Recent advances in analytical methodology in clinical chemistry make use of reagents in a dry format for quantitative analysis. Providing a reagent in a dry format for rapid use is by no means new-a familiar example is litmus paper, which dates back to the 19th century. By introducing litmus, a colored extract from several lichens, into a paper matrix, the inventor provided a dry reagent chemistry for testing the alkalinity of a solution. This simple concept provided the user with a new dimension in routine analysis. Testing for alkalinity was made simple and rapid, and the dry reagent could be stored conveniently for later use. The litmus paper principle was the basis for other dry chemistries used in that century for qualitative analysis of glucose and other reducing sugars. Although not an analytical testing device, another noteworthy dry format chemistry that made its appearance in the 19th century was the photographic plate. It provided a dry format activated photochemistry capable of recording an object’s image from its reflected light. Unlike litmus paper, the plate required treatment with liquid reagents to prepare the image. Like litmus paper, however, it introduced a previously unrealized dimension of convenience to the art of reproduction and documentation. The first major impact dry format chemistries had on clinical testing was the appearance in the 1950s of Ames Clinistix urine reagent strips for testing urinary glucose. By comparing the color developed on the reagent strip with a color chart provided on the product label, the user got a rapid qualitative glucose analysis that otherwise would require a laboratory and skilled personnel. Clinistix reagent strips provided the groundwork for other reagent strips developed by Ames and other manufacturers for testing urine constituents. The appearance of Ames Dextrostix reagent strips in the 1960s for the semiquantitative analysis of blood sugar propagated the development of dry format 498A
chemistries for testing hlocd constituents. With the later introduction of instrumentation, the era of quantitative clinical analysis for glucose with reagent strips began. In the 19709, more sophisticated dry chemistries emerged for quantitative analysis of blood constituents. Whereas many of the clinical test formats available share the common trait of requiring reconstitution of reagents prior to use, either manually or automatically, the emerging reagents have a totally new format. In all cases, a complete chemistry is miniaturized into a dispensable dry format. No prior reconstitution of reagents is required, and many manipulations are replaced simply by applying the sample. An analysis is complete in 1-7 min. For simplicity, I will refer to these analytical elements as dry reagent chemistry carriers. Each such element contains all the chemical constituents required for a specific clinical analysis. The clinical sample is applied directly onto the carrier and, as with other chemistries, the analysis is usually monitored by instrumentation. The development of dry reagent chemistries is the cumulation of several technologies. These technologies provided crucial knowledge on how to prepare quantitative chemistries in dry media such as thin films, paper matrices, and other synthetic porous materials. They also provided knowledge on how to cast thin films of desired porosity with high precision, how to make paper matrices and other porous matrices reproducibly with welldefined characteristics, and how to laminate various materials to each other. The photographic industry has made substantial contributions in advancing the technology of dry reagent chemistries (I).With the appearance of color photography, the industry had developed a technology that is based on conducting quantitative chemistries in discrete films arranged in multiple layers. This eventually resulted in instant photography. An in-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Figure 1. Basic components of dry reagent chemistry carriers
stant color print may have as many as 15layers of film, each with a specific chemical or physical function to perform in developing the photographic image. This very technology was used to develop several dry reagent chemistry formats for clinical testing. The coating and plastic industry provided a variety of techniques for precision casting of thin films (2),and it has developed lamination techniques frequently required for bonding various materials together. It also developed a variety of inert materials used in constructing dry reagent chemistries. The paper and fabric industries have developed techniques for making fibrous matrices with reproducible parameters to fita variety of unique needs (3). Some of the parameters that can be controlled easily include matrix thickness, fiber density, and solvent absorbency, which are important in generating dry reagent chemistries. Contributions from these industries led to the evolution of dry reagent chemistries that can be adapted to meet any need in the clinical laboratory. The main focus of this text will be to describe some basic features governing the structure of these carriers, how chemical reactions are monitored, and what advantages these carriers provide for clinical chemistry analysis. 0003-2700/83/0351-498A$01.50/0 0 1983 American Chemical Society
Report Bert Waiter Blood Chemistry Laboratwy
Ames Division, Miles Laboratories, Inc. Elkhart, Ind. 45615
-Film
Reagent Layer Matrices
-Paper -Synthetic ~
Fibers
Nonfibrous Porous Materials
Separation Membrane Masking Layer I Trapping LayerFlgure 2. Basic constituents used in constructing carrier reagent zones
Basic Structure of Dry Reagent Carriers The common features of most dry reagent chemistry carriers monitored by diffuse reflectance methods are a support material, a reflective zone, and a reagent zone. This is illustrated schematically in Figure 1. In some carriers these three features are distinctly defined; in others, the reflective zone may coalesce with the reagent zone or the support zone, or both. The support material usually consists of a thin, rigid plastic or a plasticlike material that may be transparent or reflective. The support material serves as a building base for the reagent chemistry carrier. The major function of the reflective zone is to reflect to a detecting system any light not absorbed by the chemistry of the carrier. The reflective zone is usually con. strueted with pigments such as TiOz or BaS04 or reflective materials such as metal foils. Where paper constitutes the reagent zone, the paper matrix itself acts as a reflective layer. The primary requirement for the reflective material is that its absorbance of light in the visible range and, if possible in the UV, be negligible. The reagent zone assembly contains all the reagents for a specific chemis-
