742
Anal. Chem. 1984, 56,742-745
Reflectance Photometer for Multilayer Dry Film Slides William E. Neeley' Division of Laboratory Medicine, Department of Pathology, School of Medicine, University of California, San Diego, California 92103
Design features and performance characterlstlcs of a cllnlcal reflectance photometer are dlscussed and compared with referencemethods. The Instrument was speclflcally deslgned for the cllnlcal laboratory and uses only 2 pL of serum or plasma on Eastman Kodak multilayer dry fllm slides. Both sample and reference solutlons were placed on a sllde, and after an lncubatlon perlod, colored spots developed. The spots were illuminated with filtered light from a xenon flashlamp and the reflected light from both spots was focused by a camera lens onto a llnear photodiode array. The analog signal from each detector was dlgltlzed and transmltted to a computer where the percent reflectance for each spot was computed. Spot reflectance was proportlonal to analyte concentratlon and spot diameter proportlonal to sample volume. Slnce spot color Intensity was not hlghly dependent upon sample volume, accurate plpettlng was not required. The Instrument Is easy to operate and Is Ideal for performlng routlne chemlcal analyses where only small sample volumes are avallable.
Chemical determinations in a dry format date back to the 19th century when litmus paper was used to estimate the alkalinity of solutions. In the 1950s dry format chemistry technology advanced and was applied on a large scale to clinical urine testing. Increasing sophistication in the 1970s led to the introduction by several companies of complex multilayer dry film slides for quantitative analyses using serum, plasma, or whole blood (1-3). Basically, a slide was saturated with sample and the analyte reacts with chemicals in the layers to form a color. The slide was illuminated with filtered light and the reflected light measured with a single photodetector. The results were subjected to a linearizing transformation to compute analyte concentration. Recently, the development of a new technique representing the amalgamation of two high technology areas employing multilayer dry film slides and a prototype image analyzer based on a photodiode array demonstrated the feasibility of accurately and precisely quantitating glucose or urea on 0.5-1.5 pL of serum or plasma in which accurate pipetting was not required (4, 5). In order for this technique to be applicable in the clinical laboratory it was necessary to design and construct a new instrument specifically to meet the requirements for everyday use.
EXPERIMENTAL SECTION Instrument. A block diagram of the instrument is shown in Figure 1. Conceptually, portions were based, in part, on earlier systems designed for other applications (5-7). A line scan camera
was used that contained a linear photodiode array (PDA) which 1024 photodiodes, 15 pm wide, on 25.4 pm centers, with an aperature of 432 pm and a 55-mm camera lens (E.G.&G. Reticon, Sunnyvale, CA). The camera was used as received with the exception that the clock count was set to 2056 and the line scan time was adjusted to 30 ms. The video output signal from the Current address: Laboratory Service, VA Hospital, La Jolla, CA 92161. 0003-2700/84/0356-0742$01.50/0
PDA sample and hold circuitry was amplified by a differential operational amplifier to eliminate the dc offset and a second operational amplifier was used to amplify and buffer the signal. The 0 to +10 V video signal was digitized by a 12-bit analogto-digital converter (Model ADC 1102, Analog Devices, Norwood, MA). The digital signals were interfaced to a computer (Model 9845B, Hewlett-Packard, Palo Alto,CA), via a 16-bit1/0 interface. Direct memory access was used to acquire data from the PDA circuitry. All programming was done in Hewlett-Packard BASIC language. A block diagram of the optical configuration is shown in Figure 2. A xenon flashlamp (Model Sunpak Auto 611, Berkey Co. Woodside, NJ), with a remote light sensor was used. The flashlamp has a maximum light output of 4500 beam candle power seconds (BCPS) and produced an adequate amount of light over the visible spectrum that could be precisely measured by the photodiodes. Two light pipes constructed from polished polystyrene and covered with aluminum foil were used to control light direction. The first light pipe gathered and directed as much light as possible from the xenon flashlamp to an interference filter located in a filter wheel and the second light pipe directed the filtered light to the specimen area from a 45O angle with respect to the specimen plane to minimize front surface reflection. The PDA camera was positioned at a 90° angle and the reflected light was focused by the camera lens onto the PDA located within the camera. A stepping motor controlled filter wheel with eight different interference fiiters provided automatic wavelength selection. Four optical limit switches detected slots in the filter wheel edge to produce a unique 4-bit filter