Fluorescence Resonance Energy Transfer-Mediated Immunosensor Based on Design and Synthesis of the Substrate of Amp Cephalosporinase for Biosensing.

The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of ß-cyclodextrin (ß-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL-1 to 70 ng mL-1, and the limit of detection can reach 0.03 ng mL-1. The spiking recoveries of PCT in human serum samples range from 98.3% to 107%, with relative standard deviations ranging from 2.14% to 12.0%. This approach was applied to detect PCT in real patient serum samples, and the results are consistent with those obtained with a commercial ELISA kit.

Phytic acid functionalized Fe3O4 nanoparticles loaded with Ti(IV) ions for phosphopeptide enrichment in mass spectrometric analysis.

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a ß-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (ß-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10-10 mol L-1. This sorbent has a high absorption capacity (53.5 µg mg-1) and shows good recoveries (90%). As many as 2145 phosphopeptides were isolated from 500 µg tryptic digest of a rat liver lysate after enrichment by Ti-PA-MNPs. This is superior to that (1568 phosphopeptides) of commercial TiO2 kit. Graphical abstract Schematic presentation of fabrication for a novel modified magnetic nanomaterial (Ti-PA-MNPs) based on the chelation of phytic acid (PA) with Ti(IV) ions. Ti-PA-MNPs were successfully applied to enriching low abundance phosphopeptides from biosamples in mass spectrometric analysis.

Label-Free Sandwich Imaging Ellipsometry Immunosensor for Serological Detection of Procalcitonin.

Analysis of trace low molecular weight (LMW) proteins in serum using the label-free imaging ellipsometry (IE) immunosensor is still a challenge due to the lack of an effective signal amplification strategy and the serious nonspecific adsorption. Herein we have developed a sandwich strategy-mediated IE (SSIE) immunosensor to enable the immunodetection of LMW protein biomarkers in serum samples. We have first found that the weak binding affinity and the insufficient surface amount density of the ligand are two important factors which hinder the detection of LMW proteins in serum using the IE immunosensor. Then we have deduced that the sandwich strategy can amplify the detection signal of IE and avoid the nonspecific adsorption in serum. As a validation of the serological detection of LMW proteins, the SSIE immunosensor has been used to accomplish the quantitative detection of procalcitonin (PCT) in serum. Compared with other PCT analysis methodologies, the SSIE immunosensor enjoys the advantages of simplicity, rapidity, and sufficient sensitivity. Furthermore, we have proposed the criteria to predict the ability of the SSIE immunosensor for the detection of LMW protein biomarkers in serum, which can make the detection of LMW proteins smart and efficient.

Enzyme-Free Amplification Strategy for Biosensing Using Fe3+-Poly(glutamic acid) Coordination Chemistry.

In this work, we outline a signal amplification strategy using the coordination chemistry between Fe3+ and poly(glutamic acid) (PGA) for biosensing applications. The theoretical calculation based on density functional theory shows that PGA has a much higher binding affinity with Fe3+ than the other metal ions. Guided by this rationale, we prepare a PGA-mediated signal probe through conjugating PGA onto polystyrene (PS) nanoparticles to form a brushlike nanostructure for Fe3+ coordination. This PGA-PS brush (PPB) has a large loading capacity of Fe3+ with a number of 1.92 × 108 Fe atoms per nanoparticle that greatly amplifies the signals for assays in an enzyme-free way. Combined with ferrozine coloration-based readout, this PPB-mediated amplification is further applied for the enzyme-free immunoassay that shows an ultrahigh sensitivity for detection of microcystins-LR (12 pg/mL), a 5-fold enhancement compared with that of traditional enzyme-linked immunosorbent assay (ELISA) (60 pg/mL). In addition, the good stability, rapid response, and long shelf life make this enzyme-free amplification strategy a promising platform for point-of-care biosensing applications.

Controllable Assembly of Enzymes for Multiplexed Lab-on-a-Chip Bioassays with a Tunable Detection Range.

