Real Biological Samples Research Articles

Poly-Phthalocyanine-Doped Graphene Oxide Nanosheet Conjugates for Electrocatalytic Oxidation of Drug Residues.

Donor and acceptor phthalocyanine molecules were copolymerized and linked to graphene oxide nanosheets through amidation to yield electrocatalytic platforms on glassy carbon electrodes. The platforms were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, UV/Vis spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The fabricated electrochemical catalytic surfaces were then evaluated toward electrocatalytic detection of ascorbic acid and tryptophan. These were characterized by a wide linear dynamic range and low limits of detection and quantification of 2.13 and 7.12 µM for ascorbic acid and 1.65 and 5.5 µM for tryptophan, respectively. The catalytic rate constant was 1.86 × 104 and 1.51 × 104 M−1s−1 for ascorbic acid and tryptophan, respectively. The Gibbs energy for catalytic reactions was −17.45 and −14.83 kJ mol−1 depicting a spontaneous reaction on the electrode surface. The sensor platform showed an impressive recovery when applied in real samples such as fresh cow milk, in the range 91.71–106.73% for both samples. The developed sensor therefore shows high potential for applicability for minute quantities of the analytes in real biological samples.

Pyrrolidinyl ligand motif-assisted bovine serum albumin molecularly imprinted polymers with high specificity

Ideal binding ligands for anchoring proteins are essential for the design and assembly of desirable molecularly imprinted polymers (MIPs). In this study, bovine serum albumin-MIPs (BSA-MIPs) were successfully prepared by orchestrating the involvement of orientation-controllable binding ligands via sequential thiol-ene click and thiol-ene-amine conjugation. We showed that the optimal thiol-ene-amine conjugates and binding ligands were decisive in determining the rebinding capacity and selectivity. The pyrrolidinyl MIPs exhibited the best adsorption capacity of 352 ± 22 mg/g and a superior imprinting factor of 4.72 among MIPs with various binding ligands. These favourable results were further studied by computational simulation and isothermal titration calorimetry (ITC). Molecular docking revealed the preferential binding free energy and H-bonds between BSA residues and the thiol-ene-amine conjugates. Meanwhile, the pyrrolidinyl ligand motif enabled entropy-favourable affinity to be achieved via hydrophobic effects with the BSA template by ITC thermodynamics. Because of these favourable bindings, the MIPs exhibited excellent adsorption specificity to BSA over competing proteins. The proof-of-concept of MIPs with orientation-controllable conjugates and proven binding ligands for target proteins demonstrates that this material is promising for use with a real biological sample.

Non-targeted Lipidomics Using a Robust and Reproducible Lipid Separation Using UPLC with Charged Surface Hybrid Technology and High-Resolution Mass Spectrometry.

Lipids play an important role in the energy storage, cellular signaling, and pathophysiology of diseases such as cancer, neurodegenerative diseases, infections, and diabetes. Due to high importance of diverse lipid classes in human health and disease, manipulating lipid abundance and composition is an important target for metabolic engineering. The extreme structural diversity of lipids in real biological samples is challenging for analytical techniques due to large difference in physicochemical properties of individual lipid species. This chapter describes lipidomic analysis of large sample sets requiring reliable and robust methodology. Rapid and robust methods facilitate the support of longitudinal studies allowing the transfer of methodology between laboratories. We describe a high-throughput reversed-phase LC-MS methodology using Ultra Performance Liquid Chromatography (UPLC®) with charged surface hybrid technology and accurate mass detection for high-throughput non-targeted lipidomics. The methodology showed excellent specificity, robustness, and reproducibility for over 100 LC-MS injections.

Structure and function encoding of a bidirectional activatable synergetic DNA machine for speeded and ultrasensitive determination of microRNAs

This work describes the unique design of a bidirectional activatable synergetic DNA machine (BAS-DNA machine) for speeded and ultrasensitive determination of microRNA-21 (miR-21), a well-known biomarker for biomedical research and early diagnosis of lung cancer. The BAS-DNA machine is composed by a pair of track strands (Track 1 and Track 2) encoding with two regions in the opposite direction for miR-21 recognition. Introduction of miR-21 can hybridize either with Track 1 or with Track 2 to activate the BAS-DNA machine with a synergistic effect for speeded amplifying the fluorescence signal. Moreover, compared with common DNA machine with only one switch for exogenous target recognition, the BAS-DNA machine with two switches for miR-21 binding allows the speeded and strong operation of the autonomous strand scission, replication, and displacement on Track 1 and Track 2 simultaneously. This behavior makes the BAS-DNA machine powerful for ultrasensitive, specific, and fast screening of miR-21 even from real biological samples, and the fluorescence signal was found to be linear from 1 pM to 10 nM with a detection limit of 703.6 fM. We envision this BAS-DNA machine with its superior assay performance will provide a new avenue for simple, sensitive, and affordable biomedical assays.

