DNA-probe-target interaction based detection of Brucella melitensis by using surface plasmon resonance

Abstract

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (K_D) and maximum binding capacity of analyte (B_max) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (K_D) and 81.02 m° (B_max) with probe 1 and 54.9 pM and 55.29 m° (B_max), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10 min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications.

Introduction

The recent bioterrorist attacks and its escalating threat have generated the urgent demand for rapid, field-deployable, and cost effective methods for the detection of biological warfare agent (BWA) in a simpler and efficient manner (Orozco et al., 2015, Adducci et al., 2016, Singh et al., 2012). Several BWA are available, among them Brucella melitensis (B. melitensis) is one of the potential BWA which causes the brucellosis and this can be transmitted from animals to human beings (Wheelis et al., 2006, Inglesby et al., 2002; Thavaselvam et al., 2010). Therefore, reliable and rapid identification of B. melitensis is highly desirable amidst the current climate of bioterrorism. B. melitensis is the gram-negative, facultative and intracellular bacteria in the shape of cocci, coccobacilli or short rods, 0.5- 0.7 μm in diameter and 0.6–1.5 μm in length. Brucellosis is a major animal infection which founds various parts of the world (particularly in the Middle East, South and Central Asia, North and East Africa, Mediterranean countries of Europe, Central and South America) (Thavaselvam et al., 2010; Pappas, et al., 2006). Brucellosis has been recognized by several terms such as Malta fever, Gibraltar fever, Bang’s disease, undulant fever and by name typhomalarial fever because of its similarity to malaria and typhoid like fevers.

Although numerous analytical methods and techniques were developed for the detection of Brucella species such as ELISA (Chikweto et al., 2013), PCR (Tomaso et al., 2010, Al-Dahouk et al., 2007, Khosravi et al., 2006), combinatorial PCR (Mirnejad et al., 2012), conventional RT-PCR (AL-Garadia et al., 2011), and combination of PCR and ELISA ( Al-Dahouk et al., 2004). However, the practical application of these methodologies are limited by the low sensitivity and selectivity, complex sample preparation, skilled personnel requirement, slow response time, false positive, non-portability, operational complexity, and difficulties in real-time monitoring.

Screening of biomolecular interaction has been progressively much useful in environment safety, drug development, food safety, medical diagnostics and molecular biology research. A broad variety of detection methods are available for bio-molecular interactions using the processes of antigen–antibody, DNA–DNA, protein–DNA, protein–protein and protein–peptide interactions. Traditional methods and techniques usually necessitate fluorescent labeling and this needs more time, more cost and it can also affect the binding signals leading to false negatives. Furthermore, fluorescent substances may generate a background signal leading to false positives due to their constant hydrophobic nature (Piliarik et al., 2007, Pollack et al., 1999).

Labeling based methods are frequently utilized to pursue interactions between molecular DNA probes attached on a sensor surface and target molecule. This labeling takes place either on the target molecules directly before interaction or at the step after binding of the target on the sensing surface. However, labeling process is a time consuming and non economical and also disturbs the binding interactions, mainly with proteins (Cooper, 2002). So as to overcome these problems, optical biosensors, particularly surface plasmon resonance (SPR) based sensors are of great interest because it offers a promising platform for monitoring biomolecular interactions in a label-free and real-time manner (Tsouti et al., 2011).

SPR is an optical technique and it employs evanescent wave at the interface between a dielectric medium typically either liquid or air on a free electron rich metal like Au or Ag and is highly sensitive to small changes in refractive index (RI). In SPR system, biomolecular interactions studied, where probes or ligands are covalently bound on the sensor surface and analyte injected, this induces perturbations of the evanescent wave because of refractive index variation. Over the past decade, the utilization of SPR has been explored extensively in many diverse potential applications, ranging from biomolecular interaction analysis to environmental monitoring. (Kaur et al., 2016, Maesawa, 2003, Stern et al., 2016; Guo, 2012; Rich and Myszka, 2007; Li et al., 2012).

DNA association with proteins is a phenomenon of extreme significance. Certainly, approximately all features of cellular function, for example chromosome maintenance, transcriptional regulation, replication and DNA repair depend on the proteins interaction with DNA. To understand above biochemical processes, one needs a capable system to describe different type of interactions like DNA–protein interactions, hence, different techniques have been developed to explain such type of interactions over past years (Dey et al., 2012, Cooper, 2003, Amano et al., 1999). SPR permits synchronized screening of hybridization dynamics of immobilized probes and target interaction in real-time label-free manner (Pennacchio et al., 2014, Pan et al., 2010, Sato et al., 2006, Xue et al., 2014). The previous investigations for the development of SPR detection methodologies for BWAs (Gupta et al., 2010a, Gupta et al., 2012, Gupta et al., 2011b) and biological toxicants (Sikarwar et al., 2014, Sikarwar et al., 2015) are also lined up.

The aim of present investigation is to develop a SPR based methodology as there is no report in the literature for the SPR sensing of B. melitensis by the interaction between unlabeled oligonucleotide complementary DNA target or DNA PCR fragment with two different immobilized DNA probes in PBS buffer. Two different DNA probes of B. melitensis were covalently attached on the different 4-MBA/Au SPR chip and their use for the detection of complementary DNA fragments of B. melitensis is conducted. The parameters influencing the SPR response were optimized and finally K_D and B_max were calculated. Furthermore, thermodynamic parameter such as Gibb's free energy change (ΔG) involved in the interaction between DNA probes and target of B. melitensis was also deduced. As will be illustrated below, the new SPR based detection strategy addresses the challenges associated with conventional detection methodology and also to find out the best sensitivity and specificity having probe for SPR sensing and this allow for more accurate calculation of affinity constants during DNA–DNA interactions with the best sensitivity.