Report Robert L. Bowman Gerald G. Vurek a Laboratory of Technical Development National Heart, Lung, and Blood Institute Building 10, Room 5D-20 Bethesda, Md. 20205
Analysis of Nanoliter Biological Samples (a)
Thermistor
Holes Copper
Ν
Ρ Anodized Aluminum Plates
Aluminum Heat Sink
(b)
Figure 1. Thermoelectrically controlled freezing point osmometer (a) Thermoelectric 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 of the thermoelectric cooler assembly. The magnified areas show details of the sample holes, of the de pression for oil, and of the thermistor on the sam ple stage. Reprinted with permission from Refer ence 6. Copyright 1963. AAAS This article not subject to U.S. Copyright Published 1984 American Chemical Society
Over the past several years, we have been developing techniques and in struments for the analysis of nanoliter samples. Most of our work has been in collaboration with the physiologists of the Laboratory of Kidney and Electro lyte Metabolism 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 partici pates in the stabilization and control of the water and solute content of blood. The mammalian kidney may have more than a million individual tubules which convert filtered blood entering the tubules at the glomeruli into urine through many active and passive transport processes. These tu bules may have a diameter of 20 μιτι and pass fluid at a rate of a few nanoliters per minute. As Jamison and Kriz have described, the anatomy of the blood vessels is very intimately con nected to the transport mechanism, not only in controlling blood flow but also in generating a countercurrent concentration system that exploits the differential permeability properties of the various segments of the tubules to achieve selective recovery of impor tant materials, including water (/). Two techniques, micropuncture and microperfusion, have been used to ob tain samples for study of the transport processes. Micropuncture, which has been summarized by Windhager, in volves putting a sharp glass pipet through the wall of an individual kid ney tubule and withdrawing a sample with a volume that may range from a few hundred picoliters to tens of nano-
liters (2). Microperfusion involves dis secting 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 collec tion end. In either case, the samples are sandwiched between segments of mineral oil to prevent water loss be tween 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 mount ing the sample pipet in a holder at tached to a stereo microscope is very helpful in sample handling. This ar rangement allows the user to keep the pipet tip always in view and, with ap propriate translating mechanisms, col lect and deliver samples with better than 1% precision. Although micro puncture yields important informa tion about the way tubule fluid is transformed in the living animal, the anatomy of the kidney makes study of all segments by micropuncture impos sible. The development of microperfu sion (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 devel oped different approaches to solving similar problems. A notable example is the work of Lechene in the develop° Present address: Sorensun Research Division of Abbott Laboratories, 4455 Atherton Dr., Salt Lake City, Utah 84107
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3,
MARCH 1984 · 391 A
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Table I. Elements, Analytical Wavelengths, and Sensitivities Obtained with Helium Glow Excitation Element
Wavelength (A)
ment of the electron microprobe (4). This elegant but expensive instrument can analyze picoliter samples for a number of elements at once. Our approach has been to make spe cial-purpose instruments suited to a single type of analysis. The areas that have concerned us have included os molality, cation analysis for sodium, potassium, etc., and other organic me tabolites. Salt and Water Osmotic forces are very important in the recovery of water from the fil tered tubule fluid. The kidney recov ers about 99% of the filtered water. Osmometry is a convenient way to measure the water activity, and freez ing 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 ther mometer. Prager and Bowman devel oped a microfreezing point apparatus with Peltier elements to control the sample temperature (6). In their ap paratus, the samples were suspended in oil held in holes in a copper bar 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 at 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 calibra tions could be carried out. This al lowed operators much greater control and measurement accuracy than did the older techniques. Sodium is the major osmotically ac tive cation, and potassium is impor tant because the ratio of the extracel lular concentration to the intracellular concentration determines the cell membrane potential. Changes in po-
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392 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
tassium concentration influence the responsiveness of heart muscle, nerves, and other excitable tissues. While these elements are easily mea sured 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 heli um plasma to excite the samples to produce their characteristic emission spectra (7, 8). The samples were dilut ed with a spectroscopic buffer of cesi um 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 demon strated femtomole sensitivity for sodi um, 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 com mon marker for water transport is inulin, a poly(fructose) that is filtered but not reabsorbed. The ratio of the plas ma to tubule fluid concentration is used to indicate changes in tubule fluid water concentrations. Inulin con centrations have been measured with 14 C-labeled material, colorimetrically with the reaction product of inulin with anthrone using a microcapillary cuvette in a standard spectrophotome ter, and by fluorometric measurement of the reaction product with dimedone (10). In the latter case, we modified a commercial filter fluorometer to ac cept capillary cuvettes that held the 2 μι, of reaction product between the nanogram amounts of inulin and re agent. In the case of isolated tubule studies, other materials may be used as water markers, such as urea, creati nine, and polymeric dyes. Measure ment of these will be discussed below in relation to the continuous-flow in struments. The principal anion in tubule fluid is chloride. Microcoulometry has been most effective for chloride measure ment. The total charge required to generate enough silver ions to precipi tate the sample chloride is easily mea sured by electronic integration. The titration endpoint is indicated by a change in the chloride indicator elec trode potential (11). Bicarbonate The kidney has an important role in the control of the pH of the blood through its excretion of bicarbonate
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and ammonium ion. The bicarbonate content of tubule fluid has been esti mated by equilibrating samples with gases of known pCC>2 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 CO2 and releases about 90 kJ/mol. Picomole amounts of CO2 can be measured by putting a single granule of LiOH on one thermistor of a matched pair used in a half-bridge (12), as shown in Fig ure 2. Nanoliter samples are injected into a small volume of sulfuric acid, and the released CO2 is swept to the thermistor chamber by a flow of Fréon. A small mercury drop acts as a leak-tight seal through which the pipet can pass. About 10 pmol of C 0 2 (0.2 nL) can be detected by this instrument. Each measurement takes about 2 min, so measurements can be 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).
396 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
The reaction (Equation 1) is essentially complete in 1 min; thus, this measurement can be done as fast as samples can be obtained from the experimental preparation. NH 4 + + alpha-ketoglutarate + NADH — glutamate + NAD+
(1)
For a fluorometer to have good sensitivity, it is necessary to have the greatest interaction of the excitation light with the sample and collect the greatest amount of the emitted light. Communications-grade optical fibers transmit the 366-nm light 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 but is not directed toward the detector. The detector is placed near the cuvette, which also has a mirror behind it 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 be exploited effective ly. In the case of the fluorometer, the sample is injected directly into the re agent 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 re agent stream. Reagent flow rate for the ammonium assay is about 4 /zL/min and 1 ^L/min for lactate, so reagent consumption is not an eco nomic factor.
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While fluorometry can have high sensitivity and can be adapted to mea surement of many materials, colorimetric procedures often are simpler. We have constructed a flow-through colorimeter with a working volume of 0.3 μι, and a path length of 1.1 cm. Two techniques made this feasible. One was to 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 be formed by a modified glassworking technique. Using a stereo microscope and ei ther a miniature gas-oxygen torch (for quartz) or a hot Pt-Ir wire (for soft glass), two right-angle bends are formed about 1 cm apart (17). A bit of extra glass is added to the bend, and it is blown out to a thin bubble (Figure 4). With careful heating, the bubble is allowed to collapse, forming a reason able window at the end of the cuvette. The imperfection of the windows makes each cuvette different from one another, but each can be calibrated. The effect of fluid refractive index changes must be measured for this sort of cuvette just as for other flowthrough cuvettes. Although the first colorimeter of this series used an in jection port similar to the CO2 analyz er, it became apparent that a hole in the side of the lead-in tubing was sim pler and eliminated the possibility of mercury contamination. Single-wave length colorimeters have been devel oped for measurement of Mg 2+ ,
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398 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
(c) Collapse bubble to form nearly flat window. Figure 4. Construction of quartz capil lary cuvette The final product has two 90-degree bends con structed as follows: The capillary is sealed at one end and connected to a 10-mL glass syringe at the other to allow the intraluminal pressure to be increased, (a) Beginning with the capillary hori zontal, the initial bend is made by heating a small section of the capillary while maintaining gentle pressure to keep the lumen open. An additional small amount of quartz is melted onto the bend to thicken the wall, (b) The quartz is then softened with the flame, and a bubble is blown at the bend. (c) The portion of the bubble that is to be in the light path is flattened by local application of heat and gradual reduction of intraluminal pressure
PO4 3 -, Ca 2+ , and urea. Recently, a system with an adjustable wavelength source has been constructed so that the colorimeter can be adapted quick ly to different methods (18). These miniature flow systems re quire very constant reagent pumping rates. We have used synchronous motor-driven syringes to draw the re agent through the cuvettes 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 alterna tive to the pump would be to use a capillary to control the flow generated (continued on p. 405 A)
by a c o n s t a n t gravitational head. W e have found t h e syringe w i t h d r a w a l p u m p convenient a n d reliable for gen erating flows u n d e r 10 μΙ_,/ηιίη used in t h e colorimeter a n d fluorometer.
Conclusion T h e need to analyze nanoliter bio logical samples has s t i m u l a t e d t h e d e velopment of new i n s t r u m e n t s a n d t h e miniaturization of conventional in s t r u m e n t s . T h e r e seems to be a con vergence of t h e u l t r a m i c r o a n d m o r e common analytical a p p r o a c h e s , par ticularly as t h e resolving power of H P L C a n d capillary electrophoresis becomes detector a n d s a m p l e volume limited. M u c h work r e m a i n s t o be done, especially in t h e field of emis sion analysis to a d a p t it t o micro-continuous-flow m e t h o d s . So m u c h of t h e power required by flame or p l a s m a torch emission p h o t o m e t e r s is used for solvent volatilization t h a t if t h e sol vent flow could be reduced t o nanoliters per second, t h e n m u c h smaller, lower powered i n s t r u m e n t s could be used. T h e solution to t h i s p r o b l e m would bring t h e benefits of c o n t i n u o u s on-line analysis of nanoliter s a m p l e s to H P L C as well as t o biological s a m ples.
Acknowledgment T h e a u t h o r s acknowledge t h e col laboration of t h e staff of t h e L a b o r a tory of K i d n e y a n d Electrolyte M e t a b olism, including R. W. Berliner, M. B . Burg, a n d t h e dozens of fellows who passed t h r o u g h t h e laboratory, work ing with us t o develop a n d perfect these i n s t r u m e n t s which have helped answer so m a n y questions in renal physiology.
(14) Vurek, G. G. Anal. Biochem. 1981, 7/4,288-93. (15) Vurek, G. G. Anal. Chem. 1982,54, 840-42. (16) Vurek, G. G.; Good, D. W. Anal. Bio chem. 1983, 130, 199-202. (17) Vurek, G. G.; Knepper, M. A. Kidney Int. 1982,22,656-58. (18) Adkinson, J. T.; Evans, J. C. Anal. Chem. 1983,55,2450-51.
Joseph B. Lambert, Editor Northwestern University Robert Bowman is chief of the Labo ratory of Technical Development of the National Heart, Lung, and Blood Institute, a laboratory devoted to the development of research instrumen tation for biomedical science. He re ceived his AB degree from Columbia University in 1938 and MD degree from NYU College of Medicine in 1942. He has contributed to instru mentation for flame photometry, spectrofluorometry, osmometry, gas chromatography detectors, chloridimetry, microanalytical methods, and several devices for circulatory sup port and catheters for interventional radiology.
