Nanomaterial-based Artificial Enzymes Research Articles

Al3+ Cofactor Evoking Iron Porphyrin-AuNP Hybrids as Oxidase-Mimicking Nanozymes with Prominent Catalytic Efficiency in a Broad pH Range

Nanomaterial-based artificial enzymes (nanozymes) have great potential for boosting their intrinsic activities to narrow the gaps against natural enzymes. Different from natural enzymes, nanozymes usually cannot work efficiently under physiological pH, which limits their application in environmental and biological fields. Here, we proposed that the originally futile tetrakis (4-carboxyphenyl) iron porphyrin (FeTCPP) compounds in the FeII-TCPP-Au nanoparticle (FeII-TCPP-AuNP) hybrid are functionalized to serve as critical active sites involved in cytochrome P450 to greatly boost the oxidase-mimicking activity by an Al3+ cofactor at neutral pH. The microenvironment around FeTCPP can be chemically optimized to provoke the oxidase-mimicking activity with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate with the help of Al3+. The activity-provoking ability of the system is tunable by changing the concentrations of Al3+, the strong ligand F–, and temperature. Based on this, the selective and sensitive colorimetric sensor of Al3+ based on nanozymes was first developed. The novelty of the present work is not only a new means to develop iron porphyrin nanozymes with superior oxidase-mimicking activity at neutral pH but is also the first example that hard acid Al3+ can serve as a robust and efficient cofactor to synergize the catalytic performance of nanozymes. Therefore, this study provides some enlightenment about the integration of artificial cofactors in biomimetic chemistry and expands the application of nanozymes in environmental fields.

Nanoclay Modulates Cation Occupancy in Manganese Ferrite for Catalytic Antibacterial Treatment.

Controllable preparation is the current frontier in the field of inorganic nanomaterial-based artificial enzymes (nanozymes). For ferrites, the factors affecting cation occupancy are very complex, making the modulation of cation occupancy extremely challenging. Herein, we report a new strategy to modulate the cation occupancy of manganese ferrite (MFO) based on the structural properties of nanoclay (i.e., montmorillonite K10). It demonstrates that montmorillonite K10 mainly changes the valence state and occupancy of manganese ions in manganese ferrite and not iron ions. K10 increases the proportion of Mn (II) in manganese ferrite and causes more manganese ions to migrate to the tetrahedral sites. As expected, the prepared new inorganic compound possesses excellent enzyme-like catalytic activities and antibacterial functional properties, which are attributed to Mn (II) accelerating Fe (III) reduction and hydroxyl radical formation. Furthermore, steady-state kinetic assays are used to study the reaction mechanism in detail. In vitro and in vivo antibacterial experiments show that the synthesized inorganic compounds exhibit satisfactory disinfection and wound skin recovery efficiencies. This work emphasizes the controllable preparation of new inorganic compounds with biomimetic activity and provides novel insight into the biological effects of inorganic nanomaterials.

Bimetallic FeMn@C derived from Prussian blue analogue as efficient nanozyme for glucose detection.

In recent decades, nanomaterial-based artificial enzymes called nanozymes have received more and more attention and have been applied in biological, chemical, medical, and other fields. In this work, bimetallic FeMn@C was synthesized by calcination from the Prussian blue analogue. The synthesized bimetallic FeMn@C exhibits efficient peroxidase-like activity. The effect of Mn doping amount, catalytic kinetics, and mechanism of FeMn@C nanozyme was further studied in detail. The results show that the peroxidase-like activity of bimetallic FeMn@C is nearly 16 times higher than that of single-metal Fe@C. The peroxidase-like activity of FeMn@C originates from its production of radicals. Compared with natural enzymes, FeMn@C nanozyme has a better affinity for the substrates.Besides, FeMn@C nanozyme has better stability than natural enzymes. Because of its strong magnetism, FeMn@C nanozyme can be recycled easily and exhibits excellent recycling performance. Based on the good affinity of FeMn@C for H2O2, a rapid and selective colorimetric assay for glucose detection is constructed, with a wide linear range of 0.01-0.75mM and low detection limit of 4.28µM. This sensor has been successfully applied to the determination of glucose in fruit juice, showing good selectivity and accuracy. The synthesis of bimetallic FeMn@C provides a feasible way to design nanozymes with excellent catalytic activity, high stability, and easy separation.

