RESUMO
We report the fabrication of polyaniline nanofiber (PANI)-modified screen-printed electrode (PANI/SPE) incorporated in a poly-dimethylsiloxane (PDMS) microfluidic channel for the detection of circulating tumor cells. We employed this device to detect melanoma skin cancer cells through specific immunogenic binding of cell surface biomarker melanocortin 1 receptor (MC1R) to anti-MC1R antibody. The antibody-functionalized PANI/SPE was used in batch-continuous flow-through fashion. An aqueous cell suspension of ferri/ferrocyanide at a flow rate of 1.5â¯mL/min was passed over the immunosensor, which allowed for continuous electrochemical measurements. The sensor performed exceptionally well affording an ultralow limit of quantification of 1 melanoma cell/mL, both in buffer and when mixed with peripheral blood mononuclear cells, and the response was log-linear over the range of 10-9000 melanoma cells/10â¯mL.
Assuntos
Técnicas Biossensoriais/instrumentação , Contagem de Células/instrumentação , Melanoma/sangue , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes/patologia , Compostos de Anilina/química , Anticorpos Imobilizados/química , Linhagem Celular Tumoral , Eletrodos , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Melanoma/patologia , Nanofibras/química , Nanofibras/ultraestrutura , Receptor Tipo 1 de Melanocortina/análiseRESUMO
An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.