Abstract
The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters. Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne–azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.
Highlights
The synthesis of dibenzocyclooctyne (DBCO)-functionalised carbon dots (CD) 2 ready to be conjugated to azido-decorated Abs started from acid functionalised CD 3, which were prepared in one pot from citric acid and ethylenediamine under microwave irradiation following a modi ed procedure by Mondal et al.[16] (Fig. 2A)
To demonstrate the versatility of our CD-based Ab labels for diagnosis applications, we have examined Glial Fibrillary Acid Protein (GFAP) in 13 formalin- xed paraffin embedded biopsy brain tumour samples from different patients (12 glioblastoma, IDH wildtype, WHO Grade 4 and 1 negative control schwannoma, WHO Grade I, see Electronic supplementary information (ESI): Table S3†) using our conjugated antibody 13a (Fig. 5)
The two-step strategy relies on the used DBCOfunctionalised CDs and azide-functionalised proteins that can be prepared by simple amide conjugation methods from suitably functionalised linkers with control over degree of functionalization
Summary
Overall less than 20% of brain tumour patients are alive 5 years a er diagnosis, in part because they present late with large inoperable tumours.[1]. There is an urgent need to develop new sensitive tests of brain tumours to help general practitioners in primary care.[2]. The most common malignant primary brain tumour called glioblastoma is characterised by abnormal blood vessels resulting in a leaky Blood–Brain Barrier (BBB).[3]. Glial Fibrillary Acid Protein (GFAP) is unique to the brain and not present in normal peripheral blood. There is evidence that GFAP crosses the leaky BBB and is an early nonspeci c peripheral blood biomarker which predates the clinical diagnosis of glioblastoma.[4]. GFAP levels are too low for routine detection by commonly used protein diagnostic tests such as ELISA, and more sensitive methods for its identi cation are needed.[5]
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