identification code. The first three bits were used for the filter code and one bit was used to detect the presence or absence of a filter. This output code was compared to a 4-bit output code from the computer. If the two 4-bit codes matched, the stepping motor remained stationary otherwise the stepping motor automatically rotated the filter wheel until the two 4-bit binary codes were identical. Three cavity interference filters with a 10-nm band-pass were used (Ditric Optics, Hudson, MA). Exposed photographic film of different optical densities served as neutral density filters in conjunction with some filters to provide coarse adjustment of light intensity. Fine adjustment of the xenon fashlamp output was digitally controlled by computer software commands. Eight parallel output lines from the computer to a D-to-A converter and its associated amplifier (Figure 3) produced an analog voltage proportional to digital input. Through a power FET this signal was used to control the light level in a light emitting diode located within an optical isolator (Model VTL5C, Vactec, Maryland Hts, MO). The other half of this optical isolator contained a photoresistor that was used to replace the photodetector within the xenon flashlamp remote light sensor. Resistance changes in the photoresistor controlled the flash duration, To obtain the maximum dynamic range for the PDA, the computer digitally selected the flash duration at each wavelength to provide as much light as possible without saturating any of the photodiodes. In the event that any photodiode became saturated, the xenon flash duration was decreased and the scan repeated. In addition, the optical isolator also protected the PDA and computer circuitry by isolating them from the high voltages in the xenon flashlamp. Also shown in Figure 3 is the trigger circuitry for the xenon flashlamp. To prevent the possibility of a series of highly repetitive pulses reaching the xenon trigger inputs, which was found to destroy the flashlamp circuitry, an optically isolated logic circuit was designed to allow the SCR to be triggered no more than once every 2 s. Two output lines from the computer were used. One output was used to enable 0 1984 Arnerlcan Chemical Society
ANALYTICAL CHEMISTRY, n
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Flgure 1. Block diagram of instrument.
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Flgure 2. Block diagram of optical system.
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Flgure 3. Simplified schematic diagram illustrating computer control of flashlamp emission and trigger circuitry.
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Flgure 4. Timing diagram for scan camera.
or disable the trigger circuitry. The second line provided a trigger pulse. A 6-v, 160 AH recharageable battery provided power for the flashlamp. The timing diagram for performing a single scan is shown in Figure 4. A request for data was initiated by the computer. The line scan time refers to the time from the start of one scan to the start of another and represents the total time period for light exposure by any single photodiode. At the beginning of a scan, each capacitor was recharged in sequential order from 1 to 1024. The photodiodes are continuously active and discharged by a photogenerated current at a rate proportional to local light intensity. After all photodiode capacitors have been recharged, the xenon flashlamp was triggered, and depending upon the amount of light required, the flash duration ranged from 0.4 to 9 ms. At the end of 30 ms each photodiode signal was sequentially integrated and retained by a sample and hold circuit to provide a sample-and-hold boxcar voltage output wave form with the voltage level being proportional to light exposure. A timing diagram in Figure 5 illustrates A-to-D conversion of the video signal for the fmt two photodiodes followed by computer handshake. The negative edge of the camera clock pulse triggered a one-shot output to produce a 3-ps output pulse. This provided
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
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Flgure 6. (A) Plot of digitized photodiode output vs. photodiode number for a scan of reference and sample spots, S 1, and scan of an area void of specimens, 52. (B) Plot of percent reflectance vs. photodiode
number for reference and sample spots. dark signals was found by making repeated scans and comparing the coefficients of variation for every 100th photodiode. After four scans the precision did not improve significantly. Therefore, the dark signals for all subsequent experiments were based on the average of four scans for dark signals. The average dark signals were stored as linear array
mi)
