RESUMO
The quantitative sensitivity and dynamic range of conventional immunohistochemistry (IHC) with 3,3'-diaminobenzidine (IHC-DAB) used in pathological diagnosis in hospitals are poor, because enzyme activity can affect the IHC-DAB chromogenic reaction. Although fluorescent IHC can effectively increase the quantitative sensitivity of conventional IHC, tissue autofluorescence interferes with the sensitivity. Here, we created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs have 100-fold greater brightness and a more than 300-fold greater dynamic range than those of commercially available fluorescent nanoparticles, quantum dots, whose fluorescence intensity is comparable to tissue autofluorescence. Additionally, a newly developed image-processing method enabled the calculation of the PID particle number in the obtained image. To quantify the sensitivity of IHC using PIDs (IHC-PIDs), the IHC-PIDs method was compared with fluorescence-activated cell sorting (FACS), a method well suited for evaluating total protein amount, and the two values exhibited strong correlation (R = 0.94). We next applied IHC-PIDs to categorize the response to molecular target-based drug therapy in breast cancer patients. The results suggested that the PID particle number estimated by IHC-PIDs of breast cancer tissues obtained from biopsy before chemotherapy can provide a score for predicting the therapeutic effect of the human epidermal growth factor receptor 2-targeted drug trastuzumab.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Diagnóstico por Imagem/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Rodaminas/química , 3,3'-Diaminobenzidina/química , Anticorpos/química , Antineoplásicos Imunológicos/uso terapêutico , Biópsia , Biotina/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diagnóstico por Imagem/instrumentação , Feminino , Fluorescência , Expressão Gênica , Humanos , Imidas/química , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tamanho da Partícula , Perileno/análogos & derivados , Perileno/química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Estreptavidina/química , Trastuzumab/uso terapêuticoRESUMO
In this study, we report label-free detection of alpha-fetoprotein (AFP), which has been used as a biomarker for hepatocellular carcinoma, by a microfluidic reflectometric interference spectroscopy (RIfS) system adopting a simple halogen light source and an inexpensive silicon-based sensor chip. Introduction of carboxy groups on a silicon nitride sensor chip to immobilize anti-AFP monoclonal antibody (anti-AFP) was carried out simply by immersion in aqueous solution containing triethoxysilylpropylmaleamic acid bearing a carboxy group and a silanol group. The RIfS system with the anti-AFP-immobilized sensor chip was found to give a reversible response through 100 on/off cycles using a regeneration buffer with high reproducibility (coefficient of variation (CV) = 5.7%). The limit of detection (LOD) of AFP was 100 ng mL(-1), and the measurement range spanned 3 orders of magnitude. Furthermore, the sensor chip showed no cross-reactivity with human serum albumin, Immunoglobulin G, transferrin, or fibrinogen at 100 µg mL(-1) without the use of blocking reagents such as bovine serum albumin. Consequently, the proposed RIfS system is a potentially effective tool for biomarker detection and in vitro diagnostics.
