Click Chemistry-Mediated Nanosensors for Biochemical Assays

Abstract

Click chemistry combined with functional nanoparticles have drawn increasing attention in biochemical assays because they are promising in developing biosensors with effective signal transformation/amplification and straightforward signal readout for clinical diagnostic assays. In this review, we focus on the latest advances of biochemical assays based on Cu (I)-catalyzed 1, 3-dipolar cycloaddition of azides and alkynes (CuAAC)-mediated nanosensors, as well as the functionalization of nanoprobes based on click chemistry. Nanoprobes including gold nanoparticles, quantum dots, magnetic nanoparticles and carbon nanomaterials are covered. We discuss the advantages of click chemistry-mediated nanosensors for biochemical assays, and give perspectives on the development of click chemistry-mediated approaches for clinical diagnosis and other biomedical applications.

Keywords: Click chemistry; bio-conjugation; nanosensor; signal amplification system..

Scheme 1

The scheme of click chemistry-mediated nanosensors for biochemical assays. (A) CuAAC combined with Au NPs for detection of Cu (II) and other targets, in which CuAAC is used for signal transformation. The “R-N=N=N-R” represents the ligands of azides, and R-C≡C-R represents the ligands of alkynes (B) Three types of nanosensors based on nanoparticles (quantum dots, QDs; magnetic nanoparticles, MNPs; and graphene) and click chemistry for detection of circulating tumor cells (CTC), pathogens, cancer biomarkers and imaging applications. Click chemistries are employed to functionalize the surface of NPs or used as bio-conjugation strategy. “Tz” presents one ligands of 1,2,4,5-tetrazines, and “TCO” presents another ligands of trans-cyclooctene.

Figure 1

CuAAC-mediated Au NPs-implemented nanosensors for detection of Cu(II) in solution-based assay. (A) Azide-and alkyne-functionalized Au NPs can be triggered to aggregate in the presence of Cu (I) by CuAAC, and the degree of color change of AuNPs is related to the concentration of Cu(II). (B) Schematic depiction of the copper-triggered aggregation of AuNPs for Cu (II) detection. (C) The colorimetric method for detection of Cu (I) based on densely functionalized DNA-Au NP conjugates and CuAAC. (D) The unmodified Au NPs combines with “alkyne-azide” clickable DNA probe for detection of Cu (II). Adapted with permission from [42, 70, 85, 86].

Figure 2

CuAAC-mediated Au NPs-implemented nanosensors for detection of Cu(II) in surface-based assay. (A) The lateral flow test based on “clickable” DNA and Au NPs for detection of Cu (II). (B) A naked-eye biosensor based on silver enhancement signal amplification strategy and CuAAC for highly sensitive detection of Cu (II). Adapted with permission from [87, 88].

Figure 3

CuAAC-mediated Au NPs-implemented nanosensors for detection of reducing agents. (A) CuAAC-mediated nanosensor for detection of the whole protein in which azide-Au NPs and alkyne-Au NPs are triggered to aggregate by CuAAC with the protein and Cu (II). (B) Naked-eye-based colorimetric detection of OPs using CuAAC (OPs: organophosphate pesticides; Au: gold nanoparticles; AChE: Acetylcholineesterase; ATCl: acetylthiocholine; CuO: CuO nanoparticles). (C) CuAAC-mediated nanosensor for detection of the ascorbic acid which can reduce Cu (II) into Cu(I). (D) CuAAC-mediated nanosensor for detection of NO(g) which can reduce Cu(II) to Cu(I) in the redox reaction. Adapted with permission from [73, 89, 90, 91].

Figure 4

CuAAC-mediated Au NPs-implemented nanosensors for immunoassays. (A) Immunoassay based on the CuO-labled antibody, CuAAC and Au NPs as the signal readout. CuO NPs are dissolved to release copper ions that trigger the aggregation of the Au NPs via CuAAC. (B) The scheme of the fluorescence sensor for detection of alpha fetoprotein (AFP) based on CuAAC and CuO NPs. (C) Colorimetric immunoassay based on ALP-triggered CuAAC between azide/alkyne functionalized Au NPs. Adapted with permission from [92, 93, 15].

Figure 5

Click chemistry-mediated functionalization of quantum dots to label cancer cells. (A) Tetrazine-functionalized EGF can bind to the surface of cancer cells that overexpressed EGFR, and the nonbornene-coated QDs (green circles) can react with tetrazine-modified EGF to form the QDs conjugates for targeted imaging. (B) Cyclooctyne-modified Fe₂Tf has been attached to azide-modified water-soluble CdSe/ZnS QDs for monitoring the uptake of fluorescent QD-transferrin conjugates in transferrin-receptors (TfR) expressing tumor cells. Adapted with permission from [35, 116].

Figure 6

Click chemistry-mediated functionalization of quantum dots. (A) Schematic illustration of the one-step synthesis of the azido-derivatized multidentate-imidazole polymer ligands (N₃-PMAH) along with the structures of QDs protected by the multidentate ligands, and the general strategy for the specific labeling of viruses with QDs via strain-promoted metal-free click chemistry. (B) Bioorthogonal labeling of H5N1p with NIR QDs and particle sizes of QDs, H5N1p, and QD-labeled H5N1p (QD-H5N1p). Adapted with permission from [120, 121].

Figure 7

Click chemistry-mediated functionalization of SMNPs. (A) The magnetic relaxation switch (MRS) sensor based on SMNPs and a two -step bioorthogonal reaction strategy for diagnosis of tumor cells. (B) Detection of pathogenic bacterium in blood samples based on SMNPs and layer-by-layer “bioorthogonal reaction” signal amplification strategy. Adapted with permission from [46, 142].

Figure 8

Click chemistry-mediated functionalization of carbon nanomaterials. (A) Schematic illustration of sequential functionalisation of GO using two-step CuAAC click reactions. (B) Schematic representation for conjugation of graphene onto Au surface via π-π interaction and CuAAC click reaction. (C) Illustration for the process of the bio-conjugation between the azide-SWNTs and alkyne-anti-IgG antibody by CuAAC. The anti-IgG antiobody-functionalized SWNTs conjugation can be used as signal recognition system in the immunosensor. (D) Schematic illustration of bio-conjugation of Fe₃O₄ NPs to the surface of multi-carbon nanotube nanomaterials by CuAAC. Azide-Fe₃O₄ can bind to the surface of alkyne- multi-carbon nanotube nanomaterials to prepare multifunctional nanoparobes, which has great potential in the fields of in vivo imaging and in vitro diagnosstics. Adapted with permission from [155, 156, 150, 157].