Novel Bioconjugate Materials: Synthesis, Characterization and Medical Applications

Abstract

Bioconjugation is a pillar of modern medicine, enabling the likes of targeted therapeutics and sensitive diagnostics by exploiting synergies between biomolecules and functional materials. Conjugation techniques have expanded to match an evolving materials discovery landscape, fueling a new wave of bioconjugates. Despite the breadth of conjugate literature, most reviews describe common and relatively simple substrates such as metal nanoparticles or polymers. This review therefore centers around novel materials including biological (e.g., viral capsids, live cells), hybrid (e.g., gold-decorated nanoparticles, covalent-organic frameworks), and synthetic (e.g., piezoelectrics, upconverting nanoparticles) substrates. Applications in cancer and viral therapy, tissue engineering, optogenetics, antimicrobials, diagnostics, advanced imaging, and related topics are explored, revealing trends in conjugation approach. This review also compares characterization techniques used to confirm and optimize conjugation before offering perspectives on the future of the field. By shedding light on the latest materials, applications, and analytical methods, we hope to empower researchers to harness bioconjugation for transformative medical innovations.

Keywords: bioconjugation; bio‐bio conjugation; diagnostics; nanozymes; novel materials; targeted therapy.

Figure 1

Visual representation of the terms bioconjugation, ligand and substrate. Categories for each are specified in writing; this review focuses on those highlighted in blue. Created in Martin, C. (2025) https://BioRender.com/p19o072.

Figure 2

Examples of novel biological, hybrid and synthetic substrates (and their ligands) that we will highlight in this review. Created in Martin, C. (2025) https://BioRender.com/i7lixcs.

Figure 3

a) Schematic of streptavidin‐GOx and streptavidin‐HRP attached to TMV surface coat proteins (CPs). A 22‐fold excess of biotin was used to conjugate every second cysteine‐modified coat protein (CP_Cys/Bio). Reproduced under the terms of the CC BY license.^{[ 8 ]} Copyright 2015, the Authors. b) Schematic demonstrating the targeting capabilities of LXY30‐ and Cy5.5‐conjugated Hepatitis E VLPs. Reproduced with permission.^{[ 21 ]} Copyright 2016, JoVE.

Figure 4

a) Schematic representation of SPAAC adaptor conjugation and the recruitment of donor DNA through base pairing for improved CRISPR‐Cas9 HDR efficiency. Reproduced under the terms of the CC BY‐NC license.^{[ 25 ]} Copyright 2020, the Authors. b) Disulfide conjugation of Npys Arg₉ peptides for enhanced cell penetration of TALENs.Adapted under the terms of the CC BY 4.0 license.^{[ 30 ]} Copyright 2014, the Authors.

Figure 5

a) Left: schematic demonstrating the extracellular labeling process for HEK293 cells transfected with pDisplay‐LAP‐CFP‐TM. The engineered cells are first ligated with reactive dienes (e.g., MeTz3, or 3‐hydroxymethyl‐6‐methyl tetrazine) via enzyme‐mediated conjugation, which is supplemented with magnesium acetate, ATP, and LplA. DAinv is then performed using TCO‐Prop‐sulfoCy5, contributing a pink fluorescence. Right: fluorescent images of live‐cell‐labelled membrane proteins. The top left image shows Cy5 fluorescence from conjugated cells, while the top right image displays the natural CFP fluorescence of the same cells. Merged images (bottom left and right) indicate a high specificity of conjugation, as demonstrated by the overlap of Cy5 and CFP signals. White arrows in each image indicate nontransfected cells serving as internal negative controls. Scale bar = 25 µm. Adapted with permission.^{[ 35 ]} Copyright 2019, American Chemical Society. b) Schematic diagram of cell surface conjugation and the stepwise formation of multicellular structures. The yellow sheet represents a fibronectin‐coated SIRM made from polydimethylsiloxane (PDMS), grey cells represent SMCs, and green cells represent HUVECs. Reproduced with permission.^{[ 38 ]} Copyright 2013, WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim. c) Top: schematic illustration of live cell biotinylation followed by secondary conjugation with streptavidin‐quantum dots (QDs) and traptavidin‐DNA‐Cy5, respectively. Bottom: fluorescent images of unconjugated neurons are shown on the left hand side (ChR2 was expressed in a construct with enhanced yellow fluorescent protein, or EYFP). The middle images show red fluorescence produced by quantum dot conjugation on the top and Cy5 conjugation on the bottom. Merged images are shown to the right, demonstrating opsin‐specific conjugation. Reproduced with permission.^{[ 39 ]} Copyright 2020, Elsevier Inc.

