Zhoumin Li

Smartphone-based visualized microarray detection for multiplexed harmful substances in milk

In this paper, we report a sensitive, simple and inexpensive analytical method for the immunoassay microarray based on a smartphone in which various harmful substances in milk could be assayed. Tetracyclines (TCs) and Quinolones (QNs) were selected as the model targets in this study. TCs and QNs antigens were immobilized in the microarray and then samples containing free of antibiotics and corresponding antibodies as well as AgNPs labeled secondary antibodies were added to the microarray. The signal of this competitive format was further amplified by silver enhancement technique based on the development reagents and achieved a visual dots in the array. The resulting microarray could be detected by the smartphone placed in the minicartridge. The limit of detection (LOD) of this novel detection platform was 1.51ngmL-1 (TCs) and 1.74ngmL-1 (QNs). To achieve one-well quantitative analysis, a series of gradient concentration mouse IgG was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentrations of mouse IgG. The results showed that a quantitative detection of TCs and QNs established were consistent with external standard curve. Compared to other methods, this method was superior in terms of detection limit, time saving, and one-well quantitative detected with smartphone which were simple sample-preparation.

Bivalent Aptasensor Based on Silver-Enhanced Fluorescence Polarization for Rapid Detection of Lactoferrin in Milk

Here we report a novel type of bivalent aptasensor based on silver-enhanced fluorescence polarization (FP) for detection of lactoferrin (Lac) in milk powder with high sensitivity and specificity. The novel two split aptamers were obtained from the aptamer reported in our previous SELEX (systematic evolution of ligands by exponential enrichment) selection, and their minimal structural units were optimized on the basis of their affinity and specificity. Also, dual binding sites of split aptamers were verified. The bivalent aptamers were modified to be linked with signal-molecule fluorescein isothiocyanate (FITC) and enhancer silver decahedral nanoparticles (Ag10NPs). The split aptamers could bind to different sites of Lac and assemble into a split-aptamers-target complex, narrowing the distance between Ag10NPs and FITC dye. As a result, Ag10NPs could produce a mass-augmented and metal-enhanced fluorescence (MEF) effect. In general, ternary amplification based on Ag10NPs, split aptamers, and the MEF effect all contributed to the significant increase of FP values. It was proved that the sensitivity of this assay was about 3 orders of magnitude over traditional aptamer-based homogeneous assays with a detection limit of 1.25 pM. Furthermore, this design was examined by actual milk powder with rapid and high-throughout detection.

Simultaneous detection of four nitrofuran metabolites in honey by using a visualized microarray screen assay

A visualized microarray sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplexed approach, metabolites of four main nitrofuran antibiotics can be detected simultaneously. Individual antigens were spotted onto 96-well plates. An indirective competitive assay format, with visualized signal response, was employed. An extraction method, based on derivatization with 2-nitrobenzaldehyde and partition into ethyl acetate, was used for screening. The limits of detection were 0.10, 0.04, 0.04, and 0.10ngg-1 for 3-amino-5-morpholino-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), and 1-aminohydantoin (AHD), respectively. The recovery rate ranged from 78% to 93% for the four targets. In addition, this method was easy to operate with low detection cost and fast speed. This microarray method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety monitoring.

Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples

In this work, we demonstrate, for the first time, the development of a disposable MoS2-arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS2-arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

A Highly-Sensitive Colorimetric Assay Method for Antibody Array Based on an Tyramide Signal Amplification System

In this work, a highly-sensitive and cost-effective detection approach based on the integration of tyramide signal amplification with a silver enhancement method (SEM-TSA) has been developed successfully. To demonstrate the feasibility of this approach, human IgG as a model target protein was employed and its concentration was assayed based on colorimetric detection. The analytical parameters including the concentrations of detection antibody, streptavidin-horseradish peroxidase, biotinyl tyramide, and streptavidin-nanogold were systematically optimized. The quantitative analysis was performed and a dynamic range was obtained from 0.18 ng/mL to 39.1 ng/mL, while no detectable images could be observed when the silver enhancement method (SEM) without TSA was used. The detection limits were 0.18 ng/mL and 21 ng/mL for SEM-TSA and SEM, respectively. The results showed that sensitivity of the presented colorimetric assay significantly increased by two-orders of magnitude. In addition, this method has been successfully applied in analyzing normal human serum samples. The results suggested the colorimetric detection method based on TSA-SEM has promising potential applications in biomedical analysis and clinical diagnosis.

A visual chip-based coimmunoprecipitation technique for analysis of protein-protein interactions

Here we report a visual chip-based coimmunoprecipitation (vChip-coIP) platform for analysis of protein-protein interactions (PPIs) by combining advantages of an antibody microarray, traditional coIP, and a silver enhancement detection method. The chip was fabricated by spotting anti-Flag antibody on aldehyde-modified slides, and the resulting platform could assay immunoprecipitate from a small amount of crude cell lysates containing Flag-bait and Myc-prey. The interaction signals are visible using biotinylated anti-Myc antibody and colloidal gold-labeled streptavidin followed by a silver enhancement detection method. It is shown that vChip-coIP is a simple, cost-effective, and highly efficient platform for the comprehensive study of PPIs.

Pharmacokinetics, distribution and excretion of PNA in rat by liquid chromatography mass spectrometry method.

The pharmacokinetic profiles, tissue distribution and excretion patterns of PNA in healthy male and female Sprague-Dawley rats following a single intra-duodenum administration were investigated by previously established LC-MS method. Absorption was rapid in rats as evidenced by a short time to maximum concentration (Cmax) of 0.050 ± 0.021, 0.072 ± 0.017 and 0.067 ± 0.018 h at the 25, 50 and 100 mg/kg dose level, respectively. The Cmax were 754.9 ± 196.1, 1187.8 ± 642.1 and 2082.1 ± 1,278.5 ng/mL and the AUC0-4 were 71.8 ± 25.9, 135.2 ± 69.9 and 303.3 ± 198.3 µg/L h, which showed linear with the intra-duodenum administration range from 25-100 mg/kg. Tissues were collected at 4 time points (3, 10 min, 1 and 3 h) after dosing at 50 mg/kg. Compare to the concentrations of plasma and other tissues, the level was particularly high in the liver and brain during 3 h. Bile/urine and feces samples were collected before dosing and till 24/48 h post-dosing. The mean cumulative excretion of unchanged PNA in bile amounted to 0.021% of the dose up to 24 h. The mean recoveries of unchanged PNA were 0.0095% and 0.043% of the dose up to 48 h in urine and feces, respectively. There was no significant difference in PNA concentration observed between male and female rats during the experiments.