCR was used to produce single-stranded PCR products. Streptavidin-polyHRP80 was employed to improve the signal gain during electrochemical detection. We optimized important experimental conditions for DNA sensing, including the streptavidin-polyHRP, the signal probe and the ion strength. Finally, we achieved a remarkable sensitivity of 10 fM synthetic DNA target, and successfully performed the analysis of PCR amplicons from as low as 0.2 pg µL(-1) of E. coli genome. Compared with traditional single stranded DNA (ssDNA) probe based detection, our present work demonstrated 3 orders of magnitude improvement in sensitivity. In addition, our electrochemical DNA biosensor was 4 orders of magnitude more sensitive than normal electrophoretic analysis of PCR products. Our work made important progress in DNA nanostructured probe-based biosensors toward application in real applications.

A Sensitive and Label-Free Pb(II) Fluorescence Sensor Based on a DNAzyme Controlled G-Quadruplex/Thioflavin T Conformation.

Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.

Wheat mitogen-activated protein kinase gene TaMPK4 improves plant tolerance to multiple stresses through modifying root growth, ROS metabolism, and nutrient acquisitions.

KEY MESSAGE: Wheat MAPK member TaMPK4 responds to abiotic stresses of Pi and N deprivations and high salinity and is crucial in regulating plant tolerance to aforementioned stresses. Mitogen-activated protein kinase (MAPK) cascades are important signal transduction modules in regulating plant responses to various environmental stresses. In this study, a wheat MAPK member referred to TaMPK4 was characterized for its roles in mediating plant tolerance to diverse stresses. TaMPK4 shares conserved domains generally identified in plant MAPKs and possesses in vitro kinase activity. Under stresses of Pi and N deprivations and high salinity, TaMPK4 was strongly upregulated and its expressions were restored upon recovery treatments from above stresses. Sense- and antisense-expressing TaMPK4 in tobacco significantly modified plant growth under the stress conditions and dramatically modified the root architecture through transcriptional regulation of the auxin transport-associated genes NtPIN3 and NtPIN9, whose downregulated expressions dramatically reduced the root growth. Compared with wild type (WT), the antioxidant enzymatic activities under the stress conditions, P accumulation under P deprivation, and N amount under N deficiency were altered dramatically in the transgenic plants, showing higher in the TaMPK4-overexpressing and lower in the TaMPK4-knockout plants, which were in concordance with the modified expressions of a set of antioxidant enzyme genes (NtPOD2;1, NtPOD9, NtSOD2, NtFeSOD, and NtCAT), two phosphate transporter genes (NtPT and NtPT2), and two nitrate transporter genes (NtNRT1.1-s and NtNRT1.1-t), respectively. Downregulated expression of above genes in tobacco largely reduced the plant growth, and Pi and N acquisitions under the stress conditions. TaMPK4 also involved regulations of plant K(+) and osmolyte contents under high salinity. Thus, TaMPK4 is functional in regulating plant tolerance to diverse stresses through modifying various biological processes.

Target-responsive, DNA nanostructure-based E-DNA sensor for microRNA analysis.

Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this work, we have demonstrated a new generation (the third generation) of E-DNA sensor for the sensitive and specific detection of microRNAs. Our third generation of E-DNA sensor can sensitively detect microRNA target (microRNA-141) as low as 1 fM. The excellent specificity has been demonstrated by its differential ability to the highly similar microRNA analogues. In our design, the use of DNA tetrahedron ensures the stem-loop structure in well controlled density with improved reactivity. The regulation of the thermodynamic stability of the stem-loop structure decreases the background signal and increases the specificity as well. The enzymes attached bring the electrocatalytic signal to amplify the detection. The combination of these effects improves the sensitivity of the E-DNA sensor and makes it suitable to the microRNA detection. Finally, our third generation of E-DNA sensor is generalizable to the detection of other micro RNA targets (for example, microRNA-21).

DNA nanostructure-based ultrasensitive electrochemical microRNA biosensor.