Multiplexed analysis of molecules with different concentrations requires assays with a tunable detection range. A strategy is outlined that uses click chemistry to assemble horseradish peroxidase in a controlled fashion to generate enzyme assemblies as probes for multiplexed bioassays. This controllable assembly of enzymes on detection antibodies allows for lab-on-a-chip immunoassays with a tunable detection range from pg mL-1 to µg mL-1 . Simultaneous, multiplexed bioassays of clinically relevant inflammatory biomarkers in serum are demonstrated in one lab-on-a-chip format, with a limit of detection of 0.47 pg mL-1 for interleukin-6, 2.6 pg mL-1 for procalcitonin, and 40 ng mL-1 for C-reactive protein. This controlled assembly technique provides a multiplexed platform for simultaneous and quantitative analyses of both low-abundance and high-abundance biomarkers with a broad detection range, which holds great promise as a point-of-care platform for biomedical diagnostics.

Magnetic Lateral Flow Strip for the Detection of Cocaine in Urine by Naked Eyes and Smart Phone Camera.

Magnetic lateral flow strip (MLFS) based on magnetic bead (MB) and smart phone camera has been developed for quantitative detection of cocaine (CC) in urine samples. CC and CC-bovine serum albumin (CC-BSA) could competitively react with MB-antibody (MB-Ab) of CC on the surface of test line of MLFS. The color of MB-Ab conjugate on the test line relates to the concentration of target in the competition immunoassay format, which can be used as a visual signal. Furthermore, the color density of the MB-Ab conjugate can be transferred into digital signal (gray value) by a smart phone, which can be used as a quantitative signal. The linear detection range for CC is 5-500 ng/mL and the relative standard deviations are under 10%. The visual limit of detection was 5 ng/mL and the whole analysis time was within 10 min. The MLFS has been successfully employed for the detection of CC in urine samples without sample pre-treatment and the result is also agreed to that of enzyme-linked immunosorbent assay (ELISA). With the popularization of smart phone cameras, the MLFS has large potential in the detection of drug residues in virtue of its stability, speediness, and low-cost.

[Development of Fluorescence Resonance Energy Transfer Sensor for Determination of Adenosine Monophosphate in Biological Drug].

The biological drug of the calf-blood dialysate has various pharmacological effects. It can promote the oxygen and glucose uptake for the hypoxia cells, and has beneficial effects on the malfunction of the blood circulation and trophic disturbances in the brain, and the impairment of peripheral blood circulation. Furthermore, it is favorable to wound healing and can regulate the central nervous system. Adenosine monophosphate (AMP) is a main active ingredient of the biological drug. In this report, a fluorescence resonance energy transfer (FRET) sensor has been developed with ß-CD-capped ZnS QDs as energy donor and 3-hydroxyflavone (3-HF) as energy acceptor. The results showed that AMP can lead to the fluorescence quenching of the FRET sensor at 526 nm, and the Stern-Volmer curve between the fluorescence quenching and the concentrations of AMP present a satisfactory linearity with the correlation coefficient of 0.996. The developed sensor has successfully applied for determination of the AMP in the biological drug.

[Interaction between three isoflavones and different isomers of human serum albumin].

The interaction mechanisms between isoflavones (Genistein, 3', 4', 7-trihydroxyisoflavone and Biochanin A) and different isomers of human serum albumin (HSA) were investigated by fluorescence spectroscopy. Various parameters (quenching rate constants, binding constants and number of binding sites) of isoflavones-human serum albumin complexes were calculated. The results showed that the isoflavones have only one binding site on human serum albumin, located at the Site I, and the binding constants were between 0.17 x 10(5) and 1.20 x 10(5) L x mol(-1). Fluorescence enhancement experiments showed that the fluorescence intensities of the drugs significantly increased after interacting with HSA, indicating that the combination of drug and HSA had occurred. The binding mechanisms between three isoflavones and HSA were discussed.

Development of dual-enhancer biocatalyst with photothermal property for the degradation of cephalosporin.

