A gold nanoparticle-based lateral flow immunoassay for atrazine point-of-care detection using a handhold scanning device as reader.

A method is described to achieve accurate quantitative detection of atrazine (ATZ) in maize by using lateral flow strips based on gold nanoparticles (GNPs) and a handheld scanning reader. GNPs of 15 nm in diameter were applied as label, and a lateral flow immune assay strip was prepared. The linear range was 5.01-95.86 ng mL-1 with a detection limit of 4.92 ng mL-1 in phosphate buffer, 4 times better than the readout by the naked eye. ATZ-spiked corn samples were also analysed. The accuracy of results of spiked samples was confirmed by ELISA and liquid chromatography-tandem mass spectrometry (HPLC), which proved the reliability of the proposed method. A handhold device with an optical scanning system was designed for on-site quantitative detection. Combined with the pretreatment, the assay could be completed in less than 20 min.

Rapid and ultrasensitive detection of DNA and microRNA-21 using a zirconium porphyrin metal-organic framework-based switch fluorescence biosensor.

Sensitive and accurate detection of nucleic acid biomarkers is critical for early cancer diagnosis, disease monitoring, and clinical treatment. In this study, we developed a switch fluorescence biosensor for simple and high-efficient detection of nucleic acid biomarkers using 6-carboxyfluorescein (FAM)-modified single-stranded DNA (ssDNA) probes (FAM-P1/P2), and zirconium porphyrin metal-organic framework nanoparticles (ZrMOF) acted as fluorescence quencher. FAM-P1/P2 probes were adsorbed on ZrMOF surface because of π-π stacking, hydrogen bonding, and electrostatic interactions. Fluorescence quenching event occurred by fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) processes, thereby achieving the "off" fluorescence status. Once the specific binding was formed between the fluorescence probes and the targets, the rigid double-stranded DNA (dsDNA) structures were released from ZrMOF surface, resulting in the recovery of fluorescence and the "on" status. Because of the superior adsorption ability of ZrMOF toward ssDNA than dsDNA, the switch of fluorescence signals from "off" to "on" allowed rapid and ultrasensitive detection of ssDNA (T1) and microRNA-21 (miR-21) within 30 min. The limit of detection (signal-to-noise ratio = 3) for T1 and miR-21 were 2 fM and 11 aM, respectively. Moreover, the proposed strategy was very simple as it worked by the facile adsorption-quenching-recovery mechanism without difficult and complicated immobilization processes. Also, this biosensor showed an excellent analytical performance in the detection of miR-21 in human serum samples. Therefore, this biosensor might be considered a potential tool for the detection of DNA and miRNA biomarkers in clinical samples.

A highly sensitive and dual-readout immunoassay for norfloxacin in milk based on QDs-FM@ALP-SA and click chemistry.

A dual-readout immunoassay based on QDs-FM@ALP-SA and click chemistry was developed for quick and sensitive detection of norfloxacin (NOR), which is an important fluoroquinolone antibiotic. In the system, the NOR-biotin conjugate (NOR-Biotin) was synthesized by click chemistry for signal transformation, and alkaline phosphatase-labeled streptavidin (ALP-SA) was attached to quantum dot fluorescence microspheres (QDs-FM) by an activated ester method to form QDs-FM@ALP-SA for signal amplification. Here, QDs-FM was a dual-functional carrier: it was used not only as a chemiluminescence signal amplification carrier but also as a fluorescent signal due to its fluorescence character. The NOR antibody was coated on a 96-well chemiluminescence microtiter plate, and NOR-Biotin was bound to the antibody specifically. Then, QDs-FM@ALP-SA was combined with NOR-Biotin to develop a direct competition chemiluminescence/fluorescence immunoassay (dc-CLIA/FIA). The IC50 values were 0.345 and 1.206 ng/mL for dc-CLIA/FIA, respectively. The linear range was 0.013-12.48 ng/mL and 0.042-39.86 ng/mL, respectively. The recovery from the standard fortified blank milk samples was in the range of 86.44%-101.3%. Therefore, this method could be a useful tool for routine screening of NOR residues in milk.

A highly sensitive method for simultaneous detection of hAAG and UDG activity based on multifunctional dsDNA probes mediated exponential rolling circle amplification.

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.

Detection of 4 quinolone antibiotics by chemiluminescence based on a novel Nor-Biotin bifunctional ligand and SA-ALP technology.

