Sensitive Colorimetric Assay Research Articles

Enhanced mimic peroxidase activity of carbon nanozyme by simultaneous phosphorus, oxygen dual-heteroatom doping and nanosheet structure construction

The carbon-based nanomaterials with peroxidase-like property show huge potential as candidates for natural enzymes. The design of carbon nanozymes with high activity can expand their potential application. Herein, the carbon nanozymes with enhanced catalytic property by simultaneous phosphorus, oxygen dual-doping and nanosheet structure construction are fabricated through one-step pyrolysis process. The dual-doped phosphorus and oxygen element is conducive to accelerate electron transfer and form active site with moderate free energy for activation of H2O2 to ·OH. Besides, the nanosheet structure with enlarged surface area is beneficial to expose more catalytic sites. Benefiting from its great peroxides-like activity, the phosphorus, oxygen dual-doped carbon nanosheet materials (POCNS) are used in colorimetric determination of H2O2 and glucose with cascade reaction of glucose oxidase and POCNS. And the sensitive colorimetric detection assay for organophosphorus pesticide of chlorpyrifos was constructed with regulation of peroxidase-like activity by aptamer. The system of POCNS and H2O2 further displays great antibacterial property in inactivation of Escherichia coli. This work provides an avenue to construct high-efficient carbon nanozymes and develop their potential application in environmental and biological field.

Detection of cysteine-induced salivary H2S to evaluate the H2S-producing capability of oral bacteria, using a simple and sensitive colorimetric assay: A preliminary study

The cysteine challenge test can be used to evaluate the ability of oral bacteria to produce malodor. But it involves administration of high concentrations of cysteine directly into the mouth, together with requiring special and expensive instruments. In this study, we detected cysteine-induced hydrogen sulfide (H2S) in saliva using two colorimetric H2S assays: a microplate cover-based assay and a silver nanoprism (Ag NPR)-coated paper-based assay. Based on the results of the microplate cover-based H2S sensing assay, salivary H2S varied with cysteine concentration, treatment time, and bacterial composition. The Ag NPR-coated H2S sensing paper assay was used to quantitatively analyze cysteine-induced H2S concentrations in 21 saliva samples treated with 20 mM cysteine for 3 min. As a result, the cysteine-induced salivary H2S level was within a range from 1.43 to 3.60 μM. And it was positively correlated with the DNA copy number of Porphyromonas gingivalis in the saliva. Therefore, we suggest that cysteine-induced salivary H2S be used as an alternative to the oral cysteine challenge test for evaluating the H2S-producing capability of oral bacteria.

Nanozyme based colorimetric detection of biogenic gaseous H2S using Ag@Au core/shell nanoplates with peroxidase-like activity.

A highly sensitive and facile colorimetric assay is introduced for detecting biogenic gaseous H2S using peroxidase (POD)-like catalytic activity of silver core/gold shell nanoplates (Ag@Au NPls). H2S can react with Ag@Au NPls to form Ag2S or Au2S on their surface, which can reduce POD-like activity of Ag@Au NPls and consequently decrease the absorbance at 650nm due to oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). For in situ and multiple detection of H2S, we utilized a microplate cover with 24 polydimethylsiloxane inner wells where Ag@Au NPls reacted with H2S gas followed by treatment with TMB/H2O2. As a result, the change in absorbance at 650nm showed a linear relationship with the H2S concentration in the range 0.33 to 2.96μM (0.36 absorbance/μM H2S in PBS, R2 = 0.994) with a limit of detection of 263nM and a relative standard deviation of 4.4%. Finally, this assay could detect H2S released from Eikenella corrodens, used as a model bacterium, in a short time (20min) or at a low number of bacteria (1 × 104 colony forming units/mL). Therefore, this assay is expected to be applied for the study of H2S signaling in bacterial physiology, as well as measure H2S production released from other oral bacteria that cause halitosis and oral diseases, leading to the subsequent diagnosis.

