Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system.

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.

The discovery of lactoferrin dual aptamers through surface plasmon resonance imaging combined with a bioinformation analysis.

An analytical method for screening aptamers for different recognition sites in lactoferrin (Lac) molecules has been developed based on Surface Plasmon Resonance imaging (SPRi), combined with the cluster classification calculation of a quasi-aptamer library strategy and molecular docking simulation analysis. Using the software simulation, a homology analysis was performed on the selected quasi-aptamer sequences, which could be divided into 8 different families. Based on the principle of biomolecular recognition, a label-free, high-throughput dual immune site screening method was established, in which the nucleic acid aptamers of recognizing ability for lactoferrin molecules were fixed onto the surface of the SPRi sensor chip and could bind to the lactoferrin molecules. Then, the aptamer candidates to be paired were introduced, and the recognition event of the second immune site was judged by observing the binding signal of SPRi. The paired SPRi signal was generated only when the site identified by the second nucleic acid molecule was different from the first immune site. Based on this principle, a pair of Lac nucleic acid aptamers (Lac-8 and Lac-25) was finally screened and confirmed using computerized simulation, and has been employed to assay Lac in milk by ELONA (Enzyme-Linked Oligonucleotide Assay).

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.

Simultaneous detection of α-Lactoalbumin, ß-Lactoglobulin and Lactoferrin in milk by Visualized Microarray.

BACKGROUND: α-Lactalbumin (a-LA), ß-lactoglobulin (ß-LG) and lactoferrin (LF) are of high nutritional value which have made ingredients of choice in the formulation of modern foods and beverages. There remains an urgent need to develop novel biosensing methods for quantification featuring reduced cost, improved sensitivity, selectivity and more rapid response, especially for simultaneous detection of multiple whey proteins. RESULTS: A novel visualized microarray method was developed for the determination of a-LA, ß-LG and LF in milk samples without the need for complex or time-consuming pre-treatment steps. The measurement principle was based on the competitive immunological reaction and silver enhancement technique. In this case, a visible array dots as the detectable signals were further amplified and developed by the silver enhancement reagents. The microarray could be assayed by the microarray scanner. The detection limits (S/N = 3) were estimated to be 40 ng/mL (α-LA), 50 ng/mL (ß-LG), 30 ng/mL (LF) (n = 6). CONCLUSIONS: The method could be used to simultaneously analyze the whey protein contents of various raw milk samples and ultra-high temperature treated (UHT) milk samples including skimmed milk and high calcium milk. The analytical results were in good agreement with that of the high performance liquid chromatography. The presented visualized microarray has showed its advantages such as high-throughput, specificity, sensitivity and cost-effective for analysis of various milk samples.

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.

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.

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.

A multiplexed screening method for agonists and antagonists of the estrogen receptor protein.

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC(50) values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10(-7) M versus 1.74~4.23 × 10(-7) M and 7.58 × 10(-8) M versus 0.89~5.84 × 10(-8) M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.

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.