Immobilization Amount Research Articles

Preparation and characterization of micro-cell membrane chromatographic column with silica-based porous layer open tubular capillary as cellular membrane carrier.

Cell membrane chromatography (CMC) is a powerful tool to study membrane protein interactions and to screen active compounds extracted from natural products. Unfortunately, a large amount of cells are typically required for column preparation in order to carry out analyses in an efficient manner. Micro-CMC (mCMC) has recently been developed by using a silica capillary as a membrane carrier. However, a reduced retention of analytes is generally associated with mCMC mostly due to a low ligand (cellular membrane) capacity. To solve this common problem, in this work a silica-based porous layer open tubular (PLOT) capillary was fabricated and, to the best of our knowledge, for the first time applied to mCMC. The mCMC column was prepared by physical adsorption of rabbit red blood cell (rRBC) membranes onto the inner surface of the PLOT capillary. The effects of the PLOT capillaries fabricated by different feed compositions, on the immobilization amount of cellular membranes (represented by the fluorescence intensity of the capillary immobilized with fluorescein isothiocyanate isomer-labeled cellular membranes) and on the dynamic binding capacity (DBC) of verapamil (VP, a widely used calcium antagonist which specific interacts with L-type calcium channel proteins located on cellular membrane of rRBC) have been systematically investigated. The fluorescence intensity of the mCMC column when combined with the PLOT capillary was found to be more than five times higher than the intensity using a bare capillary. This intriguing result indicates that the PLOT capillary exhibits a higher cellular membrane capacity. The DBC of VP in the PLOT column was found to be more than nine times higher than that in the bare capillary. An rRBC/CMC column was also prepared for comparative studies. As a result, mCMC provides similar chromatographic retention factors and stability with common CMC; however, the cellular membrane consumption for mCMC was found to be more than 460 times lower than that for CMC. Graphical Abstract Comparision of mCMC chromatograms and SEM images between bare capillary and PLOT capillary.

Grafting l -valine on polyamidoamine dendrimer-modified magnetic microspheres for enantioselective adsorption of dansyl amino acids

Abstract Chiral selector-functionalized magnetic microspheres have significantly potential in the recognition and separation of chiral compounds. In this work, novel chiral selector-functionalized magnetic microspheres ( l -Val@G3.0-PMMPs) were achieved by grafting l -valine ( l -Val) on polyamidoamine (PAMAM) dendrimer-modified magnetic microspheres, which were confirmed by various characterization methods. The synthesized l -Val@G3.0-PMMPs had clearly three-layer structure, with a mean diameter of 590 nm and exhibited good dispersion and strong magnetic response. The immobilization amounts of l -Val determined by high performance liquid chromatography with mass spectrometry (HPLC–MS) increased with increasing PAMAM generation, and the results indicated that the PAMAM dendrimer bonded onto the surface of magnetic microspheres obviously improved the immobilization amounts of l -Val. The synthesized l -Val@G3.0-PMMPs as chiral magnetic selectors were used to conduct the enantioselective adsorption of dansyl amino acids. The chiral discrimination ability of the l -Val@G3.0-PMMPs was investigated through determining the enantiomeric excess of the l -Val@G3.0-PMMPs with capillary electrophoresis. Under the optimal amount of l -Val@G3.0-PMMPs and interaction times, the enantiomeric excess of dansyl amino acids adsorbed onto the surface of the l -Val@G3.0-PMMPs was calculated to be 20.1%. The results of enantiomeric excess indicated that the l -Val@G3.0-PMMPs selectively interacted with dansyl amino acid and exhibited excellent chiral discrimination ability. In the future research, the novel l -Val@G3.0-PMMPs are possible to be an efficient and suitable chiral selector for the enantioseparation of chiral compounds.