MicroRNAs (miRNAs) are key regulators of a wide range of cellular processes, and have been identified as promising cancer biomarkers due to their stable presence in serum. As an surface-based electrochemical biosensors which offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and the surface crowding effect at the water-electrode interface. Here, we present a protocol as well as guidelines for ultrasensitive detection of miRNA with DNA nanostructure-based electrochemical miRNA biosensor. By employing the three-dimensional DNA nanostructure-based interfacial engineering approach, we can directly detect as few as attomolar (<1000 copies) miRNAs with high single-base discrimination ability. Since this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, it can conveniently and reliably analyze miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients.

Investigation on pharmacokinetics, tissue distribution and excretion of a novel platinum anticancer agent in rats by inductively coupled plasma mass spectrometry (ICP-MS).

1. DN604 is a new platinum agent with encouraging anticancer activity. The present study was to explore the pharmacokinetic profiles, distribution and excretion of platinum in Sprague-Dawley rats after intravenous administration of DN604. A sensitive and selective inductively coupled plasma mass spectrometry (ICP-MS) method was established for determination of platinum in biological specimens. The pharmacokinetic parameters were calculated by a non-compartmental method. 2. The area under concentration-time curve AUC0-t and AUC0-∞ for platinum originating from DN604 at 10 mg/kg were 25.15 ± 1.29 and 28.72 ± 1.04 µg/hml, respectively. The mean residence time MRT was 36.59 ± 6.65 h. The volume of distribution Vz was 11.42 ± 2.49 l/kg and clearance CL was 0.18 ± 0.01 l/h/kg. In addition, the elimination half-life T1/2z was 44.83 ± 9.75 h. After intravenous administration of DN604, platinum was extensively distributed in most of tested tissues except brain. The majority of platinum excreted via urine, and its accumulative excretion ratio during the period of 120 h was 63.5% ± 7.7% for urine, but only 6.94% ± 0.11% for feces. 3. The satisfactory half-life, wide distribution and high excretion made this novel platinum agent worthy of further research and development.

Development of Optical Differential Sensing Based on Nanomaterials for Biological Analysis.

The discrimination and recognition of biological targets, such as proteins, cells, and bacteria, are of utmost importance in various fields of biological research and production. These include areas like biological medicine, clinical diagnosis, and microbiology analysis. In order to efficiently and cost-effectively identify a specific target from a wide range of possibilities, researchers have developed a technique called differential sensing. Unlike traditional "lock-and-key" sensors that rely on specific interactions between receptors and analytes, differential sensing makes use of cross-reactive receptors. These sensors offer less specificity but can cross-react with a wide range of analytes to produce a large amount of data. Many pattern recognition strategies have been developed and have shown promising results in identifying complex analytes. To create advanced sensor arrays for higher analysis efficiency and larger recognizing range, various nanomaterials have been utilized as sensing probes. These nanomaterials possess distinct molecular affinities, optical/electrical properties, and biological compatibility, and are conveniently functionalized. In this review, our focus is on recently reported optical sensor arrays that utilize nanomaterials to discriminate bioanalytes, including proteins, cells, and bacteria.

Construction of an Enzyme Cascade Based on the Accurate Adjacent Arrangement of Coupled Enzymes Using a Triblock PolyA DNA Probe.

Intracellular enzyme cascades are essential for various biological processes, and mimicking their functions in artificial systems has attracted significant research attention. However, achieving convenient and efficient spatial organization of enzymes on interfaces remains a critical challenge. In this work, we designed a simple single-DNA scaffold using triblock polyA single-stranded DNA for the arrangement of coupled enzymes. The scaffold was assembled onto a gold electrode through the affinity of polyA-Au, and two enzymes (glucose oxidase and horseradish peroxidase) were captured through hybridization. The molecular distance between the enzymes was regulated by changing the length of the polyA fragment. As a proof of concept, a glucose biosensor was constructed based on the enzyme cascade amplification. The biosensor exhibited excellent detection capability for glucose in human serum samples with a limit of detection of 1.6 µM. Additionally, a trienzyme cascade reaction was successfully activated, demonstrating the potential scalability of our approach for multienzyme reactions. This study provides a promising platform for the development of easy-to-operate, highly efficient, and versatile enzyme cascade systems using DNA scaffolds.