A regenerable flow-through affinity sensor for label-free detection of proteins and DNA.
Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible-near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.
Zavali
Maria
M
Institute of Microelectronics, NCSR "Demokritos", Aghia Paraskevi, Greece.
Petrou
Panagiota S
PS
Goustouridis
Dimitrios
D
Raptis
Ioannis
I
Misiakos
Konstantinos
K
Kakabakos
Sotirios E
SE
D000818:Animals; D055910:Antibodies, Immobilized / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004867:Equipment Design; D017743:Equipment Reuse; D007163:Immunosorbent Techniques; D008027:Light; D016014:Linear Models; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D013057:Spectrum Analysis / Q000295:instrumentation* / Q000379:methods*; D005719:gamma-Globulins / Q000032:analysis / Q000378:metabolism
D055910:Antibodies, Immobilized; D009841:Oligonucleotides; D011506:Proteins; D005719:gamma-Globulins; D012822:Silicon Dioxide; D004247:DNA