A novel and label-free biosensors for uracil-DNA glycosylase activity based on the electrochemical oxidation of guanine bases at the graphene modified electrode

Uracil-DNA glycosylase (UDG) as an important base excision repair enzymes is widely distributed in organism, and it plays a crucial role in sustaining the genome integrity. Therefore, it is significant to carry out the analysis of UDG activity. In this present work, a novel and label-free electrochemical sensing platform for the sensitive detection of uracil DNA glycosylase (UDG) activity has been developed. Herein, the graphene modified glassy carbon (GC) electrode was prepared. And two complementary DNA strands were hybridized to form dsDNA (P1P2). In the presence of UDG, the uracil bases in P1P2 were specifically hydrolyzed, inducing the unwinding of the DNA duplex, and accompanied by the release of P1. Thus, the released P1 was adsorbed onto the graphene/GC electrode surface via π–π stacking. By investigating the electrochemical behavior of P1 at the graphene/GC electrode, the electrochemical oxidation of guanine bases in P1 was obviously observed. Therefore, using the current responses of guanine base in P1 as a signal indicator, UDG activity can be simply determined with high sensitivity, and the detectable lowest concentration is 0.01U/mL. This present design does not need covalent attachment of redox indicator to DNA, preventing participation of redox labels in the background. Meanwhile, the proposed strategy for the assay of UDG activity also has a remarkable sensitivity due to the excellent properties of graphene, which could increase both the immobilization amount of released ssDNA and the conductivity of the sensing system. All these elucidate that this developed protocol may lay a potential foundation for the sensitive detection of UDG activity in clinical diagnosis.

Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications.

In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O2 plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods.

Preparation and Characterization of Polysulfone-graft-4′-aminobenzo-15-crown-5-ether for Lithium Isotope Separation

A novel polymer polysulfone (PSF)-graft-4′-aminobenzo-15-crown-5-ether (AB15C5) (PSF-g-AB15C5) for lithium isotope separation was prepared from PSF and AB15C5 as starting materials via nucleophilic substitution reaction. The chemical structure and properties of PSF-g-AB15C5 polymers were characterized by FT-IR, 1H NMR, XPS, and TGA. The polymers obtained were used for lithium isotope separation by solid–liquid extraction. The effects of the immobilization amount of crown ether grafting on PSF, the type of counteranion of lithium salt, and the kind of solvent on the single stage separation factor were explored. Results showed that the single stage separation factor was only 1.002 ± 0.002 for AB15C5 in the traditional liquid–liquid extraction system of H2O–LiCl/CHCl3–AB15C5, whereas the single stage separation factor increased from 1.003 ± 0.001 to 1.015 ± 0.002 with the increase of the immobilization amount of crown ether from 0.23 to 0.79 mmol g–1 on PSF-g-AB15C5 polymers in the solid–liquid extraction system of CH3OH–LiCl/PSF-g-AB15C5 polymers. The order of the single stage separation factor obtained in different lithium salts was LiI > LiBr > LiClO4 > LiCl. Further, the maximum value of single stage separation factor was 1.031 ± 0.002 for the extraction system of CH3NO2–LiCl/PSF-g-AB15C5 polymers. Moreover, 6Li and 7Li were enriched in the polymer phase and the solution phase, respectively.

Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation.

Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

BSA Adsorption and Immobilization onto Charged Monodisperse Polymer Nanoparticles

Controlling the behavior of adsorbed and immobilized proteins is essential for protein purification and a wide range of applications, including biosensors, biocatalysis and biomedical devices. In this study, monodisperse polymer particles were synthesized by soap free emulsion polymerization and the surface functional groups were directly introduced as a monomer or chemically modified with epoxy groups to enable protein immobilization through covalent bonding. Three kinds of surface charged particles having cationic, anionic, or both, groups were synthesized and characterized. Bovine serum albumin (BSA) was selected as a model protein to study the effect of pH of a buffer solution containing protein on the adsorption and immobilization amount. Protein adsorption was found to be strongly affected by pH and matching of the global protein and polymer particle charges, respectively. When pH was below the pI of either protein (pH 3.8), negatively charged polymer particles were found to adsorb a high amount of proteins. At maximum surface coverage of BSA on negatively charged polymer particles at pH 3.8 and after subsequent rinsing of the BSA-polymer particle complex with phosphate buffer (pH 7), ~50% of BSA was desorbed. Therefore, 50% of BSA was adsorbed by ionic interaction and the remaining fraction was immobilized covalently at pH 3.8. The remaining immobilized fraction was sufficient to completely shield the anionic charge of the polymer particles. Fluorescence spectroscopy suggests that at maximum immobilized amounts, the conformation of immobilized BSA appears to be the same as in aqueous solution.

