Label free biosensors techniques and applications 1st Edition Matthew A. Cooper
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- 5. Label free biosensors techniques and applications 1st Edition Matthew A. Cooper Digital Instant Download Author(s): MatthewA. Cooper ISBN(s): 9780521884532, 0521884535 Edition: 1 File Details: PDF, 12.93 MB Year: 2009 Language: english
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- 8. LABEL-FREE BIOSENSORS Label-free biosensors use biological or chemical receptors to detect ana- lytes (molecules) in a sample. They give detailed information on the binding selectivity, affinity, and, in many cases, the stoichiometry, kinetics, and thermodynamics of an interaction. Although they can be powerful tools in the hands of a skilled user, there is often a lack of knowledge regarding the best way to utilize label-free assays to screen for biologically active molecules and to accurately and precisely char- acterize molecular recognition events. This book reviews both established and newer label-free techniques. It is intended to give both the expert user and the general reader insight into the field from expert opinion leaders and practitioners of these techniques. Chapters also contain worked examples that are written to guide the reader through the basics of experimental design, setup, assay development, and data analysis. Matthew A. Cooper is Founder and Managing Director of Cambridge Medical Innovations and Distinguished Australia Fellow at the Uni- versity of Queensland. Dr. Cooper has consulted widely for biosensor, biotechnology, and pharmaceutical companies in the United Kingdom, Europe, and the United States. He is a review panel member for Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, National Institutes of Health, National Institute of Allergy and Infectious Diseases, and Science Foun- dation Ireland and a Fellow of the Royal Society of Medicine.
- 10. Label-Free Biosensors TECHNIQUES AND APPLICATIONS Edited by Matthew A. Cooper The University of Queensland
- 11. CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 8RU, UK First published in print format ISBN-13 978-0-521-88453-2 ISBN-13 978-0-521-71151-7 ISBN-13 978-0-511-50688-8 © Cambridge University Press 2009 2009 Information on this title: www.cambridge.org/9780521884532 This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Published in the United States of America by Cambridge University Press, New York www.cambridge.org paperback eBook (EBL) hardback
- 12. Contents Contributors page vii Preface ix 1 Label-free optical biosensors: An introduction 1 Brian T. Cunningham 2 Experimental design 29 Robert Karlsson 3 Extracting affinity constants from biosensor binding responses 48 Rebecca L. Rich and David G. Myszka 4 Extracting kinetic rate constants from binding responses 85 Rebecca L. Rich and David G. Myszka 5 Sensor surfaces and receptor deposition 110 Matthew A. Cooper 6 Macromolecular interactions 143 Francis Markey 7 Interactions with membranes and membrane receptors 159 Matthew A. Cooper 8 Application of SPR technology to pharmaceutical relevant drug-receptor interactions 179 Walter Huber 9 High-throughput analysis of biomolecular interactions and cellular responses with resonant waveguide grating biosensors 206 Ye Fang, Jack Fang, Elizabeth Tran, Xinying Xie, Michael Hallstrom, and Anthony G. Frutos 10 ITC-derived binding constants: Using microgram quantities of protein 223 Richard K. Brown, J. Michael Brandts, Ronan O’Brien, and William B. Peters 11 Electrical impedance technology applied to cell-based assays 251 Ryan P. McGuinness and Edward Verdonk Index 279 Color plates follow page 36 v
- 14. Contributors J. Michael Brandts Vice President MicroCal Northampton, MA Richard K. Brown, PhD President and CEO MicroCal Northampton, MA Matthew A. Cooper, PhD Managing Director Cambridge Medical Innovations Cambridge, UK Distinguished Australia Fellow Institute for Molecular Bioscience University of Queensland St. Lucia, Australia Brian T. Cunningham Associate Professor University of Illinois Department of Electrical and Computer Engineering Urbana, IL Jack Fang, PhD Strategic Analysis Manager Corning® Epic® System Corning, Inc. Corning, NY Ye Fang, PhD Research Manager Cellular Biophysics Corning, Inc. Corning, NY Anthony G. Frutos, PhD BioAssay Development Manager Science & Technology Corning, Inc. Corning, NY Michael Hallstrom Assistant Product Line Manager Corning® Epic® System Corning, Inc. Corning, NY Walter Huber, PhD Pharmaceutical Research Discovery Technology F. Hoffmann-La Roche Basel, Switzerland Robert Karlsson Research and Development Director, System and Applications Department GE Healthcare Uppsala, Sweden Francis Markey GE Healthcare Uppsala, Sweden vii
- 15. viii Contributors Ryan P. McGuinness Senior Scientist Drug Discovery MDS Analytical Technologies Sunnyvale, CA David G. Myszka, PhD Director Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, UT Ronan O’Brien, PhD Head of Applications Research MicroCal Northampton, MA William B. Peters, PhD Applications Scientist and Head of Training MicroCal Northampton, MA Rebecca L. Rich, PhD Senior Research Scientist Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, UT Elizabeth Tran, PhD Senior Research Scientist Science & Technology Corning, Inc. Corning, NY Edward Verdonk, PhD Senior Scientist Drug Discovery MDS Analytical Technologies Sunnyvale, CA Xinying Xie Field Application Scientist Corning® Epic® System Corning, Inc. Houston, TX