References (1) Jamison, R. L.; Kriz, W. "Urinary Con centrating Mechanism: Structure and Function"; Oxford University Press: New York, N.Y., 1982. (2) Windhager, Ε. Ε. "Micropuncture Techniques and Nephron Function"; Butterworths: London, U.K., 1968. (3) Burg, M. B.; Grantham, J.; Abramow, M.; Orloff, J. J. Physiol. 1966,210, 1293-98. (4) Lechene, C. "Microprobe Analysis as Applied to Cells and Tissues"; Hall, T.; Echlin, P.; Kaufmann, R., Eds.; Academ ic: London, U.K., 1974. (5) Ramsay, J. Α.; Brown, R.H.J. J. Sci. Instr. 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. Anal. Chem. 1967,39, 1599-1601. (9) Deutsch, R. D.; Hieftje, G. M., Indiana University, unpublished work. (10) Vurek, G. G.; Pegram, S. E. Anal. Biochem. 1966,16, 409-16. (11) Ramsay, J. Α.; Brown, R.H.J.; Croghan, P. C. J. Exp. Biol. 1955,32, 822-29. (12) Vurek, G. G.; Warnock, D. G.; Corsey, R. Anal. Chem. 1975,47, 765-67. (13) Vurek, G. G. Anal. Letts. 1981, /4(A4), 261-69.
Archaeological Chemistry—III
Gerald Vurek received his BS degree from the California Institute of Tech nology in 1956, M.S.E.E. degree from Stanford in 1957, and PhD in physi ology from Stanford in 1964. He was a development engineer at the Biomed ical Engineering and Instrumenta tion Branch of the Division of Re search Services, National Institutes of Health, and then joined the Labo ratory of Technical Development, National Heart, Lung, and Blood In stitute, where he was concerned with the development and application of new microanalytical techniques to renal physiology and clinical inten sive care monitoring. In 1983, he joined the Sorenson Research Divi sion of Abbott Laboratories as a sen ior scientist.
W! Ne
Details progress in archaeological chemis try. Surveys analytical techniques such as atomic absorption, X-ray fluorescence, neutron activation, and Auger spectrosco pies and particle acceleration. Reports the findings of the Shroud of Turin study, a re liable method for direct dating of manu script ink, and reviews radiocarbon dating. Updates "Archaeological Chemistry" and "Archaeological Chemistry—II" (Advances in Chemistry Series 138 and 171). CONTENTS REE Analysis to Study Utilization and Pro curement of Soapstone · Provenance Stud ies of Middle Eastern Obsidian · Geochemical Techniques to Study Predynastic Sites in Egypt · Soil Chemistry Investigation in Illi nois Archaeology · Chemical Analysis of Ar chaeological Soils from Yagi Site, Japan · Analysis of Soil Associated with Woodland Burials · NAA and Ancient History · Major, Minor, and Trace Element Analysis of Medie val Stained Glass by Flame AAS · Prove nance and Technical Studies of Mexican Ma jolica · Provenance of Fine Orange Maya Ceramic Figurines by Flame AAS · Techno logical Examination of Egyptian Blue · Anal ysis of Medieval Pigments from Cilician Ar menia · SAM for Dating Manuscript Inks · Trace-Element Discrimination of Discrete Sources of Native Copper · MS as Historical Probe · Chemical Compositions of CuBased Roman Coins · Radiocarbon Dating by Particle Accelerators · Identification of Plant Gums in Artistic Applications · Techni cal Examination of Oriental Lacquer · Ex amination of Textile Fabric Pseudomorphism • Formation of Image on Shroud of Turin by X-rays · Examination of Various Stains and Images on Shroud of Turin Based on a symposium sponsored by the Di vision of History of Chemistry of the American Chemical Society Advances in Chemistry Series 205 487 pages (1983) Clothbound LC 83-15736 . ISBN 0-8412-0767-4 US & Canada $89.95 Export $107.95 Also available: Archaeological Chemistry Advances in Chemistry Series 138 254 pages (1974) Clothbound US & Canada $39.95 Export $47.95 Archaeological Chemistry—II Advances in Chemistry Series 171 389 pages (1978) Clothbound US & Canada $54.95 Export $65.95 Vols, l-lll ordered as a set: US & Canada $164.95 Export $197.95 Order from: American Chemical Society Distribution Office Dept. 57 1155 Sixteenth St., N.W. Washington, DC 20036 or CALL TOLL FREE 800-424-6747 and use your VISA or MasterCard.
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