Preparation of Nanomaterial-Based Artificial Enzymes Using Integrated Graphitic Carbon Nitride Nanosheet-encapsulated Metal-Organic Framework Optimized by Central Composite Design for Enhanced Electrochemiluminescence Biosensing of Gene-Specific Methylation in Thyroid Cancer Patients' Plasma

Preparation of Nanomaterial-Based Artificial Enzymes Using Integrated Graphitic Carbon Nitride Nanosheet-encapsulated Metal-Organic Framework Optimized by Central Composite Design for Enhanced Electrochemiluminescence Biosensing of Gene-Specific Methylation in Thyroid Cancer Patients' Plasma

Active nanozyme derived from biomineralized metal-organic frameworks for cholesterol detection

Metal-organic framework (MOF) derived nanozymes show significant promises in biosensing and biomedicine. Nanozymes are nanomaterial-based artificial enzymes. But how to improve the catalytic capacity of nanozymes in a simple manner is still a challenge. Herein, we demonstrated that the pore size/volume and catalytic capacity of Mn3O4 can be modulated by biomolecules biomineralized in Mn-MOF before calcination. The biomineralization-derived Mn3O4 shows larger pore size/volume and higher peroxidase/oxidase dual enzymatic activity compared to the pure Mn-MOF derived one. After loading with cholesterol oxidase, the prepared bio-inorganic hybrid nanozyme is able to detect the cholesterol in serum samples with high sensitivity. This work demonstrates that biomineralization of MOF with exogenous molecules is an ingenious and convenient strategy to synthetize nanozyme with high catalytic activity.

Nanozyme-catalyzed cascade reactions for high-sensitive glucose sensing and efficient bacterial killing

The development of nanomaterial-based artificial enzymes with multiple functions that mimic the complexity and function of natural systems is essential for the design of biomimetic devices that imitate physiological processes. In this work, a biomimetic nanozyme fabricated using single-layer graphitic carbon nitride incorporating copper was developed, which triggers catalytic cascade reactions for glucose sensing and bactericidal action. This nanozyme that mimics glucose oxidase was capable of photocatalytic production of hydrogen peroxide by coupling glucose oxidation and dioxygen reduction under visible light irradiation. The hydrogen peroxide produced in situ served as the substrate in the subsequent peroxidase-like reaction, allowing the oxidation of chromogenic substrates to complete the bifunctional oxidase-peroxidase action on the same catalyst. The bifunctional cascade catalysis was successfully applied for the real-time and colorimetric sensing of glucose with a limit of detection (LOD) of 0.71 μM. Furthermore, the system exhibited striking bactericidal performance against the gram-negative bacteria Shewanella putrefaciens (S. putrefaciens, associated with food spoilage) that was attributed to the production of detrimental free radicals via nanozyme catalysis. This study not only paves the way for the construction of enzyme-like cascade systems, but also provides an efficient route for defect engineering in food preservation.

Molecular Imprinting on Nanozymes for Sensing Applications.

As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.

Iron oxide magnetic nanoparticles exhibiting zymolyase-like lytic activity

Iron oxide (Fe3O4) magnetic nanoparticles (MNPs) were found to exhibit an intrinsic yeast lytic activity with enzyme kinetics similar to that of natural zymolyase. The Fe3O4 MNPs were synthesized by a co-precipitation method and were then employed to catalyze the disruption of yeast cells. The Fe3O4 MNPs accelerated the biocatalytic reaction to completion within 10 min. Compared to natural zymolyase, the newly discovered biocatalytic activity of Fe3O4 was outstanding and remained stable against temperature and pH changes. More importantly, Fe3O4 MNPs with excellent activity could be successfully synthesized using a high-gravity co-precipitation method promising for large scale production in industrial applications. Considering its cost-effectiveness and ease of preparation, Fe3O4 was expected to show great potential for the digestion of yeast cell walls through serving as nanomaterial-based artificial enzymes or nanozymes.