- 16. Preface Over the past two decades the benefits of biosensor analysis have begun to be recognized in many areas of analytical science, research, and development, with analytical systems now used routinely as mainstream research tools in many laboratories in many fields. Simplistically, biosensors can be defined as devices that use biological or chemical receptors to detect analytes (molecules) in a sample. They give detailed information on the binding affinity and in many cases also the binding stoichiometry, thermodynamics, and kinetics of an interaction. Label-free biosensors, by definition, do not require the use of reporter elements (fluorescent, luminescent, radiometric, or colorimetric) to facilitate measure- ments. Instead, a receptor molecule is normally connected in some way to a transducer that produces an electrical signal in real time. Other techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), and mass spectrometry require neither reporter labels nor surface-bound receptors. In all cases detailed information on an interaction can be obtained during analysis while minimizing sample processing requirements. Unlike label- and reporter- based technologies that simply confirm the presence or absence of a detector molecule, label-free techniques can provide direct information on analyte bind- ing to target molecules typically in the form of mass addition or depletion from the surface of a sensor substrate or via changes in a physical bulk property (such as the heat capacity) of a sample. Until recently, label-free technologies have failed to gain widespread acceptance due to technical constraints, low throughput, high user expertise requirements, and cost. Whereas they have proved to be powerful tools in the hands of a skilled user, they have not always been readily adapted to everyday lab use in which simple-to-understand results are a prerequisite. Despite this limitation, the potential today for label-free approaches to complement or even displace other detection technologies has never been higher. This book covers established label-free technologies and emerging develop- ments in label-free detection systems, their underlying technology principles, and end-user case studies that reveal the power and limitations of such biosen- sors. The chapters are intended to give both the expert user and the general reader interested in the technologies and applications behind label-free biosensors an insight into the field from expert opinion leaders and practitioners. As such, most chapters contain one or more worked examples that guide the reader through ix
- 17. x Preface the basics of experimental design, setup, assay development, and data analysis. The book is heavily weighted toward applications using optical biosensors and surface plasmon resonance (SPR) instrumentation. This is primarily because of the overwhelming bias in the installed base of optical biosensors due to their early commercialization and uptake. Other label-free technologies conspicuous by their absence from this volume include analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and mass spectroscopy. These will be covered as part of a new cluster of titles on bioanalytical techniques and applications. Matt Cooper Cambridge, U.K. May 2008
- 20. 1 Label-free optical biosensors: An introduction Brian T. Cunningham INTRODUCTION 1 RATIONALE FOR LABEL-FREE DETECTION 3 OPTICAL BIOSENSORS 4 PERFORMANCE METRICS 7 Sensitivity 7 Assay sensitivity 8 Resolution (or limit of determination) 8 Ease of use 9 Sensor cost 10 Detection instrumentation 10 Throughput 11 REVIEW OF OPTICAL BIOSENSOR METHODS 12 Surface plasmon resonance 12 HOLOGRAM BIOSENSORS 14 Reflectometric interference spectroscopy 16 Dual polarization interferometry 18 PHOTONIC CRYSTAL BIOSENSOR 19 WHISPERING GALLERY MODE RESONATORS 22 ACTIVE RESONATORS: DFB LASER BIOSENSOR 25 CONCLUSIONS 25 REFERENCES 27 INTRODUCTION The ability to study interactions among biomolecules, to observe the activity of cells, and to detect analytes specifically from bodily fluids, manufacturing pro- cesses, or environmental samples are cornerstones of life science research, phar- maceutical discovery, medical diagnosis, and food/water safety assurance. These capabilities and many others are enabled by the ability to perform biochemical and cell-based assays that allow scientists to ask basic questions about whether 1
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- 22. Fleming knocked at the door, and in answer to a cold "come in," entered. "Did you ring, my lord?" he said. "You know I didn't," said Yorke. "What is it? You look upset, Fleming," and he smiled the smile which is not good to see on the lips of any man, young or old, simple or gentle. "Beg pardon, my lord," said Fleming, who was genuinely attached to his master, and who had watched the change in him with sincere grief and regret, "but I thought you would want to send me somewhere, perhaps." Yorke smiled. "The best thing I could do for you would be to send you about your business!" he said. "Oh, don't say that, my lord," remonstrated Fleming. "I'm—I'm afraid something is wrong, my lord—" "Yes," said Yorke, grimly. "Something is very much wrong, Fleming. The fact is I am up a tree; cleaned out and ruined." "Ruined?" "That's it," assented Yorke, coolly. "I've been hard up, once or twice before—you know that, Fleming?" "Oh, yes, my lord." "But this is the finale, the climax, the wind up. But don't let me stand in your light. Look here, you have been a deuced good servant —yes, and a friend to me, and as it won't do you any good to be mixed up in this beastly mess you had better go at once. Lord Vinson has often told me that if I wanted to get rid of you he'd be glad to take you on. So you go to him—I'll give you a letter and—" For the first time in his exemplary life Fleming was guilty of vulgar language.
