Click Chemistry由K.Barry Sharpless、Hartmuth C.Kolb和M.G.Finn于2001年提出，用于描述快速選擇性反應(yīng)或以可預(yù)測(cè)的方式相互“點(diǎn)擊”以形成具有雜原子鏈（C-X-C）的生理穩(wěn)定產(chǎn)物的反應(yīng)。Click chemistry廣泛用于生物分子、表面、顆粒和有機(jī)化合物的改性，具有許多優(yōu)點(diǎn)1：

* 應(yīng)用范圍廣泛；

* 模塊化性質(zhì)；

* 在“小量”和“大量”反應(yīng)中均適用；

* 反應(yīng)條件溫和；

* 產(chǎn)品分離簡單（幾乎不需要純化）；

* 產(chǎn)率高，速度快；

* 無害副產(chǎn)品生成（遵循綠色化學(xué)的12項(xiàng)原則）；

* 兼容性良好，尤其在生命系統(tǒng)中（允許生物分子的化學(xué)選擇性修飾，幾乎不受干擾）2。

在大約10種不同類型的點(diǎn)擊反應(yīng)中，有幾種是在各種生命科學(xué)應(yīng)用中使用最頻繁，從“簡單”的生物分子標(biāo)記和檢測(cè)到先進(jìn)的CRISPER應(yīng)用。在此，我們重點(diǎn)介紹最重要的9種（最新）應(yīng)用：

* 生物分子標(biāo)記與檢測(cè)

*（固&液相）生物分子修飾/連接

* 構(gòu)建用于構(gòu)效關(guān)系分析的類似物庫

* 藥物先導(dǎo)化合物發(fā)現(xiàn)

* 藥物輸送

* 材料優(yōu)化（聚合物改性）

* 病毒研究探針

* CRISPER sgRNA合成和靶基因標(biāo)記

* 新應(yīng)用，包括“點(diǎn)擊發(fā)布”

1.生物分子標(biāo)記與檢測(cè)

Click chemistry十分有用的功能之一是它能夠標(biāo)記和可視化生物分子，如脂質(zhì)3、肽4、聚糖5、糖蛋白6、核酸和合成分子7,8（如紫杉醇9），并且具有最小的生理干擾性（體外和體內(nèi)）10。在進(jìn)行標(biāo)記的兩步反應(yīng)中，首先用雙正交點(diǎn)擊手柄（如炔烴或疊氮化物）標(biāo)記目標(biāo)生物分子（酶、代謝11,12或合成（請(qǐng)見圖1）9,13）。然后當(dāng)一個(gè)分子上有熒光或親和基團(tuán)的互補(bǔ)點(diǎn)擊手柄與目標(biāo)分子發(fā)生點(diǎn)擊反應(yīng)時(shí)，就會(huì)發(fā)生檢測(cè)/可視化。

Click chemistry應(yīng)用生物分子標(biāo)記與檢測(cè)13

例如，在活體發(fā)育的斑馬魚中，表面聚糖以亞細(xì)胞分辨率被觀察到；依靠基因編碼的傳統(tǒng)分子成像方法通常無法看見14。在這項(xiàng)研究中，Bertozzi等人將代謝糖工程與多色檢測(cè)策略相結(jié)合，以揭示細(xì)胞表面表達(dá)、細(xì)胞內(nèi)運(yùn)輸和整個(gè)斑馬魚胚胎發(fā)生過程中聚糖組織分布的差異。類似的研究也在小鼠中進(jìn)行，以跟蹤移植細(xì)胞和測(cè)定細(xì)胞對(duì)肽的攝取情況，這有助于結(jié)構(gòu)-活性-通透性關(guān)系優(yōu)化研究15。兩個(gè)位點(diǎn)標(biāo)記生物分子（稱為雙位點(diǎn)標(biāo)記）有效的促進(jìn)了復(fù)雜生物系統(tǒng)的研究16,17,18,19,20。

2.(固相和液相）生物分子修飾/連接

肽、核苷酸、小分子、超分子等都可以通過固相或液相點(diǎn)擊化學(xué)進(jìn)行修飾，幾乎無需使用保護(hù)基團(tuán)，也無需產(chǎn)品純化3,21,22?？傮w來講，固相合成更快，且需要更少的后處理，但是每種方法都各有優(yōu)缺點(diǎn)23,24。

