BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 19: Biosensors(continued ast time: biosensor device cla Gene array biosensor Today detection methods Detection Elements Macroscopic fluorescence, diffraction, or interference ·what Example: quantum dot-loaded microsphere capture agents QDs show size-dependent luminescence Narrow emission bands from a common excitation wavelength Stable against photobleaching Load polymer microspheres with different amounts of several colors of QDs to obtain a unique fluorescence signature colors at 10 possible intensities allows for> 10 possible Capture molecule on surface of beads grabs labeled analyte beads emiting single-color signals at 484, 508, 547, 575, and 611 nm. The beads slide, which caused a slight clustering effect. See Experimental Protocol 050100150200250300 Fluorescence intensity (arbitrary units x10 (Han et al, 2001) Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 19: Biosensors (continued) Last time: biosensor device classes Gene array biosensors Today: detection methods Detection Elements o Readout Macroscopic fluorescence, diffraction, or interference • what Optical bar-coding4 • Example: quantum dot-loaded microsphere capture agents5 QDs show size-dependent luminescence Narrow emission bands from a common excitation wavelength Stable against photobleaching Approach: Load polymer microspheres with different amounts of several colors of QDs to obtain a unique fluorescence signature • 6 colors at 10 possible intensities allows for > 106 possible ‘codes’ Capture molecule on surface of beads grabs labeled analyte (Han et al, 2001) Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Excitation of bar-code and target fluorochrome by same wavelength 1 Gow. Figure 5. Schematic illustration of DNA hybnidization assays using spectrophotometer for detection Cascade Blue. After hybridization, nonspecific molecules and excess reagents were of emission spectra from individual bead and hybridization kinetics(30 min). See legend in Figure 6 for the sequences used ptical absorption Surface plasmon resonance and SPR arra Developed commercially later 1980,s(Cooper 2002) Typically, receptor is immobilized and free ligand is passed over sensor chip Both ways possible, small ligands simply interfere with binding if immobilized Flow channa Box 2 Couping methods for reeeptor immobih Senso chp Bit ior Mrpa ii-l septum a). The alt Rom - tiny tes ef streptase eflected epitope FLAG etape an ea His I ai( have boom widcly uod for diet derale decay in the lrdl ed crocrgram Figure 2 Typical set-up for an SPR biosensor, Surface smon resonance(SPR) detects changes in the refractve dex In the mmerllata wcinty of the sl rtane layer ot a sanor chip SPR 0=° observed as a sharp shadow h the reflected Ight from the surface at anange that s dependent on the mass of materal at the surface. The SPR angle shfts (from I to ll inthe bwer left-hand diagram) when biomolecules bind to the surtace and hange the mass of the surtace layer. This change in resonant ange can be monitored non-invaslely in real time as a pbt of resonance signal (proportiona to mass change) versus time. (Cooper 2002) Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Excitation of bar-code and target fluorochrome by same wavelength Microscope-based spectrophotometer for detection of emission spectra from individual beads Optical absorption (colorimetric) • what Surface plasmon resonance and SPR arrays • Developed commercially later 1980’s (Cooper 2002) • Typically, receptor is immobilized and free ligand is passed over sensor chip Both ways possible, small ligands simply interfere with binding if immobilized (Cooper 2002) Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 GoMd-dextran surfaces Biacore sensor chips ~八5 SCOn ont eowale nt attache aminpuup.? dnul hude loa basilar aunt, wtae thiastbrn linlin reagents to flea Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Biacore sensor chips Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Dissociation Kinetics 8。8 concentraton Time (s) For3 Atypical tene ng cyc sew wm an opteD Donor A ooc t mmoodpoaonmeconporaurtocamn (Cooper 2002 Optical fiber-based Single cell analysis optical fiber probes Advantages/disadvantages Pros o Fast measurements o Sensitive o Cannot perform detection on turbid solutions Electrochemical Electrochemical readouts? o Conductometric o Measure changes in the conductance of the biological component arising between a pair of metal electrodes due to e. g. metabolism Potentiometric o Measure electrical potential difference between a sample and reference electrode o Monitor the accumulation of charge at zero current created by selective binding at the electrode surface Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Cooper 2002) Optical fiber-based • Single cell analysis optical fiber probes6 Advantages/disadvantages Pros o Fast measurements o Sensitive Cons o Cannot perform detection on turbid solutions Electrochemical Electrochemical readouts7 o Conductometric o Measure changes in the conductance of the biological component arising between a pair of metal electrodes due to e.g. metabolism o Potentiometric o Measure electrical potential difference between a sample and reference electrode o Monitor the accumulation of charge at zero current created by selective binding at the electrode surface Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 o Electrode may be selective for certain ions or gase E.g. F- I, CN-, Nat, K+, Ca2+, H+, NH4 CO2 NH3 o Amperometric o Measure current generated by electrochemical oxidation or reduction of electroactive species at a constant applied potential Electrochemical detection ISFET-ion-sensitive field-effect transistors As Se Ni membrane ep --群 0H一 Common pH-modifying enzymatic reactions glucose oxidase ource D-glucose+02-+ D-glucono-1, 5-lactone H2O2- D-gluconate H [6.31 penicillinase penicillin→ penicilloic acid+H’[6.4 enz ane pH-FET H2 NCONH2+H2O+2H一2NH4·+cO2I65] Fig. 2. Biosensor based on a pH-sensitive FET. ease(pH 9.5)D H2NCONH2+ 2H20-+ 2NH3+ HCO3+H (6.61 neutral lipids+H2o-) glycerol+ fatty acids+H[6.71 (Mulchandani and Rogers, 1998) Advantages/disadvantages Pros o Fast measurements Sensitive ow detection limits typically- 10M on Ability to perform measurements on turbid/opaque solution o PH-sensing mechanisms require weakly buffered or non-buffered solutions Calorimetric Calorimetric readouts o Measurement of heat generated by an enzymatic reaction o Typically utilize thermistors to transform heat into an electrical signal Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Electrode may be selective for certain ions or gases E.g. F-, I-, CN-, Na+, K+, Ca2+, H+, NH4+ CO2, NH3 o Amperometric o Measure current generated by electrochemical oxidation or reduction of electroactive species at a constant applied potential Electrochemical detection: ISFET - ion-sensitive field-effect transistors Common pH-modifying enzymatic reactions: (Mulchandani and Rogers, 1998) Advantages/disadvantages Pros o Fast measurements o Sensitive Low detection limits typically ~ 10-9 M o Ability to perform measurements on turbid/opaque solutions Cons o PH-sensing mechanisms require weakly buffered or non-buffered solutions Calorimetric Calorimetric readouts o Measurement of heat generated by an enzymatic reaction o Typically utilize thermistors to transform heat into an electrical signal Lecture 19 – Biosensors