= ( a ) , d(2), 4n)) where n = number of photodiodes in one scan. Before any precision studies could be performed on colored spots, it was essential to develop algorithms for computing reflectance. This computation was complicated by variations in venon flashlamp emission from one scan to another and by nonuniform illumination of the entire specimen viewing area. A solution to this problem was to make two scans. The f i s t scan was made directly across the center of two spots and the second either was taken in the same location using a different wavelength or was manually repositioned so the scan was made in an area adjacent to the specimens. The dark signals were subtracted from each photodiode signal and the data from the first scan was stored in linear array S l ( i ) = ( s l ( l ) , s1(2), ..., s l ( n ) ) e..,
and the second scan stored as S2(i) = (s2(1), s2(2), ..., s2(n))
S1 and S2 are plotted in Figure 6A. To correct for variations in light output from the xenon flashlamp, the values in array S1 were porportionally adjusted to match array S2. The average values of 10 photodiodes on each end of the scan were used as reference photodetectors. Ll,, = ( C S l ( i ) ) / l O ; for i = 1 t o 10
Rl,, = ( E S l ( i ) ) / l o ; for i = n - 9 t o n L2,, = (CS2(i))/lO; for i = 1 t o 10 R2,, = (CS2(i))/lO; for
i = n - 9 to n
An increment was computed that accounted for proportional changes in light intensity from one edge of the scan to the other. increment = ((R2,,/RlaV) - (L2,,/LlaV))/n
To proportionally correct S1 to S2, for i = 1 to n S l ( i ) = S l ( i ) x ((L2,.,/L1,,) + (increment x i)) Percent reflectance was computed by %R(i)= (Sl(i)/S2(i)) x 100; for i = 1 t o n and these results are plotted in Figure 6B. A software routine was used to locate and compute the average reflectance for 70 photodiodes located across the center of each spot. Use of Spot Diameter to Estimate Sample Volume. A ~-FL syringe was used to apply different volumes of serum to glucose slides. Because of the precise geometric arrangement of the photodiodes in a linear array, PDAs have been used extensively in industry for noncontact optical measurements. By taking advantage of this capability, we can estimate the spot diameter by scanning the spots across their
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Figure 7. Plot illustrating the relationship between spot diameter on glucose slides in terms of the number of photodiodes between the spot edges at half the peak height vs. sample volume.
centers and determining the number of photodetectors between the spot edges at half the peak height. The relationship between spot diameter and sample volume is shown in Figure 7. The values are approximations since all spots are not perfectly round and the scan may not have been taken through the exact center. An estimate of sample volume provides useful information that was used to alert the operator that lower or upper volume limits have been exceeded or if the spot was scanned off center. Precision. Basic instrument precision was studied by making 20 repeated scans of stable colored spots of differing color and intensities. Since it was impossible to produce colored spots with a high degree of color uniformity, any movement in spot position would result in a scan of a different area and pigment distribution. Thus, the spots were allowed to remain stationary and scans were performed a t two wavelengths. A series of yellow, red, and blue spots were scanned at 4001540 nm, 540/640 nm, and 6401400 nm, respectively. The overall precision from 96% to 10% reflectance for all spots yielded a coefficient of variation of less than 0.5% whereas at reflectance levels of less than 10% the coeficient of variation increased to greater than 3% at 3% reflectance. Upon measurement of patient samples, repeated analyses (n = 20) of serum samples containing different levels of analyte concentrations yielded the following for glucose: X = 830 mg/L, coefficient of variation = 1.9% within run, and 2.6% day-to-day; X = 3350 mg/L, coefficient of variation = 2.7% within run and 3.5% day-to-day. Precision studies with fluid controls evaluated by Kodak and other testing laboratories were 2.1% of the glucose concentration within their dynamic range of 200 to 6250 mg/L (9). For BUN the following results were obtained X = 91 mg/L, coefficient of variation = 2.0% within run and coefficient of variation = 3.4% day-to-day; X = 610 mg/L, coefficient of variation = 4.7% within run and 5.5% day-to-day where % coefficient of variation = standard deviation/mean x 100. Precision studies performed by Kodak and other laboratories using fluid controls yielded 3.3% of urea concentration within their dynamic range of 20-1200 mg/L (10). Correlation Studies with a Commercial Instrument. Eighty serum samples were analyzed in parallel by the proposed system and a Beckman ASTRA automated system. Regression analysis for glucose studies revealed n = 80, y = 0.9810~+ 1.822, and r = 0.9956, and BUN yielded n = 80, y = 1.016~- 0.0992, and r = 0.9916 where concentration units for both were mg/L. Projections. The main limitation to this system is that the instrument is not commercially available. A less critical limitation is that the slides must be manually positioned in the instrument for scanning. Recent work on a second generation instrument using a square photodiode array capable of scanning the entire slide in less than 100 ms should eliminate the requirement for manual alignment. Earlier systems were limited to measurements above 500 nm because of the relatively low light emission by quartz halogen lamps a t lower wavelengths. Attempts were made