3. 類似物庫的建設(shè)

類似物庫可以通過點(diǎn)擊化學(xué)快速可靠地構(gòu)建，無需太多的合成工作，然后通過原位高通量篩選（HTS）來促進(jìn)分子結(jié)構(gòu)-活性關(guān)系（SAR）分析，這是優(yōu)化和發(fā)現(xiàn)生物活性分子所必需的。已經(jīng)有許多基于click（三唑）骨架的片段庫（聚焦組合）通過此方法被構(gòu)建出來25，例如Janus激酶抑制劑ruxolitinib衍生的三唑文庫，它被用來評(píng)估JAK3抑制劑24 。

4. 用于先導(dǎo)化合物發(fā)現(xiàn)的原位點(diǎn)擊化學(xué)

原位點(diǎn)擊化學(xué)是一種（動(dòng)力學(xué)）靶點(diǎn)導(dǎo)向合成方法，Sharpless及其同事于2002年第一次提出并應(yīng)用于發(fā)現(xiàn)一種有效的乙酰膽堿酯酶抑制劑26。這種方法使用目標(biāo)生物分子本身作為支架，如果使其足夠接近并以適當(dāng)?shù)姆较蚍磻?yīng)，則結(jié)合配體在其上進(jìn)行咔噠反應(yīng)。通過這種方式，可以從帶有互補(bǔ)反應(yīng)性官能團(tuán)的片段庫中篩選出能夠與目標(biāo)物形成穩(wěn)定絡(luò)合物的最佳配體27。無需事先對(duì)文庫成員進(jìn)行合成、純化和生化評(píng)估，即可快速且經(jīng)濟(jì)高效地篩選大量化合物28,29。

碳酸酐酶30、HIV蛋白酶31、幾丁酶32、核苷酸配體33、蛋白質(zhì)-蛋白質(zhì)相互作用（通過磺基點(diǎn)擊化學(xué)）34、抗體樣蛋白質(zhì)捕獲劑35,36、轉(zhuǎn)錄因子37、通道38等的抑制劑也已被表征。

5. 藥物輸送

藥物進(jìn)入人體的控制給藥是有效藥物設(shè)計(jì)的一個(gè)重要方面。點(diǎn)擊化學(xué)已用于構(gòu)建聚合物納米和微粒藥物遞送系統(tǒng)（DDS），如聚合物膠束、脂質(zhì)體、膠囊、碳納米管等6,39。

6. 材料優(yōu)化（聚合物改性）

在材料制造領(lǐng)域，從線性聚合物和接枝聚合物到更復(fù)雜結(jié)構(gòu)（如星形聚合物、嵌段共聚物和樹狀聚合物）的合成，再到表面和界面的功能化40，點(diǎn)擊化學(xué)都產(chǎn)生了巨大的影響。例如，由于不產(chǎn)生小分子副產(chǎn)物，點(diǎn)擊化學(xué)可以最大限度地減少氣泡、空穴和不規(guī)則的形成，就像其他縮聚反應(yīng)一樣，這些氣泡、空穴和不規(guī)則會(huì)破壞新合成熱固性材料的外觀和性能41。

CuAAC click chemistry還被用作一種高效、環(huán)保的交聯(lián)策略，以改善適用于涂料和粘合劑的水性聚合物的性能42（下圖2）。廣泛適用于聚氨酯（WPU）、聚酯分散體（PED）和聚丙烯酸酯乳液（PAE），該策略優(yōu)于其他可用的交聯(lián)策略（包括基于N-羥甲基丙烯酰胺（NMA）、懸垂乙酰乙酸基團(tuán)和可逆酮酰肼反應(yīng)的自交聯(lián)系統(tǒng)）。Click交聯(lián)聚合物薄膜的機(jī)械強(qiáng)度、硬度和耐水/溶劑性能顯著提高，為工業(yè)涂料應(yīng)用中使用硬化劑提供了一種有可能降低成本的替代品。