Development of preparation of immobilized enzyme reactors in proteomics

As an important part in "bottom-up" strategy of proteomics, immobilized enzyme reactors have great significance in the development of fast and more efficient protein analytical method, owing to its advantages of high speed and enzymatic efficiency, good stability and activity, easy operation, and the possibility of hyphenating with multiple detection instruments. In this paper, the preparation methods of immobilized enzyme reactors and their applications in proteomic investigation are introduced, focusing on the nature of enzymes, the immobilization methods and the carrier materials used for immobilizing enzyme. In recent years, the investigations are focused on increasing the immobilization amounts of enzyme, keeping enzymatic activity, improving enzymatic efficiency and decreasing nonspecific adsorption. The investigation results showed that by using novel carriers such as nanomaterial and monolith, increasing of hydrophilicity of carrier and tandem hydrolysis with multiple enzymes can greatly improve the performance of immobilized enzyme reactors and increase protein identification efficiencies.

Ultrasensitive simultaneous detection of four biomarkers based on hybridization chain reaction and biotin–streptavidin signal amplification strategy

A sandwich-type electrochemical immunosensor based on redox probe tags identification technology for ultrasensitive simultaneous detection of four antigens was proposed. In this project, well-distributed graphene/gold (GR–Au) hybrid film was acquired through one-step codeposition in an electrode surface and served as the base substrate for immobilizing capture antibodies (Ab1). Hybridization chain reaction (HCR) and biotin/streptavidin (B/SA), combining with gold magnetic nanoparticles were applied to increase the immobilization amount of signal tags in detection antibody (Ab2) bioconjugates. To verify this strategy, four representative biomarkers, a-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen (CA)125 and prostate special antigen (PSA), were used as model analytes. The resulting immunosensor could simultaneously detect four antigens in single-pass differential pulse voltammetry (DPV) scan, and exhibited obviously improved senstivity compared to previous similar immunosensors, displayed good linear relationships in the ranges from 0.2 to 800pg/mL for AFP, 0.2 to 600pg/mL for CEA, 0.2 to 1000pg/mL for CA125, 0.2 to 800pg/mL for PSA and with detection limits of 62, 48, 77 and 60fg/mL, respectively.

Fast and highly-efficient removal of methylene blue from aqueous solution by poly(styrenesulfonic acid-co-maleic acid)-sodium-modified magnetic colloidal nanocrystal clusters

Magnetic colloidal nanocrystal clusters (MCNCs) modified with different amounts of poly(4-styrenesulfonic acid-co-maleic acid) sodium (PSSMA) have been prepared through simple one-step solvothermal method for removal of methylene blue (MB) from aqueous solution. The prepared MCNCs are characterized by Fourier transform infrared (FT-IR) spectra, scanning electron microscope (SEM), transmission electron microscope (TEM), thermogravimetric analysis (TGA), vibrating sample magnetometer (VSM), X-ray diffraction (XRD), nitrogen adsorption–desorption technique and dynamic light scattering (DLS). Moreover, effects of the solution pH, contact time, adsorbent dosage, ionic strength and initial dye concentration on MB adsorption onto the MCNCs are systematically investigated. The PSSMA-modified MCNCs show fast and highly-efficient MB removal capacity, which dramatically depends on the immobilization amounts of PSSMA, solution pH and adsorbent dosage. Their adsorption kinetics and isotherms exhibit that the kinetics and equilibrium adsorptions can be well-described by pseudo-second-order kinetic and Langmuir model, respectively. These magnetic nanocomposites, with high separation efficiency, low production cost and recyclable property, are promising as functional adsorbents for efficient removal of cationic organic pollutants from aqueous solution.

Highly ordered nanoporous carbon films with tunable pore diameters and their excellent sensing properties.

Ordered porous carbon films with tunable pore diameters, immobilized with glucose oxidase (GOD) have been fabricated and employed for the construction of a biosensor for glucose molecules. The as-prepared porous films have large specific surface areas and highly ordered porous structure with uniform pore sizes, which are critical for the immobilization of large amounts of GOD and support the promotion of heterogeneous electron transfer. The developed biosensors give enough room for the encapsulation of a high amount of GOD molecules and show excellent biosensing performance with a linear response to glucose concentration ranging from 0.5 to 9 mM and a detection limit of 1.5 μM. It is also demonstrated that the sensitivity of the biosensor can be easily tuned by modulating the pore size of carbon film as it dictates the amount of immobilization of GOD in the porous channels. The fabricated carbon-film-based biosensor has a good stability and a high reproducibility, which opens the gateway for the commercialization of this excellent technology.

Poly(2-vinyl-4,4-dimethylazlactone)-functionalized magnetic nanoparticles as carriers for enzyme immobilization and its application.

Fabrication of various efficient enzyme reactors has triggered increasing interests for its extensive applications in biological and clinical research. In this study, magnetic nanoparticles were functionalized by a biocompatible reactive polymer, poly(2-vinyl-4,4-dimethylazlactone), which was synthesized by reversible addition-fragmentation chain transfer polymerization. Then, the prepared polymer-modified magnetic nanoparticles were employed as favorable carriers for enzyme immobilization. l-Asparaginase was selected as the model enzyme to fabricate the enzyme reactor, and the prepared enzyme reactor exhibited high loading capacity of 318.0 μg mg(-1) magnetic nanoparticle. Interestingly, it has been observed that the enzymolysis efficiency increased slightly with the lengthened polymer chain, resulting from the increased immobilization amount of enzyme. Meanwhile, the immobilized enzyme could retain more than 95.7% activity after 10 repeated uses and maintain more than 72.6% activity after 10 weeks storage. Moreover, an extracorporeal shunt system was simulated to estimate the potential application capability of the prepared l-asparaginase reactor in acute lymphoblastic leukemia treatment.

Selective covalent immobilization of ferritin on alumina.

Selective and specific covalent immobilization and simultaneous suppression of nonspecific adsorption of the protein ferritin (FN) on the surfaces of polycrystalline α-alumina colloidal particles and single α-alumina crystals is demonstrated. FN immobilization is obtained by using a classical immobilization route and by combining either the organic silane 3-(triethoxysilyl)propylsuccinic anhydride (TESPSA) or (3-aminopropyl)triethoxysilane (APTES) with the zero-length cross linking system N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS). The combination of APTES or TESPSA with EDC/NHS leads to a stable FN binding via amide bonds. However, the authors demonstrate that the TESPSA-EDC/NHS system enables an overall higher amount of covalent immobilization and a simultaneous suppression of nonspecific FN adsorption. After TESPSA functionalization negatively charged carboxylic groups are formed and can at the same time both electrostatically repel the overall negatively charged FN proteins and react with EDC/NHS for FN covalent immobilization. Moreover, the authors show that by specifically controlling the FN concentration during the immobilization reaction, the molecule distribution and density of bound FN can be easily tuned. The approach presented enables to selectively immobilize FN at mild conditions on substrates with different geometries and is therefore relevant for the fabrication of biomimetic nanomaterials and two-dimensional FN arrays.

Synthesis and characterization of a new magnetic bromochromate hybrid nanomaterial with triethylamine surface modified iron oxide nanoparticles

Abstract We describe a novel method for the synthesis a new magnetic bromochromate hybrid nanomaterial, Fe 3 O 4 @SiO 2 @TEA@[CrO 3 Br], as a catalyst. The physical properties, morphology and magnetic investigations of magnetic bromochromate hybrid nanomaterials are identified by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM) techniques. Fourier transform infrared (FT-IR), elemental analysis, X-ray fluorescence (XRF), X-ray diffraction (XRD) were also used for structural identification. The quantity of chromium is approximately 0.38%, which confirms to the immobilization amount of [CrO 3 Br] − and is equal to 0.007 mol/100 g.