745
Anal. Chem. 1984, 56. 745-749
to extend the wavelength range by using higher wattage halogen lamps but they were also unsatisfactory because of inadequate emission at lower wavelengths and excessive heat production. A xenon flashlamp provided excellent emission over the entire visible spectrum that was controlled by the computer and produced no detectable heat. To further enhance sensitivity a PDA was chosen with a 432-pm aperature instead of the 25.4-pm aperature originally used (5-7). The Kodak Ektachem 400 is a commercially available system that uses multilayer dry slides but is not adequate for analyzing small quantities of serum samples since it has a pipet dead volume of 30 pL and requires an additional 10 pL for each test. Furthermore, additional serum must be present in the cup to prevent aspiration of air. In contrast, the proposed system uses blood collected and separated in a microhematocrit tube that provides up to 30 pL of serum or plasma. A 10-pL syringe is used to hold and protect samples from evaporation and provides a negligible dead volume. As demonstrated in earlier work ( 1 , 4 , 5 ) variations , in sample volume produce minimal changes in color intensity. Dispensing 2-pL samples from a 10-pL syringe is convenient and slight variations in pipetting volumes do not significantly alter the final results. Not all dry film chemistry slides were suitable because of poor spreading characteristics found in less sophisticated slides such as those produced by Ames that were based on a paper matrix. Placement of small serum quantities on these slides did not form distinct and uniform spots. Eastman Kodak slides work satisfactorly since they were designed to allow the sample to spread smoothly. Dry film slides from Boehringer Mannheim and Fuji Photo Film Co. were not available for evaluation. The principles set forth here open the way to performing multiple simultaneous analyses on a single slide. Performing ten analyses on a slide designed for a single test would be very cost effective and the savings would be significant. Improvements in multilayer dry film technology that result in
thinner slides would reduce the minimal sample requirements to only a fraction of a microliter and would allow a more dense sample application pattern. The ease of operation, small storage space required for slides, low power consumption, and small sample size would make the proposed system suitable for future use in a neonatal or adult intensive care unit and would avoid the costly and time-consuming sample transportation to the laboratory and problematical delivery of results to the ward. At this time ten differnet slide tests have been evaluated and are in the process of extensive clinical trials. Additional tests are becoming available. ACKNOWLEDGMENT The author wishes to thank Alfred Zettner for his invaluable sugestions and Walter Fontana for his expert technical assistance. Registry No. Urea, 57-13-6. LITERATURE CITED Curme, H. G.; Columbus, R. L.; Dappen, G. M.; et ai. Clin. Chem. (Winston-Salem, N . C . ) 1978, 24, 1335-1342. Walter, B. Anal. Chem. 1983, 55, 498A-514A. Ohkubo, A.; Kamel, S.;Yamanaka, M.; et al. Clin. Chem. (WinstonSalem, N . C . ) 1981, 27, 1287-1290. Neeley, W. E.; Zettner, A. Abstract presented at Second International Congress on Pedlatric Laboratory Medicine, Toronto, Ontario, Canada, June 1983. Neeiey, W. E.; Zettner, A. Clln. Chem. (Winston-Salem, N . C . ) 1983, 29, 2103-2105. Neeley, W. E.; Epstein, D.; Zettner, A. Clin. Chem. ( Winston-Salem, N . C . ) 1981, 27, 1665-1668. Neelev, W. E.: Zettner, A. Clin. Chem. (Wlnston-Salem, N . C . ) 1983, 29, 1038-1041. Wllllams, F. C.; Clapper, F. R. J . Opt. SOC.Am. 1953, 43, 595-599. Glucose Test Methodology, HSMD Publication MP2-8, Eastman Kodak Co., Rochester, NY, 1961. BUN/Urea Test Methodology, HSMD publication MP2-9, Eastman Kodak Co., Rochester, NY, 1981.
RECEIVED for review November 4,1983. Accepted December 23, 1983.
Selective Leaching of Trace Metals from Sediments as a Function of pH John H. Trefry* and Simone Metz Department of Oceanography & Ocean Engineering, Florida Institute of Technology, Melbourne, Florida 32901
Trace metals were leached from sediments and suspended particulates by using phthalate buffers at pH values of 2.2-6. Cadmlum, Cu, Fe, Mn, Pb, and Zn were determined In the resulting leachates by flame or flameless atomic absorption spectrometry. The fractlon of total metal removed varied wlth sample composition, flnal pH, and element determined. Analytical preclslon for the leach was generally 9 (21). Marine sediments commonly have pH values of 6-8 (21). In streams where mine 0 1984 American Chemical Society
