Dry Reagent Chemistries 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
chemistries for testing blood constituents. With the later introduction of instrumentation, the era of quantitative clinical analysis for glucose with reagent strips began. In the 1970s, 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 (1). 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-
498 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
ReagentZone
Reflective Zone Support Layer
Figure 1. Basic components of dry reagent chemistry carriers
stant color print may have as many as 15 layers 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 fit a 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 © 1983 American Chemical Society
Report Bert Walter Blood Chemistry Laboratory Ames Division, Miles Laboratories, Inc. Elkhart, Ind. 45615
in Clinical Analysis Film Reagent Layer Matrices
Paper •Synthetic Fibers Nonfibrous Porous Materials
Separation Membrane Masking Layer Trapping Layer Figure 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 car riers these three features are distinctly defined; in others, the reflective zone may coalesce with the reagent zone or the support zone, or both. The sup port material usually consists of a thin, rigid plastic or a plasticlike ma terial that may be transparent or re flective. 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 ab sorbed by the chemistry of the carrier. The reflective zone is usually con structed with pigments such as T1O2 or BaSC>4 or reflective materials such as metal foils. Where paper consti tutes the reagent zone, the paper ma trix itself acts as a reflective layer. The primary requirement for the re flective material is that its absorbance of light in the visible range and, if pos sible 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 nonfibrous materials, or a hybrid of these. The reagent zone of the carrier is the most complex region of the carrier. In addition to the detecting chemistry, this zone may provide several addi tional functions as shown in Figure 2. It may include a separation mem brane, 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 re quired assay components. For exam ple, if the constituents have to be seg regated 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 chem istry. Separation membranes and trapping layers also can be introduced as necessary between reagent layers. The cross-sectional thickness of such a carrier may be less than 100 μνα. The quantities of reagents made available during analysis are controlled by their concentration in the casting medium, the thickness of the cast film, and
Contributions from several technologies have led to the development of sophisticated testing devices for clinical use. The technology of making dry reagent chemistries is suffi ciently advanced that carriers can be developed for almost any chemical analysis. The dry reagent chemistry carrier con tains all the reagents required to conduct an analysis. Separa tion steps required by conven tional 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 o n e step. Only the sample need be applied to reconstitute the system and start the analysis. Hence, the skill of the user is less impor tant. The waste of reconstituted but unused solutions is elimi nated, providing for more effi cient and cost-effective use 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 shelf life is prolonged beyond re constituted conventional chem istries. The advantages provided by dry reagent chemistries will help ease the burden of quality control in operating labora tories.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983 • 499 A
Detector
Source I r (Diffuse Reflected Light) dent
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 re agents in the sample solvent. Typical thicknesses of reagent zones con structed with these matrices range from 0.2-0.5 mm. The porosity of the reagent zones may be further con trolled by introducing a membrane layer on top of the paper matrix. To affix these matrices to other surfaces, various lamination techniques are em ployed, the most common of which make use of double-sided adhesive layers or a mesh overlayer to anchor the matrices to a given surface.
Reflective Reaction Volume
Instrumentation
Figure 3. Principle of reflectance photometry
Detector
Source Dispersing Medium Selecting λ ex
\ e m (Fluorescence)—i
A
i
Dispersing" Medium Selecting λ em
λ ex (Monochromatic Excitation Light)
A ex
(Reflected Excitation Light)
Reflective Reaction Volume
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 ac etate. The porosity of the film is con trolled by such factors as the molecu lar weight of the polymer, the degree of cross-linking, and the concentration of the polymer in the casting medium (4). The second approach to con structing a reagent layer involves the use of a paper or a synthetic fiber ma trix. The paper matrices commonly employed consist of pure α-cellulose
with uniform fiber density and de fined thickness. Reagents are intro duced by saturating the matrix with reagent solutions followed by drying. Reagents can be introduced in a onestep saturation process or in multistep saturation processes. To prevent per turbation of reagents already deposit ed in multistep saturations, differen tial 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 avail able during an assay is controlled by the reagent concentration in the satu
500 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
The chemical reactions occurring on dry reagent carriers usually are moni tored by diffuse reflectance spectros copy (5) and, to a lesser extent, by flu orescence (6). In discussing reflec tance spectroscopy, two kinds of re flections must be considered. One is specular reflection, which is the mir rorlike reflection from a surface where the angle of incidence is equal to the angle of reflection. This type of reflec tion 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 pre dominant reflection of interest. Dif fuse reflection may result from illumi nation 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 illu minated material. Figure 3 illustrates the general principle of reflectance spectroscopy. The reaction volume of the carrier, where a chromophore is ei ther generated or degraded, is illumi nated at a suitable wavelength. The amount of diffuse light recovered with the aid of the reflective layer is a mea sure of the progress of the reaction. The intensity of light reflected by the reaction media is determined relative to a known reflectance standard. The commonly used percent reflection (%R) can be expressed as: %R = γ X R r tr where I s , I r , and R r represent the re flected light from the reagent carrier, the reflected light from the reference, and the percent reflectivity of the ref erence, respectively. The reflectance measurements are comparable to a transmittance measurement in ab sorption spectroscopy, where the con-
Xenon Flash Tube
Detector Microprocessor Test Module Instrument Keyboard
Reagent Pad
Instrument Table
Figure 5. Cross-sectional diagram of the Seralyzer reflectance photometer (Ames Division, Miles Laboratories, Inc.)
centration of the absorbing compo nent can be measured. As with transmittance, reflectance is not linear with concentration. To make the measure ment useful and convenient, several algorithms are available to convert re flectance measurements to functions that are linear with concentration (7, 8). The specific algorithm em ployed depends on the nature of the illumination, the reflection character istics of the dry reagent carrier, and the geometry of the instrument. Reactions resulting in the appear ance or disappearance of fluorescence are monitored by front-face analysis (9) in which irradiation and fluores cence 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 be tween 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 freestand ing instruments. Many of the hand
held instruments are designed to mon itor a specific dry reagent chemistry. They are microprocessor controlled, allowing the user to store calibration curves and to time the chemical reac tion 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 bG (Boehringer Mannheim Corporation), the EasyTest (Australia Bio-Transduc ers), and the Hypo-Count IIB (Hypoguard, Ltd.) used in whole-blood glu cose analysis. For more general testing, bench top manual instrumentation is available for handling more than one chemistry. Figure 5 illustrates the basic compo nents of one such instrument, the Ser alyzer 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 be tested for several analytes without recali brating test modules. The instru ment's microprocessor maintains con trol of several operations including temperature, timing of reactions, cal culations, and display of clinical re sults. In addition, the system alerts the user to malfunctions and operator errors. The instrument can be updat ed easily as new test modules and dry
502 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
reagent chemistries are made avail able. Fully automated instrumentation is also available as exemplified by the Ektachem 400 Analyzer (Eastman Kodak). The operator need only pro vide the samples and select the de sired tests. The instrument automati cally selects the reagent carrier from a cassette, calibrates the selected chem istry, applies the sample, and reports the results. It is capable of more than 500 tests per h and can hold 1600 dry reagent chemistries. The instrument can be programmed to run any of 16 currently available tests and can be updated to run additional tests as they become available. The system re ports malfunctions, diagnoses prob lems, and tells how the operator can correct them. Dry Reagent Chemistries in Clinical Analysis
Dry reagent chemistries have been described for a variety of blood ana lytes. These include serum metabo lites, enzymes, and serum electrolytes as well as therapeutic drugs. Many of these chemistries are available on the market and provide a unique ap proach to conducting quantitative analysis of serum analytes. Each dry reagent chemistry provides an inte grated assay for a specific analyte that requires only the application of the serum sample. The structure and chemistry of a few representative car riers will be described to illustrate how several conventional steps have been integrated into a single-step procedure to provide a new concept in clinical analysis. Blood Metabolites. Dry reagent chemistries are presently available for nearly all the commonly tested blood metabolites. These include glucose, blood urea nitrogen, uric acid, choles terol, triglycerides, creatinine, biliru bin, ammonia, and calcium. Analysis of many of these metabolites by con ventional means requires several steps that are done either manually or by automation. The integration of several steps into a single-step analysis is ex emplified by the dry reagent chem istries developed for whole-blood glu cose analysis. The cross sections of two such carriers developed by Boehringer Mannheim Corporation (BMC) and Ames Division, Miles Lab oratories, Inc., are illustrated in Figures 6a and b, respectively. Glucose is de tected by a glucose oxidase-peroxi dase procedure. In both cases, ap proximately 50 μL of whole blood is applied to the surface of the carrier (approximately 0.5 cm X 1 cm), where plasma containing glucose is sepa rated from red blood cells by the car rier matrix. After an allotted reaction
(a)
(b) Sample
RBC + Glucose
iiUumiHA! m Matrix: Film Membrane
Matrix: Porous Film (Alginate) Reagents: 1. Glucose Oxidase 2. Peroxidase 3. Redox Indicator
Reagent Zone
Support Layer
Reflective Zone
Matrix: Paper Reagents: 1. Glucose Oxidase 2. Peroxidase 3. Redox Indicator
Reflective Zone
Chemistry: Glucose + 0 2 + 2H 2 0
G UC0Se0xidaSe
'
2H 2 0 2 + l n d i c a t o W c e d ) ^
> Gluconic Acid + 2H 2 0 2 * 4H 2 0 + lndicator(0xJdized)
Figure 6. Cross section and chemistry of carriers for whole-blood glucose analy sis, developed by (a) Boehringer Mannheim, Inc. and (b) Ames Division, Miles Laboratories, Inc.
slide) developed by Eastman Kodak for blood urea analysis. The cross sec tion of the carrier is illustrated in Fig ure 7 (10). The carrier consists of a transparent support material, a reflec tive layer, and two reagent layers (film matrix) separated by a semipermeable membrane. Α ΙΟ-μί, sample of undi luted serum is applied to the reflective layer, which also acts as a spreading layer, to meter a uniform reaction vol ume. As the sample enters the first re agent layer, the urea is converted to ammonia and C 0 2 by the enzyme ure ase. The semipermeable membrane acts as a barrier to hydroxyl ions and allows the diffusion of the ammonia into the second reagent layer where it deprotonates a pH indicator. The re action is over in 7 min. The color de veloped 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 reagent carrier are 2.8 X 2.4 cm, with an application region of less than 0.8 cm 2 . The thickness of the carrier is less than 100 μπι.
Figure 7. Cross section and chemistry of Eastman Kodak Ektachem slide for blood urea analysis
Among the carriers incorporating catalytic centers is the dry reagent strip for blood urea analysis developed for the Seralyzer system by Ames. The cross section of the carrier is illus trated in Figure 8. The carrier consists of a support that incorporates a reflec tive zone and a paper matrix reagent layer that contains uniformly distrib uted cation exchange centers. Upon application, the sample is distributed into the reagent zone via capillary ac tion; this solubilizes the reagents. The blood urea reacts with o-phthalaldehyde to produce 1,3-dihydroxyisoindoline (DHI). The cation exchange centers catalyze the coupling of DHI to 3-hydroxy-l,2,3,4-tetrahydrobenzo(/i)quinidine (HTBQ) to form a chromogen. The rate of color develop ment, monitored by reflectance from above the carrier, is then converted into blood urea concentration units. The analysis requires 30 μί, of solu tion after a threefold dilution of a serum sample with water (10 μ!., 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.
time (usually 1-3 min), the red blood cells are removed by washing or wip ing, and the color developed is ana lyzed and translated to blood glucose concentrations. Both carriers consist of a support material that also incor porates the reflective zone. Both de vices 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
Enzymes. As with serum metabo lites, sophisticated dry reagent chem istries also are available for the analy sis of serum enzymes. Dry reagent chemistries have been described for the analysis of creatine kinase, lactate dehydrogenase, aspartate transami nase, alanine transaminase, α-amyl ase, γ-glutamyltransferase, and alka line phosphatase. Some of these car riers are available on the market. De termining enzyme activity in serum often involves complex and lengthy chemistries where several enzyme as-
Sample BUN
Mount Sample Containing BUN Is Spread Uniformly
Spreading and Reflective Layer
Reagent Layer 1 BUN + H20 Urease , pH 8.0 ' 2NH, + CO,
Matrix: Porous Film Reagents: 1. Urease 2. pH 8.0 Buffer Semipermeable Membrane: Permeable to NH
Excludes OH from Reagent Layer 2
Reagent Layer 2 Matrix: Porous Film Reagent: pH Indicator Transparent Support
H-lndicator + NH3 (Colorless) Indicator" + NH, ' (Color)
a paper matrix with a membrane. Both devices have reduced the blood glucose analysis from a laborious ef fort 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 integrat ed into carriers at any reaction step of an analysis. An example of a dry re agent chemistry employing a separa tion step to isolate a product is the multilayer film chemistry (Ektachem
504 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
—Sample
BUN
Reagent Layer Matrix: Paper Reagents: 1. o-phthalaldehyde • 2. HTBQ · • · 3. Cation Exchange j ,_ BUN + o-phthalaldehyde • Centers
• •
·
·
· •
•
· ·
·
· •
-· ·
· ·
-i-,-,^. Cation Exchange _, Ξ—yChromogen Center
DHI + HTBQ
· ·
·
_
·
. DHI
Reflective Support
· ·
Figure 8. Cross section and chemistry of the Ames Division, Miles Laboratories, Inc., Seralyzer dry reagent strip for blood urea analysis
-Sample Creatine Kinase Reagent Layer Matrix: Paper Reagents: 1.Creatine Phosphate (CrP) 2. Adenine Diphosphate (ADP) 3. Glucose 4. Hexokinase (Hx) 5. Nicotinamide Adenine Dinucleotide Phosphate (NADP + ) 6. Glucose-6-phosphate Dehydrogenase (G-6-PDH)
CrP + ADP
C eatine
: > Kinase Cr + Adenine Triphosphate (ATP)
ATP + Glucose ·
Hx
ADP + Glucose-6-phosphate (G-6-P)
G.6.p
+
NADP+
G 6 PDH
- -
>
6-Phosphogluconate+ NADPH
Reflective Support
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 con duct an analysis. The creatine kinase (CK) determination developed for the Seralyzer system by Ames is an exam ple of a multiple-coupled-enzyme dry reagent strip chemistry. The cross sec tion of the carrier is illustrated in Fig ure 9. The carrier consists of a paper matrix reagent zone fixed onto a re flective support. The chemistry is based on the Rosalki procedure (11), which couples a hexokinase and a glu coses-phosphate dehydrogenase reac tion 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 concentra tion units. The dry reagent carrier is 0.5 X 1.0 cm and 0.2 mm thick and re-
quires 30 μ!., of solution derived from the ninefold dilution of a serum speci men (3.3 μι, of undiluted serum is re quired 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 com bined in an enzyme analysis. This pro cedure excludes undesirable absorp tion of light by reagents in the chemis try during an analysis. This is readily exemplified by an Ektachem slide for α-amylase analysis developed by East man 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 mask ing layer to hide the dye. The masking layer also incorporates the reflective
506 A ·• 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 con taining fewer than 10 glucose units migrate through the masking layer into the registration layer where they become trapped. The dye is quantitated by reflectance spectroscopy at 540 nm. The quantity of dye ap pearing in the registration layer after a given time is proportional to the α-amylase concentration in the sam ple, 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 registra tion layer. The masking layer sepa rates and hides the dye-bound amylopectin from the instrumental mea surement. This dry reagent chemistry requires 10 μϋ, 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 cm 2 . Electrolytes. Dry reagent chem istries developed for electrolyte analy sis are based on ion-selective mem branes using either electrochemical or spectroscopic principles for analysis. As with metabolites and enzyme chemistries, dry reagent chemistries for electrolytes are also small, dispos able, and require a small sample vol ume. 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 + concentra tions (12). An ion-selective membrane is used to construct a K + -specific elec trode. The electrode consists of a sup port base and a silver and silver chlo ride layer that serves as the reference electrode. The salt bridge consists of KC1 deposited in a film. The K + -selective membrane, consisting of the ionophore valinomycin in a hydrophobic medium, resides on top. The dry re agent carrier consists of two such elec trodes 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 KC1 solu tion 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 con ventional electrode measurements, the sample is referenced directly to a stan dard solution. The dimensions of the carrier are 2.8 X 2.4 cm, with a thick ness of 150 μτα requiring only 10 μι, of sample and reference solution for a K + analysis. Similar dry reagent elec trochemistries have been described for Na+, CI - , and C 0 2 analysis.
Sample icMmytdse
, Α Nonpolar Phase Containing the lonophore Valinomycin
Spreading and Reagent Layer Matrix: Porous Cellulose Acetate-Ti0 2 Film Reagent: Anionic Dye Linked to Amylopectin (ADA)
α-Amylase -ADA »· Starch- Dye Fragments
Masking and Reflective Layer Matrix: Low-Porosity Gelatin Film Containing \Mhiia
D a m a n t
_Allows Diffusion of Small Starch-Dye Fragments
Registration Layer Reagent: Cationic Hydrophobic Polymer (CHP)
_Anionic Starch-Dye + CHP *• Complex
, Sample Solution Is Mixed with Anionic Dye Reagent. A Drop of This Solution Is Placed on the Test Device lonophore Binds Potassium Specifically
Transparent Support -Mount
x Dye
Enters the Nonpolar Phase to Neutralize the Charge
Figure 10. Cross section and chemistry of Eastman Kodak Ektachem slide for α-amylase analysis The Liquid Drop Is Removed
Reference Solution Well
Sample Electrode
Sample Well
Reference Electrode
Reflectance of the Bound Dye Is Measured
Bridge Hydrophobic Film Containing Valinomycin Potassium Chloride F i l m Silver Chloride Layer—Silver Layer Support Layer
Potentiometer
Figure 11. Diagram of Eastman Kodak Ektachem slide for potassium analysis by electrochemical means
Figure 12 illustrates the cross sec tion of a dry reagent chemistry devel oped by Ames Division, Miles Labora tories, Inc., for K + determination by spectroscopic methods (13). The car rier consists of a support base with a reflective zone and an ion-selective membrane. The membrane consists of a nonpolar phase containing the ionophore valinomycin. A drop of sample mixed with an anionic dye is placed on the membrane. The ionophore trans ports K + into the nonpolar phase at a rate proportional to the K + concentra tion. To maintain charge neutrality, a
dye molecule comigrates. At the end of the reaction period, the drop is re moved by washing, and the K + con centration is determined from the quantity of dye in the nonpolar phase as measured by reflectance spectros copy. The dimensions of the carrier are 1.0 X 0.5 cm and 100 ftm thick. Only 15 μΐ., of a serum sample is re quired per analysis. Therapeutic Drugs. A more recent advance in dry reagent chemistries has been the development of carriers for the analysis of therapeutic drugs in serum. Figure 13 illustrates the cross
508 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Figure 12. Cross section and chemistry of the Ames Division, Miles Laborato ries, Inc., dry reagent strip for potassi um analysis by reflectance photometry
section of a dry reagent strip devel oped by Ames Division, Miles Labora tories, Inc., for the detection of the antiasthmatic drug theophylline in serum [14). The chemistry is a com petitive protein-binding assay based on the substrate-labeled fluorescence immunoassay (15). Upon placing the sample on the carrier, a competition for antibody-binding sites is estab lished between the serum theophylline and the theophylline conjugate. The unbound conjugate remaining is pro portional to the theophylline in the sample. The conjugate is monitored by fluorescence after the removal of the galactose moiety by the action of /3-galactosidase. The conjugate, antibody, and enzyme are introduced in the paper matrix by differential solubility techniques. This prevents the prema ture interaction of the conjugate with the antibody or enzyme, yet provides a single-step, homogeneous assay. The dose response for such a carrier is il lustrated in Figure 14, where the in crease of fluorescence intensity is pro portional to the theophylline concen tration. Similar dry reagent chemistry
Sample Theophylline
TEMPERATURE CONTROL FOR LC COLUMNS Temperature control in liquid chromatography has generally been neglected. Improved resolution, efficiency, and precision often result from operation of LC columns at controlled temperatures above ambient.
OPTIONAL PREHEATER
ANOMZED ALUMINUM HEATING BLOCK