Figure 6

a) Top: cell capture by antibody ligands. The left‐hand schematic illustrates PCI‐15B cell capture on spider silk conjugated with protein G and anti‐EGFR antibody labelled with green fluorescent Alexa Fluor 488. A confocal image to the right shows PCI‐15B cells loaded with red fluorescent Calcein as they are captured onto the spider silk complex. The right‐hand schematic illustrates HT29 cell capture on spider silk conjugated with protein G and anti‐EpCAM (epithelial cell adhesion molecule) antibody. Cells were loaded with green fluorescent Calcein acetoxymethyl (Calcein AM) to allow visualization (confocal image to the right of the schematic). Control spider silk conjugated with mouse IgG2b antibody showed minimal cell capture (far right), demonstrating the cell‐specific nature of conjugation. Bottom: activity of enzyme ligands. The right‐hand graph shows enzymatic activity of LacZ conjugated to spider silk as measured by 420 nm absorption (corresponding to ONP, the product of ONPG hydrolysis by LacZ), relative to unconjugated spider silk (treated with LacZ but no mTG). The left‐hand graph shows enzymatic activity of Pfs and LuxS as measured by the presence of homocysteine (HCY), the product of LuxS activity, relative to unconjugated spider silk (treated with Pfs and LuxS but no mTG). Adapted with permission.^{[ 46 ]} Copyright 2016, Wiley Periodicals, Inc. b) Schematic illustrating cell capture by the self‐assembled network of spider silk proteins. The enlarged image to the right shows the spider silk conjugates and their captured cells in more detail. Light blue structures attached to the grey spider silk proteins represent azido groups, while the light grey arrows “clicking” into the azido groups represent DBCO. DNA, shown in blue, is attached to the DBCO groups. Attached to the Jurkat cells and shown in red is lipid‐DNA complementary to the spider silk conjugates. Adapted with permission.^{[ 47 ]} Copyright 2022, American Chemical Society.

Figure 7

a) Cellulose bioconjugation using CDAP (SEM image of BNC pictured on the top left). b) Fluorescence microscopy images of HUVECs on unmodified BNC, fibronectin‐adsorbed BNC, and fibronectin‐conjugated BNC after 1, 3 and 8 days of culturing. Cell nuclei stained with DAPI can be seen in blue and actin filaments stained with rhodamine‐phalloidin can be seen in red. As shown, the covalently conjugated surface facilitated the growth of many adhered cells with well‐defined actin filaments, acquiring a cobblestone morphology typical of endothelial cells. Adapted with permission.^{[ 55 ]} Copyright 2013, Elsevier B.V.

Figure 8

Schematic depicting the use of gold‐decorated nanopillar/antibody conjugates for topical antibiotic treatment of infected diabetic wounds. A micrograph (top center) shows a single nanopillar decorated with gold nanoparticles ranging from 2–50 nm. Adapted with permission.^{[ 64 ]} Copyright 2017, American Chemical Society.

Figure 9

a) Schematic depicting the synthesis, decoration and conjugation of hollow carbon sphere nanozymes, with applications in tumor therapy demonstrated using mice models. Reproduced with permission.^{[ 72 ]} Copyright 2020, Elsevier B.V. b) Top: full synthesis process for the nanozyme‐ChOx conjugate. Bottom: schematic representation of the conjugate‐mediated cascade resulting in TMB oxidation for the quantification of cholesterol. Reproduced under the terms of the CC BY license.^{[ 73 ]} Copyright 2023, the Authors.

Figure 10

Schematic depicting the synthesis process for the two‐component COF (COF_HD) and the COF_HD‐GOx conjugate. As can be seen, GOx is attached to the DBA components (shown in blue). Reproduced with permission.^{[ 78 ]} Copyright 2021, Partner Organizations.

Figure 11

Schematic depicting targeted photodynamic therapy by maltotriose‐MOF conjugates for cancer treatment. Reproduced with permission.^{[ 84 ]} Copyright 2021, American Chemical Society.

