Chemical Amplification in Analysis: A Review W. J. Blaedel" Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53 706
R. C. Boguslaski immunochemistry Laboratory, Ames Research and Development, Miles Laboratories, inc., Eikhart, Indiana 465 14
Amplification is tradltionally conceived to be performed In analysis by means of electronics, after the measurement step. However, amplification may also be performed chemically, before the measurement step. Such methods are beginnlng to appear Increasingly on the analytical scene. By Imparting catalytic or cycllc functions to analyte molecules, they may be determined with great sensitivity. Selectivity is obtalned through the use of enzyme or antibody reagents. Through the use of polymer and protein modification reactions, these ampllflcation systems may be localized into very small volumes or onto surfaces, giving promise of microscale technlques far below currently employed conventlonal techniques. It is slgnlflcant that most of these methods have been invented In the life science and cllnlcal science areas, In response to the special analytlcal needs of workers in those areas. There is now the need for input by analysts-for analytical research-to extend and to apply these new chemistries to all disciplines, wherever chemical measurements are involved.
Chemical amplification consists of reacting a substance with a reagent through a catalytic, cycling, or multiplication mechanism, to generate a relatively large amount of product. In this way, in principle, a trace concentration of an analyte may be caused to yield order of magnitude higher product concentrations which, for analytical purposes, may be more easily measured than the analyte itself. A catalyst is a substance which affects the rate of a chemical reaction without itself being consumed in the reaction. The catalyst may be measured by its effect on the reaction rate. For analytical purposes, the catalyst may be determined by letting a product of the catalytic reaction accumulate under controlled conditions until it becomes measurable (Figure 1). In principle, since it is not consumed, a catalyst may be determined with infinite sensitivity, limited in practice only by the rate of the reaction, and by interferences. Substances can also be determined that are themselves not catalysts, but that influence the rate of a catalytic reaction. In various ways, it is possible to cast some substances into a cycling role in a carefully designed reaction sequence. If the cycling is efficient, and if the substance is not lost from the reaction system, it may be determined with a sensitivity approaching that for a catalyst. Until now, the use of catalysis and cycling reactions for analytical purposes has been extremely limited. Recently, however, there have been great advances in the chemical modification of surfaces and macromolecules (1). These modification techniques have produced substances with unique analytical potential, since they permit the introduction of chemical functions to surfaces and molecules which extend their analytical capabilities. Enzyme catalysts may now be localized at the surface of ion selective electrodes, conferring upon them high selectivity for the determination of specific substances, such as glucose, urea, uric acid, and many others 0003-2700/78/0350-1026$01 .OO/O
(2). Also, catalytic activity may be introduced into a variety of molecules that are not themselves catalytic. For example, enzymes have been covalently linked to antibodies, which are proteins that can recognize a single substance specifically, even in the presence of structural or stereoisomers. These chemically modified antibodies form a new class of highly selective and sensitive reagents that can locate subcellular structures in tissue sections, and that can quantitate nanomolar levels of drugs and hormones in body fluids. These unique and ingenious reagents have led to methodologies that are a t the frontiers of analytical research. The purpose of this article is to describe and to classify the kinds of chemical amplification methods that are presently or potentially within our grasp, with the hope that they will be developed and applied to some of the vexing analytical problems that demand ever-increasing sensitivity and selectivity.
AMPLIFICATION BY CATALYSIS Under controlled conditions, including defined concentrations of the reagents, the rate of appearance of products is related to the catalyst activity, and may be taken as a measure of the catalyst concentration. For very low catalyst concentrations, a long time may be taken to permit the products to accumulate to measurable levels. There are of course practical limitations to the lowest catalyst concentration that can be determined: (1)A finite rate of the uncatalyzed reaction may give product levels too high to permit accurate measurement of the product formed from the catalyzed reaction. (2) Product impurities or other catalytic substances may give a blank too high t o permit accurate measurement of the product formed from the catalyzed reaction. Inorganic Catalysts. A classical example of amplification by catalysis is the determination of iodide, which catalyzes the slow reaction between ceric and arsenious ions: 2Ce( IV)
