CA1154827A - Method and apparatus for analysis - Google Patents

Abstract

"METHOD AND APPARATUS FOR ANALYSIS"

ABSTRACT OF DISCLOSURE

This invention relates to a method and to apparatus for chemical and biochemical analyses which employ an energy-transmissive core and may employ one or more sheaths which selectively absorb, react with, and/or filter an analyte or a product of an analyte. The core is transmissive to a chosen energy carrier and it has an inlet end and an outlet end.
Between these ends it has an extended length. The passage of energy through the core is modified by reason of events which occur in one or more of the sheaths or in the case where no sheath is employed, by reason of events which occur in an ambient fluid. The resulting modification of the transmitted energy is a measure of such events which in turn are a measure of the analyte. The energy may be any of several types of energy which can be transmitted through the core from end to end and which is susceptible to modification by reactions in the sheath or sheaths or ambient fluid. The energy may be electromagnetic, electrical or sonic. In the method aspect of the invention a permeable core may be used which is bare, i.e. without a sheath, and exposed directly to an environment, e.g. the air or an industrial fluid.

Description

SPECIFICATION

Many analytical techniques have been developed for chemical or biochemical purposes. Procedures that use a discrete fluid sample for analysis of a single analyte are traditionally characterized as wet chemical techniques or dry chemical tech-niques. In recent years both types of techniques have been automated in order to reduce costs and simplify procedures.
Wet chemical methods, typified by the technicon auto analyzers, utilize batches of reagent solutions, pumps and fluid controls, coupled with conventional sensors, (such as densitometric, fluorescent, colormetric (i.e. radiometric), polarographic, conductimetric, or ultrasonic). These techniques are character-ized by large equipment, generally expensive, and yenerally re-quiring a skilled operator.

Dry chemical techniques utilize reagents stored under dry conditions within a single or multi-layer flat element such that a test liquid will result in a reaction that can be radiometric-ally detected (see U.S. Patent No. 3,092,465, issued June 4, 1963 to Miles Laboratories, Inc.). These techniques are simple to use, but have traditionally yielded only qualitative results. There are several reasons for this that have been well explained in recent U.S. patents assigned to Eastman Kodak (see U.S. Patent Nos. 3,992,158, issued November 16, 1976, 4,042,335, issued August 16, 1977, and 4,066,403, issued January 31 1978). The major reasons are: non-uniform spreading of the fluid over the -;
flat surface; non-uniform penetration of the fluid or analyte into the region where the reagent is

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1 ¦ stcred; and non-uniform effects at the edges of the spread 2 ¦ liquid. The well-known "dip stick" proaucts had to utilize

3 ¦ chemical reactions that proceeded to completion because their 41 transient response characteristics were temperature dependent 5¦ and they were used in a non-thermostatted environment. A new 61 system of dry chemistry has been recently introduced by Eastman 7 ¦ Kodak claiming to overcome many of the traditional weaknesses 8 ¦ of dry chemical methods.

The Kodak technique utilizes flat, multilayexed sheets 11¦ arranged in sequence such that the top layer receives the liquid 12¦ sample and it passes downward undergoing separations and 13 ¦ reactions in a pre-arranged sequence. The sheet is designed 14 ¦ to accept a small volume of liquid and distribute it uniformly over a reprcducible area; the area is less than the total area 16 ¦ of the multi-laminar sheet. Each layer of the sheet is 17 ¦ essentially homogeneous in a direction parallel to the surface;
18 thus, once spread radially (a rapid process) the components 19 oE the liquid can move downward at rates that are essentially 20 ¦ the same in any plane that is parallel to the surface. In 21 ¦ this way uniform reactions, filtrations, etc. can occur. ;~

23 ¦ The analyte is detected in such multilayered sheets 24 by radiometric methods, carried out in a thermostatted environ~
25 ¦ ~ent. This permits one to use kinetic measurements as well 26 as static ones in order to detect analyte concentrations in 271 th liquid samp1e.-1 ~5~8Z~7 1 ¦ Radiation is caused to enter this assembly in a path 2 ¦ which is transverse to the several layers. The radiation is 3 ¦ modified by the analyte or by a com~cne~t cr product of the -¦
41 analyte. For example, the exciting radiation may be paxtially 5¦ absorbed by the analyte or by a component or product of the 61 analyte. The modified radiation may be reflected back trans-71 versely through the laminar assembly or it may pass through 81 t~.e entire assembly. In either case (reflection or transmission) 9 the path of the exciting radiation is very short and is determined by the thickness of the layer in which the exciting 11 radiation encounters the substance which is excited. Since 12 this dimension must be very small to permit rapid measurement, 13 e.g., 10 ~m to 100 /Um the degree of modification of the 14 exciting radiation is quite small. This limits the applicability of this technique to analyses wherein the analyte (or a 16 component or product of the analyte) interacts very strongly 17 with the exciting radiation or it requires the use of very 18 sensitive detecting apparatus. It has been shown to be a 19 useful method for measuring analytes in blood that exist at relatively high concentrations, e.g., glucose, BUN, cholesterol, 21 albumin.

23 Othcr analytical mcthods havc bccn developed th~t 24 utilize rapidly reversible chemical reactions in order to continuously monitor analyte concentrations in biological 26 fluids, or industrial effluent streams, ox ponds, lakes and 27 streams. For exa~le, several methods have been proposed to 23 n asure the oxygen 1evel in blood of critically ill patents.

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1 It is an object of the present invention to provide 2 improvements in analytical procedures and apparatus.
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4 It is a further and more particular object of the present invention to provide an analytical technique (both 6 method and apparatus) by which a laminar assembly of the general 7 type described above can be employed without the limitations 8 inherent heretofore.

10 The above and other objects of the invention will be `
11 apparent from the description below and the appended claims.

13 In accordance with the present invention a core is 14 provided which is transmissive to the chosen energy, which may be electromagnetic (e.g. ultravlolet or visible light), 16 electronic or scnic energy. This core is provided with one or 17 more permeable or semi-permeable sheaths (i.e. permeable to 18 a fluid sample containing the analyte, or permeable to so much 19 of the test fluid as is desired but acting to filter out unwanted components). This part of the apparatus may be referred 21 to as the "sheath structure" signifying that it may consist 22 of one or more permeable or semi-permeable sheaths. As will 23 appear more fully from the description below, there are several 2 functions that may be performed by the sheath structure any 2 one cr more of which may be performed by one or more of the 26¦ dividual sheaths;

1... c,, l 1 lXa~ ;P'7 1 ¦ The core, as .,tated, is transmissive to the chosen 2 ¦ energy and causes that energy to pass in a direction generally 3 ¦ parallel to the surface to which the test fluid is applied.
4 ¦ The core has an "active length" which, as will appear more S ¦ fully hereinafter, is that portion of its length, usually but 6 ¦ not necessarily less than the entire length of the core, 7 ¦ wherein the e~nergy passing through the core is subjected to 8 ¦ the influence of the test fluid and is modified thereby. The 9 ¦ magnitude of this active length is large, and as will appear 10¦ more fully hereinafter it is very large compared to the 11 ¦ thickness of the sheath structure and/or the core~ By this .
12 ¦ arrangement the energy passing through the core along its 13 ¦ active length is subjected to a cumulative, although not 14 ¦ necessarily uniformly cumulative, modification by reason of 15 ¦ the presence of analyte in the test fluid. .
16 l 17 ¦ As will also appear more fully hereinafter, the core, 18 ¦ if permeable to an analyte in a liquid or gaseous test fluid, 19 ¦ or to a product of such analyte, may be bare, i..e~ devoid of 20 ¦ a sheath structure and may therefore be in direc:t contact with 23 he test fluid.

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The physical shape and configuration of the device or apparatus of the invention may vary considerably. In a pre~
ferred embodiment, the device is cylindrical and consists of a central core or fiber which is transparent to the exciting radiation and is surrounded by a sheath structure consisting of one or more concentric layers of absorptive material. Alter-natively the shape in cross section may be polygonal with one or more absorptive layers surrounding the transmissive layer.

In any such configuration, the path of the carrier energy is generally parallel to, rather than transverse to the overlying layer or layers. Many of the figures in the drawings will serve to illustrate various embodiments of the invention. For the most part the carrier energy will be described as electromagnetic radiation.
The invention will be further described with reference to the accompanying drawings, in which:-Figs. 1 to 4 depict an embodiment of the invention wherein a single inner core-outer sheath configuration is used;
Figs. 5 and 6 show typical curves of the relationship of the output of electrical energy produced from the emitted light energy and the analyte concentrations; ~;
Fig. 7 depicts an embodiment of the invention wherein two parallel fiber assay devices (FAD) are used;
Fig. 8 depicts a range limitation of highly sensitive assay systems;
Fig. 9 depic-ts a multiple fiber assay device (MFAD) configuration of the invention;
FigsO 10 to 12 depict a dual FAD designed for glucose determinations;
Figs. 13 and 14 depict a continuous monitoring case for the use of a dual F~D;
Figs. 15 and 16 depict a multi-sheathed single FAD of a multiple FAD system.
Figs. 17 and 18 depict an embodiment of the invention designed for high sensitivity immunoassays;
Fig. 19 depicts a single sheath-inner core embodiment wherein the sheath has a lower refractive index than the core;
Fig. 20 depicts a wicking means for uniformly saturating a FAn;
Fig. 21 depicts a device of the invention designed for enzyme determination in fluid samples;
Figs. 22 and 23 depict embodiments of the invention wherein the fiber is incorporated into a cathe-ter structure.
In Figure ] there is shown one form of the apparatus of the present invention which is generally designated by the reference numeral 10 and which, as shown in the cross sectional view of Figure 2, consists of an inner core of fiber 11 and an outer sheath 12. The core or fiber 11 is transparent to the exciting radiation but, instead of being a conventional optical fiber such as quartz fiber, it is selected so that it is not only optically transmissive but is also permeable to components of an aqueous solution. The outer sheath 12 is of absorptive, semi-permeable material.

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1 To the outer sheath 12 is applied, for example by 2 imrnersion or dipping, a fluid containing the analyte under -3 consideration, for example blood where it is desired to measure 4 one of its low molecular weight components. If it is desired to prevent penetration by large molecules and formed elements 6 such as erythrocytes, platelets, or white cells, the material 7 of which the sheath 12 is constructed may be suitably selected 8 so as to filter out such large molecules and elements.
9 Alternatively an outer layer (not shown) may be provided which is permeable to water, and small molecules but which is 11 impermeable to large molecules and formed elements of the 12 blood. Thus, it is understood that one function of layer 12 13 is to act as an impermeable barrier to those unwanted components 14 of the test fluid.

16 When the device 10 is immersed in the test ~luid, 17 for example, in blood, the fluid will penetrate to the core 18 of fiber 11. Its presence may be detected by illuminating 19 the core with (e.g.) light of a wavelength that is selectively absorbed by the analyte. Thus the diminution of light that 21 emerges from the exit end of the fiber is proportional to 2~ ¦ e concentration of the analyte in the sample fluid.

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l In Figure 1 there is shown diagramatically a system 2 for carrying out such a determination of analyte in an aqueous 3 solvent, including a source of radiation 13, a focussing lens 14 4 and a suitable filter 15 to transmit light of the proper wavelength. At the exit end of the device is a light detector 6 16 and an electrical signal processor 17. The electrical 7 signal proces~sor amplifies the signal from the light detector 8 and may be made from any of several well known and commercially 9 available devices, and may include readout means of visual type and a recorder for a printed readout.

12 It is to be understood that the instruments of 13 Figure l that are used to introduce energy into the analytical 14 apparatus and to measure that which leaves the system can be configured in any of a number of ways depending on the use of l~ the device. The simplicity of use and intrinsically rapid 17 response of the invention suggests that one use will be to 18 make bedside measurements. In such a case a portable ~lnit 19 with rechargeable batteries would be preferred. It is to be further understood that modern electronic data processing 21 methods are so compact that one may further simplify the use 22 of the analytical apparatus by utilizing electronic corrections -23 and calibrations which permit the use of assay methods that 24 are non-linear in their response to the analyte concentration.
In fact, it is possible to assemble several analytical 26 el nts in a paral1el arrangement in a single instrument.

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1¦ The letters "a" and "b" and the lead lines therefrom 21 in Figure l signify the "active length" of energy transmissive 31 device lO. This active length is that portion of tne device 41 which is exposed to analyte, or to a product of an analyte, 51 and along which the flow of energy is cumulatively modified 61 by the analyte or a product of the analyte. The path fxom ¦
71 "a" to "b" (which may be continuous or segmented, is long 81 compared to the thickness of the element lO.

10¦ In the apparatus of Figure l the core ll may be bare, 11 i.e. devoid of a sheath. Thus where, for example, the l2¦ atmosphere or water, an industrial fluid or a biological --13¦ fluid contains the analyte of interest, e.g. a contaminant 14 ¦ such as sulfur dioxide or a nitrogen oxide in stack gas or 15 ¦ a phenolic contaminant, the material of the core ll may be 16 1 selected so that it absorbs such contaminant which modifies 17 ¦ the flow of energy through the core, e.g~ by absorption of a 18 ¦ selected wavelength of light.

20 ¦ Referring now to Figure 3, a system similar to that 21 ¦ of Figure l is shown where the fiber assay device (FAD) lO
22 ¦ shown on a larger and exaggerated scale and with the interior 23 exposed to show the several components including a core 25 24 1 (hereinafter designated by the letter "C") which is transmissive 25 ¦ to ultraviolet radiation and is impermeable to fluids; and a 26 ¦ sheath 26 of gas permea~le material containing an oxygen 27 ¦ quenchable fluorescent dye substance. The index of refraction 28 ¦ ns of sheath 26 is greater than the index of refraction nc f 29 ¦ the core. The outer sheath 27 is oxygen permeable and is 30 ¦ reflective. It may be impermeable to large molecules and to 31 ¦ formed elements such as platelets and red cells in blood.

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1 ¦ Referring to Figure 4, which is a fragmentary cross 2 ¦ section through the FAD, molecules of fluorescent dye are -3 ¦ indicated at 28. Ultraviolet light passes through the core 4 ¦ and some of it is refracted into the sheath 26 where it reacts ¦-

5 ¦ with fluorescent molecules which emit radiation in the visible

6 ¦ spectrum; such emission is isotropic. That visible radiation

7 ¦ which impinges upon the interface between the sheaths 26 and 27

8 ¦ is reflected into the core along with radiation which enters

9 ¦ the core directly. Assuming, as will be the case in practice, ¦

10 ¦ uniformity of distribution of fluorescent molecules in the

11 ¦ sheath 26, during passage through each increment of length of

12 ¦ the core ~X, (X being the distance along the active length

13 ¦ of the core) the radiation passing through the core will pick

14¦ up an increment ~i of emitted light. ~i will not, of course,

15 ¦ be constant inasmuch as the UV radiation is somewhat attenuated

16 ¦ as it passes through the core 25. Nevertheless a cumulative

17 ¦ effect will occur and the lntensity of emitted visible light

18 ¦ ~ ~i will be much greater than the value of ~i emerging from

19 ¦ a small segment of the path.

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21 ¦ When a fluid, for example blood, industrial water

22 ¦ or river wa~er con-~aining oxygen (which is th~ analy~ to be

23 ¦ determined) is applied to the outer sheath 27, it will penetrate

24 ¦ to the sheath 26. The dissolved oxygen in the fluid will quench

25 ¦ fluorescence, therefore, it will diminish the intensity of

26 ¦ fluorescent light emitted at the output end ôf the core. The

27 ¦ emitted radiation passes through the ultraviolet filter 18,

28 ~ then through an opto-electrical detector (O-E.D.) 30 which acts ~2 .

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1¦ as a transducer to convert the emitted light energy into 2 ¦ electrical energy. The emitted electrical energy is processed 31 in a unit 31 resulting in a digital or analog outpu~. A suitable 4 ¦ processor consists of amplifiers, limiters, meters, and elements 5 ¦ for electrical logic, as are well known to those in the 6 ¦ instrumentation business A molecule well known for its 7 ¦ tendency to e~hibit 2 quenching o~ fluorPscence is ~luoranthrene.
8 l 9 ¦ Figure 5 shows a curve typical of such an output.
lO¦ Advantageous features of this system include its low temperature ll ¦ sensitivity, and rapid response time.

13 ¦ Referring now to Figure 6, a plot of analyte 14 ¦ concentration against output of a similar system is shown.

16 ¦ The solid curve represents a mèan calibration curve 17 ¦ in the absence of other molecules. The dotted curve is the 18 ¦ calibration curve in the presence of a molecule that may also 19 ¦ occur in the fluid. Thus, the calibration curve shifts up or 20 ¦ down the vertical axis as a ~unction of the un~nown concentration 21 ¦ of contaminant, Cc. If CA is the concentration of the analyte, 22 we may represent this phenomeon of interference by the expression:
23 OUTPUT ~ (CA + Cc) 24 where ~ represents a proportionality. In order to obtain an output that is independent of Cc one would traditionally have 26 to remove~C by some chemical process, or measure it by an 27 independent method'. However, the present invention lends 28 itself to a double fiber assay (DFA) system which dispenses

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~ ~L~3Lt~ 7 - l 1¦ with the need for calibration, such being shown in Figure 7.
21 As shown in Figure 7 there are two FAD units one of which 33~
3¦ measures the output shown in Figure 5; the other being a unit 34 41 which is designed so as t~ measure the concentration of 5¦ contaminant C. The output of the two fibers can be detected 61 with equal sensitivity because the fibers are illuminated from 71 a single light source. The two signals can be electronically 8¦ subtracted as can be done in commercially available analogue 91 or digital devices indicated generally at 35. Therefore 10¦ without the need for calibration the output is a measure of 11¦ the concentration of analyte.
]21 13¦ Yet another limitation of many highly sensitive 14¦ assay systems (e.g. radioimmune assay) is summarized in 1~¦ Figure 8. The analyte is detected by reaction with a reagent.
16¦ The high specificity of the reaction leads to a steep dose 17 response curve; i.e. for a given reagent concentration the 18¦ output changes over its full range when traversing a narrow 19 ¦ range of analyte concentration. When the range of anticipated 20 ¦ analyte concentration is wide, as denoted by the shaded area 21 ¦ in Figure 7, one must perform several determinations, each 22 ¦ with a different reactant concentration in order to determine 2~ ¦ he ana~yte concentration.

28 ~ ' .2 ~ j, -1 Referring now to Figure 9, a multiple fiber assay 2 device (MFAD) is shown consisting on n such FAD devices, each 3 of which embodies within one of its sheaths a reagent. The 4 reagent concentrations vary from one device ~o the other. It will be apparent that when these fibers are wet by a fluid 6 that there is one reagent concentration resulting in an output 7 which is a r~lection of the analyte concentration. The other 8 fibers either over- or under-react with the analyte yielding 9 unmeasurable outputs. Assume that FADj (j being an integer from one to n) is optimum. The separate outputs of the FADs are 11 converted by O-E.D.s to electrical outputs whlch are separately 12 transmitted to a switching devlce 36 controlled by a micro-13 processor 37 to select the output of FADj and reject the others.

~eferring now to Figure l0, a dual F~ system is 16 shown intended for the determination of the glucose concentration 17 of a body fluid, for example blood. As in Figure 3, sheath 26 18 of each device contains an oxygen quenchable dye and sheath 27 19 is an oxygen permeable sheath which reflects light back into the core. The sheath 27 also functions to prevent penetration 21 of fluorescent molecules from the sample. Alternativel~, an 22 outermost sheath (not shown) may he employed for that purpose.
23 One such device 36a is the control device that measures oxygen 24 (the contaminant); and the other 36b is modi~ied by having in the sheath 27 a quantity of glucose oxidase. Both devices 26 are wetted with a sample simultaneously. Oxygen in both 227 samples penetrates'through sheath 27 to sheath 26 and quenches ~2 ~ ~ 5 4 ~
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., 1 i fluorescence. However, glucosè oxidase in sheath 26 of device 2 36b causes reaction of a port1on of the oxygen with the glucose 3 and therefore diminishes the oxygen available for quenchlng 4 fluorescence. The rate of oxidation is proportional to the concentration oE glucose. Therefore, the output of the device 6 36b will be greater than that of the device 36a and the 7 difference is~ measured by the output of the device. The 8 system includes a filter 37, OED devices 38 and a processor 39.

Referring now to Figure ll, the output of each O-ED
11 in Figure l0 is plotted as a function of time following 12 addition of a blood sample to the DFAV, presuming that the 13 oxygen level of the blood is higher than that o~ ambient air.
14 In the case of the oxygen sensor 36a the excess oxygen is lost 1~ by diffusion to the air and ul~imately the output (dotted) 16 curve reaches a steady value of fluorescence that reflects 17 equilibration with air. However, the modified fiber 36b 18 consumes the oxygen more rapidly due to its reaction with the 19 glucose in the sample, causing a greater rise in fluorescence.
When all the glucose that is present in the blood has been 21 ¦ consumed, this fiber also equilibrates with ambient air.
22 ¦ Thus, the two curves ultimately merge. Figure 12 is a curve 23 ¦ representing the integration of the space between the two 24 ¦ curves and therefore its area is a measure oE the total amount 26 ¦ f glucose in the sample.

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-1 Referring now to Figure 13, a plot is shown in the 2 case of continuous monitoring where the dual FAD device is 3 implanted within a patient. ~See also Figures 22 and 23 below 4 and the description thereof). The first portion of the curve represents normal variations in glucose level in the blood 6 and the large increase represents a large increase in glucose 7 level after, ~or example, a patient has had a meal. The 8 solid curve represents the o~tput of device 36b and the broken 9 line curve represents the output of device 36a. Figure 14 is a plot of the difference between the curves of Figure 13, 11 therefore of the variation with time of the ~lucose level of 12 the patient.

14 Referring now to Figure 15, a single FAD of a multiple FAD system is shown as an example of an immunoassaY
16 described in Figure 9 above. The FAD comprises an innermost 17 sheath 41 whose index of refraction ns is greater than the 18 index of refraction nc of the core and which contains an 19 antigen-fluorescein complex designated as A* in quantity sufficient to fully saturate an antibody located in sheath 43.
21 Sheath 42 is a sheath which is hydrophilic and contains 22 reflective particles and which is impermeable to antigen (A) 23 when dry but permcable when wet. Sheath ~3 is hydrophilic 24 and contains as a reagent, an antibody Ab to the anti~ens A*
and A. (A is the analyte of interest.) The outermost sheath 44 26 is microporous. A sample of f luid containing the antigen A

28 is added to sheath'44.

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-1 ¦ The kinetics of diffusion and reaction of the system 2 ¦ are as follows: .
3 l 4 ¦ A diffuses through sheath 44 into sheath 43 and .
5 ¦ A* diffuses through sheath 42 into sheath 43. The following 6 ¦ competing reversible reactions occur in sheath 43.
7 I v 8 (1) A* ~ Ab~_ _ A* - Ab 91 ~2) A + Ab~_ A - Ab 11 The antibody complexes A*-Ab and A-Ab cannot diffuse out of i 12 ¦ sheath 43. There is enough A* in sheath 41 to saturate the ;
13 ¦ ~b in shcath 43. ~ssuming the c~se o~ no antigen ~ in the 14 ¦ sample, reaction (1) will proceed to a state of equilibrium at which time the rate of diffusion of A* out of sheath 41 16 ¦ will equal the rate of diffusion of A* back into sheath 41.
17 At that time the output will become constant, as shown by 18 ¦ the lower curve in Figure 16. Virtually all of A* is bound 19 ¦ to Ab, yielding a low level of fluorescence. ~he middle 20 ¦ curve represents the case when the sample contains a finite 21 ¦ quantity of A; and the upper curve represents the case where 2223 A A* so that very little Ab-A* is formed.

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-1 ~ The spacing of tle curves in Figure 16 is arbitrary.
2 ¦ It will be apparent -that in actuality this spacing will depend 3 ¦ upon the concentration of A* in sheath 41 and the concentration 4 ¦ of A in the sample. By employing a bundle of EADs each 5 ¦ containing a different concentration of A* in sheath 41, one 6 ¦ of the devices will contain an optimum concentration of A*
7 ¦ such that the~ spacing of the curves is optimum. By means 8 ¦ of the logic selector 45, the output of that device will be 9 ¦ selected and the others rejected. By calibration the c~ncen-lO ¦ tration of A in the sample may be determined.

12 ¦ Yet another method for utilizing the specificity 13 and high sensitivity of immunoassays is shown by Figures 17 14 ¦ and 18. Figure 17 illustrates in detail a single element of 15 ¦ an array of several FADs, each containing different concentratio~c 16 ¦ of antibody Ab. The core c is permeable to the product Y of 17 ¦ a reaction E
18 I X~ Y
19 ¦ wherein E is an enzyme r X is high molecular weight and Y is low molecular weight. Sheath 50 is permeable to Y but not 21 to X; sheath 51 contains an antibody Ab and an antigen-enzyme 22 complex A-E wherein the enzyme E is active. This complex 23 reacts with antibody in accordance with the following reversible 2 reaction.
A -- E + Ab ~ A - E Ab 26 Enzyme E is inactive in A - E - ~b, Sheath 50 has a lower 27 index of refraction than the index of refraction of core c, 28 in order to reflect all the light along the axial path within 23 the core.

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. ~L5~8;~t7 1 This system is intended to measure analyte A, which 2 is an antigen, in a fluia sample. Sheath 51 is microporous 3 and permeable to A. When sheath 51 is wetted with a sample 4 containing the antigen A, it diffuses into sheath 51 but no ¦
further. There it reacts with Ab, displacing A-E in direct 6 proportion to their relative concentrations. The A-E that is 7 released cata-lyzes the reaction, forming Y which then diffuses 8 into the core where it is detected by light that is transmitted ¦
9 alcng the core. I~ the wavelength of light is selected ~or ~ maximum absorbance by Y and sheath 51 contains an excess of 11 X the method will be highly sensitive and specific for the 12 presence of A in ~he sample fluid. Figure 18 describes the 13 electrical output of the system following the application of 14 the sample to sheath 51. As in Figure 9 this analytical 15 method will require the use of a switching system so as to ;~
16 measure the curve for the element that contains the optimum 17 quantity of Ab-A-E. The rate of change of output from that 18 element will then be proportional to the concentration of A
19 in the sample.

21 Re~erring now to Figure 19, an FAD is shown comprising 22 a core c and a sheath 52 ha~ing a lower refractive index than 2~ the core or containing reflective material ~or surrounded by 24 a reflective sheath, not shown). The sheath 52 is permeable to analyte (i.e. an antigen, A) but not to higher molecular 26 weight components of the sampl2. The core contains antibody Ab 28 bonded to a dye D ~o form the complex Ab-D. Light at the ~" c`

1¦ absorption peak of the dye is transmitted through core c. The 21 dye e~hibits the property that its absorptive powers are -- .
31 changed when the antigen binds to Ab-D. When sample is applied 41 to sheath 55, antigen A (the analyte of interest) diffuses 5¦ through this sheath and into core c, which is selected for 61 this purpose. The reversible reaction ¦
71 ~ Ab-D ~ A---~aAb-D-A
8 ¦ occurs.
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10¦ This causes a shift in the amount of liyht transmitted 11¦ through the element. By using a set of elements with varying 12 ¦ concentrations of Ab-D one may select the optimum element for 13¦ the level of A in the fluid. Since the reaction of A with ,61 A -D is reversible this may be used for on~inuous moni~oring.

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~ 48~'7 ~ ) 1 When measuring the analyte concentration in ~ discrete 21 fluid sample, it is necessary to apply a reproducible volume 31 of fluid uniformly to the analytical element described herein.
41 While this can be achieved by a skilled operator utilizing 51 micro pipettes, it is intended here to describe another means ;
61 whereby this may be achieved in a simpler fashion. It will 71 be seen that.this is a unique feature of the elements described 81 hcrein.

10¦ Referring now to E'igure 20, a means for uniformly 11¦ saturating an FAD is shown which employs the phenomonen of 12 wicking. The FAD shown consists of a core c and a single 13¦ sheath S. The same means may be employed with FADs containing 14¦ several sheaths and two or more FADs.

16 ¦ A base 60 is shown comprising a rigid support 61.
17 ¦ Adhered to the top of the base is a hydrophilic coating 62 18 ¦ which is wetted with the sample which is used in excess to 19 ¦ that needed to wet the FAD. The sample fluid will diffuse 20 ¦ thr~ugh the base to the FAD. It will ascena by capiliary 21 1 action to the top of the F~D. Provided the height of the 22 ¦ device is not excessive, the wicking or capiliary effect will ~41 esult in a uniform wotting of the sheath S.

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1 The analytical device described herein may be adapted 2 to permit the measurement of large molecular weight analytes 3 that do not norm~l]y permoate shea~hs in a selective -Fa~shion.
4 Figure 21 describes such a device that is useful for determining the quantity of enzyme in a fluid sample. The core c is 6 pexmeable to a dye D and is transmissive to light with a 7 wavelength selected for maximum absorption by D. The dye D
8 is chemically bound to a hydrophilic polymer that is incorporated 9 into sheath 57. Sheath 58 is permeable to D, impermeable to higher molecular weight components of the fluid, and selected 11 from materials with a lower refractive index than the core.
12 The chemical bond between the dye and the polymer will be 13 selected so it can be selectively degraded by the enzyme in 14 the fluid that one wishes to detect. Thus, for example, if one wishes to measuxe the concentration of esterase in 16 plasma, utilize an ester linkage between the dye and the 17 polymer. This can be accomplished e.g. by use of any of the dyes 18 classed as acidic dyes, reacted with the hydroxyl groups in 19 gelatin~
21 When the element is wet by a sample, D will be 22 I enzymatically released and diffuse into the core where it 23 ¦ will be detected by a decrease in light transmission through 24 ¦ the element. The rate of change in light transmission will 26¦ e proportional to the concGntration of the erzyme ~ ' c ~ ) I '~4 1 ¦ Onc oE thc advantages of -the ~ib~r cmbodiment o:E ~h~
2 ¦ invention is that it can be readily incorporated in a catheter 3 ¦ for insertion into the body, for example, into a vein or an 4 ¦ artery. A suitable form of catheter-fiber structure is shown ¦
5 ¦ in Figure 22.

71 Re~érring to Figure 22, a catheter 70 is formed in 8 ¦ two parts, namely a tip portion 71 and a bod~ portion 72.
9¦ These are made of suitable material such as polypropylene, 1~¦ polyethylene, silicone rubber, polyvinyl chloride, or poly 11¦ (ethylene vinylacetate) and may have a diameter of, for example, 12¦ .5 to 1.5 mm, appropriate ~or a particular purpose such as, 13¦ for example, insertion into a vein or an artery. A cylindrical 14¦ fiber-shaped device 73 is affixed, for example in a spiral 15¦ configuration as shown, to the tip 71 such that it is exposed 16¦ to the body fluid when the catheter is implanted. The fiber 73 17 ¦ has protruding tips 73a and 73b. The exposed fiber 73 (either 18 ¦ all or a portion of it) is susceptible to penetration by an 19 ¦ analyte and is of a construction such as that shown in any of 201 Figures 2, 3, 7, 9, 10 or 19, hereinabove. The body 72 of the 21 ¦ catheter is formed with parallel passages 75 into which fiber 22 ¦ extensions 73a and 73b are inserted, these being recessed so 23 as to form sockets 77. The fiber extensions 73a and 73b may 24 ¦ be bare optical fiber or they may be coated with a protective 2~ ¦ coating. When the tip 71 and the body portion 72 are assembled 26 ¦ in operative condition the protruding tips 73a and 73b will be 27 ¦ received in the sockets 77 and will be in physical and optical 28 contact with the fiber extensions 75a, thereby providing a 291 continuous optical path from a source of exciting radiation 301 to the output end.

321 .

I - 23 - ~

IL ~ , I .
1 ¦ liith such a cathete.r continuous moni.toring of a body 2 ¦fluid is possible with an appropriate readout to inEorm the 3 ¦diagnostician, either visually or by printout or by both means.
4 I ~ `
5 ¦ Referring now to Figure 23, another form of ca-thetex 6 ¦is shown which comprises a housing 80 through which an optical 7 ¦~iber 81 pass~és and which also supports an extension 82 of the 8 ¦optical fiber having a po~tion 82a exposed to body fluids.
9 ¦'l'he exposed portion 82a may be constructed as in any of Figures 2, .::
10 13, 7, 9,-lD or 19 above. The projections 81a of fibers 81 and 82, Il ¦project into sockets 83 in a tip 84 which embodies a prism 85.
12 ¦When the tip 84 and the housing 80 are brought together a 13 ¦continuous light path is provided through the optical fiber 81 ¦t prism and the opt cal riber 8la 29 .

.. . - - - I L.. ~

~ 82'~
I ~

1 General Discussion 3 It will be apparent that the invention may be manifested 4 in numerous forms and is capable of numerous applications.
~ Structurally there are at least two elements one of which, 6 exemplified by the transmissive core is a medium for transmitting ¦
7 energy in continuous form such as electro-magnetic energy, e.g. ¦
8 ultraviolet light or visible light, electric current (AD or DC) 9 or sonic energy. The other element tor elements) is a sheath or sheaths. The configuration is preferably rod-like with a 11 transmissive core in the form of a ~iber typically about lO;~m 12 to 1 mm in diameter with one or more sheaths surrounding the 13 core typically about lOf~M to 100~uM in thickness. The active 14 length of the device (i.e., the length which is wetted by the ]5 test fluid) may vary from about 0.5cm or less to 1 meter or 16 more. In most bioassay applications the length will not exceed 17 about lOcm. Departures from such dimensions are permissible.
18 ~s stated above, other configurations, e.g. polygonal 19 configurations, are permissible.

21 The core, besides its transmissivity and shape, may 22 have the following characteristics: It may be impermeable to 1 23 aqueous liquids. If permeable it may contain a reagent, e.g.
24 a dye, or it may be devoid of a dye and be a receptor for 2 permeation by a reagent. Suitable materials for impermeable 2 cores are quartz, polymethylmethacrylate, polycarbonate, 27 polystyrene, ctc. If the core is permeable, suitable materials 2 are plasticized polyvinyl chloride, polyurethanes, polypropylene, 2 nylon, gelatin, polyvinyl alcohol, natural rubber, butyl rubber, 3 cis-polyisoprene, poly (ethylene vinyl acetate). The index of 3 refraction of the core, nc, may be greater or less than that 3 of the adjacent sheath.

1 ,, c ~ ) -1 The material and construction of the sheaths will 2 depend upon their function. In all cases when a reaction 3 occurs in a sheath or where a liquid must diffuse into, through 4 or out of a sheath, it should be permeable to water. Permeable I -sheaths may be permeable to large and small molecules and to 6 finely divided solids suspended in a liquid sample, or they 7 may be select-ive with regard to permeability such that unwanted 8 large molecules, etc. are excluded, as taught in the book by 9 Crank and Park, "Diffusion in Polymers." One or more sheaths may contain a reagent or a precursor of a reagent and such 11 reagent or precursor may be immobile or mobile and it may 12 undergo a reaction such as enzymatic cleavage to render its 13 product mobile or it may undergo a reaction such as antibody-14 antigen reaction which makes it immobile. All such physical states of sheath material and reagents are possible and methods 16 of synthesis or forming are well known to those practi~ed 17 in the art.

19 Elements of the dimensions suitable for this invention e.g. (1-100~m diameter) can be made by normal ~iber-forming 21 techniques; the Encyclopedia of Polymer Science and Technology 22 provides an adequate description of these techniques. Briefly, 23 there are three major techniques: melt forming, wet forming, 24 and dry forming. Melt forming is used for thermoplastic 2~ polymers( e.g~ polypropylene) that exhibit a low viscosity 26 when heated above their melting point. Wet process forming 27 consists of extrud~ng a solution of the poly~er in a solvent 28 and passing the fiber ~hrough a bath of a second solvent. This 2 bath solvent has the property that it ~ill dissolve the polymer ` 1-,. c ~) l~S~8~''o~

1 solvent, but nQt the polymer; thus the solvent is extracted 2 by the ba-th solvent, leaving a pure polymer fiber. Dry forming 3 consists of extruding a solution of polymer and volatile solvent 4 into a heated air stream, where the solvent evaporates.
.
6 Adaptations of these processes can be used to coat 7 the core fiber with the sheaths. If the central fiber is made 8 from a high melting point material (e.g. glass), one could 9 coat it with a melted polymer. It is more likely that one will use polymer solutions, especially when it is necessary 11 to incorporate chemicals that are used to react with analyte 12 or otherwise participate in the required chemical analysis.
13 Many such reagents degrade under conditions of high temperature.

lS If, for example, one wanted to form an assymmetric 16 microporous membrane as the outer sheath as is required for 17 the enzyme assay described above (Figure 21~, one could use 18 wet forming. The polymer and dye-polymer conjugate would be 19 dissolved in a solvent and coated onto the fiber by pulling the fiber (c, coated with sheath 58) through an orifice that 21 has the solution on the upstream side. On the downstream 22 side is a ba-th with a solvent that is selected to elute the 23 primary solvent leaving a polymer sheath 57 that has microscopic 24 holes. The bath solvent is selected so as to be a non-solven~
26¦ f oth the po1ym~r and the dye-polymer conjugate.

28 .

.
31 ~ .

115~

l Or,ce formed as a continuous, long coated fiber, it 2 will be cut into short lengths and mounted in a holder, whose 3 purpose is to align the ends of the fiber into a reproducible 4 position, to provide a simple means to insert the fiber into the instrument (cf. Figure l), and a means to protect the 6 fiber during storage and use. The housing could also be within 7 a catheter f~r use as a monitoring instrument as shown in 8 Figures 22 and 23. The catheter could be insertea into the 9 fluid or body cavity of interest; it contains highly conductive ¦
input and output fibers that are coupled to each end of the ll coated fiber assay system so as to introduce exciting radiation 12 and to recover the analyte-modified radiation. These housings 13 will generally be made of plastic material and are understood 14 to be fabricated by the standard methods available, namely, injection molding, transfer molding, extrusion, epoxy molding, 16 or heat forming.

18 Reagents, reagent pre-cursors, reflective material, 19 etc. which may be incorporated in various sheaths include the following: enzymes, O2-quenchable fluorescent molecules 21 (e.g. fluoranthrene), antibodies, dyes, fluorescen-t dyes, 22 reflec~ive materials (TiO2, SiO2, etc.), dye-polymer products.
23 These and other reagents, reagent pre-cursors and reflective 24 materials are well known to those skilled in the art.

26 It will there~ore be apparent that new and use~ul 27 apparatus and methods are provided for chemical and biochemical 29 analyses.

Claims (35)

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

1. Apparatus for analysis comprising:
(a) an energy transmissive element having a longitudinal dimension and a transverse dimension and capable of transmitting energy along said longitudinal dimension, (b) a source of energy capable of transmission through said element along its longitudinal dimension, such energy being selected so as to be capable of modification by the presence of an analyte contained in a fluid external to the element, (c) an energy sensing and processing means capable of detecting and measuring the change of transmitted energy resulting from such modification, (d) said element (a), source (b) and means (c) being arranged so that energy from the source is transmitted through the element (a) along its longitudinal dimension in a path wherein the transmitted energy is modified by the presence of analyte in an ambient fluid and so that the modified energy emerging from such path is sensed and measured by said means (c), (e) said path being sufficiently longer than said transverse dimension that there is a rapid response of modification of transmitted energy to the influence of ambient analyte, such modification being cumulative along said path.

2. The apparatus of Claim 1 wherein said element is permeable to the analyte, to the product of an analyte or to a reagent.

3. The apparatus of Claim 1 wherein said element is directly exposed to ambient analyte which permeates the element and thereby cause modification of transmitted energy.

4. The apparatus of Claim 1 wherein said element (a) is surrounded by a sheath structure including at least one permeable sheath and the apparatus includes a reagent which is reactive with an analyte or with a product of an analyte.

5. The apparatus of Claim 4 wherein the reagent is incorporated in the sheath structure.

6. The apparatus of Claim 4 wherein the reagent is incorporated in the element (a).

7. Analytical apparatus comprising: ¦

(a) a first lamina (b) a second lamina surrounding and in operative contact with the first lamina said first lamina being transmissive to energy capable of modification by events during analysis occurring in or transmitted through said second lamina, said second lamina being permeable to aqueous liquids and at least certain of their dissolved components the configuration of said apparatus being such that the dimension perpendicular to the interface of the laminae is small compared to the dimension parallel to such interface said second lamina being selected from a material which provides a zone for occurrence of events, or for transmission of events, resulting from wetting of the apparatus with a fluid sample containing an analyte, such events being capable of modifying the energy transmitted through said first lamina such modification being cumulative as energy is transmitted through the first lamina.

8. The apparatus of Claim 7 wherein the first lamina is impermeable to aqueous liquid.

9. The apparatus of Claim 7 wherein the first lamina is permeable to small solute molecules.

10. The apparatus of Claim 8 wherein the second lamina contains a reagent which is reactive with the analyte or with a product of the analyte.

11. The apparatus of Claim 7 wherein the first lamina is transmissive to electromagnetic energy and the apparatus includes in one of its laminae a reagent which is reactive with an analyte or with a product of an analyte to result in a product which modifies the output of electro-magnetic energy by the first lamina.

12. Analytical apparatus comprising:

(a) a core which is transmissive to energy (b) a sheath structure including at least one permeable sheath surrounding and in operative relation to said core, said core providing a path for transmission of energy which is large compared to the thickness of elements (a) and (b), and (c) a reagent embodied in said apparatus of a nature and so located that upon wetting of the apparatus with a fluid sample containing an analyte, a product resulting from direct or indirect action of the analyte upon the reagent causes a modification of the energy passing through said core, such modification being a measure of the concentration of the analyte and being cumulative along said path.

13. The apparatus of Claim 12 wherein the core is transmissive to electromagnetic energy and is impermeable to aqueous liquid and the reagent is located in a sheath surrounding the core.

14. The apparatus of Claim 12 wherein the core is permeable to analyte and contains the reagent.

15. The apparatus of Claim 12 wherein the core is permeable to aqueous liquid and wherein the reagent is contained in a sheath surrounding the core and results, when acted upon directly or indirectly by the analyte, in diffusion of a substance into the core which modifies the transmitted energy.

16. The apparatus of Claim 14 wherein the reagent is a dye.

17. The apparatus of Claim 15 wherein the reagent is a dye.

18. Apparatus as in Claim 12 including a plurality of successive sheaths one of which contains the reagent.

19. Apparatus of Claim 12 wherein the index of refraction of the core is less than that of the adjacent sheath, said adjacent sheath contains a reagent which is acted upon by electromagnetic energy diffracted from the core and reflective means is provided to reflect radiation back into the core including radiation of a different wavelength than incident radiation.

20. Apparatus of Claim 19 including an outermost sheath which is microporous and acts to exclude large molecules from the inner sheath or sheaths.

21. Apparatus including a pair of devices as defined in Claim 7, radiation input means for separate input of energy of the same type into each device, one such device containing reagent and the other being devoid of reagent, and means connected to the output of each device to subtract one output from the other to eliminate background output and to measure only modification of output due to analyte.

22. Apparatus including a multiplicity of devices as defined in Claim 7 each containing a reagent appropriate for the intended analysis but in different concentrations, together with means for selecting the output of that device whose output is optimum for purposes of the analysis and rejection of outputs of the other devices.

23. Apparatus including a device as defined in Claim 12 in the form of a catheter insertable into the body of a patient to monitor a bodily function and including terminals for connecting the input of the core to a source of energy and connecting the output of the core to equipment for measuring the fluctuation of such function.

24. Apparatus including a device as defined in Claim 12 and means for uniformly wetting the sheath structure with a sample by capillary means.

25. A method of analysis comprising:

(a) providing an energy transmissive element defining a path for transmission of energy, the length of said path being much greater than the transverse diminsion of the element, (b) causing energy to be transmitted through said path, such energy being selected so as to be modified by the presence of an ambient analyte, (c) modifying the thus transmitted energy by exposure of the element along said path to the influence of ambient analyte, such modification being cumulative along the length of said paths, and (d) sensing and measuring the change of energy emerging from said path caused by such exposure and modification.

26. The method of Claim 25 wherein said energy is electromagnetic energy, which is modified by one of the phenomena: absorption, fluorescence, scattering.

27. The method of Claim 26 wherein said element is in the form of a core transmissive to electromagnetic energy and is surrounded by a sheath structure which is permeable to the analyte.

28. The method of Claim 27 wherein the core permeable and the core or the sheath structure contains a reagent which is reactive with the analyte or with a product of the analyte.

29. The method of Claim 28 wherein the reagent is incorporated in the core.

30. The method of Claim 28 wherein the reagent is incorporated in the sheath structure.

31. The method of Claim 27 wherein the core is impermeable to the test fluid and a reagent is incorporated in the sheath structure which is reactive with the analyte or a product of the analyte.

32. The method of Claim 25 wherein the energy transmissive element is in the form of a transmissive core and is surrounded by a permeable sheath structure including at least one sheath and which is permeable to a fluid containing the analyte, there being a reagent incorporated in the core-sheath structure which is reactive with the analyte or with a product of the analyte.

33. A method of analysis comprising:

(a) providing an energy transmissive core surrounded by a permeable sheath, one such component containing a reagent which is reactive with an analyte or with a product of an analyte (b) the configuration and the transverse and longitudinal dimensions of the core-sheath structure being such that energy can be transmitted through the core in its longitudinal direction in a path which is greater than the transverse dimension of the core-sheath structure, such dimensions being selected so that when a fluid sample containing an analyte is applied to the sheath there is a rapid response of modification of energy transmitted through the core and such that the modification is cumulative along such path (c) exposing the core-sheath structure to a fluid containing the analyte (d) causing energy to be transmitted through the core along its longitudinal dimension, such energy being selected to be modified by events occurring as a result of such exposure and (e) sensing and measuring the resulting modification of energy.

34. The method of Claim 33 wherein two such core-sheath structures are employed one of which serves to measure analyte plus a contaminant and results in an output which does not discriminate between the analyte and the contaminant, the other such structure serving to measure the contaminant only, said method including processing the output of the two core-sheath structures and subtracting the output of one from that of the other so as to measure the analyte concentration.

35. The method of Claim 33 wherein a multiplicity n of core-sheath structures are employed each of which includes a reagent appropriate for the analyte which is to be measured, said core-sheath structures containing such reagent in n different concentrations, said method further comprising so processing the outputs of the n core-sheath structures that the output of structure j (j being an interger from 1 to n) is selected and the other outputs are rejected, the output of structure j being most suited to measurement of the analyte.