High-efficient separation of deoxyribonucleic acid from pathogenic bacteria by hedgehog-inspired magnetic nanoparticles microextraction.

Efficient separation of deoxyribonucleic acid (DNA) through magnetic nanoparticles (MN) is a widely used biotechnology. Hedgehog-inspired MNs (HMN) possess a high-surface-area due to the distinct burr-like structure of hedgehog, but there is no report about the usage of HMN for DNA extraction. Herein, to improve the selection of MN and illustrate the performance of HMN for DNA separation, HMN and silica-coated Fe3O4 nanoparticles (Fe3O4@SiO2) were fabricated and compared for the high-efficient separation of pathogenic bacteria of DNA. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are typical Gram-negative and Gram-positive bacteria and are selected as model pathogenic bacteria. To enhance the extraction efficiency of two kinds of MNs, various parameters, including pretreatment, lysis, binding and elution conditions, have been optimized in detail. In most separation experiments, the DNA yield of HMN was higher than that of Fe3O4@SiO2. Therefore, a HMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated and used to detect pathogenic bacteria in real samples. Interestingly, the HMN-based MSPE combined qPCR strategy exhibited high sensitivity with a limit of detection of 2.0 × 101 CFU mL-1 for E. coli and 4.0 × 101 CFU mL-1 for S. aureus in orange juice, and 2.8 × 102 CFU mL-1 for E. coli and 1.1 × 102 CFU mL-1 for S. aureus in milk, respectively. The performance of the proposed strategy was significantly better than that of commercial kit. This work could prove that the novel HMN could be applicable for the efficient separation of DNA from complex biological samples.

Synthesis of micron-sized magnetic agarose beads chelated with nickel ions towards the affinity-based separation of histidine-tagged/rich proteins.

Developing high-performance magnetic particles for the effective separation and purification of target proteins has become an important topic in the area of biomedical research. In this work, a simple and novel strategy was proposed for fabricating magnetic Fe3O4@agarose-iminodiacetic acid-Ni microspheres (MAIN), which can efficiently and selectively isolate histidine-tagged/rich proteins (His-proteins). Based on the thermoreversible sol-gel transition of agarose, basic magnetic agarose microspheres were prepared through the inverse emulsion method, in which the emulsion contained agarose and amine-modified Fe3O4 nanoparticles. The size of the emulsion was controlled by the emulsification of a high-speed shear machine, which improved the specific surface area of MAIN. Subsequently, the amine-modified Fe3O4 nanoparticles were covalently crosslinked with agarose through epichlorohydrin, which could avoid leakage of the magnetic source during use and increase the stability of MAIN. The microsized MAIN exhibited a clearly visible spherical core-shell structure with a diameter range from 3.4 µm to 9.8 µm, and excellent suspension ability in aqueous solution. The maximum adsorption capacity of MAIN for histidine-rich bovine hemoglobin was 1069.2 mg g-1 at 35 °C, which was higher than those of commercialized and most reported magnetic agarose microspheres/nanoparticles. The MAIN showed excellent adsorption ability and selectivity toward His-proteins in a mixture of histidine-rich bovine serum albumin (BSA) and histidine-poor lysozyme (LYZ). When the amount of LYZ was 5-fold higher than that of BSA, the recovery of BSA reached 75.0%. To prove its practicability, MAIN was successfully employed for the enrichment of histidine-tagged RSV-F0 from the cell culture medium supernatant. According to the optimized conditions, MAIN could enrich approximately 0.1 mg of RSV-F0 from 1 mL of complex biological sample. Therefore, we believe that the novel MAIN could be applicable for efficient separation and purification of His-proteins from complex biological systems.

Functionalized pyrite nanozyme probe and imprinted polymer modified with hydrophilic layer for rapid colorimetric analysis of glycoprotein in serum.

The biological molecules used in the sandwich detection method have problems such as complex extraction processes, high costs, and uneven quality. Therefore we integrated glycoprotein molecularly controllable-oriented surface imprinted magnetic nanoparticles (GMC-OSIMN) and boric acid functionalized pyrite nanozyme probe (BPNP) to replace the traditional antibody and horseradish peroxidase for sensitive detection of glycoproteins through sandwich detection. In this work, a novel nanozyme functionalized with boric acid was used to label glycoproteins that were captured by GMC-OSIMN. The substrate in the working solution catalyzed by the nanozyme labeled on the protein underwent visible color changes to the naked eye, and the generated signal can be quantitatively detected by a spectrophotometer, and the best color development conditions of the novel nanozyme under the influence of many factors were determined through multi-dimensional investigation. The optimum conditions of sandwich are optimized with ovalbumin (OVA), and it was extended to the detection of transferrin (TRF) and alkaline phosphatase (ALP) in the application. The detection range for TRF was 2.0 × 10-1-1.0 × 104 ng mL-1 with a detection limit of 1.32 × 10-1 ng mL-1, The detection range for ALP was 2.0 × 10-3-1.0 × 102 U L-1 with the detection limit of 1.76 × 10-3 U L-1. This method was subsequently used to detect TRF and ALP levels in 16 liver cancer patients, and the standard deviation of the test results of each patient was less than 5.7%.

