Nanozyme-linked Immunosorbent Assay Research Articles

Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107–2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Development and performance of NLISA for C-reactive protein detection based on Prussian blue nanoparticle conjugates.

Prussian blue nanoparticles (PBNPs), also called nanozymes, are very attractive as an alternative to horseradish peroxidase in immunoassay development due to their simple and low-cost synthesis, stability and high catalytic activity. Today, there is a method for highly effective PBNP synthesis based on the reduction of an FeCl3/K3[Fe(CN)6] mixture by hydrogen peroxide. However, there is a lack of research showcasing the use of these highly effective PBNPs for specific target detection in clinical settings, as well as a lack of comprehensive comparisons with conventional methods. To address this gap, we prepared diagnostic reagents based on highly effective PBNPs by modifying them using gelatin and attaching anti-C-reactive protein (CRP) monoclonal antibodies through cross-linking with glutaraldehyde. As a result, a solid-phase colorimetric immunoassay in a sandwich format (nanozyme-linked immunosorbent assay [NLISA]) using highly effective PBNPs as a label for CRP detection has been demonstrated for the first time. The assay demonstrated a detection limit of 21.8pg/mL, along with acceptable selectivity, precision (CV < 25%) and accuracy (the recovery index was within acceptable limits (75-125%) for LLOQ /ULOQ range. The analytical performance of this method is on par with sensitive assays developed in the last 5 years. Notably, the results obtained from NLISA align with those from an immunofluorescence assay conducted by a certified clinical laboratory. Furthermore, this study underscores the technological challenges involved in constructing an analysis that necessitate further exploration.

P-d Orbital Hybridization-Engineered PdSn Nanozymes for a Sensitive Immunoassay.

Nanozymes with peroxidase-like activity have been extensively studied for colorimetric biosensing. However, their catalytic activity and specificity still lag far behind those of natural enzymes, which significantly affects the accuracy and sensitivity of colorimetric biosensing. To address this issue, we design PdSn nanozymes with selectively enhanced peroxidase-like activity, which improves the sensitivity and accuracy of a colorimetric immunoassay. The peroxidase-like activity of PdSn nanozymes is significantly higher than that of Pd nanozymes. Theoretical calculations reveal that the p-d orbital hybridization of Pd and Sn not only results in an upward shift of the d-band center to enhance hydrogen peroxide (H2O2) adsorption but also regulates the O-O bonding strength of H2O2 to achieve selective H2O2 activation. Ultimately, the nanozyme-linked immunosorbent assay has been successfully developed to sensitively and accurately detect the prostate-specific antigen (PSA), achieving a low detection limit of 1.696 pg mL-1. This work demonstrates a promising approach for detecting PSA in a clinical diagnosis.

The development of a method to produce diagnostic reagents using LaNiO3 nanospheres and their application in nanozyme-linked immunosorbent assay for the colorimetric screening of C-reactive protein with high sensitivity.

LaNiO3 perovskite nanoparticles, especially nanospheres (LNNS), show great promise in biomedical assays due to their peroxidase-like catalytic properties. However, LNNS-based diagnostic reagents have not been tested in nanozyme enzyme-linked immunosorbent assay (NLISA) or other enzyme-linked immunosorbent assays, and there is limited data on their synthesis. To fill this gap, it is necessary to develop a method for creating LNNS conjugates with monoclonal antibodies and to investigate the reproducibility, scalability, and applicability of these diagnostic reagents in NLISA. We have successfully developed a method for producing novel diagnostic reagents utilizing LaNiO3 nanospheres. Our research demonstrates the application of these nanospheres in a NLISA specifically designed for the detection of C-reactive protein (CRP) in real serum samples. This method is both reproducible and scalable, allowing for the efficient production of nanospheres that are functionalized with monoclonal antibodies targeting CRP, with a mean diameter of approximately 270 nm. Based on the promising results obtained from our experiments, we have developed and optimized a sandwich-format NLISA for CRP detection. This assay achieved a lower limit of detection at 0.178 μg L-1, with a dynamic range from 12.5 to 0.195 μg L-1 and a linear detection range extending from 0.195 to 6.25 μg L-1, showcasing its potential for clinical applications. The new NLISA method, utilizing LaNiO3 nanospheres in a sandwich format for the detection of CRP, significantly enhances sensitivity compared to similar use horseradish peroxidase-based ELISA. In this study for the first time, the functionalization of lanthanum nickelate nanospheres with recognition elements has been demonstrated. This advancement also sheds light on the technological challenges involved in synthesizing diagnostic reagents, identifying areas that need further exploration.