Terahertz imaging with metamaterials for biological applications

Terahertz (THz) technology has become more widespread due to its diverse range of potential applications, particularly when combined with various functional metamaterials using cutting-edge nanotechnology techniques. In this report, we introduce a highly improved THz imaging technology by comparing complementary metamaterials intuitively based on Babinet’s principle. The THz reflectance spectra for the complementary metamaterials exhibit a significant and distinct association with the polarization angle. Four different polarization angles and metamaterial pattern geometries were tested for the reflectance imaging of a target image pattern. Field enhancement on the metamaterial surface was also investigated using finite element simulations to support the experimental results. Optimizing the metamaterial based on the experimental and calculation results led to high image contrast and quality. The proposed label-free imaging platform was then employed to produce clear contrast images for mouse brain tissue and HEK-293 cells, thus highlighting the potential application of this system to real biological samples.

An alternative label-free DNA sensor based on the alternating-current electroluminescent device for simultaneous detection of human immunodeficiency virus and hepatitis C co-infection

Coinfection of HIV/HCV is a significant public health issue globally, as it increases the risk of liver cancer in co-infected individuals. The point-of-care testing (POCT) device for HIV/HCV DNA detection is promptly needed for diagnosis and monitoring of the disease progression. Here, the alternating-current electroluminescence (ACEL) technique is proposed as a sensitive POCT sensing platform for HIV/HCV cDNA detection. A conductance-based light emission modulated by the hybridization between a pyrrolidinyl PNA probe and the DNA target enabled the DNA detection in a label-free format. Enhanced electroluminescence was observed in the presence of the target DNA due to the increased proton conductivity. Under the optimal conditions, the linearity range from 1 nM to 1 μM was achieved for HIV and HCV cDNA with LODs of 1.86 pM (HIV cDNA) and 1.96 pM (HCV cDNA). The spiked HIV/HCV cDNA in healthy human serum was successfully detected, demonstrating the feasibility of the developed device for the detection of cDNA in real biological samples. Additionally, simultaneous HIV/HCV cDNA detection on a single ACEL device employing a 2x2-array detection zone design. The cross-reactivity with other viral DNA was shown to be minimal due to the high specificity of the PNA probes used. Finally, the negative and positive samples from the patient's serum were tested and the results were in 100% agreement with the commercial kit based-on real-time PCR method, thus illustrating the high sensitivity and specificity of the developed sensor.

A SERS aptasensor based on porous Au-NC nanoballoons for Staphylococcus aureus detection

In this work, we developed a new approach for fabricating hollow and porous nitrogen doped carbon nanoballoons loading AuNPs (Au-NC-NBs) with a large specific surface area, a high N and Au content. The surface-enhanced Raman scattering (SERS) aptasensor based on the resulting Au-NC-NBs possess a wider linear range (10 to 107 cells/mL), a lower detection limit (3 cells/mL), better selectivity for detecting bacteria than previously reported sensors. Importantly, Au-NC-NBs SERS aptasensor also exhibits excellent performance for detecting bacteria in the real food and biological samples. This work provides a facile and versatile designing strategy for controlled construction of SERS biosensor by combination of Au nanoparticles and carbon materials, which has a great applied potential in food safety monitoring and clinical diagnosis.

Hierarchical nickel hydroxide nanosheets grown on hollow nitrogen doped carbon nanoboxes as a high-performance surface substrate for alpha-fetoprotein cancer biomarkers electrochemical aptasensing

During recent decades, we have witnessed a great improvement in the performance of aptamer-based sensors, specifically when aptamers are combined with new nanomaterials; as a platform for biosensors. The design of hollow carbon-based materials has also received a lot of attention due to its excellent properties in various applications. Herein, we aim at designing hierarchical porous Ni(OH)2 nanosheets on hollow N-doped carbon nanoboxes Ni(OH)2@N-C n-box). In this sense, we obtained the hollow N-C n-box skeletons from the Fe2O3 nanocubes template. The development of label-free electrochemical aptasensor was carried out using the covalently immobilizing NH2-functionalized aptamer on Ni(OH)2@N-C n-box as an efficient substrate. The Ni(OH)2@N-C n-box was characterized using scanning fourier transform infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Brunauer, Emmett and Teller (BET), transmission electron microscopes (TEM) and electron microscopy (FESEM). The electrochemical evaluations clarified the fact that a linear relationship exists between the alpha-fetoprotein (AFP) contents and the charge transfer resistance (Rct) (from 1 fg mL−1 to 100 ng mL−1) with a low detection limit of 0.3 fg mL−1. Moreover, regarding the aptasensor, the superior detection recoveries were experienced in real biological samples, illustrating its great detection performance and practical feasibility. Considering the aptasensor application, these studies showed that Ni(OH)2@N-C n-box possesses different enhanced electrochemical features, making it appropriate as an electrode material for aptasensor application.

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection.

The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 μL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 μL of whole blood within 1 h.

Nitrogen-doped carbon@TiO2 double-shelled hollow spheres as an electrochemical sensor for simultaneous determination of dopamine and paracetamol in human serum and saliva

As the most commonly used antipyretic and analgesic drug, paracetamol (PA) coexists with neurotransmitter dopamine (DA) in real biological samples. Their simultaneous determination is extremely important for human health, but they also interfere with each other. In order to improve the conductivity, adsorption affinity, sensitivity, and selectivity of TiO2-based electrochemical sensor, N-doped carbon@TiO2 double-shelled hollow sphere (H–C/N@TiO2) is designed and synthesized by simple alcoholic and hydrothermal method, using polystyrene sphere (PS) as a template. Meanwhile, TiO2 hollow spheres (H–TiO2) or N-doped carbon hollow spheres (H–C/N) are also prepared by the same method. H–C/N@TiO2 has good conductivity, charge separation, and the highly enhanced and stable current responses for the detection of PA and DA. The detection limit and linear range are 50.0 nmol/L and 0.3–50 μmol/L for PA, 40.0 nmol/L and 0.3–50 μmol/L for DA, respectively, which are better than those of carbon-based sensors. Moreover, this electrochemical sensor, with high selectivity, strong anti-interference, high reliability, and long time durability, can be used for the simultaneous detection of PA and DA in human blood serum and saliva. The high electrochemical performance of H–C/N@TiO2 is attributed to the multi-functional combination of different layers, because of good conductivity, absorption and electrons transfer ability from in-situ N-doped carbon and electrocatalytic activity from TiO2.

Sensitivity and intrinsic limit of detection improvement in a photonic refractive-index sensor

In this work, we study the bulk refractive index (RI) sensitivity (S) and intrinsic limit of detection (ILOD) of a micro racetrack resonator (MRR) based RI-sensor. Sensing measurements are performed with solutions of sodium chloride (NaCl)-water. The sensing device includes an array of multiple silicon-oxy-nitride (SiON) MRRs, fibre-coupled to a vertical-cavity surface-emitting laser (VCSEL) operating at 850 nm. We particularly choose 850 nm for sensing measurement, since the water absorption coefficient at this wavelength is three orders of magnitude lower than at 1550 nm (another conventional wavelength in biosensing). Different concentrations of NaCl–water solutions (spanning from 0.0% to 1.8% weight/weight), are flowed on top of the MRR surface in the cladding layer. Measuring of whispering gallery mode (WGM) resonance wavelength shifts in NaCl–water solutions, confirming a S and ILOD of 31.66 nm/RIU and 5.98×10−4 RIU, respectively. Furthermore, with exact engineering of the physical parameters of the system, the best S and ILOD of 325.85 nm/RIU and 6.14×10−5 RIU are found, respectively. The ILOD parameter delineates the minimum amount of RI or correspondingly minimum concentration of a biological solution, which can be detected by a RI-sensor. In this regard, this device can find applications in the exact measurement of NaCl in real biological sample solutions.

Titanium dioxide‐functionalized dendritic mesoporous silica nanoparticles for highly selective isolation of phosphoproteins

Selective isolation of phosphoproteins is of great significance in biological applications. Herein, titanium dioxide-functionalized dendritic mesoporous silica nanoparticles are prepared via a post-grafting method for selective capture of phosphoproteins. The fabricated nanoparticles possess a unique central-radial pore structure with a surface area of 666.66 m2 /g and a pore size of 22.2nm. The high-binding affinity of TiO2 with the phosphate groups facilitates the selective adsorption of phosphoproteins. Moreover, the open central-radial pore structure endows the dendritic mesoporous nanoparticles with better adsorption performance toward phosphoproteins with respect to the commercial titanium dioxide nanoparticles and titanium dioxide-functionalized conventional mesoporous silica nanoparticles by providing more accessible affinity sites. At pH 2, an adsorption capacity of 157.2mg/g is derived for β-casein. The feasibility of the as-prepared dendritic material in real biological sample assay is demonstrated by the selective isolation of phosphoproteins from defatted milk, as illustrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay.

Detection of ketorolac drug using pencil graphite electrode

The ketorolac (KEC) is a non-steroidal anti-inflammatory drug (NSAID) which is a water-soluble non-opioid analgesic drug that exhibits antipyretic properties that helps in the treatment concerned with pain. The reports were found on the adverse effect of overdosage of KEC on health which makes to develop the simple, sensitive, and economic electroanalytical method for the trace level detection of KEC. In the present study, pencil graphite was employed as the electrode (PGE) for the electrochemical detection of KEC. The effects of different electrochemical parameters viz., pH of electrolyte, scan rate, and concentration etc., on KEC were explored through cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The irreversible electrochemical reaction governed by the electrode process governed by the diffusion was attained by KEC at PGE. The linearity range was between 2.0 × 10−6 and 1.0 × 10−3 M, while the limit of detection was 4.59 × 10−7 M. The efficiency of the PGE was determined by analysing KEC in pharmaceutical drugs, and spiked real samples. The capability of the PGE towards the detection of KEC in presence of other chemicals was investigated by conducting an interference study. Hence, the proposed method can be applied for the determination of KEC in real biological and clinical samples.

Simultaneous determination of selected catechins and pyrogallol in deer intoxications by HPLC-MS/MS

A selective and sensitive analytical method for the determination of selected catechins (catechin, epicatechin, gallocatechin, epigallocatechin) and pyrogallol in biological matrices by HPLC-MS/MS was developed. The utilized sample preparation technique was a two-stage liquid–liquid extraction using ethyl acetate. The HPLC-system was equipped with a Phenomenex Luna Pentafluorophenyl Column (150 × 2 mm, 5 µm) and operated with an acetonitrile–water gradient as a mobile phase system. Detection was performed with a 3200 Q Trap mass spectrometer. For analysis the mass spectrometer was used in the MRM-mode with negative ionization.The method validation was performed with serum as matrix. The selectivity of the method as well as the linearity of calibration was successfully proven for all analytes. The limits of quantification were between 5.3 and 11.2 ng/mL and the recovery rates were above 50 % for all analytes.Results from the samples of three deer poisoning cases demonstrated that the developed HPLC-MS/MS method is applicable to real biological samples.

Ex-Vivo Measurement of the pH in Aqueous Humor Samples by a Tapered Fiber-Optic Sensor.

A practical demonstration of pH measurement in real biological samples with an in-house developed fiber-optic pH sensor system is presented. The sensor uses 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) fluorescent dye as the opto-chemical transducer. The dye is immobilized in a hybrid sol-gel matrix at the tip of a tapered optical fiber. We used 405 nm and 450 nm laser diodes for the dye excitation and a photomultiplier tube as a detector. The sensor was used for the measurement of pH in human aqueous humor samples during cataract surgery. Two groups of patients were tested, one underwent conventional phacoemulsification removal of the lens while the other was subjected to femtosecond laser assisted cataract surgery (FLACS). The precision of the measurement was ±0.04 pH units. The average pH of the aqueous humor of patients subjected to FLACS and those subjected to phacoemulsification were 7.24 ± 0.17 and 7.31 ± 0.20 respectively.

Specific and sensitive detection of CircRNA based on netlike hybridization chain reaction

Circular RNA (circRNA), as a new class of biomarker, plays an important role in the occurrence and development of cancer. However, the limitations of detection methods in recent years have severely restricted the related research of circRNA. Here, we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction (HCR). It combines reverse transcription-rolling circle amplification (RT-RCA) with well-designed netlike HCR to achieve dual selection and dual signal amplification, which can eliminate the interference of linear isomers. This two-dimensional netlike HCR is composed of an ingeniously designed trigger chain and two hairpin fuel probes, which can generate a stable network structure with RT-RCA products containing multiple sets of repeats at a constant temperature, thereby producing enhanced fluorescent signals. Systematic studies reveal that the optimized netlike HCR system has higher detection efficiency for DNA strands containing multiple sets of repetitive sequences, can detect circRNA as low as 0.1 pM, and has excellent selectivity. By using human tumor cell lines and tissues, it has been verified that the netlike HCR-based method can accurately detect specific circRNA in real biological samples without RNase R enrichment, which provides a simple and useful platform for detecting low-abundance circRNA. Furthermore, the proposed strategy is also a potential method for detecting some genes containing repetitive sequences, such as telomere DNA, centromere DNA and ribosomal DNA (rDNA).

Metal-Organic Framework-Derived Hollow and Hierarchical Porous Multivariate Metal-Oxide Microspheres for Efficient Phosphoproteomics Analysis.

Pre-enrichment of the biological samples is a crucial step in phosphoproteomics research. At present, metal-oxide affinity chromatography (MOAC) is one of the most recognized enrichment strategy. Therefore, the design and preparation of a MOAC-based affinity material with better enrichment properties will be of great significance for the phosphoproteomics study. In this work, we obtained a novel multivariate metal-oxide microsphere (NiFe2O4@C@TiO2) with a hollow and hierarchical porous structure through pyrolysis of TiO2-modified Fe/Ni-based metal-organic frameworks (MOFs). After pyrolysis, the carbon matrix derived from the MOFs provided support and porous properties. Meanwhile, multivariate metal oxides endowed the microspheres with an excellent magnetic response property and superior enrichment performance for phosphorylated biomolecules. The unique hollow and hierarchical porous structure greatly enhanced the diffusion of phosphorylated biomolecules. Therefore, the microspheres exhibited excellent enrichment performance for phosphorylated biomolecules: a large adsorption capacity (124 μmol g-1), excellent selectivity (α-casein/BSA, 1:5000, m/m), perfect size-exclusion performance (α-casein digests/α-casein/BSA, 1:500:500), and extremely low detection limit (2 fmol). Furthermore, the microspheres showed excellent enrichment performance in a series of real biological samples, such as nonfat milk, serum, saliva, rat brain tissue, and plasma exosomes of patients with esophageal cancer, which further demonstrated its huge application potential in MS-based phosphoproteomics research.

Removal of adsorption sites on the external surface of mesoporous adsorbent for eliminating the interference of proteins in enrichment of phosphopeptides/nucleotides

Various mesoporous adsorbents are of great promise for enriching small molecules from biological samples based on the size-exclusion effect. At present, the mesoporous adsorbents have adsorption sites distributed uniformly on the internal and external surfaces of mesopores. However, the adsorption sites on the external surface can adsorb proteins, interfering with the enrichment of small molecules. Herein, a novel immobilized-Ti4+ magnetic mesoporous adsorbent removing the adsorption sites on the external surface was facile prepared via the coupling chemistry of isocyanate with amine and consequent hydrolysis of urea linkage by urease. The adsorbent enables fast and selective enrichment of phosphopeptides and nucleotides from biological samples. In addition, sensitive detection methods for phosphopeptides and nucleotides in human serum are developed by coupling the magnetic solid-phase extraction with matrix-assisted laser desorption/ionization time of flight mass spectrometry and liquid chromatography-mass spectrometer, respectively. Under optimal conditions, response is linear (R2 ≥ 0.9923), limits of detection are low (0.41–9.48 ng mL−1), and reproducibility is acceptable (inter- and intra-day assay RSDs of≤15.0%) for six nucleotides. The developed strategy offers an effective method to eliminate the interference of proteins in the enrichment of small molecules from real biological samples.

1′-hydroxy-2′-acetonaphthone: A simple fluorescence turn-on signaling probe with high selectivity and sensitivity for Al3+ in pure water

Adverse effects of Aluminium ion (Al3+) on human health and the environment, when present above the acceptable concentration, have stimulated much attention to its detection. Many probes are being developed to detect Al3+, most of them suffer from protracted synthesis, less aqueous solubility, and limited selectivity and sensitivity. We report a highly selective and sensitive, fluorescence turn-on sensor for Al3+ detection in pure water by employing the simple, inexpensive commercial probe, 1′-hydroxy-2′-acetonaphthone (HAN). This probe displays remarkably high ~ 120 folds fluorescence enhancement upon complexing with Al3+, the highest reported so far in an aqueous solution for Al3+ sensing. The detection limit obtained was as low as 42 nM. The huge fluorescence enhancement towards Al3+ is attributed to the chelation enhanced fluorescence (CHEF) effect and consequent impediment on the excited-state tautomeric interconversion process between keto (K*) and twisted keto (KR*) tautomers of HAN associated to its ESIPT process. This Al3+ induced inhibition in the interconversion process between K* and KR* tautomers eventually lead to the complete tautomeric arrest of KR* form of the dye complexed with Al3+, a phenomenon not reported to date for the HAN dye in any system. Notably, the present system also illustrates an efficient response towards Al3+ in tap water, suggesting the practical applicability of the current sensor system in real biological and environmental samples.

The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10−5 U mL−1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.