PROCESS BIOCHEMISTRY
·
2017
·
IF 2.497
Jinsha Huang,Wei Zhuang,Ce Wei,Liwen Mu,Jiahua Zhu,Yudan Zhu,Jinglan Wu,Yong Chen,Hanjie Ying
Orientation immobilization of enzymes has attracted intensive interest owing to the retainable specific activity and stability. Specially, glycoprotein immobilized onto Concanavalin A (Con A) modified carriers induces the orientation of the enzyme. However, the effects of the interface properties of carriers and enzymes are still not well understood yet. In this study, we synthesized the activated porous poly (styrene- divinylbenzene) resin carriers with 30 nm pore sizes and 72 m 2 g −1 specific surface areas and decorated with Con A. The resultant loading capacity of NP 1 on Con A modified carriers was as high as 4.02 mg g −1 wet support as a result of strong affinity between the enzyme and Con A decorated on carriers. It was found that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on Con A modified porous carriers were significantly improved. The reduction of K m from 18.40 ± 0.55 mg mL −1 to 17.19 ± 0.51 mg mL −1 illustrated the improved substrate affinity of HA-GA-ConA-NP 1 . Moreover, Con A-affinity NP 1 exhibited the best operational stability that only 7% of its initial activity was lost even after 9 batches repeated reaction. This work demonstrates that surface property manipulation of porous carriers and its derivatives has great potential in efficient biocatalytic systems.
研究领域:
生物化学
·
酶工程
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xinyi Mao,Binger Yu,Zijing Li,Zepeng Li,Guoqing Shi
In recent years, some studies have found that oriented immobilization of antibodies to microspheres can fully expose the antigen binding sites of antibodies, which can improve the sensitivity of sandwich immunoassays for the detection of proteins. Can this antibody immobilization strategy also improve the sensitivity of competitive immunoassays for the detection of small molecules ? To answer this question, the conjugate MS-SPG-Ab (oriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres via streptococcal protein G) and the conjugate MS-Ab (nonoriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres) were prepared, and a lateral flow immunoassay (LFIA) for the detection of aflatoxin B1 (AFB1) was established. The detection performance of the two methods was compared. The results showed that under the condition that the number of “effective” antibodies immobilized on TRF-MS was similar, compared with the nonoriented immobilization strategy (IC50 = 0.21 ng mL −1 ), the LFIA method established by the oriented immobilization strategy reduced the sensitivity of AFB1 detection (IC50 = 0.37 ng mL −1 ). However, this method can obtain higher detection precision for AFB1, the CV values were all below 8%. And it has stronger tolerance to the matrix of maize and peanut samples. The bias of LFIAs based on oriented immobilization technology (−14.93%–7.92%) was lower than nonoriented immobilization technology (28.16%–34.19%) for AFB1 detection in the two sample extracts. This study suggests that the LFIA method based on the oriented immobilization of antibodies can improve the accuracy of the detection results when performing rapid screening of small molecules.
研究领域:
分析化学
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免疫学
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食品安全
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2024
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IF 3.8
Yuxiang Zhang,Yao Zhang,Luyao Zhang,Yongdong Liu
The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.
研究领域:
生物化学
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分子生物学
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生物工程
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DOI
PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
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2018
·Vol. 42
·期 11
·8458-8468
·
IF 3.201
Jiayi Song,Ting Lei,Ye Yang,Nan Wu,Ping Su,Yi Yang
The major barriers to the use of enzymes in practical applications are their insufficient stability and easy inactivation under processing conditions. In this work, we developed a promising platform that allows for the simple and effective immobilization of enzymes on hydrophilic polydopamine (PDA) modified magnetic nanoparticles through DNA directed immobilization. Taking α-chymotrypsin (ChT) as an example, the well-designed immobilized enzyme in this study exhibited excellent kinetic performance, and the apparent Vmax and catalytic efficiency (kcat/Km) values of the DNA immobilized enzyme were 15.77 mM min−1 mg protein−1 and 4.05 s−1 mM−1, which were more than 6.0- and 5.9-fold enhanced compared to that of the free enzyme, respectively. The DNA immobilized enzyme exhibited promising thermal stability, which could preserve promising enzymatic activities of about 83% after 60 min incubation at 60 °C. The DNA immobilized enzyme also exhibited excellent long-term storage and incubation stability, which could preserve more than 89% of the initial activity at 4 °C for 35 days and more than 77% after 47 h of incubation, respectively. In addition, the DNA immobilized ChT exhibited excellent reusability, which showed a high degree of activity (more than 84%) after 10 cycles. Notably, the enzyme immobilization procedure exhibited high reversibility and reproducibility, and the magnetic nanoparticle surfaces were successfully regenerated and cycled through DNA strand displacement reactions for subsequent enzyme immobilization, which could retrieve more than 98% of the enzymatic activity. A significantly improved protein digestion efficiency was achieved with this immobilized ChT within 10 min, and the obtained sequence coverages were more than 1.3-fold higher when compared to that obtained by conventional in-solution digestion for 12 h. Therefore, this work exhibits a promising alternative platform for the efficient immobilization of industrially important enzymes and their broad applications.
