Nanospherical Brush as Catalase Container for Enhancing the Detection Sensitivity of Competitive Plasmonic ELISA

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth shows great potential for the determination of disease-related biomarkers at ultralow concentrations by using sandwich formats. However, the relatively low sensitivity of this strategy using competitive formats limits its adoption for hapten detection. Herein, we present an improved competitive pELISA for ultrasensitive detection of ochratoxin A (OTA), where silica nanoparticles carrying poly­(acrylic acid) brushes (SiO₂@PAA) were used to decrease the affinity of competing antigens to anti-OTA monoclonal antibodies and amplify the signal as a “CAT container” (SiO₂@PAA@CAT). The developed competitive pELISA exhibits extremely high sensitivity for OTA with detection limits of 10^–18 and 5 × 10^–20 g/mL by the naked eye and microplate reader, respectively. These values are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (10^–11 g/mL by the naked eye) and 8 orders of magnitude lower than that of horseradish peroxidase-based conventional ELISA (10^–11 g/mL by the microplate reader), respectively. Reliability and robustness of the proposed method were evaluated using actual agricultural products and human serum samples. This study demonstrated the potential of this modified method in practical applications involving the ultrasensitive detection of mycotoxins or other haptens

Plasmonic Enzyme-Linked Immunosorbent Assay Using Nanospherical Brushes as a Catalase Container for Colorimetric Detection of Ultralow Concentrations of <i>Listeria monocytogenes</i>

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth exhibits ultrahigh sensitivity for detecting disease-related biomarkers using sandwich formats. However, the limit of detection (LOD) of this strategy for <i>Listeria monocytogenes</i> is only around 10³ CFU/mL, which considerably exceeds the amount of <i>L. monocytogenes</i> commonly present in food products (<100 CFU/g). Herein, we report an improved pELISA method for detection of <i>L. monocytogenes</i> at ultralow concentrations with the sandwich formats using silica nanoparticles carrying poly­(acrylic acid) brushes as a “CAT container” to increase enzyme loading for enhancing the detection signal. Under optimal conditions, the proposed pELISA exhibits good specificity and excellent sensitivity for <i>L. monocytogenes</i> with a LOD of 8 × 10¹ CFU/mL in 0.01 M phosphate-buffered saline, via a reaction that can be discriminated by the naked eye. The LOD obtained by this method was 2 and 5 orders of magnitude lower than that of conventional CAT-based pELISA and horseradish peroxidase (HRP)-based conventional ELISA, respectively. Coupled with large-volume immunomagnetic separation, the LOD for <i>L. monocytogenes</i>-spiked lettuce samples reached 8 × 10¹ CFU/g. The improved pELISA also exhibited a great potential in detecting a single cell of <i>L. monocytogenes</i> in 100 μL of solution

Size-Dependent Immunochromatographic Assay with Quantum Dot Nanobeads for Sensitive and Quantitative Detection of Ochratoxin A in Corn

Fluorescent microspheres are a novel luminescent nanomaterial proposed as an alternative probe to improve the detection sensitivity of competitive immunochromatographic assay (ICA). Quantum dot nanobeads (QBs) possess strong luminescence and resistance to matrix interference. Theoretically, large-sized QBs exhibit stronger luminescent intensity than small-sized QBs and are beneficial to ICA sensitivity. However, oversized QBs may reduce the sensitivity of competitive ICA. Thus, the relationship between the size and luminescent intensity of QBs and the competitive ICA sensitivity must be elucidated. In this study, QBs of different sizes (58, 124, 255, 365, and 598 nm) were synthesized. Ochratoxin A (OTA) was selected as the model analyte for competitive ICA. The effects of QB size on the detection performance of competitive ICA were then evaluated. The cutoff limit of QB-ICA for naked eye detection was used for qualitative analysis, and the half-maximal inhibitory concentration (IC₅₀) and LOD were employed for quantitative analysis. Results indicated that 124 nm QBs used as labeling probes for competitive ICA showed the optimal detection performance and the lowest cutoff value of 5 ng/mL for qualitative detection and IC₅₀ (0.39 ng/mL) for quantitative detection. Similar to commercial ELISA, QB₁₂₄-ICA displayed good accuracy, specificity, reproducibility, and practicability. In summary, 124 nm QBs can be used as a new labeling probe for competitive ICA

Fluorescent Ru(phen)₃²⁺-Doped Silica Nanoparticles-Based ICTS Sensor for Quantitative Detection of Enrofloxacin Residues in Chicken Meat

