Report
Robert L. Bowman Gerald G. Vurek a Laboratory of Technical Development National Heart, Lung, and Blood Institute Building to, Room 50-20 Eethesda. Md. 20205
Analysis of Yanol.,er Biological Samples Thermistor
Holes -Copper
Plates Aluminum Heat Sink
Figure 1. Thermoelectrically controlled freezing point osmometer (a) Thermoeiecrric Cooler assembly showing de. tails of construction of the top and bottom stages. (b) View of the cooling unit. showing the finned heat sink. the hinged lid. and placement 01 the thermoeiectric cooler assembly. The magnified areas show details of the sample holes. of the Oepression for oil. and of the thermistor on the Jample stage. Reprinted With permission from Reference 6.Copyright 1963. AAAS
This article not subject to US. Copyright Published 1984 American Chemical Society
Over the past several years, we have been developing techniques and instruments for the analysis of nanuliter samples. Most of our work has heen in collaboration with the physiologists of the Laboratory of Kidney and Electrolyte Metaholism of the National Heart, Lung, and Blood Institute. Physiologists studying the function of the kidney are faced with the task of unraveling the way the kidney partiripates in the stabilization and control of the water and solute rontent of blood. The mammalian kidney may have more than a million individual tubules whirh ronvert filtered blood entering the tubules at the glomeruli into urine through many active and passive transport processes. These t u bules may have a diameter of 20 pm and pass fluid at a rate of a few nanoliters per minute. As Jamison and Kriz have described, the anatomy of the hlood vessels is very intimately connected to the transport mechanism, not only in controlling blood tlow but also in generating a counterrurrent concentration system that exploits the differential permeahility properties uf the various segmenu of the tubules to achieve selective recovery uf impur. tant materials, including water r l j. Two techniques, mirropunrture and microperfusion, have been used tu obtain samples fur study of the transport processes. Micropuncture, which has heen summarized by Windhager. in. volves putting a sharp glass pipet through the wall of' an individual kidney tubule and withdrawing a sample with a volume thnt may range from a few hundred piroliters to wns of nano-
liters (2). Microperfusion involves dissecting a piece of a specific part of a kidney tubule and suspending it in a nutrient bath while perfusing it with micropipets inserted in each end. Samples are removed from the collection end. In either case, the samples are sandwiched between segment.? of mineral oil to prevent water loss between collection and analysis; they may be stored within a capillary or on the surface of a glass plate covered with oil. We have found that mounting the sample pipet in a holder attached to a stereo microscope is very helpful in sample handling. This arrangement allows the user to keep the pipet tip always in view and, with appropriate translating mechanisms, collect and deliver samples with better than 1%precision. Although micropuncture yields important information about the way tubule fluid is transformed in the living animal, the anatomy of the kidney makes study of all segments by micropuncture impossible. The development of microperfusion (3)has made possible much more complete control of the composition of the fluid that surrounds and flows into the tubule. The instruments and methods we describe in this REPORT for analysis of nanoliter samples are products of our laboratory, but others have developed different approaches to solving similar problems. A notable example is the work of Lechene in the develop* Present address: Sorenson Research Division of Abbott Laboratories, 4455 Atherton Dr., Salt Lake City, Utah 84107
ANALYT CAL CHEMISTRY, VOL. 56. NO. 3, MARCH 1984
391A
Table 1. Elements, Analytical Wavelengths, and Sensitivities Obtained with Helium Glow Excitation Wavelength Concenlration SB"611i"Il). = Elsmsnl
(A)
range lesled (mM)
(x 1
0 r i 4 ~ ~ 1 )
Ca
4227 0.02-2 1 3261 0.01-0.1 0.5 3724 0.01-0.1 4 Fe 2488 0.1-1 9 Hg 2537 0.01-0.1 2 K 7699 0.1-1 4 Mg 2852 0.02-2 4 Na 5896 0.1-1 6 Pb 4058 0.01-0.1 1 Zn 2139 0.01-1 5 * Sensitivity is defined as the amount of element needed to give a signal increment equal to twice standard deviation of lhe blan Cd
cu
.-.