Fe-N-C single-atom nanozyme-linked immunosorbent assay for quantitative detection of aflatoxin B1

Aflatoxin B1 (AFB1) is a prominent food contaminant known for its high toxicity. Rapid, low-cost, and sensitive detection of AFB1 is an urgent demand for food safety risk control. In the traditional enzyme-linked immunosorbent assay (ELISA) method for AFB1 detection, horseradish peroxide (HRP) is high-cost and possesses poor environmental stability. In this study, a direct competitive single-atom nanozymes (SAzymes) -linked immunosorbent assay was developed based on Fe-N-C SAzymes. The proposed methods exhibited excellent sensitivity, rapid detection, fabrication, and low cost. Under the optimal conditions, the limit of detection of the new assay was 3.3 pg mL−1, with a quantitative relationship between 0.0084 and 0.358 ng mL−1. In addition, the developed method presented good recoveries in spiked samples, ranging from 84.50% and 103.75%, with coefficients of variation below 10%. Furthermore, the developed detection method showed good consistency (R2 = 0.9852) with the reference method, liquid chromatography/mass spectrometry (LC-MS/MS). To our knowledge, this is the first report about single-atom nanozymes-based ELISA for AFB1 determination. This work provides a feasible strategy for the rapid and sensitive detection of food contaminants using SAzymes.

A nanozyme-linked immunosorbent assay based on Au@CeO2@Pt nanozymes for colorimetric and fluorescent detection of SARS-CoV-2 nucleocapsid protein

Developing powerful alternatives to natural enzymes has been a target pursued by researchers, and nanozymes are considered to be the best candidate due to their unique advantages of high stability, high activity, and easy modification. In this study, the multishell complexes of Au@CeO2@Pt nanozymes were successfully synthesized which exhibited an excellent intrinsic peroxidase-like ability to oxidize 3,3′,5,5′-tetramethylbenzidine (TMB), and resulting in a visible colorimetric signal. Interestingly, due to the internal filtration effect, the blue-emitting oxidized TMB (oxTMB) can also quench the red-emitting light from bimetallic AuAg nanoclusters. Based on the above properties, a kind of robust immunoassay for target protein detection was established. The dual-mode immunoassay is more accurate for SARS-CoV-2 detection on account of the reciprocal corroboration of the signals. More importantly, the immunoassay had high sensitivity with a low limit of detection and excellent recovery rates in simulated urine/saliva samples. Therefore, it is highly anticipated the immunoassay based on the newly developed nanozyme became an accurate instrument for diagnosing SARS-CoV-2 in the biomedical field.

Au-X (X=Pt/Ru)-decorated magnetic nanocubes as bifunctional nanozyme labels in colorimetric, magnetically-enhanced, one-step sandwich CRP immunoassay

Scientific interest in the investigation and application of multifunctional nanomaterials in medical diagnostics has been increasing. The employment of magnetocatalytic immunoconjugates as both analyte-capturing agents and enzyme-like catalytic labels may enable rapid preconcentration and determination of relevant antigens. In this work, we synthesized and comprehensively characterized two types of noble metal-decorated magnetic nanocubes (MDMCs) which were subsequently applied in the one-step, sandwich nanozyme-linked immunosorbent assay (NLISA). Magnetic cores allow for rapid separation from complex samples of biological origin. The catalytically active shell composed of Au-decorated Pt or Ru can effectively mimic the activity of horseradish peroxididase, retaining at the same time the ability to form stable bioconstructs through self-assembly of thiolated ligands. As a result, hybrid multifunctional nanoparticles were synthesized and used to detect C-reactive protein (CRP) in serum samples. We have also paid considerable attention to the mechanistic studies of the formation of sandwich immunocomplexes with nanoparticle labels by means of immunoenzymatic methods and surface plasmon resonance. Analytical parameters of the Pt-MDMCs-labeled NLISA (detection limit LOD = 0.336 ng mL−1, recovery = 98.0%, linear response window covering two logarithmic units) turned out to be superior to the classical, one-step ELISA based on a horseradish peroxidase. In addition, our method offers further possibility of sensitivity adjustment by changing the parameters of magnetic preconcentration, together with good long-term stability of MDMCs conjugates and their good resistance to common interferences. We believe that the proposed simple synthetic protocol will guide a new approach to applying metal-decorated magnetic nanozymes as versatile and multifunctional labels for application in subsequent pre-analytical analyte concentration and immunoassays towards clinical applications.