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids.

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/μL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.

Highly sensitive and selective colorimetric and fluorescent dual-readout assay for the analysis of spermine, spermidine and cysteine

Fluorescent cuprous iodide (CuI)-based coordination polymers (CuI-CPs) were widely used in the sensor field for high selectivity and sensitivity. Here, a new CuI-CP fluorescent probe was fabricated for the detection of spermine, spermidine and cysteine with dual-mode, which was capable of both convenient colorimetric analysis and ratiometric fluorescent detection. The fluorescence of CuI-CP solution was quenched and the solution changed from white to purple upon addition of spermine or spermidine, while the solution changed from white to yellow for cysteine. This probe exhibited selective, sensitive, rapid and linear response to spermine, spermidine and cysteine with a limit of detection of 0.57, 1.06 and 1.18 μM, respectively. Furthermore, cellulose acetate was used as the matrix to fabricate intelligent fluorescent film for real-time and visual monitoring the freshness of pork samples based on distinct fluorescence changes under UV light. This work realized a rapid, efficient, and profitable method for visually evaluating food quality.

Multifunctional AuAg-doping Prussian Blue-based MOF: Enhanced colorimetric catalytic activities and amplified SERS signals for bacteria discrimination and detection

New strategy of a rapid, sensitive bacteria detection is highly desirable. Constructing a dual-mode colorimetric/surface-enhanced Raman scattering (SERS) biosensor is key to obtain highly sensitive discriminating and detecting of different pathogen bacteria with high accuracy. Herein, we developed a multifunctional colorimetric catalytic nanozyme (AuAg@PB MOF) composed of AuAg-doping Prussian Blue analogue-based metal-organic framework (MOF) that fabricated by self-assembled sheet growth of Ag-etched silver-coated gold nanoparticles (Au@Ag NPs) mediated by addition of ferricyanide (K3[Fe(CN)6]). AuAg@PB MOF displays ultrahigh peroxidase-mimicking activity, enhanced colorimetric signal brightness, and notably SERS enhancement. Through optimizing its Fe-doping level, peroxidase-like activity of AuAg@PB MOF is approximately 1900-fold higher than that of horseradish peroxidase (HRP), while its 3-fold enhancement in SERS signal compared with that of gold nanoparticles (Au NPs). Integrated with nanozyme-based sandwich system that made of bacteria/4-MPBA (4-mercaptophenylboronic acid)/AuAg@PB MOF, this dual-modal biosensor achieves a highly sensitive colorimetric assay of bacteria with a lower limit of detection (6 CFU/mL), and simultaneously discriminate E. coli and S. aureus by bacterial “fingerprints” spectra. Furthermore, this proposed colorimetric assay was combined with a smartphone readout-sensing platform for determining bacteria with ultra-sensitivity (2 CFU/mL). This work exhibits a novel colorimetric nanozyme used in dual-mode colorimetric/SERS sensing of bacteria.

Facile ultrasonication-assisted synthesis of Purpald-functionalized silver nanoparticles for the rapid spectrophotometric detection of acetamiprid pesticide in food and environmental samples

In this work, we report a highly sensitive and selective colorimetric assay using purpald functionalized silver nanoparticles (P-AgNPs) as a probe for the detection of acetamiprid pesticide in food and environmental samples using Ultraviolet–visible (UV–Vis) spectrophotometric analysis. Several methods, including Fourier transforms infrared spectroscopy, X-ray diffraction spectroscopy, Transmission electron microscopy, X-ray photoelectron spectroscopy and Dynamic light scattering, were used to characterize the synthesized P-AgNPs. As prepared P-AgNPs were spherical in shape and the average particle size was determined to be 17.69 ± 0.3 nm based on TEM analysis. The surface plasmon resonance (SPR) band for P-AgNPs was observed at 445 nm (Visible spectral region). Interestingly, increased acetamiprid pesticide concentration in the sample solution, shows decreased SPR band absorbance due to aggregation of P-AgNPs by electrostatic repulsion. Results showed an excellent linearity range 2.49 to 82.06 µM and the lowest detection limit was 36 nM, the present method is applied to detect acetamiprid pesticide in a variety of environmental and food samples, including soil samples, tomato, carrot, bean, cabbage and cucumber samples, as well as tap and drinking water. Results indicate good accuracy and precision. Therefore, the presented method is proven to be a simple, fast, low-cost and efficient method for analysis of pesticides in food and environmental samples.

