DNA-probe-target interaction based detection of Brucella melitensis by using surface plasmon resonance.
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 (KD) and maximum binding capacity of analyte (Bmax) 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 (KD) and 81.02m° (Bmax) with probe 1 and 54.9pM and 55.29m° (Bmax), 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 10min. 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.
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India.
Bhavna
B
Sikarwar
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India.
Virendra V
VV
Singh
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India.
Pushpendra K
PK
Sharma
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India.
Ashu
A
Kumar
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India. Electronic address: dtselvam@drde.drdo.in.
Duraipandian
D
Thavaselvam
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India. Electronic address: boopathi@drde.drdo.in.
Mannan
M
Boopathi
Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior 474002, India.
Beer
B
Singh
School of Studies in Biochemistry, Jiwaji University, Gwalior 474011, India.
Yogesh K
YK
Jaiswal
D001565:Benzoates / Q000737:chemistry; D017347:Brucella melitensis / Q000235:genetics / Q000302:isolation & purification*; D002006:Brucellosis / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000097:blood / Q000235:genetics; D005798:Genes, Bacterial; D006046:Gold / Q000737:chemistry; D006801:Humans; D061925:Immobilized Nucleic Acids / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D013816:Thermodynamics
D001565:Benzoates; D015342:DNA Probes; D004269:DNA, Bacterial; D061925:Immobilized Nucleic Acids; D013438:Sulfhydryl Compounds; C013594:4-mercaptobenzoate; D006046:Gold
Brucella melitensis; DNA probes; DNA target; Kinetic parameters; Real serum; Surface plasmon resonance