The abuse of cephalosporins poses a serious threat to human health and the ecological environment. In this work, cephalosporinase (AmpC enzyme) and Prussian blue (PB) crystals were encapsulated into ZIF-8 metal-organic frameworks (MOFs), and a photothermal AmpC/PB@ZIF-8 MOFs (APZ) nanocatalyst was prepared for the catalytic degradation of cephalosporin. The temperature of the APZ catalytic degradation system can be regulated by irradiation with near infrared light due to the photothermal effect of PB, and then, the activity of the APZ biocatalyst is significantly enhanced. Thereby, the degradation efficiency of cefuroxime can reach to 96%, and the degradation kinetic rate of cefuroxime augmented 4.5-fold comparing with that catalyzed by free enzyme. Moreover, encapsulation of the enzyme and PB can increase the affinity and charge transfer efficiency between APZ and substrate molecules, which can also improve the degradation efficiency of cephalosporins. Catalytic degradation pathways for three generations of cephalosporins were proposed based on their degradation products. The dual-enhancer biocatalyst based on the photothermal effect and immobilization of the PB and enzyme can significantly enhance the activity and stability of the enzyme, and it can also be recycled. Therefore, the biocatalyst has potential applications for the effective degradation of cephalosporins in the environment.

Development of dual-ligand titanium (IV) hydrophilic network sorbent for highly selective enrichment of phosphopeptides.

A hydrophilic metal-organic network based on Ti4+ and dual natural ligand, tannic acid (TA) and phytic acid (PA), has been developed to enrich phosphopeptides from complex bio-samples prior to liquid chromatography-mass spectrometric analysis. Due to the strong chelation ability of TA and PA, abundant Ti4+ can be immobilized in the material, forming hydrophilic network by one-step coordination-driven self-assembly approach. The sorbent, TA-Ti-PA@Fe3O4, exhibited satisfactory selectivity for the phosphopeptides in the tryptic digest of ß-casein, and can eliminate the interference components in 1000-fold excess of bovine serum albumin. The adsorption capacity of the sorbents for phosphopeptides was up to 35.2 mg g-1 and the adsorbing equilibrium can be reached in 5 min. The adsorbing mechanism has been investigated and the results indicated that the Ti4+ in forms of [Ti(f-TA)(H2O)4]2+, [Ti(f-PA)(H2O)4]2+ and Ti(f-PA)2(H2O)2 may play an important role in the adsorption process. The sorbent of the TA-Ti-PA@Fe3O4 has been applied to enrichment of the phosphopeptides in tryptic digest of rat liver lysate, and 3408 phosphopeptides have been identified, while the numbers of the identified phosphopeptides were 2730 and 1217 when the sample was enriched by the commercial TiO2 and Fe3+-IMAC kit, respectively. This work provides a strategy to enrich phosphopeptides from complex samples and shows great potential application in phosphoproteome research.

Biodegradation pathway of penicillins by ß-lactamase encapsulated in metal-organic frameworks.

The pollution caused by the abuse of antibiotics has posed a serious threat to the ecological environment and human health, so development of effective strategies for degradation and disposal of antibiotic residues is urgently needed. In this work, penicillinase, a kind of ß-lactamase, was immobilized into zeolitic imidazolate framework-8 (ZIF-8) by self-assembly method and the catalytic performance of the ß-lactamase@ZIF-8 porous materials for degradation of penicillins has been investigated by high performance liquid chromatography coupled with mass spectrometry. The results illustrated that the catalytic activity of the encapsulated enzyme was significantly enhanced comparing with that of free enzyme. Meanwhile, the ß-lactamase@ZIF-8 exhibited excellent stability under denaturing conditions including high temperature, organic solvent and the enzyme inhibitor. The catalytic degradation mechanism of the ß-lactamase@ZIF-8 for penicillins has been probed and verified, and it has been found that the Zn (II) ion on ZIF-8 frameworks could form the complex with the target molecule, which weakened the bond of the four-membered ß-lactam ring in the penicillin molecule, and thus enhanced the degradation efficiency of the enzyme. This work provided a promising strategy for eliminating the penicillin residues in water environment.

Sorption induced structural deformation of sodium hexa-titanate nanofibers and their ability to selectively trap radioactive Ra(II) ions from water.

