An organic substrate based magnetoresistive sensor for rapid bacteria detection

Abstract

A point-of-care diagnostic system has been developed to detect pathogenic bacteria rapidly, of which system contains a magnetoresistive (MR) sensor in cooperation with a magnetic bead coated by specific antibody against bacteria. MR sensor with Teflon passivation layer has been fabricated on organic substrate, being flexible and low cost material, and passivated by Teflon layer for maintaining flexibility. The performance of the MR sensor is demonstrated using Magnetospirillum magneticum AMB-1 and its detection limit was found to be 1.3×10⁸ cells/ml. Further, Escherichia coli is captured by immobilised anti-E. coli antibodies on the surface of the sensor and detected using magnetic bead labelled with anti-E. coli antibody. The detection limit of E. coli was found to be 1.2×10³ cells/ml. The technique is simple, rapid, sensitive and does not require pre-treatment of the sample and can detect a variety of microorganisms. The high performance of sensor fabricated on flexible organic substrate may allow its future use for bio-applications in implantable types of devices.

Introduction

Detecting pathogenic bacteria in food, water or clinical samples traditionally requires culturing the bacteria in different media, observing and confirming the pathogen by microscopy or biochemical and serological tests. Although the traditional methodology is precise, it is laborious, time-consuming, costly and requires highly trained personnel. Biosensors, which combine biological recognition and physical transduction, are the best alternative option (Lu et al., 2008). Biosensors either eliminate or simplify sample preparation, and are also highly sensitive and specific, where they may be optical (Cooper Matthew, 2002), mechanical (Arlett et al., 2011), thermal (Ramanathan and Danielsson, 2001), acoustic (Rocha-Gaso et al., 2009), electrochemical (Wang, 2005), piezoelectric (Muramatsu et al., 1987) or magnetic biosensors (Baselt et al., 1998).

Sensing methods rely on nucleic acid or antibody detection. Nucleic acid biosensors detect either DNA or RNA from target cells. In this process, polymerase chain reaction (PCR) is required to amplify the target nucleic acid sequence; the signal arises from the hybridisation of the probe and the target DNA (Heo and Hua, 2009). The basis for immunosensing is the interaction between the antigen of the target cell and a surface-immobilised antibody. This antigen–antibody interaction can be detected using fluorescence (Lazcka et al., 2007), electrochemical impedance (Yang and Bashir, 2008), a quartz crystalline microbalance (Skottrup et al., 2008) and by various magnetic methods (Mujika et al., 2009, Xiao et al., 2008, Fal-Miyar et al., 2008, Blanc-Beguin et al., 2009, Chen et al., 2011).

Sensitivity is a major issue for all rapid methods, which can be improved by a pre-concentration step. Immunofunctionalised magnetic particles/beads and immunomagnetic separation have been widely used in biosensing applications (Xiao et al., 2008, Fal-Miyar et al., 2008, Blanc-Beguin et al., 2009, Chen et al., 2011). Bacterial detection with magnetic beads is usually based on the classical sandwich assay format, where magnetic separation is usually followed by various detection techniques, such as colorimetric or various fluorescent and chemiluminiscent methods. Magnetic beads, which can bind specifically to biomolecules, are currently used in diagnostic procedures. The simplicity of their use in separation assays and their potential for use in automated processes (Mujika et al., 2009) also makes them ideal for other research applications.

Magnetoresistive (MR) sensors are promising for many applications in both industrial and basic academic research as well as for point-of-care diagnostics, because they are highly sensitive, easily scalable, inexpensive, portable and use little power. To date, magnetic beads have been used to detect pathogens in dilute samples. There is a dearth of reports on the use of magnetic sensors for the detection of bacteria. Hitherto, various highly sensitive MR sensors were successfully developed for biochip applications (Besse et al., 2002, Gaster et al., 2011, Oh et al., 2011a, Mujika et al., 2009). Single biomolecule resolution is possible when the biomolecule-labelled magnetic particles/beads can be precisely controlled and placed on the sensor surface. The sensor infers the presence of magnetic beads by detecting magnetic fringe fields from the magnetic beads, with the number of detected beads indicating the concentration of the specific target. Sensors that are able to effectively detect magnetic beads, may also be compact and interface with automated systems, thereby providing a rapid and easy-to-use point-of-care diagnostic tool (Lee et al., 2009, Schotter et al., 2009).

Compared with other detection methods, such as those based on fluorescent markers or electrochemical measurements, magnetic beads that are coupled with MR sensors can detect biomolecules at very low sample concentrations and have an extensive linear dynamic range (Gaster et al., 2009). Additionally, other compounds present in the test samples do not show any magnetic behaviour, thus minimising the noise and possible interference in the measurements by the MR sensor (Larsson et al., 1999).

Flexible devices have garnered significant interest over the last decade, because they can assume different geometries, which will be useful for a variety of new applications (Fernández et al., 2012, Chen et al., 2008). Magnetic sensor devices on flexible substrates could enable the fabrication of smart biomedical systems, where large-angle folding of micrometer-sized functional elements is required for successful implementation (Melzer et al., 2011). Notably, there are no reports of a flexible MR sensor operating in solution state which is pre-requisite condition for real implantable types of devices.

In this work, we developed a MR sensor (PHR: Planar Hall Resistance) with Teflon passivation layer coated on organic substrate, that can be used to detect the bacteria. To check the sensor performance, Magnetospirillum magneticum AMB-1 (magnetotactic bacteria) is detected using MR sensor on the basis of stray field from the magnetosomes present within the bacteria. Escherichia coli is the pathogenic bacteria employed in the chemical binding method. E. coli is the most extensively used pathogen in biosensor development because it is ubiquitous and known to be responsible for various food and water-transmitted infections (Laczka et al., 2011).