One-step synthesis of thermally stable artificial multienzyme cascade system for efficient enzymatic electrochemical detection

Recently, metal-organic framework (MOF)-based multienzyme systems integrating different functional natural enzymes and/or nanomaterial-based artificial enzymes are attracting increasing attention due to their high catalytic efficiency and promising application in sensing. Simple and controllable integration of enzymes or nanozymes within MOFs is crucial for achieving efficient cascade catalysis and high stability. Here, we report a facile electrochemical assisted biomimetic mineralization strategy to prepare an artificial multienzyme system for efficient electrochemical detection of biomolecules. By using the GOx@Cu-MOF/copper foam (GOx@Cu-MOF/CF) architecture as a proof of concept, efficient enzyme immobilization and cascade catalysis were achieved by in situ encapsulation of glucose oxidase (GOx) within MOFs layer grown on three-dimensional (3D) porous conducting CF via a facile one-step electrochemical assisted biomimetic mineralization strategy. Due to the bio-electrocatalytic cascade reaction mechanism, this well-designed GOx@Cu-MOF modified electrode exhibited superior catalytic activity and thermal stability for glucose sensing. Notably, the activity of GOx@Cu-MOF/CF still remained at ca. 80% after being incubated at 80 °C. In sharp contrast, the activity of the unprotected electrode was reduced to the original 10% after the same treatment. The design strategy presented here may be useful in fabricating highly stable enzyme@MOF composites applied for efficient photothermal therapy and other platform under high temperature.

Fabrication of an efficient and sensitive colorimetric biosensor based on Uricase/ Th-MOF for uric acid sensing in biological samples

Recently, nanomaterial-based artificial enzymes have expected abundant consideration because of cheap, accessibility, and respectable stability. In this study, we report a Th-MOF artificial peroxidase, which can oxidize 3,3,5,5-tetramethylbenzidine by hydrogen peroxide to yield a blue product. The catalytic behavior of Th-MOF tracked Michaelis–Menten equation and the affinity of this nanozyme to the substrate was higher than horseradish peroxidase as a natural enzyme. The absorbance value of oxidized TMB is linearly associated with the hydrogen peroxide concentration. Since, hydrogen peroxide is the oxidative end product of uric acid (UA) by uricase, an efficient and sensitive approach for uric acid determination was also established. Results showed that Km value of Th-MOF with TMB as a substrate is much lower than that of other mentioned catalysts. The linear regression equations for uric acid substrate was stated as A = 0.0039C + 0.0519 with a correlation coefficient of 0.9955. The linear range for uric acid was from 4.0 to 70 μM, and the LOD was measured as 1.15 μM. Furthermore the absorbance of assay reaction was approximately constant in the following four cycles, demonstrating that Th-MOF catalyst has outstanding stability. Results showed that the public interfering substances had no obvious absorbance values and it was less than 0.1. Results indicated that the recoveries for UA in serum fluids were between 93.10% and 99.04%. The relative standard deviation (RSD, n = 3) at each concentration value was less than 4.3%. UA determination in serum fluids has been confirmed through a comparison between the recommended technique and tedious clinical approaches. Good recovery and accuracy of UA measurement indicated that this established colorimetric sensing system is appropriate for UA revealing in actual experimental samples.

