ACS Appl Mater Interfaces. 2016 Aug 17;8(32):20570-82.

Microfluidic Immuno-Biochip for Detection of Breast Cancer Biomarkers Using Hierarchical Composite of Porous Graphene and Titanium Dioxide Nanofibers.

Md. Azahar Ali, Kunal Mondal, Yueyi Jiao, Seval Oren,Zhen Xu,Ashutosh Sharma,§ and Liang Dong,

  • Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa 50011, United States
  • Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States
  • §Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India

 

Abstract

We report on a label-free microfluidic immunosensor with femtomolar sensitivity and high selectivity for early detection of epidermal growth factor receptor 2 (EGFR2 or ErbB2) proteins. This sensor utilizes a uniquely structured immunoelectrode made of porous hierarchical graphene foam (GF) modified with electrospun carbon-doped titanium dioxide nanofibers (nTiO2) as an electrochemical working electrode. Due to excellent biocompatibility, intrinsic surface defects, high reaction kinetics, and good stability for proteins, anatase nTiO2 are ideal for electrochemical sensor applications. The three-dimensional and porous features of GF allow nTiO2 to penetrate and attach to the surface of the GF by physical adsorption. Combining GF with functional nTiO2 yields high charge transfer resistance, large surface area, and porous access to the sensing surface by the analyte, resulting in new possibilities for the development of electrochemical immunosensors. Here, the enabling of EDC−NHS chemistry covalently immobilized the antibody of ErbB2 (anti-ErbB2) on the GF−nTiO2 composite. To obtain a compact sensor architecture, the composite working electrode was designed to hang above the gold counter electrode in a microfluidic channel. The sensor underwent differential pulse voltammetry and electrochemical impedance spectroscopy to quantify breast cancer biomarkers. The two methods had high sensitivities of 0.585 μA μM−1 cm−2 and 43.7 kΩ μM−1 cm−2 in a wide concentration range of target ErbB2 antigen from 1 × 10−15 M (1.0 fM) to 0.1 × 10−6 M (0.1 μM) and from 1 × 10−13 M (0.1 pM) to 0.1 × 10−6 M (0.1 μM), respectively. Utilization of the specific recognition element, i.e., anti-ErbB2, results in high specificity, even in the presence of identical members of the EGFR family of receptor tyrosine kinases, such as ErbB3 and ErbB4. Many promising applications in the field of electrochemical detection of chemical and biological species will derive from the integration of the porous GF−nTiO2 composite into microfluidic devices. 

PMID: 27442623

 

Supplementary

Overexpression (∼30%) of several receptor tyrosine kinases in epidermal growth factor receptor 2 (ErbB2) is associated with increasing breast cancer metastasis [1]. The ErbB2 gene belongs to the mammalian EGFR family and encodes a 185 kDa transmembrane glycoprotein and a receptor tyrosine kinase with intrinsic tyrosine kinase activity. The excessive signaling of ErbB2 is a critical factor in the development of malignant cancerous tumors. A recent statistical survey [2] shows that in the U.S., the second most common cancer is the breast cancer after skin cancer, and a huge number of cases (246,660) for invasive breast cancer are predictable to be diagnosed along with 61,000 cases for non-invasive breast cancer in women. Though several methods such as enzyme-linked immunoabsorbent assay (ELISA), immunoblotting, and immunohistochemistry have been greatly employed for the detection of cancer biomarkers, they require complex purification and pretreatment processes or are used as semi-quantitative methods. In addition, X-ray mammography, ultrasonic and magnetic resonance imaging techniques are most widespread tools for detection of breast cancer. However, their early diagnostics, expatiations, and costs are major concerns. Therefore, low-cost, high-sensitivity, point-of-care devices for the rapid detection of breast cancer biomarkers at an early stage are extremely desirable for clinical diagnosis and treatment.

A team of researchers at the Laboratory of MEMS and Biochips at Iowa State University, in collaboration with the North Carolina State University and the Indian Institute of Technology Kanpur, developed a new biosensor for detection and quantification of breast cancer biomarkers. The sensor utilizes porous graphene foam modified with titanium dioxide nanofibers as an immunosensor bio-scaffold integrated inside a microfluidic channel. The interior and exterior surfaces of the bio-scaffold is functionalized with a specific bio-recognition antibody of ErbB2 via antigen–antibody interactions (Fig. 1). The bio-scaffold is placed above and in parallel with the counter and reference electrodes in the channel. This immunosensor is coupled with electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) to output signals associated to the specific antigen-antibody interactions at the sensor.

 

 

fig1

Figure 1 (a) Schematic of the microfluidic immunosensor with porous GF electrode modified with carbon-doped titanium oxide nanofibers for the detection of breast cancer biomarkers. (b) Photo of two microfluidic immunosensors.

 

Their measurement results (Fig. 2) have demonstrated high sensitivities of 0.585 μA μM−1 cm−2 and 43.7 kΩ μM−1 cm−2 for the EIS and DPV methods, respectively, in a concentration range from 1 femtomolar to 100 nanomoles of ErbB2 antigens. The limit of detection concentration is one femtomolar, which is 500 times lower than that of the conventional ELISA method for breast cancer biomarker detection [3]. The sensor has also demonstrated high selectivity in presence of nonspecific antigen molecules such as ErbB3 and ErbB4. The sensor requires about ten microliters of analyte solution, and can complete measurements within three minutes. The high performances of the sensor are originated from the new sensing electrode design with porous graphene foam and titanium oxide nanofibers. The graphene foam provides large surface area, superior electroactivity, and efficient conduction paths for rapid generation and transfer of electrons due to the detection of specific biomarkers. The high porosity of GF also makes it suitable for integration with titanium oxide nanofibers to obtain enhanced surface area, chemical stability, and loading capacity of recognition molecules, thus improving the detection efficacy of the device.

 

 

dl-fig2-jpg-2

Figure 2 (a) DPV response curves of the microfluidic immunosensor in detecting different ErbB2 concentrations. (b) Response calibration plot of the peak DPV current as a function of ErbB2 concentration. (c) EIS spectra of the microfluidic immunosensor with the BSA/anti-ErbB2/GF–nTiO2 electrode for detecting ErbB2 at different concentrations. (d) Response calibration plot of Rct as a function of [ErbB2]. The linear test range of [ErbB2] was 1 fM–0.1 mM.  In both the DPV and EIS measurements, [ErbB2] ranged from 0.1 µM to 1.0 fM in PBS solution of pH 7.4 and incubation time of 5 min. In each experiment, ErbB2 was exposed to the sensor and then washed with PBS solution. The error bars were derived from the relative standard deviation of three measurements at each concentration.

 

References

[1] Zhang, H., Berezov, A., Wang, Q., Zhang, G., Drebin, J., Murali, R., Greene, M. I. (2007) ErbB receptors: from oncogenes to targeted cancer therapies. J Clin Invest 117, 2051–2058.

[2] http://www.breastcancer.org/symptoms/understand_bc/statistics.

[3] Gauchez, A. S., Ravanel, N., Villemain, D., Brand, F. X., Pasquier, D., Payan, R., Mousseau, M. (2008) Evaluation of a manual ELISA kit for determination of HER2/neu in serum of breast cancer patients. Anticancer Res 28, 3067-73.

 

 

 

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