Antibody Quantum Dot Conjugates Developed via Copper-Free Click Chemistry for Rapid Analysis of Biological Samples Using a Microfluidic Microsphere Array System

Abstract

Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features towards realization of this goal such as high photostability, brightness and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation and stability issues. We report a facile yet effective strategy to conjugate anti epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin-streptavidin schemes. The Fc and Fab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/ml. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood.

Quantum dot surface engineering: Toward inert fluorophores with compact size and bright, stable emission

“…Much more controllable reactions can result from “click” chemistry approaches that can allow the generation of conjugates without the need for coupling reagents or catalysts [270]. Norbornene-tetrazine cycloaddition reactions [217, 218] and strain-promoted azide-cyclooctyne cycloaddition reactions [189, 273] have been found to be fully compatible with QDs.…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…[42] The specific, fast, efficient, stable, facile, modular and aqueousphase conjugation strategy of the various classes of "click" reactions has proven to be a reliable and powerful technique that is employed widely in nanomaterials chemistry. [47] Click chemistry mainly involves reactions of high stereospecificity and conjugation efficiency occurring under a wide range of reaction conditions. The copper I catalyzed alkyne-azide cycloaddition (CuAAC) was developed by the Sharpless and Meldal groups independently in 2002, one of the widely used click chemi stry reaction.…”

“…Such Cufree reactions are typically undertaken at room temperature using strained alkynes such as dibenzocyclooctyne (DBCO) and azides which allow highly selective reaction, resulting in high yields with minimal purification steps (Figure 7). [47] The toxicity issues associated with Cu catalyst-assisted click reaction between azide and alkyne functional groups can be minimized by complexing the Cu using a chelator such as (tris[(4-carboxyl-1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine). [45,46,92] It has also been reported that CuAAC for NP-biomolecule coupling reactions can lead to the protein modification or its complete degradation.…”

“…The conjugates were tested in human breast cancer tissue and showed significant sensitivity for detection of EGFR. [47] An advantage of the click chemistry strategy is that the reaction can be conducted in parallel with other conjugation strategies owing to the orthogonality of the chemistries and functional groups involved. This can be important when specific functionalities are required on nanoparticle surfaces.…”

The stereodynamics of macrocyclic succinimide‐thioethers

“…The formation of succinimide thioethers via thiol 1,4‐addition is frequently used to form bioconjugates or to immobilize bioactive compounds on surfaces . It is the preferred method for the formation of antibody‐drug conjugates .…”