Biomimetic compartmentalization immobilization of multi-enzyme system

doi:10.12211/2096-8280.2024-025

Abstract

Abstract:

Biomimetic compartmentalization immobilization of multi-enzyme system is a frontier for in vitro synthetic biology, focusing on the spatial and temporal separation of reactions. Compared with simple co-immobilization, biomimetic compartmentalization immobilization can form substrate channels and promote the transmission of intermediates for sequential or coupling reaction. By controlling the relative positions of the enzymes on carriers, this method improves system stability, productivity, and purity. In this paper, we summarized the recent advances of carriers for biomimetic compartmentalization immobilization of multi-enzyme systems, including metal-organic frameworks (MOFs), polymer vesicles and polymer capsules. Metal-organic frameworks (MOFs) are porous coordination materials which are composed of metal ions as nodes and organic linkers. MOFs possess unique characteristics including high porosity, large specific surface area and tunable structure, which are suitable for multi-enzyme systems. The strategies involving the hierarchically porous MOFs, MOF-on-MOF and multi-MOF combinations construct compartmentalized environments for efficient catalytic reactions in vitro. Polymer vesicles are hollow nanostructures composed of amphiphilic block copolymers. The membrane structure of polymer vesicles, similar to the natural phospholipid bilayers, has good mechanical stability and biocompatibility for protecting enzyme molecules, and provides unique microenvironment for sequential reactions. Multiple small vesicles were encapsulated into the larger vesicles to form a "vesicle-in-vesicle" by mimicking the structure of cellular organelles. Polymer capsules with a core-shell spherical nanostructure are formed by the templating method, and have structural stability and excellent shape controllability. Multilayered core-shell structures created by layer-by-layer self-assembly are applied for compartmentalized immobilization of multi-enzyme. In the future, the integration of microfluidic technologies with biomimetic compartmentalization immobilization of multi-enzyme is expected to provide highly efficient and stable multi-enzyme catalytic systems for in vitro synthetic biology and green biomanufacturing.

Key words: multi-enzyme coupling, compartmentalized biomimetic, immobilized enzyme, metal-organic frameworks, polymer vesicles, polymer capsules

摘要：

仿生分区室固定化多酶耦联是体外合成生物学的前沿技术，目的是实现多酶分区室固定化和反应的时空分离。与简单共固定化不同，仿生分区室固定化技术通过控制酶在载体上的空间分布，形成底物通道促进中间产物传递，并提高串联或耦联反应的系统稳定性、产率和产物纯度。本文综述了近年来仿生分区室固定化多酶体系的进展，包括金属有机框架（MOFs）、聚合物囊泡和聚合物胶囊等固定化策略。MOFs具有结构可控、功能易调控等优点，采用分级多孔、MOF-on-MOF和多种MOF组合等仿生策略构建分区室微反应器，实现高效的体外多酶耦联催化反应。聚合物囊泡的膜结构可模拟天然磷脂双分子层，将多个小囊泡包封到大囊泡形成“囊泡中囊泡”模仿细胞器分区室固定化酶。聚合物胶囊是通过模板法形成核壳纳米球体结构，结构稳定性优异，进一步通过层层自组装能够形成多层核壳结构实现分区室固定化。将来，微流控等技术与仿生分区室固定化多酶技术融合，将促进体外合成生物学和绿色生物制造等领域的发展。

关键词: 多酶耦联, 仿生分区室, 固定化酶, 金属有机框架, 聚合物囊泡, 聚合物胶囊

CLC Number:

Q814.3

Lingling DONG, Feixuan LI, Hangbin LEI, Qidi SONG, Shizhen WANG. Biomimetic compartmentalization immobilization of multi-enzyme system[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-025.

董玲玲, 李斐煊, 雷航彬, 宋启迪, 王世珍. 仿生分区室固定化多酶体系[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-025.

