Employing Gold Nanoparticles Research Articles

Grain boundary engineering for efficient and durable electrocatalysis

Grain boundaries in noble metal catalysts have been identified as critical sites for enhancing catalytic activity in electrochemical reactions such as the oxygen reduction reaction. However, conventional methods to modify grain boundary density often alter particle size, shape, and morphology, obscuring the specific role of grain boundaries in catalytic performance. This study addresses these challenges by employing gold nanoparticle assemblies to control grain boundary density through the manipulation of nanoparticle collision frequency during synthesis. We demonstrate a direct correlation between increased grain boundary density and enhanced two-electron oxygen reduction reaction activity, achieving a significant improvement in both specific and mass activity. Additionally, the gold nanoparticle assemblies with high grain boundary density exhibit remarkable electrochemical stability, attributed to boron segregation at the grain boundaries, which prevents structural degradation. This work provides a promising strategy for optimizing the activity, selectivity, and stability of noble metal catalysts through precise grain boundary engineering.

Magnetic-gold nanoparticle-mediated paper-based biosensor for highly sensitive colorimetric detection of food adulteration

Food adulteration presents a significant challenge due to the evasion of legal oversight and the difficulty of identification. Addressing this issue, there is an urgent need for on-site, rapid, visually based small-scale equipment, along with large-scale screening technology, to enable prompt results without providing opportunities for dishonest traders to react. Colorimetric reactions offer advantages in terms of speed, visualization, and miniaturization. However, there is a scarcity of suitable colorimetric reactions for food adulteration detection, and interference from colored food impurities and easily comparable color results affects accuracy. To overcome limitations, this study introduces a novel approach utilizing polydopamine magnetic nanoparticles to enrich DNA in food samples, effectively eliminating interfering components. By employing gold nanoparticles to generate magnetic-gold nanoparticles, a single magnetic bead achieves simultaneous enrichment, impurity removal, and detection. The use of paper-based biosensors and visualization equipment allows for the visualization and digital analysis of results, achieving a low detection limit of 4.59 nmol mL−1. The method also exhibits high accuracy and repeatability, with a RSD ranging from 1.6 % to 4.0 %. This innovative colorimetric method addresses the need for rapid, miniaturized, and large-scale detection, thus providing a solution for food adulteration challenges.

Customized Self-Assembled Gold Nanoparticle-DNA Origami Composite Templates for Shape-Directed Growth of Plasmonic Structures.

The metal plasmonic nanostructure has the optical property of plasmon resonance, which holds great potential for development in nanophotonics, bioelectronics, and molecular detection. However, developing a general and straightforward method to prepare metal plasmonic nanostructures with a controllable size and morphology still poses a challenge. Herein, we proposed a synthesis strategy that utilized a customizable self-assembly template for shape-directed growth of metal structures. We employed gold nanoparticles (AuNPs) as connectors and DNA nanotubes as branches, customizing gold nanoparticle-DNA origami composite nanostructures with different branches by adjusting the assembly ratio between the connectors and branches. Subsequently, various morphologies of plasmonic metal nanostructures were created using this template shape guided strategy, which exhibited enhancement of surface-enhanced Raman scattering (SERS) signals. This strategy provides a new approach for synthesizing metallic nanostructures with multiple morphologies and opens up another possibility for the development of customizable metallic plasmonic structures with broader applications.

Nucleic acid amplification-free biosensor for sensitive and specific cfDNA detection based on CRISPR-Cas12a and single nanoparticle dark-field microscopy (DFM) imaging

Circulating cell-free DNA (cfDNA) is known to be an attractive biomarker for liquid biopsy. In this work, a rapid and sensitive biosensor was achieved for cfDNA detection based on CRISPR-associated nuclease 12a (CRISPR-Cas12a) and single nanoparticle dark-field microscopy (DFM) imaging. The biosensor employed gold nanoparticles (AuNPs) as signal source, DFM as readout system, and Meanshift algorithms as the image processing systems, respectively. The presence or absence of cfDNA caused the different existing states (monomer or aggregated state) of AuNPs. AuNPs monomers could be effectively distinguished from the aggregated ones under DFM since the AuNPs aggregation could induced the green-to-yellow or green-to-red changing of scattering color. The monomer ratio easily obtained by Meanshift and partial least-square (PLS) algorithms was used for quantitative analysis. Using this strategy, breast cancer gene-1 (BRCA-1), a representative of cfDNA biomarker for breast cancer could be measured with high sensitivity in 40 min and has a low detection limit of 0.081 fM. It is hoped that this nucleic acid amplification-free sensor could be also utilized to detect other nucleic acid biomarkers, and thus, provides a novel strategy for biomedical image analysis.