Revealing the Active Site of Gold Nanoparticles for the Peroxidase-Like Activity: The Determination of Surface Accessibility

Despite the fact that the enzyme-like activities of nanozymes (i.e., nanomaterial-based artificial enzymes) are highly associated with their surface properties, little is known about the catalytic active sites. Here, we used the sulfide ion (S2−)-induced inhibition of peroxidase-like activity to explore active sites of gold nanoparticles (AuNPs). The inhibition mechanism was based on the interaction with Au(I) to form Au2S, implying that the Au(I) might be the active site of AuNPs for the peroxidase-like activity. X-ray photoelectron spectroscopy (XPS) analysis showed that the content of Au(I) on the surface of AuNPs significantly decreased after the addition of S2−, which might be contributed to the more covalent Au–S bond in the formation of Au2S. Importantly, the variations of Au(I) with and without the addition of S2− for different surface-capped AuNPs were in good accordance with their corresponding peroxidase-like activities. These results confirmed that the accessible Au(I) on the surface was the main requisite for the peroxidase-like activity of AuNPs for the first time. In addition, the use of S2− could assist to determine available active sites for different surface modified AuNPs. This work not only provides a new method to evaluate the surface accessibility of colloidal AuNPs but also gains insight on the design of efficient AuNP-based peroxidase mimics.

Facile strategy to prepare a metalloporphyrin-based hydrophilic porous organic polymer with enhanced peroxidase-like activity and high stability for colorimetric detection of H2O2 and glucose.

Nanozymes, nanomaterial-based artificial enzymes, have attracted researchers' enormous interest due to their unique properties compared with natural enzymes. To mimic the catalytic function of natural enzymes, designing high-efficient, novel nanozymes is crucial yet challenging task. In this article, we described the synthesis and functions of a metalloporphyrin-based porous organic polymer, namely FePPOPs-SO3H. FePPOPs-SO3H was synthesized effortlessly via an extensive aromatic electrophilic substitution and the following sulfonation reactions. This strategy was cost-efficient without the participation of precious metal catalysts. The resultant FePPOPs-SO3H is intriguing since the framework itself is constructed by covalently linked porphyrin units, which could serve as a built-in catalyst and strengthen the stability of polymer. With sulfonic acid side groups, FePPOPs-SO3H is well water-dispersive. Owing to these unique characteristics, FePPOPs-SO3H exhibited excellent peroxidase-like activity toward a classical peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue product only within 20 s. The peroxidase-mimicking performance of FePPOPs-SO3H outperforms the ferric porphyrin monomer and normal Fe3O4 nanoparticles. Based on the excellent catalytic activity of FePPOPs-SO3H, two visual colorimetric sensors for ultrafast detecting H2O2 and glucose, respectively, were constructed with a wide linear range of 50-1800 μM (for H2O2) and 200-1500 μM (for glucose), as well as a relative lower limit of detection (LOD) [26.70 μM (for H2O2) and 16.38 μM (for glucose)]. Our strategy highlights opportunities for the design of new metalloporphyrin-based porous organic polymers with built-in catalytic skeletons and inherently excellent peroxidase-mimicking performance.

Redox Recycling-Triggered Peroxidase-Like Activity Enhancement of Bare Gold Nanoparticles for Ultrasensitive Colorimetric Detection of Rare-Earth Ce3+ Ion.

Although it has been demonstrated that rare-earth elements (REEs) disturb and alter the catalytic activity of numerous natural enzymes, their effects on nanomaterial-based artificial enzymes (nanozymes) have been seldom explored. In this work, the influence of REEs on the peroxidase-like activity of bare gold nanoparticles (GNPs) is investigated for the first time, and a new type of Ce3+-activated peroxidase mimetic activity of GNPs is obtained. The introduced Ce3+ can be bound to the bare GNP surface rapidly through electrostatic attraction, after which it donates its electron to the bare GNP. As H2O2 is a good electron scavenger, more •OH radicals are generated on the surfaces of the bare GNPs, which can considerably enhance TMB oxidation. Due to its redox cycling ability, the activation effect of Ce3+ is proved to be more efficient in comparison to those of the other reported metal ion activators (e.g., Bi3+, Hg2+, and Pb2+). In addition, it is determined that Ce3+ should directly contact with the gold core to trigger its activation effect. When the surface states of the bare GNPs are altered, the Ce3+-stimulated effect is strongly inhibited. Furthermore, a novel colorimetric method for Ce3+ is developed, on the basis of its enhancing effect on the peroxidase mimetic activity of bare GNPs. The sensitivity of this newly developed method for Ce3+ is excellent with a limit of detection as low as 2.2 nM. This study not only provides an effective GNP-based peroxidase mimic but also contributes in realizing new applications for nanozymes.

