Promoting the healing of infected diabetic wound by nanozyme-containing hydrogel with anti-bacterial inflammation suppressing, ROS-scavenging and oxygen-generating properties.

Bacterial infections already pose a significant threat to skin wounds, especially in diabetic patients who have difficulty healing wounds. However, wound or bacterial infections are known to produce excess reactive oxygen species (ROS), and hypoxia may further hinder wound healing and the development of chronic wounds. In this study, a multifunctional hydrogel for ROS scavenging and bacterial inhibition was developed by cross-linking polyvinyl alcohol (PVA) and sodium alginate (SA) with graphene oxide (GO) loaded with silver-platinum hybrid nanoparticles (GO@Ag-Pt). The PVA/SA hydrogel loaded with GO@Ag-Pt exhibited the ability to scavenge different types of ROS, generate O2, and kill a broad spectrum of bacteria in vitro. The silver-platinum hybrid nanoparticles significantly increased the antibacterial ability against Escherichia coli and Staphylococcus aureus compared with silver nanoparticles (AgNps). GO@Ag-Pt loaded hydrogel was effective in treating infections caused by S.aureus, thereby significantly promoting wound healing during the inflammatory phase. Hydrogel therapy significantly reduced the level of ROS and alleviated inflammation levels. Notably, our ROS-scavenging, antibacterial hydrogels can be used to effectively treat various types of wounds, including difficult-to-heal diabetic wounds with bacterial infections. Thus, this study proposes an effective strategy for various chronic wound healing based on ROS clearance and bacteriostatic hydrogels.

Preparation of micron-sized benzamidine-modified magnetic agarose beads for trypsin purification from fish viscera.

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe3O4@agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 µm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g-1 at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g-1 min-1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl2, pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl2, pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems.

Fabrication of micron-sized boronate-decorated polyethyleneimine-grafted magnetic agarose beads for specific enrichment of ribonucleic acid.

Exploiting high-performance magnetic beads for specific enrichment of ribonucleic acid (RNA) has important significance in the biomedical research field. Herein, a simple strategy was proposed for fabricating boronate-decorated polyethyleneimine-grafted magnetic agarose beads (BPMAB), which can selectively isolate cis-diol-containing substances through boronate affinity. The size of the basic magnetic agarose beads was controlled through the emulsification of the water-in-oil emulsion with a high-speed shear machine, which enhanced the specific surface area of BPMAB. Subsequently, to modify more boronic acid ligands, branched PEI with excellent hydrophilicity and numerous reaction sites was grafted. 2,4-Difluoro-3-formylphenyl boronic acid (2,4-DFPBA) was covalently immobilized for selectively capturing cis-diol-containing substances under physiological condition (pH 7.4). The BPMAB with a diameter range from 1.86 µm to 11.60 µm possessed clearly spherical structure, and excellent magnetic responsiveness and suspension ability in aqueous solution. ß-Nicotinamide adenine dinucleotide (ß-NAD), a short-chain cis-diol carrying agent, was selected as a target molecule for evaluating the adsorption property of BPMAB and the maximum adsorption capacity of BPMAB for ß-NAD could reach 205.11 mg g-1. In addition, the BPMAB as adsorbent was used to selectively enrich RNA from mammalian cells. The maximum adsorption capacity of BPMAB for RNA was 140.50 mg g-1. Under optimized conditions, the BPMAB-based MSPE successfully enriched the high-quality total RNA with 28S to 18S ribosomal RNA ratios ranging from 2.06 to 2.16. According to the PCR analysis of GADPH gene, the extracted total RNA was successfully reverse transcribed into cDNA. Therefore, we believe that the BPMAB-based MSPE could be applicable for the specific enrichment of RNA from complex biological systems.

Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

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.

Anastomotic leak risk factors following colon cancer resection: a systematic review and meta-analysis.

