SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Yiyu Chen,Huiting Lian,Bin Liu,Guangming Liu,Xiaofeng Wei
Food allergy diagnosis requires a highly sensitive detection of allergen-specific immunoglobulin E (sIgE) in the serum. Enzyme-linked immunosorbent assay (ELISA) is a classical method widely used for sIgE detection but had to rely on costly and time-consuming assay procedures, skilled technicians, and bulky equipment. Here, a multiplexed origami paper analytical device (moPAD) that is hexahistidine-tagged and integrated with a nitrilotriacetic acid-functionalized surface with nickel (Ni-NTA) was developed to achieve low-cost and high-efficiency immunosensing of multiple allergen-specific IgEs in serum. The major oyster allergens, Cra a 1, Cra a 2, and Cra a 4, were used to detect sIgE in human serum. The feasibility of the moPAD-based immunosensing strategy was successfully demonstrated by desirable sensitivities for multiple targets. The detection results of moPAD for 72 serum samples were 100% consistent with clinical test results, while 69 of these results showed no significant differences from ELISA results, demonstrating the accuracy and stability of this method. A fully integrated moPAD reduces detection time and achieves highly sensitive quantification using a simple smartphone-based readout. Efficient allergen modification of paper devices has great applicability in allergen component-resolved diagnostics. The implementation of moPAD can make the process of allergy diagnosis simpler for patients, physicians, and healthcare systems alike.
研究领域:
免疫学
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生物医学工程
·
诊断技术
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过敏研究
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产品关联
DOI
高影响因子
Nanoscience and Nanotechnology Letters
·
2018
·Vol. 10
·期 3
·435-439
·
IF 2.917
Xu Hongpan,Sun Tingting,Huang Yuanfu,Song Lian,Li Zhiyang,Wang Wei
Placing of affinity tags at desired position of the proteins has been widely used to facilitate protein separation. One of the most commonly method for protein separation is nickel ion (Ni2+) affinity chromatography. Complex steps and time consuming in nickel ion affinity
chromatography have caused high demand for alternative purification methods that allow direct and rapid separation of His-tagged proteins. In the present study, magnetic core–shell beads enriched with Ni2+-nitrilotriacetate (Ni-NTA) on their surface were prepared in this study
by precipitation polymerization, with a uniform size of about 10 μm. The presented Ni2+ amount was 2.23%, which endowed the spheres with excellent magnetic responsivity and dispersity. We compared the magnetic beads enrichment method and nickel ion affinity chromatography
method for extraction of His-tagged protein sample with different concentrations. The results revealed that, when compared with nickel ion affinity chromatography, extraction efficiency, purity of the extracted protein, and regeneration effort were much higher with the magnetic beads enrichment
method. By using magnetic beads enrichment method, samples could be separated within 20 s, and purified within 1 h, with unlimited flow velocity and sample capacity. In addition, the magnetic beads enrichment method was particularly promising for the separation of His-tagged proteins in less
samples.
研究领域:
生物化学
·
蛋白质组学
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DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2022
·
IF 3.705
Liya Zhou,Yaping Ouyang,Weixi Kong,Teng Ma,Hao Zhao,Yanjun Jiang,Jing Gao,Li Ma
In this study, a novel kind of Ni-NTA modified monodispersed SiO 2 nanoflowers (Ni-NTA@SiO 2 nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO 2 nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)− 3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO 2 nanoflowers was 40.17 mg/g support and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of C C bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.
研究领域:
生物化学
·
酶工程
·
纳米材料科学
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产品关联
DOI
PubMed
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2021
·Vol. 44
·期 10
·2193-2204
·
IF 3.21
He Song,Wu Xiaomei,Ma Baodi,Xu Yi
The novel Ni-NTA-functionalized magnetic chitosan microspheres (MCS-NTA-Ni) were prepared via amino functionalization of MCS with epichlorohydrin and ethylenediamine, followed by the introduction of the aldehyde groups and NTA in turn, and nickel (II) ions were chelated in the end. MCS-NTA-Ni contained numerous long-armed NTA-Ni surface groups, ensuring high enzyme loading and providing more space and flexibility to attach enzymes and maintain their activity. This microsphere can have highly selective adsorption of his-tagged recombinant protein. The his-tagged recombinant Microbacterium esterase of E. coli BL21 (DE3)/pET21a-EstSIT01 was first immobilized on MCS-NTA-Ni by affinity fixation, giving high immobilization yield (90.1%) and enzyme loading (120 mg/g). Compared with free esterase, the immobilized esterase was found to exhibit higher pH stability and thermal stability. In addition, the immobilized esterase had excellent reusability for the synthesis of key chiral intermediate of d -biotin and the substrate conversion could still keep 100% after 8 cycles continuously.
研究领域:
生物化学
·
酶工程
·
合成生物学
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产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2025
·Vol. 15
·期 32
·26362-26370
·
IF 4.6
Han Wu,Jiayao Fang,Jiao Chen,Yaoqi Wang,Bo Zheng
Protein display technology enables high-throughput screening and plays an important role in protein discovery and engineering. Conventional in vivo display methods face challenges such as inefficient gene transformation and complex cell proliferation dynamics, while in vitro display methods are often limited to affinity-based selection and suffer from expression bias due to homogeneous reaction conditions. Here, we present a hydrogel particle-based protein display method enabled by particle-templated emulsification. This approach uses functionalized polyacrylamide hydrogel particles as isolated microreactors, incorporating DNA primers for genotype immobilization and Ni-NTA groups for capturing histidine-tagged protein phenotypes. Displayed proteins are synthesized via cell-free protein expression within isolated droplets, overcoming the limitations of in vivo cell culture and enabling compartmentalized screening, which is challenging in conventional homogeneous in vitro systems. Using particle-templated emulsification, single hydrogel particles can be rapidly encapsulated with individual DNA templates into isolated water-in-oil droplets within 30 seconds, without the need for specialized instrumentation. Up to 109 particles can be emulsified in a standard 50 ml conical tube. Compared to conventional droplet microfluidics, particle-templated emulsification achieves higher single-particle encapsulation and improved one-to-one particle–DNA pairing efficiency, reducing reagent consumption and minimizing DNA library loss caused by improper pairing. Digital PCR and cell-free protein expression are sequentially performed within droplets, with both the amplified DNA and the expressed protein immobilized on the same particle, thereby establishing a stable genotype–phenotype linkage. This method eliminates the need for cell handling, enables compartmentalized functional screening, and provides a fast, scalable, and user-friendly workflow for protein display, offering strong potential in directed evolution and protein engineering.
研究领域:
生物化学
·
生物技术
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蛋白质工程
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合成生物学
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分子生物学
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微流控技术
·
定向进化
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产品关联
DOI
PubMed
高影响因子
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