try. This zone may be constructed with a paper matrix, a synthetic fiber matrix, film layers, other porous non fibrous materials, or a hybrid of thes The reagent zone of the carrier is the most complex region of the carrier. I1 addition to the detecting chemistry, this zone may provide several additional functions as shown in Figure 2 It may include a separation membrane, a masking layer, or a trapping layer. Two distinct approaches have been used to construct the layers containing the assay reagents. One approach makes use of film casting techniques. The layers are constructed by casting a porous film containing all the required assay components. For example, if the constituents have to be segregated before use. the reagents may be deposited in several films. Hence, a reagent zone may be constructed of multiple film layers, each containing portions of a specific detection chemistry. Separation membranes and trapping layers also can he introduced as necessary between reagent layers. The cross-sectional thickness of such a carrier may be less than 100 Km. The quantities of reagents made available during analysis are controlled hy their concentration in the casting medium, the thickness of the cast film, and
*
Contributions from several technologies have led t o the development o f sophisticated testing devices for clinical use. The technology of making dry reagent chemistries is sumciently advanced that carriers can be developed for almost any chemical analysis. The dry reagent chemistry carrier contains all the reagents required to conduct an analysis. Separation steps required by conventional analysis are integrated into the analytical element. This offers several advantages to the user. The most obvious is the reduction of multiple-step procedures to one step. Only the sample need be applied to reconstitute the system and start the analysis. Hence, the skill of the user i5 less important. The waste of reconstitute but unused solutions is eliminated, providing for more efficient and cost-effectiveuse of reagents. The small size of the carriers means little space is required for storage. Since the dry reagent chemistries are stored dry until use, their she1 life is prolonged beyond reconstituted conventional che istries. The advantages provi by dry reagent chemistries wil
-
-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4. APRIL 1983
49BA
ration solution, the thickness of the matrix, the fiber density of the matrix, the absorptivity of the solution by the fibers, and the solubility of the reagents in the sample solvent. Typical thicknesses of reagent zones constructed with these matrices range from 0.2-0.5 mm. The porosity of the reagent zones may he further controlled by introducing a membrane layer on top of the paper matrix. T o affix these matrices to other surfaces, various lamination techniques are employed, the most common of which make use of double-sided adhesive layers or a mesh overlayer to anchor the matrices to a given surface.
b
1
gure S. Principle of
I
reflectance photumesy
sourc Dispersing Medium Selecting Aex
A ..(Fluorescence) -
Dispersing-
A ex
(Reflected Excitation Light)
Figure 4. Principle of front-face fluorescence analysis
their solubility in the sample solvent. Film matrices commonly consist of such polymers as cross-linked gelatin, agarose, alginate, polyvinylalcohol, polyvinylpyrrolidone, and cellulose acetate. The porosity of the film is controlled by such factors as the molecular weight of the polymer, the degree of cross-linking, and the concentration of the polymer in the casting medium (4). The second approach to constructing a reagent layer involves the use of a paper or a synthetic fiber matrix. The paper matrices commonly employed consist of pure a-cellulose 500A
with uniform fiber density and defined thickness. Reagents are introduced by saturating the matrix with reagent solutions followed by drying. Reagents can be introduced in a onestep saturation process or in multistep saturation processes. T o prevent perturbation of reagents already deposited in multistep saturations, differential solubility techniques are used. The solvents for successive deposition of reagents are chosen so that reagents already laid down are not redissolved. The quantity of reagents made available during an assay is controlled by the reagent concentration in the satu-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Instrumentation The chemical reactions occurring on dry reagent carriers unually are monitored by diffuse reflectance spectroscopy (5) and, to a lesser extent, by fluorescence (6). In discussing reflertance spectroscopy, two kinds of reflections must be considered. One is specular reflection, which is the mirrorlike reflection from a surface where the angle of incidence is equal to the angle of reflection. This type of reflection is of limited value in monitoring dry reagent chemistries. The second is diffuse reflection, which is a reflection from a matte surface. This is the predominant reflection of interest. Diffuse reflection may result from illumination of the reaction volume of the carrier with diffuse light or diffusion of light in the illuminated reaction volume. Diffuse reflectance is not a surface phenomenon, but the result of light interacting with various chemical and physical factors in the reaction volume of the carrier. These factors include the absorption, transmission, and scattering properties of the illuminated material. Figure 3 illustrates the general principle of reflectance spectroscopy. The reaction volume of the carrier, where a chromophore is either generated or degraded, is illuminated at a suitable wavelength. The amount of diffuse light recovered with the aid of the reflective layer is a measure of the progress of the reaction. The intensity of light reflected by the reaction media is determined relative m a known reflectance standard. The commonly used percent reflection (%R) can be expressed as:
%R = I .x R. 1,
where I,, I., and R, represent the reflected light from the reagent carrier, the reflected light from the reference, and the percent reflectivity of the reference, respectively. The reflectance measurements are comparable to a transmittance measurement in absorption spectroscopy, where the con-
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XenonFlasn lube +Detector
I
I
Reagent Pad
I
Instrument Table
Figure 3. woss-sectional diagram ur the Seralyzer reflectance photometer (An,==
Division, Miles Laboratories, Inc.)