High-Indexed Intermetallic Pt3Sn Nanozymes with High Activity and Specificity for Sensitive Immunoassay

Great efforts have been made to expand the application fields of nanozymes, which puts forward requirements for nanozymes with both superior catalytic activity and specificity. Herein, we reported the high-indexed intermetallic Pt3Sn (H-Pt3Sn) with high peroxidase-like activity and specificity. The resultant H-Pt3Sn exhibits a specific activity of 345.3 U/mg, which is 1.82 times higher than Pt. Moreover, H-Pt3Sn possesses negligible oxidase-like and catalase-like activities, achieving superior catalytic specificity toward H2O2 activation. Experimental and theoretical calculations reveal both the splitting energy for adsorbed H2O2 and the energy barrier for the rate-determining step of H-Pt3Sn are significantly decreased compared with Pt3Sn and Pt. Finally, a nanozyme-linked immunosorbent assay is successfully developed, achieving the sensitive and accurate colorimetric detection for carcinoembryonic antigen with a low detection limit of 0.49 pg/mL and showing practical feasibility in serum sample detection.

SERS Resolving of the Significance of Acetate on the Enhanced Catalytic Activity of Nanozymes

Understanding the structure-activity correlation and reaction mechanism of the catalytic process in an acetic acid-sodium acetate (HAc-NaAc) buffer environment is crucial for the design of efficient nanozymes. Here, we first reported a lattice restructuration of Au-LaNiO3-δ nanofibers (NFs) after acidification with the HAc-NaAc buffer to show a significantly enhanced oxidase-like property. Surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT) calculation confirm the direct evidence for the formation of specific enhanced intermediate O-O species after acidification, indicating that the insertion of the carboxyl group in the A-Au/LaNiO3-δ NFs plays crucial roles in both producing vacancies in HAc-NaAc solution from its dissociation during the catalytic process and the protection of the vacancies, which can be directly interacted with oxygen in the environment to produce O-O species, realizing the enhanced oxidation of substrate molecules. The insertion of the carboxyl group increased the oxidase-like catalytic activity by 2.38 times and the SERS activity by 5.27 times. This strategy offers a way to construct an efficient nanozyme-linked immunosorbent assay system for the diagnosis of cancer through the highly sensitive SERS identification of exosomes.

Porphyrin NanoMOFs as a catalytic label in nanozyme-linked immunosorbent assay for Aflatoxin B1 detection

Food safety is a global problem, and methods to reliably and sensitivity detect contaminants could be transformative. Herein, we synthesized a novel Porphyrin NanoMoFs (NanoPCN-223(Fe)) with excellent dispersion and peroxidase-like activity. The Km value of NanoPCN-223(Fe) was 2.0 × 10−4 M toward the H2O2 substrate during the catalytic process. We use the NanoPCN-223(Fe) to construct an enhanced dispersion MOF-linked immunosorbent assay (Ed-MOFLISA) to sensitive detect AFB1 in milk. The optimized Ed-MOFLISA displayed a broad quantitative range from 0.05 to 10 ng/mL and a limit of detection of 0.003 ng/mL. Spiked peanut and soy milk recovery ranged from 91.22% to 97.63%. Intra-assay and inter-assay coefficient of variation values ranged from 0.78 to 3.85%, demonstrating the outstanding reproducibility and accuracy of Ed-MOFLISA. We applied the Ed-MOFLISA assay to test the milk samples, demonstrating its potential use for monitoring food quality.

Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs.

CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence that activates Cas enzymes to cleave reporter molecules. Currently, most CRISPR-based diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here we show the combination of a CRISPR-Cas-based reaction with a nanozyme-linked immunosorbent assay, which allows for the quantitative and colorimetric readout of Cas13-mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate that CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilize this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme will serve as a universally applicable signal catalyst for CRISPR-based diagnostics, which will expand the spectrum of targets for preamplification-free, quantitative detection.

Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood.

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

An In Situ Generated Prussian Blue Nanoparticle-Mediated Multimode Nanozyme-Linked Immunosorbent Assay for the Detection of Aflatoxin B1