I
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ment of the electron microprobe ( 4 ) . This elegant hut expensive instrument :an analyze piculiter samples for a number of elements a t once. Our approach has been to make special-purpose instruments suited to a ,ingle type of analysis. The areas that have concerned us have included osmolality, cation analysis for sodium, potassium, etc., and other organic metabolites.
I:
We can help with application advice, we can help with column selection, we can help with technical trouble-shooting or with an accessory that will do the trick, or even a custom made column for your unique needs.
Salt and Water Osmotic forces are very important in the recovery of water from the filtered tubule fluid. The kidney recovers about 99% of the filtered water. Osmometry is a convenient way t o measure the water activity, and freezing point depression can be used if millidegree sensitivity can be achieved. Ramsay developed a microfreezing point depression apparatus in which samples within a capillary were observed with a microscope as the ice bath in which they were suspended was slowly warmed ( 5 ) .The operator had to decide when the last bit of ice was about to melt and then read the temperature of the bath to the nearest
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millidegree with a Beckmann thermometer. Prager and Bowman developed a microfreezing point apparatus with Peltier elements to control the sample temperature (6). In their apparatus, the samples were suspended in oil held in holes in a copper har which formed the top link of a twostage cooler. A thermistor on the link was part of a servo loop that allowed the operator to hold the temperature a t any point while inspecting each sample with a microscope. Figure 1 shows this arrangement. In this way, the smallest ice crystal could be held in equilibrium with the fluid portion, and the true melting point could be estimated with good precision. Also, standards could be put in adjacent holes so that simultaneous calihrations could he carried out. This allowed operators much greater control and measurement accuracy than did the older techniques. Sodium is the major osmotically active cation, and potassium is important because the ratio of the extracellular concentration to the intracellular concentration determines the cell membrane potential. Changes in po-
C
iOH Gram
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392A
Figure 2. Arrangement for the extraction and measurement of picomole amounts of C 0 2 from nanoliter samples
ANALYTICAL CHEMISTRY, VOL. 56, NO.
3. MARCH 1984
4i The VG PlasmaQuad is an inductively coupled plasmasource mass spectrometer Mat isdesigned for both elemental and isotopic analysis. The ICP-MS technique is creating more interest among analysts 3nb sgectro; scopists than any new method of analysis in the past10 years, and for good reason: V G s PlaamaQtladcan analyze more elements in thrperiodic table than any other method, at lower OonanrtretiOW (sotneat partsper&Uibn)adwera wider tan@ of conoent~oas
The PlasmaQuad has been designed with routine day to day analysis in mind-ease of operation, reliability, high m p l e thruput rmd proven ac. curacy. An example of its cost effectiveness is that a PlasmaQuad wNl replace mora than one type of instrument in your laboratory. No wonder that ICP-MS is creating interest as the analyticai tool for elemental analysis of the w60%. There are additional masons why you should consider the VG PlasmaQuad and we would ilke to pregent these to
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tassium concentration influence the responsiveness of heart muscle, nerves, and other excitable tissues. While these elements are easily measured by flame emission photometry, to use this approach with our samples would require water with parts-pertrillion purity levels. We explored the use of a radio-frequency-excited helium plasma to excite the samples to produce their characteristic emission spectra (7,s).The samples were diluted with a spectroscopic buffer of cesium nitrate and ammonium phosphate to swamp interelement interferences, and aliquots of the diluted samples were put on an iridium wire. The water evaporated, leaving the sample behind. The plasma was initiated while the wire was heated to volatilize the sample into the discharge. Table I shows that the technique has demonstrated femtomole sensitivity for sodium, potassium, and a number of other elements. Recently, Hieftje and Deutsch have done additional work along this line (9). An important piece of information about the concentration process is the extent to which water and solutes are transported by active or passive (osmotically driven) processes. A common marker for water transport is inulin, a poly(fructose) that is filtered but not reabsorbed. The ratio of the plasma to tubule fluid concentration is used to indicate changes in tubule fluid water concentrations. Inulin concentrations have been measured with 1%-labeled material, colorimetrically with the reaction product of inulin with anthrone using a microcapillary cuvette in a standard spectrophotometer, and by fluorometric measurement of the reaction product with dimedone (IO).In the latter case, we modified a commercial filter fluorometer to accept capillary cuvettes that held the 2 fiL of reaction product between the nanogram amounts of inulin and reagent. In the case of isolated tubule studies, other materials may be used as water markers, such as urea, creatinine, and polymeric dyes. Measurement of these will be discussed below in relation to the continuous-flow instruments. The principal anion in tubule fluid is chloride. Microcoulometry has been most effective for chloride measurement. The total charge required to generate enough silver ions to precipitate the sample chloride is easily measured by electronic integration. The titration endpoint is indicated by a change in the chloride indicator electrode potential (11).
Bicarbonate The kidney has an important role in the control of the pH of the blood through its excretion of bicarbonate 39SA
Flgure 3. Flow-through microfluorometer
and ammonium ion. The bicarbonate content of tubule fluid has been estimated by equilibrating samples with gases of known pCO2 and measuring the pH of the samples with micro pH electrodes, by precipitating the fluid with barium chloride and using an electron probe to measure the barium, and by microcalorimetry. Microporous lithium hydroxide reacts with COz and releases about 90 kJ/mol. Picomole amounts of COz can be measured by putting a single granule of LiOH on one thermistor of a mawhed pair used in a half-bridge (IZJ, as shown in Figure 2. Nanoliter samples are injected into a small volume of sulfuric acid, and the released CO:! is swept to the thermistor chamber by a flow of Freon. A small mercury drop acts as a leak-tight seal through which the pipet can pass. About 10 pmol of COz (0.2 nL) can he detected by this instrument. Each measurement takes about 2 min, so measurements can he made during the experiments.
Fluorometry The development of a flow-through fluorometer and a colorimeter with submicroliter-volume cuvettes has opened a range of new methods for picomole analysis (13-15). The first application of the fluorometer was for the determination of ammonium ion using a standard enzymic assay (16).
ANALYTICAL CHEMISTRY, VOL. 56. NO.
3. MARCH 1984
The reaction (Equation 1)is essentially complete in 1min; thus, this measurement can be done as fast as samples can be obtained from the exuerimental preparation.
+
NH4+ alpha-ketoglutarate NADH -glutamate NAD+ (1)
+
+
For a fluorometer to have good sensitivity, i t is necessary to have the greatest interaction of the excitation liaht with the sample and collect the greatest amount of the emitted light. Communications-grade optical fibers transmit the 366-nm liaht from a mercury lamp used to excite the NADH fluorescence. As shown in Figure 3, the fiber is aimed along the axis of the cuvette so that the light, which is emitted in a nearly collimated beam, very effectively excites the fluorescence hut is not directed toward the detector. The detector is placed near the cuvette, which also has a mirror behind i t to direct more light to the detector. This instrument has demonstrated a sensitivity for ammonium of less than 0.3 pmol. Other metabolites can be assayed with the fluorometer. For example, by adding urease to the ammonium reaction, urea can be measured. Lactate production by isolated tubule fragments can be assayed with a NADH coupled enzyme reagent. In this sys-
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tem, the increase in fluorescence is proportional to the amount of lactate, in contrast to the small decrease in fluorescence used in the ammonium assay. An advantage of working with these small samples-is that surface tension can sometimes he exploited effectively. In the case of the fluorometer, the sample is injected directly into the reagent stream without going through a valve or seal. The injection port is merely a small hole in the side of the tubing carrying the reagent from the reservoir to the cuvette. As with many of these instruments, the sample pipet is mounted in a holder attached to a binocular microscope so that the user can always see the pipet tip and place it precisely in the middle of the reagent stream. Reagent flow rate for the ammonium assay is about 4 pL/min and I pL/min for lactate, so reagent consumption is not an economic factor.
Colorimetry While fluorometry can have high sensitivity and can he adapted to measurement of many materials, colorimetric procedures often are simpler. We have constructed a flow-through colorimeter with a working volume of 0.3 FL and a path length of 1.1 cm. Two techniques made this feasible. One was M make the optical windows from the capillary which formed both the cuvette and connecting tubes. The other was to coat the outside of the tube with a light-absorbing substance. This step prevents light that travels in the glass, bypassing the sample, from reaching the photosensor. Although it is difficult to attach flat windows to small capillaries, acceptable windows can he formed by a modified glassworking technique. Using a stereo microscope and either a miniature gas-oxygen torch (for quartz) or a hot Pt-lr wire (for soft glass), two right-angle bends are formed about 1 cm apart (17). A hit of extra glass is added to the bend, and it is blown out to a thin huhhle (Figure 4). With careful heating, the huhhle is allowed to collapse, forming a reasonable window at the end of the cuvette. The imperfection of the windows makes each cuvette different from one another, hut each can he calibrated. The effect of fluid refractive index changes must he measured for this sort of cuvette just as for other flowthrough cuvettes. Although the first colorimeter of this series used an injection port similar to the CO? analyzer, it became apparent that a hole in the side of the lead-in tubing was simpler and eliminated the possibility of mercury contamination. Single-wave. length colorimeters have been developed for measurement of Mg2-,
LMU
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
b
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Figure 4. Construction of quartz capil-
lary cuvette The final product has two Sodegree bends constructed as follows: The capillary is sealed at one end and connected to a tO-mL glasa syringe at the othw to allow he imralminal pressure to be increased. (a) &ginning with the capillary horizontal, he initial bend is made by heating a small section of he capillary while maintaininggentle pressure to keep lhe lumen open. An addnional m a i l amwnt of qwnz is melted om0 he bend to thicken he wall. (b) The quam is then soflened with lhe flame. and a bubble is blown at the bend. ( c )The pntion of the bubble he1 is to be in the light path is flanened by local application of heat and gradual reduction of intraluminal presswe
Pod3-, Ca2+, and urea. Recently, a system with an adjustable wavelength source has been constructed so that the colorimeter can he adapted quickly to different methods (18). These miniature flow systems require very constant reagent pumping rates. We have used synchronous motor-driven syringes to draw the reagent through the cnvettes from the reservoirs. Flow variations as small as 1%peak to peak are detectable, so the screws and gears of the pump drive must have good precision. An alternative to the pump would he to use a ' capillary to control the flow generated (continued on p. 405 A )
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by a constant gravitational head. We have found the syringe withdrawal pump convenient and reliable for generating flows under 10 pL/min used in the colorimeter and fluorometer.
Conclusion The need to analyze nanoliter biological samples has stimulated the development of new instruments and the miniaturization of conventional instruments. There seems to be a convergence of the ultramicro and more common analytical approaches, particularly as the resolving power of HPLC and capillary electrophoresis becomes detector and sample volume limited. Much work remains to be done, especially in the field of emission analysis to adapt it to micro-continuous-flow methods. So much of the power required by flame or plasma torch emission photometers is used for solvent volatilization that if the solvent flow could be reduced to nanoliters per second, then much smaller, lower powered instruments could be used. The solution to this problem would bring the benefits of continuous on-line analysis of nanoliter samples to HPLC as well as to biological samples. Acknowledgment The authors acknowledge the collaboration of the staff of the Laboratory of Kidney and Electrolyte Metabolism, including R. W. Berliner, M. B. Burg, and the dozens of fellows who passed through the laboratory, working with us to develop and perfect these instruments which have helped answer so many questions in renal physiology. References (1) Jamison. R. L.; Kriz, W. "Urinary Concentrating Mechanism: Structure and Function"; Oxford University Press: New York, N.Y., 1982. (2) Windhager, E. E. "Micropuncture Techniques and Nephron Function"; Butterworths: London, U.K.. 1968. (3) Burg, M. B.; Grantham. J.; Abramow, M.: Orlaff. J. J. Phvsrol. 1966.210. . . 1293-98. (4) Lechene, C. "Microprobe Analysis as Applied to Cells and Tissues"; Hall, T.; Eehlin. P.; Kaufmann. R., Eds.; Academic: London. U.K.. 1974. (5) Ramsay, J. A,; Brown. R.H.J. J. Sei. Inslr. 1955.32,372. (6) Prager, D. J.; Bowman, R. L. Science 1963,142.237-39. (7) Vurek. G. G.; Bowman R. L. Science 1965,149,448-50. (8) Vurek, G. G. A n d . Chem. 1967,39. 1599-1fiOl. (9) Deutsch, R. D.; Hieftje. G. M.. Indiana
University, unpublished work.
(IO) Vurek, G. G.; Pegram, S. E. Anal. Rioehem. 1966,16.409-16. (11) Ramsay. J. A,; Brown, R.H.J.; Crophan, P. C. J. Exp. R i d . 1955,32.822-29. (12) Vurek, G.G.; Warnwk. D. G.; Corsey, R. Anal. Chem. 1975.47.765-67. (13) Vurek. G. G. Anal. Letts. 19RI, 14(A4), 261-69.
(14) Vurek. G.G.Anal. Rioehem. 1981, 114.2~8-93. (15) Vurek, G.G. Anal. Chem. 1982.54, YALA?
(16) Vurek. G . G.; G o d , D. W. Anal. Rioehem. 1983,130,199-202. (17) Vurek, G. G.; Knepper. M. A. Kidney Inl. I982,21,fi5fi-58. (IS) Adkinson. J. T.; Evans. J. C. Anal. Chem. 1983.55,2450-51.
Archaeological --Chemisftry-111 /
iph B. Lam ert, Editor hwartnm I I
Robert H w r n o n is chiiwdhis H S degree from the California Institutr of Technology i n 19.56, M.S.E.E. degree from Stanford i n 19.57, and PhD i n physid o g y from Stanford in 1964. He was a development engineer at the Riomedical Engineering and Instrumentation Rranch o f the Diuision of Research Services, National Institutes of Health, and then joined the Laboratory of Technical Deuelopment, National Heart, Lung, and Rlood Institute, where he was concerned with the deuelopment and application of nrw microanalylical techniques t o renal physiology and clinical intensive care monitoring. In 1983, h e joined the Sorenson Research Diuiaion o f Abbott Laboratories as a senior scientist. ANALYTICAL CHEMIST
DetaIIs progress In a r c h w o l ~ l c aChemls. l try. Surveys analytical techniques such as atomic absorption. X-ray Iluorercence. neutron activation. and Auger rpectropies and particle acceleration. Reparts the lindlngs of the Shroud 01 Turin study, a reliable method lor direct datina of manuscript ink and reviews radioiarbon datln Updates "'Archaeological Chemistry" an? "Archaeological Chemistry-11'' (Advances in Chemistry Series 138 and 171). CONTENTS
Based on a symwsium sp~nsoredby HM Dc visslon of Hslstory of Chernlslry Of the Arne&*" Chemical Smiefy Advances in Chemist Series205 487 pages (1983) 8othbound LC 83-15736 ISBN 0-841247674 US 8 Canada 189.95 Expn $107.95 Also available: Archaeological ChmlAry Advances in Chemistr Series 138 254 pages (1974) dothbound US 8 Canada 139.95 Exmn 147.95 Archaeological Chemlstry-ll Advances 8n Chemlslr Series 171 389 pages (T978) l h h b o u n d US 8 Canada $54.95 Expan 565.9! Vols I-Ill Ordered as B set: Expan 5197.95 US 8 Canada $164.95
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