centration of the absorbing component can be measured. As with transmittance, reflectance is not linear with concentration, To make the measurement useful and convenient, several algorithms are available to convert reflectance measurements to functions that are linear with concentration (7,8). The specific algorithm employed depends on the nature of the illumination, the reflection characteristics of the dry reagent carrier, and the geometry of the instrument. Reactions resulting in the appearance or disappearance of fluorescence are monitored hy front-face analysis (9) in which irradiation and fluorescence are from the same surface of the carrier. The general principle of frontface fluorescence analysis is illustrated in Figure 4. For irradiation, a specific wavelength, dictated by the chemistry, is selected by passing light through a filter or light-dispersing device such as a monochromator. With the aid of the carrier’s reflective layer, a portion of the total light (fluorescence diffuse reflected excitation light) is collected by the detection system. The angle of irradiation is chosen so as to minimize specular reflection of the excitation light into the detector. The detection system segregates fluorescence from residual reflected excitation light by a filter or monochromator placed hetween the detector and the reaction surface. This allows the transmission of the fluorescence light only. Unlike reflectance, measured fluorescence is linear with fluorophore concentrations in the absence of self-quenching. Instrumentation for dry reagent carriers ranges in size from hand-held dedicated devices to large freestanding instruments. Many of the hand-
+
502A
held instruments are designed to monitor a specific dry reagent chemistry. They are microprocessor controlled, allowing the user to store calibration curves and to time the chemical reaction on the carrier. They also provide the user with a clinical value after a measurement. Examples are the Glucometer reflectance photometer (Ames Division, Miles Laboratories, Inc.), the Accu-Chek hG (Boehringer Mannheim Corporation), the EasyTest (Australia Bio-Transducers), and the Hypo-Count IIB (Hypoguard, Ltd.) used in whole-blood glucose analysis. For more general testing, bench top manual instrumentation is available for handling more than one chemistry. Figure 5 illustrates the basic components of one such instrument, the Seralyzer reflectance photometer (Ames Division, Miles Laboratories, Inc.). The instrument requires that each chemistry and sample be introduced individually onto the instrument’s table by the operator. Each test has a specific module that contains part of the computer memory for the test, the algorithm, and the optical interference filter for that test. Analysis time may range from 30 s to 4 min, depending on the nature of the chemistry. After initial Calibration for each chemistry of interest, a specimen can he tested for several analytes without recalibrating test modules. The instrument’s microprocessor maintains control of several operations including temperature, timing of reactions, calculations, and display of clinical results. In addition, the system alerts the user to malfunctions and operator errors. The instrument can he updated easily as new test modules and dry
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
reagent chemistries are made available. Fully automated instrumentation is also availahle as exemplified by the Ektachem 400 Analyzer (Eastman Kodak). The operator need only provide the samples and select the desired tests. The instrument automatically selects the reagent carrier from a cassette, calibrates the selected chemistry, applies the sample, and reports the results. I t is capable of more than 500 tests per h and can hold 1600 dry reagent chemistries. The instrument can he programmed to run any of 16 currently available tests and can be updated to run additional tests as they become available. The system reports malfunctions, diagnoses prohlems, and tells how the operator can correct them.