Linking peptide-oriented surface imprinting magnetic nanoparticle with carbon nanotube-based fluorescence signal output device for ultrasensitive detection of glycoprotein.

Determination of trace glycoprotein has important guiding significance in clinical diagnosis and is usually achieved by immunoaffinity. However, immunoaffinity possesses inherent drawbacks, such as poor probability of high-quality antibodies, instability of biological reagents, and harmfulness of chemical labels to the body. Herein, we propose an innovative method of peptide-oriented surface imprinting to fabricate artificial antibody for recognition of glycoprotein. By integrating peptide-oriented surface imprinting and PEGylation, an innovative hydrophilic peptide-oriented surface imprinting magnetic nanoparticle (HPIMN) was successfully fabricated with human epidermal growth factor receptor-2 (HER2) as a model glycoprotein template. In addition, we further prepared a novel boronic acid-modified/fluorescein isothiocyanate-loaded/polyethylene glycol-covered carbon nanotube (BFPCN) as fluorescence signal output device, which was loaded with numerous fluorescent molecules could specifically label the cis-diol of glycoprotein at physiological pH via boronate-affinity interaction. To prove the practicability, we proposed a HPIMN-BFPCN strategy, in which the HPIMN first selectively captured the HER2 due to the molecular imprinted recognition and then the BFPCN specific labeled the exposed cis-diol of HER2 based on the boronate-affinity reaction. The HPIMN-BFPCN strategy exhibited ultrahigh sensitivity with limit of detection of 14 fg mL-1 and was successfully used in the determination of HER2 in spiked sample with recovery and relative standard deviation in the range of 99.0%-103.0% and 3.1%-5.6%, respectively. Therefore, we believe that the novel peptide-oriented surface imprinting has great potential to become an universal strategy for fabrication of recognition units for other protein biomarkers, and the synergy sandwich assay could become a powerful tool in prognosis evaluation and clinical diagnosis of glycoprotein-related diseases.

Fabrication of self-healing magnetic nanoreceptors for glycoprotein via integrating boronate-affinity-oriented and sequential surface imprinting.

Molecularly imprinted polymers (MIPs) as artificial receptors have been widely applied in various fields. However, construction of MIPs for precise recognition of glycoprotein still remains a rather challenging task. To overcome this problem, we first fabricated boronate-affinity-oriented and sequential-surface imprinting magnetic nanoparticles (BSIMN) through integrating the boronate-affinity-oriented and sequential surface imprinting. The boronate-affinity-oriented immobilization of glycoprotein template endowed the BSIMN with homogeneous imprinted cavities. In addition, the polydopamine (PDA) imprinted layer was introduced by self-polymerization of dopamine in the first imprinting process, and then the phenylboronic acid (PBA) imprinted layer was introduced by boronate-affinity interaction in the second imprinting process. Surprisingly, the PBA imprinted layer possessed self-healing property due to the presence of pH-dependent boronate-affinity interaction between two imprinted layers. Therefore, the fabricated BSIMN exhibited excellent selectivity toward glycoprotein templates. To quantitatively detect glycoproteins in biological samples, the BSIMN was linked with hydrophilic rhodamine B-loaded/boronic acid-modified graphene oxide (HRBGO), which could selectively label glycoprotein and output amplified signal. In quantitative analysis, target glycoproteins were firstly captured by BSIMN and then specifically labeled by HRBGO; subsequently, the releasing agent was added to release numerous rhodamine B from HRBGO, and the corresponding fluorescence signal was used for further quantitative analysis. The proposed strategy showed ultrahigh sensitivity for ovalbumin, carcinoembryonic antigen and alpha fetoprotein with limit of detection of 4.5 fg mL-1, 3.6 fg mL-1 and 4.2 fg mL-1, respectively, and was successfully applied in determination of these glycoproteins in serum samples.

Construction of PEGylated boronate-affinity-oriented imprinting magnetic nanoparticles for ultrasensitive detection of ellagic acid from beverages.