BACKGROUND: Despite improved surgical techniques, anastomotic leakage is still a serious complication that can occur after colon cancer resection, resulting in increased morbidity and mortality. The aim of this study was to evaluate the risk factors for anastomotic leakage after colon cancer surgery, provide a theoretical basis for reducing its occurrence, and guide the practice of clinicians. METHODS: A systematic review of PubMed, Ovid, Web of Science and Cochrane Central Register of Controlled Trials databases was conducted by using a combination of subject terms and free words for online searches. The databases were searched from their inception to 31 March 2022, and all cross-sectional, cohort or case‒control studies examining the risk factors for the development of anastomotic fistula after surgery for colon cancer were identified. RESULT: A total of 2133 articles were searched for this study, and 16 publications were ultimately included, all of which were cohort studies. A total of 115,462 subjects were included, and a total of 3959 cases of anastomotic leakage occurred postoperatively, with an incidence of 3.4%. The odds ratio (OR) and 95% confidence interval (CI) were used for evaluation. Male sex (OR = 1.37, 95% CI: 1.29-1.46, P < 0.00001), BMI (OR = 1.04, 95% CI: 1.00-1.08, P = 0.03), diabetes (OR = 2.80, 95% CI: 1.81-4.33, P < 0.00001), combined lung disease (OR = 1.28, 95% CI: 1.15-1.42, P < 0.00001), anaesthesia ASA score (OR = 1.35, 95% CI: 1.24-1.46, P < 0.00001), ASA class ≥ III (OR = 1.34, 95% CI: 1.22-1.47, P < 0.00001), emergency surgery (OR = 1.31, 95% CI: 1.11-1.55, P = 0.001), open surgery (OR = 1.94, 95% CI: 1.69-2.24, P < 0.00001) and type of surgical resection (OR = 1.34, 95% CI: 1.12-1.61, P = 0.002) are risk factors for anastomotic leakage after colon cancer surgery. There is still a lack of strong evidence on whether age (OR = 1.00, 95% CI: 0.99-1.01, P = 0.36) and cardiovascular disease (OR = 1.18, 95% CI: 0.94-1.47, P = 0.16) are factors influencing the occurrence of anastomotic leakage after colon cancer surgery. CONCLUSIONS: Male sex, BMI, obesity, coexisting pulmonary disease, anaesthesia ASA score, emergency surgery, open surgery and type of resection were risk factors for anastomotic leakage after colon cancer surgery. The effect of age and cardiovascular disease on postoperative anastomotic leakage in patients with colon cancer needs further study.

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%.

Bioconjugation of nanozyme and natural enzyme to enable a one-step cascade reaction for the detection of metabolites.

Nanozyme, with enzyme-mimicking activity and excellent stability, has attracted extensive attention. However, some inherent disadvantages, including poor dispersion, low selectivity, and insufficient peroxidase-like activity, still limit its further development. Therefore, an innovative bioconjugation of a nanozyme and natural enzyme was conducted. In the presence of graphene oxide (GO), histidine magnetic nanoparticles (H-Fe3O4) were first synthesized by a solvothermal method. The GO-supported H-Fe3O4 (GO@H-Fe3O4) exhibited superior dispersity and biocompatibility because GO was the carrier and possessed outstanding peroxidase-like activity because of the introduction of histidine. Furthermore, the mechanism of the peroxidase-like activity of GO@H-Fe3O4 was the generation of •OH. Uric acid oxidase (UAO) was selected as the model natural enzyme and covalently linked to GO@H-Fe3O4 with hydrophilic poly(ethylene glycol) as a linker. UAO could specifically catalyze the oxidation of uric acid (UA) to generate H2O2, and subsequently, the newly produced H2O2 oxidized the colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue ox-TMB under the catalysis of GO@H-Fe3O4. Based on the above cascade reaction, the GO@H-Fe3O4-linked UAO (GHFU) and GO@H-Fe3O4-linked ChOx (GHFC) were used for the detection of UA in serum samples and cholesterol (CS) in milk, respectively. The method based on GHFU exhibited a wide detection range (5-800 µM) and a low detection limit (1.5 µM) for UA, and the method based on GHFC exhibited a wide detection range (4-400 µM) and a low detection limit (1.13 µM) for CS. These results demonstrated that the proposed strategy had great potential in the field of clinical detection and food safety.

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.

Bioconjugation of nanozyme and natural enzyme for ultrasensitive detection of cholesterol.

