RESUMO
Highly sensitive, multiplexed detection of soluble cancer protein biomarkers can facilitate early cancer screening as well as enable real-time monitoring of patients' sensitivity and resistance to therapy. Current technologies for detection of soluble cancer protein biomarkers, e.g., enzyme-linked immunosorbent assay, however, suffer from limited sensitivity, as well as the requirement of expensive monoclonal antibodies, which undergo the quality variability. Herein, we propose a sensitive, cheap, and robust surface-enhanced Raman scattering technology to detect a panel of soluble cancer protein biomarkers, including soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1) and soluble epithermal growth factor receptor (sEGFR), which are related to disease progression and treatment efficacy. In this assay, gold-silver alloy nanoboxes that have strong Raman signal enhancement capability were used as plasmonic nanostructures to facilitate highly sensitive detection. In addition, nanoyeast single-chain variable fragments were utilized as mAb alternatives to allow specific and stable protein capture performance. We successfully detected sPD-1, sPD-L1, and sEGFR with a limit of detection of 6.17 pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. We further tested the detection of these three soluble cancer protein biomarkers in human serum and achieved recovery rates between 82.99% and 101.67%. We believe our novel platform that achieves sensitive, multiplexed, and specific detection of soluble cancer protein biomarkers could greatly benefit cancer treatment and improve patient outcome.
Assuntos
Ligas/química , Biomarcadores Tumorais/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Proteínas de Neoplasias/metabolismo , Prata/química , Anticorpos de Cadeia Única/química , Análise Espectral Raman/métodos , Detecção Precoce de Câncer , Ensaio de Imunoadsorção Enzimática , Humanos , Limite de Detecção , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Neoplasias/diagnósticoRESUMO
Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.
Assuntos
Técnicas de Cultura de Células , Descoberta de Drogas , Avaliação Pré-Clínica de MedicamentosRESUMO
Phage display methodologies offer a versatile platform for the isolation of single-chain Fv (scFv) molecules which may be rebuilt into monoclonal antibodies. Herein, we report on a complete workflow termed PhageXpress, for rapid selection of single-chain Fv sequences by leveraging electrohydrodynamic-manipulation of a solution containing phage library particles to enhance target binding whilst minimizing non-specific interactions. Our PhageXpress technique is combined with Oxford Nanopore Technologies' MinION sequencer and custom bioinformatics to achieve high-throughput screening of phage libraries. We performed 4 rounds of biopanning against Dengue virus (DENV) non-structural protein 1 (NS1) using traditional methods (4 week turnaround), which resulted in the isolation of 19 unique scFv clones. We validated the feasibility and efficiency of the PhageXpress method utilizing the same phage library and antigen target. Notably, we successfully mapped 14 of the 19 anti-NS1 scFv sequences (â¼74%) with our new method, despite using â¼30-fold less particles during screening and conducting only a single round of biopanning. We believe this approach supersedes traditional methods for the discovery of bio-recognition molecules such as antibodies by speeding up the process for the development of therapeutic and diagnostic biologics.
Assuntos
Anticorpos Antivirais , Sequenciamento por Nanoporos , Biblioteca de Peptídeos , Anticorpos de Cadeia Única , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Vírus da Dengue/química , Humanos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Proteínas não Estruturais Virais/químicaRESUMO
Whilst recent advances in nanotechnology have yielded many new biosensing capabilities, innovative biological attachment and detection modalities remain relatively underdeveloped. Bi-specific antibodies (bsAbs)--which exhibit binding capability for two separate targets--offer an inherent advantage over conventional antibody reagents by significantly simplifying sensor surface preparation. Herein, we report the deployment of bsAbs for simultaneous attachment to a polymer-coated transducer and label-free, electrochemical (EC) detection of target antigens.
Assuntos
Anticorpos Biespecíficos/metabolismo , Técnicas Biossensoriais , Polietilenoglicóis/metabolismoRESUMO
Targeted nanomaterials promise improved therapeutic efficacy, however their application in nanomedicine is limited due to complexities associated with protein conjugations to synthetic nanocarriers. A facile method to generate actively targeted nanomaterials is developed and exemplified using polyethylene glycol (PEG)-functional nanostructures coupled to a bispecific antibody (BsAb) with dual specificity for methoxy PEG (mPEG) epitopes and cancer targets such as epidermal growth factor receptor (EGFR). The EGFR-mPEG BsAb binds with high affinity to recombinant EGFR (KD : 1 × 10(-9) m) and hyperbranched polymer (HBP) consisting of mPEG (KD : 10 × 10(-9) m) and demonstrates higher avidity for HBP compared to linear mPEG. The binding of BsAb-HBP bioconjugate to EGFR on MDA-MB-468 cancer cells is investigated in vitro using a fluorescently labeled polymer, and in in vivo xenograft models by small animal optical imaging. The antibody-targeted nanostructures show improved accumulation in tumor cells compared to non-targeted nanomaterials. This demonstrates a facile approach for tuning targeting ligand density on nanomaterials, by modulating surface functionality. Antibody fragments are tethered to the nanomaterial through simple mixing prior to administration to animals, overcoming the extensive procedures encountered for developing targeted nanomedicines.