Electroanalytical Methods Of Biological Materials | Anna Brajter-toth,

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Electroanalytical Methods Of Biological Materials

ABSTRACT

This text details contemporary electroanalytical strategies of biomolecules and electrical phenomena in biological systems. It presents significant developments in sequence-specific DNA detection for more efficient and cost-effective medical diagnosis of genetic and infectious diseases and microbial and viral pathogens. The authors discuss the late

TABLE OF CONTENTS

chapter 1|4 pages

Charge Migration Through the DNA Double Helix

chapter |1 pages

Figure 1 Reactions used to study electron transfer in DNA with photophysical methods. (A) Photoinduced reduction of Rh(phi)bpy by *Et [12]. (B) Triplet

chapter |1 pages

[14–15]. (D) Photoinduced oxidation of G by adenine analogues (*A [16]).

chapter |3 pages

mediated electron transfer reactions to base stacking. (A) Photoinduced electron transfer between *Et and Rh(phi) bpy is attenuated by the

chapter |7 pages

Figure 3 Sensitivity of DNA-mediated long-range chemical reactions to base stacking. (A) The long-range oxidation of distal guanine doublets is decreased in the presence of intervening bulges disrupting base stacking [18]. (B) Long-range repair of thymine dimers is attenuated in the presence of bulges [22].

chapter |2 pages

Figure 5 Series of DM-modified duplexes constructed to evaluate long-range electron transfer through DNA films. (Adapted from Ref. 24.) Figure 6 The presence of a CA mismatch between electrode and intercalator prohibits the electrochemical reduction of crosslinked DM (Ref. 24.)

chapter |3 pages

Figure 7 The electrochemical response of DM noncovalently bound to a DNA film decreases when the immobilized duplexes contain a CA mismatch. (Adapted from Ref. 25.)

chapter |3 pages

Figure 9 Detection of a CA mismatch with electrocatalysis at a DNA film. (Adapted from Ref. 25.)

chapter 2|1 pages

Electrochemical DNA Biosensors

chapter |3 pages

Figure 1 Major processes involved in any biosensor system: analyte recognition, signal transduction, and readout.

chapter |3 pages

monolayer in a solution containing the target DNA. (From Ref. 8.)

chapter |2 pages

Table 1 Electroactive indicators commonly used for the biosensing of DNA hybridization

chapter |1 pages

Figure 5 Voltammetric hybridization response of a biosensor based on a electropolymerizable oligonucleotide-substituted polypyrrole, to increasing levels of the DNA target: 0(a,b), 66(c), 165(d), and 500(e) nmol. (From Ref. 27.)

chapter |4 pages

Figure 6 An ion-channel sensor based on a PNA probe immobilized on gold electrode, and detection of the hybridization based on the electrostatic repulsion of a negatively charged redox marker (shown as an octahedron). (From Ref. 32.)

chapter 3|3 pages

Amplified and Specific Electronic Transduction of DNA Sensing Processes in Monolayer and Thin-Film Assemblies

chapter |1 pages

Figure 1 (A) Equivalent circuit corresponding to the impedance features of a DNA-modified electrode interfaces in the presence of a redox probe. (B) Schematic faradaic impedance spectra presented in the form of a Nyquist plot for: (a) A modified electrode where the impedance is controlled by diffusion of the redox probe (low frequencies) and by the interfacial electron transfer (high frequencies). (b) A modified electrode where the impedance is

chapter |1 pages

controlled by diffusion of the redox probe. (c) A modified electrode where the impedance is controlled by the interfacial electron transfer within the entire range of the applied frequencies.

chapter |3 pages

for: (a) An electrode of low resistance. (b) A modified electrode exhibiting high resistance.

chapter |1 pages

Figure 3 (A) Chronocoulometric transients measured in the presence of 50 µM Ru(NH ) in 0.01 M Tris-

chapter |1 pages

buffer, pH 7.3, for: (a) A bare Au-electrode; (b), (c), and (d) After modification of the electrode in the presence of (1), 5 µM, for 60, 90 and 180 min, respectively. (B) Surface coverage of (1) on the electrode derived by chronocoulometry (∆), and by microgravimetric experiments (•), at different time intervals of modification with (1).

chapter |2 pages

Figure 4 Schematic amplified analysis of a target DNA using: (A) An oligonucleotide-enzyme conjugate and a biocatalyzed transformation as amplification route. (B) An oligonucleotide-functionalized particle (liposome or nanoparticle) as an amplifying unit.

chapter |5 pages

Figure 5 Cyclic voltammogram of cytochrome c at the Au-electrode functionalized with the primer 5′-ACGGATGCTCC-(CH )SH that was

chapter |3 pages

Figure 8 Biocatalyzed transformations leading to the precipitation of insoluble products on the transducer interfaces. Figure 9 Stepwise assembly of a DNA-sensing electrode by the functionalization of the conductive support with the sensing oligonucleotide (13), interaction with the target DNA (14) pre-hybridized with the biotinylated oligonucleotide (15), and the association of the avidin-HRP conjugate (16). The biocatalyzed precipitation of the insoluble product (8) is used as an amplification route for the electrochemical transduction of the biorecognition event.

chapter |2 pages

oligonucleotide (13) on the interface; (c) after the formation of the complex (2 h interaction) between the sensing interface and the target DNA analyte, (14), 5.8×10 mg·mL, pre-

chapter |3 pages

Figure 11 Time-dependent frequency changes resulting upon the biocatalyzed precipitation of (8) onto an Au-quartz crystal modified with the (13)-oligonucleotide sensing monolayer, which was interacted with the target DNA, (14), 5.8×10

chapter |1 pages

Figure 13 (A) Faradaic impedance spectra of (a) the (17)-functionalized Au electrode, (b) after interaction of the sensing electrode with (18) (5×10

chapter |2 pages

interaction with the (19)-functionalized liposomes. Inset: Faradaic impedance spectra of (a) the (17)-modified electrode, (d) after interaction with (18a) (5×10 M), (e) after treatment

chapter |1 pages

Figure 14 (A) Time-dependent frequency changes of the (17)-

chapter |3 pages

functionalized Au-quartz-crystal upon: (a) Interaction with (18), 5×10 M. (b)

chapter |2 pages

Figure 15 (A) Faradaic impedance spectra of (a) The (17)-functionalized Au electrode. (b) After interaction of the sensing electrode with (18) (5×10

chapter |1 pages

Figure 17 (A) Time-dependent frequency changes of a (22)-functionalized Au/quartz crystal upon interaction with: (a) The analyte DNA, (23), 2×10 M. (b) After treatment of

chapter |3 pages

and (23) with the (24)-functionalized Au-nanoparticles. (c) After treatment of the sensing interface with (23a), 5×10 M. (d) After treatment of the

chapter |1 pages

Figure 19 (A) Faradaic impedance spectra corresponding to: (a) A bare Au electrode. (b) The (26)-functionalized electrode. (c) After interaction of the sensing electrode with the analyte DNA, (27), 6.5×10

chapter |2 pages

After interaction with avidin, 200 ng·mL . (f) After interaction of the

chapter |2 pages

probe corresponding to (a) The Au-bare electrode. (b) The oligonucleotide (26)-functionalized electrode. (c) After interaction of the sensing electrode with the analyte DNA, (27), 6.5×10

chapter |3 pages

functionalized electrode; (b) after ligation of (30), 1×10 M, with the

chapter |3 pages

Figure 24 Chronocoulometric transients for (a) A bare Au-electrode. (b) The oligonucleotide (33)-modified Au-electrode. (c) The (33)-modified Au-electrode after hybridization with M13φ, 2.3×10 M, for 1.5 hours. (d)

chapter |1 pages

Figure 27 (A) Faradaic impedance spectra corresponding to the amplified sensing of the vesicular stomatitis virus (VSV) RNA: (a) The (35]-functionalized electrode. (b) After hybridization with the VSV-RNA, 1×10 M, in a solution consisting of

chapter |3 pages

NaCl, 80% formamide, pH=7.5. (c) After the reverse transcription for 45 minutes (80 U·mL ) in the presence of

chapter |1 pages

Figure 28 Amperometric transduction of the formation of a double-stranded complementary DNA complex using a SBP-DNA conjugate as an electrobiocatalytic amplifier.

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Figure 29 Chronoamperometric responses of the (36)-functionalized redox-hydrogel-modified electrode upon the hybridization and the sensing of DNA, (37), and its mutants, (37a) and (37b), at variable temperatures: (a) the analyte, (37); (b) the single-base mismatched DNA mutant, (37b); (c) the four-bases mismatched DNA mutant, (37a). (A) At 45°C. (B) At 57°C. The amperometric responses originate from the electrobiocatalyzed

chapter |2 pages

reduction of HO, 1×10 M, E=−0.06

chapter |1 pages

Figure 31 (A) Faradaic impedance spectra corresponding to (a) the (38)-modified electrode; (b) the (38)-modified electrode upon interaction with (39), 3×10 mole·mL; (c) the

chapter |3 pages

double-stranded (38)/(39)-functionalized electrode after interaction with the Klenow fragment, 20 U·mL in a Tris-buffer, pH=7.8.

chapter |7 pages

functionalized Au-quartz crystal with (40), 3×10 mole·mL, followed by

chapter 4|7 pages

Direct Electrochemistry of Proteins and Enzymes at Electrodes

chapter |1 pages

k=2.2×10 cm/s, α= 0.31, and D =D=1.1×10 cm/s. Solid line is

chapter |2 pages

Figure 3 (A) Cyclic voltammetry of cytochrome c a tin doped indium oxide optically transparent electrode after purification. Solutions contained [Cyt c]=73 mM, 0.21 M Tris and 0.24 M cacodylic acid, pH 7.0, electrode area=0.71 cm . Potential scan rates in

chapter |2 pages

[Cyt c]=199 M, 0.05 M NaSO,

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ferricytochrome c at 275 mM; (b) ferrocytochrome c at 310 M. Solution is pH 7.0 Tris/cacodylic acid buffer.

chapter |3 pages

Figure 6 Background-subtracted cyclic voltammetry of metmyoglobin: (A) 86 µM met-myoglobin, pH 7.0; (B) 67 mM metmyoglobin, pH 7.0, with excess cyanide to give [Mb(III)H O]:[CN] of 1:300. Scan

chapter |2 pages

Figure 8 Model of the cytochrome c oxidase immobilized electrode surface. The octadecyl mercaptan anchor molecules contain sulfur, biological amphiphiles have two tails, and Cyt is cytochrome c.

chapter |4 pages

Figure 9 Cell used in preparing and characterizing cytochrome c oxidase in the electrode-supported bilayer membrane. Cell dimensions are 2×5×5 cm. The QCM electrode and the two cell parts are clamped together.

chapter |1 pages

Figure 12 Flow injection analysis of reduced cytochrome c reacting at a cytochrome c oxidase modified electrode. Three sequential 83 µL injections of 100 µM reduced cytochrome c. Applied potential is 472 mV vs NHE, solution contains 0.1M phosphate buffer, pH 7.4, and the electrode area is 0.2 cm .

chapter |3 pages

Figure 13 QCM-FIA data of oxidized cytochrome c at a lipid bilayer modified electrode containing no cytochrome c oxidase: (a) 10 µM cytochrome c in 0.10 M phosphate buffer; (b) 2.5 µM cytochrome c in 0.010 M phosphate buffer; (c) 5 µM cytochrome c in 0.010 M phosphate buffer; and (d) 10 µM cytochrome c in 0.010 M phosphate buffer. Buffer pH is 7.4 and the electrode area is 0.2 cm .

chapter |3 pages

Figure 15 Tapping mode-scanning force microscopy images of cytochrome c oxidase immobilized in a lipid bilayer membrane on an electrode, (a) topographic image, (b) phase contrast image.

chapter 5|1 pages

Voltammetric Investigations of Iron-Sulfur ur Clusters in Proteins

chapter |2 pages

Figure 1 Structures of the major classes of Fe-S clusters. The larger clusters are found in nitrogenase: [8Fe-7S] and [8Fe-7S] are oxidized and

chapter |4 pages

Figure 2 The oxidation levels that are possible for 2Fe, 3Fe, and 4Fe clusters. Oxidation levels having an odd number of d-electrons are usually easy to detect by EPR.

chapter |1 pages

Figure 3 Interactions of protein molecules with an electrode. Voltammetric responses expected for (A) diffusion to an electrode densely

chapter |3 pages

packed with productive sites; (B) diffusion to an electrode having such a low population of productive sites that they behave as micro-electrodes; (C) a homogeneous adsorbed layer of noninteracting protein molecules.

chapter |1 pages

Figure 5 Voltammograms of solutions of 7Fe ferredoxins at a PGE electrode: (A) Azotobacter vinelandii, (B) Sulfolobus acidocaldarius. In each case, proteins are 80 µM in 20 mM

chapter |2 pages

Mes, 0.1M NaCl, 2mM neomycin, 0.1 mM EGTA, pH 6.4. Scan rate 5 mV s , temperature 0°C.

chapter |1 pages

Figure 6 Cyclic voltammograms of 7Fe ferredoxins immobilized at a PGE

chapter |2 pages

electrode with polymyxin as co-adsorbate, pH 7.6, temperature 0°C. Conditions (not optimized to reveal couples in clearest detail) are as follows. Top: Azotobacter vinelandii FdI; scan rate 100 mV s ; Center:

chapter |1 pages

Figure 3.21 Biocatalyzed ligation, replication, and scission of single- and double-stranded DNA on electronic transducers. Figure 5.4 The structure of Azotobacter vinelandii 7Fe ferredoxin

chapter |1 pages

and views of the two clusters, [3Fe-4S] and [4Fe-4S]. Figure 5.8 Structure in the vicinity of the buried [3Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin, showing the residues that may assist proton transfer. Upper view shows important residues and distances; lower view shows the same view but through a van der Waals surface; note the cluster is buried.

chapter |1 pages

Figure 5.23 Structures of fumarate reductase and hydrogenase showing the Fe-S “wires.” (From coordinates published with Refs. 93 and 94.)

chapter |1 pages

Figure 7.1 Surface representation of HRP colored by calculated ET rates (blue<10 s, red>10 s). The

chapter |2 pages

Figure 7.2 Representative orientations of HRP taken from docking calculations of (A) nonglycosylated HRP and (B,C) glycosylated HRP. (B) Extended carbohydrates, solution conditions and (C) folded-back carbohydrate, dry conditions. The surface of graphite used in the calculations is represented by a black plane in the bottom of the diagram. Coloring and molecular representation (surface, ribbon, and ball and stick) are identical to those used in Figure 7.1. The result of 250 docking runs suggests that the molecules come to rest on the surface in nonrandom orientations with preferentially maximal surface area in contact.

chapter |1 pages

Figure 9 Voltammograms of Azotobacter vinelandii 7Fe ferredoxin D15N and native forms, each commenced from the oxidized state, showing how the oxidation reaction is ‘gated’ in different time domains (*) by proton transfer. Proton transfer is retarded in the D15N mutant, thus re-oxidation is not observed at 1Vs but

chapter |1 pages

the proton does not arrive at the cluster during the cycle. For the native protein, re-oxidation is not observed at 100 V s .

chapter |2 pages

Figure 10 Representative “Trumpet Plots” for D15N and native A.v. FdI. Those for D15N also show the fits based on k=2.5 s. Note the region

chapter |3 pages

to Table 2. (Reproduced by permission from Nature (Ref. 62). Copyright 2000 Macmillan Magazines Ltd.) Table 2 Thermodynamic and Kinetic Parameters for Redox-Coupled Proton Transfer at A.v. FdI and Mutant Forms, Extracted from Protein Film Voltammetry Experiments

chapter |2 pages

Figure 13 Proposed formation of a rapid intermediate in the oxidation of the hyper-reduced [3Fe-4S] cluster.

chapter |3 pages

Figure 15 Cartoon showing coordination of the [3Fe-4S] cluster in P.f. Fd and D.a. FdIII and the reaction with metal ions such as Fe to form a cubane adduct. The labile metal ion may be coordinated by the carboxylate group of the aspartate that replaces cysteine in the normal motif.

chapter |1 pages

[1989]. Copyright, The Biochemical Society.) Figure 17 Formation of [M3Fe-4S] cubane adducts (M=Fe, Zn, and Cd) in

chapter |3 pages

D.a. FdIII adsorbed as a film on a PGE electrode with neomycin as co-adsorbate. Left-hand side shows successive cycles (oxidative direction only) at 470 mV s : [Fe]=300 µM;

chapter |1 pages

Figure 19 Top: Cyclic voltammograms of C.p. 8Fe ferredoxin adsorbed on a PGE electrode, scanned before and after applying a 3-second oxidative pulse at 720 mV vs SHE (result shown is third cycle (100 mVs ) after the pulse. Voltammetry

chapter |2 pages

Graph showing how the ratio of [3Fe-4S] to [4Fe-4S] clusters depends on the pulse potential. Pulse duration 3 seconds; cluster populations measured on the third cycle.

chapter |1 pages

Figure 21 Voltammograms showing reversible binding of an exogenous ligand (RS= ethanol 2-thiolate) to the

chapter |1 pages

transformed [Fe3Fe-4S] cluster in Desulfovibrio africanus FdIII, as observed by increasing the scan rate. The protein is adsorbed to approximately mono-layer coverage on an edge-oriented pyrolytic graphite electrode, using neomycin as coadsorbate, and the electrolyte contains 0.2M NaCl at pH 8.0, with 0.1 mM Fe(II). Temperature 0°C.

chapter |2 pages

Figure 22 Square scheme depicting the reversible binding of an exogenous ligand to a [4Fe-4S] cluster; represented as [Fe3Fe-4S]. Data are as determined by simulation for binding of 1-hydroxyethane-2-thiolate at the transformed cluster of Desulfovibrio africanus FdIII, at 0°C. Note that RS

chapter |1 pages

Figure 24 Voltammograms of a film of E. coli fumarate reductase (approximately mono-layer) adsorbed on a PGE electrode in the presence of polymyxin at pH 9.0. Top. No substrate, baseline corrected. FAD signal is clearly observed above the Fe-S clusters. Voltammogram also detects the low-potential (medial)

chapter |6 pages

[4Fe-4S] cluster. Bottom. In presence of fumarate (0.2mM) with electrode rotating at 3000 rpm. Note the boost that is observed at a potential corresponding to the [4Fe-4S] cluster. (Adapted with permission from J. Am. Chem. Soc. 1997, 119, 11628–11638. Copyright 1997 American Chemical Society.)

chapter 6|2 pages

Polyion and Surfactant Films on Electrodes for Protein Electrochemistry

chapter |2 pages

Figure 2 Structures of surfactants and lipids that form bilayers.

chapter |1 pages

buffer containing no protein on a bare PG electrode; (b) 25 µM Mb purified by ultrafiltration in buffer on bare PG; (c) 20 µm Mb-DDAB film on PG electrode in buffer, no Mb in solution. (Adapted from Ref. 17 with permission. Copyright 1995 American Chemical Society.)

chapter |1 pages

Figure 4 Conceptual model of structures of films cast from insoluble surfactants and myoglobin.

chapter |2 pages

Figure 5 Thin-layer cyclic voltammograms of approximately 0.5 µm Mb-DLPC film on PG electrode at 2Vs and 25°C in buffers of different

chapter |1 pages

(Adapted from Ref. 26 with permission. Copyright 1995 American Chemical Society.)

chapter |1 pages

Figure 7 Influence of electrode material on formal potential (E) of

chapter |4 pages

Figure 8 Influence of pH on formal potential for films of Mb-DDAB from CV (○) and SWV (+), Mb-DMPC from CV (□) and SWV (●), and Mb-DLPC from CV (+) and SWV (∆). CV values are averages for scan rates from 0.1 to 2Vs , SWV values are

chapter |1 pages

Figure 10Cyclic voltammograms on basal plane PG electrodes at 0.1Vs

chapter |3 pages

Table 2 Electrochemical Parameters for Cyt P450 at pH 7

chapter |2 pages

Figure 13 Illustration of the method of layer-by-layer adsorption for constructing polyion-protein films.

chapter |1 pages

Figure 15 Polycations and polyanions used for layered film formation.

chapter |1 pages

Figure 16Cyclic voltammogram at scan rate of 0.5Vs of Au-

chapter |3 pages

Figure 17 QCM frequency changes for dried films at each step of assembly of a Mb/DNA film on a smooth Au-MPS underlayer. DNA was double-stranded calf thymus (ds-CT). Measurements after adsorption cycles 9–11 were omitted. (Adapted from Ref. 81 with permission. Copyright 1998 American Chemical Society.)

chapter |1 pages

Figure 19 Cyclic voltammograms at 0.3Vs in pH 5.5 buffer of

chapter |1 pages

Figure 20 Influence of number of bilayers deposited on the amount of electroactive myoglobin in films constructed on rough PG with various polyanions. PSS and ds-CT DNA were adsorbed from 0.5M NaCl solutions

chapter |4 pages

Figure 21 Cyclic voltammograms showing effect of catalytic reduction of TCA by Mb in DMPC films at 0.1V/s and pH 5.5. Numbers on curves show TCA concentration in mM; dashed curve to the far right is for reduction of TCA on a DMPC-coated electrode without Mb. (Adapted with permission from Ref. 25. Copyright 1997 Elsevier.)

chapter |7 pages

Figure 25Cyclic voltammograms at 0.2Vs in pH 7.4 buffer in a sealed

chapter 7|11 pages

Electrochemistry of Peroxidases

chapter |8 pages

Figure 3 Calibration plots for glucose in the presence (○) and in the absence (●) of 0.1 mM acetaminophen. The calibrations were recorded with graphite electrodes modified with bienzyme layer of (A) native HRP and glucose oxidase or (B) recombinant HRP and glucose oxidase. The measurements were done in a flow-injection mode, with a flow carrier of 20 mM phosphate buffer, pH 7 containing 0.1M KCl. Applied potential was -50 mV vs. Ag/AgCl/0.1 M KCl.

chapter 8|1 pages

Mechanisms and Kinetics of Neurotransmission Measured in Brain Slices with Cyclic Voltammetry

chapter |4 pages

Figure 1 Anatomy of a neuron.

chapter |5 pages

Electrode carbon fiber radius was 6 µM.

chapter |2 pages

Figure 5 Schematic of presynaptic mechanisms involved with the uptake of dopamine (DA). MAO, monoamine oxidase (located on the surface of the mitochondrion); VMAT, vesicular monoamine transporter DAT dopamine transporter. Cellular machinery has been enlarged to show detail.

chapter 0|3 pages

V and was initially to positive potentials. The upper cyclic voltammogram was obtained by subtracting the average of five scans immediately following the first stimulus pulse from the average of five scans during the baseline preceding the stimulus pulse. The lower cyclic voltammogram was obtained by subtracting the average of five scans 13 s after the stimulus was applied from the average of five scans obtained during the prestimulus baseline. (Reproduced from Neuroscience with permission [17].)

chapter |3 pages

was given at 1, 5, 10, 30, or 60 s after the first pulse. Inset: Rate of recovery measured as the height of the second pulse relative to the first pulse for the control response (○; n=5) and the response in the presence of 2 µM sulpiride (●; n= 4). Solid lines in the inset are double exponentials. For data with sulpiride present, the relative percentage was 100×[1/2 exp (−1.1t)+1/2 exp (−0.15t)]. For control data the relative percentage was 100×[1/2 exp (−1.1t)+1/2 exp (−0.4t)]. (Reproduced from Journal of Neurochemistry with permission [6].)

chapter |5 pages

Figure 9 Effects of amphetamine (10 µM) on release and uptake of DA. Measurements were made 30 min after drug application. DA release was evoked in the mouse caudateputamen by a single electrical impulse. Measured concentrations were recorded at 100 ms intervals. Concentrations were measured before drug application (●) and 30 min after 10 µM amphetamine (▲). Solid lines represent simulations obtained with the best-fit parameters: before drug, [DA] =9.0 µM, V=3.5 µM/s,

chapter 9|5 pages

Electrochemical Monitoring of Exocytosis from Individual PC12 Cells in Culture

chapter |1 pages

current scale (C). Data was obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. Figure 2 Amperograms of individual vesicular exocytosis events from PC12 cells. (A) Amperogram of a control experiment with the tip of the working electrode 200 µm from the cell. When the electrode tip is bathed with 30 nL of stimulating solution (1 mM nicotine

chapter |1 pages

in 105 mM K balanced salt solution), a slow change in the background amperometric response is observed. (B) Amperogram of vesicular exocytosis induced by bathing a cell with stimulating solution (described above), with the electrode placed on top of the cell. Narrow current transients are clearly observed at times corresponding to the first two chemical stimulations. Only background current is observed following a third cell stimulation. It appears that the vesicles responsible for the release of catecholamines are depleted with multiple stimulations on this time scale. (C) A one-second portion of data from the first stimulation in B is displayed here with an expanded time scale. All data were obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. (Reproduced from Anal. Chem. with permission [15].)

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Figure 3 Frequency distribution of the amount of catecholamine contained in individual current transients from single cells. The bins are divided into 66 zmol intervals. Numbers on the x-axis represent the mean of each interval. Data shown have been obtained from the first stimulation of 13 individual cells; 1912 spikes have been included. (Reproduced from Anal. Chem. with permission [15].)

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Figure 4 Q and Q histograms from a single bovine chromaffin cell. (A) Area histogram for spikes obtained during 15 repetitive 60 mM K

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Figure 5 Q histograms for

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Figure 6 Fast-scan cyclic voltammetry data collected during stimulated exocytosis of a single PC12 cell. Cyclic voltammetry was performed in the two- electrode mode using an SSCE reference electrode for all measurements. A scan rate of 300 V/s was employed using a triangular wave form that was ramped between −0.5 and +1.0 V. (A) Average current within a 60-mV window of the peak oxidation potential of DA. Individual current spikes represent faradaic current. (B) Enlargement of the region between scans 600 and 800 showing two current transients used to generate voltammograms. (C) Background-subtracted voltammograms generated by subtracting the average signal of

chapter |2 pages

scans 687–690 from the signal of scan 693. (D) Background-subtracted voltammogram of a standard 25 µM DA solution collected using the flow system described in the text. The same buffer medium is used for both cellular experiments and for standards. (Reproduced from Anal. Chem. with permission [13].)

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Beveling a carbon fiber on a 45° angle creates an elliptical surface with major and minor radii of about 3.5 and 2.5 µm, respectively. It is apparent that a large difference exists between the vesicular area and that of the total electrode. (Reproduced from Anal. Chem. with permission [13].)

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for 1 hr. Cyclic voltammetry was performed in the two-electrode mode using an SSCE reference electrode for all measurements. A scan rate of 300 V/s was employed using a triangular wave form that was ramped between −0.5 and +1.0 V. (A) Average current within a 60-mV window of the peak oxidation potential of DA. Individual current spikes represent faradaic current. (B) Enlargement of the region between scans 1010 and 1225 showing multiple current transients used to generate voltammograms. (C) Background-subtracted voltammogram generated by subtracting the average signal of scans 1020–1023 from the signal of scans 1025–1026. (D) Background-subtracted voltammogram of a standard 25 µM DA solution. (Reproduced from Anal. Chem. with permission [13].)

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Figure 9 Apparent distributions of vesicular concentrations detected by fast-scan rate cyclic voltammetry following K-stimulated exocytosis.

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(A) Distribution generated from control PC12 cells (n=29 cells, 77 total events). (B) Distribution generated from L-DOPA-exposed (100 µM for 1 hr) PC12 cells (n=11 cells, 145 total events). For each data set, the peak current of the anodic wave of all events is matched to its respective calibration plot to determine the detected concentration. All data are collected into bins having increments of 0.25 µM and plotted as the percent of the total number of release events detected. (Reproduced from Anal. Chem. with permission [13].)

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Figure 11 Normalized histogram of calculated vesicle radius (open bars) overlaid with the known radius histogram (shaded bars) from Schubert et al. (1980) (14 bins, 10 nm/bin each). (Reproduced from J. Neurosci. Meth. with permission [31].)

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Figure 12 Vesicular DA concentration histogram (20 bins, 49 mM/bin) calculated from Eq. (4) where each r

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release of ACh detected from a rat magnocellular basal forebrain neuron (C). In this example, the detector consists of a patch of rat skeletal muscle membrane expressing embryonictype nicotinic ACh receptors voltage-clamped to −80 mV (C). Unitary channel currents are visible on the decaying phase of the response. The expanded portion of the data shows a small artifact preceding the first channel opening. This is due to extracellular pickup of the somal action potential by the patch electrode and conveniently can be used to estimate the delay between stimulation and transmitter release. (Reproduced from Trends Neurosci. with permission [33].)

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the microsensor (shown by the short bar). In panel A, release from a single cell was evoked by histamine exposure and resulted in the amperometric spikes due to catecholamine oxidation (lower panel). At the same time, Ca

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Figure 15 Current-time traces for exocytosis at single PC12 cells. A6s ejection of stimulant [105 mM K (A),

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Figure 16 Distributions of latencies observed between stimulation and secretion. The time between the application of the stimulus and the detection of released dopamine for each exocytosis event in the first 40 s of release is plotted versus the percentage of the total number of events observed for each type of stimulus. The differences in the time courses of release stimulated by 105 mM K (■; n=17 cells; 475 events), 1

chapter |1 pages

events), and 1 mM muscarine (□; n=19 cells; 439 events) are apparent. All data were obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. (Reproduced from J. Neurochem with permission [18].) Figure 17 Differentiated PC12 cells with varicosities. Bright-field microphotograph of 4-day-old PC12 cells that have been exposed to 100 ng/mL levels of β-NGF continually from initial subculturing observed through the 40X objective of an IX70 Olympus microscope. The arrow points to a varicosity from which

chapter |2 pages

dopamine release is monitored. This structure is completely covered by the electrode when it is placed on the varicosity.

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of 9 of the 16 cells. The second stimulation and release indicates that the varicosity is still responsive after initial stimulation. (B) Representative data from one of the seven cells that exhibited a delay between the time of stimulation with 105 mM potassium chloride and secretion of DA. The mean vesicle content in both immediate and delayed release is 178 zmol. The 5 µm carbon fiber electrode is held at a potential of 0.65 V vs. SSCE for all measurements. The inset is an example of a typical current transient observed at a varicosity displayed on an expanded time scale. The release time (3 ms rise-time of the current spike) is similar to that observed at an undifferentiated cell (Reproduced from Brain Res. with permission [3].)

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Figure 19 Distribution of vesicle content for potassium-stimulated release at varicosities plotted as the

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cubed root of catecholamine released. Plots of the percent of total events observed in the first 40 s following initiation of release vs. the cubed root of the amount of catecholamine released upon elevated potassium stimulation for (A) 17 undifferentiated PC12 cells (475 total release events) and (B) 16 differentiated PC12 cells (156 total release events). (Reproduced from Brain Res. with permission [3].)

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Figure 20 Comparison of single PC12 cells cotransfected with reporter pEGFP and either control pcDNA3 plasmid (A), pBoNT/C1 (C) or a nontransfected cell from this condition (B). Cells were transfected with 20 µg pEGRP and either 20 µg control pcDNA3 or 15 µg pBoNT/C1 and 5 µg pcDNA3. Four to six days post transfection, cells were examined by fluorescence microscopy. A

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fluorescent control transfectant (A), a nonfluorescent untransfected cell from the toxin transfection dish (B), and a fluorescent toxin-transfected cell (C) were identified. Cells were loaded with dopamine and stimulated with a3s pulse of 100 µM ATP at 100 s following the start of the amperometric recording. All amperometric data were obtained using a 5 µm carbon fiber electrode held at 0.70 V vs. Ag/AgCl (Reproduced from Pflugers Arch. with permission [5].)

chapter |1 pages

release for (A) a control cell and (B) a cell that has been incubated in 10 µM d-AMPH for 10 min. Cells were stimulated by local perfusion (30 nL of 1 mM nicotine in 105 mM KCl saline) to induce vesicular exocytosis. (C) A histogram displaying the percentage of peak sizes in paired control and AMPH-treated cells. Peaks are combined in intervals of 30,000 molecules. The lower limit of each bin size is shown on the abscissa. All data were obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. (Reproduced from J. Neurosci. with permission [52].)

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Figure 22 (A) Amperometric trace from a PC12 cell incubated for 40 min in 50 µM L-DOPA. Individual quanta are represented by the peaks after stimulation and the quantal size is determined by the charge of each peak. The sharp inflection in the baseline is due to a6s perfusion application of the stimulation saline (arrow). The width at half height (t) is determined

chapter |2 pages

sizes by percentage. L-DOPA exposure shifts the distribution of quantal sizes to the right. The lower limit of each bin size is displayed on the abscissa. Data sets are not significantly different from normal distribution (Kolmogorov-Smirnov normality test; p>0.05). All data were obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. (Reproduced from J. Neurochem. with permission [16].)

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Figure 23 Effect of reserpine on quantal release from PC12 cells. In the

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first column, a single PC12 cell was repeatedly stimulated with 80 mM KCl for 3 s at the arrows; amperometric spikes were elicited in each case. In the second column, a different cell was treated identically except that at 2 min following the first stimulation, 1 µM reserpine was added to the medium. Subsequent stimulations indicate that quantal release is abolished by reserpine. All data were obtained using a 5 µm carbon fiber electrode held at 0.65 V vs. SSCE. (Reproduced from Anal. Chem. with permission [13].)

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Figure 24 Summary of spike characteristic ratio values created at individual PC12 cells. Under control conditions, there was an average of approximately 194,000±13 molecules released per vesicle. For the ratios

chapter |1 pages

presented, an average of 169±27 and 102±12 amperometric values were used to determine pre and post means, respectively. Bars represent the mean ±SEM of spike characteristic ratio values for the different experimental conditions (control, n=6; L-DOPA, n=7, reserpine, n=5; reserpine and L-DOPA, n=5). Values marked with * are statistically different, with p<0.001; indicates a significant difference, with p<0.01; and * indicates a significant difference, with p<0.05 vs. control (ttest). (Reproduced from J. Neurosci. with permission [57].) Figure 25 Representative TEM images from single PC12 cells following treatment with (A) physiological

chapter |5 pages

saline, (B) 100 µM L-DOPA, (C) 100 nM reserpine, and (D) 100 µM L-DOPA and 100 nM reserpine for 90 min. A portion of the nucleus can be seen for the cells shown in A and B (at the top and bottom, respectively). Scale bars in each figure represent 500 nm. (Reproduced from J. Neurosci. with permission [57].)

chapter 10|5 pages

Milestones of Electrochemical Immunoassay at Cincinnati

chapter |1 pages

Figure 1 The protocol for metal-label based immunoassay for human serum albumin (HSA) using releasable In.

chapter |6 pages

Figure 2 Differential pulse anodic stripping voltammograms from an HSA immunoassay as illustrated in Figure 1 for a series of HSA standards of increasing concentration (A–F) in 0.1M citrate pH 1.5. Voltammetry conditions: hanging mercury drop electrode (volume 0.37 µL), deposition time 5 min, deposition potential −800 mV vs. Ag/AgCl scan rate 2 mV s,

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Figure 6 General protocol for competitive and sandwich enzyme immunoassays.

chapter |6 pages

Assay conditions were as follows: 10 µg/mL primary antibody concentration, 1/125 digoxin-ALP conjugate dilution, 4-h antigen/antibody incubation interval, and 40-min substrate reaction time. (From Ref. 24.)

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Figure 11 Reaction of the activated carboxyl group of the protein with the amino groups on the inner surface of the capillary (From Ref. 3.)

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Figure 13 Schematic of the overall sandwich immunoassay based on paramagnetic beads. (From Ref. 3.)

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Figure 14 Schematic of the experimental setup for the rotating disk electrode (RDE) detection in a bead-based immunoassay using the ALP-PAPP enzyme-substrate pair.

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Figure 15 Current-time plots for a bead sandwich immunoassay with RDE detection for the model analyte Bugbead. (From Ref. 52.)

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Figure 16 Schematic representation of scanning electrochemical microscopy (SECM) for imaging heterogeneous enzyme immunoassay microsurfaces.

chapter |2 pages

Figure 17 (A) Schematic representation of bead deposition. (1) Drop of bead suspension is extruded out of a positive-displacement micropipet; (2) after concentrating the beads at the bottom of the drop, the drop is reduced to 0.3 µL; (3) the drop is set on the hydrophobic surface; (4) the drop will stay at the pipet tip if the pipet is retracted slowly from the surface, leaving the beads behind. (B) SEM images of bead domains produced using 1 µL of (1) 4.1×10 ,

chapter |1 pages

Concentration of mouse IgG in ng/mL: (a) 1150, (b) 115, (c) 11.5. (From Ref. 61.)

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Figure 19 An automated meso-scale electrochemical immunosensor system with the components (a) microbead reservoirs, (b) rinse buffer reservoir, (c) conjugate (Ab*) reservoir, (d) enzyme substrate reservoir, (e) valves, (f) electromagnets, (g) central micro-fluidic silicone channel, (h) rotary pump.

chapter |5 pages

Figure 20 Current-time plots of a mouse IgG immunoassay done in the meso-scale immunosensor shown in Figure 19.

chapter 11|8 pages

Electrochemical Detection of Peptides

chapter |3 pages

Figure 3 Detection scheme for peptides containing cystine (RSSR) and cysteine (RSH) employing a dual gold amalgam working electrode system.

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Figure 4 Typical potential step sequence in (A) pulsed amperometric detection (PAD) and (B) integrated pulsed amperometric detection (IPAD) of peptides.

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Figure 5 Reaction of NDA with a primary amine. (From Ref. 131.)

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Figure 6 Reaction of OPA with a primary amine and a thiol. (From Ref. 132.)

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Figure 7 Reaction of 6-AQ with a primary amine. (From Ref. 132.)

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Figure 8 Reaction of DNPT with a primary amine. (From Ref. 140.)

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Figure 9 Reaction of NDTE with a primary amine. (Adapted from Ref. 141.)

chapter |1 pages

Figure 10 Reaction of Cu(II) with a peptide. (Adapted from Ref. 147.) Figure 11 Application of a dual-electrode electrochemical detector to products of the biuret reaction.

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Figure 12 Half-wave potential ranges for tyrosine, tryptophan, and a number of peptide classes versus Ag/AgCl. Here XXD represents a peptide composed of any amino acid in the first and second position with Asp in the third position.

chapter 12|2 pages

Microfabrication of Electrode Surfaces for Biosensors

chapter |1 pages

Figure 1 Enzymes as signal transducers. As drawn, an enzyme specific for only one substrate is immobilized in close proximity to the electrode surface. The substrate (e.g., glutamate) is oxidized by the enzyme to the corresponding enzyme product (e.g., α-ketoglutarate) with concurrent reduction of a cofactor in a1:1 ratio. (NAD is reduced to NADH for

chapter |1 pages

“transduced” to an electroactive species by the enzyme. Barring introduction of NADH (or peroxide) to the solution, any increase in faradaic current may then be attributed to the presence of the enzyme substrate alone. Figure 2 Amperometric biosensor configurations. Detailed descriptions are in the text. (A) Addition of substrate to a solution containing enzyme and cofactor. (B) Immobilized

chapter |3 pages

enzyme “upstream” from sensing electrode. (C) Enzyme immobilized directly on detecting surface.

chapter |10 pages

tip, and the outer surface of the fiber is usually chemically treated to remove any epoxy, etc. This cleaning has been found to hinder electron transfer as compared to an electrochemically pretreated disk electrode. To make a disk electrode (B), the end of a pulled electrode is first cleaved with a scalpel, then polished/beveled on polishing wheel. Following electrochemical pretreatment, for example pre-anodization/pre-cathodization [8], this surface exhibits facile electron transfer and much better time resolution than the barrel configuration.

chapter 13|2 pages

Electrocatalytic Determination of Biochemical Compounds

chapter |2 pages

Figure 1 Cyclic voltammetry of (A) 5.6 mM cysteine and (B) blank electrolyte at a glassy carbon electrode coated with a film of mvRuOx. Electrolyte, 0.2MK SO adjusted to

chapter |1 pages

electrode with 0.2M KSO at pH 2.0

chapter |1 pages

Figure 3 Hydrodynamic voltammogram for the oxidation of 0.58 µM insulin with a mvRuOx-coated glassy carbon electrode. (From J.A.Cox and T.J.Gray, Anal. Chem. 61, 2462, 1989, with permission of the publisher). The conditions are those in Figure 2 except that the currents are corrected for the component from the oxidation of water.

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Figure 4 Flow-injection analysis of 95 nM bovine insulin (MW 5733) by oxidation at a mvRuOx-coated electrode. (From J.A.Cox and T.J.Gray, Anal. Chem, 61, 2462, 1989, with permission of the publisher). The conditions are those in Figure 2 with an applied potential of 0.96 V vs. Ag/AgCl.

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Figure 7 Structure of phosphomolybdate ion, a Keggin-type polyoxometalate.

chapter |2 pages

Figure 8 Cyclic voltammogram of 5 mM PMo O in a mixed organic-aqueous solvent (1:1 acetonitrile-water by volume) containing 0.5M HSO.

chapter |1 pages

Figure 9 Cyclic voltammetry at a glassy carbon electrode in (a) 1.8 mM Rh(OAc) and (b) 1.5 mM RhPOM

chapter |1 pages

absence (---) of 3.7 mM methionine. Supporting electrolyte, 0.5M phosphate buffer at pH 7.0; scan rate, 0.25 V s .

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Table 2 Separation of Selected Peptides Followed by Amperometric Detection at a Composite Electrode Doped with RhPOM

chapter |3 pages

Figure 10 Structure of a pentaerythritol-based metallodendrimer with Ruterpyridine

chapter 14|3 pages

Electrodes Based on the Electrical “Wiring” of Enzymes

chapter |1 pages

concentrations measured with an Accu-Chek glucose meter.

chapter |2 pages

sensors, one implanted intravascularly in the jugular vein and one implanted subcutaneously in the rat’s back, with the measured glucose concentration in withdrawn blood samples, following an intravenous insulin injection. The prediction of a model translating the subcutaneously measured glucose concentration to the blood glucose concentration is also shown.

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Figure 4 Composition of the film on the immunosensing cathode.

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Figure 5 The reaction cycles leading to the observed current.

chapter |1 pages

established between the SBP-heme centers and the electrode via the redox polymer. This contact enables the electrocatalytic reduction of H O to

chapter |2 pages

amounts of the probe oligonucleotide. The probe was deposited for (a) 1 minute, (b) 2.5 minutes, (c) 5 minutes, and (d) 10 minutes.

chapter |1 pages

Figure 10 Increase in the catalytic current of microelectrodes at 25°C,

chapter 45|4 pages

°C, and 57°C after adding the SBP-labeled fully complementary or partially mismatched targets. (A) perfectly matched target; (B) target with a single mismatched base; (C) target with four mismatched bases. The dashed lines represent the best fit of the data to Equation 8. Figure 11 Current-time plot of the catalytic current of a microelectrode coated with the probe-bearing redox polymer. An aliquot of the SBP-labeled target with four mismatched bases was introduced at A, then an aliquot of the SBP-labeled perfectly matching target at B.

chapter 15|2 pages

Capillary Electrophoresis/ Electrochemistry

Instrument Design and Bioanalytical Applications

chapter |2 pages

Figure 1 CEEC systems with (A) off-column detection and (B) end-column detection.

chapter |2 pages

Figure 2 Electropherogram of 10 nM each (1) dopamine, (2) norepinephrine, and (3) isoproterenol. (Reprinted with permission from Ref. 15.)

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Figure 3 HDVs recorded for dopamine and catechol using end-column detection at capillary-to-electrode distances of 20 and 60 µm without (A)

chapter |4 pages

and with (B) a fracture decoupler. Symbols: dopamine () and catechol (■) at 20 µm; dopamine (●) and catechol (▲) at 60 µm. Open and closed symbols represent different trials. (Reprinted with permission from Ref. 26.)

chapter |1 pages

Figure 6 Dual-electrode configurations for CEEC: (A) ring-disk, (B) tubular-wire, and (C) wire-wire.

chapter |1 pages

Figure 7 Simultaneous detection of thiols and disulfides with Hg/Au amalgamated tubular-wire dual electrode. Detection of (1) 50 µM

chapter |1 pages

cysteine, (2) 100 µM glutathione, and (3) 500 µM glutathione disulfide. (Reprinted with permission from Ref. 47.)

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Figure 8 Schematic of a microchip CEEC device with dual working electrodes.

chapter |1 pages

Figure 9 Electropherogram showing dual-electrode detection in microchip CEEC using gold microelectrodes. Detection of 100 µM tyrosine, HIAA, and catechol. (Reprinted with permission from Ref. 58.)

chapter |1 pages

Figure 10 Metabolism of leu-enkephalin in plasma monitored by copper complexation and CEEC: (A) plasma blank, (B) t=30 min, (C) t=60 min, (D) t=90 min. Peak identification: (1) tyrosine, (2) leu-enkephalin, (3) des-tyr leu-enkephalin. (Modified with permission from Ref. 60.)

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Figure 11 Diagram of a typical off-line microdialysis sampling system.

chapter |2 pages

Figure 12 Typical microdialysis probes for various tissues of rats or similar-size animals. (A) intracerebral guide cannula and rigid concentric cannula probe for brain tissue, (B) linear probe used for sampling peripheral tissues such as liver, muscle, dermis, tumor, kidney, (C) vascular probe for sampling from the jugular vein, and (D) shunt probe for sampling from the bile duct. Unlabeled arrows indicate direction of dialysate flow into and out of probes. (Courtesy of Bioanalytical Systems Inc.)

chapter |2 pages

Figure 13 Electropherogram of dialysate sample taken after intraperitoneal tryptophan administration. Peak identification: kynurenine (KN) and tryptophan (Trp). (Reprinted with permission from Ref. 67.)

chapter |1 pages

isoproterenol. (Reprinted with permission from Ref. 69.) Figure 15 Schematic of an on-line microdialysis-CEEC system. Figure 16 Electropherograms of transdermally delivered nicotine in the microdialysate at various time points. Arrows indicate peak corresponding to nicotine. (Reprinted with permission from Ref. 72.)

chapter |5 pages

Figure 17 Time course of nicotine in the microdialysate following patch administration. (Reprinted with permission from Ref. 72.)

chapter 16|3 pages

Ultrahigh Sensitivity Analysis of Amino Acids and Peptides by Capillary Liquid Chromatography with Electrochemical Detection

chapter |1 pages

configurations coupled to capillary liquid chromatography columns. (A) End-column or “wall jet” electrode configuration with the working electrode positioned proximal to the fused silica capillary outlet. (B) On-column electrode showing a cylindrical carbon fiber positioned in the fused silica capillary outlet forming a thin annular layer between the fiber surface and the inner capillary wall.

chapter |1 pages

Figure 2 Chromatographic efficiency (A) and a chromatogram (B) obtained using an end-column electrochemical detector coupled to an OTLC column. (A) Experimentally measured data of

chapter |2 pages

the OTLC system (plotted points) is compared to the theoretical efficiency as predicted by the Golay equation (solid curve) for a column diameter of 14 µm and diffusion coefficient of 10

chapter |1 pages

Figure 3 Flow injection analysis of 1 nM of a9.5 kilobase oligonucleotide at a Cu electrode with sinusoidal voltammetry. Upper graph shows the signal at different frequencies after Fourier transform of the voltammograms for single-stranded (ss) and double-stranded (ds) DNA. Lower trace shows a flow injection trace for the ds DNA at the sixth

chapter |3 pages

harmonic. The excitation sine was from 0.05 to 0.55 V at 2 Hz. The FIA flow rate was 0.5 mL/min and the electrolyte was 0.1M NaOH. (Adapted from Ref. 23.) Figure 4 Derivatization and scavenging reactions. Top: Derivatization of amino acid (GABA) by OPA with t-buSH as a nucleophile. Middle: Elimination of excess OPA by addition of glycine (GLY). Bottom: Elimination of excess t-buSH by addition of iodoacetamide (IAA).

chapter |1 pages

Figure 5 Comparison of chromatograms for different volume injections but constant mass (800 amol) of met-enkephalin (peak indicated by arrow). Injections were 10 nL of 80 nM (A), 100 nL of 8.0 nM (B), and 1000 nL of 0.80 nM (C). Capillary column was 8 cm long, 25 µm i.d. fused silica capillary packed with 5 µm Alltima C-18 reversed-phase particles. After loading sample, the column was rinsed with 5% solvent B-95% solvent A for 5 min before gradient elution started. Gradient elution started at 5% solvent B, linearly changed to 50% solvent B in 5 min, kept at this composition for 2 min, and then stepped back to 5% solvent B. Solvent A was 1 mM phosphate buffer in 10 mM sodium sulfate adjusted to pH 7.0. Solvent B was 40% (v/v) of solvent A with 60% acetonitrile. Detection utilized a 9 µm diameter by 2 mm long C-fiber electrode inserted into the outlet of the column. Detector electrode potential

chapter |3 pages

was set at +1.0 V versus Ag/AgCl reference. Table 1 Effect of Large Injection Volume on Characteristics of Met-Enkephalin Peak

chapter |1 pages

Figure 7 High sensitivity detection of biuret peptide complexes by CLC-EC. Chromatograms are from injection of 1 µL of 100 pM bradykinin (B), 100 pM vasopressin (V), 400 pM oxytocin (O), and 1 nM neurotensin (N). (A) Raw chromatogram. (B) Chromatogram after median filtering. (C) Blank after median filtering. Chromatographic conditions same as Figure 5. Capillary column was 15 cm long by 25 µm i.d. fused silica capillary packed with 5 µm Astec C-18. The aqueous portion of

chapter |4 pages

the mobile phase (solvent A) was 40 mM sodium carbonate buffer in 0.75 mM sodium potassium tartrate and 0.25 mM cupric sulfate adjusted to pH 10.5 with sodium hydroxide. The organic phase (solvent B) consisted of 40% (v/v) of solvent A with 60% acetonitrile. Gradient elution started at 5% solvent B, linearly changed to 35% solvent B in 5 min, kept at this composition for 2 min, and then stepped back to 100% solvent A. Other conditions similar to Figure 5.

chapter |1 pages

Figure 8 Demonstration of chemical noise produced by amine derivatization coupled with gradient elution. (A) Blank of deionized, HPLC-grade water without intentionally added amines after OPA-buSH derivatization. (B) Blank after derivatization and scavenging using IAA followed by cysteic acid/sulfite. (C) 25 nM amino acid standard derivatized as in (B). All sample injection volumes were 100 nL from a 500 µL sample. Chromatography was performed using a 48 µm×40 cm packed capillary column (5 µM Alltima C8) and a linear, binary gradient of mobile phase A (50 mM phosphate pH 6.8w/1 mM

chapter |1 pages

EDTA) and B (35% mobile phase A and 65% acetonitrile (v/v) with 1 mM EDTA) from 35% to 70% B using a slope of 2% B/min using a flow rate of 0.3 µL/min. Detection performed using a 9 µm×1.5 mm cylindrical carbon, fiber microelectrode held at+0.75 V (vs. Ag/AgCl reference). Amino acids: L-aspartate (ASP), L-glutamate (GLU), L-asparagine (ASN), L-histidine (HIS), L-glutamine (GLN), L-citrulline (CIT), L-arginine (ARG), L-serine (SER), L-threonine (THR), glycine (GLY), L-tyrosine (TYR), L-alanine (L-ALA or ALA), taurine (TAU), and GABA. Figure 9 Microscale analysis of amino acid standards and 10 s microdialysate fractions (120 nL sample collected from live rat brain). Blank of saline (bottom trace) and an amino acid standard (top trace) were diluted to 2 µL and derivatized in a polypropylene microvial. Concentrations of the amino

chapter |1 pages

acids (nM): ASP (10), GLU (20), ASN (20), HIS (40), GLN (500), CIT (20), ARG (50), SER (100), O-phosphoelthanolamine (PEA, 20), THR (50), GLY (100), TYR (20), L-ALA (100), β-alanine (β-ALA, 25), TAU (50), and GABA (10). Inset is expanded portion of the chromatogram where GABA elutes. For all experiments, an injection volume of 250 nL was used. Separation and detection conditions are as described in Figure 8 except the mobile phase pH was 6.51.

chapter |2 pages

Figure 10 System configuration for the automated, capillary-scale amino acid analyzer. Liquid chromatography is performed using a high pressure syringe pump (Isco 100-DM, Lincoln NE). The mobile phase exits the syringe pump through a set of check valves (C) and 0.2 µm stainless steel filters (F) prior to mixing. The volumetric flow from the pump is reduced from 6 to 0.3 µL/min using a fused silica capillary splitter and carried from the splitter to the autosampler using a short length of 50 µm i.d. capillary. The 50 µm i.d. packed capillary column is mounted directly to the low-dead volume injection valve. The electrochemical detector is similar to that described in previous figures.

chapter |1 pages

detection parameters are as described in Figure 9.

chapter |1 pages

Figure 12 Chromatographic resolution and analysis time for mobile phase gradients designed to produce different average capacity factor. Amino acid separations using an average capacity factor (k″ of 23 (A), 11.6 (B), and 5.8 (C). The gradient amplitude (35– 63%B), flow rate (0.3 µL/min), and injection volume (250 nL) for all the separations were held constant while

chapter |2 pages

the gradient slope was varied. Analyte and retention order the same as in Figure 9.

chapter |6 pages

Figure 13 Design of recycling electrochemical detector. (A) Top view of finished electrodes on Pyrex and SiO . (B) Cross-section of assembled