Figures/Tables 6

References 100

1	SPERL J M, SIEBER V. Multienzyme cascade reactions—status and recent advances[J]. ACS Catalysis, 2018, 8(3): 2385-2396.
2	GIANNAKOPOULOU A, GKANTZOU E, POLYDERA A, et al. Multienzymatic nanoassemblies: recent progress and applications[J]. Trends in Biotechnology, 2020, 38(2): 202-216.
3	SCHOFFELEN S, VAN HEST J C M. Chemical approaches for the construction of multi-enzyme reaction systems[J]. Current Opinion in Structural Biology, 2013, 23(4): 613-621.
4	SCHOFFELEN S, VAN HEST J C M. Multi-enzyme systems: bringing enzymes together in vitro [J]. Soft Matter, 2012, 8(6): 1736-1746.
5	HAMMES G G, WU C W. Regulation of enzyme activity[J]. Science, 1971, 172(3989): 1205-1211
6	KÜCHLER A, YOSHIMOTO M, LUGINBÜHL S, et al. Enzymatic reactions in confined environments[J]. Nature Nanotechnology, 2016, 11(5): 409-420.
7	ZHU Z G, KIN TAM T, SUN F F, et al. A high-energy-density sugar biobattery based on a synthetic enzymatic pathway[J]. Nature Communications, 2014, 5: 3026.
8	RÖCKER J, SCHMITT M, PASCH L, et al. The use of glucose oxidase and catalase for the enzymatic reduction of the potential ethanol content in wine[J]. Food Chemistry, 2016, 210: 660-670.
9	SOJITRA U V, NADAR S S, RATHOD V K. A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification[J]. Food Chemistry, 2016, 213: 296-305.
10	LIU X, QI W, WANG Y F, et al. A facile strategy for enzyme immobilization with highly stable hierarchically porous metal-organic frameworks[J]. Nanoscale, 2017, 9(44): 17561-17570.
11	LI D, XIONG Q R, LIANG L, et al. Multienzyme nanoassemblies: from rational design to biomedical applications[J]. Biomaterials Science, 2021, 9(22): 7323-7342.
12	LIU Y, DU J J, YAN M, et al. Biomimetic enzyme nano complexes and their use as antidotes and preventive measures for alcohol intoxication[J]. Nature Nanotechnology, 2013, 8(3): 187-192.
13	ZHANG L Y, SINGH R, D S, et al. An artificial synthetic pathway for acetoin, 2,3-butanediol, and 2-butanol production from ethanol using cell free multi-enzyme catalysis[J]. Green Chemistry, 2018, 20(1): 230-242.
14	BECKER M, NIKEL P, ANDEXER J N, et al. A multi-enzyme cascade reaction for the production of 2'3'-cGAMP[J]. Biomolecules, 2021, 11(4): 590.
15	YIN L, GUO X, LIU L, et al. Self-assembled multimeric-enzyme nanoreactor for robust and efficient biocatalysis[J]. ACS Biomaterials Science & Engineering, 2018, 4(6): 2095-2099.
16	SHI T, HAN P P, YOU C, et al. An in vitro synthetic biology platform for emerging industrial biomanufacturing: bottom-up pathway design[J]. Synthetic and Systems Biotechnology, 2018, 3(3): 186-195.
17	SCHOONEN L, VAN HEST J C M. Compartmentalization approaches in soft matter science: from nanoreactor development to organelle mimics[J]. Advanced Materials, 2016, 28(6): 1109-1128.
18	THINGHOLM B, SCHATTLING P, ZHANG Y, et al. Subcompartmentalized nanoreactors as artificial organelle with intracellular activity[J]. Small, 2016, 12(13): 1806-1814.
19	MARGUET M, BONDUELLE C, LECOMMANDOUX S. Multicompartmentalized polymeric systems: towards biomimetic cellular structure and function[J]. Chemical Society Reviews, 2013, 42(2): 512-529.
20	PALEOS C M, TSIOURVAS D, SIDERATOU Z. Preparation of multicompartment lipid-based systems based on vesicle interactions[J]. Langmuir, 2012, 28(5): 2337-2346.
21	VAN DONGEN S F M, VERDURMEN W P R, PETERS R J R W, et al. Cellular integration of an enzyme-loaded polymersome nanoreactor[J]. Angewandte Chemie International Edition, 2010, 49(40): 7213-7216.
22	TANNER P, ONACA O, BALASUBRAMANIAN V, et al. Enzymatic cascade reactions inside polymeric nanocontainers: a means to combat oxidative stress[J]. Chemistry, 2011, 17(16): 4552-4560.
23	XU C, HU S, CHEN X Y. Artificial cells: from basic science to applications[J]. Materials Today, 2016, 19(9): 516-532.
24	BALASUBRAMANIAN V, CORREIA A, ZHANG H B, et al. Biomimetic engineering using cancer cell membranes for designing compartmentalized nanoreactors with organelle-like functions[J]. Advanced Materials, 2017, 29(11): 1605375.
25	LIU J, YANG Q H, LI C. Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors[J]. Chemical Communications, 2015, 51(72): 13731-13739.
26	KRACHER D, KOURIST R. Recent developments in compartmentalization of chemoenzymatic cascade reactions[J]. Current Opinion in Green and Sustainable Chemistry, 2021, 32: 100538.
27	WONG B, BOYER C, STEINBECK C, et al. Design and in situ characterization of lipid containers with enhanced drug retention[J]. Advanced Materials, 2011, 23(20): 2320-2325.
28	ELANI Y, LAW R V, CES O. Protein synthesis in artificial cells: using compartmentalisation for spatial organisation in vesicle bioreactors[J]. Physical Chemistry Chemical Physics, 2015, 17(24): 15534-15537.
29	HINZPETER F, GERLAND U, TOSTEVIN F. Optimal compartmentalization strategies for metabolic microcompartments[J]. Biophysical Journal, 2017, 112(4): 767-779.
30	LEE C H, LIN T S, MOU C Y. Mesoporous materials for encapsulating enzymes[J]. Nano Today, 2009, 4(2): 165-179.
31	GKANIATSOU E, SICARD C, RICOUX R, et al. Metal–organic frameworks: a novel host platform for enzymatic catalysis and detection[J]. Materials Horizons, 2017, 4(1): 55-63.
32	SUN Q M, SHI J Q, SUN H, et al. Membrane and lumen-compartmentalized polymersomes for biocatalysis and cell mimics[J]. Biomacromolecules, 2023, 24(11): 4587-4604.
33	QIAO J, MA Q, CHENG C, et al. Fabrication of dual-stimuli-responsive polymer vesicles for regulation of enzymolysis efficiency in a cascade reaction[J]. Chemistry-an Asian Journal, 2023, 18(12): e202300285.
34	BELLUATI A, THAMBOO S, NAJER A, et al. Multicompartment polymer vesicles with artificial organelles for signal-triggered cascade reactions including cytoskeleton formation[J]. Advanced Functional Materials, 2020, 30(32): 2002949.
35	ZHOU L L, FAN Y X, LIU Z, et al. A multiresponsive transformation between surfactant-based coacervates and vesicles[J]. CCS Chemistry, 2021, 3(12): 358-366.
36	LIAN X Z, CHEN Y P, LIU T F, et al. Coupling two enzymes into a tandem nanoreactor utilizing a hierarchically structured MOF[J]. Chemical Science, 2016, 7(12): 6969-6973.
