A disposable immunosensor device for point-of-care test of tumor marker based on copper-mediated amplification

Abstract

A paper-based immunodevice was fabricated for point-of-care test (POCT). The array device was simple and easily assembled. It comprised as many as 4×10 detection points on a single paper array. The immunosensor array was prepared by covalently immobilizing capture antibodies on corresponding working zone on a disposable paper array. With a sandwich-type immunoreaction, the CuO nanoparticles (CuO NPs)-labeled secondary antibody (Ab₂) bioconjugates were captured in each working zone. The coordination of dithizone (DZ) at the surface of CdTe quantum dots (CdTe QDs) could strongly quench the green emission of CdTe QDs by a fluorescence resonance energy transfer (FRET) mechanism. After the Cu²⁺ was released from CuO NPs-Ab₂, the fluorescence of CdTe QDs–DZ was “turn-on”. The fluorescence intensity would increase with the increasing of analytes. The calibration plot showed a good linear relationship between the fluorescence intensity and the logarithm value of the analytes concentration with the low detection limit. The immunosensor array was performed for cancer screening. The high throughput, low-cost, acceptable stability, reproducibility, sensitivity and accuracy showed good applicability of the proposed multiplex immunoassay in clinical diagnosis. The results indicated that the device could be applied to comprehensive sample and point-of-care detection.

Introduction

The sensitive detection of cancer biomarkers plays a crucial role in early disease diagnosis, cancer screening, disease recurrence, and therapeutic treatment efficacy (Ahn et al., 2012, Sardesai et al., 2011, Huang et al., 2011). Conventional detection methods for cancer biomarkers include enzyme-linked immunosorbent assay (Laing et al., 2011), surface-enhanced Raman scattering (Wang et al., 2011), chemiluminescence (Fu et al., 2008), electrophoretic (Zhang et al., 2012), and mass spectrometric immunoassays (Hu et al., 2007). However, these conventional immunoassays method are complicated, time-consuming, expensive, and labor-intensive. Moreover, these methods are not suitable for point-of-care test (POCT). Thus, it is urgently needed to develop an accurate, highly sensitive, inexpensive, and rapid detection method for the POCT. Considerable efforts have been made worldwide to develop and improve clinical immunoassays with the aim of making portable and affordable devices (Zong et al., 2012, Akter et al., 2012, Ge et al., 2012c). As an alternative to the conventional immunoassay procedures, the fluorescence immunosensors (Yan et al., 2012b, Qian et al., 2010) receive a particular attention because of its simple operation, fast response time, high throughput and POCT.

To improve the detection efficiency, multianalyte assays can be performed in two dominant modes: multilabel modes (Tang et al., 2011, Lai et al., 2011) and spatial-resolved modes (Zong et al., 2012, Deiss et al., 2009, Luchansky and Bailey, 2011, Wu et al., 2012). Multilabel mode needs to lable different signal molecule, and simultaneously detects various signal, therefore, often gets into trouble in specificity because of the signal cross interference and the differences of assay conditions. To overcome these shortcomings, several multilabel resolution strategies have been reported (Fu et al., 2006, Fu et al., 2007). However, it is difficult to obtain the simultaneous detection of different protein markers due to the limited available labels. Spatial-resolved mode with a single label can simultaneously detect different targets in different immunoreaction areas on one substrate. Various spatial-resolved arrays, such as electrochemical (Wu et al., 2012, Wilson and Nie, 2006) and optical (Zong et al., 2012, Luchansky and Bailey, 2011) sensor arrays, have appeared in the past decade.

The paper-based analytical device has been reported in our lab, which not only retains the simplicity, low-cost, portability and disposability of paper-based analytical devices, but also provides new opportunities and directions in the development of precise and sensitive diagnostic devices (Wang et al., 2012b, Ge et al., 2012a, Yan et al., 2012a, Zang et al., 2012). Research in this area has aimed at developing simple, inexpensive, portable, disposable, and easy-to-use POCT platforms for developing countries, resource-limited, and remote regions. This technology holds great promise and it has gained more and more attention and interest over recent years. Protein array-based biosensing is a wide attention research area with strong prospect for the development of novel, multiplexed diagnostic assays. In this technology, fluorescent signals are used to estimate analyte concentration after each assay step. Quantum dots (QDs) offer superior fluorescent properties that can be applied for optical biosensing (Crivat et al., 2010, Biju et al., 2012, Sarkar et al., 2010, Jiang and Zhang, 2010, Jiang et al., 2009). QDs have been applied into many assays by fluorescence resonance energy transfer (FRET). QDs-FRET assays have been reported for the detection of a wide variety of analytes ranging from nucleic acids, proteins, and drugs, to biological processes such as phosphorylation and proteolysis. However, the assembly of FRET-based QDs sensors is usually a complicated chemical process involving the modification of the QDs surface, the immobilization of biological receptors, and the adsorption of a chosen dye quencher. Therefore, the QDs fluorescence “turn-on” assay for the detection of analytes is achieved only under a harsh condition and is never reported for the cancer screening in a paper array. “turn-on” assay can greatly improve the sensitivity due to the efficient decrease of background signals from probe signals themselves (Lu et al., 2010, Xia et al., 2011, Yin et al., 2010).

In this work, dithizone was thus bound onto the surface of CdTe QDs through a surface coordinating reaction. The fluorescence of CdTe QDs was quenched by a FRET due to the spectral overlap between the emission of CdTe QDs and the absorption of the surface dithizone (DZ)–Cd complex. The CuO NPs-Ab₂ was captured on the array by sandwich immunoreaction. Then, Cu²⁺ was released into the sensing sites by HCl, which resulted in the fluorescence recovery of CdTe QDs–DZ. The enzyme-free immunoarray based on a hybrid CdTe QDs–DZ was a newly developed analytical method for the rapid and high-throughput biological assay, with the advantages of high sensitivity and good selectivity immunoassay. The enzyme-free immunoassay could bear the harsh environment, save cost and time. Combined with the paper array, the immunosensor was able to simultaneously detect multiple analytes. Preliminary results demonstrated that this immunosensor could be used to screen tumor marker for POCT.