Urinalysis device and dry reagent for quantitative urinalysis

Abstract

A method of quantitatively determining the concentration of at least one analyte in a sample by: (i) adding a portion of the sample to a first analyte assay formulation and to an analyte assay reference formulation to generate a first analyte sample and analyte reference sample and determining the concentration of the at least one analyte in the sample; and/or (ii) adding a portion of the sample to a second analyte assay formulation and determining the concentration of the at least one analyte in the sample, as well as formulations, kits of parts, systems and computer implemented methods associated with the method.

Claims

1. A formulation for use in analysis of a concentration of creatinine in a sample, the formulation comprising: a strong base in a concentration of from about 40 to about 80 g/L; a buffer in a concentration of from about 50 to about 250 g/L; at least two surfactants in a concentration of from about 0.1 to about 20 g/L; a compound that reacts with creatinine to generate a product; and water; wherein the at least two surfactants comprises an anionic surfactant in a concentration from about 0.1 to about 10 g/L and a cationic surfactant in a concentration from about 0.1 to about 10 g/L, and wherein the cationic surfactant includes a quaternary ammonium moiety.

2. The formulation of claim 1, wherein the compound that reacts with creatinine to generate a product is dinitrobenzoic acid or picric acid.

3. The formulation of claim 2, wherein a concentration of the compound that reacts with creatinine to generate a product is from about 1 to about 5 g/L.

4. The formulation of claim 1, wherein the compound that reacts with creatinine to generate a product is dinitrobenzoic acid.

5. The formulation of claim 4, wherein a concentration of the compound that reacts with creatinine to generate a product is from about 1 to about 5 g/L.

6. The formulation of claim 1, wherein a ratio of cationic surfactant:anionic surfactant is 1:5.

7. The formulation of claim 1, wherein the formulation is lyophilised.

8. The formulation of claim 7, wherein the formulation further comprises a bulking agent in an amount of from about 1 to about 40 weight % of the formulation.

9. The formulation of claim 8, wherein the bulking agent is selected from one or more of the group consisting of sugar-mannitol, lactose, and trehalose.

10. The formulation of claim 1, wherein a concentration ratio of the buffer:strong base in the formulation is from 0.5:1 to 2.5:1.

11. The formulation of claim 1, wherein the cationic surfactant is selected from one or more of the group consisting of cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide, and hexadecyltrimethylammonium bromide.

12. The formulation of claim 1, wherein the anionic surfactant is selected from one or more of the group consisting of sodium dodecyl sulfate, polystyrene sulfonates, linear alkylbenzene sulfonate, secondary alcohol sulfonate, alcohol olefin sulfonate, and alcohol sulfate.

13. The formulation of claim 1, wherein the buffer is selected from one or more of the group consisting of K.sub.2HPO.sub.4, Na.sub.2HPO.sub.4, and borate.

14. The formulation of claim 1, wherein the strong base is NaOH and/or KOH.

15. The formulation of claim 1, wherein a ratio of cationic surfactant:anionic surfactant is from 1:2 to 1:10.

16. The formulation of claim 1, wherein a ratio of cationic surfactant:anionic surfactant is from 1:3 to 1:7.

Description

DESCRIPTION OF THE ATTACHED DRAWINGS

(1) FIG. 1: Sensing principle of albumin

(2) FIG. 2: Sensing principle of creatinine

(3) FIG. 3: Dry reagent preparation

(4) FIG. 4: Cuvette cartridge and cuvette holder

(5) FIG. 5: Sample introduction device

(6) FIG. 6: Sample introduction structure

(7) FIG. 7: Cuvette holder and optical module

(8) FIG. 8: Assay method

(9) FIGS. 9A-9C: BCG dry-reagent: Compensation of pH effect

(10) FIGS. 10A-10C: BCG dry-reagent: Compensation of temperature effect

(11) FIG. 11: Calibration curve of BCG dry-reagent

(12) FIG. 12: Calibration curve of BCG dry-reagent with real urine sample before data processing

(13) FIG. 13: Graph of A.sub.S versus A.sub.R

(14) FIG. 14: Calibration curve of BCG dry-reagent with real urine sample after data processing

(15) FIG. 15: Calibration curve of DNBA dry-reagent

(16) FIG. 16: A system for generating a calibration curve for use in quantitatively determining the concentration of albumin in a urine sample executes a calibration process, according to an embodiment of the invention

DESCRIPTION OF INVENTION

(17) It is desired to provide a simple and compact point-of-care urinalysis system using wet, or more particularly, dry reagents. In particular, the reagents may be suitable for use with a point-of-care analysis system that can simultaneously measure a number of analytical samples.