Sodium hexa-titanate (Na(2)Ti(6)O(13)) nanofibers, which have microporous tunnels, were prepared by heating sodium tri-titanate nanofibers with a layered structure at 573 K. The void section of the tunnels consist of eight linked TiO(6) octahedra, having a quasi-rectangular shape and the sodium ions located in these tunnel micropores are exchangeable. The exchange of these sodium ions with divalent cations, such as Sr(2+) and Ba(2+) ions, induces moderate structural deformation of the tunnels due to the stronger electrostatic interactions between di-valent ions Sr(2+) and Ba(2+) and the solid substrate. However, as the size of Ba(2+) ions (0.270 nm) is larger than the minimum width (0.240 nm) of the tunnel, the deformation can lock the Ba(2+) ions in the nanofibers, whereas Sr(2+) ions (0.224 nm) are smaller than the minimum width so the fibers can release the Sr(2+) ions exchanged into the channels instead. Therefore, the hexa-titanate (Na(2)Ti(6)O(13)) nanofibers display selectivity in trapping large divalent cations, since the deformed tunnels cannot trap smaller cations within the fibers. The fibers can be used to selectively remove radioactive Ra(2+) ions, which have a similar size and ion-exchange ability to Ba(2+) ions, from wastewater for safe disposal.

Differentiation and dating of red ink entries of seals on documents by HPLC and GC/MS.

A novel approach for differentiation and dating of red ink entries of seals on documents was developed based on ion-pairing HPLC (IP-HPLC) and GC/MS. Sixty-nine red ink pastes of seals were collected and the chromatographic conditions for separation of the dye components by IP-HPLC and the volatile additives by GC/MS in the ink entries were optimized. According to the dye components and additives, the ink entries were classified by HPLC with a multi-wavelength UV detector. The volatile components of the inks were identified by GC/MS and the classification of the ink entries was also investigated based on these volatile additives. The results showed that most of the ink entries of the seals can be differentiated by combining HPLC with a multi-wavelength detector and GC/MS methods. The degradation of the standard dye mixtures and the compositional changes of the ink entries of seals were investigated in light or natural aging conditions. The results indicated that the dye components decomposed in light or natural storage conditions, while the rates of the degradation depended on the structures of the dye components, the aging conditions, even the additives of the ink pastes. The results also showed that there existed good relationships between the compositional changes of the ink entries and the aging time, which can provide scientific evidences and valuable clues for dating of the ink entries.

[Spectroscopic investigation on the interaction of protocatechuic acid and veratric acid with biomacromolecules].

Protocatechuic acid (P) and veratric acid (V) are phenolic acidic compounds and have a wide biological and pharmaceutical activities, and their interaction with biomacromolecule has been a hot topic. The interaction mechanism of P and V with fsDNA was investigated by fluorescence and UV absorption spectroscopic methods. The UV results showed that P and V have three strong absorption bands at 190-230 nm (K band), 230-270 nm (B band) and 270-310 nm (R band) respectively. When the excitation wavelength was 280 nm, the fluorescence emission bands of P and V were at 338 and 334 nm, respectively, while the fluorescence emission band of DNA was very weak and had little influence on those of the P and V. The fluorescence intensities of the P and V were strongly quenched after interacting with fsDNA, and their Stern-Volmer quenching rate constants were 1.03 x 10(12) and 0.61 x 10(12) L x mol(-1) x s(-1), respectively. It was illustrated that the fluorescence quenching was mainly static and the complex was formed between the drug and fsDNA. When the concentration of DNA was high, their Stern-Volmer curves were not linear, and it was indicated that the quenching mechanism was complex and may contain dynamic quenching process. Their binding constants were calculated based on the static fluorescence quenching, with K(fsDNA/P) = 6.22 x 10(6) L x mol(-1) and K(fsDNA/v) = 1.57 x 10(4) L x mol(-1). The investigation showed that the molecular ratio of V-fsDNA was 1 : 1, while that of P-fsDNA was 1: 2. It was demonstrated that the protocatechuic acid can bind with two bases of fsDNA, which was related to the two hydroxyl groups on the drug molecule. The results showed that the structure of P and V greatly influenced their binding mode with DNA molecules.

Application of matrix solid-phase dispersion and high-performance liquid chromatography for determination of sulfonamides in honey.