- 23. "I'm damned if I do!" he said. "I beg your pardon, my lord, I humbly beg your lordship's pardon, but I'm not that kind of a man—I'm not, indeed;" and there was something very much like water in the honest fellow's eyes. "I shouldn't think of leaving your lordship while you were up a tree, as your lordship puts it. I should never look myself in the face again. I'm much obliged to Lord Vinson; but no, my lord. I'm not the man to desert a good and kind master in misfortune. I beg your lordship's pardon, but I thought—" He hesitated respectfully. "Think away," said Yorke, lighting another cigar and tilting his hat back. "Perhaps your thinking will be more valuable than mine. I've been thinking, and can see no way out of the mess." "The—the duke, my lord," suggested Fleming. "I'm sure he—" "So am I, Fleming; but the duke has left for the Continent, and I don't know where he has gone, and this paper says that I've got to show up at the court in the city at once." "And it will all be in the newspapers!" said Fleming aghast. To be 'in the newspapers' was the direct disgrace and calamity in the eyes of that worthy man. "Just so," said Yorke, knocking the ashes off his cigar. "You see, Fleming, I am in a hole out of which it is impossible to pull me. Never you mind; after all, it doesn't matter." "Doesn't it matter, my lord?" echoed Fleming, startled. "You—you who are so well known to—to appear in court!" "And get six months—is it six months or six weeks? I don't know—I don't know anything; but I suppose I shall, and pretty quickly. Never mind. Look here; see that man in the next room has all he wants." "Oh, yes; all right, my lord," said Fleming, with a touch of impatience, "All he wants is beer, and I've given him half a dozen bottles." Yorke laughed and leaned back in the chair.
- 24. "All right. Bring any letters that may come; I should like to know the worst." Fleming went out, but appeared again in a few minutes. "Will you want me for half an hour or three-quarters, my lord?" he said, in a thoughtful, troubled kind of way. "No. Going after that place, Fleming? Better." Fleming colored and opened his lips; but he did not say anything; and Yorke, left alone again, leaned his head on his hand and gave himself up to gloomy reverie. A man in possession in the next room, a summons to appear in a debtors' court, his name in the newspapers as a ruined man! It was all bad enough, but he scarcely felt it. He had endured the maximum of suffering when he had become convinced that Leslie had jilted him, and this—well, this was, so to speak, almost a relief and a diversion. And yet the disgrace! He passed a very bad half hour in that dressing-room—a half hour in which there rose the specter of an ill-spent past in which follies marched in ghostly procession before him, and all, as they promenaded by, whispered hoarsely, "Ruin!" And yet, through it all he saw more plainly than anything else the sweet face of Leslie, the only woman he had ever loved— the woman who had seemed to him an angel of truth and constancy, but who had deserted him the moment she had heard that he was not a duke. Fleming, meanwhile, had put on his hat and sallied into the street. He had left his beloved master utterly reckless and indifferent, and therefore it rested with him, the devoted servant, to display all the more energy. That he should sit still and see Lord Yorke drift into utter ruin and destruction was simply impossible. "Something's got to be done," he said to himself, "and I've got to do it. He isn't going to appear at any court; not if I know it! What! my guv'nor, the cousin of a duke, to come up before a beak—some miserable city alderman?" Fleming's ideas of the city law courts were, like his master's, hazy. "Certainly not—not if I have to move
- 25. heaven and earth! Now, if the duke was at home I could see Mr. Grey, and we could arrange this little matter between us; but as he isn't, why, the thing to do is to go to the next person, and that is, naturally, Lady Eleanor Dallas. It isn't likely that she'd see Lord Yorke in such a hole as this without helping him out; and she's rich, and richer than ever lately. I'll try her!" He called a hansom and had himself driven to Kensington Palace Gardens. "Anyhow, her ladyship can only refuse to see me," he said to himself. "But I don't think she will;" and "he winked the other eye." Oh! my friends, do you think our servants are deaf, and dumb, and blind? They know all our little secrets and our little difficulties; all our little entanglements. There is scarcely a letter we receive that, unless we lock it up securely, they do not read. No friend ever visits us but they know all about him and his, and whom his daughter is engaged to, or why the engagement is broken off. Therefore let us be grateful to a kind Providence for the servants who are also devoted and trusty friends, such as was Fleming. When Fleming reached Kensington Palace Gardens he was told by one of the footmen that Lady Eleanor was engaged. "You've come with a message from Lord Auchester, Mr. Fleming, I suppose?" said the footman. Fleming was an 'upper servant' and was always addressed by those beneath him as 'Mr.,' and he was very much respected on his own account as one who had saved money and was in 'good society.' "Well, no, I haven't," said Fleming, gravely, and a little pompously. "I've come on business of my own." The footman took his name into the boudoir where Lady Eleanor was sitting with no other than Mr. Ralph Duncombe. She flushed slightly. "It is Lord Auchester's valet," she said.
- 26. Ralph Duncombe looked up with a slight start. "I do not wish him to see me, Lady Eleanor," he said. "No, no; oh, no! I understand," she said nervously. "And yet I should like to know what he has to say." Lady Eleanor pointed to a large four-fold Japanese screen which cut off one of the corners of the room. "He will not be here many minutes," she said. Ralph Duncombe went behind the screen, and Lady Eleanor rang the bell and told the footman she would see Fleming. He came in, looking rather nervous and embarrassed, for it was a bold thing he was going to do, and he knew that Lady Eleanor could look and speak haughtily and sternly when she was displeased. "You want to see me, Fleming?" she said, graciously enough. "Is it a message from Lord Auchester?" "No, my lady," he said, and like a man of the world he went straight to the point. "No, my lady, his lordship does not know that I have come, and if he had known I was coming I'm sure he would have forbidden me; but I ventured to intrude on your ladyship, knowing that you and my master were old friends, if I may say so." "Certainly you may say so, Fleming," said Lady Eleanor, pleasantly, and looking as if she were expecting anything but bad news. "Well, my lady, my master is in a terrible trouble," he said, plunging still further into the business. "In terrible trouble?" echoed Lady Eleanor; and her face flushed. "What do you mean, Fleming?" "It's money matters, my lady," said Fleming, gravely, and looking around as if he feared an eavesdropper. "His lordship—I'm obliged to speak freely, my lady, or else you won't understand; but it's out of no disrespect to his lordship, who has been the best of masters to me—"
- 27. "Say what you have to say quite without reserve," said Lady Eleanor, in a low voice. "Well, my lady, I was going to say that his lordship has always been hard up, as you may say. There's always been a difficulty with the money. It's usual with high-spirited gentlemen like Lord Yorke," he said, apologetically. "They don't know, and can't be expected to know, the value of money like common ordinary folk, and so they— well, they outrun the constable." "Lord Auchester is in debt?" said Lady Eleanor, guardedly. "It's worse than that, my lady," said Fleming. "That would be nothing, for ever since I've been in his service he has been in debt. But now the people he owes money to want him to pay them." He gave the information as though it were the most extraordinary and unnatural conduct on the part of any creditor of Lord Auchester that he should want payment. "People who owe money must pay it some time, Fleming," suggested Lady Eleanor. "Yes—ah, yes, my lady, some time," admitted Fleming, "but not all at once. It seems as if the people my lord owes money to had joined together and resolved to drop upon him in a heap. There's a man in possession in Bury Street, my lady." "A man in possession!" repeated Lady Eleanor, as if she scarcely understood. "Yes, a bailiff, my lady, sitting there in his lordship's sitting-room; and I daresn't throw him out of the window." Lady Eleanor looked down. "And—and Lord Yorke, Fleming—I suppose he is in great trouble about this?" Fleming hesitated.
- 28. "Well, my lady, he is in great trouble; but if you mean is he cut up about this money matter, I can't say that he is. He don't seem to care one bit about it, and takes it as cool and indifferent as if—well, as if nothing mattered. But he is in great trouble for all that, and he has been for weeks past—" He hesitated. Lady Eleanor looked up. "You had better tell me everything, I think, Fleming," she said, in a low voice. "Well, my lady, it's just thus: His lordship had a blow—a disappointment of some kind. It isn't money, it isn't betting, or card- playing, or I should have heard of it, for his lordship generally makes some remarks, such as 'I've had a good day, Fleming,' or, 'I'm stone broke, Fleming,' so that I know what kind of luck he's had; it isn't that. It's something worse—if there is anything worse," he put in philosophically. "A little while ago his lordship was in the very best of spirits; I never saw him in better, and he's a bright-hearted gentleman, as you know, my lady. I'm speaking of the time when he came back from that place in the country where he and his grace the duke were—Portmaris." Lady Eleanor leaned her head on her hand so that her face was hidden from him. "Then all of a sudden a change came, and his lordship got bad, very bad. It was dreadful to see him, my lady. Eat nothing, cared for nothing; scarcely even spoke. Nothing but smoke, smoke, all day, and wander in and out looking like the ghost of himself. And he, who used to be so bright and cheerful, with the laugh always ready! I'd have given something to have spoken a word, and asked him what was the matter; but—well, my lady, with all his pleasantness, my master's the last gentleman to take a liberty with." "You don't know what it was, this terrible disappointment?" said Lady Eleanor, almost inaudibly.