研究领域:
生物化学
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纳米生物学
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酶工程
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DOI
高影响因子
Journal of Materials Chemistry B
·
2020
·Vol. 8
·期 36
·8467-8475
·
IF 5.344
Hao Shen,Xuelian Zheng,Zixin Zhou,Wenting He,Mengqi Li,Ping Su,Jiayi Song,Yi Yang
Various organelles (e.g., mitochondria and chloroplasts) have a multicompartment structure, providing superior function of material transformation, selective segregation and energy conversion. Enlightened by the elegant evolution of nature, intended isolation of the biochemical process by cooperative multicompartments in cells has become an appealing blueprint to construct bioreactors. In this study, we develop a “soft separation” way to establish a delicate multicompartment multienzyme system (MMS) with polyphenol-encapsulated enzyme–DNA conjugates, which are anchored on magnetic Janus particles, providing a biomimetic catalysis network with the model cascade reactions in confinement. The well-designed MMS exhibits preferable bioactivity benefitting from the dependable DNA bridges and the oriented immobilization of enzymes, while the polyphenol shell further protects the anchored enzymes from exterior attacks, such as heat and enzymatic degradation. Moreover, by applying the MMS as nanomotors, the asymmetrical distribution of enzymes on Janus particles is found to improve mutual elevation between the self-driven locomotion and enzyme-mediated reactions, delivering enhanced dispersal ability and bioactivity. Owing to the excellent enzymatic activity, promoted stability and satisfying biocompatibility, the assembled MMS is proved to be promising for the in vitro and intracellular sensing of glucose, showing significant potential for biochemical analysis applications.
研究领域:
生物化学
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纳米医学
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催化科学
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES B-BIOINTERFACES
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2021
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IF 5.268
Tongle Liu,Yuan Rao,Wenfeng Zhou,Wei Zhuang,Lei Ge,Rijia Lin,Wenxia Zhang,Jinglan Wu,Chenjie Zhu,Hanjie Ying
Adenylate cyclase (AC) can efficiently catalyze the conversion of adenosine triphosphate (ATP) to cyclic adenosine-3′, 5′-monophosphate (cAMP). However, AC directly immobilized on substrate is not desirable due to enzyme inactivation. Herein, bio-inspired adhesive of polydopamine and polyethyleneimine (PDA/PEI) was used as flexible chains to graft on graphene oxide (GO), and the AC was directionally immobilized through affinity between metal ions and his-tags of AC. The properties of modified GO and the activity of immobilized AC were studied in detail. PDA/PEI layers have been proved to improve the amino density of GO surface for affinity groups decoration and adjust the interaction between AC and support. And modified GO by this novel method contributes to subsequent grafting and immobilization of AC by affinity. AC immobilized on modified GO exhibited high activity recovery with about 90 % of free AC, while enzyme immobilized on unmodified GO has been inactivated. This study offers a versatile approach for support modification and enzyme oriented immobilization. PDA/PEI functionalized GO can be used as a promising carrier to immobilize other his-tagged enzymes.