A Ru­(phen)₃²⁺-doped silica fluorescent nanoparticle (FN)-based immunochromatographic test strip (ICTS) sensor was developed for rapid, high sensitivity, easy to use, and low cost quantitative detection of enrofloxacin (ENR) residues in chicken meat. The fluorescence signal intensity of the FNs at the test line (FI_T) and control line (FI_C) was determined with a prototype of a portable fluorescent strip reader. Unique properties of Ru­(phen)₃²⁺ doped silica nanoparticles (e.g., large Stokes shift, high emission quantum yield, and long fluorescence lifetime) were combined with the advantages of ICTS and an easy to make portable fluorescent strip reader. The signal was based on FI_T/FI_C ratio to effectively eliminate strip to strip variation and matrix effects. Various parameters that influenced the strip were investigated and optimized. Quantitative ENR detection with the FNs ICTS sensor using 80 μL sample took only 20 min, which is faster than the commercial ELISA kit (that took 90 min). The linear range of detection in chicken extract was established at 0.025–3.500 ng/mL with a half maximal inhibitory concentration at 0.22 ± 0.02 ng/mL. Using the optimized parameters, the limit of detection (LOD) for ENR using the FNs ICTS sensor was recorded at 0.02 ng/mL in chicken extract. This corresponds to 0.12 μg/kg chicken meat which is two (2) orders of magnitude better that the maximum residue limits (MRLs) imposed in Japan (10 μg/kg) and three (3) orders of magnitude better than those imposed in China. The intra- and inter-assay coefficient of variations (CVs) were 6.04% and 12.96% at 0.5 ng/mL, 6.92% and 12.61% at 1.0 ng/mL, and 6.66% and 11.88% at 2.0 ng/mL in chicken extract, respectively. The recoveries using the new FNs ICTS sensor from fifty (50) ENR-spiked chicken samples showed a highly significant correlation (<i>R</i>² = 0.9693) with the commercial enzyme-linked immunosorbent assay (ELISA) kit. The new FNs ICTS sensor is a simple, rapid, sensitive, accurate, and inexpensive quantitative detection of ENR residues in chicken meat and extracts

Folic Acid Targeting for Efficient Isolation and Detection of Ovarian Cancer CTCs from Human Whole Blood Based on Two-Step Binding Strategy

Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FRs) on the CTC surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin-coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTC isolation. The streptavidin–biotin-system-mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than that of FA-decorated magnetic nanoparticles based on the one-step CTC separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA–biotin–CTC complex. Furthermore, when the system was applied for the isolation and detection of CTCs in ovarian cancer patients’ peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting, in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation, is a promising tool for CTC enrichment and detection of early-stage ovarian cancer

Immunochromatographic Assay for Ultrasensitive Detection of Aflatoxin B₁ in Maize by Highly Luminescent Quantum Dot Beads

Highly luminescent quantum dot beads (QBs) were synthesized by encapsulating CdSe/ZnS and used for the first time as immunochromatographic assay (ICA) signal amplification probe for ultrasensitive detection of aflatoxin B₁ (AFB₁) in maize. The challenges to using high brightness QBs as probes for ICA are smooth flow of QBs and nonspecific binding on nitrocellulose (NC) membrane, which are overcome by unique polymer encapsulation of quantum dots (QDs) and surface blocking method. Under optimal conditions, the QB-based ICA (QB-ICA) sensor exhibited dynamic linear detection of AFB₁ in maize extract from 5 to 60 pg mL^–1, with a median inhibitory concentration (IC₅₀) of 13.87 ± 0.16 pg mL^–1, that is significantly (39-fold) lower than those of the QD as a signal probe (IC₅₀ = 0.54 ± 0.06 ng mL^–1). The limit of detection (LOD) for AFB₁ using QB-ICA sensor was 0.42 pg mL^–1 in maize extract, which is approximately 2 orders of magnitude better than those of previously reported gold nanoparticle based immunochromatographic assay (AuNP-ICA) and is even comparable with or better than the conventional enzyme-linked immunosorbent assay (ELISA) method. The performance and practicability of our QB-ICA sensor were validated with a commercial ELISA kit and further confirmed with liquid chromatography tandem mass spectrometry (LC–MS/MS). Given its efficient signal amplification performance, the proposed QB-ICA offers great potential for rapid, sensitive, and cost-effective quantitative detection of analytes in food safety monitoring

Targeted Detoxification of Aflatoxin B₁ in Edible Oil by an Enzyme–Metal Nanoreactor

Mycotoxin contamination is an important issue for food safety and the environment. Removing mycotoxins from food without losing nutrients and flavor components remains a challenge. In this study, a novel strategy was proposed for the targeted removal of aflatoxin B1 (AFB1) from peanut oil using an amphipathic enzyme–metal hybrid nanoreactor (PL-GOx-Fe3O4@COF) constructed with covalent organic frameworks (COFs) which can selectively adsorb AFB1. Due to the confined space provided by COFs and the proximity effect between GOx and Fe3O4, the detoxification of AFB1 is limited in the nanoreactor without affecting the composition and properties of the oil. The detoxification efficiency of AFB1 in the chemoenzymatic cascade reaction catalyzed by PL-GOx-Fe3O4@COF is six times higher than that of the combination of free GOx and Fe3O4. The AFB1 transformation product has nontoxicity to kidney and liver cells. This study provides a powerful tool for the targeted removal of mycotoxins from edible oils