- 29. Fleming hesitated and glanced at her; then he coughed discreetly behind his hand. It was sufficient answer, and Lady Eleanor's face grew red. "Whatever it was that made him so happy and cheerful, it was knocked on the head and put an end to, my lady," he said. "And so it is that this regular smash-up of affairs—I mean these summonses and man in possession—don't seem to affect him. You see, my lady, he was as low down as he could be already. Sometimes—" He stopped, and looked down at the carpet very gravely and anxiously. "Well?" "Well, my lady, it isn't for me to say such a thing, but I've been almost afraid to let him out of my sight in the morning, and I've been truly thankful to see him come in at night." Lady Eleanor drew a long breath and shuddered. "You mean—" "Men, when they're down as low as my master, they do rash things sometimes, my lady," said Fleming, in a solemn whisper. Lady Eleanor's face went white, and she put her hand to her delicate throat as if she were suffocating. "You—you should not say—hint—at such terrible things, Fleming," she panted. "I—I beg your ladyship's pardon," he said, humbly, "but it's the truth and—and I thought I ought to tell you, being his lordship's friend." "Yes—yes, I am his friend," she said, as if she scarcely knew what she was saying. "And I will try to help him." Fleming's face brightened. "Oh, my lady!" he said, gratefully. "Stop!" she said. "Your master, Lord Yorke, must not know;" and her face grew crimson again.
- 30. "Oh, no, no, my lady! Certainly not! Why, if his lordship ever knew that I'd come to you—" He stopped and shook his head. "I understand," said Lady Eleanor. "No, Lord Yorke must never know —no one must know—" "I should have gone to the duke, my lady, but his grace is abroad, as no doubt your ladyship knows." Lady Eleanor turned her head aside. She and Ralph Duncombe had timed the attack on Yorke for the moment when the duke should be beyond reach. "His grace would have helped my master, I know; and I'd have made bold to write to him, but there isn't time." Lady Eleanor shook her head. "No, no," she said. "He must not know—no one must know. You need not be anxious any longer, Fleming. You were right in coming to me and—and—" She sunk into the chair. Fleming heaved a sigh of relief. "Very well, my lady. I don't know much about it, but the person who seems the principal in this set upon his lordship is a man named Duncombe—a money-lender, I expect. They take all sorts of names. I wish I had him to myself for a quarter of an hour. I'd teach him to put a man in possession—begging your ladyship's pardon," he broke off. Lady Eleanor's face reddened, and she glanced toward the screen. "You had better go back now, Fleming," she said, "and—and don't leave Lord Auchester more than you can help. And, remember, not one word that might lead him to guess that you have been to me." "You may be sure I shall be careful for my own sake, my lady," said Fleming, with quiet emphasis; and, with a bow in which gratitude and respect were fairly divided, he left the room.
- 31. Ralph Duncombe came from behind the screen and stood looking down at Lady Eleanor, whose proud head was bowed upon her hands. "What are you going to do?" he asked. She looked up. "Set him free—at once—at once!" she responded with feverish impetuosity. "Did you not hear the man? That he actually feared his master would—" She shuddered. "This must come to an end at once. It will drive him mad!" Ralph Duncombe smiled grimly. "I heard the man say that it was not the money trouble that was affecting Lord Auchester," he said. "It seems to me, Lady Eleanor, that we have taken a great deal of trouble for nothing. This marriage which you so much dreaded was broken off before any plans to prevent it were put in operation. The—the young lady had disappeared—" She looked up suddenly as he stopped and bit his lip. "Disappeared? How do you know?" she exclaimed breathlessly. His face was as pale as hers, but was set and stern. "Well, I thought I had better run down to this place, Portmaris, and see for myself how matters were going," he said, in a kind of business-like coolness and indifference, "and—and I found that Miss —what is her name?" he asked, as if he had forgotten. "Lisle—Leslie Lisle," said Lady Eleanor. "Ah, yes! Miss Lisle had flown." "Flown?" "Yes, flown and disappeared. Disappeared so completely that all my efforts to discover her track failed." He still spoke calmly and with affected indifference, but if she herself had not been so agitated she would have noticed the pallor of his face and the restless movement of his hands.
- 32. "What—what do you think it means?" she asked, in a whisper. He shrugged his shoulders. "A lovers' quarrel—but no; it is more shame than that. Yes; I should say that the engagement was broken off for some reason or other, so that you have had all this trouble and expense for nothing, Lady Eleanor." "And you can not find her? Disappeared?" He took up his hat. "Disappeared," he repeated, grimly. "And that is why he is wretched and unhappy," she said, with a sigh. "How—how he must love her after all!" and her head drooped. Ralph Duncombe moistened his lips. "Yes," he said. "But perhaps she did not care for him. Any way, you see it is she who has left him, not he who has left her." "Yes," she said, and she pushed the hair from her fair forehead with an impatient gesture. "Oh, I cannot understand it! The engagement broken off! Disappeared! But there must be an end to these law proceedings now, Mr. Duncombe." "There can be only one way of terminating them," he said. "And that?" "Is by paying the money into court," he said. "The thing has gone too far." "I see," she said. Then she held out her hand. "I will send or come to you in the morning. I am too confused and—and upset even to think at this moment." Fleming hastened back to Bury Street and found Yorke sitting as he had left him, with the formidable-looking letters and papers littered around him.
- 33. Fleming picked them up and put them away, and got out Yorke's dress clothes. "Don't trouble, Fleming, I shall dine at home," said Yorke; but Fleming went on with his preparations. "Very sorry, my lord, but the kitchen grate is not in order." He didn't intend that his master should eat his dinner in company with a man in possession. "Better go and dine at the club, my lord, if I may make so bold." Yorke got up with a grim smile. "Perhaps you're right, Fleming," he said, listlessly. "I suppose they never have anything the matter with the kitchen grate at Holloway, or whatever other quod it is they send people who can't pay their debts. And what about these clothes, Fleming? Perhaps our friend in the next room will object to my walking out in them." "I'd punch his head if he was to offer a remark on the subject," said Fleming, fiercely. "I beg your lordship's pardon—if I might say a word, my lord, I'd implore your lordship not to take this business too much to heart; I mean not to worry too much over it. You never can tell what may turn up." Yorke laughed drearily as he allowed Fleming to dress him. "I won't," he said. "To tell you the truth, I don't feel so cut up as you'd imagine, or as I ought, Fleming. I feel"—he stopped and looked round absently—"well, as if I were another fellow altogether, and I was just looking on, half sorry and half amused." "Yes, that's right. Keep feeling like that, my lord," said Fleming, cheeringly. "Depend upon it, it will come out right." Yorke shrugged his shoulders. "I dare say," he said, indifferently. "Don't sit up for me. I may be late." He came in a little after two in the morning, and Fleming could have been almost glad if his beloved master had showed signs of having
- 34. spent a 'warm' night; but Yorke was 'more than sober,' and looked only weary and sick at heart, as he had done for weeks past. "Oh, by the way, Fleming," he said, as he took off his coat, and as if he had suddenly remembered it, "you must call me pretty early to- morrow. I have to be down in the city, you know." That was all.
- 35. CHAPTER XXXII. BOUGHT AND PAID FOR. A city law court is not exactly the place in which to spend a happy day—unless you happen to be a lawyer engaged in a profitable case there—and Yorke, as he entered the stuffy, grimy, murky chamber, looked round with a feeling of surprise and grim interest. Upon the bench sat the judge in a much-worn gown and a grubby wig. A barrister was drowsing away in the 'well' of the court, and his fellows were sleeping or stretching and yawning round him. The public was represented by half a dozen seedy-looking individuals who all looked as if they had not been to bed for a month and had forgotten to wash themselves for a like period. There was an usher, who yawned behind his wand, one or two policemen with wooden countenances, and two or three wretched-looking individuals, who were, like Yorke, defendants in various suits. The entrance of this stalwart, well-dressed and decidedly distinguished and aristocratic personage created a slight sensation for a moment or two; then he seemed to be forgotten, and he stood and looked on, and wondered how soon his case would be heard, and whether he would be carried away to jail forthwith. He waited for a half hour or so, feeling that he was growing dirty and grimy like the rest of the people round him, and gradually the sense of the disgrace and humiliation of his position stole over him. Great heavens, to what a pass he had come! He had lost Leslie. He was now to lose good name and honor—everything! Would it not be better for himself and everybody connected with him if he went outside and purchased a dose of prussic acid?
- 36. The suspense, the stuffy court, the droning voice of the counsel began to drive him mad. He went up to the usher. "Can you tell me when my case comes on?" he said. The man looked at him sleepily. "Your case—what name?" he asked, without any 'sir,' and with a kind of drowsy impertinence, which seemed to be in strict harmony with the air of the place. "Auchester!" said Yorke. "I am the—the defendant." "Horchester? Don't know. Ask the clerk," said the man. With a sick feeling of shame Yorke went up to the man pointed out by the usher and put the same question to him. "Auchester? Duncombe versus Auchester; Levison versus Auchester; Arack versus Auchester?" said the clerk, in a dry, business-like way. "Yes, I dare say that's it," said Yorke, hating the sound of his own name. The clerk looked down a list, then raised his eyes with the faintest of smiles. "Scratched out," he said, curtly. "Scratched out?" echoed Yorke, blankly. "Yes, sir—my lord," said the clerk, who, while looking at the list, had come upon Yorke's title. "The cases have been removed from the list. Settled." "Settled? I don't understand," said Yorke, staring at him. "I've only just come down—I've paid nothing." "Some one else has, then, my lord," said the clerk. "Wait a moment till this case is heard; it will be over directly, and I'll explain." Yorke, feeling like a man in a dream, stepped into a corner and waited. Presently the court adjourned for luncheon, and the clerk
- 37. came toward him. "This way, my lord." He led Yorke into an office. "Now, my lord. Yes, all the cases have been discharged from the list—been settled this morning." "This morning?" echoed Yorke, mechanically, still with a vast amazement. "But—but who—I don't know who could have done this. I have not, for the best of all reasons. I came down here prepared to go to prison, or wherever else you sent me." The clerk raised his brows and shook his head gravely. "Yes, you would have been committed, my lord, for a certainty," he said. "You see, you let things slide too long. But there is no fear now. The money, all of it, has been paid. You are quite free, quite. I congratulate your lordship." "But—but"—stammered Yorke, and he put his hand to his brow —"who can have done it—paid it? Is it the Duke of Rothbury?" Could Dolph have heard of it in some extraordinary way and sent the money? The clerk went into the inner office for a few minutes, then he came back with a slip of paper in his hand. "I don't know whether I am doing right, my lord," he said, gravely, and even cautiously. "Perhaps I ought not to give you this information, but I trust to your lordship's discretion. You won't get me into a scrape, my lord?" "No, no!" said Yorke, "who is it?" The clerk handed him the slip of paper. It was a check on Coutts' for a large—a very large—sum, and it was signed "Eleanor Dallas." "Eleanor!" The name broke in a kind of sigh from Yorke's lips, and his face reddened. But it was pale again as he handed the check back to the
- 38. clerk. "Thank you," he said. He stood and looked vacantly before him as if he had forgotten where he was; then he woke with a start. "Then I can go?" he said. "Certainly, my lord," said the clerk. "As I said, you are quite free. There are no actions against you now; everything is squared—paid." Yorke thanked him again, wished him good-day, and got outside. Everything paid—and by Eleanor! He repeated this as he walked from the city to the west; as he tramped slowly, with downcast head, across Hyde Park. He told himself that he ought to be grateful; that he could not feel too grateful to the woman who had come to his aid and saved him from ruin and disgrace. But he knew why she had done it, and he knew what he ought to do in return. The least he could do would be to go and kneel at her feet, and ask her to accept the life which she had snatched from disgrace. And why shouldn't he? The only woman he had ever loved had proved false, and mercenary, and base, and there was nothing now to prevent him asking Lady Eleanor to be his wife; and yet, alas! he could not get that other face out of his mind or heart. He thought of her—she haunted him as he walked along; the clear gray eyes, so tender one moment, so full of fire and humor the next; the dark hair, the graceful figure, the sweet voice. "Oh, Leslie, Leslie! if you had but been true!" was the burden of his heart's wail. He looked up and found himself close upon Palace Gardens; unconsciously his feet had moved in that direction. He rang the bell of Lady Eleanor's door. Yes, her ladyship was at home, the footman said, and said it in that serene, confident tone which a servant uses when he knows that his
- 39. mistress will be glad to see the visitor. Yorke followed the man to the small drawing-room. Lady Denby was there tying up some library books. She started slightly as she saw his altered appearance, but she was too completely a woman of the world to let him see the start. "Why, Yorke!" she said, "what a stranger you are! We were only speaking of you this morning at breakfast, and wondering where you were. Have you been away? Sit down—or tie up those tiresome books for me, will you? They slip and slide about in the most aggravating way. I'll go and tell Eleanor; I fancy she was going out." She met Lady Eleanor in the hall, and drew her aside. "Yorke is in there, Eleanor," she said. "Yorke!" Lady Eleanor repeated the name and started almost guiltily, almost fearfully. "Yes, I came to tell you, and—well, yes—prepare you. I don't want you to do as I did—jump as if I'd seen a bogey man. He has been ill, or up to some deviltry or other, and he looks—well, I can't tell you how he looks. It gave me a shock. I thought I'd prepare you." Lady Eleanor touched her hand. "Thank you, dear. No, I won't look shocked. He looks very ill?" "Very ill, oh! worse than ill. Like a man who has robbed a church and been found out, or lost everything he held dear." Lady Eleanor put her handkerchief to her lips. They were trembling. "I don't mind what he has been doing," she said. "Oh, my dear Eleanor!" "No, I don't. I'll go in now. Don't let any one disturb us. He—he may have come to see me to talk about something."
- 40. She went into the room, and Yorke turned to meet her. It was well that she had been forewarned of the change in his appearance. As it was, she could scarcely suppress the cry that rose to her lips. "Well, Yorke," she said, with affected lightness, "tying up aunt's books? That is so like her. No one can come near her without getting employed. What a shame to worry you!" "It doesn't worry me," he said. He leaned against the table and looked down at her. There is a picture of Millais's—it is called, I think, 'A Hot-house Flower'—which Lady Eleanor might have sat for that morning, so delicate, so graceful, so refined and blanche was her beauty. She wore a loose dress of soft cashmere, cream in color, almost Greek in fashion. Her hair was like gold, her eyes placid yet tender, with a touch of subdued sadness and anxiety in them. A charming, an irresistible picture, and one that appealed to this man with the storm-beaten heart aching in his bosom. She glanced up at him, saw the haggard face, the dark rings round the eyes, that indescribable look which pain and despair and utter abandonment produce as plainly as the die stamps the hall-mark on the piece of silver, and her heart yearned for him, for his love— yearned for the right to comfort and soothe him. Ah! if he would only have it so—if he would only let her, how happy she would make him! All this, and much more, she felt; but she looked quite placid and serene—like a dainty lily unstirred by the wind—and said in her soft voice: "We were thinking of advertising for you Yorke. Have you been away?" He might have answered: "Yes, I have been in the Valley of Sorrow and Tribulation, on the Desert of Dead Love and Vain Hope," but instead he replied: "No, just here in London; but I have been busy." She looked up and smiled.
- 41. "Busy! That sounds so strange, and so comic, coming from you!" "And yet it is true," he said. "I have been busy thinking." If there was a touch of bitterness in his voice she did not notice it. "And that's hard work for me—it's so new, you see." There was silence for a moment. He held the string with which he had been tying up the books in his hands, and fidgeted with it restlessly. Lady Eleanor dropped into small-talk. Had he been to the chrysanthemum show at the Temple? Had he noticed that the Duchess of Orloffe was not going to give her autumn ball? Did he— He broke in suddenly as if he had not been listening, his voice hoarse and thick: "Eleanor, why did you do it?" "Why did I—do what, Yorke?" she said. "Why did you fling so much money away upon a worthless scamp?" His face went white, then red. "Who told you?" she breathed. "They told me down at the court where I had gone to be disgraced," he said, "and you saved me! How can I thank you, Eleanor? How can I? And you would have done it in secret, would have kept it from me?" "Yes, oh, yes," she murmured, her head drooping. "Don't—don't say anything about it. It was nothing—nothing!" She looked up at him eagerly, pleadingly. "Yorke, you will not think badly of me because I did it? Why shouldn't I? I am rich—you don't know how rich—and what better could I do with the stupid money than give it to a—a friend who needed it more—ten thousand times more—than I do or ever shall! Don't be angry with me, Yorke." "Angry!" The blood flew to his face and his eyes flashed. He drew nearer to the chair in which she sat, he knelt on one knee beside her.
- 42. "Eleanor, I am utterly worthless—you know that quite well. I was not worth the saving, but as you have saved me, will you accept me? Eleanor, will you be my wife?" Her face went white with the ecstasy which shot through her heart. Ah, for how long had she thirsted, hungered for these words from his lips! And they had come at last! "Will you be my wife, Eleanor? I will try to make you happy. I will do my best, Heaven helping, to be a good husband to you! Stop, dear! If you act wisely you will send me about my business! There are fifty —a hundred better men who love you; you could scarcely have a worse than I, but if you will say 'yes,' I will try and be less unworthy of you. All my life I will never forget all that I owe you—never forget that you saved me from ruin and disgrace. Now, dear, I—" She put out her hand to him without a word; then as he took it her passion burst through the bonds in which she thought to bind it, and she swayed forward and dropped upon his breast. "Yorke, Yorke, you know"—came through her parted lips—"you know I love you—have always loved you!" "My poor Eleanor!" he said, almost indeed, quite pityingly. "Such a bad, worthless lot as I am!" "No, no!" she panted. "No, no; the best, the highest to me! And— and if you were not, it—it would be all the same. Oh, Yorke, be good, be kind to me, for you are all the world to me!" They sat and talked hand in hand for some time, and once during that talk he said: "By the way, Eleanor, how did you hear I was in such a mess—how did you come to know?" It was a very natural question under the circumstances; but Lady Eleanor started and turned white, absolutely white with fear. "No, no; not one word will I ever say or let you say about this stupid money business!" she exclaimed. Then she took his hand and
- 43. pressed it against her cheek. "Why, sir, what does it matter? It was only—only lending it to you for a little time, you see. It will all be yours soon." Lady Denby came in after a discreet cough outside; but Lady Eleanor did not move or take her hand from Yorke's. "Oh!" said Lady Denby. "Eleanor has made me very happy, Lady Denby," he said, rising, but still holding Lady Eleanor's hand. "Oh!" said Lady Denby again. "What do you want me to say? That you deserve her? No, thank you, I couldn't tell such an obvious fib. What I'm going to say in the shape of congratulation is that she is much too good for you." "That is so," he said with a grim smile. "You'll stay to dinner?" murmured Lady Eleanor. "You will stay, Yorke?" "Yes," he said, bending down and kissing her—"yes, thanks. But I must go and change my things. I'm awfully dirty and seedy." She went with him to the door, as if she begrudged every moment that he should be out of her sight, and still smiled after he had left her and had got half-way down the Gardens. Then suddenly he stopped and looked round him with a ghostly look. And yet it was only the face of Leslie that had flashed across his mental vision. Only the face of the girl who had jilted him! "My God! shall I never forget her?" he muttered, hoarsely. "Not even now!"
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