37	ZHAO M Y, LI Y, MA X J, et al. Adsorption of cholesterol oxidase and entrapment of horseradish peroxidase in metal-organic frameworks for the colorimetric biosensing of cholesterol[J]. Talanta, 2019, 200: 293-299.
38	LIAN X Z, ERAZO-OLIVERAS A, PELLOIS J P, et al. High efficiency and long-term intracellular activity of an enzymatic nanofactory based on metal-organic frameworks[J]. Nature Communications, 2017, 8(1): 2075.
39	LIU H J, DU Y J, GAO J, et al. Compartmentalization of biocatalysts by immobilizing bienzyme in hollow ZIF-8 for colorimetric detection of glucose and phenol[J]. Industrial & Engineering Chemistry Research, 2020, 59(1): 42-51.
40	MAN T T, XU C X, LIU X Y, et al. Hierarchically encapsulating enzymes with multi-shelled metal-organic frameworks for tandem biocatalytic reactions[J]. Nature Communications, 2022, 13(1): 305.
41	WU G H, LI M, LUO Z G, et al. Designed synthesis of compartmented bienzyme biocatalysts based on core-shell zeolitic imidazole framework nanostructures[J]. Small, 2023, 19(7): e2206606.
42	LI Y, WEN L Y, TAN T W, et al. Sequential co-immobilization of enzymes in metal-organic frameworks for efficient biocatalytic conversion of adsorbed CO₂ to formate[J]. Frontiers in Bioengineering and Biotechnology, 2019, 7: 394.
43	CHEN S J, WEN L Y, SVEC F, et al. Magnetic metal–organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis[J]. RSC Advances, 2017, 7(34): 21205-21213.
44	TIAN D P, HAO R P, ZHANG X M, et al. Multi-compartmental MOF microreactors derived from Pickering double emulsions for chemo-enzymatic cascade catalysis[J]. Nature Communications, 2023, 14(1): 3226.
45	ZHENG J, XU H, LI B Z, et al. Spatially segregated MOF bioreactor enables versatile modular glycoenzyme assembly for hierarchical glycan library construction[J]. ACS Applied Materials & Interfaces, 2023, 15(16): 19807-19816.
46	LIANG J Y, MAZUR F, TANG C Y, et al. Peptide-induced super-assembly of biocatalytic metal-organic frameworks for programmed enzyme cascades[J]. Chemical Science, 2019, 10(34): 7852-7858.
47	ZHU D L, AO S S, DENG H H, et al. Ordered coimmobilization of a multienzyme cascade system with a metal organic framework in a membrane: reduction of CO₂ to methanol[J]. ACS Applied Materials & Interfaces, 2019, 11(37): 33581-33588.
48	XU Z L, XIAO G W, LI H F, et al. Compartmentalization within self-assembled metal–organic framework nanoparticles for tandem reactions[J]. Advanced Functional Materials, 2018, 28(34): 1802479.
49	LIU J, GUO Z Y, LIANG K. Biocatalytic metal-organic framework-based artificial cells[J]. Advanced Functional Materials, 2019, 29(45): 1905321.
50	YOON J, LEE S H, TIEVES F, et al. Light-harvesting dye–alginate hydrogel for solar-driven, sustainable biocatalysis of asymmetric hydrogenation[J]. ACS Sustainable Chemistry & Engineering, 2019, 7(6): 5632-5637.
51	WANG Y X, ZHAO Q C, HAAG R, et al. Biocatalytic synthesis using self-assembled polymeric nano- and microreactors[J]. Angewandte Chemie International Edition, 2022, 61(52): e202213974.
52	GODOY-GALLARDO M, LABAY C, TRIKALITIS V D, et al. Multicompartment artificial organelles conducting enzymatic cascade reactions inside cells[J]. ACS Applied Materials & Interfaces, 2017, 9(19): 15907-15921.
53	VAN DONGEN S F M, NALLANI M, CORNELISSEN J J L M, et al. A three-enzyme cascade reaction through positional assembly of enzymes in a polymersome nanoreactor[J]. Chemistry, 2009, 15(5): 1107-1114.
54	ELANI Y, LAW R V, CES O. Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways[J]. Nature Communications, 2014, 5: 5305.
55	KLERMUND L, POSCHENRIEDER S T, CASTIGLIONE K. Biocatalysis in polymersomes: improving multienzyme cascades with incompatible reaction steps by compartmentalization[J]. ACS Catalysis, 2017, 7(6): 3900-3904.
56	PETERS R J R W, MARGUET M, MARAIS S, et al. Cascade reactions in multicompartmentalized polymersomes[J]. Angewandte Chemie International Edition, 2014, 53(1): 146-150.
57	QU Q L, ZHANG X L, YANG A Q, et al. Spatial confinement of multi-enzyme for cascade catalysis in cell-inspired all-aqueous multicompartmental microcapsules[J]. Journal of Colloid and Interface Science, 2022, 626: 768-774.
58	BÄUMLER H, GEORGIEVA R. Coupled enzyme reactions in multicompartment microparticles[J]. Biomacromolecules, 2010, 11(6): 1480-1487.
59	SHI J F, ZHANG L, JIANG Z Y. Facile construction of multicompartment multienzyme system through layer-by-layer self-assembly and biomimetic mineralization[J]. ACS Applied Materials & Interfaces, 2011, 3(3): 881-889.
60	ZHANG P, FISCHER A, OUYANG Y, et al. Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems[J]. Chemical Science, 2022, 13(25): 7437-7448.
61	KREFT O, PREVOT M, MÖHWALD H, et al. Shell-in-shell microcapsules: a novel tool for integrated, spatially confined enzymatic reactions[J]. Angewandte Chemie International Edition, 2007, 46(29): 5605-5608.
62	DIAMANTI E, ANDRÉS-SANZ D, ORREGO A H, et al. Surpassing substrate–enzyme competition by compartmentalization[J]. ACS Catalysis, 2023, 13(17): 11441-11454.
63	BHATTACHARYA A, BREA R J, SONG J J, et al. Single-chain β-D-glycopyranosylamides of unsaturated fatty acids: self-assembly properties and applications to artificial cell development[J]. The Journal of Physical Chemistry B, 2019, 123(17): 3711-3720.
64	DOULIEZ J P, MARTIN N, GAILLARD C, et al. Catanionic coacervate droplets as a surfactant-based membrane-free protocell model[J]. Angewandte Chemie International Edition, 2017, 56(44): 13689-13693.
65	GARENNE D, BEVEN L, NAVAILLES L, et al. Sequestration of proteins by fatty acid coacervates for their encapsulation within vesicles[J]. Angewandte Chemie International Edition, 2016, 55(43): 13475-13479.
66	LARRAÑAGA A, LOMORA M, SARASUA J R, et al. Polymer capsules as micro-/ nanoreactors for therapeutic applications: current strategies to control membrane permeability[J]. Progress in Materials Science, 2017, 90: 325-357.
67	ARASTE F, ALIABADI A, ABNOUS K, et al. Self-assembled polymeric vesicles: focus on polymersomes in cancer treatment[J]. Journal of Controlled Release, 2021, 330: 502-528.
68	BALASUBRAMANIAN V, HERRANZ-BLANCO B, ALMEIDA P V, et al. Multifaceted polymersome platforms: spanning from self-assembly to drug delivery and protocells[J]. Progress in Polymer Science, 2016, 60: 51-85.