Nanozyme: new horizons for responsive biomedical applications.

Nanozymes are nanomaterial-based artificial enzymes. By effectively mimicking catalytic sites of natural enzymes or harboring multivalent elements for reactions, nanozyme systems have successfully served as direct surrogates of traditional enzymes for catalysis. With the rapid development and ever-deepening understanding of nanotechnology, nanozymes offer higher catalytic stability, ease of modification and lower manufacturing cost than protein enzymes. Additionally, nanozymes possess inherent nanomaterial properties, providing not only a simple substitute of enzymes but also a multimodal platform interfacing complex biologic environments. Recent extensive research has focused on designing various nanozyme systems that are responsive to one or multiple substrates by tailored means. Catalytic activities of nanozymes can be regulated by pH, H2O2 and glutathione concentrations and levels of oxygenation in different microenvironments. Moreover, nanozymes can be remotely-controlled via different stimuli, including a magnetic field, light, ultrasound, and heat. Collectively, these factors can be adjusted to maximize the diagnostic and therapeutic efficacies of different diseases in biomedical settings. Therefore, by integrating the catalytic property and inherent nanomaterial nature of nanozyme systems, we anticipate that stimuli-responsive nanozymes will open up new horizons for diagnosis, treatment, and theranostics.

Engineering Nanozymes Using DNA for Catalytic Regulation.

DNA treatment of metal nanoparticles provides a potent tool for tuning their native properties and constructing advanced materials. However, there have been limited studies on interactions between DNA and nanomaterial-based artificial enzymes (nanozymes) to influence their intrinsic peroxidase-like properties. Here, we present the utilization of DNA as a capping ligand to engineer various bio-nanointerfaces for high-precise and adjustable regulation of catalytic behaviors of nanozymes toward the oxidation of substrates. The treatment of stiff double-stranded DNA only induced a negligible enhancement of the catalytic activity of nanozymes, and both coil-like single-stranded DNA and hairpin DNA-capped nanoparticles produced a medium signal increase. Interestingly, hybridization chain reaction (HCR) product-treated nanoparticles showed the highest peroxidase-like activities among four DNA structures. Furthermore, significant parameters that influence HCR process and the modulation of catalysis, such as the concentration of the hairpin DNA, the ionic strength, and the amount of nanozyme, were also systematically investigated. On the basis of HCR amplification and iron oxide (Fe3O4) nanoparticles, we develop a simple, fast, label-free, and sensitive colorimetric strategy for sensing of a Yersinia pestis-relevant DNA sequence with a detection limit as low as 100 pM as well as single nucleotide polymorphism discrimination. These results highlight DNA engineering as a facile strategy to regulate the catalytic activities of nanozymes and understand the interactions between metallic nanoparticles and nucleic acids for biosensing applications.

Electrospun nanofibrous materials: A versatile platform for enzyme mimicking and their sensing applications

Abstract Nanomaterial-based artificial enzymes (nanozymes) are a type of newly-developed artificial enzymes owing to their significant merit of environmental stability and low cost. Recently, electrospun nanofibrous materials have been demonstrated as efficient and promising nanozymes due to their high aspect ratio, large surface area to volume, and tunable compositions and structures. This review presents the recent progress of the electrospun nanofibrous materials for enzyme mimicking and their sensing applications. After a brief introduction, this article introduces the electrospinning technique and the fantastic properties of electrospun nanofibrous materials. Next, a large variety of electrospun fibrous nanomaterials with controlled morphology, composition and chemical structure for peroxidase, oxidase and catalase mimicking are described. Herein, the strategies for the enhanced enzyme-like activity are discussed in detail. After that, the catalytic mechanism and promising applications of electrospun nanofibrous materials-based nanozymes for ultrasensitive sensing of H 2O2, ascorbic acid, dopamine, l -cysteine, glutathione, sulfite and phenol have been presented. Finally, the current challenges and future research opportunities in utilizing electrospun nanofibrous materials for enzyme mimicking are addressed.