A simple and effective direct competitive chemiluminescence immunoassay for the detection of 4 kinds of quinolone antibiotics in milk was established using Nor-Biotin (biotin-modified norfloxacin [NOR]) bifunctional ligand and alkaline phosphatase-conjugated streptavidin signal amplification technology. The polyclonal antibody was obtained after the immunization of New Zealand White rabbits using norfloxacin-derived antigen. "Click chemistry" was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive chemiluminescence assay method was developed and used for sensitive detection of 4 kinds of quinolone drugs (NOR, pefloxacin, ciprofloxacin, and danofloxacin). The IC50 of the 4 kinds of quinolone drugs ranged from 7.35 to 24.27 ng/mL, and the lowest detection limits ranged from 0.05 to 0.16 ng/mL, which were below their maximum residue levels, approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the 4 quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.

Simultaneous detection of diethylstilbestrol and estradiol residues with a single immunochromatographic assay strip.

An immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneously rapid and sensitive detection of diethylstilbestrol (DES) and estradiol (E2) in milk and tissue samples. For this purpose, two monoclonal antibodies raised against those two estrogens were conjugated to gold nanoparticles and were applied to the conjugate pads of the test strip. The competitors of the DES-BSA/E2-BSA conjugates were immobilized onto a nitrocellulose membrane at two detection zones to form T1 and T2, respectively. The immunochromatographic assay had a visual detection limit of DES at 30 ng/g in milk powder, 25 ng/g in liquid milk, and 25 ng/g in shrimp tissue, respectively, and the results can be judged within 7-10 min. The visual detection limit of E2 was 75 ng/g in milk powder, 65 ng/g in liquid milk, and 60 ng/g in shrimp tissue, respectively, and the results can be judged within 3-4 min. It had advantages in easy operation without requiring sophisticated equipment and specialized skills. By testing thirty milk and shrimp tissue samples from the local market, the method was compared with the HPLC-MS / MS method, and there was no statistical difference between the two methods. Furthermore, the immunochromatographic assay had good specificity, simple procedure, and low cost. This protocol was well suited for the food safety monitoring and early warning.

Effects of bisphenol A and nanoscale and microscale polystyrene plastic exposure on particle uptake and toxicity in human Caco-2 cells.

Microplastics are abundant in oceans, lakes, soils and even air, and can pose potential threats to human health through food or respiratory intake. Moreover, microplastics have synergistic toxicity to the body after absorbing organic pollutants. In this study, laser scanning confocal microscope and flow cytometry were used to observe the intake of colonic cancer Caco-2 cells to polystyrene plastic with five different particlesizes (300 nm, 500 nm, 1 µm, 3 µm, 6 µm). The uptake rates of microplastics with different particle sizes were 73%, 71%, 49%, 43%, and 30%, respectively. Then, High Performance Liquid Chromatography (HPLC) was used to analyze the adsorption differences of polystyrene plastic with different particle sizes to bisphenol A (BPA). Finally, the proliferation toxicity of polystyrene microplastics with different particle sizeson Caco-2 cells before and after adsorption of BPA was compared. MTT experiments confirmed that microplastics caused an increase in cytotoxicity. This result may be related to increased cellular oxidative stress and mitochondrial depolarization. This hypothesis has been confirmed in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) assays because nanoscale microplastics cause a large amount of ROS on Caco-2 cells after microplastic exposure, and micron-scale microplastics cause a significant decrease in MMP. At the same time, nanoscale microplastics can cause further depolarization of mitochondria due to their large specific surface area adsorption of BPA, which leads to enhanced cytotoxicity of microplastics after BPA adsorption. The results of this study are of great significance in the evaluation of the safety of microplastics in the human body.

A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level.