+ As(111)
I-+
2Ce( 111)
+ As( V )
(1)
Under controlled conditions and selected initial concentrations of Ce(1V) and As(III), the rate of disappearance of Ce(1V) (measurable spectrophotometrically or by other means) is related to the iodide concentration. The mechanism involves several steps and several intermediate oxidation states of iodine. Amounts of iodide approaching the 10-ng level may be determined by the method. There are interferences, among which are ruthenium and osmium, which also catalyze the reaction. A good number of other inorganic catalysts (mostly metallic elements, but also some nonmetallic elements) may be determined in ways similar to that for iodide ( 3 , 4 ) . The thyroid hormones thyroxine (T4)and triiodothyronine (T3)can be made to catalyze Reaction 1,through loss of some of the iodine via an oxidation reaction. An assay system for these hormones in blood serum based on the cerate-arsenate reaction has recently been reported (5): it permits the determination of as little as 0.2 ng of T, and 0.1 ng of T4. Numerous other organic and inorganic substances have pronounced inhibitory or activating effects on chemilumi0 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Figure 1. Concept of catalysis
REGION FOR ENZYME DETERMINATION S-K,
I
> REGION FOR SUBSTRATE DETERMINATON S e K ,
SUBSTRATE MOLARITY, S [L
Flgure 2. Dependence of velocity of an enzyme catalyzed reaction upon substrate concentration. (At a constant level of enzyme activity)
nescent reactions. These effects have been used to determine submicromolar levels of metal ions, hydrogen peroxide, potassium ferricyanide, aminoacids, hemes, and organophosphorus compounds (6, 7), down to nanomolar concentration levels. Enzyme Catalysts. Analytical Properties of Enzymes. Determination of Substrates (2,4,8,9). Enzymes are protein catalysts for reactions that occur in living systems. A general mechanism for the reaction of a reagent (substrate, S)in the presence of an enzyme (E) to give a product (P) is:
E + S % E S Gk P + E k2
The rate of disappearance of substrate, or of appearance of product is
(3) or
(4) In Equation 4, V, represents the maximum velocity achievable a t high substrate concentrations, and K , is the Michaelis constant, that substrate concentration for which the velocity is equal to half of V,. Figure 2 shows the dependence of the reaction rate upon substrate concentration, together with the parameters V , and K,. The outstanding analytical characteristic of enzymes is their chemical selectivity. Some are almost specific. For example, glucose oxidase catalyzes the oxidation of P-D-glucose by oxygen almost exclusively. A study of 60 oxidizable sugars and their derivatives showed that only one, 2-deoxy-D-glucose, was oxidized a t a rate comparable to that for @-D-glucose.All of the other sugars are oxidized at rates less than a few percent of the rate a t which P-D-glUCOSe is oxidized. Even the optical isomer, a-D-glucose, is oxidized a t a rate less than 1%of the
1027
rate a t which P-D-glucose is oxidized. Urease, which catalyzes the hydrolysis of urea to ammonia is stated to be even more selective than glucose oxidase. On the other hand, there are many enzymes that react with classes of substrates, rather than single compounds. D-Aminoacid oxidase catalyzes the oxidation of most D-aminoacids-from tyrosine, which is oxidized most rapidly, to histidine, which is oxidized a t only a few percent of the rate at which tyrosine is oxidized. A most important analytical application of enzyme catalyzed reactions is the determination of substrate concentration. Usually, S is best determined at concentrations below K,, and a t high enzyme concentration for maximum sensitivity (see Figure 2 and Equation 4). Enzyme catalyzed reactions have also been used to determine substances which activate or inhibit the enzymes. Under properly controlled conditions, and usually at high substrate concentrations, enzyme catalyzed reactions are used widely to determine the activity of the enzymes themselves (see Figure 2 and Equation 3). The use of enzyme based assays for the determination of substrates, inhibitors, activators, and enzymes themselves in research is enormous. However, the routine use of these assays in the clinical analytical laboratory to provide diagnostic information on the health of humans and animals is perhaps even greater. A typical example of substrate determination is the measurement of ethanol in blood serum. Simple chemical analytical methods for ethanol are insufficiently selective. Enzymatically, however, ethanol may be made to react with the cofactor nicotinamide adenine dinucleotide (NAD) in the presence of the enzyme alcohol dehydrogenase (ADH):
Ethanol
+ NAD'
ADII __+
pyruvate
+ NADH + H"
(5)