Dry Reagent Chemis4tries In Clinical Analysis Dry reagent chemistries have been described for a variety of blood analytes. These include serum metabolites, enzymes, and serum electrolytes as well as therapeutic drugs. Many of these chemistries are available on the market and provide a unique approach to conducting quantitative analysis of serum analytes. Each dry reagent chemistry provides an integrated assay for a specific analyte that requires only the application of the serum sample. The structure and chemistry of a few representative carriers will be described to illustrate how several conventional steps have been integrated into a single-step procedure to Drovide a new conceDt in clinical anhysis. Blood Metabolites. Drv reaaent chemistries are presently available for nearly all the commonly tested blood metabolites. These include glucose, blood urea nitrogen, uric acid, cholesterol, triglycerides, creatinine, bilirubin, ammonia, and calcium. Analysis of many of these metabolites by conventional means requires several steps that are done either manually or by automation. The integration of several steps into a single-step analysis is exemplified by the dry reagent chemistries developed for whole-blood glucose analysis. The cross sections of two such carriers developed hy Boehringer Mannheim Corporation (BMC) and Ames Division, Miles Laboratories, Inc., are illustrated in Figures 6a and b, respectively. Glucose is detected by a glucose oxidase-peroxidase procedure. In both cases, approximately 50 pL of whole blood is applied to the surface of the carrier (approximately 0.5 cm X 1 cm), where plasma containing glucose is separated from red blood cells by the carrier matrix. After an allotted reaction
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I I I I
p
support
Layer
.
Chemistry: Glucose 2H,O,
+ 0, + 2H,O
Glucose Oxidase
-
+ IndicatortRe,u~,lPeroxidase
+ 2H,O,
*Gluconic Acid 4H,O
+ Indicatort,,x,d,,,l
Figure 6. Cross section and chemistry of carriers for whole-blood glucose analysis, developed by (a) Boehringer Mannheim, Inc. and (b) Ames Division, Miles Laboratories, Inc.
;ample Mount Sample Containing BUN
Isspread Uniformly
BUN
+ H,O
ZNH,
+ CO,
Urease F z
Excludes OH- from Reagent Layer 2
H-Indicator (Colorless)
Indicator(Color)
+ NH,+ NH,'
Dure 7. Cross section and chemistry of Eastman Kcdak Ektachem slide for bloc ea analysis time (usually 1-3 min), the red blood cells are removed by washing or wiping, and the color developed is analyzed and translated to blood glucose concentrations. Both carriers consist of a support material that also incorporates the reflective zone. Both devices depend on a film layer to exclude red blood cells from the reagent zone and quantitatively meter a sample volume. In one case, the reagent zone is part of the film matrix (BMC); in the other, the reagent zone consists of 504A
a paper matrix with a membrane. Both devices have reduced the blood glucose analysis from a laborious effort of removing red blood cells, either by centrifugation or by precipitation prior to analysis, to one simple step. Separation steps or immobilized catalytic centers also can be integrated into carriers at any reaction step of an analysis. An example of a dry reagent chemistry employing a separation step to isolate a product is the multilayer film chemistry (Ektachem
ANALYTICAL CHEMISTRY, VOL. 55. NO. 4, APRIL 1983
slide) developed by Eastman Kodak for hlood urea analysis. The cross section of the carrier is illustrated in Figure 7 (IO). The carrier consists of a transparent support material, a reflective layer, and two reagent layers (film matrix) separated by a semipermeable membrane. A 1O-pL sample of undiluted serum is applied to the reflective layer, which also acts as a spreading layer, tu meter a uniform reaction volume. As the sample enters the first reagent layer, the urea is converted to ammonia and COz by the enzyme urease. The semipermeable membrane acts as a barrier to hydroxyl ions and allows the diffusion of the ammonia into the second reagent layer where it deprolunates a pH indicator. The reaction is over in 7 min. The color developed is monitored by a reflectance meter from below the carrier, and the results are expressed as concentration of blood urea. The dimensions of the dry reiyent carrier are 2.8 X 2.4 cm. with an application region of less than 0.8 cm2. The thickness of the carrier is less than 100pm. Among the carriers incorporating Catalytic centers is the dry reagent strip for blood urea analysis developed for lhe Seralyzer system by Ames. The croys section of the carrier is illustrated in Figure 8. The carrier cunsists of a support that incorporates a reflective zone and a paper matrix reagent layer that contains uniformly distributed cation exchange centers. Upon application, the sample is distrihuted into the reagent zone via capillary action: this sulubiliees the reagents. The blood urea reacts witho-phthalaldehyde to produce 1.3-dihydroxyisoindoline (DHI). The cation exchange centers catalyze the coupling of DHI to 3-hydroxy-1.2.3,4-tetrahydrubenzoth)quinidine (HTBQ) to form a chromogen. The rate of color development, monitored by reflectance from above the carrier, is then converted into blmd urea concentration units. The analysis requires 30 pL of solution after a threefold dilution of a serum sample with wawr (IO p L of undiluted serum is required per assay). The dimensions of the carrier are 0.5 X 1.0 cm. with a thickness of less than 0.5 mm. Enzymes. As with serum metabolites, sophisticawd dry reagent chemistries also are available for the analysis of serum enzymes. Dry reagent chemistries have been descrihed for the analysis of creatine kinase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, n-amyl. ase, 1 -glutamsltransferase, and alkaline phosphatase. Some of these carriers are available on the market. De. termining enzyme activity in serum often involves complex and lengthy chemistries where several enzyme nj-
F STORAQE
.
ii
.