Multiplex DNA methylation and glycosylation are ubiquitous in the human body to ensure the normal function and stability of the genome. The methyltransferases and glycosylases rely on varied enzymes with different action mechanism, which still remain challenges for multiple detection. Herein, we developed a tri-functional dsDNA probe mediated exponential amplification strategy for sensitive detection of human DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG) activities. The tri-functional dsDNA probe was rationally designed with M-DNA and U-DNA. M-DNA contains the 5'-GCmGCGC-3' site for Dnmt1 recognition. U-DNA possesses one uracil as the substrate of UDG and a primer sequence for initiating the amplification reaction. M-DNA was complementary to partial sequence of U-DNA. In the presence of Dnmt1 and UDG, BssHⅡ and Endo Ⅳ were used to nick the 5'-GCGCGC-3' and AP sites respectively, resulting in the release of single-stranded DNA sequence (primer sequence), respectively. After magnetic separation, the released primer sequence hybridizes with padlock DNA (P-DNA), initiating exponential rolling circle amplification to produce numerous G-quadruplexes for recordable signals. The strategy exhibited the limit of detection as low as 0.009 U mL-1 and 0.003 U mL-1 for Dnmt1 and UDG, respectively. Meanwhile, this strategy was successfully applied to detect Dnmt1 and UDG activities in living cell samples at single-cell level and assay the inhibitors of Dnmt1 and UDG. Therefore, the strategy provided a potential method to detect Dnmt1 and UDG activities in biological samples for early clinic diagnosis and therapeutics.

Highly sensitive detection of ochratoxin A based on bio-barcode immunoassay and catalytic hairpin assembly signal amplification.

Herein, a highly sensitive ochratoxin A (OTA) detection strategy was developed based on a bio-barcode immunoassay with catalytic hairpin assembly (CHA). Two nanoprobes were designed for the assay: one was a gold nanoparticle (AuNP) harbouring numerous bio-barcode DNA and antibodies, and the other was an antigen-functionalized magnetic nanoparticle (MNP). In the presence of target OTA, the antigens of the MNPs competed with OTA for binding to the antibodies on the AuNPs. After magnetic separation, the unbound AuNPs and target OTA were washed away. Dithiothreitol (DTT) was then added to the MNP-bound AuNPs to elute the bio-barcode DNAs of AuNPs, which triggered the CHA reaction. Under optimal conditions, the proposed method could sensitively detect target OTA ranging from 0.001 to 10000 ng/mL. The limit of detection (LOD, 3 N/S) and limit of quantification (LOQ, 10 N/S) for OTA were 0.54 pg/mL and 1.80 pg/mL, respectively. The bio-barcode immunoassay was used to analyse food samples (corn, wheat, and peanut), and the recovery and relative standard deviations (RSD) ranged from 93.30% to 108.80% and from 3.2% to 6.9%, respectively. The total assay time was 6 h. Therefore, the proposed strategy will provide a new approach for the detection of mycotoxins and other small molecule analytes and can be applied for quality control to ensure food safety.

Ultrasensitive detection of staphylococcal enterotoxin B in foodstuff through dual signal amplification by bio-barcode and real-time PCR.

This study developed an ultrasensitive method involving (1) gold nanoparticles encoded with a double-stranded DNA as the first signal amplification and goat anti-staphylococcal enterotoxin B (SEB) polyclonal antibody and (2) magnetic microparticles coated with anti-SEB monoclonal antibody to detect SEB. Both functionalized nanoparticles can capture SEB in a sandwich system. The released DNA barcodes were then characterized through SYBR Green real-time polymerase chain reaction and resulted in the second signal amplification. Under optimized experimental conditions, an ultrasensitive bio-barcode for SEB detection was established, and its detection limit could reach 0.269 pg mL-1, which was 1000-fold lower than that of conventional enzyme-linked/immunosorbent assay. The proposed method shows great potential in food security.

High-specificity double-stranded DNA detection with a "humanoid" molecular beacon and TALEs.

Owing to the limitation of DNA-specific recognition, improving the sensitivity of bacteria detection without additional culture poses a considerable challenge. Herein, a sensitive, novel, and high-specificity fluorescence detection method was developed using transcription activator-like effectors (TALEs) as the specifically recognized element. Based on the property of TALEs for recognizing double-stranded DNA, the detection method was first proposed. Its sensitivity was further improved by isothermal amplification of RPA. The "humanoid" molecular beacon (H-MB) was designed to achieve signal output. The steric hindrance of the protein opens the stem, enhancing the fluorescence intensity of an attached FAM/BHQ-1 pair. This method can be used to detect Escherichia coli O157:H7 with single-base mismatch specificity and high sensitivity (detection limit of 3 cfu mL-1) and can be applied in actual sample detection by boiling the sample for 10 min. TALEs can be assembled according to the target, so the method can be used to detect other foodborne pathogens and viruses in the food safety and clinical fields.

Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10-3 ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.