A novel method for simultaneous determination of 8 sulfonamide residues (sulfathiazole, sulfapyridine, sulfadiazine, sulfamerazine, sulfamonome-thoxine, sulfachloropyridazine, sulfamethoxazole, and sulfadimethoxine) in honey samples by high-performance liquid chromatography (HPLC) has been developed on the basis of precolumn derivatization with 9-fluorenylmethyl-chloroformate (FMOC-Cl). Sulfonamide residues in honey samples were extracted and purified by matrix solid-phase dispersion with C18 as the solid support. The residues were derivatized by FMOC-CI, and the FMOC-sulfonamide derivatives were further purified by solid-phase extraction with silica gel as the solid support prior to HPLC analysis. The average recoveries for most sulfonamide compounds at different spiking levels (from 10 to 250 microg/kg) were > 70% with relative standard deviations < 16%, and their limits of detection were 4.0 microg/kg. The established analytical method has high sensitivity and repeatability and can be applicable for determining the sulfonamide residues in various honey matrixes.

Development of graphite carbon nitride based fluorescent immune sensor for detection of alpha fetoprotein.

A novel fluorescent immunosensor for determination of alpha fetoprotein (AFP) in serum samples has been developed based on the nano graphite carbon nitride (g-C3N4) as fluorophore and immunomagnetic beads (MBs) as separation material. The bulk g-C3N4 was obtained by thermal polymerization of melamine, and then carboxylated and exfoliated to acquire the carboxylated nano g-C3N4 (c-n-g-C3N4), which has been characterized and the results showed that it had excellent fluorescent properties. The antibodies of AFP (Ab1, Ab2) were conjugated to the MBs and the c-n-g-C3N4, respectively. In assay of AFP detection, the magnetic part of the immunosensor, MBs-Ab1, would form the sandwich type complex with the signal part of the sensor, c-n-g-C3N4-Ab2. The developed immunosensor could simplify the process of separation due to the MBs. The results illustrated that proposed approach held a good linearity between the fluorescence intensity of the sensor and the AFP concentration ranging from 5-600ng/mL with the limit of detection as low as 0.43ng/mL, and its spiking recoveries ranged from 98.2% to 105.9% with RSD from 2.1% to 3.5%. The fabricated fluorescent immunosensor possesses the merits of good sensitivity, excellent selectivity, high biocompatibility and low cost, and the results provide a novel clue to develop immunosensor for determination of the biomarkers in complex matrices.

Phosphate-imprinted magnetic nanoparticles using phenylphosphonic acid as a template for excellent recognition of tyrosine phosphopeptides.

The tyrosine phosphorylation of proteins and peptides plays a vital role in cell signal transduction pathways, and it is very important to assay them for understanding their action mechanism. Due to the low levels of the tyrosine phosphopeptides (pTyr) in cells, it is a challenge to enrich them with traditional sorbents, therefore, development of specific and selective sorbents is urgent and necessary. In this work, the phosphate-imprinted magnetic nanoparticles (PMNPs) to enrich the pTyr with high efficiency and selectivity have been fabricated using the phenylphosphonic acid as a template for the "epitope" of pTyr. The magnetic nanoparticles have been functionalized with TiO2 and then the imprinting silica shells have been coated on the surface of the functional core to obtain the PMNPs sorbents. The PMNPs can obviously shorten the enrichment time and improve the adsorption efficiency for pTyr, and the epitope imprinting films provide an excellent selective recognition ability to target. The recognition capability of PMNPs for pTyr is 90.3 µg/mg and the imprinting factor of the sorbents can reach 24.4. The results indicate that the PMNPs can enrich the pTyr from the tryptic digest of ß-casein samples with high specificity, and the spiking recoveries of the pTyr range from 85.1% to 93.8% with the RSD from 0.04 to 3.73. With the high adsorption capacity, rapid separation, excellent specificity and recyclability, the PMNPs sorbents show great potential for analysis of the phosphorylation of peptides in biological and medical fields.

Characterization of the interaction between human serum albumin and morin.

The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13+/-0.11x10(-5) L Mol(-1) in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug-protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA-morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein alpha-helix and beta-sheet structures.

Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids.

KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90±0.16)×106Lmol-1, (0.93±0.21)×106Lmol-1 and (1.16±0.45)×106Lmol-1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

Poly-L-lysine brushes on magnetic nanoparticles for ultrasensitive detection of Escherichia coli O157: H7.

We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19µg/mg and 16µg/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816µg/mg, protein µg per mg of MPLLBs) and Ab (387µg/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8cfu/mL) and shortened the analysis time (within 2h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples.