6: Labchip technology Application * Cellular/Particle Analysis (Ret of Cell+Particle, Slit-Type Filtration, Weir-Type Filtration, Cell Adhesion, Polymer Entrapment,

3D Flow Control, Optical Trapping, DEP, Cell Culture, Electroporation, Chip Cleaning+ Sterilization) - Filtration of 6m latex beads by slit-type filter. 500mwide/5.7-m-deep ch. fabric. on 400m-thick Si wafer. Slit structure of 5m-wide slits bet 73m-wide posts fabric. by deep reactive ion etching -Lateral percolation in microfabr. filter: Liq. vertically enter microfabr. cubes attached to underlying substr., drawn laterally to sides of array thru interconnected ch, part. >1.5-m chans sep. cubes, sim. to axial filter. Entrance to ch blocked by particle, liq can flow under part., alt routes for liq. to enter bed, migrate thru filter. -Specific filter capacity: particle filtered/cm2 on filter face. Eq. to high-capacity axial filtration system -Microfilter design: 1)Filter pillar in ch, 2)Ch Widened, 3)Filter pillar define square rxn chamber where beats collect -Porous microfluidic ch After irradiated w ions of low en. -> beam det. Length of tracks allowing perforation of top layer -Weir type filter: Ch In Si anisotropically etched w EPW -> V-shaped grooves (stop surface tension lock 0.1um), lid is pyrex glass attached by anodic bonds

reagent flow from middle ch become increasingly strong

-Cell scanning& noise filtering: 1)fluores. yeast cell gg thru detection windo generate peaks over bkgrd. 2)Clearer peak after filter noise (>2.5Hz) 3)Narrower detection window on other cells distinguish mother from daughter yeast cell by peak & shoulder -BkGrd correction: raw data minus bkgrd baseline (fluores.) 1)Peak envelope. -Math modeling for fluorescein diacetate (FDA) metabolism in single cell: 1) Yeast cell control influx of FDA, hydrolysis of FDA, form fluorescein, efflux of fluorescein due to stimuli of pH & glucose 2)Intracellular conc. of FDA & fluorescein measured 3)Curve fitting: Stripes=amt of fluorescein; dotted/ solid lines= amt of intracellular FDA& fluorescein, (calibrated w fluorescent bead) RFI: relative fluorescent intensity; 1% = fluores. from full hydrolysis pdts from 61019 mol FDA -Microfluidic vortex device w streamlines (follow EOF in large ch, follow PF in small ch) EOF and PF condition such that net flow rate ~0. Pair of symmetr. vortices generated at ends of trapping ch, flow recirculates bet. diverging & converging ch.

-Pumping PCR chip where sol. mixture pumped back & forth over 3 temp zones (90, 55, and 72 oC). Glass wafer consist of etched region to house 3 PZT disks for piezoelectric actuation. Si wafer consists of 3 heating region which are thermally isolated by the recess structures. Rxn chamber: heater, temp sensor, optical fiber ,pn diode -HIV genotyping: 3 temp zones: Storage, Reaction, Hybridization. RNA purifiation from a serum lysate (extraction); R2, RTPCR; R3, nested PCR; R4, DNase frag. & dephosphorylation; R5, terminal transferase labeling; R6, dilution and hybridization, washing, phycoerythrin staining, washing -Continuous-flow PCR &RTPCR. 2 inlets 4 PCR, 2 inlets for reverse transcription, 5 outlets for pdt collectn at 20,25,30,35,40 cycle. Chip over 4 heating blocks: denat-uration, extension, annealing for PCR, RT. -Microbeads conjugated with BT-TATA, BT-PBS, BT-NFB. FC, fluorescein-labeled target; BT, biotinylated probe; PBS, NFB, RAD, TATA =DNA -DNA seq. + capture chamber (CC). Injc. modified include CC inlet/outlet wells for oligonucleotide purifications. CE waste & cath. give V access pts 4 EK injc./ CE anal. 4 tapered turns = elongated serpentine sep. ch. geometry (15.9 cm) turninduced dispers. High-V anode well at bottom

-Dissociation of AbTNB-Fl at 6 det. Pt on sep. CE ch. by rotary fluorescent scanner -Flow & diffusion of T-sensor at 1:1:1 ratio. Ref sol. enter from left, det sol from mid, particle-laden sample stream enter from right. Inset= original flow bound-aries, ref stream, part-laden sample stream, diffu of detector subs to ref stream, diffu of ref subs to det stream, det. stream, diffu of sample analyte to det stream, diffu of detector subs to sample stream, det window

PDMS: Elastomeric: deformed reversibly/ repeatedly w/o distortion or relaxation of features, Moldable at scale suitable for optic appli. (0.110 m) w high fidelity, optically transparent to ~300 nm, durable, inert, non-toxic, com. Avail, affordable, Bonds readily PDMS: Require master for molding/ fabri., hydrophobic surface not fully compatible w aq buffer, low EO flow w modification, costly (VS PMMA) -Microcontact printing (CP): 1)planar substr w planar PDMS stamp 2)planar substr w rolling stamp 3)curved substr w planar stamp -MIMIC, TM, replica molding:

create high aspect ratio (100:1 depth:width) structure - Embossing: decorate or raised design, Electroforming: Electrode-position in plating bath over base form - Miniaturized plate-type heat exchanger fabricated by LIGA technique & housing: Struct. height: 300m, Mat.: Ni/Cu, Al2O3, Al. Al2O3 + Al heat exchanger realized by embossing w embossing tool or embossing tool in stainless steel fabr. by die sinking (micro spark erosion or electric. discharge machining-EDM) w LIGA electrodes -Why not Si? 1) Lower mechanic strength 2) Poorer heat transfer -Test rxn & characterization of mixing micromixer array: 2 concurrent rxns: 1. Acid with sodiumacetate (ultrafast) 2. Iodide (J-) & iodate (JO3-) ions yield iodine (fast). If good mixing, no iodine in micromixer (no H+)= better mixing than Tpieces -Integrated microrxn w mixing unit & heat exchanger: 1) photoetchable glass made by photolitho and wet -Surface acoustic wave (SAW)= IDT: interdigitated etching 2)Special alloy made by micro spark erosion transducer (Electric. discharge machining-EDM grinding) -Bonded ch:1)adhesive bonding 2)contact areas only -Device for multiphase reactions:

Integrated sensing syst made, Efficiencies limited by V breakdown, characteristics of device, Injc. not as easy for cap. Adv: Small vol. of sample, Enhanced performance ( speed, efficiency), Integration & automation (synthesis/sample prep/ analysis on chip), Easy construction of parallel syst. (high thruput appli.), Pumping mechanism is pulseless & generates no back pressure, Valveless liq. handling, Very amenable to miniaturization, Miniaturization= surface-to-olume ratio, Better heat dissipation, High V, Faster sep. -Photolitho: 1)Positive resist: exposure to UV changes cheml structure of resist = solubility in - Solute interaction in MEKC using SDS and CD developer, 2)Negative resist: exposure to UV causes (competition bet micellar solubilization & inclusion): it to polymerize (solubility) -> Sim. to semiconductor processing on Si, Typically use glass substr. -Micromoulding: PDMS: polydimethy-lsiloxane, Use photolitho to make Si mold (master), Use master to make polymeric devices

-Whole blood = dark (reflect Light), after filter of blood cell, plasma cross barrier & fill bottom ch -Schematic diagram of the weir-based device for physical trapping of cells. And The cross section of the device showing how the cells are retained in chamber, with the fluid flowing over the two barriers

-Chrono seq. of viability assay in single Jurkat T cell: 1)Live cell perfused w trypan blue dye. Alive cell not stained 2)Cell perfused with MeOH -> cell membrane permeabilization -> cell death 3)Permeable cell perfused with dye; rapidly (< 5s) stained. Total=2min -Docking& alignment on microchip: 1)Fluorescence image alignment of mammalian HL-60 cells 2)Single layer of HL-60 cells with good integrity. Cells stained w acridine orange (yellow -> cell viable after docking. Cells stained w ethidium bromide (red) -> dead cell -Cumulus removal process in IVF: 1)Cumulus oocyte complex (CC) reoriented as it pass thru constricted region. 2)CC at left removal port. 3)Cumulus rotates to bottom port. 4)Remaining cumulus removed -> clean oocyte -Fused Si glass microchip for immune-sorbent assay on trapped polystyrene beads (4.5 m)

-Microbarcode/bead sorting plate w weir-type filter. Loading of microbeads stacked at center of sorting plate. After spin, micro-beads destacked, collect at perimeter

-Light-addressable potentiometer sensor (LAPS) chip integrated w flow ch 4 study of adherent cells

-8-ch. LAPS chip w cover slip (w adherent cells), LEDs, fluid connections -Laminar flow pattern in PDMS sealed to polystyrene Petri dish. 1) By fluores. 2)Substr. placed at top&bottom inlet, PBS at middle, gravity flow (5min) + 3-min PBS wash; form pattern of bigger cells (outer lanes). 3)Suspension of E. coli placed in middle inlet, PBS in inlets 1&3 gravity flow 10min + 3-min PBS wash; creates pattern of smaller cells (middle lane). Both cell adhere to Petri dish by nonspecific adsorption. Cells visualized w Syto 9 (15M in PBS). Phase-contrast of BCE cells w trypsin/EDTA. Cells adhere and spread in a fibronectin-treated capil. network for 6h, nonadherent cells removed by washing. Trypsin/EDTA and media flow from desig. inlet for 12min. - Phase contrast micrograph of cardiac myocytes patterned in acrylic ch.:1)Myocytes placed in laminar flows of buffer, 2)Localized delivery of 100-M octanol to upper part -Hydrogel microstructure: 1)Microch. filled w fibroblasts & hepatocytes, 2)Green fluores.= fibroblasts 3)Blue fluores.= hepatocytes -3D flow control to cell retention: 1)2D ch flow field created by mid ch flow. Zero speed point (ZSP)= flow speed ~0. ZSP in middle. Fluid potential , shape of flow field change, ZSP move left. No reagent flow from mid ch 2) 3D flow field cause balanced cell on arc slope by upward force exerted by the liquid flow 3) Downward gravity& reaction force on sloping wall. 5)Cell position change as

-Microvortices gen. in microfluidic syst. to study rotation of particle -Recirculation flows :sharp corners, circular cavities (rotors), side chamber, interconnect chambers. stable centers vortices needed. Adjacent microchambers create counter-rotating flows (fluidic gears) Scale: 30/60m -Optical gradient force (flow switch): Micro-spheres flow to downstream ch. from top reservoir, captured by optical trap, directed manually(left/right) After optical trap release, microsphere follow fluid stream. Laser source=850-nm vertical cavity surfaceemitting laser (VCSEL): create sufficient output power & trapping strength, laser spatial mode = Laguerre mode -Cultivation of E. coli cells in microchamber fabr. on glasschip of SU-8 struc. (5m high) -Latex part.(15-m) confined from part. jet from dielectrophoresis (DEP). High field by 10 V (1 MHz) across 20-m electrode gap (0.5MV/m) -DEP w non-planar cylindrical electrode: 1)Cell-array chip. Dynamic lumines. Info used to sort indiv. cells by control syst. to fraction collectors. Scale: 20m 2)Pseudocolored scanning electron micrograph (SEM) of 4 electroplated gold electrode arranged trapezoidally along w substrate interconnect 3)SEM of 18 array of DEP traps. Sc100m -DEP on microchip: Streaming DEP 200-nm fluorescent latex spheres. Glass microch. 7-m deep, array of 36-m-square posts w 63-m spacing. DC field (80 V/mm) applied over 2 ends. Resulting EO from top to bottom -Bright field epi-fluores. image of 2 polymorphonuclear white cells 1) Hoechst stain 2)Diff. in conformation bet nuclei: Cell (top) stuck at entrance to ch, cell (bottom) deformed to ch. Ch coated w polyurethane to reduce cell adhesion &enhance white cell penetration -Erythrocytes flow thru microch. w bkgrd subtraction. Single, doub, triple transits -CE sep. of fluores. dyes from indiv. cells. Cell lysis & dye sep. by AC field (Vpp of 450 V/cm & 50% dutycycle) with DC offset (675 V/cm). DC offset maintain sep., AC field avoid gas bubble form -Hydrodynamic focusing & sorting of highly conc. sus. of fluorescent beads. Beads enter output ch. w equal probability. Activation of perpend. EO flow deflects suspension into desired output ch. (based on electrode polarity) -Aerodynamic focusing: when flow injection rate of syringe pump, width of water column -Non-coaxial shealth flow: Vertical pos. inlet positions sample flow in center. Horizontal control ports used to pos sample flow w horizontal shift. Seq. pairwise fusion of liposomes: dyed liposome w membrane fluorescent dye-DiO (distribute evenly) -Electroporation chip: 1)DNA attraction & targeting, 2)Electro-poration processes *Nucleic Acid Analysis -Dual Peltier:

-Mask design 16-ch. CE chip for parallel DNA seq. Wafer =10cm dia, thickness= 1.1mm. 16 identical 250-m twin-T injector used. Width of lines on mask =10m. Final ch. width 110m obtained w depth =50m. Reserv formed by access holes (1.4mm for sample+waste, 2.1mm for cath+ anode) drilled on etched wafer. (1.7-L+3.8-L vol. reserv) Alignment holes (2.1mm). Scan line= scanning location of detector. - Mask pattern for 96-ch. radial cap. affinity EP (CAE) microplate (10cm). Sep. ch w 200-m doubleT injectors masked to 10m width etched to form 110-m-wide by 50-m-deep ch. (1.2mm). dist. from injector to detection point=33mm -T-ch. for DNA sep. Large ch. 100 m, narrows to 5m at Tjunction. Depth=3m. Elastomer ch. Bow+pinch off by sealing directly to glass. Add support pillars. -Brownian ratchet: Flow angle=10.8 & obst Nanostruc reg. for size-exclusion-type sep: 1) narrow (small mol)+ wide gap - Microch.w nanopillars to sep. big DNA.

-Fluores. image of labeled Ag bound to immobilized Ab at intersect of microchip. Ab flow from bottom ch confined by left/right ch. Ag intro from left confined by top/bottom ch. Info on Bkgrd & non-specific binding extracted. Quantitation by averaging fluores. intensity in a small area in reg. of interest -Microfluidic netwk (FN): Serial dil. of analytes (Ag-containing serum) using serum blank (BSA). Dilution assist by chaotic advection mixer. Each mixer includes 4 cycles of herringbone patterns (4mm) Dil. Ab interact w spatially segregated Ag, immobilz on polycarbonate Ag-presenting membrane -PMMA CD disk for ELISA w 24 assays & single assay (waste; det. 1st Ab, washing; blocking protein; Ag/sample; 2nd Ab; substr) -Protein sep: 1) SDS dil. well connected to sides of dil. intersect. 2)Sep. buffer, waste wells, load wells. Others contain sample -Microfluidic structure for assay of mixing an enzyme w inhibitor, followed by mixing w a substr, and det. Enzyme, inhibitor, substr loaded in reserv connected to ch After release by cap burst valves, enzyme+ Inhibitor mixed in meandering 100-m-wide ch. After released by burst valve, enzyme-inhibitor mixed w substr in meandering ch. Mixture emptied in cuvette -Phosphorylation of kemptide by PKA in reagent well on glass chip: Successive electropherograms of unphosphorylated (substr) and phosphory-late fluorescein-labeled kemptide (pdt) 600s. dimethyleth-ylammonium propane sulfonate= zwitterionic to prevent enzyme adsorption; ATP allow phosphate transfer to serine res. of kemptide dur phosphorylation; cAMP= enable funct. of PKA (cAMP-depend kinase); DTT= protect enzyme frm EC damage -Microfluidic device w H-filter for cell lysis/fractionation coupled to T-sensor for det. of intracellular enzyme (-galacto sidase) Pump rates controlled at all inlet+1 outlet. In H-filter, lytic agent diffu. into cell sus. (lyses cell) Intracellular enzyme diffuse away frm cell stream, some brought to det. ch. In Tsensor, -Gal det by fluores.

-Bonding of PDMS thin glue layer (A): -Bonding of hard substrates w PDMS prepolymer (B)

-H2 syst. w microreactor & immobilization of Pd cat.:

-Soft lithography process: Microcontact printing, Micromolding in cap., Microtransfer molding,Replica molding, Bonding, Microchip CE, Paper microfluidics -Microcolumn sep. syst.: Controlled dil. (EK injc, Applic: AA, Oligonucleotides, V switchingsample withdrawal, DNA seq, Enzymatic digestion), Synchronized cyclic CE, Field flow EP, HPLC chip -Microreactor: Micro-pump, heat exchange, - SEM PDMS-bonded glass fluidic ch contrast= mixer,membrane module, Microrxn syst. (H2 w charging effect -improvement: air escaping ch. immobilized Pd Cat.) To remove trapped air 4: Column & Buffer for CE -Integrated Chip-Based Microcolumn Sep : *COLUMN TECH: Uncoated col., Coated col., Fluores. recorded at T junct. after controlled dil of Packed col., Gel -filled col., Dynamic sieving CE, CE fluorescein sample. Dil. controlled bystepwise on chip reduction of potential V2 -Uncoated fused Si cap. used for sep. of -V switch for electr field charge/size ratio, Coating stop adsorption, Packed control withdraw of precol. Used for CEC, Gels used for size sieving, selected sample zones Buffers optimize selectivity at T-intersect: For with-COATED col: Coating: Polyethylene glycol; drawal of single oligo Polyacrylamide w siloxane(Si-O)bond to Si, -Electric field controlled Polyacrylamide w Si-C bond Si(more pH stable than enzymatic digestion of Si-O), Polyethyeneimine, LC stat. phase, GC stat. DNA: phase, charged-reversed coating, biopolymer - Alt. to polyimide coated fused Si cap.: Rectangle tubing pathlength, UV trans-parent outer coating for det., Polymeric tubing (PTFE): not UV transparent -Procedure for deactivation Si wall:

-Crown Ethers: Synthetic macrocyclic polyethers capable of forming stable inclusion complex w various organic & inorganic cation -Complex formation based on ion-dipole interactions bet host & guest molecule -Structure of complex formed bet 18-crown-6 & protonated primary -Hybrid CE Chip fabr.: Photolitho+micro-molding: amino compound: - Complex Equilibrium: ION + LIGAND = COMPLEX K = B/A A: molar fraction of ions in free sol B: molar fraction of ions w ligand - Observed = A(ep,cation + EOF ) + B observed, LIGAND *Systematic Evolution of Ligands by Exponential -Microfabr. CE injection: Enrichment (SELEX) - Procedure: 1) Binding a random nucleic-acid library to target in CE cap (CESELEX), 2) Sep. bound & unbound nucleic acids (CE in CESELEX), 3) Amplifying bound nucleic acid by polymerase chain rxn (PCR) for next round of selection (if PCR done in the last round of selection, process called non-SELEX), 4) After each round of selection, smaller pool of nucleic acid seq. binding to target is retained and unbound nucleic acid discarded 5) Aptamers cloned and sequenced - Non-SELEX selection of aptamers: EP mig. of nave DNA library & respective protein or eq mixture under optimized condition for estimation of bulk affinity & det. of aptamer-collection window for noneq cap EP of eq mixtures (NECEEM) partitioning -Non-eq cap. EP of eq. mixture (NECEEM): (a) Components of equilibrium mixture (EM): free ligand (L), free target (T), ligand-target complex (C). -Microfabr. (b) NECEEM-based separation of L, T, and C: Short CE-LIF plug of the eq. mixture injected in cap at t0. High V Detection.: applied. Assume T migrate faster than L; C has intermediate mobility. Eq. fractions of free L & T migrate as indiv. Zones (do not change in time). The eq. fraction of C continuously dissociates during sep. (time t1 and t2), leaving smears of L and T. By time t2, only fraction of C remain intact - Effective dissociation constant: Kd =

-Short nanopillar-free regions placed at reg interval for detection w/o fluorescent light scattering Fluores. using nano-sphere- containing med & double pressurization - Nanostructured region for size-exclusion-type separation. (a) Principle of separation in the nanostructure region consisting of narrow and wide gaps. Larger molecules move in the wide gaps, while smaller ones move in narrow and wide gaps. -Schematic representation of microch with nanopillars for separation of large DNA molecules. Each nanopillar (a black dot) diameter, spacing, and height used here were 500, 500, and 2700nm. Short nanopillar-free region placed at regular intervals for det w/o problem of fluorescent light scattering

-Others: DNA seq, digested pdts(Hinfl), post-column rxns (microfabr. w quartz), post-column derivatizations, synchronized cyclic CE (diff mobilities), field flow EP(charge) [FFE device:2 buffer inlet, sample inlet, side bed inlet, 2 side bed outlet, side bed w Pt -Preparing vinyl-bound polyacrylamide-coated cap: electrodes, outlet] -HPLC chip: a)ch. syst, b)cross-sec of ch axis, c)cross-sec of det. cell axis. IS: sample/ mobile ph inlet, S: split injc, OS: outlet for rejected sample/ mobile ph, C: sep ch, F: frit, D: optical det. cell, OD: waste outlet, P: positioning grooves for optical fiber

*Protein Analysis (Immunoassay, Prot. Sep., Enzymic Array) -6 rxn cell/sep ch in glass SPIDY for immunoassay. Rxn cell has reservoir for sample, antibody, sample waste, run buffer. Contains mixer in rxn cell to mix Ab & Ag, T injector design for loading sample from reactor to sep column, smooth turn at sep ch., ch pattern at buffer waste reserve used for sep. Thicker lines identify ch segments 300m wide, thinner lines are 50-m-wide seg., laser beam swept across det zone. 2 outermost ch= optical alignment ch filled with fluores. dye

- H2O2+NADH for det. of glucose& EtOH: 1) control mixture 2)sample glucose 3)EtOH+ ADH+NAD+ 4)sample of glucose+ EtOH+ GOx+ ADH+ NAD+. Injc. pot. +2500V (3s); sep. pot. +2000V. Ep buffer, phosphate buffer (pH7.8). Amperometric det. Conductedw gold-plate screen-printed electrode at +1V -PEG-based hydrogel micropatches for glucose det: 1)2 micropatches w diff. enzymes immobilized in 2 17-m-high/135-m-wide ch w photomask 2) Optical micro-graph 2 hydrogel micropatches enclosed in single larger horizontal ch. (32m high/200 m wide) Hydrogel (left) w GOx + HRP, (right) only HRP. 3)Fluores. micrograph 2 micropatches after flow (1L/min) of glucose, O2, Amplex Red. Strong fluores. only in micropatch w both enzymes -Microchip for on-chip rxn, sep, post-column label: Fluid reservoir: substr, enzyme/ DTT, buffer, sample waste, NDA, waste

-Electrolyte syst.: EP buffer, Micellar media (MEKC), Inclusion additive, Complexing additive, Others -EP buffers:- pH ranges: phosphate(I) 1.1 3.1, acetate 3.8 5.8, phosphate(II) 6.2 8.2, borate 8.1 10.1; zwitterionic: MES (2-(Nmorpholino) ethane sulfonic acid) 5.5 6.7, Tris (tris(hydroxyl-methylamino-methane)) 7.3 9.3 - Choice depend on: Solubility, stability of analytes in electrolyte, degree of ionization, Influence of anion & cation on electromigration of solutes, pH effect, Effect of organic modifiers & other additives, Dissipation of heat -Ionization of analytes: For weak acids: KHA = ( CH+ CA- ) / CHA A = CA- / CA = KHA / ( KHA + CH+) A = A A For weak base: -Eg. chitosan-coated cap. w pH 4.36 Na acetate, 2nl KBH+ = ( CH+ CB ) / CHB+ B = CBH+ / CB prot. Mix to 0.4pg of each protein Injection -Paper Microfluids (Lab-on-paper): a) Pattern SU- PACKED col.: Pressure required to push liq. thru = CH+ / ( KBH+ + CH+) 8 photoresist embedded into paper b)modified for packed particles, Inlet &outlet frit sintered in col. by B= BH CH+ /(KBH+ + CH+)= BH B bioassays packing mat., Mono-lithic used; req lower pressure -Conc. distribution or peak shape related to relative mobility (r) to push liq. -Micellar Electrokinetic Chromatography (MEKC) Capacity factor: k = (tM t0)/[t0 (1tM/tmc)] Resolution: -Gell filled col.: Gel prep. bifunctional agent (3methyacryloxypropyltrimethoxysilane): one end of bifunct. reagent carry reactive funct. grp that bind chemically to silano groups, other end has 2nd reactive grp capable of forming covalent bond with polymeric gel material, pore size of poly-acrylamide (PAGE) gel det. by %T & %C -Gel prep. w/o bifunct. agent: low V (< 300V/cm) appli., Non-crosslinked PAGE: easier to empty & refill, resolving power, Agarose gels: allow UV det. at (232nm) than PAA, but less stable -Resolution & efficiency of gel col.: R = t / 4 t, N = ( t / t )^2 (t=mig. time, t=diff in mig time bet 2 consec peak, t is std width of single peak) -Polymer matrices for CGE: 1) Crosslinked polymer: PAA= Oligonucleotides, DNA seq, native & SDS bound prot., 2)Linear polymer: PAA, polyvinyl alcohol, dextran= Restriction fragments, Oligonucleotides, DNA seq, protein., 3)Agarose: Restriction fragments, protein -Dynamic sieving CE: Use size sieving sol. (soluble, linear polymer: methylcellulose & polyethylene glycol), Convenient: sol easily removed frm col. than gel, Viscosity should not be too high to permit filling & removal, efficiencies than gel-filled col (higher diffusivities (N 1/D)] - Cap. array w sheath flow cuvette 96 cap detected simultaneously -Optical det. syst. that shines focused light in tubes and det. amt & color of emitted light -CE on a Chip: Device (chip) fabric. by photolitho, Sep. ch, etched onto glass plate or molded w polymer, Microch. w dimension comparable to cap., where s is the separation factor given by k2/k1. -Resolution for CZE: R = 0.177 ( ep,1 ep,2) {V/[D(ep + eo)]}^1/2 where ep,1/ ep,2 = EP mobility for2 solutes and ep = average EP mobility. eo = EO mobility. D = diffusion coeff, V = applied voltage -Ion-exchange/ion-pairing: Velocity of analyte solute, vs: vs = vEO + Fvep + (1-F) v ep(p) where vep = EP velocity of analyte, vep(p) = EP velocity of polymer ion, F = fraction of analyte ion free from polymer ion: F = [S-]/{[S-] + [S- P+]} = 1/(1+Kip [P+]) Kip = [S- P+]/{[S-][P+]} where Kip = ion-pair formation constant; [S-], [P+] & [S- P+] = conc. of solute, polymer ion, ion pair *Cyclodextrins (CD) -Cyclic oligosaccharide molecule built of D-(+) glucopyranose unit bonded by - (1,4) linkages -Known as Schardinger dextrins, Cycoglucopyranoses, cycloamyloses or cycloglucans -slightly soluble in water, but cavities non-polar -, , & -cyclodextrins = 5, 7 & 8 glycopyranose unit, diameter of 0.47-0.52,0.60-0.64, 0.75-0.83 nm - Selectively include guest organic & inorganic molecule/ions into cavity - Capacity for highly hydrophobic solute in CDMEKC: k = nmc/nCD = KVmc/VCD where nmc & nCD = total amt of solute included by micelle & CD, Vmc & VCD = Vol of CD & micelle, K = distribution coeff - Ratio of solute incorporated in micelle depend on: hydrophobicity, but inclusion complex formation w CD depend on matching solute molecular size w cavity diameter of CD in addition to hydrophobicity

*COLUMN TECH.: coated col. (prevent adsorption), packed col.(stat. ph. for CEC), gel filled col./dynamic sieving (size-based sep.), CE on chip (photolitho & micromolding, pinched & gated injection, laser induced fluores. det.), *BUFFERS AND ADDITIVES: pH ranges, micelles: neutral cpd, others ( selectivity): ion pairing/ion exchange cpd, inclusion complexes: cyclodextrins, crown ether, aptamers 3: CE Instrumentation *Sample intro.: Electromigration, Hydrostatic, Pneumatic injection *Detection: UV/Vis, Fluorescence, Conductivity, Electrochemical, Radioactive, Post col. rxn - Sample Intro:

-Vol. of sample injc depend on magnitude & duration of vacuum applied, viscosity, cap. dimension. After injc, cap. placed back into inlet buffer vial and electric field applied -Hydrodynamic injection by GRAVITY (siphoning): Cap. placed into sample vial. Sample vial & cap. raised a distance, H, above destination vial, causing sample sol. to siphon into cap. -Vol. of sample injc. depend on H, duration vial is raised, viscosity, cap. dimension. After injection, cap. placed back into inlet buffer vial and electric field applied *Electrokinetic (electromigration) injection : Voltage applied while cap. inserted into sample vial - Procedure: 1) Cap. & anode placed in sample vial, 2) Voltage applied cause sample ions to migrate into cap. due to EO & EP mobility, 3) Amt of sample injc. depend on EP mobility of solute, EO flow rate, applied voltage, cap. dimension, solute conc, 4) After sample injc., cap. & anode placed back into source vial & voltage applied - Length of sample zone: l = (vep + veo) ti = ( ep + eo) Viti / L Where Vep & Veo = EP & EO velocities, ti = injc. time, ep & eo = EP & EO mobilities, Vi = injc. voltage, L = length of col. - Amt of sample injc: w = r^2 l C where r = radius of cap., C = sample conc., l = length of samp. zone, w = r^2 C (ep + eo) Viti / L - For best reso. & peak shape, optimum conc. of injc. sample = 100 times less than buffer conc. (dil. sample) - Amt of sample intro. by electrokinetic injc depend on its migration velocity (sample bias due to voltage effect), whereas amt intro. by hydrodynamic injc does not - Stacking: EP velocity at high field hence analytes rapidly reach boundary bet. sample zone & bkgrd buffer (efficiency + preconcentrate sample) - Amt injc. controlled by variations in injc time & injc voltage. During EK injc, 2 types of bias in relative amt of different species in sample injc occur - Sample bias due to differences in mobilities of species (more mobile components injected in larger quantities) & electrical conductivity (EO & EP differ) - For sample solution containing species 1 and 2, sample bias (same sample, different species): w(1) / w(2) = b C (1) / C (2) where b = bias factor: b = ( (1) + eo) /( (2) + eo) - When b=1, eo is large (no bias) - Migration time (time for species i to reach detector at distance z frm injc end of cap.)= tm,i = z / vtot,i = z / [((i) + eo) E] - For 2-species syst., bias factor is: b = t m,2 / t m,l - Bias factor calculated from migration time - For 2 diff. samples: w(i;s1) / w(i;s2) = vtot(i;s1)C(i;s1) / vtot(i;s2)C(i;s2) w = mol of sample, v = vol (mL), C = conc. (mol/mL), i = sample component i, s1,s2 = sample 1 or 2 - Field-Amplified sample injc (FASI): plug of sample in buffer of lower ionic strength injc in col. w buffer of higher ionic strength - Sample ions migrate rapidly to boundary bet. sample & run buffer under applied voltage Stacking in front of sample plug - Comparison of amt injc & effective plug length for EK injc & FASI: 1) Conventional: Amt injc: (EP + EOF)ACi Et Plug length: (EP + EOF)Et 2) FASI: Amt injc: ( EP + EOF) ACi Et Plug length (EP + EOF / )Et where = ratio of conc. of bkgrd buffer to sample buffer, A = crosssection area -Larger : more sample injc & plug length shorter when FASI used. For FASI, samples prepared in low conductivity sol.(water). Further enhancement achieved by injecting plug of water before sample intro. to field strength. To pre-concentrate negative ions, FASI w polarity switching used - vEP = EP E, vEP = EP E - Large E (large V): fast migration - No stacking: Injection sample dissolved in buffer w same conductivity as EP run buffer., Top: Sample injc., Mid: Volt. applied & sample zone migrates thru cap. under influence of EOF and EP mobility, Low: Solutes elute in zone w slightly larger than, and proportional to the original sample plug

field strength abruptly drops, solute's EP velocity slows. Meanwhile, solutes at mid to rear of injc zone still exposed to high field strength and continue to move forward at "full" speed. Hence, ions in injc band continue to narrow, until all migrate into BGE. Negatively charged counterions stack up at rear of injc zone. - Stacking of anions in low-pH buffer: (EP +ve & EO -ve): Negatively charged anions migrate toward anode and cross boundary bet. injc sol. and BGE at rear of injection zone. -Anti-Stacking: When sample w high ionic strength relative to BGE injc, electric field over injc zone declines as defined by Ohm's law. When positive ion EP into BGE, it is exposed to high field strength over BGE. Hence, cation accelerate away frm those cations, still remaining in injc zone substantial band broadening. - pH Mediated Stacking: When volt. applied, negatively charged peptide migrate toward anode. As peptide at rear of band enter acidic buffer (low pH), charge flips and direction of migration reverse. Peptides at front of band still migrate toward anode. Band collapses on itself (neutralized). All material in narrowed band become positively charged and migrate toward cathode (sep. buffer: 10 mM citrate, pH 2.5; injc buffer: 10 mM ammonium hydroxide) - Stacking of Neutral Solutes (MECC): Electric field applied, negatively charged micelle migrate into injc zone and exposed to high field. Micelles stack and enrich neutral solute. As surfactant conc. increase, field strength declines until it equals that over BGE, stacking ceases, and sep. proceeds

-Monolithic integrated polycarbonate device for DNA assay (PCR & DNA hybridization): serpentine PCR ch, 7-L hybridization ch, Pluronics valve, Pluronic trap, hydrophobic air-permeable membrane, PCR reagent-loading holes, sample driving syringe pump (SP), waste-withdrawing SP, wash SP -PCR-CE mask w microfabr. heaters & resistance temperature detector(RTD). Leads of heater electroplated w gold. 4-lead RTD =accurate temp. VS 2-lead RTD: sep. Joule heating to actual T - 2 glass layer + rel. alignment - Sex det.: PCR-CE microdevice: DNA sizing ladder -Flow-thru PCR chip: Thermostated copper blocks, 3 temp zones (95, 60, 77 oC) for melting, annealing, extension of DNA. Glass chip has 20 identical cycles. 1st cycle: 3-fold in time 4 DNA melting by longer serpentine ch. length

-Chromatography paper patterned w photoresist: 1)Patterned paper after absorb Water-man red ink (5 mL) by cap action. Central ch absorb sample by cap action & pattern direct sample to 3 sep. test area 2)Complete assay after spotting reagent 3) Negative control for glucose & prot.by artificial urine(5 mL) 4) Positive assay for glucose & protein sol. w 50mm glucose & 7 mm BSA in artificial urine(5mL). Control well spotted w KI, not enzyme 5) Glucose & prot. det assay by varying conc. of glucose & BSA - (Phenomenological def.) *Microreactors for: 1) Fast single rxn= Continuous flow syst. w relatively short miniaturized path, 2) long single rxns= Semibatch syst. include valves, 3) multiple parallel rxn 5: Soft Lithography (fast or long)= Semi-batch syst. sim. to titerplates photolith: Thin film of organic photoresists against -Immunoassay chip: connect to micro- dispensing syst. & sep/ analysis etching highly developed for Si, 2/ 3D patterns -Microfabr. cap. for immunoassay: Serpentine cap. w unit -(Funct. Def.): 1) microrxn components= reproduce in Si w high precision by bulk &surface 22cm sep. length. Path of rotary scanning objective Single (unit) operation, (mixing, heat exchange, rxn, for fluorescent detection presented w 6 detection pts micromachining, Si batch-fabr. by tech. used for sep.) 2) Integrated microrxn syst. for single rxn= fabricating integrated circuit, Si/SiO2 stable Single rxn w several (unit) operations 3)Integrated chemically/thermally microrxn syst. for multiple-step rxn= Multiple rxn w photolith:High purity Si exp, brittle, opaque in several (unit) operation *Microreactors UV/visible reg., Surface chem of Si complicated, -LIGA: litho, galvano-forming, plastic moulding costly (req. clean room) - Inclusion complex migrate w EOF, while micelles (Use of high en. radiation (X-ray or short UV)

-In CE, sample intro by on-column injc part of sep col. serve as injc., Injc vol. small (1 50 nL) VS several L in GC & HPLC, Length of injc sample plug (injc sample vol) should be as small as possible to minimize possibility of zone spreading (loss of efficiency & reso) except for on-column preconcentration/stacking of dilute sample -Hydrodynamic (Hydrostatic) injc: gravity flow, pressure or vacuum suction - NO sample bias 1) Gravity flow injection: vol. injc (Hagen-Poiseuelle Law) q = g r^4 h ti / (8L) = B h ti B = g r^4 / (8L) - Amt injc: w = g r^4 C ti / (8L) = B h ti C Where = density of sample sol, g = grav., r = internal radius of cap., h = height diff. bet liq. level in sample & buffer reservoir, ti = injc time, is viscosity, L = cap. length, C = conc. b. Pressure or vacuum injection: - Amount injected: w = r^4 PC ti / (8L) = B P ti C where P = pressure diff. (P = g h) -Correction (usually small) for cap. travel time bet. reservoir in hydrodynamic injection: wtot = wi + 2 wT = h C (ti + 2 tT) B Where wtot = total amt injc, wi = amt injc during actual injc, wT = amt injc during travel time b4 or after injc, h = height difference, C = sample conc., ti = actual injc time, tT = travel time - Hydrodynamic injc. by PRESSURE: Sample vial pressurized, forcing sample sol. into cap. - Vol. of sample injc. depend on magnitude & duration of pressure applied, viscosity, cap. dimension After injc, cap. placed back into inlet buffer vial and electric field applied -Hydrodynamic injection by VACUUM: Vacuum applied to destination vial, pulling sample into cap.

- Salt-mediated Stacking of Neutral Solutes: Electric field over injc plug reduced relative to over BGE due to high salt content. At high pH, EOF directed toward cathode, pushes everything in that direction. Countermigrating negatively charged surfactant micelles enter front of injection plug, exposed to low field strength in that region. Their countermigration toward anode slows,and they stack up at head of zone. Neutral solutes pushed to catho by EOF. At front of injection zone, they encounter a very high concentration of micelles. Since neutrals bind to micelle through hydrophobic interaction, solute concentration build up at zone boundary. - Whole Capillary Injection: Entire cap. or large portion of it filled w anionic sample dissolved in water. When negative volt. applied, anions migrate toward positive electrode and stack at boundary bet. injc zone and enter BGE. EOF naturally drives overall migration velocity toward negative electrode. Simultaneously, water electroosmotically exit cap. at negative-electrode injc end. As water exits, BGE enter cap. at opposite end. As analyte encounter BGE, migration ceases (no field strength over highionic-strength BGE as volt. drop occurs over water) When water is nearly out of cap. (shown by current reading), power supply polarity reversed. Analytes quickly separated and eluted. - Sweeping: picking and accumulating of analyte by pseudostationary phase that fill sample zone during appli. of volt. - Evolution of analyte zones in EKC under sweeping condition: A. Starting situation. injc of sample sol. S with length linj conditioning w BGS. a1* and a2* analyte molecule farthest from S/BGS boundary, and mc* = first batch of micelles that enter S zone after appli. of negative volt. B. Micelles frm cathodic vial enter S zone and sweep analytes into narrower band (ka1 > ka2), where k = retention factor = ratio of no. of moles of solute in mobile micellar ph. to stat. aq. ph. C. First batch of micelles that enter S zone reach interface bet. S and BGS zone D. Sep. of zones based on MEKC: dmc = linj dai = linj (kai) /(1+kai) lsweep = dmc dai = linj /(1+kai) Csweep = Cinj (1+kai) * CE DETECTION 1) UV/Visible absorbance det.:Widely available, commonly used in HPLC, suitable for analytes w chromophore 2) Indirect det.: Decrease in bkgrd signal detected, useful for det. species which are difficult to detect (do not absorb UV/vis) 3) Laser induced fluores. det.: Highly sensitive (require fluorophore for excitation by laser source). Derivatization necessary to intro suitable fluorophore -Stacking: Sample stacking w sample dissolved in 4) Mass spectrometry (MS): Useful for structure det. sol. (water) that has lower conductivity than EP run & cpd identification, only volatile buffer used for buffer. Top: Sample injected, Mid: Volt. applied and compatibility w vacuum operation, high cost since electric field in sample sol. is higher rest of - UV/VIS: A = C l cap., cations migrate more rapidly thru sample sol. - Variable UV/Vis det.: White light of all until reaching boundary w buffer, and stacked at diffracted to indiv. by grating. Grating turned to boundary., Low: Stacked sample migrate thru cap. select that impinges on cap. After light passes thru as narrow plug cap., its intensity measured by photodetector and -Stacking w polarity switch: A: Fill cap. w sep. detector converts intensity to absorbance units. buffer, measure current (I). Large vol. of sample hydrodynamically injc., B: Negative volt. applied at inlet, stacking samp., force out sample sol., C. When most sample sol. removed (current ~90% original), polarity reversed., D. Stacked anions sep by CZE

- Small cap., light deviate more frm straight path, smaller linear range Extended Light Path Cap.: Bubble cell (pathlength 3x, Too wide = peaks merge), High-sensitivity cell (pathlenth 1.2mm) Z-cell (pathlength to 3mm) - Indirect UV det.: Mobile ph. contain component that provide actual response (open circles). When - A 100-nL injc = ~10% of cap. vol. Compared w a 5- analytes (solid circles) reach det, displacement nL injc, limit of detection improved by factor of 13. results in in bkgrd signal (egative peak) Even w stacking buffer, some injection-mediated -Conc. limit of det. : Clim = Cm / (DR x TR) band broadening occur. While a 20-fold LOD where Cm = conc. of added bkgrd species (UV improvement as expected, improvement only a active, electroactive, or fluorescent), DR = dynamic factor of 13 reserve = ratio of bkgrd signal to noise, TR = no. of -Stacking of cations in low-pH buffer: (Both EP & bkgrd molecule displaced or replaced by 1 analyte EO towards cathode). When positively charged molecule (Transfer ratio) solute migrate out of injc zone and encounter BGE,

- Preferable to have small Clim -5% C give smallest pore sizes for all %T - Indirect det. almost universal; does not require - Initiation: chemical peroxide (ammonium persulfate chromophore, fluorophore or electrophore in sample w N,N, N,N-tetramethylethlenediame, TEMED, as catalyst, or UV photochemical) - Polymer sol. forming macromolecular network used as size sieving media in CE (replaceable gels) - Linear Range of Sep. for Nucleic Acids on Denaturing PAA Gels - Denaturing gel: contain additives (Urea) that maintain the molecules to be sep. in denatured state - Compared to conventional slab gel, CGE = faster (minutes vs hrs); peaks (area - digital) more quantitative than bands in slab gels (visual) * MEKC (MECC): Based on solute partitioning bet. the micellar ph. & the sol. ph. (or relative solubilities - Fluorescence detection: Find for optimum in the 2 phases). Permits sep. of neutral & charged excitation and maximum fluores., highly sensitive species. Micelles form in sol. when a surfactant is specific - Laser-induced fluores.: Light from laser focused added to sol. (water) in conc. above its critical micelle conc. (CMC) by lens onto cap. Emitted light collected at right - Micelles consist of aggregates of surfactant angles, filtered, det. by photomultiplier molecule w typical lifetimes of <10s. Most common used surfactant in MEKC is sodium dodecyl sulfate (SDS) (CMC=0.008 M, aggregate no. = 58 at 298K) which is an anionic surfactant (Anionic head + hydrocarbon tail). Anionic micelles move at slower rate than bulk flow (EOF) thru cap. due to attraction by anode - Neutral molecules partition in & out of micelles based on hydrophobicity (Very hydrophilic neutral molecules (MeOH) spend very little time in micelles -Eg: He-Cd laser, 442 nm whereas very hydrophobic molecules (Sudan III; -CE with ESI-MS detection commonly used micelle marker), spend nearly all the -CE-MS: A = electrically insulating sampling box; B = time inside micelles & later migrate relatively slowly anode and sample injc reservoir; C = fused Si cap.; - Micelles often referred as pseudostationary phases D = cathode and electrospray needle; E= electro- Order: polar compounds; followed by more hydrospray; F = focusing ring; G = nozzle; H = skimmer; I phobic molecules vx = veo + [ k*/(1+k*)]vmc where = rf only quadrupole; J = ion entrance aperture; K = vx = electromigration (EO + EP) velocity of solute, quadrupole MS; L = channeltron electron multiplier veo = EO velocity, Vmc= migration velocity of micelle (-ve), k* = phase capacity ratio = moles of X in micelle/moles of X in buffer - Surfactant: 1) Anion: SDS, cholic acid, 2) Cationic: CTAB (Cetyltrimethylammonium bromide), 3) Zwitterionic: CHAPS, CHAPSO, 4)Non-ionic: Triton X100 *CEC: based on distribution equilibria. Utilizes packed or coated cap. Packing material enhance EOF due to charges on their surfaces. Not widely -Electrospray ionization (ESI) interface setup used since manufacture & use of packed cap. showing liquid sheath electrode: impose practical difficulties - Enhanced veo since each particle bears own electrical double layer. Additional selectivity due to interaction with stationary phase *CIEF: Sep. of amphoteric species (positive or negative depending on pH) based on differences in isoelectric points (or pI). Commonly used to sep. proteins. Protein samples & sol. that forms pH gradient placed inside cap (mixture of ampholytes). Anodic end of cap. placed in acidic sol. (anolyte) - On-Column Conductivity Detector for CZE: Constructed by fixing platinum wires thru diametric whereas cathodic end placed in basic solution (catholyte) opposite holes in 50- or 75-m-i.d. fused-Si cap. tubing. These 40-m i.d. holes were made w comp- - Under an applied electric field, charged proteins controlled C02 laser. Under a microscope, two 25- migrate thru medium until they reside in region of pH m-o.d. Pt wires placed in holes exactly opposite to where they become electrically neutral & stop each other to minimize potential diff. bet. electrodes migrating. Zones focused until steady state reached. After focusing, zones migrate (mobilized) from cap. when high electrical field strength applied. hydrodynamically (by gravity or pressurized flow), or - Contactless Conductivity Detection for CE: by adding salt to anolyte (by principle of electroelectrodes made of conducting silver varnish or metallic syringe cannulas w the cap assembled thru. neutrality, sodium ions exchange for protons in tube, - C^4D uses transmitter electrode to subject sample generate a pH imbalance gradient which cause region to large amplitude, high freq. electromagnetic migration of components chemical mobilization) - Cathodic Mobilization (proteins move to cathode): signal. A corresponding, attenuated, AC signal register at receiver electrode. Size of received signal Competing ion added (Cl-) to one of focusing reagents (catholyte) disrupting eq attained during affected by conductivity of sample. Received AC focusing. Addition of competing ion causes pH shift signal deconvoluted to convert amplitude into DC analog voltage signal appropriate for data collection that swept entire length of cap., effectively mobilizing - Procedure: As analyte ions pass into det. region, focused protein zones. - Anodic Mobilization (proteins move towards they cause small changes to overall sample anode): Competing ion added (Na+) to one of the conductivity. Continous monitoring of conductivity signal show series of peaks, areas (or heights) that focusing reagent (anolyte) disrupting the eq. attained during focusing. Addition of competing ion causes a are related to analyte conc. Signal processed like conventional chromatogram. C4D electrodes do not pH shift that swept entire length of cap., effectively mobilizing focused protein zones. make direct contact w sample and are electrically *CITP: Performed in a discontinuous buffer syst. isolated frm sample (ideal for EP det.), electrode fouling eliminated. Since vast majority of analytes for (different buffer at 2 end of cap.) CE are ionic, a C4D system functions as a 'universal' - Sample components condense bet. leading & terminating electrolytes, producing a steady state method of det. requiring minimal sample prep. migrating config. composed of consecutive zones Sensitivity similar to UV-Vis. - Electropherograms from CITP appear as series of 2: Principles of Separation in CE -Modes: 1) CZE capillary zone, 2) CGE capillary steps, w each step representing an analyte zone; measured zone length, (proportional to amt of gel, 3) MEKC micellar electrokentic chrom., 4) sample present) used for quantitation CEC capillary electrokinetic chrom., 5) CIEF -On-column Transient Isotachophoretic Preconc: capillary isoelectric focusing, 6) CITP capillary Inject large plug, Concentrate to small plug, More isotachophoresis, 7) Additional separation mech:. conc. higher sensitivity Inclusion complexation, chiral CE, CD-MEKC *CZE: Separation of charged species, Base on differences in EP mobilities. Sep. mechanism based ondifferences in solute size & charge at a given pH. Migration velocities of ionic species depend on EO& EP flows -Order: Cations; then neutral; then anions vx = veo + vep - Electroosmotic flow (EOF) arises from charges on inner wall of cap. & serves as bulk flow - Electrophoretic (EP) flow is the migration of a charged species under the influence of a voltage - If EOF > EP flow, all species regardless of charges migrate along w EOF A: Injection step: sample injected into col. filled w *CGE: Sep. based on differences in solute size as leading electrolyte, B: Focusing step: terminating analytes migrate through the pores of gel-filled cap., electrolyte placed in reservoir and voltage applied, molecular sieving mechanism C: Separation step: reservoir replaced w leading - Gels serve as anti-convective media which electrolyte, and sep. voltage applied. minimise peak broadening due to diffusion, prevent - Additional sep. mechanism: ion exchange solute adsorption to wall of cap. & eliminate EO interaction, complexing interactions, charge (unlike in sep. of ions by CZE where EOF used to association, particle interaction, formation of reduce analysis time, EOF is not favorable in CGE inclusion complex, chiral sep. for achieving purely sizebased sep.; also, EOF may - Inclusion complexation w Cyclodextrins (CD): destroy gel) CD have cavities which can form inclusion complex, - Gels must exhibit temp. stability & suitable pore CD can be charged additional selectivity size. CGE capable of extremely high efficiencies (30 - CD-MEKC: Analytes can form inclusion complex w m theoretical plates per m of cap.). PAA common CD or partition into micelles migrate more slowly, - Pore size of PAA determined by total gel (acryl(samples interacting w CD more strongly migrate amide) conc., % T, & conc. of crosslinking agent %C faster). Combination of CD w micelles provide - PAA, Acrylamine, Acrylic Acid: additional selectivity. - Chiral separation by CE: 1. CE: (a) Indirect sep. (b) Direct sep.: CD, crown ether, carbohydrate, macrocyclic antibiotic, protein, ligand-exchange (le), chiral ion-pairing reagent, chiral surfactant, ionic liq., - %T = [bis + acryl]/V*100% dual selector syst., chiral selector, 2. Cap. electro%C = [bis/(bis+acryl)]*100% where bis = weight chromatography (open tubular cap., packed cap., of bisacrylamide, acryl = acrylamide weight, V = total monolithic phases - si-based, polymer-based, vol. -%T, Pore size particle-fixed), 3. imprinted chiral phase, 4. non-aq.

CE, 5. miniaturized technique, 6. Det. sensitivity, 7. on-line chiral CEMS coupling, 8. Miscellaneous Chiral CE: (vs HPLC): high reso., low cost (no chiral col.), simple and versatile (chiral selector) Chiral CE: Microscale (difficult fraction collection) Low UV sensitivity (short path length) - Importance of Chiral Sep.: Living organisms made of chiral biomolecule (AA, sugar, protein, nucleic acids). Many natural biomolecules exist in only one enantiomeric form (AA in L-form, sugar in D-form) Living organisms show different biological response to one enantiomers in drugs, pesticides etc. Enantiomers have different pharmacological activities, pharmacokinetic and pharmacodynamic effect. Body interacts w each racemic drug differently, metabolize each enantiomer by sep. pathway to produce diff. pharmacological activity. One isomer may produce desired therapeutic activities, while the other inactive or, produce unwanted effect. *Examples: 1) Positively charged CD: 2-O-(2aminoethyl-imino-propyl)- -Ohydroxypropyl- CD as chiral selector in 50mM NaH2PO4, (CDen): ethylenediamine derive. substituted in primary pos., (THCMH): cysteamine-bridged hemispherodextrin 2) Carbohydrate: (HS-Cys): Highly sulfated cyclosophoraoses, 3) Ionic liquids: Organic salts w melting pt. < 100 C. Soluble in polar & nonpolar sol. 4) Chelate complexing -CD deriv.: Mixed complexes of histamine subst. -CD w tryptophan enantiomers 5) Polymer-based monolith: Chiral selector (+)-1-(4- aminobutyl)-(5R,8S,10R)-terguride attached to epoxy group at surface of monolith 6) Particle-fixed monolith: particle-loaded teicoplanin aglycone monolith, 7) Miniaturized tech: (a) integrated catalysis/analysis chip to study cleavage rate of substrate glycidyl phenyl ether by different enzymes. Heptakis-6-sulfato--CD used as chiral selector. (b) single-wall carbon nanotubes-BSA conjugates via two-step process of diimide-activated amidation in polymethylmethacrylate chips using DLtryptophan as model compound, (c) ITPCZE coupling system for sample preconc. and clean-up in chiral pharmaceutical analysis, 8) Misc: Device for applying a radical field w respect of controlling EK potential and EOF used for chiral sep. of terbutaline. -Power dissipation: Cap. tube contain EP medium behaves as cylindrical ohmic conductor (V=IR, volt. =current x resistance). To attain high efficiencies in CE, necessary to ensure efficient heat dissipation. Electricpower dissipated per unit length of cap.: P/L = Cr2 V2 /L2 where P = power, L = cap. length, = molar conductance of sol., C = buffer conc., r = col. radius & V applied volt. - Effect of Adsorption on Peak Shape and Efficiency: Adsorption results in peak distortion and irreproducibility. Large molecule (proteins) vulnerable. Effect of adsorption on efficiency: Had = 2 C2 (1-C) [ep + eo] E tad where Had = contribution frm adsorption to plate height, E = electric field, C = fractional conc. of free solute, tad = mean residence time of adsorbed solute. - Fronting Peak: Solute anion mobility higher than run buffer anion mobility

-EOF = Ionization of Si-O-H+ grp or adsorption of ion frm buffer results in negative charges on inner surface of cap. Negatively charged surface attracts positively charged cations diffuse double layer. Layer which is more tightly held by negatively charged groups (e.g. Si-O-) Fixed layer. Layer which is NOT held tightly is called the mobile layer. - When electric field applied, mobile layer pushed toward negatively charged cathode, resulting in electroosmotic flow (EOF). Plane bet. fixed & mobile layer is Stern plane. Imaginary plane in mobile layer that separate mobile fluid frm fluid that remains attached to surface is the slipping plane. Potential difference at interface bet. Fixed and mobile layer (relative to the bulk fluid) is zeta potential, - EOF proportional to zeta potential, results in sol. flow w/o use of mechanical pumps - Zeta potential proportional to thickness of double layer: = 4 e/ where = thickness of diffuse double layer, e = charge per unit surface area, = dielectric constant : Ratio of electrical energy stored/capacitance of a material to that of vacuum (Vacuum: 1, MeOH: 30, H2O: 80) -EOF profile: driving force (charge along cap. wall), no pressure drop, flow velocity uniform across cap. does not contribute to band broadening like pressure-driven flow in LC - Hydrodynamic flow profile: Frictional forces at col. wall cause pressure drop across col. CE = sharper flow profile (sharp peaks) sep. efficiency - Electroosmotic mobility ( EOF or eo) EOF = where = zeta potential = dielectric constant, = viscosity of buffer Mobility of EOF depend on buffer charact, and independent of applied electric field - Velocity of EOF ( vEOF or veo) vEOF = where E = applied electric field (= volt./length of cap.) depends on applied electric field (or voltage/distance), buffer pH, buffer conc., organic solvent and surface of capillary - Effect of applied voltage: volt. EOF: migration times, faster sep. - voltage sep. efficiencies provided Joule heating of buffer solution dissipated (Joule heating cause convection and/or bubbles) Joule heat proportional to power (watts): P = VI - Max. voltage used determined by Ohms law Plot (current vs voltage)

ernary amines, Hydrophobic end of attached quarternary amines associate w other quarternary amines in solution. Their exposed positive charges attract negatively charged anions frm buffer. Solvated anions migrate to anode.

Example Calculation of EOF at Two pH Values A certain solution in a capillary has a electroosmotic mobility of 1.3 x 10-8m2/Vs at pH 2 and 8.1 x 10-8 m2/Vs at pH 12. How long will it take a neutral solute to travel 52 cm from the injector to the detector with 27 kV applied across the 62 cm long tube?

- Hystersis: forward and reverse paths do not coincide! - To minimize effect: New capillary: Flush with MeOH, water, 1N NaOH, water (510min each), Flush 20min run buffer, Flush w 1N NaOH 5 min, wait 5min, water 5min, run buffer 2030min, conditioning bet. runs, first try flushing w buffer only, try not to expose the cap. to pH extremes, flush neutral or basic buffer w 0.1 N NaOH, buffer bet. runs, flush acidic buffers w 0.1M phosphoric acid and buffer bet run -Effect of EOF on reso: EOF sep. time sep. efficiencies but reso - Reso from EP:

-EP mobility: - q/r, EP mobility -EP velocity:

- EOF increases w pH ! - Effect of pH buffer: Buffer pH changes zeta potential, At high pH, more dissociation of Si-OH to Si-O- greater zeta potential greater EOF. At lower pH, less surface ionization, & lower zeta potential. At pH < 2, NO EOF - EOF decreases w buffer conc. - Inorganic/Organic buffer: EOF decreases linearly with ln (ionic strength) - Tailing Peak: Solute anion mobility lower than run - Effect of buffer conc.: If temp controlled, buffer buffer anion mobility conc. (or ionic strength) EOF. If temperature not - To obtain symmetrical peak shape, mobility of run controlled, buffer conc. will EOF (current, buffer should match that of solutes temp, viscosity of buffer 1: Introduction - Effect of temperature - Joule Heating :Result of - Capillary Electrophoresis (CE): modern anac resistance of sol. to flow of current (electrical power: technique used to separate different species based P = VI = I2R). If heat is not sufficiently dissipated , on differences in their EP mobilities in a suitable resulting temp. and density gradients reduce sep. medium inside a small capillary efficiency. Heat dissipation key to CE: Power per - Basic CE Instrument: High-voltage (30 000V), capillary, 2 buffer reservoir, detector simple, low unit capillary P/L r^2 - For smaller capillaries heat dissipated due to large cost, easily miniaturized. surface area to volume ratio: capillary internal volume =r2 L End result: if heat dissipated, high potential can be applied for extremely fast separations (30kV) - Effect of organic solvent: Hard to predict effect of organic solvent on EOF. Depends on which solvent & how much added - 1) Adding MeOH to water: viscosity up to 50% MeOH, then (H-bond), 2) Adding acetonitrile to water: viscosity frm 0 to 100% acetonitrile - Effect of organic solvent on EOF proportional to - Zeta potential: Inside wall cap. covered by silanol ratio : / groups (SiOH) that are deprotonated (SiO-) at pH > - Control of Electroosmotic Flow by Chemical 2. SiO- attracts cations to inside wall of cap. Modification of capillary inner surface: Distribution of charge at surface described by Stern Chemicals bonded to cap. wall or simply dissolved in double-layer model. buffer (dynamic coating). Chemical modification results in EOF due to: Shielding of charge on cap. wall, Increased viscous drag, Polymers (PAA & polyvinylalcohol). For dynamic coating, surfactants & hydrophilic polymers used (cetyltrimethylammonium bromide (CTAB) cationic surfactant) - Measurement of EOF: 1) Injection of uncharged cpd, neutral marker (nm): VEOF = where le = effective length of cap. (distance from inlet to detector), tnm = migration time of neutral marker - Criteria for neutral marker: Uncharged at buffer pH , detectable, Pure, no interaction with cap.wall, -EOF = Excess cations in diffuse Stern double layer Soluble in buffer (Eg. MeOH, mesityl oxide, flow to the cathode. Net flow occurs as solvated formamide) cations drag along sol. - Method 2: Gravimetric method: (weighing buffer being flushed out of cap. for a given period of time Accurate balance needed, Prevent evap. - Reversing EOF: When analysing ANIONS, it is desirable to reverse direction of EOF, & polarity of applied volt., since this arrangement would cause anions to migrate out faster & sep. time reduced. - How to reverse EOF: Quanternary amines (CTAB or CTAC), TTAB or TTAC (tetradecyltrimethylammonium bromide or chloride), Protein (alphalactalbumin) - Reversal of electroosmotic flow by addition of quarternary amine to buffer: Negatively charged SiO- grp at cap. wall attract positively charged quart capillary internal surface area = 2 r L

Tutorial 1 1. Typical diameter and length of a capillary in CZE: 10 to 100 m and 30 to 100 cm long. 2. Two main forces in CZE: EP & EO 3. Surface of quartz cap. in buffer or water: H+ ions dissociate, surface becomes negatively charged. 4. Net migration proceed to the cathode when a potential is applied to cap. 5. What causes electro-osmosis? Electro-osmosis results when mobile cations in electrical double layer due to the surface silanol groups on the Si cap. surface migrate in bulk to cathode when a positive volt. applied. 6. Control rate of mobility in electrophoretic portion? The voltage. 7. To resolve substances with extremely small differences in mobility:The best resolution obtained when EOF just balances EP migration (R is large when EOF~ - Ave). 8. At pH 10, wall of bare cap. negatively charged w Si-O- grps and there is strong EOF to cathode. At pH 2.5, wall nearly neutral w Si-OH grp and there is almost no EOF. The few -Si-O- groups left give slight flow to cathode.The aminopropyl cap. has positive flow at pH 10, but rate ionly about half as great as that of bare cap. Negative charge reduced because most of -Si-O- grp converted to -SiCH2CH2CH2NH2 although some of aminopropyl grp may be deprotonated (-Si-CH2CH2CH2NH- ) at pH 10. At pH 2.5, all of the aminopropyl groups are protonated. Net charge on wall is positive and flow is reversed. 9. Based on the van Deemeter eq, explain how CE theoretically can provide unprecedented resolution and extremely sharp peak profile: a. By using an open tubular column, we can eliminate plate height contributions due to multiple flow paths. b. Without having to use stationary phases, the mass transfer term that comes from the finite time needed to equilibrate between the mobile and stationary phases can be eliminated. c. The only fundamental source of broadening under ideal conditions is longitudinal diffusion. H = A + B/u + cu Tutorial 2 1) DNA Sequencing in CE: Sanger sequencing (chain termination or dideoxy method) involves using enzymatic procedure to synthesize DNA chains of varying length (according to the template or sample) in 4 different rxn, stopping DNA replication at pos occupied by one of the 4 bases, and then determie resulting fragment lengths. Each chain terminators of the fragments labelled w fluorescent dyes, each w different of fluores. depending on which base it has. Different fragments migrate out at different times based on size differences, and detection performed at 4 . 2) DNA Fingerprinting in CE: Short Tandem Repeats (STR): STR: repeated sequences of 3-5 base pairs (loci) which can be identified in a known database. Useful in DNA analysis because they show great variability among indiv. Newer and more precise method yielding errors of about 1 in 1029. Does not require very much DNA, can couple w PCR. Variable (polymorphic) nature of STR regions intensifies discrimination bet. one DNA profile and another. Likelihood that any two individuals (except identical twins) will have the same 13-loci DNA profile can be as high as 1 in 1 billion or greater. - STR Analysis: Extraction of nuclear DNA from cell of forensic sample of interest. Amplification of specific polymorphic regions of extracted DNA by PCR. Analysis of these DNA regions by either gel electrophoresis or CE. Determination of no. of repeats of STR sequence in question. Use fluorescent dyes for CE - CE: DNA sample run thru thin Si polymer-coated cap., DNA travel thru cap. via an induced electric field. Fragments migrate according to charges and sizes. Sample identification done by use of fluorescent tracker tags attached during PCR. Multiple samples analyzed simultaneously (multiplex analysis) 3) Laminar VS Turbulent Flow: In turbulent flow, extensive mixing of molecules/ions prevent separation of components into discrete zones/peaks. 4) For a small molecule with a diffusion coefficient of 106cm2/s, how far would it diffuse after flowing over 1 cm at a flow rate of 10 cm/s? Ld= 2Dt = 2 x 10 -6cm2/s x 0.1cm = 0.00045 cm = 4.5 m Tutorial 3 1. Estimate migration times and no. of theoretical plates for sep. of the following ions by CZE, assuming that the EO mobility is 40 x 10^ -9. Ion Electrophoretic mobility (x 10 -9): Ion 1 = 25, Ion 2 = 20 Effective length of cap. is 50 cm and applied voltage is 20 kV. Assume diffusion coefficients are equal to 1 x 10^-9.