When nanozymes are used in biological analysis, higher activity can improve the detection sensitivity, and better selectivity can eliminate other interference. To improve the specificity and sensitivity, we fabricated an innovative bioconjugated nanozyme with natural enzyme (BNNZ), in which natural ChOx was immobilized onto histidine-modified Fe3O4 (His-Fe3O4) with hydrophilic poly(ethylene glycol) (PEG) as a linker. ChOx could specifically catalyze the oxidation of cholesterol to generate H2O2 molecule, and then the newly formed H2O2 oxidized the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue ox-TMB by peroxidase-like His-Fe3O4. According to the above cascade reaction, the BNNZ-based colorimetric strategy was proposed for the detection of cholesterol. Wherein, natural enzymes specifically catalyzed substrates, which endowed BNNZ with excellent specificity for target molecules; meanwhile, the introduction of histidine on His-Fe3O4 effectively increased the peroxidase-like activity of BNNZ, which provided a guarantee for sensitivity. Furthermore, BNNZ after reaction could be rapidly separated by an external magnetic field without interfering with colorimetric quantitative detection. The proposed strategy exhibited excellent sensitivity with limit of detection of 0.446 µM and was successfully used for the detection of cholesterol in spiked human serum sample with recovery and relative standard deviation in the range of 97.9-103.5% and 2.5-4.0%, respectively. This work indicates that the bioconjugation of nanozyme and natural enzyme may be a universal strategy for synthesis of high-performance enzyme-nanozyme systems, and the new-type BNNZ will be widely used in biological detection and disease treatment.

Improving performance of cell imprinted PDMS by integrating boronate affinity and local post-imprinting modification for selective capture of circulating tumor cells from cancer patients.

Efficient capture of circulating tumor cells (CTCs) from cancer patients is an important technique that may promote early diagnosis and prognosis monitoring of cancer. However, the existing systems have certain disadvantages, such as poor selectivity, low capture efficiency, consumption of antibodies, and difficulty in release of CTCs for downstream analysis. Herein, we fabricated an innovative PEGylated boronate affinity cell imprinted polydimethylsiloxane (PBACIP) for highly efficient capture of CTCs from cancer patients. The antibody-free PBACIP possessed hierarchical structure of imprinted cavities, which were inlaid with boronic acid modified SiO2 nanoparticles (SiO2@BA), so it could specifically capture target CTCs from biological samples due to the synergistic effect of boronate affinity and cell imprinting. Furthermore, PEGylation was accurately completed in the non-imprinted region by the template cells occupying the imprinted cavity, which not only retained the microstructure of original imprinted cavities, but also endowed PBACIP with hydrophilicity. The artificial PBACIP could efficiently capture human breast-cancer cells from biological sample. When 5 to 500 SKBR3 cells were spiked in 1 mL mice lysed blood, the capture efficiency reached 86.7 ± 11.5% to 96.2 ± 2.3%. Most importantly, the PBACIP was successfully used to capture CTCs from blood of breast cancer patients, and the captured CTCs were released for subsequent gene mutation analysis. The PBACIP can efficiently capture and release CTCs for downstream analysis, which provides a universal strategy toward individualized anti-tumor comprehensive treatments and has great potential in the future cell-based clinical applications.

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.

Hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide nanocomposite as a substitute of enzyme-labeled second antibody for ultrasensitive detection of antibodies.

Enzyme-labeled secondary antibody is often used to amplify the output signal in the process of antibody detection. However, its preparation process is complex and time-consuming. Herein, we fabricated an innovative hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide (HRBGO) nanocomposite, used as a substitute of enzyme-labeled second antibody. The synthetic HRBGO was loaded with generous rhodamine B and modified with boronic acid. Therefore, the HRBGO could selectively label the carbohydrate chains of Fc fragment of primary antibody through specific boronate affinity recognition, and then perform signal output and amplification by releasing rhodamine B. To verify the practicability of HRBGO, trastuzumab as a humanized monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2) was selected as model antibody. A glycosylation site-blocked / HER2-immobilized magnetic nanoparticles (GHMN) was also prepared for selectively capturing trastuzumab from complex samples via specific immunoaffinity. Because the glycosylation sites of HER2 can also be labeled with the HRBGO by boronate affinity recognition, these sites were blocked by a masking agent to minimize the background signal. For specific and ultrasensitive detection of trastuzumab, the integration of GHMN and HRBGO was proposed and optimized in detail. Trastuzumab detection based on HRBGO consisted of three steps: specific capture, selective labeling, and output signal. The proposed strategy provided ultrahigh sensitivity with limit of detection of 0.35 fg mL-1 and was successfully applied in the detection of trastuzumab in spiked serum sample with recovery and relative standard deviation in the range of 98.7-103.8% and 3.8-6.0%, respectively. To assess universal applicability, the HRBGO was also successfully used for the determination of anti-SARS-COV2 RBD antibody in human serum sample.