Hierarchical CNFs/MnCo2O4.5 nanofibers as a highly active oxidase mimetic and its application in biosensing

Recently, much attention has been paid on the nanomaterial-based artificial enzymes due to their tunable catalytic activity, high stability and low cost compared to the natural enzymes. Different from the peroxidase mimics which have been studied for several decades, nanomaterials with oxidase-like property are burgeoning in the recent years. In this paper, hierarchical carbon nanofibers (CNFs)/MnCo2O4.5 nanofibers as efficient oxidase mimics are reported. The products are synthesized by an electrospinning technique and an electrochemcial deposition process in which the CNFs are used as the working electrode where MnCo2O4.5 nanosheets deposit on. The resulting binary metal oxide-based nanocomposites exhibit a good oxidase-like activity toward the oxidations of 3,3′,5,5′tetramethylbenzi-dine (TMB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) salt and o-phenylenediamine (OPD) without exogenous addition of H2O2. The system of CNFs/MnCo2O4.5-TMB can be used as a candidate to detect sulfite and ascorbic acid via a colorimetric method with a high sensitivity. This work provides the efficient utilization and potential applications of binary metal oxide-based nanocomposites with oxidase activities in biosensors and other biotechnologies.

Co4N Nanowires: Noble-Metal-Free Peroxidase Mimetic with Excellent Salt- and Temperature-Resistant Abilities.

Compared to natural enzymes, nanomaterial-based artificial enzymes have attracted immense attention because of their high stability and cost-effectiveness. In this study, cobalt nitride (Co4N) as a noble-metal-free artificial enzyme exhibiting highly intrinsic peroxidase-like activity and good stability was reported. Kinetic studies revealed that the resultant Co4N nanowires (NWs) exhibited a stronger affinity for 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 than HRP. Compared to Co3O4 NWs, Co4N NWs exhibited highly improved catalytic activities, with H2O2 exhibiting an apparent Km approximately 2 orders of magnitude less than that of Co3O4. In particular, the peroxidase-like activity of Co4N was maintained well over a wide range of temperatures and ionic strength. A Co4N-based method was further developed for the detection of glucose with good sensitivity and reliability. Because of advantages such as easy storage, cost-effectiveness, high sensitivity, and outstanding stability, Co4N NWs demonstrate the potential for replacing noble-metal-based peroxidase mimetics in a wide range of promising applications.

Fabrication of CeO2/rGO nanocomposites with oxidase-like activity and their application in colorimetric sensing of ascorbic acid

Nanomaterial-based artificial enzymes(nanozymes) have shown great potential for application in colorimetric sensing. In this paper, the nanocomposites of CeO2 and reduced graphene oxide(rGO) were fabricated via a one-pot hydrothermal process, in which CeO2 nanoparticles were well anchored on the surface of rGO. The prepared CeO2/rGO nanocomposites exhibited excellent catalytic activity for the oxidation of 3,3′,5,5′-tetra-methylbenzidine(TMB) in the presence of O2. Compared with the individual CeO2 nanoparticles, the nanocomposites had a higher affinity and adsorptivity towards the substrate TMB, which was due to the combination of rGO. In addition, based on the oxidase-like activity of CeO2/rGO nanocomposites, a facile and sensitive colormetric sensor for ascorbic acid(AA) was investigated. The detection limit of AA was low(0.15 μmol/L) and the linear detection range was between 0.5 μmol/L and 40 μmol/L. Furthermore, the colorimetric system was used for the detection of AA in medicine and food analysis, such as tablets, beverage and milk powder.

Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus.

Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H2O2) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively.