Fluorescent Immunochromatographic Assay Research Articles

Preparation and Application of Time-Resolved Fluorescent Nanospheres (TRFNs) for Immunochromatography of Ferritin

Time-resolved fluorescent nanospheres (TRFNs) are polymer nanospheres with special functional groups on the surface loaded with rare earth complexes which emit fluorescence when irradiated by ultraviolet light. TRFNs offer spherical morphology, homogeneity, good dispersity, large specific surface area, easy chemical modification, high loading capacity and good biocompatibility. The rare earth complexes possess unique properties that render them highly sensitive and capable of effectively excluding unspecific fluorescence. Hence, they constitute an excellent labeling material for the time-resolved fluorescence immunochromatographic assay (TRFIA). The developed method has a linear response to ferritin from 6 to 1000 ng/mL with a detection limit of 3.5 ng/mL, good reproducibility, and good accuracy compared with commercial chemiluminescence measurements of serum. These results suggest that the reported TRFN has utility for time-resolved fluorescence immunochromatographic assays.

Development of time-resolved fluorescence immunochromatography and ic-ELISA based on monoclonal antibodies specifically recognizing fumonisin B1

Fumonisins (FBs) are mycotoxins primarily synthesized by Fusarium moniliformis. Among these, FB1 exhibits not only high toxicity towards humans and animals but also carcinogenic properties. The global prevalence of FB1 contamination in cereals and related products, particularly maize, is alarmingly significant. Consequently, the accurate determination of FB1 levels in cereals holds immense importance. In this study, highly sensitive monoclonal antibodies specifically targeting FB1 were prepared and utilized for the establishment of a time-resolved fluorescence immunochromatographic assay (TRFIC) to detect FB1. The parameters of antibody labeling with time-resolved fluorescent microspheres were optimized. The detection time was significantly reduced to 6 min. The limits of detection (LOD) for corn, rice, and feed were determined as 0.496–0.844 μg/kg, and the quantification (LOQ) was 0.788–1.322 μg/kg. In addition, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was successfully developed. Under optimized conditions, the half inhibitory concentration (IC50) value for FB1 was determined as 2.137 μg/L. A strong correlation between the results obtained from these two methods and HPLC-MS/MS analysis was observed in the same samples tested. In conclusion, both immunological methods developed in this work are highly suitable for rapid FB1 detection in real field samples.

Rapid identification of A29L antibodies based on mRNA immunization and high-throughput single B cell sequencing to detect Monkeypox virus

ABSTRACT With the large number of atypical cases in the mpox outbreak, which was classified as a global health emergency by the World Health Organization (WHO) on 23 July 2022, rapid diagnosis of mpox and diseases with similar symptoms to mpox such as chickenpox and respiratory infectious diseases in the early stages of viral infection is key to controlling the spread of the outbreak. In this study, antibodies against the monkeypox virus A29L protein were efficiently and rapidly identified by combining rapid mRNA immunization with high-throughput sequencing of individual B cells. We obtained eight antibodies with a high affinity for A29L validated by ELISA, which were was used as the basis for developing an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobeads (SiTQD-ICA). The SiTQD-ICA biosensor utilizing M53 and M78 antibodies showed high sensitivity and stability of detection: A29L was detected within 20 min, with a minimum detection limit of 5 pg/mL. A specificity test showed that the method was non-cross-reactive with chickenpox or common respiratory pathogens and can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. This antibody identification method can also be used for rapid acquisition of monoclonal antibodies in early outbreaks of other infectious diseases for various studies.

Development of a time-resolved fluorescent immunochromatographic assay for the quantitative detection of cardiovascular disease by MPO and Lp-PLA2

Development of a time-resolved fluorescent immunochromatographic assay for the quantitative detection of cardiovascular disease by MPO and Lp-PLA2

Development of ZnCdSe/ZnS quantum dot-based fluorescence immunochromatographic assay for the rapid visual and quantitative detection 25⁃hydroxyvitamins D in human serum.

Vitamin D deficiency is associated with various diseases such as obesity, digestive problems, osteoporosis, depression, and infections, and has therefore emerged as a topic of great interest in public healthcare. The quantitative assessment of 25-hydroxyvitamin D (25-OH VD) in human serum may accurately reflect the nutritional status of vitamin D in the human body, which is significant for the prevention and treatment of vitamin D-deficient patients. In this study, we developed an assay for quantitative detection of 25-OH VD based on the 25-OH VD monoclonal antibody (mAb), and identified the optimal process parameters. The following process settings were found to be suitable for the test strips: pH of 7.6, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) ratio of 1:2000, and the anti-25-OH VD mAb ratio was 1:8. The equilibration time of the immune dynamic assay was 15min. Under optimal conditions, the quantum dot nanoparticle-based fluorescent immunochromatographic assay (QDs-FICA) exhibited dynamic linear detection of 25-OH VD in PBS, from 5ng/mL to 100ng/mL, and the strip quantitative curve could be represented by the following regression equation: y = -0.02088 logx)+1.444 (R2 = 0.9050). The IC50 of the QDs-FICA was 39.6 ± 1.33ng/mL. The specificity of the QDs-FICA was evaluated by running several structurally related analogues, including 25-OH VD2, 25-OH VD3, 1,25-OH2VD3, 1,25-OH2VD2, VD2, and VD3. The coefficients of variation were all below 10%. The shelf life of the test strips in this study was about 160 days at room temperature. Briefly, this study is the first to perform QDs-FICA for the rapid visual and quantitative detection of 25-OH VD, with great potential significance for clinical diagnosis of vitamin D-associated diseases.

Fluorescent Immunochromatographic Assay (FICA) for Monkeypox Virus

Monkeypox, a rare zoonotic disease classified as a category B infectious disease, is caused by the monkeypox virus (MPXV), which has a broad host range. Since May 2022, human MPXV infection outbreak has spread to more than 100 countries, and China has reported 106 MPXV infections between June 2 and 30, 2023. Therefore, rapid and effective MPXV detection is essential to control and prevent its transmission. In this study, a rapid, cost-effective, and convenient fluorescent immunochromatographic assay (FICA) for MPXV antigen A35R detection was developed. Under the optimal reaction conditions, the visual detection limit of the proposed assay was 7.5 ng/mL and was completed within 15 min. There was no cross-reactivity with norovirus (NV) or dengue virus (DENV) controls. Additionally, the clinical application of the proposed method was demonstrated by testing human serum samples spiked with the MPXV antigen A35R. FICA was faster and more convenient than traditional enzyme-linked immunosorbent assays (ELISA) or polymerase chain reaction (PCR), and hence suitable for the early screening of MPXV infection. In summary, FICA could realize rapid, low-cost, sensitive, and specific MPXV detection hold potential application prospects in clinical settings.

Rapid detection of zearalenone in cereals using La3+-doped upconversion nanoparticles-based immunochromatographic assay

A novel fluorescent immunochromatographic assay (ICA) with excellent specificity and sensitivity for evaluating trace residues of zearalenone (ZEN) in cereal samples was successfully developed. La3+-doped upconversion nanoparticles (UCNPs) were assessed as antibody labels, following optimization of doping ratios. Our results indicated that 20% La3+-doped UCNPs had superior morphological structure and fluorescence characteristics, making them the optimal UCNPs. After labeling 20% La3+-doped UCNPs with ZEN monoclonal antibodies (mAbs), optimal polyacrylic acid amount, coupled mAbs amount, detection time, and fluorescent immunoprobe concentrations were determined as 2.0 mL, 20 μg, 10 min, and 8 mg/mL, respectively. Under optimized detection conditions, the limit of detection (LOD) for ZEN standard solutions was 0.25 ng/mL with coefficients of variation (CVs) ＜ 8.67% and a high linear correlation (R2 = 0.9944). Furthermore, specificity analysis demonstrated that prepared test strips were highly specific for the target mycotoxin. The LODs of ZEN in wheat and maize were both 0.5 μg/kg in spiking recovery experiments, and the recoveries were 87.65%–99.26% and 84.12%–95.12% with the CVs being 1.29%–7.14% and 4.03%–9.01%, respectively, confirming that the optimal UCNPs-ICA was efficient, straightforward, and suitable for rapid on-site screening of ZEN in cereals.

A highly sensitive and accurate dual-channel fluorescent immunochromatographic assay for simultaneous quantitative detection of influenza A virus and adenovirus antigens

Sensitivity and accuracy are the key performance metrics for evaluating immunochromatographic assay (ICA). In this study, we have developed a quantum dot nanobeads-based dual-channel fluorescent ICA (QBs-DICA) designed for the sensitive and accurate detection of both influenza A virus (IAV) and human adenovirus (ADV) antigens. Following systematic assay optimization, we achieved a remarkable improvement in sensitivity, with gains of up to two orders of magnitude at the best. To enhance signal accuracy for IAV and ADV testing, we implemented a temperature-controlled dual-channel detector during sample incubation and the chromatographic process, resulting in an approximate increase of 34.2% and 36.7%, respectively. The Limit of Detection (LOD) for IAV and ADV testing was robustly established at 165 copies/mL and 805 copies/mL, respectively. Furthermore, in the case of IAV-spiked and ADV-spiked samples, we achieved consistently high recoveries ranging from 94.5% to 104.8% and 94.7% to 106.4%, with impressively low coefficients of variation (CV) of below 7.16% and 6.25%, respectively. Our established QBs-DICA system has exhibited a remarkable enhancement in both sensitivity and accuracy, making it exceptionally well-suited for supporting the rapid advancement of clinical diagnosis and treatment strategies.

The instantly blocking-based fluorescent immunochromatographic assay for the detection of SARS-CoV-2 neutralizing antibody.

At present, there is an urgent need for the rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs) to evaluate the ability of the human body to resist coronavirus disease 2019 (COVID-19) after infection or vaccination. The current gold standard for neutralizing antibody detection is the conventional virus neutralization test (cVNT), which requires live pathogens and biosafety level-3 (BSL-3) laboratories, making it difficult for this method to meet the requirements of large-scale routine detection. Therefore, this study established a time-resolved fluorescence-blocking lateral flow immunochromatographic assay (TRF-BLFIA) that enables accurate, rapid quantification of NAbs in subjects. This assay utilizes the characteristic that SARS-CoV-2 neutralizing antibody can specifically block the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2) to rapidly detect the content of neutralizing antibody in COVID-19-infected patients and vaccine recipients. When 356 samples of vaccine recipients were measured, the coincidence rate between this method and cVNT was 88.76%, which was higher than the coincidence rate of 76.97% between cVNT and a conventional chemiluminescence immunoassay detecting overall binding anti-Spike-IgG. More importantly, this assay does not need to be carried out in BSL-2 or 3 laboratories. Therefore, this product can detect NAbs in COVID-19 patients and provide a reference for the prognosis and outcome of patients. Simultaneously, it can also be applied to large-scale detection to better meet the needs of neutralizing antibody detection after vaccination, making it an effective tool to evaluate the immunoprotective effect of COVID-19 vaccines.

Development of a Fluorescent Immunochromatographic Assay Based on Quantum Dot-Functionalized Two-Dimensional Monolayer Ti3C2 MXene Nanoprobes for the Simultaneous Detection of Influenza A Virus and SARS-CoV-2.

Accurate and rapid detection of the influenza A virus (FluA) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can effectively control their spread. We developed a colorimetric and fluorescent dual-functional two-channel immunochromatographic assay (ICA) biosensor to simultaneously detect the above-mentioned viruses. A unique two-dimensional Ti3C2-QD immunoprobe was established by adsorbing dense quantum dots (QDs) onto the light green monostromatic Ti3C2 MXene surface, resulting in light green colorimetric and superior fluorescence signals and guaranteeing high sensitivity, stability, and excellent liquidity for ICA detection. Rapid visual screening for FluA and SARS-CoV-2 infections was applicable via a green colorimetric signal. Sensitive and quantitative detection of viruses in their early stages of infection was performed by using the fluorescence signal. Our proposed Ti3C2-QD-ICA biosensor can simultaneously detect 1 ng/mL or 2.4 pg/mL FluA and 1 ng/mL or 6.2 pg/mL SARS-CoV-2 via its colorimetric or fluorescence signals, respectively, with a short testing time (20 min), good reproducibility, specificity, and accuracy. In addition, this method demonstrated sensitivity higher than that of the conventional AuNP-based ICA method in throat swab samples. Hence, our proposed Ti3C2-QD-ICA method can be potentially applied for the rapid, ultrasensitive, and multiplex detection of respiratory viruses.

Colorimetric–fluorescent dual-signal enhancement immunochromatographic assay based on molybdenum disulfide-supported quantum dot nanosheets for the point-of-care testing of monkeypox virus

The sudden outbreak and worldwide spread of monkeypox suggests an urgent demand for more effective on-site diagnostic technology to counter the silent spread of highly pathogenic virus. This paper reports a dual-signal readout immunochromatography assay (ICA) with colorimetric–fluorescence co-enhanced ability for the ultrasensitive and flexible detection of the monkeypox virus (MPXV) antigen in different scenarios. A two-dimensional film-like nanotag with a molybdenum disulfide (MoS2) core and multilayered quantum dot (QD) shells was customized for dual-signal ICA to provide superior signal responses, larger reaction interface, and better stability than conventional spherical nanolabels. A portable dual-mode reading instrument was developed for the simultaneous identification of two signals and the processing of data. The integrated platform, in which the colorimetric signal supported the rapid screening of MPXV and the fluorescent mode allowed the quantitative detection of the target virus with detection limits down to 0.1 and 0.0024 ng/mL, greatly broadens the application range of existing ICA techniques.

Comparison of IgG and Neutralizing Antibody Response After Pfizer BioNTech COVID-19 Vaccine Among Iraqi Individuals

SARS-C0V-2 has quickly caused a pandemic. The distribution of vaccines is presently underway in an effort to stop the viral transmission and stop fatalities, several vaccinations have been created to decrease COVID virus disease, in 2019, the development of vaccine-induced population immunity is an important global strategy. Review of the anti-spike protein receptor-binding domain (S-RBD) a person's level of immune response to antibody strategy can be used to assess SARS-CoV-2 viral infection in 2019. Their efficacy, safety, and immunogenicity in various population are not thoroughly understood. The objective of this study was to evaluate the vaccinations adverse effects as well as determine the immunogenicity of the messenger ribonucleic acid (mRNA) BNT162b2 vaccines through the production of IgG and neutralizing antibodies against the protein s subunit. A total of 41 vaccinated individuals with Pfizer-BioNTech COVID-19 vaccine, as well as (10) non vaccinated were included in the study. Measurements of Neutralizing antibody (Nab) levels and CoV-IgG levels were tested by fluorescence Immunochromatographic assay. IgG and Nab levels showed a significant difference between its level in the sera of vaccinated and controls (p<0.05). This means the majority of immunization recipients between the ages of 18 -50 years can develop an immunological response to the SARS-Cov-2 vaccine.

A Europium Nanosphere-Based Time-Resolved Fluorescent Immunochromatographic Assay for the Rapid Screening of 4,4′-Dinitrocarbanilide: Aiming at Improving Strip Method Performance

Considering that the strip method is simple and convenient for users, a Europium nanosphere-based time-resolved fluorescent immunochromatographic assay (TRFICA) for the rapid screening of 4,4'-dinitrocarbanilide (DNC) was developed to improve the performance of strip assays. After optimization, TRFICA showed IC50, the limit of detection, and cut-off values of 0.4, 0.07, and 5.0 ng mL-1, respectively. No significant cross-reactivity (CR < 0.1%) with 15 DNC analogs was observed in the developed method. TRFICA was validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 77.3% to 92.7%, with coefficients of variation of <14.9%. Moreover, the time needed for the detection procedure, including the sample pre-treatment, was less than 30 min for TRFICA, which had never been achieved before in other immunoassays. The newly developed strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique for DNC analysis in chicken muscle.

Europium Nanoparticles-Based Fluorescence Immunochromatographic Detection of Three Abused Drugs in Hair.

Drug abuse is becoming increasingly dangerous nowadays. Morphine (MOP), methamphetamine (MET) and ketamine (KET) are the most commonly abused drugs. The abuse of these drugs without supervision can cause serious harm to the human body and also endanger public safety. Developing a rapid and accurate method to screen drug suspects and thus control these drugs is essential to public safety. This paper presents a method for the simultaneous quantitative detection of these three drugs in hair by a europium nanoparticles-based fluorescence immunochromatographic assay (EuNPs-FIA). In our study, the test area of the nitrocellulose membrane was composed of three equally spaced detection lines and a quality control line. The test strip realized the quantitative analysis of the samples by detecting the fluorescence brightness of the europium nanoparticles captured on the test line within 15 min. For the triple test strip, the limits of detection of MOP, KET and MET were 0.219, 0.079 and 0.329 ng/mL, respectively. At the same time, it also showed strong specificity. The strip was stable and could be stored at room temperature for up to one year, and the average recovery rate was 85.98-115.92%. In addition, the EuNPs-FIA was validated by high-performance liquid chromatography (HPLC) analysis, and a satisfactory consistency was obtained. Compared to the current immunochromatographic methods used for detecting abused drugs in hair, this method not only increased the number of detection targets, but also ensured sensitivity, improving detection efficiency to a certain extent. The approach can also be used as an alternative to chromatography. It provides a rapid and accurate screening method for the detection of abused drugs in hair and has great application prospects in regard to public safety.

A Simplified Amplification-Free Strategy with Lyophilized CRISPR-CcrRNA System for Drug-Resistant Salmonella Detection.

Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drug-resistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug-resistant Salmonella has an ultra-sensitive detection limit of 84 CFUmL-1 within 30min, with good linearity in the range of 102 -106 CFUmL-1 . In real-world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC-TRFIA offers a cost-effective, sensitive, and virtually equipment-independent platform for preventing foodborne illnesses, screening for drug-resistant Salmonella, and guiding clinical use.

A helper antibody based competitive fluorescence immunochromatographic assay for quantitative detection of florfenicol in poultry eggs.

Florfenicol (FF) is a chloramphenicol analogue used in animals, and florfenicol amine (FFA) is the main metabolite of FF. However, their residues in agricultural products are harmful to human health. A highly specific and sensitive assay for FF/FFA detection needs to be developed since the traditional detection methods are low sensitive. In this study, a new method for rapid quantification of FF/FFA in poultry eggs by fluorescent immunochromatographic assay (HAFIA) was established. The triple antibodies including a primary monoclonal antibody (mAb) specific to the targets FF and FFA, a secondary polyclonal antibody (pAb) labeled with Europium nanoparticles (EuNPs) and a helper monoclonal antibody (hAb) reacting with pAb but not react with mAb or target antigen are designed, which can form structural aggregation complexes in microwells with a single step of reactions. By loading the reaction sample solution, the triple-antibodies (mAb-pAb-hAb)-EuNPs complexes migrate to the test (T) line on nitrocellulose membrane of testing strip, which are competitively captured by the immobilized FF-BSA conjugates on the membrane and the FF/FFA targets in sample solution. Fluorescence on T line is read by a portable fluorescent strip reader in 10 min, and the result is given by the ratio of fluorescent intensity on T line than control (C) line. This new fluorescent testing strip with triple-antibody complexes' amplification signal has 50-fold higher sensitivity than conventional CG-LFIAs, and can detect as low as 0.01 ng/mL florfenicol and 0.1 ng/mL florfenicol amine targets from egg samples. The developed competitive fluorescent immunochromatography method based on auxiliary antibodies has the advantages of high sensitivity and specificity for the rapid and quantitative detection of FF/FFA in poultry eggs.

Highly selective monoclonal antibody-based fluorescence immunochromatographic assay for the detection of fenpropathrin in vegetable and fruit samples

Fenpropathrin (FPT) is a typical pyrethroid pesticide that can cause chronic toxicity to humans. Herein, an anti-FPT monoclonal antibody (mAb) was elicited via a novel hapten synthesized by introducing a carboxyl-containing spacer arm in the cyclopropane moiety of FPT. Characterized by enzyme-linked immunosorbent assay (ELISA), the mAb exhibited high affinity and selectivity to FPT with a half-maximal inhibitory concentration of 31.05 μg/L and negligible cross-reactivities with analogs of pyrethroids. Based on the mAb, a fluorescence immunochromatographic assay (FICA) for FPT detection was firstly developed. The detection limit of the FICA is 0.012 mg/kg which is much lower than the maximum residue limit of FPT for food samples. The average recoveries of FPT from spiked food samples by the FICA were 85.0–105.0%, and the obtained results were in good agreement with those of gas chromatography-tandem mass spectrometry. Overall, this work provided a reliable tool suitable for the detection of FPT residue for large-scale samples in a rapid and cost-effective manner.

Ratiometric fluorescent immunochromatography for simultaneously detection of two nitrofuran metabolites in seafoods

Competitive immunochromatographic assay (ICA) has been widely used in food safety on-site monitoring. However, traditional competitive ICA relies on the change of single-signal intensity to discriminate the results, leading to limited sensitivity and accuracy of naked-eye visual inspection. In this study, we developed a sensitive ratiometric fluorescent immunochromatographic assay (RFICA) judging results based on the color changes of fluorescence. Two different color quantum dot nanobeads (QBs) were introduced as signal labels for rapid and accurate detection of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in seafoods. Under the optimal conditions, the visual detection limit for 2-NPAOZ and 2-NPSEM reached up to 1 and 0.5 ng/mL, which were 5- and 14-fold lower than that of traditional single-signal response fluorescent ICA strips. In addition, the RFICA strips were applied to real samples with acceptable recoveries of 81.33 ∼ 99.23 %. These results demonstrate that the developed RFICA platform possesses considerable potential in the on-site detection.

Rapid and sensitive quantification of capsaicinoids for edible oil adulteration by immunomagnetic solid-phase extraction coupled with time-resolved fluorescent immunochromatographic assay

Immunochromatographic methods are acknowledged analytic assay to analyze capsaicinoids. Immunomagnetic solid-phase extraction (IMSPE) coupled with time-resolved fluorescence immunochromatographic assay (TRFICA) was proposed to quantify capsaicinoids in oil samples. Monoclonal antibodies (mAb) were synthesized with CNBr–Magnetic Crystarose 4B particles (CNBr–MCPs) under mild condition. The resultant CNBr–MCPs@mAb were conjugated high affinity mAbs on its surface, which was utilized to extract capsaicinoids from lipid matrices via antibodies-antigens capture. Under the optimized conditions, the whole IMSPE procedure was achieved within 15 min, and quantified by TRFICA strips. The results showed coefficients up to 0.9975 and the visual detection limit as low as 0.6 µg kg−1. The recoveries were ranging from 88.3 % to 112.4 % with the intra-day and inter-day precision lower than 11.6 %. Finally, the proposed IMSPE-TRFICA method was successfully used to detect capsaicinoids in lipid matrices, which has great utility to quantify capsaicinoids and adulteration detect vegetable oils.

Carboxylated fluorescent microsphere based immunochromatographic test strip enabled sensitive and quantitative on-site detection for florfenicol in eggs

Florfenicol (FF), used popularly in prevention and treatment of virus infections in livestock and poultry, has widely been found in eggs and harmful to human health. In this work, a sensitive and quantitative on-site detecting solution, monoclonal antibody-based carboxylated fluorescent microsphere immunochromatographic test strip assay (FM-ICTS), is design and applied for FF detection. The proposed method can sensitively detect FF in low detection limit of 0.030 ng/g and quantitatively measure its concentration from 0.1 ng/mL to 8.1 ng/mL (R2 = 0.9991) with high repeatability (CV<8.0 %). In addition, the established FM-ICTS method exhibited high measurement accuracy in FF samples as compared with HPLC-MS analysis and demonstrated satisfied recoveries (99.1–101.3 %). More importantly, the quantitative FF test strip demonstrate ultra-high stability, which presents approximately equivalent detection ability to the fresh one after stored at 4 °C for more than one year or stored at 37 °C for 60 days. Therefore, the proposed method is a promising solution for rapidly and sensitively quantitative determination of FF in eggs.