Sensitive and Label-Free Colorimetric Detection of Glyphosate Based on the Suppression Peroxidase-Mimicking Activity of Cu(II) Ions.

The sensitive and accurate determination of glyphosate (Glyp) is urgently demanded because it is closely correlated with human health and environmental safety. In this work, we proposed a sensitive and convenient colorimetric assay by employing copper ion peroxidases for the detection of Glyp in the environment. Free Cu(II) ions displayed high peroxidase activity and can catalytically oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB, resulting in an obviously visible discoloration reaction. Once the Glyp is added, the ability of copper ions to mimic peroxidase can be largely suppressed because of the generation of Glyp-Cu2+ chelate. The favorable selectivity and sensitivity were demonstrated in the colorimetric analysis of Glyp. Furthermore, this rapid and sensitive method was successfully applied in the accurate and reliable determination of glyphosate in the real sample, holding promising applications in pesticide determination in the environment.

Rapid and sensitive smartphone non-enzymatic colorimetric assay for the detection of glucose in food based on peroxidase-like activity of Fe3O4@Au nanoparticles

A low-cost and reliable analytical method based on the combination of a newly designed Fe3O4@Au as peroxidase mimetics, supported on smartphone analysis software package was proposed for the determination of glucose content in food samples. The nanocomposite was prepared by self-assembling technique, and the characterization was carried out using transmission electron microscopy (TEM), Fourier transforms infrared, and X-ray diffractometer. Record the color change of the solution with a smartphone camera and optimize the operation parameters and reaction conditions. A smartphone with a free self-developed app was accustomed live the RGB (red–greenblue) values of color intensity within the Fe3O4@Au system and were processed with Image J software before computationally convert them glucose concentrations. At the optimization experiment, reaction temperature of 60 °C, reaction time of 50 min and the amount of addition of Fe3O4@Au 0.0125 g was the optimal combination of detecting glucose smartphone color detection system. Hereon, the accuracy of the proposed method was evaluated by comparison between smartphone colorimetry and UV–vis spectrophotometer, a linear calibration in the range of 0.25 ∼ 15 mmol/L glucose was obtained with minimum detection limit of 1.83 and 2.25 μmol/L, respectively. The proposed method was applied effectively to the detection of glucose in actual samples. The results were in accordance with the conventional UV–vis spectrophotometer method.

DNA-Programmed Tuning of the Growth and Enzyme-Like Activity of a Bimetallic Nanozyme and Its Biosensing Applications.

Nanozymes, which combine the merits of both nanomaterials and natural enzymes, have aroused tremendous attention as new representatives of artificial enzyme mimics. However, it still remains to be a great challenge to rationally engineer the morphologies and surface properties of nanostructures that lead to the desired enzyme-like activities. Here, we report a DNA-programming seed-growth strategy to mediate the growth of platinum nanoparticles (PtNPs) on gold bipyramids (AuBPs) for the synthesis of a bimetallic nanozyme. We find that the preparation of a bimetallic nanozyme is in a sequence-dependent manner, and the encoding of a polyT sequence allows the successful formation of bimetallic nanohybrids with greatly enhanced peroxidase-like activity. We further observe that the morphologies and optical properties of T15-mediated Au/Pt nanostructures (Au/T15/Pt) change over the reaction time, and the nanozymatic activity can be tuned by controlling the experimental conditions. As a concept application, Au/T15/Pt nanozymes are used to establish a simple, sensitive, and selective colorimetric assay for the determination of ascorbic acid (AA), alkaline phosphatase (ALP), and the inhibitor sodium vanadate (Na3VO4), demonstrating excellent analytical performance. This work provides a new avenue for the rational design of bimetallic nanozymes for biosensing applications.

