CA1040078A - Method for constituent analysis with thin-layer reactant mixture - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention provides an analysis procedure in which reagents and a sample solution in an optically-thin layer chemically react to produce a reaction product that is a known measure of a constituent in the sample solution. Use is made of fibrous sheets which are essentially non-absorbing and uniform in structure within and contiguously beyond the reaction site to generally eliminate the need of using confined test sites. The layer is examined under electromagnetic radiation with a sensing means that responds in a linear manner to the concentration of a selected constituent-manifesting product of the reaction. The invention enables such measurements to be made repeatedly with precision even when the spatial distributions of the material being analysed and of the reaction-producing reagents over the thin layer both are not uniform and differ from one another, provided that the distributions are the same for successive samples.

Description

34(~8 BACKGROUND OF THE INVENTION
This invention relateq to a method for measuring a soluble constituent in a material such as a biological fluid.
Moxe particularly, the invention provides a constituent-measuring method in which the sample material being analyzed is subjected to a constituent-manifesting chemical reaction in an optically-thin layer of the reactants. The layer is examined under electromagnetic radiation with sensing means that responds in a linear manner to the concentration of a selected constituent-manifesting product of the reaction.
The invention enables such measurements to be made repeatedly with precision even when ~he spatial distributions of the material being analyzed and of the reaction-producing reagents over the thin layer both are not uniform and differ from one another, provided that the distributions are the same or successive samples. Moreover, the procedures of the invention provide highly sensitive measurements of constituent concentrations as contrasted to the prior art.

Prior art regarding the invention includes the teachings in the U.S. Patents Nos.:
Yagoda 2,129,754 Natelson 3,036,893 Natelson 3,216,804 Natelson 3,219,416 Natelson 3,260,413 Natelson 3,261,668 Natelson 3,331,665 Natelson 3,368,872 Natelson 3,502,438 Rey 3,526,479 Findl 3,526,480 Fetter 3,552,925 An object of the invention is to provide an improved method for measuring constituents in a biological fluid con-tained in a fibrous cuvette.

L~
~,`, ., , '78 1 A further object is to provide a procedure for measuring one or more soluble constituents in a fluid material contained A ' in a fibrous eY~e~ and which provides higher sensitivity than the prior art.
Another object is to provide a method of the above character that can employ as the cuvette a fibrous sheet member that is free of material-constraining structure, such as a disk of confined size or constraining rings on the sheet member.

Another object of the invention is to provide a method of the above character in which the cuvette medium can have one or more test sites that are unbounded.
It is also an object of the invention to provide a method of the above character capable of precise and accurate constituent measurements.
A further object of the invention is to provide a method of the above character that requires only a minute volume of the sample material, typically less than 3~ microliters.
Another object of the invention is to provide a method of the above character capable of providing analyses rapidly, and further with minimal setup.

A ~urther object of the invention is to provide a method of the above character that can perform both rate-reaction measurements and end-point measurements.
Other objects of the invention will in part be obvious and will in part appear hereinafter.
The invention accordingly comprises the several steps and` the relation of one or more of such steps with respect to each of the others thereof as exemplified in the procedures hereinafter disclosed, and the scope of the invention is indicated in the claims.

- 2 -SU~RY OF THE INVENTION

In brief, the invention provides an analysis procedure i.n which reagents and a sample solution in an optically-thin layer chemically react to produce a reaction product that is a known measure of a constituent in the sample solution. Upon illumi-nation of the layer with electromagnetic energy of a selected wavelength, a radiation detector or other energy-sensing means having a llnear response provides a linear measure of the concentration of the constituent of interest.

The reagents and the sample solution are spatially distributed so that, ideally, the amounts of the various reactants are balanced with each other throughout the test area for optimum chemical interaction to produce the reaction product of interest. A fibrous sheet preferably is used to contain .. the reactants and serve as the reaction cuvette With this arrangement, the balanced di.stribution of reactants often involves delivering the reactants to the fibrous cuvette in a selected sequence, and not necessarily with uniform or identical dis tr ibuti ons.
Also, in at least many instances of the practice of the invention, the reaction sites do not require a confining structure such as a Yagoda ring. Instead, the reaction site can be unbounded.
The invention can be practiced with the apparatus des-cribed in the commonly-assigned United States Patent No.3,844,717 entitled "Press For Progressive Compression Of Liquid-Bearing Absorbent Article" of L. Sodic~son and F. Lim The press structure of that patent can be used to deliver a sample solution such as blood serum ~' .

1 for analysis in accordance with the invention described herein from a sample of whole blood. Further, the fluorometer structure of the patent can also be used to advantage in the practice of the invention described herein.
One advantage of performing chemical constituent analyses in accordance with the invention is that it requires little time, particularly as compared to prior techniques. Further, the practice of the invention requires a minimal amount of equipment.

For example, in the lactate analysis of whole blood with a fibrous cuvette already treated with analysis reagents as described below, the desired serum of the blood sample can be transferred to the cuvette by means of the ultrafiltration press described in the patent identified above, and the lactate analysis completed, in a matter of five minutes or so.
For a fuller understanding of the nature and object of the invention, reference should be made to the following detailed description.

D T~ILED DESCRIPTION

The invention typically is practiced by introducing a liquid sample solution containing the material to be analyzed as a solute to a selected site on a fibrous sheet that serves as the analysis cuvette. Multiple chemical reagents are in-troduced to the fibrous cuvette either prior or subsequent to introduction of the sample solution. The sample solution and each reagent are introduced to the fibrous cuvette so that each has substantially the same spatial distribution of concentration for every performance of a given test or analysis. That is, in performing an analysis on multiple samples in accordance with the invention, the spatial distribution of different samples 7~3 1 across the several fibrous cuvettes is the same, i.e. repeatable.
So also the spatial distribution of each reagent is the same for testing each sample.
The cuvette contains the sample solution and the reagents in an optically-thin layer. This makes it possible to measure the constituent of interest by examination of the reaction mixture with electromagnetic energy and to secure a response that-is linearly related to the unknown constituent concentration.
- As used herein, the layer of sample solution and reagents is considered to be optically thin when its transmission of the incident and detected output measuring energies at every point in the field of view of the measuring instrument vary essentially linearly with concentration of the constituent of interest up to the maximum concentration that is to be measured.
The constituent-manifesting reaction product of interest produced in the reaction mixture under the foregoing conditions is preferably measured with a fluorometer or other radiant-energy responsive instrument that has a linear response to the concentration of the substance of interest.
More particularly, in the examples set forth below, tl~e reaction mixture of the invention produces a fluorescent reaction product with a concentration responsive to the con-centration of the unknown substance being measured. Upon illumination from the fluorometer source, at every point on the cuvette in the fluorometer field of view the constituent-measuring reaction product in the reaction mixture fluoresces with an intensity that is linearly proportional to concentration so long as the layer is optically thin. The fluorometer de-tector has a linear response to this radiant fluorescence '1~34U~
1 from every point In its field of view and hence produces an electrical signal that is a linear function of the product of the fluorescent energy and the fluorometer sensitivity profile integrated over the area of the cuvette surface within the fluorometer field of view. The fluorometer sensitivity profile is the spatial distribution of the instrument's response over its field of view to fluorescence from its lamp. Hence the profile is a composite of the spatial characteristics of the fluorometer lamp and of the fluorometer detector.
As noted above, the United States Patent number

3,844,717 describes a fluorometer construction suitable for use in practicing this invention. The fluorometer of that patent has equal spectral angles for the incident energy and for the measured fluorescent energy. This is desired to provide the instrument with a composite, i.e. lamp and detector, sensitivity profile which is symmetrical and approximately uniform about the detector boresight axis, albeit with some variation radially rom this axis, to measure all points in the fluorometer field of view uniformly. ~hat is, with the fluoro-meter angle o~ incidence equal to the angle of sensed radiation, the total optical path from the fluorescent source of the reaction layer and then to the fluorometer detector is essentially uniform, at least to a first order, for all points within the fluorometer field of view.
Alternative to a fluorometer, the invention can be practiced with an oscillometer that measures the interaction of reaction mixture components with an oscillating electromagnetic field. For example, the cuvette sheet can be placed between two flat electrodes to form in effect a flat-plate capacitor.
3~
The instrument is calibrated to measure the change in capacitance ! ~
~;`~ .. :. .

1 due to the reaction product of interest at a selected frequency.
The fibrous material that forms the reaction cuvette for practice of the invention is preferably of fibers that are inert to the sample solution and to the analysis reagents, and which are non-absorbing and are transparent to the electro-magnetic wavelengths involved in the measurement. However, these requirements are not mandatory, rather they facilitate factors such as calibration and measuring precision, and enhance measuring sensitivity. By way of example, the invention has been successfully practiced with fibers of cellulosic material as well as of fiberglass.
Fibers of glass, i.e. fiberglass, tend however to be sufficiently fragile so that they break upon being compressed with the press described in the patent noted above. Accordingly, where such a press or other structure that subjects the fibrous material to stress is to be employed, it is considered preferable that fragile fibers not be used or at least that the fibrous sheet have a mixture with less ragile, e.g. cellulosic, fibers.
For measurement of a rate reaction, the sheet of fibers preferably has a fiber structure such that all liquid reagents involved cease spreading within the sheet in a time that is short compared to the time during which the reaction of interest proceeds linearly. That is because the linear portion of the reaction should proceed in large part after all the reactants have ceased spreading due to capillary action of the cuvette fibers. In the measurement of a typical rate reaction, it is desirable that approximately three-quaters of the linear portion of ths reaction time take place after significant spreading ceases.

1~4U(~78 1 With further regard to the spreading of fluid materials within the fibrous cuvette, it is desired that all materials move through the sheet at the same rate. Otherwise, the distributions of various reactants tends to become unbalanced at different locations within the cuvette area. In view of the vastly different flow characteristics of materials typically involved in constituent analyses, this objective often may not be realized to the desired extent. However, it has been found that disparate spreading of different materials can in many 10 instances be limited by applying the materials to the fibrous cuvette in a selected sequence. In particular, it has been found that the application of large-molecule reagents, with or without drying, prior to the application of reagents of smaller molecules substantially diminishes the spreading of the reagent applied first by the latter-applied reagent. That is, it has been found that where a solution of small-molecule constituents is delivered ~o a fibrous cuvette followed by a solution of large molecule constituents, the heavy molecule material tends to spread and push the lighter molecule material out ahead of it with the result that the small-molecule material is concentrated in an annular ring outside of the area of maximum concentration of the large molecule material. This condition is undesired and results in significantly lesser measuring sensitivity than where the procedure is reversed, as further detailed hereinafter.
As example of fibrous sheets suitable for the practice o~ the invention, Schleicher & Schuell test papers Nos. 903, 903-C, 404, 410 and 25, and Whatman test papers GF/A, GF, B, and G~/C have all been used successfully. In some instances Yagoda-type s~ot confining rings have been found desirable.
In general, it appears that these rings are more desirable on )4~78 1 non-fiberglass papers and on thinner papers. In particular, wax confining rings have been used to advantage with the Schleicher & Schuell 903-C and 410 papers, whereas good results, including high sensitivity, have been obtained with Schleicher & Schuell 404 paper without confining rings.
The foregoing comments concerning the sequence of applying reagents applies also to the addition of the sample solution.

The term sensitivity is used in connection with this invention to describe the magnitude of rate of change in sensed energy, e.g~ fluorescence intensity, for a given concentration of the constituent being measured.

EXAMPLE I
As a first example of the practice of the invention, blood is tested to measure the concentration of glucose. The analysis uses the known hexokinase reaction HK
Glucose ~ ~TP ~ G-6-P + ADP

G-6-P ~ NADP~ 6 PG ~ NADPH -~ H

the conventional practice of which is described for example in the brochure "Diagnostic Test Combinations, Operating Instructions"
distributed by Boehringer Mannheim Corporation.
In the simplest procedure, a solution containing all necessary rea~ents is obtained by dissolving one Smith Kline Eskalab*Glucose tablet in 0.5 milliliter of distilled water.
Ten lambda of the sample serum to be analyzed is deposited on a fibrous sheet such as S&S No. 903-C. The sample is deposited continuously, rather than drop-by-drop, at the center of the reaction site, as with a pipette. This is followed by depositing * Trade Mark _ 9 _ ~1 ..!., ~s~
1 ten lambda of the dissolved ~=~L~i solution in the same con-, ~ .
tinuous manner at the same spot. The order of addition is critical, as the observed reaction rate is three times greater in the above case than if the solution is added before the serum.
The reaction site is then monitored with a fluorometer which illuminates the reaction site at 340 nanometers wavelength O~ser~S
and~63~ the fluorescence with a detector responsive to the 460 nanometers wavelength of maximum NADPH emission. The fluorometer output signal is measured during the linear portion of the reaction. The measurement preferably is of the rate of NADPH production rather than of the total amount of NADPH
production to facilitate the measurement of the fluorescence from the NADPH separate from the background fluorescent radiation from other materials at the test site as well as the cuvette sheet itself. The latter radiation is essentially time invariant, whereas the fluorescence from the NADPH increases with the increase in NADPH production. Also, care is taken to avoid contact of the fluorometer end window with the cuvette and the reaction mixture to avoid variations that otherwise arise in the detected fluorescence.

The foregoing glucose analysis has been performed successfully with a variety of fibrous sheets for ~he cuvette, including S&S 404, 595 and 25 papers, and the Whatman GF/A, GF/B and GF/C papers.
EXAMPLE II
Blood serum is tested for lactate concentration with a - 10 ~

~4~ 78 1 pre-prepared fibrous cuvette, as o~ S&S gO3-C paper with a wax ring about the test site. The cuvette is prepared by first depositing, at the center of the test site, LDH in an ammonia suspension, such as Sigma Type III-Beef Heart. This is followed by deposition at the same point of ten lambda of a water solution of fifteen milligrams of pyradine nucleotide (NAD) per milliliter with a trace of surfactant such as Brij*.
Where fresh serum is to be tested, the pre-prepared fibrous cuvette is first treated by depositing ten lambda of glycine-hydrazine buffer at a pH of nine. This is followed by the deposition of ten lambda of the serum sample.

It has been found to be important in the preparation of the fibrous cuvette that the LDH solution be added prior to the NAn solution, otherwise the lactate analysis occurs with significantly lesser sensitivity. It is believed tha-t this is because the relatively heavy LDH molecules establish a bond with the cuvette fibers or otherwise resist spreading upon the subse~uent addition oE the lighter NAD molecules so that the two reagents largely occupy the same portion of the fibrous ~0 sheet. When the rea~ents are deposit~d in reverse order, it is believed that the lighter NAD molecules are moved laterally outward from theFoint of deposition by the heavier LDH molecules with the result that the cuvette has a concentration of LDH
molecules within a predominantly annular concentration of NAD
molecules. The sensitivity is also observed to be diminished when the se~uence of adding buffer and serum is reversed, i.e. when the sample serum is adde,d before the buffer. (Note that * Trade Mark - a series of polyoxyethylene fatty alcohol derivatives made by Atlas Powder Co.

4t3~7~3 1 the addition last of sample serum for the lactate analysis of this example is opposite to the preferxed sequence for the glucose analysis of Example I.) EXAMPLE III

As a third example, a fibrous cuvette is again prepared in the manner of Example II, but the serum to be tested is available as a dried blot of whole blood carried on S&S 903-C
test paper. The paper containing the whole blood sample is placed over the pre-prepared reagent sheet with an intervening filter sheet, as described in the above-noted patent, and placed in the press described in that patent. Prior to closing the press, the blood stain is reconstituted. Where the stain is fresh, this is done by the addition of twenty lambda of saline (0.9 percent normal NaCl solution). The press is then closed to transfer the serum through the intervening filter sheet to be fibrous cuvette.
Alternatively, where the blood stain is not fresh, it is preferably reconstituted by the addition of twenty lambda of water with a trace of Brij or like surfactant, and then subjected to pressure to transer it to the reaction cuvette.
In either case, the press is left closed for approximately one minute. The press transfers a known portion of the lactate in the sample to the reaction cuvette, but typically insufficient liquid is transferred to provide sufficient molecular mobility for the reaction to proceed. Accordingly, the sheet that initially carried the blood sample and the intervening filter sheet are stripped from the reagent sheet, and twenty lambda of the glycine-hydrazine buffer (pH of nine) is applied to wet the lowermost reaction sheet. Again, the rate of NADH

~4 ;,7~3 production is monitored with a fluorometer as set forth above.
For all of the foregoing tests, the fluorometer field of view is approximately a one square centimeter circular area centered on the point at which the reagents are deposited on the reaction cuvette.

EXAMPL~ IV

As an illustration of the application of the invention - to a more complex chemical reaction for blood analysis, a fibrous cuvette of S&S 903-C paper is prepared as follows to test blood serum for the concentration of triglyceride. Again the basic chemical reactions for this analysis are known, as described for example in the above-noted brochure of Boehringer Mannheim Corporation under the heading "Neutral Fat (Tri-glycerides) and Glycerol". However, lipase is used to hydrolize the neutral fat to glycerol.
The first step in the preparation of the fibrous cuvette is to apply to the center of a reaction site on S&S
903-C paper twenty lambda of lipase (Schwartz-Mann No. 25) as a continuous stream or single droplet and allow it to dry.
This is followed by the deposition at the same spot of twenty lambda of the enzyme solution glycerokinase-glycerophosphate dehydrogenose, and again the fibrous sheet is allowed to dry.
Thereafter, ten lambda of a co~factor solution containing NAD
and ATP, and the Mg+~ activator for glycerokinase and Ca +
activator for lipase in Brij water solution of at least three milligrams per milliliter is deposited.
It should be noted that the three reagents so far added to the fibrous sheet have been added in the order of decreasing molecular weight. The remaining preparation of the ~L~4(~(i'7E~
fibrous sheet is to increase the concentration of the combined enzyme solution by successive depositions, so as not to unduly spread the material but rather to concentrate it over a limited area, e.g. one square centimeter, of the fibrous sheet.
Accordingly, before the co-factor solution dries, another ten lambda of the enzyme solution is deposited on the sheet and the sheet is then allowed to dry. Thereafter, ten lambda of a glycine-hydrazine buffer of pH ~ is deposited on the sheet at the center of the reaction site followed by another ten lambda of the enzyme solution and another drying step. The fibrous reaction sheet is now ready for use, and can be stored until needed.
To make a triglyceride analysis with the reagent sheet prepared in the foregoing manner, ten lambda of the glycine-hydrazine buffer is first deposited on the sheet ~ollowed by ten lambda of the blood serum being tested. The reaction sit~
is then examined with a fluorometer for the rate of NADH
production. A second test is performed with ~he same pre-prepared reaction sheet in the same manner except that ten lambda of saline are deposited in place of the sample serum, and the rate of NADH production measured in the same manner.
The differenae between the two rates of NADH production, as measured one with the sample and the other with saline, is the desired measure of the triglyceride concentration. It is believed that such a differential measurement is needed to attain high accuracy due to impurities in the reagents and/or in the fibrous sheet forming the cuvette, and hence with pure materials the second, blank test can be eliminated.

~ 14 -~4~1)7~3 Although the foregoing examples have involved analyses in which NADH is produced, the invention can equally be used in performing analyses in which a fluorophor is consumed, and the rate of consumption is monitored.
It will thus be seen that the ob]ects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in carrying out the above method without departing from the scope 10 ' of the invention, it is intended that all matter contai.ned in the above description shall be interpreted as illustrative and not in a limiting sense.
It is also to be understood that the follo~ing claims are intended to cover all of the generic and specific features of the invention harein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

. . ~ ., ,

Claims (15)

The embodiments of the present invention in which an exclusive property or privilege is claimed are defined as follows:

1. In apparatus for measuring a selected product of chemical reactants at a test site on a fibrous medium and having means for subjecting said site to oscillating electromagnetic energy and for sensing a selected parameter of energy which is responsive to said oscillating energy and to the presence of said selected reaction product, the improvement comprising A. measuring means for sensing product-responsive energy for a selected interval during which said reactants produce said selected product at a substantially linear rate, and B. a substantially uniform fibrous structure in said fibrous medium within and contiguously beyond said reaction site, said structure consisting essentially of non-absorbing fibrous material, and being characterized by the substantial termination of spreading therein of said reactants in liquid solution prior to the completion of said sensing of product-responsive energy, said uniform fibrous structure being further characterized by the substantial termination of spreading therein of said reactants in liquid solution significantly prior to the termination of said selected measuring interval.

2. In apparatus as defined in claim 1, the further improvement wherein said measuring means has a field of view on said test site with a sensitivity profile which is substantially symmetrical and uniform about an axis along which said oscillating energy is directed.

3. In apparatus as defined in claim 1, the further improvement wherein said measuring means is free of contact

Claim 3 continued ...

with said fibrous medium, at least over the field of view from which energy is sensed.

4. In the constituent analysis of sample material on a fibrous medium by reaction with at least one reagent to produce a constituent-manifesting reaction product, the improvement comprising the steps of A. distributing said sample material and said reagent in liquid state over a selected analysis site of said medium, B. producing said reaction product at said analysis site with only an optically-thin concentration, C. illuminating said analysis site with incident electromagnetic radiation, D. sensing, from a field of view within said analysis site, electromagnetic radiation that is resultant from said incident radiation and responsive to the concentration of said reaction product, and E. producing a measure of the constituent in response to radiation sensed from the same area within said site at at least two different times between which said reaction produces said reaction product.

5. In a method as defined in claim 4, the further improvement in which said step of producing a measure includes, measuring the rate of change of said radiation sensed during the production of said reaction product.

6. In a method as defined in claim 5, the further improve-ment in which said step of producing a measure includes measuring the rate of change of said radiation from a time after the cessation of spreading of liquids in said fibrous medium.

7. In a method as defined in claim 5, the further improvement in that said radiation is sensed substantially continuously during production of said reaction product, said distributing step includes balancing the amounts of said sample material and said reagents at said analysis site to produce a substantially uniform rate of reaction, and the further step of timing said measurement of rate of change to coincide with at least a portion of the duration of said uniform rate of reaction.

8. In a method as defined in claim 5, the further improvement in that said step of producing said measure responds to a function of the difference between the radiation sensed at said different times.

9. In a method as defined in claim 4, the further improve-ment in which said sensing step comprises sensing fluorescence from said reaction products.

10. In a method as defined in claim 4, the further improve-ment in which said medium has an unbounded test site.

11. In a method as defined in claim 4, the further step of providing said medium with substantially non-absorbing fibers at said analysis site.

12. In a method as defined in claim 4, the further improve-ment in which said distributing step includes distributing said sample material and said reagent with substantially repeatable spatial distributions of concentration over a selected analysis site of said medium, with at least one reactant selected from said sample material and one said reagent being provided at said site with a nonuniform spatial distribution of concentration.

13. In a method as defined in claim 12, the further improvement in which said step of producing a measure of the constituent includes producing a measure of the concentration of the constituent in said sample in response to the integral of said sensed radiation over the area of at least a selected region of said test site.

14. In a method as defined in claim 12, the further improvement A. in which said step of distributing includes:
(1) forming a repeatable concentration distribution over a test site of a porous support member of each chemical reactant that produces, upon chemical reaction with said sample, a reaction product identifying the concentration of said constituent, with at least one reagent distribution being nonuniform, and (2) forming a repeatable concentration distribution over said test site of said sample with a sufficiently low concen-tration to produce an optically-thin distribution of reaction product and allowing said sample to react, in liquid solution, with said reagents at said test site to produce said reaction product, and B. said steps of sensing and producing include:
(1) sensing electromagnetic radiation emitted from said test site in response to incident energy and to the concentration of said reaction product thereat, (2) integrating said sensed radiation emitted from at least a selected region of said test site, and (3) producing an output measure in response to a change in said integral of sensed radiation resultant from said production of said reaction product.

15. A method as defined in claim 14 in which said first-mentioned distribution forming step comprises applying at least some of said reagents to said test site in sequential order according to the molecular size of an active constituent of each reagent, starting with the reagent having the largest molecular constituent.