tm = L/v = L^2/ [( eo + ep )V], N = L^2/^2 = (eo + ep )V/2D eo = 40 x 10 ^-9., D = 1 x 10^-9 , L = 50 cm, V = 20 kV ep,1 = 25 x 10 ^-9 ep,2 = 29 x 10^ -9 tm,1 =(50 x 10 -2)2/ [(40x10^ -9 + 25 x10^ 9)x20000]= 192s tm,2 =(50 x 10 -2)2/ [(40x10^ -9+ 20 x10^ 9)x20000]= 208s N1 = (40x10^ -9 + 25 x10^ -9)x20000/2x10-9 = 650,000 N1 = (40x10^ -9 + 20 x10^ -9)x20000/2x10-9 = 600,000 2. (a) Migration orders in CZE for (i) Uncoated fused Si cap.: cation, neutral, anion, (ii) Coated fused Si cap. (assume no EOF): only cations, (iii) Coated fused Si cap. w reversed EOF and negative voltage: anions, neutral, cations (b) Suitable buffer system for separation of group of anions: 50 mM chromate: UV background, 0.05 mM CTAB: EOF modifier; 100 mM NaOH and 1/10 v/v nitric acid to adjust pH 3. Explain effect of n-butanol (EOF modifier together with quarternery ammonium salt) on the EOF: Both butanol and Q+ adsorbed on cap. surface by dynamic equilibrium. Adsorbed butanol molecules occupy part of surface and shift the Q+ solutionsurface equilibrium so that the surface receives a net positive charge at a lower Q+ than it does in the absence of butanol. Lecture Questions (2) 1) Origin of EOF: OH grps lose H+. Some H+ in fixed layer. Remaining cations (H+) in mobile layer pushed by the +ve HV. 2) Why EO mobility >>> EP mobility? Many Si-OH grps, a lot more negative charges on Si wall than typical molecule 3) Why doe EOF VS pH plot show hysteresis? Starting from the acidic pH, H+ is gradually removed as pH. Starting from high pH, H+ is gradually added as pH . (Competition bet. anions due to absorption and anionic exclusion due to surface potential repulsion). 4) Comment on plot of no. of theoretical plates VS applied voltage: N with Volt. Overloading for nonzero intercept, negative deviation from linearity of high V/I may be result of T causing convection (Joule Heating) 5) Cap. Coated with trimethylchorosilane reduce EOF to give better sep. & reso. (3) 1) How can stat. ph. particles be packed in cap. w/o being eluted out? Use Frits. 2) Focused zones can detect w/o mobilization by scanning whole cap. w/o polyamide coating 3) coating cap. to eliminate EOF b4 CIEF: stable focusing, need to mobilize samples after focusing or use non-std det. 4) Isotachophoresis: same velocity (4) 1) Qualitative info from CITP: relative step height (RSH) counted as a ratio of the step height of analyte to step height of terminator 2) Quantitative info from CITP: zone length, conc. can be calculated by comparing the step length of the cpd w calibration curve of the std sol. (6) 1) Why EK injc. used in sample intro for CGE sep. of oligonucleotide? Hydrodynamic injc. would displace or extrude gel filled inside cap. 2) In sample stacking, diff bet two types of physiochem properties bet. Sample buffer and run buffer exploited: Electrical conductivity & pH 3) Why pointed tip used in electrospray interface? Highly localized electric field gradient formed at curvature of pointed tip 4) In sep. of explosives in MEKC, explain why cpds detected as negative peak? Commonly used explosive not fluorescent. Indirect laser induced fluores. used. Fluorophore added in CE buffer to produce constant baseline signal. At det zone, since analytes displace fluorophores, this will cause fall in constant baseline leading to negative peak. 5) The noise of a good detector is typically 5x10^-5 AU. A modest chromophore has a molar absorptivity of 5000. For a 50-um cap., what is the estimated conc. limit of detection (CLOD), assuming a signal to noise ratio of 5. C(lim) = Cm/ (DR x TR) CLOD = A / (a/b) = (5 x 5 x 10^-5) / (5000 x 5 x 10^-3) = 1x 10^-5 M

Adv of CE:

Disadv of CE:

(7) 1) In SDS-PAGE separation of proteins, what is the function of SDS? Sodium dodecyl sulfate (SDS) is an anionic surfactant which denatures proteins by wrapping around their polypeptide backbone (in a mass ratio of 1.4:1). In so doing, SDS confers negative charges to the polypeptide inproportion to its length, which means the denatured polypeptides become rods of negative charge cloud with equal charge-to-size ratios. So the SDS-protein complexes will be separated by different sizes, not charge-to size ratios. 2.There is a limit of DNA size in CGE separation of DNA. Describe 2 conventional methods and a microchip method to overcome such a limit? A.pulsed electric field time required for large DNA molecules to change their direction of migration in response to changing direction of electric field depends on size; B.voltage gradient increase voltage for separation of larger DNA; 3) Microchip method to overcome the limit: Nanofluidic separation device with many entropic traps. (A) Cross-sectional schematic diagram of the device. Electrophoresed DNA molecules are trapped whenever they meet a thin region, because their radius of gyration (Ro) is much larger than the thin region depth (here, td and ts are the thick and thin region depths, respectively). (B) Top view of the device in operation. Trapped DNA molecules eventually escape, with a probability of escape proportional to the length of the slit that the DNA molecule covers (wa and wb). Larger molecules have a higher escape probability because they cover wider regions of the slit (wb > wa). (C) Experimental setup. Reservoirs are made at both ends of the channel and filled with DNA solution. (8) 1) What are the main adv. and disadv. of aptamers compared with antibodies?

2. What are the main advantages and disadvantages of CE-SELEX compared with conventional SELEX? SELEX 1)Conventional partitioning methods - timeconsuming and resource-consuming - leads to DNA structures that bind to the surfaces of the filters or chromatographic support used rather than to the target, 2)Too many rounds of selection - very limited number of unique aptamer sequences obtained at their output, 3) If efficiency of partition is too low, SELEX can completely fail to select aptamers CE-SELEX 1) NECEEM partitioning method elimination of kinetic bias as there is no washing step 2) Partitioning efficiency exceeds by at least two orders of magnitude, which decrease number of rounds from 10 to 1 3. 3) Facilitates determination of binding parameters 4) May not inject the whole library of DNA due to small volume of injection . To inject 10^13 sequences in 50 nL, concentration of nucleic acids in library must be 2.3 mM - a very large nucleic acid concentration which may degrade peak shape in CE separation. 3. What are the main advantages and limitations of non-SELEX? Advantages of non-SELEX of PCR amplification are avoided respect to different oligonucleotides is avoided -amplifiable libraries Limitations of non-SELEX -Lower number of DNA sequences for sampling allows only a small fraction of the collected ligands to be sampled each round strong affinities (9) 1.Describe six problems associated with the use of polymeric chips? a)A. different polymeric materials show different UV transmittances. b)Polymeric materials have low dielectric breakdown voltages which prevent the use of high electric fields in CE c)Deep polymer channels are prone to collapse under high suction d)Many polymer (e.g. PDMS) surfaces are hydrophobic and cause problems in liquid filling of aqueous buffers into the channels. e)EOF in polymeric channels is ~10-fold less than that in glass chips f)Polymers can easily be swollen by organic solvents g)Thermal conductivities of polymeric chips are low, and this may have a problem dissipating heat

2. Native PDMS channels are hydrophobic. What are the different methods used for filling these channels? a)In order to fill aqueous solutions into PDMS channels, they need to be primed with polar solvents, e.g. thanol (up to 5%), Tween 20 (0.1% v/v), or BSA (1% in PBS). b)PDMS chips can be filled by immersing in buffer solution in an ultrasonic bath for 5-10 min. c)The channels can also be primed with CO2 gas, and so after solution filling, CO2 bubbles disappear since the gas is readily dissolved in aqueous solutions. 3. Describe three methods to generate liquid flows within microchannels? a)Hydrodynamic flow or pressure-driven flow can be achieved by a syringe pump or by suction. b)Electrokinetic flow, which includes electroosmotic and electrophoretic flow, is driven by an electric field applied along the microchannel c)Centrifugal flow in microchannels is generated by rotating a disk consisting of the microchannels that are arranged in the radial direction. The fluids are loaded at the center of the disk, and the flow moves from the center to the rim of the disk 4. What is the difference between a photomask and an etch mask in glass micromachining? An etch mass is formed on top of the glass substrate to protect the area that is not intended for etching; whereas a photomask is placed on top of the etch mask to transfer the pattern on it by UV exposure 5. Compare and contrast positive and negative photoresists. Both poitive and negative photoresists are used to transfer the pattern for etchig onto a substrate by photolithography. For positive resists, exposure to UV light weakens the chemical structure of the resist so that it becomes more soluble in, and is washed away by, the developer solution. The photomask, therefors, contains an exact copy of the pattern which is to be fabricated on the substrate. Negative resists behave in just the opposite manner. Exposure to the UV light causes the negative resist to become polymerized, and thus more difficult to dissolve. Therefore, the negative resist remains on the surface wherever it is exposed, and the developer solution removes only the unexposed portions. Photomasks used for photoresists, therefore, contain the inverse or photographic negative of the pattern to be transferred. (10) 1) Laminar flow is prevalent in microfluidic channels. Describe one advantage and one disadvantage of such a physical phenomenon. Laminar flow inside a microchannel is advantageous since this allows easy transport of different streams of solutions without mixing, and can produce a contact zone between two different liquids where isolated reactions or solvent extraction can take place. The disadvantage is that mixing between different streams of solution is solely based on diffusion, which is a slow process for the chemical reaction of the bulk liquid to complete. 2) In immunoassay, what are antigen and antibody? Can an antibody act like an antigen? Antigen: a foreign macromolecule that does not belong to the host organism and that elicits an immune response. Antibody: an anitgen-binding immunoglobin that functions as the effector in an immune response. In some cases, antibodies can act as antigens which elicit their antibodies, e.g. anti-IgG, where IgG is itself an antibody but acts an antigen.

Selectivity