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
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Calorimetric detection glucose-6-phosphate ADP pyruvate kinase H +NADH NADX Schematic diagram of a calorimetric biosensor. The sampl pyrus rence thermistor(e)and into the packed bed bioreactor(f, 1ml volume), containing the biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor(g)and the oxyger difference in the resistance, and hence temperature between the http://www.sbu.ac.uk/biology/enztech/calorimetric.html thermistors Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Calorimetric detection: QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a Graphics decompressor are needed to see this picture. http://www.sbu.ac.uk/biology/enztech/calorimetric.html Schematic diagram of a calorimetric biosensor. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). From there, it flows past the reference thermistor (e) and into the packed bed bioreactor (f, 1ml volume), containing the biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor (g) and the solution passed to waste (h). External electronics (l) determines the difference in the resistance, and hence temperature, between the thermistors. Lecture 19 – Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric, 9 Bs如 quaitzerystlmicrobslan ce detecion 0cyB0。减ad叫食qban。adw d加 mation induced as dipoles in crystals如a! with d rec ion ofelechict Dm10100mA呲吗 infreq range d1-10M oa Bindng of analye to surace changes mass of crystal and ater osc aion frequency Figure below from: www-bond. chem. monash. edu. au/theses/ Graeme%/20Snook/Chapterl. pdf Piezoelectric detection Quartz crystal microbalance Analyte solution B http://ww.g-sense.com/main.gcmdtech.html Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric8,9 Based on quartz crystal microbalance detection Based on quartz crystal microbalance detection o Crystal is oscillated at a defined frequency by an oscillating applied voltage Shear deformation induced as dipoles in crystal seek to align with direction of electric field Shear deformation induced as dipoles in crystal seek to align with direction of electric field Deformation typically 10-100 nm for AT-cut crystals operating in freq. range of 1-10 MHz ystals operating in freq. range of 1-10 MHz oBinding of analyte to surface changes mass of crystal and alter oscillation frequency of crystal and alter oscillation frequency Figure below from : www-bond.chem.monash.edu.au/theses/ Graeme%20Snook/Chapter1.pdf Piezoelectric detection: Quartz crystal microbalance Analyte solution http://www.q-sense.com/main.qcmd_tech.html Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric detection Quartz crystal microbalance Detecting H/V virions 命命 Figure 3. Signal linearity with particle numbers. (A)Serial 10-fold ssociation ofasipesentative REVS spect gle virion in PBS from (Cooper et al. 2001) Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric detection: Quartz crystal microbalance Detecting HIV virions: (Cooper et al. 2001) Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 10 SPR Arrays External analysi ctIOn Optical method Cell- and tissue-based biosensors(Stenger 2001, Gross 1997) General concepts Why cell-based biosensors? o Known ultrasensitivity of cells: Olfactory neurons respond to single odorant molecules Retinal neurons triggered by single photons T cells triggered by single antigenic peptides(Irvine 2002) DICAra oN Calcium sigr P 风 Time relative to Ca* sgnal PE fjoreeceneo Potential for single PE nucnescenoe signal In TAPg ntertege molecule sensitvity 白 g Teel rogerian offorelgn pepNde shown at rght Positon如m Complex evaluation of 405067080 o ability to'integrate cellular or tissue response to compound ne et al.,2002] Detect functionality of compound in addition to its chemical presence e. tell the difference between a dead and live virus Cell-based biosensors are based on a primary transducer( the cell) and secondary transducer(device which converts cellular/biochemical response into a detectable signal) on Secondary transducer may be electrical or optical Detection of arbitrary targets on Transfect cells with receptors to introduce responsiveness of e.g. neuronal cells to a chosen compound Basis of electrical secondary transducers Electrically-excitable cells Example cell types Neurons Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 SPR Arrays10 External analysis/detection Optical method Cell- and tissue-based biosensors (Stenger 2001, Gross 1997) General concepts Why cell-based biosensors? o Known ultrasensitivity of cells: Olfactory neurons respond to single odorant molecules Retinal neurons triggered by single photons T cells triggered by single antigenic peptides (Irvine 2002) o Ability to ‘integrate’ cellular or tissue response to compounds Detect functionality of compound in addition to its chemical presence • i.e. tell the difference between a dead and live virus Cell-based biosensors are based on a primary transducer (the cell) and secondary transducer (device which converts cellular/biochemical response into a detectable signal) o Secondary transducer may be electrical or optical Detection of arbitrary targets o Transfect cells with receptors to introduce responsiveness of e.g. neuronal cells to a chosen compound Basis of electrical secondary transducers o Electrically-excitable cells Example cell types • Neurons Lecture 19 – Biosensors
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 o Non-sensory neurons grown in culture outside of normal homeostasis and the insulation of the blood-brain barrier behave in a 'sensory manner(Gross 1997) Generate electric signals in a substance-specific and concentration-dependent manner Signals generated can be monitored by microelectrodes Cardiomyocytes 1. clrengrs in rmal ive adfrvily pallets Neuronal cells synaptically active(e. g. nerve]agents 2. changes in nett得?li的俱时和s lon canned blockers and toxins s paroxysntalrespDnses due fa pathological membrane MUWLJIIIN Baseline newwark activ + LIlauitlliiuil Acivity fallowing exposure nf 八 netwark ta 2 ply trimethylol。 pane phosphate Measures changes in extracellular acidification rate: pH changes associated with alterations in ATP consumption by cells(metabolism) AMpLy sensitive readout of changes in cell metabolism EXAMPLE OF HARDING MCCONNELL'S WORK WITH T CELLS Relative advantages and disadvantages of cell-based sensors Pros o Cell-based sensors may utilize the ability of cells to respond to complex mixtures of signals in a unique on May provide alternatives to animal testing in the future o Issues of maintaining cell viability and reproducibility in measurements Patterning cells for sensing13 Techniques used o Photolithography o Microcontact printing(soft lithography) o Microfluidic pat herning o Membrane lift-off Lecture 19-Biosensors
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Non-sensory neurons grown in culture outside of normal homeostasis and the insulation of the blood-brain barrier behave in a ‘sensory’ manner (Gross 1997) • Cardiomyocytes Generate electric signals in a substance-specific and concentration-dependent manner Signals generated can be monitored by microelectrodes o Microphysiometer11,12 Measures changes in extracellular acidification rate: pH changes associated with alterations in ATP consumption by cells (metabolism) Extremely sensitive readout of changes in cell metabolism EXAMPLE OF HARDING MCCONNELL’S WORK WITH T CELLS Relative advantages and disadvantages of cell-based sensors Pros o Cell-based sensors may utilize the ability of cells to respond to complex mixtures of signals in a unique way o May provide alternatives to animal testing in the future Cons o Issues of maintaining cell viability and reproducibility in measurements 13 Patterning cells for sensing Techniques used: o Photolithography o Microcontact printing (soft lithography) o Microfluidic patterning o Membrane lift-off Lecture 19 – Biosensors