The reduced form of NAD can be determined with great sensitivity spectrophotometrically (with a detection limit below 1 pM,by measuring the absorbance a t 340 nm). NAD may also be determined electrochemically (10), and fluorometrically, down to about 10 nM. In practice, the sample containing the ethanol is mixed with controlled amounts of NAD' and ADH in a buffered solution, and the reaction is permitted to occur for a fixed time, or to go to completion. The NADH produced is measured and taken as an estimate of the ethanol content of the sample. In this way, submicromolar concentrations of ethanol may be determined in blood serum (11-13). Enzymatic substrate determinations do not involve amplification techniques. Rather, the advantage that the analyst derives from their use lies in the conversion of a difficultto-measure analyte (alcohol in Equation 5 ) to a product (NADH in Equation 5 ) than can be more sensitively and more selectively determined than the original substrate. Determination of Enzymes ( 2 , 4 , 8 , 9). Since an enzyme is a catalyst, it is determinable by amplification, provided that a substrate or product is measureable. The estimate of the enzyme activity must be based on a measure of the rate of reaction. The reaction is permitted to proceed under controlled conditions, including controlled concentrations of the reagents, and the measured rate is taken as proportional to the enzyme activity. Conventionally, for ease and simplicity, the amount of product built up over a fixed time is measured and taken as proportional to the rate and therefore to the enzyme activity, or the time required to produce a fixed amount of product is measured and taken as inversely proportional to the reaction rate and therefore to the enzyme activity. Enzyme activity is seldom expressed in conventional concentration units, but rather in terms of the amount of substrate used or product produced per unit of time (Le., micromoles per minute) under carefully controlled conditions. The measurement of the serum level of the enzyme lactate dehydrogenase (LDH) is very useful for differentiating chest
1028
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8 , JULY 1978
ACTIVE) SITE
+(DRUG)
COVALENT ONDlNG
+I
ENZYME
ENZYME LYSOZYME
i
MORPHINE
\
W)\
ENZYME
LYSOZYME LABELLED MORPHINE
1-
\
ENZYME IS INACTIVE
Flgure 3. Preparation of the enzyme labeled drug conjugate
pain due to myocardial infarction from other sources of chest pain. LDH catalyzes the conversion of pyruvate to lactate, with NAD as a coenzyme:
pyruvate
+ NADH + H+2L-lactate + NAD'
(6)
At controlled pH and at fixed concentrations of pyruvate and NADH, the activity of the enzyme is directly proportional to the rate of decrease in the absorbance of NADH at 340 nm. Serum levels of many other enzymes are measured similarly (11-1 3). Enzyme Immunoassays. In an enzyme immunoassay (EIA), an analyte or an antibody to an analyte is covalently linked to an enzyme, and is used in some form of the specific binding reaction to measure the analyte concentration in a sample. The EIA relies on a binding protein for specificity, and on the enzyme label for sensitivity through amplification. In principle, the EIA is applicable to the quantitation of a great variety of substances with high sensitivity in biofluids and tissues (14). The enzyme multiplied immunoassay technique (EMIT) is one type of EIA that is capable of high sensitivity and high selectivity. In the EMIT, a sought-for analyte (X) competes with a known amount of a specially prepared conjugate reagent composed of analyte covalently linked to an enzyme (E-X) for a limited number of binding sites on an antibody. At equilibrium, the enzyme-labeled conjugate is distributed between two forms-an antibody-bound form, and a free form:
Ab'X-E bound
+ X + X-E + Ab*X
(7)
free
A necessary requirement for the EMIT assay is that the degree of enzyme activity expressed by the bound and free forms of the conjugate be greatly different, so that they may be easily distinguished. Under the competitive binding condition, the higher the level of X in the sample, the higher will be the level of free X-E present at equilibrium. Since the bound and free forms of the conjugate have different degrees of enzyme activity, there is no need to separate them prior to conducting an enzyme assay to determine the level of free X-E. This is a marked advantage over the radioimmune assay. The estimation of morphine in urine is illustrative of a typical EMIT assay, and in fact is available in kit form for routine use. The requirements for preparation of the enzyme labeled conjugate, schematically illustrated in Figure 3, are quite stringent. Likewise, the antibody to morphine must be specially prepared in animals (15). In this particular assay, the labeling enzyme is lysozyme, which catalyzes the hydrolysis of certain components in bacterial cell walls, causing them to lyse (i.e., to break down). The free morphine lysozyme conjugate can also catalyze the lysis, but the antibody complex with the enzyme labeled morphine is virtually inactive enzymatically. The competitive binding reaction on which the EMIT assay is based is depicted simplistically in Figure 4. The free malyte morphine in the sample competes with the enzyme labeled morphine for a limited number of binding sites. At equi-
ENZYME IS ACTIVE
Figure 4. Enzyme immunoassay for morphine (EMIT)
ISITE
ANTIGENIC
ENZYME LABELLED ANTIBODY
Figure 5. Schematic representation of staining with an enzyme labeled antibody.
librium, the higher the morphine content of the sample, the greater the concentration of free (non-antibody bound) enzyme labeled morphine in the mixture. The free enzyme labeled morphine may be quantitated by its lysing action on a bacterial cell suspension ( M . lysodeikticus) which is measurable spectrophotometrically by a decrease in turbidity. Over-all, the higher the drug concentration in the sample, the greater the lysis, and the greater the decrease in turbidity per unit time in the bacterial cell suspension. Submicromolar concentrations of morphine in urine have been determined by this assay. A number of other drugs and drug metabolites have also been determined by this technique. EMIT assays for thyroxin and digoxin possessing subnanomolar sensitivities have been reported (16, 17). In another older form of EIA, the enzyme linked immunosorbent assay (ELISA), it is not necessary that the free and antibody bound forms of the enzyme labeled conjugate have different degrees of activity, because these forms are separated prior to enzyme assay. Here, analyte and enzyme labeled analyte compete in binding to an antibody immobilized on a solid phase. At equilibrium, the antibody bound form may be easily separated from the free form by filtration or centrifugation, thereby permitting a determination of the level of enzyme activity in either separated phase. The amount of enzyme activity in the free form (i.e., not bound to antibody immobilized on the solid phase) is proportional to the analyte concentration in the sample. Various modifications of this technique have led to assays with sensitivities at the nanomolar level for a number of different substances (e.g., estradiol and hepatitis B, antigen). A recent report describes an EIA for the measurement of attomole mol) levels of ornithine &aminotransferase (18). Staining Techniques with E n z y m e Labeled Antibodies. Just as analytes may be labeled with enzymes, so may antibodies capable of binding with specific chemical entities (antigenic sites within a specimen) be covalently linked with
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8 , JULY 1978
= -KETOGLUTARATE+,"$
x
GLUTAMATE
I
f NADPH
\
NADP+
x I
t
G6PDH
f, 6-P-GLUCONATE C6PG)
\
GLUCOK-6-PkDSFHATE CG6P)
Figure 6. Amplification of NADP by cycling
enzymes. In fact, the localization of normal and abnormal components within the cellular structure of tissue specimens was one of the first uses of enzyme labeled antibodies. As illustrated in Figure 5 , a particular antigenic site can be detected in a specimen fixed to a microscope slide by exposing the specimen to an antibody that has been labeled with an enzyme. The enzyme labeled antibody combines at the specific antigenic site, inimobilizing the enzyme at that site. Upon development with appropriate substrate and dye solutions, the immobilized enzyme catalyzes the conversion of soluble substrate and dye in a developer solution into insoluble colored reaction products which are deposited at the antigenic site. Observation may be made with a light microscope or, if the deposit is electron dense, an electron microscope may be used. These staining procedures are highly selective and sensitive, being capable of detecting 0.1-1 ng/mm2 of antigen in a properly prepared tissue section (19,ZO). They require only a few hours to perform, and have been widely applied in the fields of microbiology, pathology, histology, and immunology. They have been used to detect viruses, bacterial antigens, and abnormal proteins in cells. They have also been used to localize normal cellular constituents, such as enzymes, structural proteins, receptors, and hormones. Other Highly Sensitiue Immunoassays. All immunoassays derive their great selectivity from the antigen-antibody reactions on which they are based. In addition, the EIA exhibits great sensitivity, owing to the enzyme catalyzed reaction on which the measurement method is based. T h e r w ' immunoassays based on competitive binding principles that derive their high sensitivity through measurement of labels that are radioactive, fluorescent, or chemiluminescent (21-24). In some of these assays, a separation of the antibody-bound and free forms of the label must be made prior to measurement of the label. In other assays, however, the antibody-bound and free forms of the label exhibit markedly different measurement sensitivities, and separation is not necessary. AMPLIFICATION BY CYCLING Cycling Reactions. An interesting approach for achieving amplification is through a reaction mechanism that casts the analyte itself into a cycling role, so that a stoichiometric amount of product is produced and accumulated each time that the analyte is cycled or turned over. If the cycling is efficient, without loss of the analyte to side reactions, and if the measurable product is stable enough to permit accumulation, the amplification achieved by this approach may be large. T o date, mainly cofactors for enzyme catalyzed reactions have been amplified in this way, but in principle cycling is also generally applicable to other substances. A classical example of cofactor amplification is the determination of nicotinamide adenine dinucleotide phosphate (NADP) by cycling between two enzyme catalyzed reactions to yield a product which is determined by a third enzyme catalyzed reaction (25). The cycling reaction is shown in
1029
Figure 6. In outline, the sample containing NADP is added to a mixture containing nonlimiting concentrations of the three substrates (a-ketoglutarate, ammonium ion, and G6P) and controlled amounts of the two enzymes (glutamate dehydrogenase (GDH), and glucose-+phosphate dehydrogenase (GGPDH)). Examination of Figure 6 shows that NADP cycles between its oxidized and reduced forms, and that 6phosphogluconate (6PG) and glutamate build up in the reaction mixture at a rate related to the NADP concentration in the original sample. After the cycling sequence has proceeded long enough to build up a measurable concentration of 6PG, the mixture is heated to destroy the enzymes and to stop the cycling reaction. The 6PG is then measured by an indicator reaction (Equation a), after adding controlled levels of oxidized NADP and of 6-phosphoglutonate dehydrogenase (6PGDH). The reduced NADPH is measured fluorimetrically, and is related to the level of 6PG produced by cycling, and therefore to the NADP content of the original sample.
GPGDH
6-P-gluconate + NADP' ribulose-5-P + C 0 2 + H+
NADPH
+ (8)
With high levels of the two enzymes, the reaction systems shown in Figure 6 can cycle at rates as high as 20 000 cycles per hour. With the cycling step repeated, using the NADPH formed by the first stage, another factor of 20 000 can be obtained, to give an over-all amplification of more than lo8. Two stage determinations have beenperformed on amounts of NADP as low as mol (i.e., 1p L of 1 nM solution). The lowest detectable amount is limited mainly by the blank. In principle, two-stage determinations should permit the assay of as little as mol of NADP. A similar procedure has been devised for NAD, but of somewhat lower sensitivity. Amplification assays have also been designed for ATP. Cycling procedures are not widely used for analytical purposes, because they require considerable transfer and manipulation, often with exacting techniques, and the large number of reagents may require considerable effort at purification. Through indirect procedures that couple into cofactor amplification, many different substances may in principle be measured by amplification. Typical is a scheme that permits measurement of mol of prostaglandin El (PGE1) (26). The PGEl in the sample is first oxidized in a reaction catalyzed by a prostaglandin dehydrogenase, with concomitant reduction of NAD+. At the end of this reaction, any residual reagent NAD+ is destroyed by treatment with sodium hydroxide, which does not destroy the NADH that is formed. The NADH is then amplified by cycling for 60 min, followed by measurement of the accumulated product. A similar assay has been described for certain steroids (27). The ability to amplify cofactors by cycling has led to a novel use as labels in competitive binding assays (28). A derivative of NAD (6-(2-aminoethylamino) purine dinucleotide) was bonded to the analytes biotin and 2,4-dinitrofluorobenzene, to give conjugates in which the NAD could function actively as a cofactor in a cycling system. However, when the conjugates were treated with binding proteins that were specific for either biotin or the dinitrophenyl residue, the resulting complexes were virtually inactive as NAD cofactors in cycling systems. The inhibition was relieved in a competitive manner by the presence of free analyte at levels as low as 50 nM. Catalytic (Cycling) Electrode Reactions ( 4 ) . If an electron transfer that occurs at an electrode is preceded or followed by a chemical reaction that regenerates the electroactive material, a limiting current may be obtained that is in excess of that obtained from the electron transfer reaction alone. Such currents are called catalytic currents. A general
1030
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978 "0
ELECTRODE
=-(
I
\
NAD+
4
0
NADH
f
\
DIALYSIS MEMBRANE
1
-- -- - -
--
SOLUION =rod
zox
Figure 7. A catalytic electrode reaction
mechanism is depicted in Figure 7, as occurring through the following equations: electrode
-
LACTATE
An accepted method for the determination of lactate is based on its oxidation in the presence of lactate dehydrogenase (LDH):
O+ne-R
R
+ Zo,
solution
0
+ ,Z,
The 0 product in Equation 10 is reduced at the electrode as illustrated in Equation 9, to add to the current that would normally be produced from the reaction of Equation 9 above. Rather exacting conditions are required in order to obtain appreciable catalytic currents by this mechanism: (1) The working potential of the electrode must be set sufficiently cathodic to reduce 0, but not to reduce Zox. Since Z,, is a stronger oxidizer than 0, it is necessary that Z,, have a higher overvoltage for reduction than 0 has-otherwise Z,, itself will be electrochemically reducible a t potentials that are required to reduce 0. (2) In conventional electrode systems, much of the 0 produced by the reaction depicted in Equation 10 may be too far removed from the electrode to permit efficient reduction according to Equation 9, and the cycling efficiency will be low. High ratios of catalytic current to normal current are not generally obtained. Even so, molybdate (substance 0) has been determined at 5 pg/L in the presence of HzOz (substance Zox). Traces of vanadium, tungsten, uranium, and oxygen may be similarly determined. Although a good number of other substances exhibit catalytic waves, this approach has not been put to analytical use in any significant way. In another mechanism, some organic substances bond hydrogen in a way such that it can be reduced more easily (at a lower cathodic potential) than the normal hydrated protons that exist in aqueous solutions. The mechanism is complex, with the organic compound acting as a catalyst, and with Co(I1) being involved. The current-voltage curves of such systems exhibit a catalytic hydrogen wave proportional to the concentration of the organic compound, but at lower potentials than the normal hydrogen wave. Analytical use has been made of catalytic hydrogen waves for the determination of several classes of organic compounds, including amino acids, alkaloids, and proteins. For example, cystine can be determined a t micromolar concentration levels. Other mechanisms have also been described that yield catalytic currents. Coupling of Electrochemical a n d Biochemical Reaction Systems. By the immobilization of enzymes and cofactors at an electrode surface, it should be possible to couple an electron exchange reaction at the electrode surface into a biochemical enzyme catalyzed reaction sequence, without the losses that accompany conventional catalytic electrode reactions. Analytically, and in general, this should permit the high sensitivity, control, and convenience that characterize electrochemical techniques to be applied to the measurement of biochemical substances. While enzymatic amplification and cycling techniques should yield to such electrochemical control, no such systems have yet been reported in the literature. However, the feasibility of such coupling has been demonstrated with a reagentless lactate electrode (29).
PYRUVATE
Figure 8. A reagentless lactate electrode
lactate
+ NAD'-
LDH
pyruvate
+ NADH + H'
(11)
As performed conventionally, the NAD cofactor must be supplied as a reagent. However, a system may be devised in which NAD and LDH are both immobilized at an electrode surface to which a potential is applied. When the potential is sufficiently oxidizing, any NADH formed by the enzyme catalyzed reaction is reoxidized to NAD+. With sufficiently controlled conditions, the steady state current is related to the lactate concentration. Figure 8 is a schematic of a reagentless lactate electrode. With properly controlled conditions, the steady state current is related to the lactate concentration. There are several considerable analytical advantages to this device: (1) No reagents are required. (2) The reaction rate may be measured by the oxidation current, which in principle is a simpler procedure than spectrophotometric or fluorometric procedures. (3) The enzyme catalyzed reaction may be switched on or off by applying the proper potential to the electrode. Various procedures may be used for immobilization: (1) LDH and NAD can be copolymerized with glutaraldehyde on a dialysis membrane, which is then laid across the face of the electrode. (2) A slurry of LDH and agarose-NAD (Type I, P and L Biochemicals, Milwaukee, Wis.) can be confined between the electrode surface and a dialysis membrane. Other immobilization modes are being investigated. Although the reagentless electrode has been shown to be feasible, it has a number of shortcomings-its response does not depend linearly upon the lactate concentration, and it deteriorates significantly after a few days of use. A practical electrode still needs to be developed. Wingard and co-workers have succeeded in bonding riboflavin onto graphite and glassy carbon (30). The bonded riboflavin is electrochemically and enzymatically active, which might provide the basis for reagentless electrodes or electrochemical amplification systems for substrates requiring FAD as a cofactor. AMPLIFICATION B Y MULTIPLICATION Inorganic Systems. By reaction with various reagents in a series of repetitive steps, the amount or concentration of a substance may be multiplied greatly. For example, small amounts of C 0 2 in an inert gas stream may be multiplied by. passing the sample through heated alternate layers of carbon and copper oxide in a reaction tube. C
COz + 2 C 0 Multipli-
cation: 2 X
C
2 C 0 2 + 4CO
T
--J CUO
4co2 + etc. (12)
M Y ; - 4x cation'
The multiplication proceeds geometrically, by a factor of 2
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
per step, and can proceed indefinitely, limited only by the number of layers and the length of the tube. Of course, oxygen and other reactive gases in the sample interfere in the method. A good number of multiplication reaction systems involve iodine because it exhibits so many intermediate oxidation states. A classical example is the Leipert reaction for the determination of iodide. First, the iodide is oxidized to iodate with excess bromine. The excess bromine is removed by boiling, and the iodate is then treated with excess iodide to yield iodine, giving a multiplication factor of 6:
+ 3Br, + 3 H 2 0 + IO; + 6Br- + 6Ht IO; + 51- + 6H' 3 1 2 + 3HzO I-
+
T o repeat the step, the iodine may be extracted away from the reaction mixture with carbon tetrachloride and then put back into aqueous solution. Through several steps, amplification factors exceeding two orders of magnitude have been achieved. Belcher (31) has reviewed multiplication reactions, along with reaction systems yielding very high stoichiometric ratios of product to reagent. The capabilities of direct multiplication reactions are extendable through indirect methods. Metal ion multiplication has been described. By and large, however, multiplication reaction systems have not been applied widely to analysis. The procedures require considerable manipulation and transfer, because the reactions that constitute a step are usually performed in physically separated systems. Bacterial Systems. The presence and type of bacteria in a specimen are revealed by their ability to thrive and multiply when supplied with media containing selected nutritional requirements. Conversely, bacterial growth rates have also been used to detect and quantitate particular nutrients or inhibitors in a specimen. For example, microbiological assays for vitamin B12have been developed in which growth rates of certain organisms are dependent upon the concentration of B12supplied to the growth media. Also, many antibiotic assays depend on the inhibition of growth or multiplication of selected organisms. A unique use has been made of bacteriophage systems for the monitoring of competitive binding assays. Bacteriophages are a group of bacterial viruses possessing the ability to infect and bring about lysis of growing bacterial cultures. The lysis is accompanied by the liberation of more phage, which in turn can infect additional bacteria. The reagent (X-P) is a phage that has been modified by covalently binding an analyte to the protein coat of the phage in such a way that the phage is still viable and infectious. However, upon reaction with an analyte specific binding protein (Ab), the phage is rendered inactive and incapable of lysis. Addition of analyte (X) to the system inhibits the inactivation, and the extent of inhibition is related to the concentration of the added analyte:
Ab*X-P + X + Ab*X+ X-P (inactiw)
(active)
The key requirement in the procedure is the inactivation of the phage conjugate. The inactivation occurs when the specific binding protein (Ab) complexes with the phage conjugate and sterically prevents the attachment of the modified phage to the bacteria. For the interference to occur, the analyte must be coupled to the phage near the region where it attaches to bacteria. Lysis is usually monitored by counting the number of plaques (clear areas resulting from lysis) formed in the bacterial culture. There are several versions of the viroimmunoassay procedure, all being dependent upon phage multiplication and its inhibition. These methods are sensitive to subnanomolar concentrations of analyte.
1031
OMPLEMENT
ANALYTE-LABELLED LIPOSOME W r H ENTRAPPED WRKER
LYSED LIPOSOME WITH RELEASED MARKER
Figure 9. Lysis of an analyte labeled liposome by antibody and complement, with consequent release of entrapped marker
The bacteriophage method has been used to assay insulin (32),as well as other substances (33). For a typical insulin assay, a specified quantity of antiserum to insulin is mixed with the insulin sample and left to stand for 24-48 h at 4 "C. A measured amount of an insulin bacteriophage conjugate is added, and the reaction mixture is permitted to stand for several hours at 37 OC. The mixture is then treated with soft agar containing a high level of bacteria, and the resultant solution is poured onto plates containing a bottom layer of agar. Plaques are counted after 10-18 h at 37 "C, the count being related to the insulin content of the original sample. Amounts of insulin as low as 0.05 ng/mL are detectable. Liposome Systems. Liposomes are tiny spherical assemblies of concentric lipid bilayers. The bilayers are separated by aqueous layers that may contain a marker, such as a spin label, a substrate, or an enzyme. The analyte of interest may be inserted into the membrane matrix, or covalently linked to the lipid membrane. When antibody binds to the analyte in the presence of complement, disruption of the bilayer occurs, and the marker is released as illustrated in Figure 9. Complement is a complex biological system found in serum. In one assay procedure for the glycolipid known as Forssman's hapten, the analyte specimen was first treated with excess heat inactivated specific antiserum (heat destroys any endogenous complement), so that all available analyte is antibody bound (34). The remaining level of unreacted antibody is determined from the spin labeled marker released in the presence of complement, from a measured quantity of liposomes containing hapten added to the assay mixture. The amount of marker released is inversely proportional to the analyte concentration in the original sample. Forssman's hapten is detectable at levels as low as 2.6 pmol by the liposome technique. In a recent version of this reaction, sheep erythrocyte ghost cells containing trimethylphenylammonium ion were lysed by complement to free the trimethylphenylammonium ion, which was measured potentiometrically with a monovalent cation-selective electrode (35). This reaction was employed to estimate the concentration of hemolysin antibody or of complement under properly controlled conditions. Lysis by complement is also the basis of some older techniques. In one method, red blood cells are coated with an analyte specific antibody. Lysis occurs upon exposure to analyte and complement, releasing the hemoglobin marker. In other methods, coated red blood cells, polystyrene, bentonite, and charcoal particles have been used to detect the analyte-antibody reaction through agglutination of the particles. Amplification occurs through aggregation of the particles. CONCLUSIONS In this article, we have presented examples of highly sensitive analytical procedures based upon chemical amplification methods, such as catalysis, cycling, and multiplication. Historically, most of these methods have been developed in the biochemical and clinical areas, in response to the special needs of workers in these areas. However, the potential uses of these procedures extend into all disciplines, wherever chemical measurement is involved. Research on these methods and their further development and application
1032
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
constitute an important frontier of chemical analysis.
D. S. Skeliey, L. P. Brown, and P. K. Besch, Clin. Chem. ( Winston-Salem,
N . C . ) , 19, 146 (1973). J. F. Burd, R. C. Wong, J. E. Feeney, R. J. Carrico, and R. C. Boguslaski, Clin. Chem. ( Winston-Salem, N.C.),23, 1402 (1977). H. R. Schroeder, P. D. Vogelhut, R. J. Carrico, R. C. Boguslaski, and R. T. Buckler, Anal. Chem., 48, 1933 (1976). A. Kawamura, "Fluorescent Antibody Techniques and their Applications", University Park Press, Baltimore, Md., 1969. 0. H. Lowry, J. V. Passonneau, D. W. Schulz, and M. K. Rock, J . Biol. Chem., 236, 2746, 2756 (1961). E. Anzgard, F. M. Matschinsky, and 8 . Samuelson. Science, 163, 479
LITERATURE CITED T. M. S. Chang, Ed., "Biomedical Applications of Immobilized Enzymes and Proteins", Plenum Press, New York, N.Y., 1977, Vol. I. G. G. Guilbautt, "Handbook of Enzymatic Methods of Analysls", M. Dekker, New York, N.Y., 1976. K. B. Yatsimirskii, "Kinetic Methods of Analysis", Pergamon Press, New York. N.Y.., 1966. ... H.-B.'Mark and G. A. Rechnitz, "Kinetics in Analytical Chemistry", John Wilev. New York. N.Y.. 1968. G. Knapp, H. Spitsy, and H. Leopold, Anal. Chem., 48, 724 (1974). U. I.Isacsson and G. Wettermark, Anal. Chim. Acta, 88, 339 (1974). W. R. Seitz and M. P. Neary, Anal. Chem., 48, 188 (1974). W. J. Blaedeland G. P. Hicks, Adv. Anal. Chem. Instrum., 3, 105 (1964). L. D. Bowers and P. W. Carr, Anal. Chem., 48, 544A (1978). W. J. Biaedel and R. C. Engstrom, Anal. Chem., 5 0 , 476 (1978). N. W. Tietz, "Fundamentals of Clinical Chemistry", Saunders, Philadelphia, Pa., 1970. F. Lundquist, Methods Biochem. Anal., 7 , 217 (1959). M. D. Smith and C. L. Olson, Anal. Chem., 47, 1074 (1975). G. B. Wisdom, Clin. Chem. (Winston-Salem, N.C.), 22, 1243 (1976). V. P. Butler, Jr., J . Immunol. Methods, 7 , 1 (1975). A. F. Rosenthal, M. G. Vargas, and K. S . Klass, Clin. Chem. (Winston-Salem, N.C.). 22, 1899 (1976). R. S.Galen and D.Forman, Clin. Chem. ( Winston-Salem, N . C . ) , 23,
(1969).
M. Harkonen, H. Adlercreutz, and E. V. Groman, J . Steroid Biochem., 5 , 717 (1974). R. J. Carrico, J. E. Christner, R. C. Boguslaski, and K. K. Yeung, Anal. Blochem., 72, 271 (1976). W. J. Blaedel and R. A. Jenkins, Anal. Chem., 48, 1240 (1976). L. Wingard, Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pa., private correspondence, November 1977. R. Belcher, Talanta, 15, 357 (1968). J. Haimovich, E. Hurwitz, N. Novik, and M. Sela, Biochlm. Biophys. Acta, 207, 125 (1970). M. Sela, Triangle(€ngl. Ed.), 11, 61 (1972). R. Wei, C. R. Alving, R. L. Richards, and E. S. Copeland, J . Immunol. Methods, 9, 165 (1975). P. D'Orazio and G. A. Rechnitz, Anal. Chem., 49, 2083 (1977).
119 (1977).
K. Kato, Y. Hamaguchi, S. Okawa, B. Ishikawa, K. Kobayashi, and N. Katunuma, Lancet, 1, 40 (1977). S. Avrameas, Int. Rev. Cyfol., 27, 349 (1970). S. Avrameas. Hlstochem. J . , 4, 321 (1972).
RECEIVED for review January 27, 1978. Accepted March 16, 1978. This work was supported in part by a grant (No. CHE76-15128) from the National Science Foundation.
Separation and Chemical Characterization of Finely-Sized Fly-Ash Particles J. A. Campbell,* J. C. Laul, K. K. Nielson, and R. D. Smith Physical Sciences Department, Battelle, Pacific Northwest Laboratories, Richland, Washington 99352
The concentrations of 43 major, mlnor, and trace elements were measured by x-ray fluorescence, atomlc absorption, and instrumental neutron activation for nine well-defined size fractions, with mass median diameters of 0.5 p to 50 pm, of fly ash from a western coal-fired steam plant. There was generally good agreement in concentrations of elements analyzed by more than one technique. Concentration profiles as a function of mean particle size were established for various elements. Based on the concentration profiles, the elements can be divided into three distinct groups. One group consists primarily of the volatile elements or elements partially volatilized during coal combustion (examples include As, Se, Zn, Ga, etc.), and their concentrations decrease with increasing particle slze. A second group, which shows a minor or direct dependence on particle size, as in the case of Si, is apparently associated prlmarily with the fly-ash matrix. The last group of elements, which includes Ca, Sr, Y, and the rare earths, shows small changes in their concentration profiles with a maximum in concentration at approximately 5 pm.
It has been estimated that particulate emissions from coal combustion in the United States will amount to approximately 5 X lo6 tons/year by the year 2000 ( 1 ) . A detailed characterization of fly-ash emissions is thus important to the development of technology for both reducing fly-ash emissions 0003-2700/78/0350-1032$01 .OO/O
and dealing with medical and environmental problems associated with fly-ash release. It is known that fly-ash particles emitted from coal-fired steam plants show an enrichment of several toxic trace metals (2). The submicron particles, which show the greatest enrichment for many toxic trace elements, are of particular concern since they are not efficiently removed by modern particle collection devices. In addition, these smaller particles (