Formation of click cross-linked waterborne polymers42

此外，各種（1D、2D、3D）生物材料（如水凝膠）的合成在組織工程43,44,45,46再生醫(yī)學(xué)47、藥物輸送48和基因治療領(lǐng)域49也越來越受到重視。

7. 病毒研究探針

在過去幾十年中50，與病毒相關(guān)的研究，包括病毒（蛋白質(zhì)、核酸或病毒粒子）追蹤51,52、抗病毒設(shè)計(jì)53,54、診斷55,56,57和基于病毒的傳遞系統(tǒng)58,59都使用了點(diǎn)擊化學(xué)。例如，通過將疊氮化物修飾的病毒粒子連接到由二苯并環(huán)辛烯（DBCO）衍生的量子點(diǎn)（QD），使用無銅點(diǎn)擊反應(yīng)來標(biāo)記包膜病毒（痘苗病毒（VACV）和A病毒（H9N2））。標(biāo)記效率達(dá)到80%以上，不干擾病毒的感染能力，熒光強(qiáng)度足以實(shí)現(xiàn)單個(gè)病毒粒子的跟蹤60。

8. CRISPER-sgRNA合成與靶基因標(biāo)記

Click chemistry現(xiàn)在可以在CRISPR工具箱中找到合成單個(gè)或多個(gè)單一導(dǎo)向RNA（sgRNA）的位置，繞過了與（更長）寡聚體長度相關(guān)的現(xiàn)有合成限制，并縮短sgRNA設(shè)計(jì)和應(yīng)用之間的時(shí)間。

Click chemistry（被稱為“分裂和點(diǎn)擊”）不是一次性制造整個(gè)sgRNA，而是簡單地連接兩個(gè)更?。ǜ菀缀铣桑┑钠危阂粋€(gè)按需制備的高純度~20-mer（crRNA）靶向序列和一個(gè)通用的可大規(guī)模生產(chǎn)的79-mer CRISPR內(nèi)切酶蛋白（Cas9）序列（tracrRNA）。結(jié)果發(fā)現(xiàn)，帶有三唑鍵的~99-聚體能夠在體外和細(xì)胞內(nèi)有效地進(jìn)行Cas9介導(dǎo)的DNA切割，其靶向性與體外轉(zhuǎn)錄的sgRNA相當(dāng)61。

點(diǎn)擊化學(xué)也被用于標(biāo)記靶基因（稱為sgRNA點(diǎn)擊（sgR CLK））62。該技術(shù)包括在體外轉(zhuǎn)錄的CRISPR-sgRNA的3′端安裝點(diǎn)擊手柄，以形成疊氮化物標(biāo)記的三元復(fù)合物（由dCas9、sgRNA和靶基因組成）。然后通過與炔烴對(duì)應(yīng)物的點(diǎn)擊反應(yīng)實(shí)現(xiàn)該三元絡(luò)合物的功能化。

此外，點(diǎn)擊化學(xué)還用于設(shè)計(jì)一種柔性樹枝狀聚合物，用于傳遞鋅指、TALEs和CRISPR/dCas9平臺(tái)。使用該方法具有高轉(zhuǎn)染效率和較大的處理量63。

9. 包括“點(diǎn)擊發(fā)布”的新應(yīng)用

除了連接，點(diǎn)擊化學(xué)現(xiàn)在正在探索解封或“點(diǎn)擊釋放”應(yīng)用，這使得探針激活和治療傳遞的新策略成為可能64,65,66。例如，利用逆電子需求Diels-Alder-噠嗪消除反應(yīng)在體外和腫瘤小鼠中激發(fā)阿霉素從抗體-藥物結(jié)合物（ADC）中的快速釋放67。

點(diǎn)擊化學(xué)還被用于開發(fā)新的的微芯片和毛細(xì)管系統(tǒng)68，如微流控“點(diǎn)擊芯片”69和基于石墨烯的“點(diǎn)擊芯片”70。此外，“電點(diǎn)擊”接合方法已被用于固定酶（用于生物傳感器）、制備電化學(xué)免疫傳感器以及在空間和時(shí)間上控制蛋白質(zhì)接合71,72,73。

參考文獻(xiàn)

1. Sharpless et al. (2001) Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40(11): 2004-2021.

2. Liu, B., Kenry. (2019) Bio-orthogonal Click Chemistry for In Vivo Bioimaging. Trends in Chemistry 1(8): 763-778.

3. Best, M. D., et al. (2019) Labeling of phosphatidylinositol lipid products in cells through metabolic engineering by using a clickable myo-inositol probe. Chembiochem 20(2): 172-180.