69	PALIVAN C G, FISCHER-ONACA O, DELCEA M, et al. Protein-polymer nanoreactors for medical applications[J]. Chemical Society Reviews, 2012, 41(7): 2800-2823.
70	TANNER P, EGLI S, BALASUBRAMANIAN V, et al. Can polymeric vesicles that confine enzymatic reactions act as simplified organelles?[J]. FEBS Letters, 2011, 585(11): 1699-1706.
71	LU A X, OH H, TERRELL J L, et al. A new design for an artificial cell: polymer microcapsules with addressable inner compartments that can harbor biomolecules, colloids or microbial species[J]. Chemical Science, 2017, 8(10): 6893-6903.
72	卞康晴, 郭灵怡, 迟文雅, 等. 聚合物囊泡的稳定性及H⁺透膜特性考察[J]. 药学实践与服务, 2024, 42(1): 12-17.
	BIAN K Q, GUO L Y, CHI W Y, et al. Study on the stability and H⁺ permeable membrane properties of polymersomes[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(1): 12-17.
73	HU X L, ZHANG Y Q, XIE Z G, et al. Stimuli-responsive polymersomes for biomedical applications[J]. Biomacromolecules, 2017, 18(3): 649-673.
74	LIMA A L, GRATIERI T, CUNHA-FILHO M, et al. Polymeric nanocapsules: a review on design and production methods for pharmaceutical purpose[J]. Methods, 2022, 199: 54-66.
75	KREFT O, SKIRTACH A G, SUKHORUKOV G B, et al. Remote control of bioreactions in multicompartment capsules[J]. Advanced Materials, 2007, 19(20): 3142-3145.
76	XU W N, STEINSCHULTE A A, PLAMPER F A, et al. Hierarchical assembly of star polymer polymersomes into responsive multicompartmental microcapsules[J]. Chemistry of Materials, 2016, 28(3): 975-985.
77	WU M, WANG Y Y, YAN N, et al. Self-assembly of polymeric nanovesicles into hierarchical supervesicles and its application in selectable multicompartmental encapsulation[J]. Macromolecules, 2021, 54(4): 1905-1911.
78	BJÖRNMALM M, CUI J W, BERTLEFF-ZIESCHANG N, et al. Nanoengineering particles through template assembly[J]. Chemistry of Materials, 2017, 29(1): 289-306.
79	ZHANG Z, ZHANG S S, SU R R, et al. Controlled release mechanism and antibacterial effect of layer-by-layer self-assembly thyme oil microcapsule[J]. Journal of Food Science, 2019, 84(6): 1427-1438.
80	KIM B, JEON T Y, OH Y K, et al. Microfluidic production of semipermeable microcapsules by polymerization-induced phase separation[J]. Langmuir, 2015, 31(22): 6027-6034.
81	TAGUCHI Y, ITO D, SAITO N, et al. Preparation and characterization of microcapsules containing particulate phosphorescent agent with suspension polymerization[J]. Polymers for Advanced Technologies, 2017, 28(3): 379-385.
82	ISHIZUKA F, KUCHEL R P, LU H X, et al. Synthesis of microcapsules using inverse emulsion periphery RAFT polymerization via SPG membrane emulsification[J]. Polymer Chemistry, 2016, 7(46): 7047-7051.
83	DE GEEST B G, SANDERS N N, SUKHORUKOV G B, et al. Release mechanisms for polyelectrolyte capsules[J]. Chemical Society Reviews, 2007, 36(4): 636-649.
84	SHI J F, WU Y Z, ZHANG S H, et al. Bioinspired construction of multi-enzyme catalytic systems[J]. Chemical Society Reviews, 2018, 47(12): 4295-4313.
85	LIANG J Y, GAO S, LIU J, et al. Hierarchically porous biocatalytic MOF microreactor as a versatile platform towards enhanced multienzyme and cofactor-dependent biocatalysis[J]. Angewandte Chemie International Edition, 2021, 60(10): 5421-5428.
86	SCHMIDT S, CASTIGLIONE K, KOURIST R. Overcoming the incompatibility challenge in chemoenzymatic and multi-catalytic cascade reactions[J]. Chemistry, 2018, 24(8): 1755-1768.
87	ROCHA-MARTIN J, VELASCO-LOZANO S, GUISÁN J M, et al. Oxidation of phenolic compounds catalyzed by immobilized multi-enzyme systems with integrated hydrogen peroxide production[J]. Green Chemistry, 2014, 16(1): 303-311.
88	ROCHA-MARTÍN J, DE LAS RIVAS B, MUÑOZ R, et al. Rational co-immobilization of Bi-enzyme cascades on porous supports and their applications in bio-redox reactions with in situ recycling of soluble cofactors[J]. ChemCatChem, 2012, 4(9): 1279-1288.
89	CHUNG J, HWANG E T, KIM J H, et al. Modular multi-enzyme cascade process using highly stabilized enzyme microbeads[J]. Green Chemistry, 2014, 16(3): 1163-1167.
90	HU C, BAI Y X, HOU M, et al. Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis[J]. Science Advances, 2020, 6(5): eaax5785.
91	FORNERA S, KUHN P, LOMBARDI D, et al. Sequential immobilization of enzymes in microfluidic channels for cascade reactions[J]. ChemPlusChem, 2012, 77(2): 98-101.
92	OBST F, MERTZ M, MEHNER P J, et al. Enzymatic synthesis of sialic acids in microfluidics to overcome cross-inhibitions and substrate supply limitations[J]. ACS Applied Materials & Interfaces, 2021, 13(41): 49433-49444.
93	CHU L L, ZHANG X Y, LI J N, et al. Continuous-flow synthesis of polysubstituted γ-butyrolactones via enzymatic cascade catalysis[J]. Chinese Chemical Letters, 2024, 35(4): 108896.
94	PATIL P D, SALOKHE S, KARVEKAR A, et al. Microfluidic based continuous enzyme immobilization: a comprehensive review[J]. International Journal of Biological Macromolecules, 2023, 253(Pt 6): 127358.
95	HE S, LIAN H T, CAO X G, et al. Cascaded enzymatic reaction-mediated multicolor pixelated quantitative system integrated microfluidic wearable analytical device (McPiQ-μWAD) for non-invasive and sensitive glucose diagnostics[J]. Sensors and Actuators B: Chemical, 2022, 369: 132345.
96	LI Z Y, UNO N, DING X, et al. Bioinspired CRISPR-mediated cascade reaction biosensor for molecular detection of HIV using a glucose meter[J]. ACS Nano, 2023, 17(4): 3966-3975.
97	BRAHAM S A, SIAR E H, ARANA-PEÑA S, et al. Effect of concentrated salts solutions on the stability of immobilized enzymes: influence of inactivation conditions and immobilization protocol[J]. Molecules, 2021, 26(4): 968.
98	MATEO C, PALOMO J M, FERNANDEZ-LORENTE G, et al. Improvement of enzyme activity, stability and selectivity via immobilization techniques[J]. Enzyme and Microbial Technology, 2007, 40(6): 1451-1463.
99	RODRIGUES R C, BERENGUER-MURCIA Á, CARBALLARES D, et al. Stabilization of enzymes via immobilization: multipoint covalent attachment and other stabilization strategies[J]. Biotechnology Advances, 2021, 52: 107821.
100	SHORTALL K, OTERO F, BENDL S, et al. Enzyme immobilization on metal organic frameworks: the effect of buffer on the stability of the support[J]. Langmuir, 2022, 38(44): 13382-13391.

