Contains reports on summary of research and one research project.Joint Services Electronics Program (Contract DAAB07-75-C-1346)National Institutes of Health (Grant 5 S05 RR07047-10)National Institutes of Health (Grant 5 PO1 HL14322-05)National Institutes of Health (Grant 1 ROI GM22633-01

Abrams, Jerome H.

Chong, Augustine J-Y.

King, John G.

Villars, Frederick

Weaver, James C.

DSpace@MIT

GENERAL PHYSICSI. -\IOI LCULE MICROSCOPYAcademic and Research StaffProf. John G. tKing Dr. James C. WeaverDr. John Nt. Peterson Jerome H. AbramsGraduate StudentsScott P. Fulton Dusan G. Lysy Bruce R. SilverJoseph A. Jarrell Allen Ai. Razdon Peter W\. StephensA. SUMIiIARY OF RESEARCH PROGRESS JSEPJoint Services Electronics Program (Contract DAAB07-75-C-1346)National Institutes of Health (Grants 5 SO5 RR07047-10, 5 PO1 HL14322-05,and 1 RO1 GAl22633-01)John G. King, James C. WeaverWe are continuing the development and application of molecule microscopy whichmakes possible the mapping of spatial variations of such sample properties as permea-bility, diffusion, binding of neutral molecules, and/or temporal distribution of enzymes.1. Scanning Pinhole Molecule IMicroscopy (SPMRI)During the past six months much time has been spent studying various aspects ofthe SPII system which are common to both the vascular smooth muscle experimentsand any form of in vitro molecule microscopy. Our objective is to measure small con-centrations of dissolved molecules such as oxygen or carbon dioxide. Even with semi-permeable membranes as a vacuum/sample interface, however, there is a large fluxof water into the vacuum system. These water molecules may undergo a variety of reac-tions in the electron bombardment ionizer, depending on the material and temperatureof the filaments, and the potentials of the ionizer electrodes. Thus, for example, whenusing thoria-coated iridium filaments, we observed a false oxygen signal, approximately60 of the water signal. The exact mechanism is not understood, but this effect does nottake place to any measurable degree with tungsten filaments, although the well-known"water cycle" does. Preliminary results also lead us to suspect that atomic oxygenfrom the water molecules combines with either carbon impurities in the tungsten orresidual carbon on the chamber walls to give carbon dioxide.Another problem that has been studied is the variation of the electron multiplier gainwith differing species and input current. These effects are particularly important whenprecise quantitative measurements of rates of change of several species are wantedsimultaneously.We have done some work on trying to understand the basic signal-to-noise limitationsof quadrupole mass spectrometers. We have designed improved driver circuitry for the JSEPPR No. 118(I. MOLECULE MICROSCOPY)JSEP scanning stage of SPMM that should substantially reduce vibrations and allow minimumscanning increments of 6 X 10-7 cm.2. Scanning Desorption Molecule Microscopy (SDMM)H. F. Dylla (now at the Princeton Plasma Physics Laboratory), M. J. Cardillo (nowat Bell Laboratories) and J. G. King are completing the analysis of the experiments car-ried out last year by H. F. Dylla, and are preparing a paper for publication. The workis best summarized by the draft abstract.The adsorption of CO on highly polished (planar) surfaces and oxygen-etched Si (111)and Si (100) surfaces has been studied by a combination of Scanning Electron Microscopy(SEM), Auger Electron Spectroscopy (AES), Electron Stimulated Desorption (ESD), andflash desorption mass spectrometry techniques. Average sticking probabilities, satura-tion coverages, Arrhenius parameters for desorption, and the effect of reaction etchpits on each of these have been determined. In contrast to previous findings in otherlaboratories, we find sticking probabilities of CO on planar Si surfaces and high satura-tion coverages. The presence of pits dramatically increases the sticking probabilities,as well as the characteristic desorption temperatures. The low preexponential factorin the Arrhenius equation for CO desorbing from the planar and pitted surfaces, as wellas the anomalous lack of either AES or ESD signals for all CO coverages (up to 1 mono-layer), is discussed in terms of CO penetration below the surface.3. Desorption Experiments Related to the Scanning DesorptionMolecule Microscope (SDMM)B. R. Silver has investigated electron-stimulated desorption of such molecules asDzO and ethanol from surfaces such as platinum and platinum coated with glycogen and-15 2BSA. He has found cross sections as large as 10 1 5 cm and investigated their energydependence. These cross sections (as much as 10 4 times larger than cross sectionsfor more strongly bound molecules) are very encouraging from the standpoint of reso-lution and sensitivity in the application of the SDMM.D. G. Lysy has continued his studies of the thermal desorption of water from plati-num and platinum coated with BSA, glycogen, poly-L-Alanine, poly-L-Lysine Hydro-bromide, and Tristearin. It is encouraging to see that the flash desorption spectra varyconsiderably and we can foresee preparation of an extensive catalogue of spectra of com-binations of staining molecules and biological materials and interpretation of the resultsat various levels of detail and rigor. The apparatus used by Lysy is well suited alsofor studies of denaturation. We are now almost ready to introduce biological materialinto the SDMM developed by Dylla, using a suitable sample holder that is still to bedesigned and to observe the ESD and/or thermal desorption of sample molecules orJSEP staining molecules applied to the sample.PR No. 118(I. MOLECULE MICROSCOPY)4. Molecule Fluxes in TissueWe have also been exploring the application of mass spectrometry methods to thestudy of fluxes of volatile molecules through biological tissues and membranes. Thiswork is now being performed by S. J. Rosenthal in the laboratory of Dr. Alvin Essigat Boston University Medical School. Recent experimental results show that essentiallysimultaneous measurement of 02 consumption and CO 2 productions are feasible; thisapproach can be extended to the nonvolatile species, lactate, by use of the VEP method.5. Volatile Enzyme Product MethodWe have completed an initial VEP apparatus and have begun to characterize it.Preliminary data on passive, continuous removal of preexisting volatile contaminantsand on enzyme assays are in fair agreement with experiment, and therefore very encour-aging. Specifically, with a nonoptimal arrangement, we have been able to detect andmeasure 7 X 10 9 enzyme molecules (urease) in ~102 seconds. A more detailed discus-sion of this technique follows in Section I-B.B. PROGRESS WITH THE VOLATILE ENZYME PRODUCT(VEP) METHODNational Institutes of Health (Grants 1 ROI GM22633-01 and 5 S05 RR07047-10)Jerome H. Abrams, James C. Weaver, Frederick Villars, Augustine J-Y. ChongRecent work on the first apparatus devoted to the volatile enzyme product (VEP)method is described in this report. This method, which combines the specificity ofenzymes with the sensitivity of mass spectrometry, offers a potential for rapidassays of substrates, cofactors, enzymes, and inhibitors for many enzyme-catalyzed1, 2reactions. The method interfaces a solubilized enzyme or an immobilized enzymereaction to a mass spectrometer by means of a synthetic semipermeable membrane, atpresent, dimethyl silicone. Depending on the exact configuration, much or most ofthe volatile product of the reaction enters a region of relative vacuum where a massspectrometer counts individual, volatile-product molecules with an efficiency ET-53 X 10During the past year we have focused our work on the design and construction of anapparatus that can provide a low background at the mass peak or peaks of interest (e. g.,CO 2 , ethanol, and acetic acid), that will have a high total counting efficiency, ET , forrapid counting of individual molecules and the flexibility to accommodate many antici-pated design changes. The apparatus based on these considerations is shown in Fig. I-i.This system features an approximately 50-liter stainless-steel vacuum system pumpedPR No. 118(I. MOLECULE MICROSCOPY)Fig. I-1. First apparatus devoted to the VEP method.by an oil diffusion pump (1200 1/s nominal pumping speed). The main pump is backedwith a second oil diffusion pump and forepump; all pumps are separated from the mainvacuum system by a liquid nitrogen trap. The mass spectrometer, which is suspendedfrom the central top flange, is an Extranuclear quadrupole mass filter spectrometer(QMS) supplied by Extranuclear Laboratories, Pittsburgh, Pennsylvania. It has an axialionizer with a stated efficiency of approximately 10-3. The membrane, and a smallchannel that brings a liquid sample to the membrane, are located on a flange at thebottom of the apparatus (Fig. I-2). Most of our recent work has been concerned withthis sample-handling region.The vacuum system has not yet been thoroughly baked at 400 C, a procedure thatshould provide an essentially permanent reduction by a factor of approximately 30 inthe CO 2 background. Other features, such as continuous passive degassing of pre-existing volatile contaminants (e. g., CO ), have been investigated but have not yet beenused in the present apparatus. Our results are therefore subject to these limitations.PR No. 118 4_ IL(I. MOLECULE MICROSCOPY)RELATIVEVACUUM OF QMS STAINLESS/ STEELSYRINGE PUMP SYRINGE PUMPBUFFER + SUBSTRATE BUFFER ONLY~7 I M EMRNZYMEINJECTIONSTIRRING BARFig. 1-2. Sample-handling portion of the apparatus.- PURE GASM M4- __ _ _h hSb)(b)Fig. 1-3. Configuration for continuous, passive degassing of asample tested (see Fig. I- 4 a) by substitution of therelative vacuum of the QMS for the pure gas region.-1V is the total volume flow rate (ml-sec-1 ) of the sam-ple, h is the thickness of the degassing channel, w isits width, and M is a dimethyl silicone membrane.(a) Side view. (b) End view.Much effort has been directed toward testing and characterizing the apparatus. Forexample, the total counting efficiency ET was determined to be ~3 X 10 - 5 by directinjection of a steady flux of CO 2 just above the membrane in the sample region (Fig. 1-2).This efficiency is somewhat greater than two orders of magnitude better than thatobtained in the first prototype apparatus2, 3 but still less than that obtainable in anoptimal configuration. We have also tested our ability to recover volatiles from aPR No. 118(I. MOLECULE MICROSCOPY)flowing stream in a thin channel (Fig. 1-3). A simple model suggests that if V is thevolume flow rate, and DV the diffusion constant of the volatile molecule in water, thenthe concentration of a preexisting volatile CV(0), should fall off approximately exponen-tially asCV(y) = CV(0) exp(-y/X), (1)where X = hV/wDV. If f(V) is the fraction of the preexisting volatile that leaves theflowing sample stream by permeating the membrane and escaping into the counterflowof pure gas, and if Ay = total membrane-channel length, thenf(V ) z 1 - exp(-wDVAy/hV). (2)The prediction of this simple model has been tested for CO 2 . In these experiments, avacuum and the QMS have been substituted for the pure gas counterflow region to mea-sure directly the flux of CO 2 across the membrane. We expect the steady-state countrate in this case to bens = ETf(V) VCv(0) (3)Several experiments have been performed on the membrane-channel configurations shownin Fig. I-4; the experimental results are shown in the curves. As expected, the simplecalculation does not hold for large V, but in the small V region agreement with theoryis good. Both membrane-channel configurations show a region at low V where 1 is0 0 snearly proportional to V, which indicates that in this region f(V) i. This result con-firms our expectations that we ought to be able to degas an incoming sample streamefficiently by using a continuous and essentially passive method and, after passing thesample past an immobilized enzyme region or introducing a solubilized enzyme, recovermost of the volatile product into the mass spectrometer in a second thin-channel mem-brane section.We have also made some tests by varying the concentration of the preexistingvolatile, in this case, dissolved CO 2 and bicarbonate. Although a multiple equilibriumexists, we may calibrate the apparatus by varying the concentration of HCO 3 and usingEq. 3.We have also observed a transient response correlated with injection of the sample.This effect seems to be attributable to the transient increase in pressure occurringduring injection which is such that V increases and, by Eq. 3, the count rate alsoincreases. Recent sample-handling improvements have reduced but not eliminatedthis artifactual, transient response.Finally, using urease, we have recently obtained some preliminary results onPR No. 118SIMPLE THEORYFOR Vf(V),NORMALIZEDTO DATAVACUUM =_4 _h 10 cm2 - i W--,'1-2 4 6 8 10 12103 V (ml-s -l)(a)36 SIMPLE THEORYFOR /f( (), oNORMALIZED32 TO DATA o28 02.4VACUUM 420 h=-2cm1.6 -1.2 o f(9) = Iea/9with8 a= DvR2h4r R=6.45xIO cm0 1O 2 4 6 8 10 12103 V (ml-s -)(b)Fig. 1-4. (a) Comparison of experiment and theory (Eqs. 2 and 3)for the channel shown. A . 001 inch thick dimethyl-5silicone membrane was used, and CO 2 (DV = 2 X 102 -1cm -s ) in a solution buffered at pH 7. 0 was the testvolatile molecule. In Eq. 2, Ay = 5 cm.(b) Similar comparison for a related, radial flow geom-etry. CO 2 was the test volatile, but with pH 5. 8, alarger membrane area, and different QMS tuning thanin (a).1091081 i07106105 L103 10-2 10-ACa (u-ml -1 )Fig. I-5. Preliminary data for enzyme assay using urease(EC 3. 5. 1. 5) and CO 2 as the volatile productmolecule. 0 is from the run of 4/29/76, andEl from 5/27/76, both with pH 7. 0.PR No. 118(I. MOLECULE MICROSCOPY)enzyme assay. A typical response curve is shown in Fig. 1-5. Note that these datawere obtained with a fundamental time response that is obscured by the artifactualtransient associated with injection of the sample. With this limitation, an interval ofapproximately 10 s was required to measure the change in count rate, An, followingan enzyme injection that produced a change, ACa, in enzyme activity concentration.At the lower levels, the measured response can be attributed to the detection of-1 9 -1N E  VAC vT t 7 X 10 urease molecules (v 3 X 10 Z molecules-s is the turn-over number for urease, and tR  10 is the longer approximate response time causedR 0 -1by the artifactual response) in a solution volume of A V = Vt R = 10 ml, i. e. , a mini--10mum concentration detection of enzyme of ~10 M. The observed sensitivity, ~5 X108 cps-ml-u - 1 , is in reasonable agreement with a simple calculation, which predictsa sensitivity of 2 X 108 cps-ml-u - 1As this work continues, attention will be directed toward obtaining thinner membranesso that other volatile products, such as acetic acid and ethanol, can be used more effec-tively. For such exploratory work on different membranes and channels, we have con-structed a simple, separate test vacuum system, so that when a membrane bursts orleaks, no damage to the QMS will occur. After this has been accomplished, we shallturn our attention to other enzyme systems such as DOPA decarboxylase (EC 4.1.1.26),isocitrate dehydrogenase (EC 1. 1. 1. 42), carbonic anhydrase (EC 4. 2. 1. 1), pyruvatedecarboxylase (EC 4. 1. 1. 1), lactate dehydrogenase (EC 1. 1. 1. 27), and acetylcholin-esterase (EC 3. 1. 1.7).During the course of this work we have enjoyed many stimulating discussions withother members of our group, particularly with J. G. King, J. A. Jarrell, A. M. Razdowand J. W. Peterson, and also with C. L. Cooney of the Al. I. T. Nutrition and Food Sci-ence Department. This work was supported in part by the Francis Friedman Chair ofPhysics.References1. J. C. Weaver, Quarterly Progress Report No. 114, Research Laboratory of Elec-tronics, M.I. T., July 15, 1974, pp. 8-10.2. J. C. Weaver, M. K. Mason, J. A. Jarrell, and J. W. Peterson, "BiochemicalAssay by Immobilized Enzymes and a Mass Spectrometer," Biochim. Biophys. Acta438, 296-303 (1976).3. M. K. Mason, S.B. Thesis, Department of Physics, M. I. T., June 1974.4. 1 Unit = 1 u = 1 International Unit of Enzyme Activity = 1 Fmol-min 1 = 106-lmolecules-sec-1 of product by the enzyme catalyzed reaction; see T. E. Barman,Enzyme Handbook, Vol. 1 (Springer-Verlag, New York, 1969).PR No. 118

Molecule Microscopy

http://dspace.mit.edu/bitstream/1721.1/56620/1/RLE_PR_118_I.pdf

Molecule Microscopy

Abstract

Similar works

Full text

Available Versions

DSpace@MIT