A gold nanomaterial-integrated distance-based analytical device for uric acid quantification in human urine samples.

In this article, we present the first demonstration of a distance-based paper analytical device (dPAD) for uric acid quantification in human urine samples with instrument-free readout and user-friendliness for the rapid diagnosis and prognosis of various related diseases. By employing gold nanoparticles (AuNPs) as a peroxidase-like nanozyme, our proposed technique eliminates the utilization of horseradish peroxidase (HRP), making the device cost-effective and stable. In our dPAD, uric acid in the sample is oxidized by the uricase enzyme and subsequently catalysed with AuNPs in the sample zone, generating hydroxyl radicals (˙OH). Then, the produced ˙OH reacts with 3,3'-diaminobenzidine (DAB) to form poly DAB (oxDAB), resulting in a coloured distance signal in the detection zone of the dPAD. The variation of the distance of the observed red-brown colour is directly proportional to the uric acid concentration. Our sensor exhibited a linear range from 0.50 to 6.0 mmol L-1 (R2 = 0.9922) with a detection limit (LOD) of 0.25 mmol L-1, covering the clinical range of uric acid in urine. Hence, there is no need for additional sample preparation or dilution. Additionally, this assay is highly selective, with no interferences. We also found that this approach could accurately and precisely determine uric acid in human control samples with the recovery ranging from 99.37 to 100.35 with the highest RSD of 4.05%. Our method is comparable with the use of a commercially available uric acid sensor at a 95% confidence interval. Consequently, the developed dPAD offers numerous advantages such as cost-effectiveness, simplicity, and ease of operation with unskilled individuals. Furthermore, this concept can be applied for extensive biosensing applications in monitoring other biomarkers as an alternative analytical point-of-care (POC) device.

Enhancing the sensitivity and stability of electrochemical aptamer-based sensors by AuNPs@MXene nanocomposite for continuous monitoring of biomarkers

Electrochemical aptamer-based (E-AB) sensors offer exciting potential for real-time tracking of various biomarkers, such as proteins and small molecules, due to their exceptional selectivity and adaptability. However, most E-AB sensors rely on planar gold structures, which inherently limit their sensitivity and operational stability for continuous monitoring of biomarkers. Although gold nanostructures have recently enhanced E-AB sensor performance, no studies have explored the combination of gold nanostructure with other types of nanomaterials for continuous molecular monitoring. To fill this gap, we employed gold nanoparticles and MXene Ti3C2 (AuNPs@MXene), a versatile nanocomposite, in designing an E-AB sensor targeted at vascular endothelial growth factor (VEGF), a crucial human signaling protein. Remarkably, the AuNPs@MXene nanocomposite achieved over thirty-fold and half-fold increases in active surface area compared to bare and AuNPs-modified gold electrodes, respectively, significantly elevating the analytical capabilities of E-AB sensors during continuous operation. After a systematic optimization and characterization process, the newly developed E-AB sensor, powered by AuNPs@MXene nanocomposite, demonstrated both enhanced stability and heightened sensitivity. Overall, our findings open new avenues for the incorporation of nanocomposites in E-AB sensor design, enabling the creation of more sensitive and durable real-time monitoring systems.

High Specific and Rapid Detection of Cannabidiol by Gold Nanoparticle-Based Paper Sensor.

In order to facilitate monitoring of cannabidiol (CBD), we devised a gold immunochromatographic sensor based on a specific monoclonal antibody (mAb). To prepare the antigen, a novel hapten with CBD moiety and a linear carbon chain was employed. By utilizing hybridoma technology, a specific mAb was screened and identified that exhibited a 50% maximal inhibitory concentration against CBD ranging from 28.97 to 443.97 ng/mL. Extensive optimization led to the establishment of visual limits of detection for CBD, achieving a remarkable sensitivity of 8 μg/mL in the assay buffer. To showcase the accuracy and stability, an analysis of CBD-spiked wine, sparkling water, and sports drink was conducted. The recovery rates observed were as follows: 88.4-109.2% for wine, 89.9-107.8% for sparkling water, and 83.2-95.5% for sports drink. Furthermore, the coefficient of variation remained impressively low, less than 4.38% for wine, less than 2.07% for sparkling water, and less than 6.34% for sports drink. Importantly, the developed sensor exhibited no cross-reaction with tetrahydrocannabinol (THC). In conclusion, the proposed paper sensor, employing gold nanoparticles, offers a user-friendly and efficient approach for the precise, rapid, and dependable determination of CBD in products.

Development of an impedimetric sensor for susceptible detection of melatonin at picomolar concentrations in diverse pharmaceutical and human specimens

Our investigation aimed to create and manufacture an electrochemical impedance sensor with the purpose of improving the detection efficiency of melatonin (ME). To achieve this objective, we employed gold nanoparticles coated on polydopamine formed in glassy carbon electrodes (AuNPs/PDA/GCE) as a means to enhance the sensor's capabilities. A novel approach employing the signal-off strategy and electrochemical impedance spectroscopy (EIS) technique was utilized to determine ME. When the AuNPs/PDA/GCE electrode was immersed in a buffered solution containing ME, and the oxidation current of AuNPs was recorded, it was observed that the oxidation current of AuNPs decreased upon the introduction of ME molecules. The decrease in electrical current can be ascribed to the inhibitory impact of ME molecule adsorption on the electrode surface with applying −0.2 V for 150 s in acetate buffer solution (ABS) (pH, 5) through various mechanisms, which hinders the electron transfer process crucial for AuNPs oxidation. Consequently, by utilizing EIS, various concentrations of ME were quantified spanning from 1 to 18 pM. Moreover, the ME sensor achieved an impressive detection limit of 0.32 pM, indicating its remarkable sensitivity in detecting low concentrations of ME. Importantly, these novel sensors demonstrated exceptional attributes in terms of sensitivity, specificity, stability, and repeatability. The outstanding performance of these sensors, coupled with their desirable attributes, establishes their considerable potential for a wide range of practical applications. These applications encompass various fields such as clinical diagnostics, pharmaceutical analysis, environmental monitoring, and industrial quality control, where accurate and sensitive detection of ME is of utmost importance.

Biorecognition elements appended gold nanoparticle biosensors for the detection of food-borne pathogens - A review

Food-borne diseases emanated from bacterial contamination pose serious threats to human food safety and well-being. Microbes such as Clostridium perfringens, Escherichia coli (E. coli), Listeria monocytogenes (L. monocytogenes), Norovirus, Pseudomonas aeruginosa (P. aeruginosa), Salmonella ssp, Shigella ssp, Staphylococcus aureus (S. aureus), are key food-borne pathogens, hazardous to human health. Conventional methods undertaken for the detection of the microbes include colorimetry, electrochemical analysis, surface-enhanced Raman spectroscopy (SERS), fluorescent spectroscopy, and lateral flow assay. The pathogen discovery methods employ gold nanoparticles (Au-NPs), upheld by their specific optical properties, conductivity, biocompatibility, and high surface area-to-volume ratio. Au-NPs double up as colorimetric nanoparticles, enzyme mimics, fluorescence resonance energy transfer (FRET) quenchers and signal enhancers. In conjugation with biomolecules like nucleic acids, aptamers, antibodies, cells and phages, Au-NPs can discretely discern and bind to target pathogens. This treatise presents a comprehensive review of recent (2015–2021) developments on biorecognition elements appended to Au-NPs, and their deployment in identification of food-borne microorganisms.

Gold nanoparticles stabilized by sulfonated imidazolium salt for the manufacture of modified electrodes in order to electrochemical detection of indomethacin

Modified carbon paste electrode (MCPE) was manufactured employing gold nanoparticles (Au-NPs) stabilized by 1-(3-sulfonatopropyl)imidazolium. The modification of carbon paste electrode (CPE) was carried out -quickly and simply- without separating the Au-NPs from the reaction medium. The Au-NPs were characterized by routine techniques such as UV–visible spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS) and, zeta potential (ZP). The Au-NPs obtained are quasi-spherical with a size of around 10 nm and a high surface area which made excellent NPs with catalytic properties.The MCPE was used as an electrochemical sensor for commercial indomethacin (Ind) detection. Under optimum conditions, the sensor presented a linear response in the 1.0 × 10-7 – 1.0 × 10-4 M concentration range, good detection sensitivity and, a low detection limit of 0.68 nM of Ind. The proposed sensor -with good stability and sensitivity- was also employed in capsules and injectables of commercial origin with excellent percentage recoveries.The electrochemical sensor modified by employing Au-NPs have a high confidence level and this was simple to make, convenient, cost-effective, and traceable as an alternative method for the determination of Ind in commercial samples.

Theoretical study of distance-dependent optical fiber SPR sensor based on MoS2 nanosheets

Sandwich assay employing gold nanoparticles and DNA is a common signal amplification strategy designed for surface plasmon resonance sensor. To further improve the sensitivity employing sandwich assay, sandwich-structured optical fiber surface plasmon resonance sensors based on AuNPs and MoS2 nanosheets has been theoretically studied. Au film deposited on fiber core is the bottom nanomaterial of sandwich structure while AuNPs and MoS2 nanosheets are optional used as top nanomaterials. Top and bottom nanomaterials are separated by double stranded DNA, which allows solvent to perform as a spacer. The thickness of spacer is able to be tuned by the number of base pairs of double stranded DNA. Spacer thickness and nanomaterials play important roles to regulate the performance of optical fiber surface plasmon resonance sensor. The refractive index sensitivity has been found to be enhanced by the presence of MoS2 nanosheets compared to AuNPs. Large spacer thickness supported by long double stranded DNA further improves the performance of sensor. This study yields new insight into the structural design of optical fiber surface plasmon resonance sensor enhanced by sandwich structure and will open exciting avenues to apply sandwich-like assay for biosensing.

Colorimetric biosensor for the naked-eye detection of ovarian cancer biomarker PDGF using citrate modified gold nanoparticles

Ovarian cancer is one of the fatal diseases in women. However, it can be cured if the early-stage diagnosis and suitable treatment is given on time. The goal of the research is to produce nano-biosensing technology for early identification of fatal ovarian cancer. In this regard, a rapid and colorimetric nano-biosensor was constructed employing gold nanoparticles to target platelet-derived growth factor (PDGF), a circulating biomarker that is up-regulated in plasma in prevalent ovarian cancer. The findings were supported by spectroscopy analysis and characterized the nanoparticles using SEM (Scanning electron microscopy & XRD (X-ray diffraction). In this proposed principle, gold nanoparticles (AuNPs) are mixed with PDGF specific aptamer and employed to identify PDGF by screening changes in the color as well as absorbance of the Aptamer and AuNPs caused by aggregation. The AuNPs color changes from pinkish to light purple at higher level of concentration. Signal-output exhibited for PDGF was in the linear range of 0.01–10 μg/ml under the optimum conditions. The change in color of gold nanoparticles was seen when the concentration of PDGF was as low as 0.01 μg/ml. Also, the developed technique was successfully applied in artificial serum which have many components of biofluids. The results we presented in this research can imply the practical application of aptamer and AuNPs in cancer diagnosis, exhibiting its benefits of reliability, selectivity, and reproducibility.

Trimethyl-Chitosan Coated Gold Nanoparticles Enhance Delivery, Cellular Uptake and Gene Silencing Effect of EGFR-siRNA in Breast Cancer Cells.

Purpose: Despite the promising therapeutic effects of gene silencing with small interfering RNAs (siRNAs), the challenges associated with delivery of siRNAs to the tumor cells in vivo, has greatly limited its clinical application. To overcome these challenges, we employed gold nanoparticles modified with trimethyl chitosan (TMC) as an effective delivery carrier to improve the stability and cellular uptake of siRNAs against epidermal growth factor receptor (EGFR) that is implicated in breast cancer. Methods: AuNPs were prepared by the simple aqueous reduction of chloroauric acid (HAuCl4) with ascorbic acid and coated with synthesized TMC. EGFR-siRNA was then complexed with the AuNPs-TMC via electrostatic interaction to make AuNPs-TMC/EGFR-siRNA with a w/w ratio of 10:1. Nanoparticles were assessed for physicochemical characteristics and in vitro cellular behavior on MCF-7 breast cancer cell line. Results: Spherical and positively charged AuNPs-TMC (67 nm, +45 mV) were successfully complexed with EGFR-siRNA (82 nm, +11 mV) which were able to retard the gene migration completely. Confocal microscopy and flow cytometry analysis demonstrated complete cellular uptake of Cy5 labeled AuNPs-TMC in the MCF-7 cells after 4 h incubation. MTT test after 48 h incubation showed that the AuNPs-TMC were safe but when combined with EGFR-siRNA exert significant cytotoxicity while the cell viability was about 50%. These nanocomplexes also showed a high gene expression knockdown (86%) of EGFR and also a high apoptosis rate (Q2 + Q3 = 18.5%) after 24 h incubation. Conclusion: This study suggests that the simply synthesized AuNPs-TMC are novel, effective, and promising nanocarriers for siRNA delivery, and AuNPs-TMC/EGFR-siRNA appears to be a potential therapeutic agent for breast cancer treatment.

Dynamic Protein Corona of Gold Nanoparticles with an Evolving Morphology.

Much has been learned about the protein coronae and their biological implications within the context of nanomedicine and nanotoxicology. However, no data is available about the protein coronae associated with nanoparticles undergoing spontaneous surface-energy minimization, a common phenomenon during the synthesis and shelf life of nanomaterials. Accordingly, here we employed gold nanoparticles (AuNPs) possessing the three initial states of spiky, midspiky, and spherical shapes and determined their acquisition of human plasma protein coronae with label-free mass spectrometry. The AuNPs collected coronal proteins that were different in abundance, physicochemical parameters, and interactive biological network. The size and structure of the coronal proteins matched the morphology of the AuNPs, where small globular proteins and large fibrillar proteins were enriched on spiky AuNPs, while large proteins were abundant on spherical AuNPs. Furthermore, the AuNPs induced endothelial leakiness to different degrees, which was partially negated by their protein coronae as revealed by confocal fluorescence microscopy, in vitro and ex vivo transwell assays, and signaling pathway assays. This study has filled a knowledge void concerning the dynamic protein corona of nanoparticles possessing an evolving morphology and shed light on their implication for future nanomedicine harnessing the paracellular pathway.

Development of a novel biosensor for Creatine Kinase (CK-MB) using Surface Plasmon Resonance (SPR)

In this paper, we report a new biosensor for detection of the cardiac biomarker Creatine Kinase (CK-MB) using Surface Plasmon Surface (SPR) technique, with the purpose of the diagnosis of Acute Myocardial Infarction (AMI). For this, we employed gold nanoparticles (AuNP) stabilized with Cysteamine (Cys), and functionalized with Creatine Phosphate (Pcrea), which promote an increase in the sensitivity and accuracy for creatine kinase (CK-MB) detection in saliva and urine samples. The characteristics of the electronic transfer of the sensor could be observed by Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV), that showed significant redox peaks on electrode surface evidencing the high electron exchange process and low resistance charge transfer between the electrolyte solution and the modified surface. The morphological characteristics were confirmed by Atomic Force Microscopy (AFM), micro-Raman and Contact Angle (CA), as well as the adsorption and interaction processes between the materials, which could be observed by Surface Plasmon Resonance (SPR). The SPR analytical curve, ranged from 5.0 ng mL−1 to 100.0 ng mL−1, provided some important parameters, such as limit of detection (LOD) of 0.209 ng mL−1, limit of quantification (LOQ) of 0.696 ng mL−1, and sensitivity of 8.67 × 10–3 ± 0.17 × 10–3 (°) mL ng−1. The specific interaction between functionalized substrate and CK-MB demonstrates a high affinity of 15.14 ng mL−1 based on the Michaelis-Menten fitting.

Solid-state colorimetric polydiacetylene liposome biosensor sensitized by gold nanoparticles.

Polydiacetylene (PDA), a conjugated polymer, has attracted attention for realization of a label-free real-time colorimetric biosensor because it exhibits large and rapid colorimetric responses upon the binding of biomolecules. This is due to the conformational distortion of its conjugated backbone. However, solid-state PDA biosensors for point-of-care diagnosis remain unexplored. We describe a highly sensitive solid-state biosensor based on PDA liposomes. We employed gold nanoparticles (AuNPs) on PDA liposomes as the molecular-binding-signal sensitizer, which provides additional conformational distortion in the backbone structure of PDA by exerting steric repulsion to the attached biomolecules. To prove the concept, AuNPs and a thrombin-binding-aptamer were individually functionalized on PDA liposomes, which were attached to a substrate for the detection of thrombin. We found that the sensitivity was enhanced 2.5 times in the presence of AuNPs compared with the case without AuNPs. Because the steric repulsion of the AuNPs is target-independent, we believe that our solid-state biosensor provides a path toward advanced solid-state biosensors.