Design and development of a novel colorimetric assay and a portable optical system for the detection of aminoglycoside antibiotics

In recent years, environmental contamination with antibiotics has become a severe threat to humankind requiring urgent attention. Presence of incompletely metabolized antibiotics in water bodies, leads to their entry into food chain via live stock and serve as a severe threat to human health. Hence it is imperative that the antibiotics are detected at the source itself. Aminoglycosides are broad spectrum antibiotics known to be more toxic and resistant to multidrug resistant (MDR) organisms compared to other classes of antibiotics. Accordingly in the present work a selective, sensitive and low-cost detection technique for aminoglycoside based antibiotics has been developed. An iron-dependent lipid peroxidation has been explored for the first time to develop a rapid and sensitive colorimetric assay for detecting aminoglycoside antibiotics. A lab-on-chip model (LoC) was designed and developed to miniaturize the colorimetric assay based model for its applications in the real-time analysis of antibiotics. The LoC was integrated with optical fibers, electronic components and associated signal processing circuitry to construct a portable device for antibiotic quantification. Interestingly a selective response towards gentamicin amongst the tested antibiotic was observed. The formation of a colored product within five minutes of reaction time enables a swift and less cumbersome analysis and compared to existing sophisticated analytical techniques. The detection method was validated for gentamicin spiked environmental samples and showed a recovery of more than 90%. This method can serve as a suitable alternative in limited laboratory setups and provides a generic platform for developing a rapid, user-friendly and economical detection methods for environmental monitoring of antibiotics.

Au–Fe3O4 nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing

The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au–Fe3O4 nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au–Fe3O4 nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL−1) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.

Kanamycin triggered nanozyme for homogeneous and amplified colorimetric detection of T4 polynucleotide kinase

It is of significance to develop efficient methods for detecting the activity of T4 polynucleotide kinase (T4 PNK) due to its essential role in the modulation of different life activities. In this work, we constructed a novel nanozyme using Kanamycin (KANA) as a trigger for the [Fe(CN)6]3- coordinated Cu2(OH)3NO3 (Cu2(OH)3NO3/[Fe(CN)6]3-) nanorods, and designed an amplified colorimetric method to detect T4 PNK. That was, the free KANA efficiently triggered the peroxidase-like activity of Cu2(OH)3NO3/[Fe(CN)6]3-, while the bound KANA by its aptamer lost the stimulative capability for the nanomaterials. On the basis of the bioreaction regulated generation of the KANA aptamer, a highly sensitive colorimetric assay aided by the rolling circle amplification (RCA) reaction for the detection of T4 PNK was realized. Results showed that this assay can detect T4 PNK from 1.0 × 10−3 to 10.0 U/mL, with a limit of detection (LOD) of 1.42 × 10−4 U/mL. The assay also showed acceptable performance in the detection of T4 PNK in serum samples. In addition to the satisfactory sensitivity and selectivity, the displayed T4 PNK assay also presented merits of operational convenience, without labeling or immobilization process and did not require costly instrument. It is expected that the KANA as a stimulator would have extended biosensing applications by coupling various bioreactions that can produce the KANA aptamer.

Colorimetric detection of phosphate-based on iron complexing catechol-displacement assay in eutrophicated water bodies

A simple, sensitive, and inexpensive colorimetric indicator-displacement assay based on Fe3+-catechol complex has been developed to determine PO43-. The Fe3+ and catechol formed a greenish color complex, upon the addition of PO43- to the complex, it exhibited a visual color change immediately from greenish to colorless along with UV–vis spectral change. This change was mainly attributed to the high binding affinity of PO43- with Fe3+ by displacing catechol from the Fe3+-catechol complex. The developed sensor showed good sensitivity for the determination of PO43- in a wide linear range from 2 − 1000 µM with a limit of detection (LOD) of 0.5 µM which was found to be lower than the permissible limit. The sensor presented good selectivity towards the detection of PO43- in the existence of other potential interfering anions. Furthermore, the method was successfully applied to determine PO43- in eutrophicated water bodies (river and lake water) with good recoveries and without showing any significant matrix effect.

A Silver Nanoparticles-Based Selective and Sensitive Colorimetric Assay for Ciprofloxacin in Biological, Environmental, and Commercial Samples

The wide-spread usage of ciprofloxacin (CIP) resulted in its presence in different parts of the ecosystem. Thus, a simple, reliable, on-spot detection method for CIP is required in environmental context. Herein, a colorimetric assay is developed for the detection of CIP based on the branched polyethyleneimine (PEI) conjugated silver nanoparticles (PEI-AgNPs). AgNPs are prepared using PEI as stabilizing agent following a simple one-pot two-phase procedure. The prepared PEI-AgNPs are subsequently used for an efficient and selective detection of CIP. The characteristic yellow colour of PEI-AgNPs changed to colourless when CIP was added which was further confirmed by quenching in the intensity of the SPR (surface plasmon resonance) band (hypochromic shift). The proposed method is efficient for the quantitation of CIP in a linear dynamic range (LDR) of 0.1–200 µM with a limit of detection (LOD) of 0.038 µM, and limit of quantification (LOQ) of 0.12 µM. The developed method is selective, efficient, and sensitive to CIP in the presence of numerous interfering species and in real biological, environmental, and commercial pharmaceutical samples. Excellent performance of the proposed method compared to UV-Vis spectroscopy and UPLC in environmental, biological, and commercial pharmaceutical samples is demonstrated.

One-step self-assembly of multilayer graphene oxide via streamlined click reactions for sensitive colorimetric assays

Hemin-loaded graphene oxide with excellent peroxidase-like activity shows great potential for biosensing applications. However, the detection sensitivity of biosensors based on such catalytic methods is limited by the lack of a signal amplification technique. In this work, we developed a simple and rapid signal amplification method based on streamlined click reactions enabling one-step assembly of multilayer graphene oxide nanosheets on magnetic beads to immobilize large amounts of hemin serving as active catalysts, which allowed for the highly sensitive detection of various biological targets, including copper ions, DNA sequences and proteins. With this method, we achieved detection limits down to 13.74 nM, 4.89 pM and 7.77 pg/mL for Cu2+, Ebola virus DNA sequences, and carcinoembryonic antigen, respectively. The designed platform holds great promise in the self-assembly of graphene-based nanozymes and sensitive colorimetric biosensing in a wider range of applications.

Modifier-free gold nanoparticle colorimetric sensing for detecting sulfanilamide in the presence of dopamine

In this study, we have utilized a simple and sensitive colorimetric assay using unmodified gold nanoparticles to detect sulfanilamide in the presence of dopamine.

A simple strategy based on combinatorial gold nanoparticle sizes for enhanced sensitivity in colorimetric bioanalysis.

Taking advantage of salt-induced aggregation, gold nanoparticles (AuNPs) have been intensively employed in non-cross-linking strategies (NCLs) for colorimetric bioanalysis. Such a classical method is popular due to its simplicity and cost-effectiveness, but it still suffers from poor sensitivity in analytical practice. To address this issue, we simply mixed the four sizes of non-functional AuNPs (10 nm/20 nm/30 nm/40 nm) to establish a highly sensitive combinatorial system in a non-cross-linking strategy (cNCL). For comparison, we also designed four independent systems with individual sizes of AuNPs (10 nm, 20 nm, 30 nm and 40 nm, respectively) as typical non-cross-linking strategies (tNCLs). Interestingly, the cNCLs were found to have significantly enhanced sensitivity compared to each of the tNCLs in analytical performance. TEM and theoretical calculation were employed to explore this phenomenon, and it is indicated that the aggregation behaviours of cNCLs show more compact morphology by particle-to-particle stacking. We then tuned the size proportions of various AuNPs in cNCLs to evaluate the contribution of each size of the AuNPs. It appears that 10 nm AuNPs are mainly responsible for minimizing background intensity, and 40 nm AuNPs for maximizing signal intensity. Moreover, with the well-studied effect of combinatorial AuNP sizes in cNCLs, an excellent signal-to-background (S/B) ratio can be obtained, achieving at least 500-fold and 2.5-fold improvement in the aspects of optical and visual sensitivity, respectively. Such a combinatorial AuNP size-based NCL (cNCL) strategy is modification-free towards AuNPs, and the whole process can be accomplished within 10 min. The aggregation behaviour significantly impacts the optical properties and morphology, and therefore improves analytical sensitivity. With these findings, valuable insight is provided in developing sensitive and versatile colorimetric assays based on classic AuNP aggregation.

Highly resistant and sensitive colorimetric immunochromatographic assay for sibutramine (SBT) illegally adulterated into diet food based on PDA/AuNP labelling.

A gold nanoparticle (AuNP) based immunochromatographic assay strip is a valuable tool for monitoring chemicals in foods. However, the sensitive ICA strip for SBT is rarely reported due to the fact that monoclonal antibodies (mAbs) against SBT with high affinity are commercially unavailable. Herein, a monoclonal antibody against SBT was prepared through a designed hapten with a carboxyl end-capped space arm. The obtained mAb showed high affinity for SBT and N-desmethylsibutramine, a metabolite of SBT. Furthermore, a series of core-shell NPs, polydopamine (PDA) coated AuNPs (PDA/AuNPs) with controlled shell thickness and packing density were synthesized. The obtained PDA/AuNP-mAb conjugate demonstrated high tolerance to salt and good stability in a wide pH range, which is beneficial for improving the matrix interference common in ICA. As a result, PDA/AuNP-based ICA could quantify SBT in the range of 3.39-69.60 ng mL-1 with a limit of detection (LOD) of 0.98 ng mL-1. This novel ICA improved the sensitivity of the traditional AuNP-based ICA by nearly 12 times. Method validation was conducted with spiked samples of slimming food and human serum and compared with HPLC-MS/MS. Consistent results indicated that high sensitivity, accuracy, and reliability of the PDA/AuNP-based ICA approach were achieved. To the best of our knowledge, this study reported the most sensitive immunoassay for SBT thus far.

Sensitive colorimetric assay of T4 DNA ligase by the oxidase nanozyme of LaMnO3.26 coupled with a hyperbranched amplification reaction.

The development of efficient methods for the detection of T4 DNA ligase is extremely important for public health. The present work demonstrates the integration of engineerable oxidase nanozyme of LaMnO3.26 nanomaterials for the colorimetric detection of T4 DNA ligase. Specifically, the LaMnO3.26 nanomaterials exhibited oxidase-like activity, oxidizing o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 3,3',5,5'-tetramethylbenzidine (TMB) to their corresponding oxidation products, which featured maximum absorption wavelengths at 450, 417 and 650 nm, respectively, while pyrophosphate ion (PPi) caused an obvious decrease in the oxidase-like activity of LaMnO3.26 through its surface coordination with the surface-exposed Mn element and induced aggregation of the nanozyme. Attributed to the PPi regulated oxidase nanozyme activity, LaMnO3.26 served as a colorimetric probe for the quantitative detection of T4 DNA ligase assisted by a hyperbranched amplification reaction for signal amplification. The T4 DNA ligase was detected with a linear range of 4.8 × 10-3 to 6.0 U mL-1, achieving a detection limit of 1.6 × 10-3 U mL-1. The outcome indicated that the developed nanozyme might be extended to a broad range of practical applications.