研究领域:
生物化学
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酶工程
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产品关联
DOI
PubMed
高影响因子
COMPOSITES PART B-ENGINEERING
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2021
·
IF 9.078
Wenfeng Zhou,Yuan Rao,Wei Zhuang,Lei Ge,Rijia Lin,Ting Tang,Jinglan Wu,Ming Li,Pengpeng Yang,Chenjie Zhu,Hao Wang,Hanjie Ying
The precision design of surface properties of supports and immobilization methodologies is essential for improving the biocatalytic activity of immobilized enzymes. In this study, we proposed a surface heterogeneity regulation strategy of graphene oxide (GO) to oriented immobilize adenylate cyclase (AC). The grafted flexible chain of polyethylene glycol amine (PEG-NH 2 ) and maleic anhydride (MAH) on GO surface can adjust the interaction between support and AC. The achieved immobilized AC can eliminate the non-specific adsorption and introduce cushioning effect for the AC's catalytic reaction under extreme conditions. Therefore, the active center of the enzyme can be retained. The introduction of PEG-NH 2 improves the activity recovery with 113%. Compared to GO-Ni 2+ @ AC, 8 arm-PEG-NH 2 modified GO immobilized AC has 117 times higher catalytic activity . The method proposed in this study provides a possibility to address the issues in enzyme application, such as the purification of enzyme, low enzyme activity caused by cross-linking, and non-specific adsorption due to molecular force on the surface of the supports. The strategy should provide deeper understanding of the oriented immobilization and support modification for improving the activity and stability of the immobilized enzyme.
研究领域:
生物化学
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材料科学
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酶工程
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DOI
高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 11
·1-12
·
IF 5.3
Wen Peng,Su Hao,Yin Wen-jia,Hu Jian-cheng,Wang Yu,Yang Jin-yi,Xiao Zhi-li,Xu Zhen-lin,Shen Yu-dong,Wang Hong,Hammock Bruce D.
A new type of label-free electrochemical immunosensor for the high-sensitivity determination of parathion was developed based on the oriented immobilization of nanobody (VHH9) on a gold nanoparticle-loaded polyvinyl alcohol/citric acid nanofiber membrane-modified electrode. The morphology characterization and assembly process of the modified materials were investigated using scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Under the optimum conditions, the label-free electrochemical immunosensor for parathion exhibited a linear range of 0.0015–6400 ng/mL and a low detection limit of 0.48 pg/mL, the signal response of which was 10 times higher than that of the randomly immobilized VHH9. The immunosensor possessed high selectivity, good repeatability and reusability (keeping above 90% of its initial activity after repeating 8 times), and stability (remaining 90% after 9 weeks of storage). Finally, the average recoveries of parathion from food samples were 93.76–105.73% with the coefficient of variation being 2.65–6.85%, showing good correlation with UPLC ( R 2 = 0.9950). Therefore, our nanobody immobilization protocol is simple and effective and proves the potential to be utilized as a promising candidate for sensing platform. Graphical
研究领域:
生物传感
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环境监测
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食品检测
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2025
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IF 9.8
Yu-Min Zhang,Hong-Ming Yang,Chong-Mei Xu,Jia-Yi Ju,Tian-Shui Xue,Yan Jiang,Zi-Yu Jia,Xiao-Ming Gong,Xian-Zhong Zeng,Jin-Bao Tang
Aflatoxin B 1 (AFB 1 ) is one of the most common and harmful toxins in agricultural products, and the development of a sensitive, simple, and accurate detection method for AFB 1 is crucial for ensuring continuous monitoring and safeguarding food safety. Herein, we describe a facile and effective electrochemical immunosensor for ultrasensitive detection of AFB 1 . In this framework, a novel Fc-specific cysteine (Cys)-modified antibody was first fabricated by employing photoaffinity labeling technique, which enables antibodies to be directionally immobilized onto a bare gold electrode via Au-sulfhydryl interaction. The proposed immunosensor exhibited detection limits (LODs) of 0.054 and 0.019 ng mL −1 for direct competitive and sandwich-type competitive assays, respectively. Notably, these LODs were approximately 8-fold and 10-fold lower, respectively, compared to those achieved with randomly immobilized antibodies. The proposed immunosensor demonstrated excellent stability and reproducibility, along with high sensitivity, accuracy, and precision in AFB 1 detection, suggesting its significant potential for applications in food safety applications.