(18) For example, such a system may comprise the use of a contact image sensor (CIS) module having a light emitting diode (LED) to emit light, the wavelength of the light is preconfigured to match the absorption wavelength of the reaction product of a urinalysis reagent and target analyte, an optical cuvette having the reflective surface at the far side of the cuvette from the CIS module, a freeze-dried urinalysis reagent being disposed in the optical cuvette, a urine distribution structure having reservoir to distribute sample urine into individual optical cuvette and having solution stopping structure to control the volume of distributed solution separately.

(19) Chronic Kidney Disease (CKD) can be diagnosed in its early stages by measuring microalbuminuria. Such tests can be performed by determining urine microalbuminuria and creatinine concentrations and their corresponding albumin/creatinine ratio (ACR).

(20) While the tests used herein may be used to analyse a urine sample, the tests are also suitable for analysing samples from other sources (e.g., saliva, blood etc). As such, the term urine microalbuminuria may be replaced throughout the specification by the term albumin.

(21) In order to detect ACR, two different reagents are used for urine microalbuminuria and creatinine measurements, respectively. Bromocresol green (BCG) is used for urine microalbuminuria detection, while 3,5-dinitrobenzoic acid (DNBA) is used for urine creatinine detection.

(22) Microalbuminuria present in a urine sample reacts with BCG to form a colored complex (FIG. 1). The intensity of the color, measured at a wavelength of 625 nm, is directly proportional to the microalbuminuria concentration in the urine sample.

(23) For the microalbuminuria assay, two types of wet or, more particularly, dry reagents are prepared for sampling and referencing. The mixtures of the dry reagents (sampling and referencing) and the urine sample are measured concurrently by colorimetric assay. The sampling reagent is used to measure the intensity of the color obtained by the BCG-albumin complex. The referencing reagent is used to reduce or remove pH and temperature effects and interference by applying a protein denaturing method to the urine sample. That is, the microalbuminuria assay applies a protein denaturing method, where the dry reagent for referencing contains a surfactant to denature albumin, so that denatured albumin doesn't react with the BCG present in the referencing reagent.

(24) Creatinine present in a urine sample reacts with DNBA in alkaline medium to form a purple-red complex (FIG. 2). The rate of complex formation at wavelength of 525 nm is directly proportional to the concentration of creatinine in the urine sample. Alternatively, DNBA may be replaced by picric acid.

(25) The creatinine assay only uses a sampling reagent for measuring creatinine. The creatinine sampling reagent applies a buffer addition method to minimise the interference from urine pH, while the temperature effect is compensated by a temperature factor in the calibration curve and the urine background effect is eliminated by measuring the rate of complex formation. Current, devices are tailored so as to operate within the reaction parameter of conventional formulations. This may result in a more complicated automated device, as differing sensors may need to be used to analyse the reaction products of the different reagents used to detect analytes in urine (e.g., colourimetric analysis).

(26) As described in more detail below, it is important to control the kinetics of the interaction between DNBA and creatinine. If the reaction between DNBA and creatinine is too fast or too slow for the device being used, then the device will not be able to provide an accurate analysis. In addition, as the mixture of DNBA and creatinine has a tendency to form bubbles, the analysis may not be accurate due to light scattering. The formulations disclosed herein has been formulated in order to overcome such problems associated with the analysis of the sample and the reagent.

(27) Dry Reagent for Creatinine Assay

(28) A buffer addition method is applied to the DNBA dry-reagent to remove the effect of urine pH. DNBA reacts with creatinine under alkaline conditions, and a purple-colored complex is generated. The sampling reagent contains DNBA, a strong base, a buffer, a bulking agent, an anionic surfactant and a cationic surfactant. The specific chemicals used can be chosen from the list below (Table A), and the concentration of each chemical can be within the specified range.

(29) TABLE-US-00001 TABLE A Range of Concentration Chemical Category Creatinine-Sampling reagent Strong base 40-80 g/L Buffer 50-250 g/L Bulking agent 1-40 wt % (10-400 g/L) Anionic surfactant 0.1-10 g/L Cationic surfactant 0.1-10 g/L DNBA 1-5 g/L

(30) Chemical List for creatinine assay 1) Strong base; NaOH, KOH. 2) Buffer; K.sub.2HPO.sub.4, Na.sub.2HPO.sub.4, Borate. 3) Bulking agent; Sugar (e.g., mannitol, lactose, trehalose). 4) Anionic surfactant; especially anionic sulphated/sulphonated surfactant; Sodium dodecyl sulfate (SDS), Polystyrene sulfonates (PSS), linear alkylbenzene sulfonate (LAS), secondary alcohol sulfonate, alcohol olefin sulfonate, alcohol sulfate. 5) Cationic surfactant; Cetylpyridinium chloride (CPC), Benzalkonium chloride (BAC), Benzethonium chloride (BZT), Dimethyldioctadecyl-ammonium chloride, Cetrimonium bromide, Dioctadecyldimethyl-ammonium bromide (DODAB), Hexadecyltrimethylammonium bromide (CTAB).

(31) Without wishing to be bound by theory, it is suspected that the addition of a buffer to the strong base results in the kinetic modulation of the chemical reaction between DNBA or picric acid and creatinine. As will be appreciated, the ratio of buffer and sodium hydroxide may be selected to suit the analytical device. This enables the device to produce a more accurate reading. For example, the concentration ratio (in Molar) of the buffer:strong base may be from 0.5:1 to 3.2:1, such as from 0.8:1 to 2.5:1 (e.g., from 1.5:1 to 2.5:1), such as from 1:1 to 1.5:1 (e.g., 1.25:1). For example, when used with the device described in FIGS. 4 to 7 of the current application, a buffer:strong base ratio of less than 0.5:1 may result in the complex formation becoming too quick to accurately measure the rate of its formation.

(32) In addition, without wishing to be bound by theory, it is believed that the addition of surfactants helps to reduce the formation of bubbles in the reaction mixture, thereby reducing scattering and increasing accuracy. In particular, it has been found that the use of a cationic surfactant that includes a quarternary ammonium moiety is useful in controlling the formation of bubbles when used in combination with an anionic surfactant as a solvation assistant. This is because the quarternary ammonium surfactant is not able to dissolve in sufficient quantity in the buffered solution without a solvation aid (e.g., an anionic surfactant), while the anionic surfactant does not affect bubble formation when used alone. Therefore, in order to control bubble formation, it is useful to use a specific ratio of anionic and cationic surfactants in the creatinine assay reagent. The ratio of the cationic surfactant:anionic surfactant may be from 1:2 to 1:10, such as from 1:3 to 1:7 or, more particularly, 1:5.

(33) Dry Reagent for Microalbuminuria Assay

(34) A protein denaturing method is applied for the BCG dry-reagent to remove the interference from the urine components, such as urine pH, sample temperature and urine background. Albumin reacts with BCG and the color changes, but denatured albumin does not react with BCG and the color remains the same. The 1.sup.st reagent is for sampling and the 2.sup.nd reagent is for referencing. Both reagents contain the same concentrations of BCG, Strong base, buffer, non-ionic surfactant and preservative. However, only the 2.sup.nd reagent contains an anionic surfactant to denature albumin in the reference sample. The chemicals can be chosen from the list provided below (Table B), and the concentrations can be within the specified range.

(35) TABLE-US-00002 TABLE B Range of Concentration Albumin-Sampling Albumin-Referencing Chemical Category reagent reagent BCG 0.1-1.5 g/L 0.1-1.5 g/L Strong base 10-50 g/L 10-50 g/L Buffer 50-250 g/L 50-250 g/L Non-ionic surfactant 2 1-20 wt % 1-20 wt % (10-200 g/L) (10-200 g/L) Non-ionic surfactant 1 1-20 g/L 1-20 g/L Preservative 0.1-3 g/L 0.1-3 g/L Anionic surfactant 0 w % 0.1-5 w % (1-50 g/L)

(36) Chemical List for albumin assay 1) Strong base; NaOH, KOH. 2) Buffer; ACES (N-(1-Acetamido)-2-aminoethanesulfonic acid), Acetate (Sodium acetate), ADA (N-(2-Acetamido)-iminodiacetic acid), AMP (2-Amino-2-methyl-1-propanol), AMPD (2-Amino-2-methyl-1,3-propanediol), AMPSO (N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethane-sulfonic acid), Bicarbonate (Sodium hydrogen carbonate), Bicine (N,N-Bis-(2-hydroxyethyl)-glycine), Bis-Tris (Bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane), Bis-Tris-Propane (1,3-Bis-[tris-(hydroxymethyl)-methylamino]-propane), CAPS (3-Cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CHES (2-(N-Cyclohexylamine)-ethanesulfonic acid), Citrate (tri-Sodium citrate), DIPSO (N,N-Bis-(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), HEPES (4-(2-Hydroxyethyl)-pipera-zine-1-ethanesulfonic acid), HEPPS (4-(2-Hydroxyethyl)-piperazine-1-propanesulfonic acid), HEPPSO (4-(2-Hydroxyethyl)-piperazine-1(2-hydroxy)-propane-sulfonic acid), MES(2-Morpholinoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), MOPSO (3-Morpholino-2-hydroxypropanesulfonic acid), PIPES (Piperazine-1,4-bis-(2-ethanesulfonic acid)), POPSO (Piperazine-1,4-bis-(2-hydroxy-propanesulfonicacid)), TAPS (N-[Tris-(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid), TAPSO (N-[Tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxypropanesulfonic acid), TES0 (N-[Tris-(hydroxymethyl)-methyl]-2-aminoethanesulfonic acid), Tricine (N-[Tris-(hydroxymethyl)-methyl]-glycine), Tris (Tris-(hydroxy-methyl)-aminomethane), Succinic Acid. 3) Non-ionic surfactant; Poly(propylene glycol) (PPG), Polyethylene glycol hexadecyl ether (Brij), Triton X-100, Tween, ZonylFSN fluorosurfactant, ALKANOL6112 surfactant, Polyethylene Glycol (PEG). 4) Preservative; Sugar, sorbic acid, benzoic acid, calcium propionate, sodium nitrite, sodium azide, sulfites, disodium EDTA, Antioxidants. 5) Anionic surfactant; Sodium dodecyl sulfate (SDS), Polystyrene sulfonates (PSS), linear alkylbenzene sulfonate, (LAS), secondary alcohol sulfonate, alcohol olefin sulfonate, alcohol sulfate.
Method of Preparing Dry Reagent (FIG. 3)

(37) The prescribed chemical components are mixed and filtered through a 0.45 m cellulose acetate filter disk. The filtered mixture is dispensed into an appropriate sample well by pipette or dispenser. The sample well can be an eppendorf tube, a 96 well plate, a commercial cuvette, or any other equivalent reagent holder. For example, the sample well may be a cuvette cartridge (102) as depicted in FIG. 4. The sample well is inserted into a freeze-dryer and freeze-dried under a specific profile, which consists of freezing, annealing, freezing, primary drying and secondary drying processed. The freeze-drying process can be performed under moisture-less conditions to avoid contact with moisture.

(38) The use of other reagent holders may require optimization of the primary drying duration (e.g., extension of duration).

(39) An example of the freeze-drying profile and the range of parameters is provided in the table below. These conditions may be particularly suited to the cuvette cartridge depicted in FIG. 4.

(40) TABLE-US-00003 Freeze Drying Duration Vacuum Process Temp [ C.] [hr] [mTorr] Freezing 20 to 80 0.5 to 5 N/A Annealing 10 to 30 1 to 5 N/A Freezing 20 to 80 0.5 to 5 N/A Primary drying 10 to 30 5 to 50 50-300 Secondary drying 0 to 60 1 to 20 50-300
Packaging

(41) The dried reagent should be stored in the dark and in dry conditions to prevent light, moisture and oxygen exposure. The dried reagent can be packed into moisture barrier bags and vacuum sealed in a glove box (i.e., under an inert atmosphere). If necessary, a desiccant or an antioxidant can be inserted into the package.

(42) Cuvette Cartridge, Introduction Device and Assay Method

(43) A cuvette cartridge system and sample introduction device can be utilized with the dry reagent (e.g., as depicted in FIGS. 4 to 7). The width of the cartridge may be calculated based on the path length of each analyte. Volume can be the same in each cuvette cartridge (102) when the assay is for multiple analytes. For example, the volume can be 0.5 mL to 5 mL, preferably 1 mL. For example, if the volume is 1 mL, then the cuvette width can be 5 mm for the Albumin assay, and 2 mm for the creatinine assay. The cuvette cartridge (102) can be made of transparent material, such as PMMA (Poly-methyl methacrylate, PC (Polycarbonate), COC (Cyclic Olefin Copolymer). To block stray light, the color may be black.

(44) Liquid reagent is dispensed into a cuvette cartridge (102) by dispenser (e.g., a Musashi Dispenser), and freeze-dried by a Freeze-dryer (VirTis AdVantage Plus Freeze-dryer). For example, using the method of preparing and storing the dry reagent used above. It will be appreciated that the reagent may be used in wet form, that is, without freeze-drying. The cuvette cartridge (102) is transferred into a cuvette holder (100; FIG. 4).

(45) As shown in FIG. 4, the cuvette holder (100) can hold 4 cuvette cartridges (102). For example 4 cuvettes can be arranged in 2 lines separated by a light blocking structure (101). 2 cuvette cartridges (102) can be used for one analyte, and the other 2 cuvette cartridges can be used for the other analyte. As shown in FIG. 7, two optical modules (700) can be arranged to each side of the cuvette holder (100). It will be appreciated that the cuvette holder (100) also serves to maintain the distance between the cuvette (102) and the light module (700), so that a certain path length is maintained during sample measurement.

(46) Urine is collected from a subject into a collection cup, sucked up into a syringe and introduced into a sample well (501; FIG. 5) through a sample introduction port (500) in a sample well cover (502). Urine is dispensed into each cuvette (102) through a sample introduction hole (600; FIG. 6). Urine and dry reagent are mixed in the cuvette cartridge by shaking, magnetic stirring, vibration, sonication, or in any other equivalent way. Optical measurement is performed using one or more optical modules (700; FIG. 7). As shown in FIG. 8, the absorbance value is measured for each cuvette, and, where necessary, the difference between the sample and reference is calculated. By utilizing a calibration curve which has been prepared beforehand, the analyte concentration can be calculated, and the albumin to creatinine ratio can be calculated thereafter.

(47) FIGS. 9-11 shows a calibration curve for the microalbuminuria assay based upon synthetic standards (e.g., a synthetic urine matrix). These albumin standards are prepared with artificial or pooled urine and a certain concentration of albumin. FIGS. 9A and 10A use the sampling reagent, FIGS. 9B and 10B use the referencing reagent, FIGS. 9C, 10C and 11 show the calibration curve, which results from the difference between the sampling reagent and referencing reagent when contacted with the standards. Table 1 shows the results obtained before normalization of the results in FIG. 9 for pH variations, while Table 2 shows the results obtained after normalization using equation (1) below. Table 3 shows the results before normalization found in FIG. 10, while Table 4 provides the data normalized to compensate for temperature effects.

(48) TABLE-US-00004 TABLE 1 Estimated [Albumin] in mg/L [Albumin] pH 5.08 pH 6.65 pH 8.07 0 55.81 3.93 54.19 20 39.66 20.00 77.35 50 8.89 49.91 104.96 100 32.82 100.77 151.79 200 139.15 200.43 241.71

(49) TABLE-US-00005 TABLE 2 Estimated [Albumin] in mg/L [Albumin] pH 5.08 pH 6.65 pH 8.07 0 0.51 2.73 0.32 20 24.12 19.21 23.94 50 49.40 50.14 55.97 100 94.49 100.51 101.62 200 196.62 198.94 201.81

(50) TABLE-US-00006 TABLE 3 [Alb] 25 C. 28 C. 31 C. 34 C. 0 2.20 6.83 17.56 36.10 20 18.78 27.32 37.32 55.61 50 48.78 58.29 67.80 85.85 100 103.90 110.98 120.00 137.07 200 198.29 204.63 213.17 229.51

(51) TABLE-US-00007 TABLE 4 [Alb] 25 C. 25 C. 31 C. 34 C. 0 1.05 1.05 1.77 5.36 20 19.46 17.41 19.72 19.46 50 46.64 47.41 49.72 53.31 100 106.38 102.54 104.33 106.64 200 198.18 195.36 196.13 197.15
A.sub.sA.sub.R=A*C.sub.Alb+B(1)

(52) Where A.sub.s refers to the sampling reagent, A.sub.r refers to the referencing reagent, C.sub.Alb refers to the concentration of albumin and A and B are constants. While the resulting calibration curve was found to be accurate for subjects with medium to high levels of albumin (e.g., 30-300 mg/L), it was found that the results obtained for low levels of albumin (e.g., 0-30 mg/L) could vary significantly. These results are depicted in FIG. 12 and in Table 5 (i.e., the unspiked urine samples should have an average concentration of around 0 mg/L). It is believed that this inaccuracy is caused by a urine matrix side-effect, which results in greater variation at lower concentrations.

(53) TABLE-US-00008 TABLE 5 Quantified [Albumin] Spiked Vol. # Un-spiked 100 mg/L 1 14 102 2 18 116 3 8 104 4 5 101 5 3 99 6 6 104 7 14 107 8 15 105 9 8 105 10 1 101 Average 9 104 Stdev 6.20 4.77

(54) In order to improve the accuracy of the assay at low albumin concentrations in real human samples, it was necessary to conduct further processing, as described below.

(55) Original calibration curve: A.sub.sA.sub.R=A*C.sub.Alb+B

(56) For blank sample (normal urine; 0 mg/L albumin):
A.sub.S.sup.oA.sub.R.sup.o=A*C.sub.Alb.sup.o+B(2)

(57) For spiked sample:
A.sub.S.sup.*A.sub.R.sup.*=A*C.sub.Alb.sup.*+B(3)

(58) If the result of the spiked sample is normalized with the blank sample (i.e., equation (3) minus equation (2)), equation (4) is obtained.
(A.sub.S.sup.*A.sub.S.sup.o)(A.sub.R.sup.*A.sub.R.sup.o)=A*(C.sub.Alb.sup.*C.sub.Alb.sup.o)(4)

(59) Assuming that A.sub.R.sup.*=A.sub.R.sup.o, i.e., the absorbance of the referencing reagent is the same for the Blank and Spiked samples, then equation (4) can be rewritten as equation (5):
A.sub.S.sup.*A.sub.S.sup.o=A*(C.sub.Alb.sup.*C.sub.Alb.sup.o)(5)

(60) While it is possible to measure and A.sub.S.sup.* and A.sub.R.sup.*, A.sub.S.sup.o is an unknown. However, a strong correlation is found between A.sub.S.sup.o and A.sub.R.sup.o, which counters the urine matrix effect in different real urine samples as shown in FIG. 13 and equation (6).
A.sub.S.sup.o=a*A.sub.R.sup.o+b=a*A.sub.R.sup.*+b(6)

(61) By substituting equation (6) into equation (5), a new calibration curve for the albumin assay can be obtained using equation (7) (wherein C.sup.o.sub.Alb is taken as being 0 mg/L).
A.sub.S.sup.*(a*A.sub.R.sup.*+b)=A*C.sub.Alb.sup.*(7)

(62) The normalised curve calibration curve of FIG. 14 is more accurate across the concentration range, and especially at low concentrations of albumin. This is illustrated in Table 6.

(63) TABLE-US-00009 TABLE 6 Quantified [Albumin] Spiked Vol. # Un-spiked 100 mg/L 1 1 89 2 9 110 3 1 100 4 1 100 5 6 93 6 0 101 7 2 95 8 2 94 9 2 102 10 8 96 Average 0 98 Stdev 4.69 5.81

(64) The above-described calibration process may be implemented as one or more software modules executed by a standard computer system such as an Intel IA-32 based personal computer system, as shown in FIG. 16. However, it will be apparent to those skilled in the art that at least parts of the calibration process could alternatively be implemented in part or entirely in the form of one or more dedicated hardware components, such as application-specific integrated circuits (ASICs) and/or field programmable gate arrays (FPGAs), for example

(65) As shown in FIG. 16, a system 1600 for generating a calibration curve for use in quantitatively determining the concentration of albumin in a urine sample executes a calibration process, as described above, which is implemented as one or more software components 1630 stored on non-volatile (e.g., hard disk, solid-state drive, or flash memory) storage 1602 associated with a standard computer system. The system 1600 includes standard computer components, including random access memory (RAM) 1604, at least one processor 1606, and external interfaces 1608, 1610, 1612, all interconnected by a bus 1614.

(66) The external interfaces include universal serial bus (USB) interfaces 1608, at least one of which is connected to a keyboard 1616 and a pointing device such as a mouse 1618, a network interface connector (NIC) 1610 which can be used to connect the system 1600 to a communications network such as the Internet, and a display adapter 1612, which is connected to a display device such as an LCD panel display 1620. The system 100 also includes a number of standard software components, including an operating system 1628 such as Linux or Microsoft Windows, and a statistical software package 1629 such as Matlab or R.

(67) The system 1600 stores, on non-volatile storage 1602, absorbance data 1640, which comprise first absorbance data representing the albumin-free sample and reference absorbances A.sub.S.sup.o and A.sub.R.sup.o, and second absorbance data representing the spike-in sample and reference absorbances A.sub.S.sup.* and A.sub.R.sup.*. The absorbance data 1640 may be stored from previous experiments, or may be obtained in real time from absorbance measurements carried out on albumin-free and spike-in samples using optical modules 700 and communicated to system 1600 via NIC 1610, for example.

(68) The software components 1630 may comprise a first fitting component configured to computationally fit, using statistical software package 1629 for example, the functional relationship described in Eq. (6) between the albumin-free sample absorbances and the albumin-free reference absorbances to obtain adjustment parameters a and b. An absorbance-adjusting component of components 1630 may be configured to adjust the reference absorbances using the adjustment parameters, to thereby obtain adjusted reference absorbances A.sub.R=a*A.sub.R.sup.*+b; and a second fitting component of components 1630 may be configured to computationally fit, again using statistical software package 1629, the functional relationship described by Eq. (7) between the sample absorbances, the adjusted reference absorbances and the known (spike-in) concentrations to obtain the parameters a, b and A of the calibration curve. Accordingly, when it is desired to calculate an albumin concentration C.sub.Alb for a sample under test, given measured absorbances A.sub.S and A.sub.R for the sample, C.sub.Alb can be calculated according to A.sub.S(a*A.sub.R+b)=A*C.sub.Alb using the calibration parameters a, b and A obtained by the calibration software components 1630 of system 1600.

(69) FIG. 15 shows the calibration curve for the creatinine assay. The kinetics of the DNBA/creatinine reaction is measured and a calibration curve for creatinine is generated by plotting the rate of reaction vs. creatinine concentration. Creatinine standards are prepared with artificial or pooled urine and certain concentration of creatinine.

(70) The creatinine assay requires 5 min to 10 min and microalbuminuria assay requires around 5 min.

(71) For the creatinine assay, it will be appreciated that a kinetic measurement may start from the time that the sample is added to the creatinine assay formulation, or that the measurement may be delayed (e.g., for 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes or 5 minutes) before the kinetic measurements are started. Therefore, the actual window of sensing time where the measurements are taken may be from 1 minute to 10 minutes. The time interval for data acquisition during the kinetic measurements in the creatinine assay may be from 0.1 seconds to 15 seconds (e.g., 5 seconds).

(72) Total assay time from sampling to provide ACR value can be less than 10 min, and this is a rapid measurement.

(73) Advantages of Dry Reagent Assay System

(74) The dry-reagents are very easy to be reconstituted by addition of a urine sample.

(75) The reagents allow for a one-step sensing protocol, which significantly improves user-friendliness.

(76) As mentioned hereinbefore, the formulations prevent the formation of air-bubbles adhered to the surface of the cuvette wall upon the reconstitution of the dry-reagent with a urine specimen, allowing direct optical measurement. That is, the formulation disclosed herein allows for fast optical measurement, without having to remove bubbles from the solution (if even possible), the removal of bubbles from the reconstituted formulation in urine also allows for greater accuracy of measuring absorbance, as it reduced light scattering.

(77) When supplied as a dry-reagent, the formulations discussed herein are easy to store and transport, as compared with the wet-reagents.

(78) Operative Variations and Alternatives

(79) The present urinalysis system described above, applying a buffer addition method and/or a protein denaturing method can also be applied for urinary components other than creatinine or albumin. Examples of such analytes include, but is not limited to, water, ions (e.g., sodium, potassium, chloride, phosphoric acid, phosphorus, sulfur, bromide, fluoride, iodide, calcium, magnesium, iron, lead, mercury, etc.), proteins (e.g., haptoglobin, transferrin, immunoglobulin, lactadehydrogenase, gamma-glutamyl transferase, alpha amylase, uropepsinogene, lysozyme, urokinase, etc.), sugars (e.g., glucose, phenylpyruvate, arabinose, xyloseribose, fucose, rhammose, ketopentose, galactose, mannose, fructose, lactose, sucrose, fucosylglucose, raffinose, etc.), amino acids (e.g., alanine, carnosine, glycine, histidine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, hydroxyloproline, galactosylhydroxylyzine, xylosylserine, etc.), hormones (e.g., epinephrine, norepinephrine, dopamine, serotonin, hCG, EPF, estradiol, gonadotropin, corticotropin, prolactin, oxytocin, vasopressin, thyroxine, catecholamines (epinephrine, norepinephrine, dopamine), insulin, erythropoietin, corticosteroids (aldosterone, corticosterone, cortisone), testosterone, progesterone, estrogen, etc.), vitamins (e.g., vitamin B1, vitamin B2, vitamin B6, 4-pyridoxique acid, nicotinic acid, vitamin B12, biopterine, vitamin C, etc.), drugs or other bioactive substances (e.g., caffeine, cocaine, etc.), metabolic wastes (e.g., urea, uric acid, creatine, choline, piperidine, bilirubin, allantoin, etc.), ketones, folic acid, and the like.

(80) Urinalysis can be performed for human urine with a ranging pH from 5 to 8. Most people have a urine pH that is within this range, but some may be out of the range.

(81) The sensing temperature can be from 15 C. to 40 C.

(82) The dry reagent can be packed within a moisture-proof bag and stored at room temperature for a year at least.

(83) The dry reagent system includes a reagent for reference sample to lessen the effect of pH, temperature and interferences. For creatinine, albumin or other target assay, to include chemical with buffering effect into reagent for reference sample is effective. Especially for albumin assay, denaturing albumin by surfactant in reference sample is highly effective to measure reference sample.

(84) Wet reagents may be used instead of dry reagents. In this case, the wet reagent for measuring creatinine doesn't require a bulking agent, and the wet reagent for measuring microalbuminuria doesn't require non-ionic surfactant 2 as shown in Tables 7 and 8 below.

(85) TABLE-US-00010 TABLE 7 Range of Concentration Chemical Category Creatinine-Sampling reagent Strong base 40-80 g/L Buffer 50-250 g/L Anionic surfactant 0.1-10 g/L Cationic surfactant 0.1-10 g/L DNBA 1-5 g/L

(86) TABLE-US-00011 TABLE 8 Range of Concentration Albumin-Sampling Albumin-Referencing Chemical Category reagent reagent BCG 0.1-1.5 g/L 0.1-1.5 g/L Strong base 10-50 g/L 10-50 g/L Buffer 50-250 g/L 50-250 g/L Non-ionic surfactant 1 1-20 g/L 1-20 g/L Preservative 0.1-3 g/L 0.1-3 g/L Anionic surfactant 0 w % 0.1-5 w % (1-50 g/L)

(87) In addition the microalbuminuria/creatinine assay can be separated. That is, the microalbuminuria assay may be run alone or the creatinine assay may also be run alone.