固定化策略	MOFs	酶	稳定性	参考文献
孔道分区	PCN-888	GOx和HRP	4次循环后，GOx&HRP@PCN-888活性保持不变	[36]
孔道分区	PCN-333(Al)	HRP 和胆固醇氧化酶（ChOx）	4℃下保存20天后，GOx&HRP@PCN-333仍可检测到50%酶活	[37]
孔道分区	PCN-333(Al)	超氧化物歧化酶（SOD）和过氧化氢酶（CAT）	SOD&CAT@FNPCN-333在储存7天后仍可检测到酶活	[38]
MOF-on-MOF	ZIF-8	GOx和 HRP	HRP@H-ZIF-8-GOx 储存7天后仍可检测到70%酶活	[39]
MOF-on-MOF	ZIF-8	GOx和HRP	GOx@ZIF-8@HRP@ZIF-8 在4℃储存10天后仍可检测到93.96%酶活	[40]
MOF-on-MOF	ZIF-8	GOx 和HRP	/	[41]
MOF-on-MOF	Amine-MIL-101(Cr) and HKUST-1	CA，FDH和GDH	10个循环后，产率达到1077.7%	[42]
MOF-on-MOF	HKUST-1	GOx 和HRP	HRP@GOx@HKUST-1@Fe₃O₄重复使用10次后仍可检测到80.6%酶活	[43]
MOF-on-MOF	MOF-74	脂肪酶（CALB）and GOx	5个循环后仍可检测到79.3%酶活	[44]
多MOFs组合	ZIF-90	N-乙酰己糖胺-1-激酶（NahK），尿苷二磷酸-N-乙酰半乳糖胺焦磷酸酶（GlmU）和多磷酸激酶（PPK）	/	[45]
多MOFs组合	ZIF-8	GOx，HRP 和β-Gal	/	[46]
多MOFs组合	ZIF-8	FDH，GDH 和NADH	FDH&GDH&NADH/ZIF-8使用12小时后保留50%酶活	[47]
多MOFs组合	UiO-66	HRP和GOx	GOX@MOF-Cs和 HRP@MOF-Cs 3个循环后酶活性不变	[48]
多MOFs组合	ZIF- L and MPN	GOx和HRP	GOx–ZIF-L和HRP-ZIF–L在4℃保存30天仍可检测到酶活	[49]