研究领域:
生物分析
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食品科学
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免疫学
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Jin Li,Xintong Li,Yulian Wang,Qianxu Zhou,Guoqing Shi
The method for immobilizing antibodies on nanoparticles significantly impacts the analytical performance of lateral flow immunoassay (LFIA). As both Fc-binding peptide (FcBP) and Staphylococcal Protein A (SPA) enable the oriented immobilization of antibodies in a similar manner, do LFIA methods based on these two modes exhibit differences in analytical performance? To address this question, AFB1 antibodies were immobilized on Europium nanoparticles (EuNPs) using non-oriented immobilization, oriented immobilization through SPA and Fc-binding peptide FYEILHGAC, generating the immunoprobes Ab@EuNP, Ab-SPA@EuNP, and Ab-FcBP@EuNP, respectively. The corresponding LFIA methods were developed and the performance of the three methods was compared. The results show that the Ab-FcBP@EuNP method immobilizes more “effective antibodies” compared to Ab-SPA@EuNP and Ab@EuNP. When an equivalent amount of “effective antibodies” is immobilized on EuNP, the IC50 value of the LFIA method based on Ab-FcBP@EuNP is significantly lower (2.58 and 3.61 times) than that of the LFIA methods based on Ab-SPA@EuNP and Ab@EuNP, respectively. The LFIA method utilizing Ab-FcBP@EuNP displays detection biases for AFB1 spiked in rice and maize matrices ranging from −8.53% to 3.90%, and biases in the detection results for certified reference materials (CRMs) of rice and maize containing AFB1 range from −11.06% to 6.33%. These results suggest that the LFIA method employing FcBP-based antibody immobilization exhibits superior sensitivity, accuracy, and interference resistance compared to the SPA-based method. The outcomes of this study validate that EuNPs functionalized with FcBP streamline antibody immobilization, minimize antibody usage, and improve the analytical performance of the LFIA, thus demonstrating favorable potential for practical implementation.
研究领域:
食品安全检测
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免疫学
·
分析化学
✓ 已验证
产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Xintong Li,Jiale Cui,Jin Li,Yulian Wang,Guoqing Shi
Gastrin 17 (G-17) is a major biomarker for the diagnosis of gastric cancer. As the serum concentrations of gastrin are low, highly sensitive detection methods are needed. Previous studies have shown that oriented immobilization of detection antibodies can improve the sensitivity of lateral flow immunoassay (LFIA) methods. Herein, the antibody binding capacity of four Fc-specific peptides reported in different studies was evaluated, and two peptides with better binding potency were further modified with cysteine or the peptide GAC at their C-terminus. The peptide FYWHCLDEGAC exhibited the highest antibody binding capacity and was applied to directly immobilize the anti-G-17 antibody on microspheres with carboxyl-functionalized surfaces and europium-based time-resolved fluorescence. An LFIA method was developed to detect G-17 in human serum, and the limit of detection (LOD) of this method for G-17 was 0.08 pmol L -1 , which was 10 times less than that of the nonoriented immobilization method. The working range of this method is 0.27 ∼ 600 pmol L -1 , with a recovery rate of 85.2 % −106.7 % and a CV value less than 12.4 %. This method is easy to use, and the total detection process takes less than 20 min. The test results for the actual serum samples were consistent with the commercial kit results (R 2 > 0.95), demonstrating that the method can be utilized for point-of-care testing of G-17. This approach to orientedly immobilize antibodies on nanomaterials can be used to develop sensitive LFIA methods for various molecules.
研究领域:
生物医学工程
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分析化学
·
癌症研究
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分子生物学
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产品关联
DOI
高影响因子
MOLECULES
·
2023
·Vol. 29
·期 1
·118
·
IF 4.6
Jian Zhang,Yuanyuan Li,Lin Chen,Zhihong Zheng,Chunye Liu
A facial and efficient method for the screening of acetylcholinesterase (AChE) inhibitors by capillary electrophoresis was developed. Based on the specific affinity of concanavalin A (Con A) for binding to the glycosyl group of AChE, enzyme molecules were oriented-immobilized on the surface of gold nanoparticles (AuNPs@Con A@AChE). Then, these modified nanoparticles were bounded to the capillary inlet (about 1.0 cm) by electrostatic self-assembly to obtain the oriented-immobilized enzyme microreactor (OIMER). Compared to an IMER with a free enzyme, the peak area of the product obtained by the OIMER increased by 52.6%. The Michaelis–Menten constant (Km) was as low as (0.061 ± 0.003) mmol/L. The method exhibits good repeatability with a relative standard deviation (RSD) of 1.3% for 100 consecutive runs. The system was successfully applied to detect the IC50values of donepezil and four components from Chinese medicinal plants. This work demonstrates the potential of this method as a low cost, simple, and accurate screening method for other enzyme inhibitors.Keywords:capillary electrophoresis;oriented-immobilized enzyme microreactor;acetylcholinesterase;gold nanoparticles;enzyme inhibitor screening
研究领域:
分析化学
·
生物化学
·
纳米技术
·
药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）