Molecularly imprinted polymers (MIPs) can exhibit antibody-level affinity for target molecules. However, the nonspecific adsorption of non-imprinted regions for non-target molecules limits the application range of MIPs. Herein, we fabricated PEGylated boronate-affinity-oriented ellagic acid-imprinting magnetic nanoparticles (PBEMN), which first integrated boronate-affinity-oriented surface imprinting and sequential PEGylation for small molecule-imprinted MIPs. The resultant PBEMN possess higher adsorption capacity and faster adsorption rate for template ellagic acid (EA) molecules than the non-PEGylated control. To prove the excellent performance, the PBEMN were linked with hydrophilic boronic acid-modified/fluorescein isothiocyanate-loaded graphene oxide (BFGO), because BFGO could selectively label cis-diol-containing substances by boronate-affinity and output ultrasensitive fluorescent signals. Based on a dual boronate-affinity synergy, the PBEMN first selectively captured EA molecules by boronate-affinity-oriented molecular imprinted recognition, and then the EA molecules were further labeled with BFGO through boronate-affinity. The PBEMN linked BFGO (PBPF) strategy provided ultrahigh sensitivity for EA molecules with a limit of detection of 39.1 fg mL-1, resulting from the low nonspecific adsorption of PBEMN and the ultrasensitive fluorescence signal of BFGO. Lastly, the PBPF strategy was successfully employed in the determination of EA concentration in a spiked beverage sample with recovery and relative standard deviation in the range of 96.5 to 104.2% and 3.8 to 5.1%, respectively. This work demonstrates that the integration of boronate-affinity-oriented surface imprinting and sequential PEGylation may be a universal tool for improving the performance of MIPs.

Poly(caffeic acid)-coated molecularly imprinted magnetic nanoparticles for specific and ultrasensitive detection of glycoprotein.

Molecularly imprinted polymers (MIPs) are artificial chemical receptors, and can recognize template molecules with a high selectivity and affinity. As "antibody mimics", MIPs have been widely studied in various fields. However, the general applicability of MIPs is limited by the type of functional monomers. Herein, we developed caffeic acid (CA, a natural polyphenol) as novel a functional monomer. An innovative poly(caffeic acid)-coated molecularly imprinted magnetic nanoparticles (PCA-MIMN) with transferrin (TRF) as a model glycoprotein template was fabricated by autoxidation of CA with hexamethylenediamine (HMDA) in an aerobic environment as imprinted layer. The successful fabrication of PCA-MIMN was proved in detail by diversified characterization. The PCA-MIMN exhibited not only outstanding binding affinity and specificity for target glycoprotein, but also excellent hydrophilicity due to the externally generous hydrophilic groups. To evaluate the preeminent performance, the PCA-MIMN was linked with pH-triggered allochroic-graphene oxide (AGO), which was used for determination of TRF in real samples. The proposed PCA-MIMN linked AGO strategy exhibited ultrahigh sensitivity with limit of detection of 0.38 pg mL-1 for TRF. Finally, the proposed strategy was successfully applied in determination of TRF in spiked human serum sample with recovery and relative standard deviation in the range of 97.2%-103.9% and 4.6%-5.8%, respectively. This work demonstrates that the "autoxidation of CA with HMDA" may be a universal tool for synthesis of highly specific MIPs, and the type of functional monomers will increase exponentially due to the presence of numerous polyphenols in nature.

PEGylation of boronate-affinity-oriented surface imprinting magnetic nanoparticles with improved performance.

High specific selectivity is the continuous goal of exploit glycoprotein-imprinted materials. Boronate-affinity-oriented surface imprinting can limit the heterogeneity of imprinted cavities, and PEGylation can reduce the nonspecific adsorption of imprinted materials towards non-target molecules. However, there are no reports on the integration of the above two advantages. Herein, we first integrated the boronate-affinity-oriented surface imprinting and PEGylation, and fabricated PEGylated boronate-affinity-oriented surface imprinting magnetic nanoparticles (PBSIMN) with horseradish peroxidase (HRP) as a model glycoprotein template. The successful synthesis of PBSIMN was demonstrated in detail by various characterization. Compared with non-PEGylated control, the PBSIMN showed greater adsorption capacity for HRP, and faster adsorption rate. To evaluate the improved performance, the PBSIMN was linked with hydrophilic boronic acid-modified/fluorescein isothiocyanate-loaded graphene oxide (BFGO), and used for the detection of HRP in real samples. Because PEGylation led to decrease of non-specific binding on PBSIMN, the proposed strategy provided ultrahigh sensitivity with limit of detection of 6.0 fg mL-1 for HRP, which were an order of magnitude lower than the non-PEGylated counterparts. When spiked with 0.05, 0.5 and 5.0 mg mL-1, recoveries of HRP were in the range of 97.4%-101.8% with relative standard deviation (RSD) no more than 5.4% for mouse serum, and between 98.2% and 103.2% with RSD no more than 5.0% human serum. This work indicates that the boronate-affinity-oriented surface imprinting and PEGylation can improve the performance of imprinted materials.