4. Li, H., Aneja, R., Chaiken, I. (2013) Click Chemistry in Peptide-Based Drug Design. Molecules 18(8): 9797-9817.

5. Prescher, J. A., Dube, D. H., Bertozzi, C. R. (2004) Chemical remodelling of cell surface in living animals. Nature 430: 873–877.

6. Agnew B. et al. (2011) Metabolic Labeling and Click Chemistry Detection of Glycoprotein Markers of Mesenchymal Stem Cell Differentiation. In: Vemuri M., Chase L., Rao M. (eds) Mesenchymal Stem Cell Assays and Applications. Methods in Molecular Biology (Methods and Protocols), vol 698. Humana Press.

7. Fantoni, N. F., El-Sagheer, A. H., Brown, T. (2021) A Hitchhiker’s Guide to Click Chemistry with Nucleic Acids. Chemical Reviews Article ASAP.

8. Das, S. R., Paredes, E. (2010) Click Chemistry for Rapid Labeling and Ligation of RNA. ChemBioChem 12(1): 125-131.

9. Lei, X. et al. (2013) A Bioorthogonal Ligation Enabled by Click Cycloaddition of o-Quinolinone Quinone Methide and Vinyl Thioether. J. Am. Chem. Soc. 135(13): 4996-4999.

10. Takayama, Y., Kusamori, K., Nishikawa, M. (2019) Click Chemistry as a Tool for Cell Engineering and Drug Delivery. Molecules 24(1):172.

11. Salic, A., Mitchison, T. J. (2008) A Chemical Method for Fast and Sensitive Detection of DNA Synthesis in Vivo. Proc. Natl. Acad. Sci. USA 105: 2415-2420.

12. Neef, A. B., Luedtke, N. W. (2011) Dynamic Metabolic Labeling of DNA in Vivo with Arabinosyl Nucleosides. Proc. Natl. Acad. Sci. USA 108: 20404-20409.

13. Pickens, C. J. et al. (2018) Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition. Bioconjugate Chem. 29(3): 686-701.

14. Bertozzi, C. R. (2008) In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320(5876): 664-667.

15. Partridge, A. W. et al. (2021) NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay. ACS Chem. Biol. 16(2): 293-309.

16. Devaraj, N. K. et al. (2013) Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. ChemBioChem 14: 205-208.

17. Lemke, E. A. (2014) Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy. Angew. Chem. Int. Ed. Engl 53(8): 2245–2249.

18. Zhang, X. et al. (2015) Second generation TQ-ligation for cell organelle imaging. ACS Chem. Biol. 10: 1676-1683.

19. Chin, J. W. et al. (2014) Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc. 136: 7785-7788.

20. Xie, H. Y. (2017) Integrating two efficient and specific bioorthogonal ligation reactions with natural metabolic incorporation in one cell for virus dual labeling. Anal. Chem. 89(21): 11620-11627.

21. Castro, V., Rodriguez, H., Albericio, F. (2016) CuAAC: An Efficient Click Chemistry Reaction on Solid Phase. ACS Comb. Sci. 18(1): 1-14.

22. Gehringer, M., Forster, M., Laufer, S. A. (2015) Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide-Alkyne Cycloaddition. ACS Comb. Sci. 17(1): 5-10.

23. Chang, Y-T. et al. (2011) Solid phase combinatorial synthesis of a xanthone library using click chemistry and its application to an embryonic stem cell probe. Chem. Commun. 47: 7488-7490.

24. Meier, M. A. R. et al. (2019) Direct comparison of solution and solid phase synthesis of sequence-defined macromolecules. Polym. Chem. 10: 3859-3867.

25. Zhan, P. et al. (2016) Discovery of bioactive molecules from CuAAC click-chemistry-based combinatorial libraries. Drug Discovery Today 21(1): 118-132.