This work aims to develop a novel multimode (photothermal/colorimetric/fluorescent) nanozyme-linked immunosorbent assay (NLISA) based on the in situ generation of Prussian blue nanoparticles (PBNPs) on the surface of magnetic nanoparticles (MNPs). Being considered the most toxic among the mycotoxins, aflatoxin B1 (AFB1) was chosen as the proof-of-concept target. In this strategy, MNPs, on which an AFB1 aptamer was previously assembled via streptavidin-biotin linkage, are anchored to 96-well plates by AFB1 and antibody. In the presence of HCl and K4Fe(CN)6, PBNPs formed in situ on the MNP surface, thereby achieving photothermal and colorimetric signal readout due to their photothermal effect and intrinsic peroxidase-like activity. Based on fluorescence quenching by MNPs, Cy5 fluorescence was recovered by the in situ generation of PBNPs to facilitate ultrasensitive fluorescence detection. Photothermal and colorimetric signals allow portable/visual point-of-care testing, and fluorescent signals enable accurate determination with a detection limit of 0.54 fg/mL, which is 6333 and 28 times lower than those of photothermal and colorimetric analyses, respectively. We expect that this proposed multimode NLISA can not only reduce the false-positive/negative rates through the multisignal crossdetection in AFB1 monitoring but also provide a universal way of sophisticated instrumentation-free, easy-to-use, cost-effective, and highly sensitive detection of other food hazards.

A direct competitive nanozyme-linked immunosorbent assay based on MnO2 nanosheets as a catalytic label for the determination of fumonisin B1.

A direct competitive nanozyme-linked immunosorbent assay (dcNLISA) based on MnO2 nanosheets (MnO2 NSs) as a nanozyme label was developed for the highly sensitive determination of fumonisin B1 (FB1). MnO2 NS-labeled fumonisin B1-bovine serum albumin was easily synthesized as a competing antigen for the dcNLISA. And color changes derived from the MnO2-3,3',5,5'-tetramethylbenzidine (TMB) system were exploited as the output signals of the dcNLISA. Several experimental parameters including the concentrations of the coating antibody, pH values, ionic strength and methanol concentration were optimized. Under the optimal conditions, the proposed method demonstrated a linear range (1.17-20.74 ng mL-1) with a reliable correlation coefficient (R2 = 0.9989), a satisfactory limit of detection (0.63 ng mL-1) and high selectivity for the detection of FB1. The recoveries of FB1 in spiked corn and wheat samples were in the range of 85.31-108.16% with coefficients of variation (CVs) ranging from 6.14% to 9.23%. Meanwhile, the testing results showed good consistency (R2 = 0.9892) between the developed dcNLISA and the reference method, liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) method. The proposed method was proven to be simple, sensitive, cost-effective and reliable for the screening of FB1 in cereals.

A nanozyme-linked immunosorbent assay based on metal–organic frameworks (MOFs) for sensitive detection of aflatoxin B1

In order to avoid the occurrence of false positives and false negatives caused by conventional enzyme-linked immunosorbent assay (ELISA), we established a novel indirect competitive MOF-linked immunosorbent assay (MOFLISA) method for the high throughput and high sensitive detection of aflatoxin B1. This method replaces the natural enzyme with functional MOFs to catalyze a chromogenic system. As a result, the limit of detection (LOD) of the MOFLISA method was 0.009 ng·mL−1 with a linear working range from 0.01 to 20 ng·mL−1. The developed MOFLISA method for AFB1 has a 20-fold improved LOD value compared with the conventional ELISA. The recoveries and relative standard deviations (RSD) ranged from 86.41 to 99.74% and 2.38–9.04%, respectively. The results demonstrate that the recovery rate and accuracy of this detection method is better than that of conventional ELISA, reducing risks offalsepositive andfalsenegativeresults.

One-pot synthesized citric acid-modified bimetallic PtNi hollow nanospheres as peroxidase mimics for colorimetric detection of human serum albumin.

The combination of Pt with low-cost transition metal is an effective way to diminish the bulk utilization of costly Pt and to design new nanostructured materials with improved enzyme-like activity. In the present work, citric acid-functionalized platinum-nickel hollow nanospheres (CA@PtNi hNS) were synthesized through a simple one-pot wet chemical method, which involves the galvanic replacement reaction between the Ni nanoparticles and the Pt precursor that leads to the formation of hollow nanostructures. Transmission electron spectroscopic images revealed the uniformity of the CA@PtNi hNS, with an average diameter of 10.3±2nm. Moreover, zeta potential, FTIR, and XPS measurements confirmed the existence of citric acid in the CA@PtNi hNS. During synthesis, the use of citric acid not only facilitates monodispersity but also provides a negative surface charge (-11mV) to the CA@PtNi hNS that electrostatically attracts the 3,3',5,5'-Tetramethylbenzidine (TMB) substrate. As-prepared CA@PtNi hNS possessed excellent peroxidase-like activity due to rich Pt surfaces, large surface area, and heterogeneous interaction between Pt and Ni atoms. Furthermore, a nanozyme-linked immunosorbent assay (NLISA) for human serum albumin (HSA) detection was developed by replacing the enzyme in a standard enzyme-linked immunosorbent assay with CA@PtNi hNS. The CA@PtNi hNS based-NLISA showed sensitive detection of HSA concentrations ranging from 0 to 400ngmL-1 with a LOD of 0.19ngmL-1 and an average of 112% recovery of HSA from the spiked human plasma samples. The outcomes of the present study confirm the applicability of CA@PtNi hNS as substitutes for natural enzymes.