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.

Visual detection of hepatocellular carcinoma cells with cell imprinted substrate and pH-sensitive allochroic-graphene oxide.

Herein, we integrate cell-imprinted substrate (CIS) and allochroic-graphene oxide (AGO) for specific visualization sorting of hepatocellular carcinoma cells. The state-of-the-art-of detection method relies on the enzyme linked immunosorbent assay (ELISA)-like sandwich strategy with hierarchical recognition. The target tumor cells are first selectively captured by the CIS based on cell imprinted recognition, and then specifically labeled with AGO by boronate affinity recognition between boronic acid on AGO and cis-diols on the surface of target cells. The selectively recognition of CIS for target template cells is verified by cell function experiments. It is also worth mentioning that the AGO can specifically recognize target tumor cells under physiological pH, and then perform signal amplification and output through pH-triggered allochroism. The CIS linked AGO for cell assay (CIS-AGO-CA) is successfully used for visualization detection of human hepatocarcinoma HLE cells from hepatocyte suspension. When the hepatocyte suspension is spiked with 1.0 × 105 cells, the recoveries of CIS-AGO-CA are 80.67 ± 4.33% for target HLE cells, and only 12.00 ± 1.00% for non-target Hep3B cells. It is worth emphasizing that the CIS-AGO-CA process is antibody-free. Therefore, this novel ELISA-like sandwich strategy is high specificity, cost-efficient and easy-to-use, and exhibits great prospect in the visualization sorting of tumor subpopulation.

Advances and applications of in-tube solid-phase microextraction for analysis of proteins.

In-tube solid-phase microextraction (IT-SPME) with capillary column as extraction device is a well-established green extraction technique with a lot of applications in the fields of biomedicine, food and environment. This article reviews the research contributions of IT-SPME for analysis of proteins. The paper first briefly describes the history of IT-SPME. Then, the development and principle of IT-SPME for analysis of proteins are introduced, in which capillary column configurations of IT-SPME and instruments for quantitative analysis of proteins are summarized. Subsequently, the synthesis strategy and recognition principle of different recognition units, including antibodies, aptamers, molecularly imprinted polymers, and boronate affinity materials, are discussed in detail. This part also introduces several rare recognition units, including lectins, restricted access materials, lysine modified with ß-cyclodextrin and cell membrane. The development trend and possible future direction of IT-SPME for analysis of proteins are mentioned.

Preparation of teamed boronate affinity magnetic nanoparticles for extraction of polyphenols from Flos Lonicerae Beverage under neutral pH prior to their determination by high-performance liquid chromatography-mass spectrometry.

Traditional boron affinity materials usually capture cis-diol-containing molecules under alkaline condition, but some cis-diol-containing molecules, such as polyphenols, are unstable and easy to be oxidized and degraded under alkaline condition. Teamed boronate affinity (TBA) can specifically capture cis-diol-containing molecules under neutral condition. However, the report about combination of TBA and magnetic nanoparticle for the extraction was rare. Here, we fabricated two kinds of teamed boronate affinity magnetic nanoparticles (TBAMP), including Fe3O4@TBAP and Fe3O4@SiO2@TBAP. Adsorption capacities of cis-diol-containing molecules on the latter were similar to these on the former, but the latter possessed more superior regeneration performance than the former. Therefore, the TBAMP with more superior regeneration performance was used as magnetic solid-phase extraction (MSPE) adsorbent for capturing polyphenols under neutral condition. The TBAMP MSPE was optimized in detail, and combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) for the simultaneous determination of 13 kinds of polyphenols from Flos Lonicerae Beverage. The proposed method showed low limit of detection between 0.01 and 0.20 ng mL-1. In blank Flos Lonicerae Beverage, 11 kinds of polyphenols ranged from 0.54 ng mL-1 to 52.99 ng mL-1 were detected. In the standard addition method, recoveries of cis-diol-containing polyphenols were between 85.7% and 102.1% with intra-day and inter-day relative standard deviation ranging from 3.2% to 5.1% and 5.3% to 7.3%, respectively.