固定化策略	MOFs	酶	稳定性	参考文献
孔道分区	PCN-888	GOx和HRP	4次循环后，GOx&HRP@PCN-888活性保持不变	[36]
孔道分区	PCN-333(Al)	HRP 和胆固醇氧化酶（ChOx）	4℃下保存20天后，GOx&HRP@PCN-333仍可检测到50%酶活	[37]
孔道分区	PCN-333(Al)	超氧化物歧化酶（SOD）和过氧化氢酶（CAT）	SOD&CAT@FNPCN-333在储存7天后仍可检测到酶活	[38]
MOF-on-MOF	ZIF-8	GOx和 HRP	HRP@H-ZIF-8-GOx 储存7天后仍可检测到70%酶活	[39]
MOF-on-MOF	ZIF-8	GOx和HRP	GOx@ZIF-8@HRP@ZIF-8 在4℃储存10天后仍可检测到93.96%酶活	[40]
MOF-on-MOF	ZIF-8	GOx 和HRP	/	[41]
MOF-on-MOF	Amine-MIL-101(Cr) and HKUST-1	CA，FDH和GDH	10个循环后，产率达到1077.7%	[42]
MOF-on-MOF	HKUST-1	GOx 和HRP	HRP@GOx@HKUST-1@Fe₃O₄重复使用10次后仍可检测到80.6%酶活	[43]
MOF-on-MOF	MOF-74	脂肪酶（CALB）and GOx	5个循环后仍可检测到79.3%酶活	[44]
多MOFs组合	ZIF-90	N-乙酰己糖胺-1-激酶（NahK），尿苷二磷酸-N-乙酰半乳糖胺焦磷酸酶（GlmU）和多磷酸激酶（PPK）	/	[45]
多MOFs组合	ZIF-8	GOx，HRP 和β-Gal	/	[46]
多MOFs组合	ZIF-8	FDH，GDH 和NADH	FDH&GDH&NADH/ZIF-8使用12小时后保留50%酶活	[47]
多MOFs组合	UiO-66	HRP和GOx	GOX@MOF-Cs和 HRP@MOF-Cs 3个循环后酶活性不变	[48]
多MOFs组合	ZIF- L and MPN	GOx和HRP	GOx–ZIF-L和HRP-ZIF–L在4℃保存30天仍可检测到酶活	[49]

材料	酶	稳定性	参考文献
聚合物囊泡（聚甲基丙烯酸酯PMA、聚赖氨酸PLL）	GOx和HRP	4℃储存2天后，仍能检测双酶级联到活性	[52]
聚合物囊泡（异氰肽与苯乙烯的嵌段共聚物）	GOx，HRP和CALB	/	[53]
聚合物囊泡（聚(2-甲基恶唑啉)-嵌段-聚(二甲基硅氧烷)-嵌段-聚(2-甲基恶唑啉)PMOXA-PDMS-PMOXA）	GOx，HRP和β-Gal	/	[54]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	N-酰基-D葡萄糖胺-2-烯丙基酶（AGE），N-乙酰神经氨酸醛缩酶（NAL）和CMP-唾液酸合成酶（CSS）	/	[55]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	PAMO，CALB和ADH	/	[56]
聚合物胶囊（天然多糖）	黄嘌呤氧化酶，尿酸酶和过氧化物酶	7个循环后产率是游离体系的2倍以上	[57]
聚合物胶囊（生物聚合物和碳酸钙）	β-葡萄糖苷酶（β-Glu），GOx和HRP	在4℃保存一个月以上仍可以检测到活性	[58]
聚合物胶囊（藻酸盐和鱼精蛋白）	FDH和FalDH	可循环使用8次以上	[59]
聚合物胶囊（核酸功能化羧甲基纤维素水凝胶）	GOx和β-Gal	在4℃下保存三天仍可检测到活性	[60]
聚合物胶囊（聚苯乙烯磺酸盐(PSS)和聚烯丙胺盐酸盐(PAH)）	人血清蛋白（HSA）	/	[61]
聚合物胶囊（聚烯丙胺盐酸盐和碳酸钙微粒）	(S)-3-羟丁酰辅酶A脱氢酶（DH）和黄素依赖型NADH氧化酶（NOx）	催化反应可进行72小时	[62]