26. Sharpless, K. B. et. al. (2002) Click Chemistry in Situ: Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a Femtomolar Inhibitor from an Array of Building Blocks. Angew. Chem., Int. Ed. 41, 1053– 1057.

27. Oueis, E., Sabot, C., Renard, P.-Y. (2015) New insights into the kinetic target-guided synthesis of protein ligands. Chem. Commun. 51: 12158-12169.

28. Hirsch, A. K. H. et al. (2018) Druggability Assessment of Targets Used in Kinetic Target-Guided Synthesis. J. Med. Chem. 61(21): 9395-9409.

29. Kolb, H. C. et al. (2004) In Situ Click Chemistry: Enzyme Inhibitors Made to Their Own Specifications. J. Am. Chem. Soc. 126(40): 12809-12818.

30. Kolb, H. C. et al. (2005) In Situ Click Chemistry: Enzyme-Generated Inhibitors of Carbon Anhydrase II. Angew. Chem. Int. Ed. 44(1): 116-120.

31. Fokin, V. V. et al. (2006) Inhibitors of HIV‐1 Protease by Using In Situ Click Chemistry. Angew. Chem. Int. Ed. 45(9): 1435-1439.

32. Hirose T, Sunazuka T, Omura S. (2010) Recent development of two chitinase inhibitors, Argifin and Argadin, produced by soil microorganisms. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 86(2):85-102.

33. Dervan, P. B., Poulin-Kerstien, A. T. (2003) DNA-Templated Dimerization of Hairpin Polyamides. J. Am. Chem. Soc. 125(51): 15811-15821.

34. Manetsch, R. (2011) Screening of protein-protein interaction modulators via sulfo-click kinetic target-guided synthesis. ACS Chemical Biology 6(7): 724–732.

35. Heath, J. R. (2009) Iterative in situ click chemistry creates antibody-like protein-capture agents. Angew. Chem. Int. Ed. 48(27): 4944-4948.

36. Heath, J. R. et al. (2013) In situ click chemistry: from small molecule discovery to synthetic antibodies. Integr. Biol. 5(1): 87-95.

37. Deprez, B et al. (2010) Exploring Drug Target Flexibility Using in Situ Click Chemistry: Application to a Mycobacterial Transcriptional Regulator. ACS Chem. Biol. 5: 1007-1013.

38. Fokin, V. V. et al. (2012) Generation of Candidate Ligands for Nicotinic Acetylcholine Receptors Via in Situ Click Chemistry with a Soluble Acetylcholine Binding Protein Template. J. Am. Chem. Soc. 134: 6732-6740.

39. Fernandez-Megia, E. et al. (2012) Click Chemistry for Drug Delivery Nanosystems. Pharm. Res. 29: 1-34.

40. List-Kratochvil, E. J. W. et al. (2020) Utilizing Diels-Alder “click” chemistry to functionalize the organic-organic interface of semiconducting polymers. J. Mater. Chem. C. 8: 3302.

41. Serra, À. et al. (2020) The Use of Click-Type Reactions in the Preparation of Thermosets. Polymers 12(5):1084.

42. Yang, J. et al. (2016) Click Cross-Linking-Improved Waterborne Polymers for Environment-Friendly Coatings and Adhesives. ACS Appl. Mater. Interfaces 8(27): 17499-17510.

43. DeForest, C. A., Anseth, K. S. (2011) Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions. Nat. Chem. 3(12): 925-931.

44. Becker, M. L. et al. (2013) Postelectrospinning “click” modification of degradable amino acid-based poly (ester urea) nanofibers. Macromolecules, 46(24): 9515-9525

45. Chen, H.-Y. et al. (2016) Multifaceted and route-controlled “click” reactions based on vapor-deposited coatings. Biomater. Sci. 4(2): 265-271.

46. Xu, Z., Bratlie, K. M. (2018) Click Chemistry and Material Selection for in Situ Fabrication of Hydrogels in Tissue Engineering Applications. ACS Biomater. Sci. Eng. 4(7): 2276-2291.

47. Shi, L. et al. (2021) Click chemistry-based biopolymeric hydrogels for regenerative medicine. Biomedical materials 16(2): 022003.

48. Fu, Q. et al. (2017) Clickable and imageable multiblock polymer micelles with magnetically guided and PEG-switched targeting and release property for precise tumor theranosis. Biomaterials 145: 138-153.