Synthesis, characterization and morphology of new magnetic fluorochromate hybrid nanomaterials with triethylamine surface modified iron oxide nanoparticles

Magnetic fluorochromate Fe3O4@SiO2@TEA@[CrO3F] was synthesized by ethylene glycol route. The structures, morphologies and properties of the catalyst were characterized by using X-ray diffraction (XRD), Fourier transform infrared (FT-IR), elemental analysis, X-ray fluorescence (XRF), solid state UV–vis, transmission electron microscopy (TEM), scanning electron microscope (SEM), N2 adsorption–desorption (BET and BJH) and vibrating sample magnetometer (VSM). The quantity of chromium is approximately 0.32%, which confirms the immobilization amount of [CrO3F]− which is equal with 0.006mol/100g.

Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase

Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%.Hydrolysis’ products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries.

Electrochemical immunosensor for detecting the spore wall protein of Nosema bombycis based on the amplification of hemin/G-quadruplex DNAzyme concatamers functionalized Pt@Pd nanowires

In this work, an ultrasensitive electrochemical immunosensor for detecting the Pebrine disease related spore wall protein of Nosema bombycis (SWP N.b) was fabricated based on the amplification of hemin/G-quadruplex functionalized Pt@Pd nanowires (Pt@PdNWs). The synthesized Pt@PdNWs possessed large surface area, which could effectively improve the immobilization amount of hemin/G-quadruplex DNAzyme concatamers produced via hybridization chain reaction (HCR). In the presence of SWP N.b, the hemin/G-quadruplex labeled Pt@PdNWs bioconjugations was captured on electrode surface and thus obtained electrochemical signal. After the addition of NADH into the electrolytic cell, hemin/G-quadruplex firstly acted as an NADH oxidase to locally produce H2O2 in the presence of dissolved O2. Then, the generated H2O2 would be quickly reduced via hemin/G-quadruplex as a horseradish peroxidase mimicking (HRP-mimicking) DNAzyme, which finally promoted the self-redox reaction of hemin/G-quadruplex and a greatly enhanced electrochemical signal was obtained. Furthermore, Pt@PdNWs with excellent electrocatalytic performance could also amplify electrochemical signal. With these amplification factors, the electrochemical immunosensor exhibited a wide linear range from 0.001ngmL−1 to 100ngmL−1 with a detection limit (LOD) of 0.24pgmL−1, providing a new promise for the diagnosis of Pebrine disease.

Dual Matrix-Based Immobilized Trypsin for Complementary Proteolytic Digestion and Fast Proteomics Analysis with Higher Protein Sequence Coverage

In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage.

An immobilized Thiobacillus thioparus biosensing system for monitoring sulfide hydrogen; optimized parameters in a bioreactor

A microbial biosensing system for detection of hydrogen sulfide has been developed by using immobilized Thiobacillus thioparus TK-m in poly vinyl alcohol matrix, together with a dissolved oxygen sensor. Parameters of immobilization (poly vinyl alcohol concentration and amount of wet cell) were optimized by using statistical software. The obtained values for concentration of poly vinyl alcohol and wet cell weight were 11.3% (w/v) and 45mg, respectively, where the response time of biosensor was 80s. Calibration of oxygen concentration based on hydrogen sulfide concentration was investigated between 1mg/L and 20mg/L. The effect of pH and temperature were investigated in specific range of experimental conditions as well. Some parameters including operational stability and detection limit were studied in detail for characterization of biosensing system. In order to determine the operational stability, bio-sensing system at optimized working conditions was used to distinguish viability of microorganisms in polymer beads in period of time.

Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification

In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)32+ ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)32+ binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)32+–amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)32+ interlaced into the formed amplicons within a fixed Ru(phen)32+ amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10fM for OTA was achieved. The aptasensor also has good reproducibility and stability.