SAFETY CODE
CLOMWC
NOTHING COULD BE SIMPLER Laboratories have long dep
on the Fisher reagent label its clear-cut purity grades, dt analyses, applicable OS1 DOT. and ANSI data, first ai, instructions. Now this distinguished label becomes more useful than ever. Now it will also carry the Fisher ChemAlert Guide. ChemAlert is an instant guide to storing and handling the reagent in question. A useful supplement to your regular safety program. It condenses a wealth of authoritative material into a uniquely simple three-part format.
U One part tells you, with a large uncluttered color bar, how to store the reagent. (For the color-blind, this bar also identifies itself by letter. i.e., for flammable storage it uses an "R" as well as the color red.)
0 One part rates potential hazards numerically, inside a big bold NFPA "diamond," a symbol selected for its universality. (In fact. many industries. hospitals. and municipalities now require it.)
I
0 One part demonstrates how to handle the reagent. What protective gear to wear. Whether to use a fume hood.
CIRCLE 53 ON READER SERVICE CARD
Right on the label - there's no more logical place to remind users - old and new - of the correct way to deal with chemicals. ChemAlert was designed to do this reminding in the most effective way possible. Watch for it on your familiar Fisher reagents. Use it. forsafety's sake. For a detailed brochure on new ChemAlert. circle our number on the reply-card. Or give us a call at (412) 562-8543.
Fisher Scientific An &LLIEU
Company
n
P
Matrix: Paper Reagents: 1. ophthaialdehyde 2. HTBQ 0 3. Cation Exchange
UN
0
Hi
+ o-phthaiaidehyde
DHi
Cation Exchange + HTBQ-Chromoge center
0
Figure 8. Cross section and chemistry of the Ames Division, Miles Laboratories, Inc.. Seralyzer dry reagent strip for blood urea analysis
Sample CrP + ADP Creatine
Matrix: Paper Reagents:l.Creatine Phosphate (CrP) 2Adenine Diphosphate (ADP)
3.Glucose 4. Hexokinase (Hx) 5. Nicotinamide Adenine Dinucleotide Phosohate (NADP+) 6.Giucose-Ephosphaie Dehydrogenase (G%PDH)
I
Cr
Kinase ' + Adenine Triphosphate (ATP)
ATP
Hx + Glucose +
ADP
+ GiucoseB-phosphate (G-6-P)
G4-p + NADP+
GB-PDH 6-Phosphogiuconate+ NADPH
Figure 9. Cross section and chemistry of the Ames Division, Miles Laboratories, Inc., Seralyzer dry reagent strip for creatine kinase analysis says must be coupled in order to conduct an analysis. The creatine kinase ICKJ determination develoned for the Seralyzer system by Ames & an example of a multiple-coupled-enzyme dry reagent strip chemistry. The cross section of the carrier is illustrated in Figure 9. The carrier consists of a paper matrix reagent zone fixed onto a reflective support. The chemistry is based on the Rosalki procedure ( 1 I), which couples a hexokinase and a glucose-6-phosphate dehydrogenase reaction to the ATP produced by creatine kinase to generate a creatine kinase assay. This results in the generation of NADPH that can be monitored at 340 nm by reflectance spectroscopy and converted into enzyme concentration units. The dry reagent carrier is 0.5 X 1.0 cm and 0.2 mm thick and re506A
quires 30 rrL of solution derived from the ninefold dilution of a serum specimen (3.3 VI.of undiluted serum is required per assay). The assay duration is 4 min, which includes a two-andone-half-minute lag period. Masking is another technique that allows several assay steps to be combined in an enzyme analysis. This procedure excludes undesirable absorhtion of linht - bv.reaeents " in the chemistry during an analysis. This is readily exemplified by an Ektachem slide for a-amylase analysis developed by Eastman Kodak. The cross section of such a carrier is illustrated in Figure 10. The element consists of a spreading layer containing a dye-bound amylopectin substrate followed by a masking layer to hide the dye. The masking layer also incorporates the reflective
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
~
zone. Next to the masking layer is the registration layer, which rests on a transparent support. When a serum sample is applied to the carrier, the resulting hydrolysis of the substrate generates small molecular weight dyebound starch units. Starch units containing fewer than 10glucose units migrate through the masking layer into the registration layer where they become trapped. The dye is quantitated by reflectance spectrmopy at 540 nm. The quantity of dye appearing in the registration layer after a given t,ime is proportional to the a-amylase concentration in the sample, and the results are expressed as enzyme concentration units. During the reaction, neither the enzyme nor the large molecular weight dye-bound substrate can diffuse into the registration layer. The masking layer separates and hides the dye-bound amylopectin from the instrumental measurement. This dry reagent chemistry requires 10 NL of an undiluted serum sample per analysis. The carrier is 2.8 X 2.4 cm, with an application region of less than 0.8 cm2. Electrolytes. Dry reagent chemistries developed for electrolyte analysis are based on ion-selective membranes using either electrochemical or spectroscopic principles for analysis. As with metabolites and enzyme chemistries, dry reagent chemistries for electrolytes are also small, disposable, and require a small sample volume. Figure 11 shows a diagram of the electrodes found in an Ektachem slide for K+, a dry reagent electrochemistry carrier developed by Eastman Kodak for measuring serum K+ concentrations (12). An ion-selective membrane is used to construct a K+-specific electrode. The electrode consists of a support base and a silver and silver chloride layer that serves as the reference electrode. The salt bridge consists of KCI deposited in a film. The K+-selective membrane, consisting of the ionophore valinomycin in a hydrophobic medium, resides on top. The dry reagent carrier consists of two such electrodes joined by a bridge, where only a portion of the ion-selective membrane is exposed. The sample is deposited in one opening and a reference KCI solution of known concentration is placed in the other. The K+ concentration is determined from the potentiometric difference measured between the two electrodes in the carrier. Unlike conventional electrode measurements, the sample is referenced directly to a standard solution. The dimensions of the carrier are 2.8 X 2.4 cm, with a thickness of 150 &m requiring only 10 pL of sample and reference solution for a K+ analysis. Similar dry reagent electrochemistries have been described for Na+, CI-, and CO2 analysis.
An
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The Fisher/Alnsworth SC-200. It may be the one electronic balance that can tempt you to replace your triple-beams. Unlike them, it has no bouncing beams, no sliders, no dials to twist. no weiahts to iuaale. Yet it has the same ruggedduyability as'ilhe classic triple-beam,for day-in, day-out weighing. Plus a very affordable price tag. Consider the convenience. Just tap the toggle to turn it on; tap again for one-second taring, up to 49.9 grams. Weigh samples from 0.1 to 200 grams in two seconds flat. And with 20.1 gram precision - thanks to the electronic filter that corrects automatically for environmental variables. including vibration. Examine the extras. The SC-200is -so itnalight and compact, you can take
And it operates on a single 9-volt battery, so it's independent of the power lines. Perfect for quality control right on the production line or weighing in the field. When you're finished, it turns itself off automatically to conserve the battery. Now, enjoy the price. Just $349 for a full-featured electronic balance that can do the job of three mechanicals. In less time. And with absolutely no fuss. Concerned about capacity? Meet the SC-2000. Same speed, portability and reiiability as the SC-200.But with 2000 gram capacity, precisionto ?1 gram. And only $449. Weigh all the facts. For latest literature, just check with your Fisher Representative; 'rcle our number on the reply card; or call (412)562-8543.And learn how these iuick. UncomDlicated balances can Lspeed your everyday weighing chores. I
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scientific An &ED ON R E A M R SERVICE CARD
Company
, ..Nonpolar Phase -ADA
pAmylase
Containing the lonophore Valinomycin
Starch-Dye Fragments
.Allows Diffusionof Small StarCh-Oye Fragments
.Anionic Starch-Dye + CHPComplex
--Mount
Figure 10. Cross section and chemistry of Eastman Kodak Ektachem slide for a-amylase analysis
Sample Electroc Sample Well
-
4 >Containing Valinomycin, Potassium Chloride FilmSilver Chloride Layer Silver Layer Support Layer
A
Figure 11. Diagram of Eastman Kodak Ektachem slide for potassium analysis by
electrochemical means Figure 12 illustrates the cross section of a dry reagent chemistry developed by Ames Division. Miles Lahoratories, Inc.. for Kf determination by spectroscopic methods (13).The carrier consists of a support base with a reflective zone and an ion-selective membrane. The membrane consisrs of a nonpolar phase containing the ionophore valinomycin. A drop of sample mixed with an anionic dye is placed on the membrane. The ionophore transports K + into the nonpolar phase at a rate proportional to the K + concentra tion. T o maintain charge neutrality, a 508A
dye molecule comigrates. At the end of the reaction period, the drop is removed by washing, and the K+ concentration is determined from the quantity of dye in the nonpolar phase as measured by reflectance spectroscopy. The dimensions of the carrier are 1.0 X 0.5 em and 100 pm thick. Only 15 p L of a serum sample is required per analysis. Therapeutic Drugs. A more recent advance in dry reagent chemistries bas been the development of carriers for the analysis of therapeutic drugs in serum. Figure 13 illustrates the cross
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Figure 12. Cross section and chemistry of the Ames Division, Miles Laboratories, Inc., dry reagent strip for potassium analysis by reflectance photometry section of a dry reagent strip developed by Ames Division, Miles Laboratories, Inc., for the detection of the antiasthmatic drug theophylline in serum (14). The chemistry is a competitive protein-binding assay based on the substrate-labeled fluorescence immunoassay (15).Upon placing the sample on the carrier, a compbtition for antibody-binding sites is established between the serum theophylline and the theophylline conjugate. The unbound conjugate remaining is proportional to the theophylline in the sample. The conjugate is monitored by fluorescence after the removal of the galactose moiety by the action of &palactosidase. The conjugate, antibody, and enzyme are introduced in the paper matrix by differential solubility techniques. This prevents the premature interaction of the conjugate with the antibody or enzyme, yet provides a single-step, homogeneous assay. The dose response for such a carrier is illustrated in Figure 14,where the increase of fluorescence intensity is proportional to the theophylline concentration. Similar dry reagent chemistry
I
. .
Your scientists will never be emptyhanded.. .
if you rely on FASTBACK: It's our computerized orderinghnventory control system that took 15 years and millions of dollars to develop. We use it to handle more than half-abillion dollars in sales a year. Now it's available as an invaluable service to Fisher customers. FASTBACK is the sure-fire way to keep your inventory current - month after month, year after year. You have access to more than 80,000items. And our on-line computer system instantly gives you up to the minute information on availability, quantity and price. Your data terminal may already be suitable to plug into the FASTBACK system (if not, you can purchase the proper equipment inexpensively from Fisher). Place orders via your teleprinter and simplify recordkeeping with instantaneous printouts. Forget about ordering errors, too FASTBACK will immediately signal any mistake in catalog number, size, etc. Whether you need glassware, reagents, instruments, furniture, apparatus or whatever, the FASTBACK system ties you into our central computer with access to all 28 strategically located Fisher distribution centers (and 33 million cubic
feet of lab supplies). Delivery IS guaranteed to be fast and dependable. If storeroom space is a problem, take advantage of our Customer Reserve Quantity (CRQ) program in conjunction with FASTBACK. When you make a long-term order, we reserve the merchandise exclusively for you in the Fisher warehouse closest to your lab. You can forget about backorders - and empty-handed scientists. FASTBACK is available as a service to Fisher customers at various levels of sophistication. You can benefit whether you're a crack computer expert or just starting out. For more information, just contact your Fisher rep or nearest branch. Or call (412)562-8543.It's that simple.
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/Sample
1EMPERATURE CONTROLFOR LC COLUMNS Temperature control in quid chromatography has enerally been neglected. nproved resolution, effi. iency, and precision often 3sult from operation of LC olumns at controlled tern. eratures above ambient.
I Matrlx: Paper Reagents: 1. Antibody for Theophylline (i 2. @-Galactosidas (&Gal) 3. &GalactosylUmbelliferone Theophylline (@-Gut) 4. Buffer pH 8.3
+
Ab + @-Gut Theophylllne (T) + Ab-(@-Gut)+ Ab-T @-Gut &Gut-
@-Gal
+
UrnbelliferoneTheophylline (Fluorescent)
I
'Iective Support
gure 13. Cross section and chemistry of the Ames Division, Miles Laboratories, :., dry reagent strip for theophylline analysis rriers have been described for the tertiun of antitiiotics (gentamirin, iikarin. and tobramyrin) and anti. nvulsant drugs (carhamazepine, tenytoin. and primidone).
wforrnance
Bioanalytical System Dffers a low cost columi heating system based on metal block heater ani solid state controller. Tht versatile controller can b, used with other h e a t h elements and probes for variety of laboratory appl Eations
The fate uf any new technology in e r l i n i r a l chemistry cummunity dends on its performanre vis-&vir esthlished methodologies. Although ace does not permit the comparisun e w r y dry reagent chemistry with e variuur established methodologies. 'ew representative examples will ilitrate that prrformanre of dry reent chemistries is comparable t') eshlished procedures. 'Cable I summa:es the performan(e o f the Eastman jdak Kktachem slide for glurose alysis and compares i t tu the perfur3nre of the I h Puiit Automatic inical Analyzer (ACAJ.a highly re. rded cumparative system. W i t h the nical decision level for upper nor31 being 120 mg,dL. the dry reagent rrier shows a dynamic range of ' -625 mg/dL glucose. Correlating the 'tachem slide with the ACA using i0 rlinical samples yields a regression ie . y 1 ; ~ = ~ 0.9dG6xA,-n ~ ~ h ~ ~ -O.% g/dL, a standard error uf 4 . 1 mg/dL, id a rorrelation rueffirient 01'0.999
6).
BlOA NA LYTIC1 SYSTEMS INC.
Purdue Research Park 1205 Kent Avenue West L a f a y e t t e . IN 47906
(317) 463-2505
D r y reagent chemistries for rlinical ialysis of enzymes also demonstrati, ,reptable performance. Tahle II immarizes thr pertormance uf the ~ a l y i e dry r rraycnt strip for C'K inlysis. developed by Ames, and Nmptlres i t to the performanre o f the u Punt ACA iur C'K. W i t h a clinical !cisiun level fiir upper nurmal nf iuut 100 I'iL at 37 T,the dry re. lent ;trip has 3 dynamic range of I000 IJiL of enzyme Correlating le CK drv reagent cheinistry with a
>IRCLE 31 ON READER SERVICE CAI
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 4. APRIL 1983
i
Figure 14. Dose-response curve for theophylline generated with Ames Division, Miles Laboratories, Inc., reagent strips for theophylline
commercially available automated analyzer using 87 clinical samples yields a regression line ylesgentstrip = 0.98xR-lri 5.5 WL, a standard error of 16 UIL, and a correlation coefficient o f 0.999. D r y reagent chemistries for therapeutic drug analysis also compare favorably w i t h established methodologies. Table I11 summarizes the performance o f Ames dry reagent strips for theophylline determination and compares it t o Syva's Enzyme Multiplied Immunoassay Technique-antiasthmatic drug (Emit-aad) procedure for
+
c1 I
areyoarbpi4 solidorgas samples? Fisher's Automatic K-F litrimeteP provides the answers quickly and dependably. Performs precise volumetric moisture determinations with pushbutton ease. Results accurate to 1%. System uses microprocessor-controlleddigital buret and amperometric endpoint detectionto provide sensitinty of ZOFg H20. Features proportional endpoint approach. Automatic endpoint confirmation. Pushbutton solvent delivery and purging of waste from reaction vessel. Standard Karl Fischer reagent boffles attach directly without transfer. You can also use pyridine-freetitrant (Hydranal'). You're prolecled. Effectively sealed system safeguards operator and lab from toxic, noxious vapors. Universal use. The Karl Fischer method is a world standard for moisture testing of a great variety of materials including: Chemicals Plastics Petroleumderivatives Cosmetics Textiles Pharmaceuticals Foods Tobacco Paper products Fisher applications engineers have developed specific time-saver moisture proceduresincluding: Amines. Butter and margarine. Grain alcohol. Quench oils. Pharmaceuticaltablets. Polyester resins. Tall oil. And more. Yours for the asking. Flexible optlons. Simply plug-in our pre-programmed Cakulator/Printer accessory for enhanced automation, documentation, automatic correction for parasitic reactions. Other modules open up a full range of titration capabilities. You're supported. Design and manufacture by Fisher means you gel immediate help when it's needed: equipment, Merature, technical backup, supplies,service. As close as your Fisher branch. 7iydmalisrsgutereo Vademarkol Riedeldeliaiin A 0
For further information, circle reply card number shown, or phone (412) 562-8543 Circle 56 for literatwe Circle 57 fw a demoMlratitiD"
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Table 1. Comparison of Precision of the Dry Reagent Chemistry Ektachem for Glucose with the Du Pont ACA RKuon (%CY
olucor
-
x loo)
IYl1-n
472.4 I
-
20
0.8
Gluoose test memodolog,lw Du Pcni ACA
rabie ii. Comparison of Precision of the Seraiyrer Dry Reagent Strips for CK with the Du Poni ACA Sram w y z e r dry reagent strip for C . - -
cf*.1w
R n w o n (%CV
58 158 357 58 158 357 161 684 133 680
-
8DIIn.M x D."40.d."
I
mthh M
180 180 177
60
6.3 3.1 2.6 -
60 59 20 20
1 . 1
-
-
3.2 4.4
kku(U/L)
-
20 20
loo)
-
6.5 3.4 2.8
* A m Division. Miles Labwmwies. inc.. product padrage I mlw CK feagem swips W test mmadoiooy tu Du Pont ACA
I Table 111. Comparison of Precision of Dry Reagent Strips for Theophylline Analysis with Syva's Emit-aad nnaphyllln
SWM
Syva Emn for meophylllne
(WmU 5.0 15.0 25.0 5.0 15.0
(I
30 30 30 10 10
R.*Mn (%CY = solnun x loo) mtMM D.Y-lO4.Y
8.4 3.8 3.6
25 0
10 ..
-
10.0 7.5 25.0
20