材料	酶	稳定性	参考文献
聚合物囊泡（聚甲基丙烯酸酯PMA、聚赖氨酸PLL）	GOx和HRP	4℃储存2天后，仍能检测双酶级联到活性	[52]
聚合物囊泡（异氰肽与苯乙烯的嵌段共聚物）	GOx，HRP和CALB	/	[53]
聚合物囊泡（聚(2-甲基恶唑啉)-嵌段-聚(二甲基硅氧烷)-嵌段-聚(2-甲基恶唑啉)PMOXA-PDMS-PMOXA）	GOx，HRP和β-Gal	/	[54]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	N-酰基-D葡萄糖胺-2-烯丙基酶（AGE），N-乙酰神经氨酸醛缩酶（NAL）和CMP-唾液酸合成酶（CSS）	/	[55]
聚合物囊泡（聚苯乙烯-b-聚(3-(异氰基-丙氨酰-氨基乙基)噻吩PS-b-PIAT）	PAMO，CALB和ADH	/	[56]
聚合物胶囊（天然多糖）	黄嘌呤氧化酶，尿酸酶和过氧化物酶	7个循环后产率是游离体系的2倍以上	[57]
聚合物胶囊（生物聚合物和碳酸钙）	β-葡萄糖苷酶（β-Glu），GOx和HRP	在4℃保存一个月以上仍可以检测到活性	[58]
聚合物胶囊（藻酸盐和鱼精蛋白）	FDH和FalDH	可循环使用8次以上	[59]
聚合物胶囊（核酸功能化羧甲基纤维素水凝胶）	GOx和β-Gal	在4℃下保存三天仍可检测到活性	[60]
聚合物胶囊（聚苯乙烯磺酸盐(PSS)和聚烯丙胺盐酸盐(PAH)）	人血清蛋白（HSA）	/	[61]
聚合物胶囊（聚烯丙胺盐酸盐和碳酸钙微粒）	(S)-3-羟丁酰辅酶A脱氢酶（DH）和黄素依赖型NADH氧化酶（NOx）	催化反应可进行72小时	[62]