Figure 12

a) Schematic depicting use cases for the larger Janus nanoparticle. The left‐hand side depicts the Janus nanoparticle in extracellular medium, where it can operate in freestanding mode (using magnetically‐actuated locomotion and steering of the Au/Ni half) or cell‐attached mode when flipped (using G‐protein‐coupled inwardly rectifying potassium channel 2, or GIRK2, antibodies interspersed among barium titanate on the other half). The middle image depicts the neuromodulation process: BTNPs generate piezoelectric potentials in the extracellular fluid ⁽¹⁾, which depolarize the cell membrane ⁽²⁾ to activate voltage‐gated ion channels ⁽³⁾, inducing neural stimulation ⁽⁴⁾. The right‐hand side depicts the orientation that results in maximum neural stimulation, which can be configured through magnetic actuation. Reproduced under the terms of the CC BY 4.0 license.^{[ 91 ]} Copyright 2024, the Authors. b) Top row: confocal images of HeLa cells labelled with primary antibodies specific to HLA class I molecules, followed by labeling with secondary antibody‐piezoelectric conjugates (SHRIMPs). From left to right: SHG‐marked HLA molecules in green, fluorescent calcein AM‐stained cells in red, transmission images, and merged SHG/fluorescent images. Bottom row: control samples in which cells were not labelled with primary antibody. Reproduced with permission.^{[ 92 ]} Copyright 2009, Elsevier Ltd.

Figure 13

Schematic illustrating the self‐evaluation process for photodynamic therapy via caspase‐3‐induced restoration of Cy3 fluorescence. Reproduced with permission.^{[ 103 ]} Copyright 2021, Chinese Chemical Society.

Figure 14

a) Comparison of bone tissue repair after 8 and 16 weeks using a negative control (NC), lone CMC, CMC conjugated to GO, and CMC and BMP‐2 conjugated to GO. As can be seen, the conjugates (and particularly the dual conjugate) significantly accelerate regeneration. b) Schematic illustrating the formation of dual conjugate BMP2‐GO‐CMC combined with stem cell seeding. Adapted with permission.^{[ 126 ]} Copyright 2016, WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 15

Graphic depicting conjugation approaches for material types at large. Note that some of the approaches specified for “inorganics with no standard functional groups” are not expanded upon in this review. Approaches used for biomolecules are grouped so those providing higher specificity are located to the right. The top left corner applies to ligands; the remaining figures apply to substrates.

Figure 16

a) Raman spectra of CdSe/ZnS quantum dots before and after bioconjugation with mouse ovarian cancer antibodies. Colored lines at the top correspond to different bioconjugate samples. Adapted with permission.^{[ 173 ]} Copyright 2012, WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim. b) TGA curves of graphene‐coated iron nanoparticles (Fe@C, black line), Fe@C conjugated with polyethylenimine (PEI) (GEINS1.1, red line), and Fe@C conjugated with PEI under different reaction conditions to GEINS1.1, yielding a higher ligand density (GEINS2.2, blue line). Thermal decomposition of PEI begins around 220 °C and completes around 600 °C. Reproduced with permission.^{[ 82 ]} Copyright 2016, the Royal Society of Chemistry. c) EDX of coenzyme A‐conjugated silver nanoparticles. Top left: SEM micrograph; top middle: silicon; top right: silver; bottom left: phosphorous; bottom middle: sulfur; bottom right: carbon. Overlaps from biological elements such as phosphorous, sulfur and carbon against silver suggest successful conjugation. Reproduced with permission.^{[ 189 ]} Copyright 2023, the Royal Society of Chemistry. d) Left: AFM height images of APTES/succinic anhydride functionalization (top), APTES/glutaraldehyde functionalization (middle), and plasma polymerized acrylic acid (PPAA) functionalization (bottom) of silicon wafers. Right: AFM height images in which protein G is conjugated to each respective functionalized surface. Reproduced with permission.^{[ 195 ]} Copyright 2016, Elsevier B.V.

Figure 17

Schematic illustrating the various ligand orientations as they are referred to in this section. Created in Martin, C. (2025) https://BioRender.com/ibx5ioe.

Figure 18

MTT reduction by mitochondrial hydrogenase and other cellular reductases, leading to the formation of the formazan product (and indicating cell viability via a color change from yellow to purple). Created in Martin, C. (2025) https://BioRender.com/ydqpyig.