Identification of a Profile of Neutrophil-Derived Granule Proteins in the Surface of Gold Nanoparticles after Their Interaction with Human Breast Cancer Sera.

It is well known that the interaction of a nanomaterial with a biological fluid leads to the formation of a protein corona (PC) surrounding the nanomaterial. Using standard blood analyses, alterations in protein patterns are difficult to detect. PC acts as a “nano-concentrator” of serum proteins with affinity for nanoparticles’ surface. Consequently, characterization of PC could allow detection of otherwise undetectable changes in protein concentration at an early stage of a disease, such as breast cancer (BC). Here, we employed gold nanoparticles (AuNPsdiameter: 10.02 ± 0.91 nm) as an enrichment platform to analyze the human serum proteome of BC patients (n = 42) and healthy controls (n = 42). Importantly, the analysis of the PC formed around AuNPs after their interaction with serum samples of BC patients showed a profile of proteins that could differentiate breast cancer patients from healthy controls. These proteins developed a significant role in the immune and/or innate immune system, some of them being neutrophil-derived granule proteins. The analysis of the PC also revealed serum proteome alterations at the subtype level.

Bioconjugation of AuNPs with HPV 16/18 E6 Antibody through Physical Adsorption Technique

Abstract Gold nanoparticle (AuNP) bioconjugates are increasingly being utilised in biomedicine due to their low toxicity on biological tissues and unique electronic and chemical properties. They have been utilised in several biological applications namely manufacture of nanomaterials, biosensing, electron microscopy and drug delivery systems. Particularly, immuno-assays often employ gold nanoparticles (AuNPs) to enhance detection of a biological component. This paper presents a study on the bioconjugation of AuNPs with an antibody against an oncoprotein that is overexpressed in cervical carcinoma progression through physical adsorption. The study successfully developed a HPV 16 E6/18 E6-HRP (CP15)-AuNPs bioconjugate. The study also demonstrated that the antibody pI, gold colloidal solution pH and amount of antibody determine the generation of stable Antibody–AuNPs bioconjugates. These results further demonstrate that physical adsorption can be used to produce functional Antibody–AuNPs that could be employed in diagnostic immunoassays.

A Spherical Nucleic Acid Probe Based on the Au-Se Bond.

Spherical nucleic acid probes (SNAPs) are 3D nucleic acid nanostructures with multiple superiorities over bare nucleic acid chains. Au-based SNAPs that employ gold nanoparticles (AuNPs) as cores and densely modified nucleic acid chains (commonly via Au-S bonds) as shells have been extensively investigated for the diagnosis and therapy of diverse diseases. However, abundant biothiols in living cells can severely displace nucleic acid chains from AuNPs and restrict their theranostic performance. Herein we report the design and preparation of a selenol terminal-functionalized molecular beacon (MB-SeH), which was further employed to prepare a Au-Se bond-based SNAP (SNAP-Se) for bioimaging. A series of experiments proved the successful preparation of MB-SeH and SNAP-Se, and the obtained nanoprobe could avoid biothiol interference and eliminate the false positive signals during biomarker imaging in living cells. This work will open a new avenue for the design and application of SNAPs.

Quantification of shellfish Arginine kinases by double-enhanced immunoassay employing magnetic beads and gold nanoparticles as carrier

Arginine Kinases (AK), known as the major allergen in shellfish, has gained increasing concerns in industrialized countries. Here, we proposed a simple but accurate analytical immunoassay that employed gold nanoparticles (AuNPs) and magnetic beads (MBs) for tracing and quantification of AK in shellfish. The limit of detection (LOD) was 3.91 ng ml−1 (S/N = 3), while the limit of quantitation (LOQ) was 13.04 ng ml−1 (S/N = 10), respectively. Moreover, promising recoveries (94.6%–106.8%) were obtained for the determination of AK in foodstuffs, indicating the good accuracy of this method. Furthermore, the results demonstrated that this method is capable of quantification of AK from nine different shellfish species. Therefore, the double-enhanced immunoassay with simple sample preparation procedures and user-friendly operation showed high applicability for allergen detection in shellfish related foodstuffs and production lines. Furthermore, the developed method can be employed to identify the adulteration of shellfish products using AK as biomarker due to the less conserved sequences of AK in phylogenetically different species.