Disposable microfluidic immuno-biochip for rapid electrochemical detection of tumor necrosis factor alpha biomarker

Highlights

• An electrochemical immuno-biochip for TNFα biomarker has been developed.

• Integration of fluidics and sensing parts in a disposable design.

• A detection limit of 4.1 ng/mL has been achieved.

• Successful determination in human serum has been demonstrated.

Abstract

This paper presents a disposable microfluidic electrochemical immunosensor for rapid, cheap and quantitative detection of biomarkers. Dual screen-printed carbon electrodes were biofunctionalized with specific antibodies and subsequently encapsulated with an all-disposable polymeric microfluidic cell. The electrochemical detection was carried out by means of differential pulse voltammetry (DPV) using a portable potentiostat. One of the two working electrodes was employed as an on-chip integrated negative control. The system was optimized and characterized for the detection of tumor necrosis factor alpha (TNFα), an important inflammation biomarker, with a limit of detection (LOD) of 4.1 ng/mL. Successful experiments in real human serum were also carried out.

Introduction

The development of cheap and fully disposable lab-on-a-chip devices for specific protein immunosensing is highly desirable because of their wide application spectrum in clinical diagnosis [1], as well as in environmental [2] and food analysis [3]. Among the many protein quantification methods, electrochemical techniques stand out due to high sensitivity, fast analysis, low cost, multiplexing possibilities and potential miniaturization [4]. The use of screen printed electrodes (SPEs) is especially interesting from the application point of view due to their mass-production possibilities with high inter-batch reproducibility at a very low cost [5]. The combination of the sensing parts with microfluidics is also attractive because it offers direct control over low sample volumes and potential integration of preparation steps. Low-cost disposable polymeric microfluidics are especially desirable when working with real clinical samples because cumbersome cleaning protocols and cross-contamination are avoided.

Tumor necrosis factor alpha (TNFα) is a widely-studied pleiotropic cytokine with a central role in many immune and inflammatory processes. This important multifunctional protein biomarker is involved in several high-incidence high-impact diseases such as inflammatory bowel disease, rheumatoid arthritis and many other inflammatory processes including cancer [6]. Interestingly, several biological drugs acting as TNFα activity blockers have proved their effectiveness [7]. Elevated levels of TNFα in serum are found in patients suffering some kind of the above-mentioned diseases (see, e.g., [7], [8], [9]).

The gold standard for TNFα assaying, as for many other protein biomarkers, is the enzyme-linked immunosorbent assay (ELISA). However, for a long time, the disadvantages of this method (e.g., long assaying times, large sample volumes or the necessity of expensive equipment and trained personnel) have motivated research on a wide variety of different formats such as optical [10], [11], [12], acoustic [13] or electrochemical biosensors [14], [15], [16], [17], [18], [19] which also present potential for integration and miniaturization.

In previous papers, we have reported highly-sensitive TNFα biosensors based on surface plasmon resonance [10] and amperometric magnetoimmunoassay [14] demonstrating several advantages over the conventional ELISA. However, more research must be carried out towards reducing the inherent complexity of the assays (including laborious protocols, use of expensive and critical instrumentation, long incubation times, etc.). As a result, the development of rapid, simple and sensitive methods for biomarker detection has become crucial for their use in real clinical applications. A cost-effective and disposable lab-on-a-chip can incorporate all this characteristics in a single device which is considered the best contender technology for this task [20].

In this paper we report the development of a simple and fully disposable lab-on-a-chip for electrochemical detection of TNFα. The low-cost chips integrate specifically modified electrodes for signal transduction and polymeric fluidics for sample control. The specificity, limit of detection (LOD) and linear range of the immunoassay have been characterized and TNFα in 25% spiked human serum samples has been successfully quantified.