Prussian blue nanoparticles with peroxidase-mimicking properties in a dual immunoassays for glycocholic acid

Prussian Blue nanoparticles (PBNPs) were utilized in a lateral flow immunoassay (LFA) and in an indirect competitive nanozyme-linked immunosorbent assay (icELISA), respectively, for their intense blue color and peroxidase (POx) -like activity. The PBNPs with good POx-like activity was linked to the antibody. Under the optimal parameters, both the PBNP-icELISA and PBNP-LFA perform very well. The icELISA has an IC50 value of 190 ng/mL, the working range extends from 29 to 1200 ng/mL, and the limit of detection is 22 ng/mL. The visual cut-off limit is 10 ng/mL. The dual immunoassay was used to quantify glycocholic acid in spiked human urine. Excellent recoveries and correlation between the two methods were observed.

Ratiometric Surface Enhanced Raman Scattering Immunosorbent Assay of Allergenic Proteins via Covalent Organic Framework Composite Material Based Nanozyme Tag Triggered Raman Signal "Turn-on" and Amplification.

The exploration of nanomaterials with mimic enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in an enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au-S bond and electrostatic force to further produce a Raman "hot spot". Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm-1 was weakened, and a new signal at 1591 cm-1 generated by 4-ATP was turned on, leading to the generation of a ratiometric SERS signal. Based on this performance, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I1591/I1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65-6.2 × 104 ng/mL, covering more than 3 orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery, and lower cross-reactivity, providing new insight into the application of SERS technology for trace target analysis.

Ratiometric fluorescent immunoassay for the cardiac troponin-I using carbon dots and palladium-iridium nanocubes with peroxidase-mimicking activity.

A nanozyme-linked immunosorbent assay is described for cardiac troponin I which is a biomarker for myocardial infarction. The method is based on the use of Pd-Ir nanocubes with excellent peroxidase-like activity. The nanocubes catalyze the oxidization of nonfluorescent o-phenylenediamine (OPD) by H2O2 to form a yellow fluorescent product (oxOPD) with excitation/emission maxima at 400/570nm. Carbon dots are added as areference fluorophore. Under the same excitation wavelength, they display blue fluorescence (450nm). The ELISA uses the Pd-Ir nanocubes as a label for the secondary antibody and OPD as substrate. The ratio of fluorescence intensities at 570 and 450nm increases in the 1pg·mL-1 to 1ng·mL-1 cardiac troponin I concentration range, and the detection limit is 0.31pg·mL-1. The method was applied to analyze spiked serum samples, and the results compared well with those obtained by a commercial chemiluminescence assay. Graphical abstract Schematic presentation of the ratiometric fluorescence immunoassayforcardiac troponin-I.Pd-Ir nanocubes were employed to fabricate nanozyme-based signal labels for its excellent peroxidase-mimicking activity.

A nanozyme-linked immunosorbent assay for dual-modal colorimetric and ratiometric fluorescent detection of cardiac troponin I

To develop a robust method for the detection of disease biomarkers is always the pursued goal in the field of biomedical research. Multi-modal immunoassay strategy will bring tremendous potential for the purpose. In this study, a kind of robust nanozyme-linked immunosorbent assay has been presented, which can achieve ratiometric fluorescent and colorimetric dual-modal sensing performance. Here, o-phenylenediamine (OPD) as an organic substrate can be oxidized and converted into 2,3-diaminophenazine (oxOPD) by the robust nanozyme of nanoceria with peroxidase-like properties in the present of H2O2. An exogenous fluorescence signal source of graphitic carbon nitride quantum dots (g-C3N4 QDs) is introduced, and the corresponding fluorescence can be efficiently quenched by oxOPD owing to the hydrogen bonding and π-π stacking interactions, yielding a fluorescence spectral line with dual emission peaks while only irradiated by an excitation wavelength. Meanwhile, a visible color change was derived from the oxOPD formation, which can be recorded by colorimetric method. As a proof-of-study demonstration, the detection of human cardiac troponin I was done. This method affords robust, reliable and sensitive detection performance due to the combination of the merits of ratiometric fluorescent assay and colorimetric assay. It is no doubt that our proposed approach will bring remarkable inspirations for biomedical researchers.