49. Lin, C. et al. (2020) Bioreducible crosslinked cationic nanopolyplexes from clickable polyethylenimines enabling robust cancer gene therapy. Nanomedicine: Nanotechnology, Biology and Medicine 24: 102144.

50. Ren, L. et al. (2018) Recent trends in click chemistry as a promising technology for virus-related research. Virus research 256: 21–28.

51. de Haan, C. A. et al. (2012) Visualizing coronavirus RNA synthesis in time by using click chemistry. Journal of virology 86(10): 5808–5816.

52. Kalveram B, Lihoradova O, Indran SV, Head JA, Ikegami T. (2013) Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis. Journal of Visualized Experiments: Jove.

53. Miura Y. et al. (2017) Design of glycopolymers carrying sialyl oligosaccharides for controlling the interaction with the influenza virus. Biomacromolecules 18(12):4385–4392.

54. Wang, C., Zhu, W., Wang, B.Z. (2017) Dual-linker gold nanoparticles as adjuvanting carriers for m*lent display of recombinant influenza hemagglutinin trimers and flagellin improve the immunological responses in vivo and in vitro. Int. J. Nanomed. 12: 4747-4762.

55. Donolato, M. et al. (2015) Quantification of NS1 dengue biomarker in serum via optomagnetic nanocluster detection. Scientific Reports 5: 16145.

56. Samitier J. et al. (2015) Label-free electrochemical DNA sensor using "click"-functionalized PEDOT electrodes. Biosens. Bioelectron. 74:751-756.

57. Carell, T. et al. (2020) Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry. ChemBioChem 21(15): 2214-2218.

58. Iyer, K. S. et al. (2017) Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA. Chemical science 8(4): 2923–2930.

59. Chu, Y., Oum, Y. H., Carrico, I. S. (2016) Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction. Virology 487: 95–103.

60. Xie, H. et al. (2012) A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click Chemistry. Analytical chemistry 84: 8364-8370.

61. Brown, T. et al. (2019) An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA. Nat Commun 10: 1610.

62. Srivatsan, S. G. et al. (2020) Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9. J. Am. Chem. Soc. 142(32): 13954-13965.

63. Iyer, K. S. et al. (2017) Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA. Chemical science 8(4): 2923–2930.

64. Carlson, J. C. T., Mikula, H., Weissleder, R. (2018) Unraveling Tetrazine-Triggered Bioorthogonal Elimination Enables Chemical Tools for Ultrafast Release and Universal Cleavage. J. Am. Chem. Soc. 140(10): 3603-3612.

65. Royzen, M. et al. (2016) In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma. ACS Cent. Sci. 2(7): 476-482.

66. Peplow, M. (2019) Click chemistry targets antibody-drug conjugates for the clinic. Nature Biotechnology 37: 835-837.

67. Robillard, M. S. et al. (2016) Triggered Drug Release from an Antibody-Drug Conjugate Using Fast “Click-to-Release” Chemistry in Mice. Bioconjugate Chem. 27(7): 1697-1706.

68. Chen, C. et al. (2019) Click chemistry at the microscale. Analyst 144: 1492-1512.

69. Reichert, D. E. et al. (2015) Development of a microfluidic “click chip” incorporating an immobilized Cu(I) catalyst. RSC Adv. 5: 6142-6150.

70. Aran, K. et al. (2018) Graphene-based biosensor for on-chip detection of bio-orthogonally labeled proteins to identify the circulating biomarkers of aging during heterochronic parabiosis. Lab Chip 18: 3230-3238.

71. Pingarrón, J. M. et al. (2020) Design of electrochemical immunosensors using electro-click chemistry. Application to the detection of IL-1β cytokine in saliva. Bioelectrochemistry 133: 107484.

72. Ono, T. et al. (2019) Enzyme immobilization in completely packaged freestanding SU-8 microfluidic channel by electro click chemistry for compact thermal biosensor. Process Biochemistry 79: 57-64.

73. Shi, X-W. et al. (2013) Protein addressing on patterned microchip by coupling chitosan electrodeposition and ‘electro-click’ chemistry. Biofabrication 5(4): 041001.