阅读说明：本技术 生物样品中分析物和/或蛋白质的测量方法 (Method for measuring analytes and/or proteins in biological samples ) 是由 P.多赫蒂 K.基尔斯佩尔 G.法拉斯 M.V.耶拉米利 于 2019-01-09 设计创作，主要内容包括：本公开涉及用于测量生物样品中单独或与总蛋白质组合的分析物的方法。更具体地,本公开涉及使用单独或与蛋白沉淀试剂组合的一种或多种比色试剂测量分析物和/或总蛋白质的方法。(The present disclosure relates to methods for measuring an analyte in a biological sample, alone or in combination with total protein. More specifically, the present disclosure relates to methods of measuring analytes and/or total protein using one or more colorimetric reagents, alone or in combination with a protein precipitation reagent.)

1. A method of measuring the concentration of an analyte or total protein in a sample, the method comprising:

contacting the sample with a colorimetric reagent to obtain a treated sample;

focusing a camera comprising an aperture on the processed sample;

opening the aperture for a period of time sufficient to collect a predetermined amount of light from the sample;

measuring the time period; and

correlating the time period to the concentration of analyte or total protein in the sample.

2. A method of measuring the concentration of an analyte or total protein in a sample, the method comprising:

contacting the sample with a colorimetric reagent to obtain a treated sample;

focusing a camera comprising an aperture on the processed sample;

opening the aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample during the predetermined period of time;

measuring the size of the aperture; and

correlating the size of the aperture to the concentration of analyte or total protein in the sample.

3. A method of measuring the concentration of an analyte and total protein in a sample, the method comprising:

contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate;

determining the concentration of the analyte in the treated sample by:

focusing a camera comprising an aperture on the processed sample;

opening the aperture for a period of time sufficient to collect a predetermined amount of light from the processed sample;

measuring the time period; and

correlating the time period to the concentration of the analyte in the sample; and

the concentration of total protein was determined by:

generating an image of the processed sample by a camera;

measuring a number of pixels associated with protein deposits in the image;

correlating the number of pixels to a standard curve to obtain the concentration of total protein in the sample.

4. A method of measuring the concentration of an analyte and total protein in a sample, the method comprising:

contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate;

determining the concentration of the analyte in the treated sample by:

focusing a camera comprising an aperture on the processed sample;

opening the aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample;

measuring the size of the aperture; and

correlating the size of the aperture with the concentration of analyte in the sample; and

the concentration of total protein was determined by:

generating an image of the processed sample by a camera;

measuring a number of pixels associated with protein deposits in the image;

correlating the number of pixels to a standard curve to obtain the concentration of total protein in the sample.

5. A method of measuring the concentration of an analyte and total protein in a sample, the method comprising:

contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate;

determining the concentration of the analyte in the treated sample by:

focusing a camera on the processed sample;

opening an aperture to collect an amount of light from the processed sample;

measuring the intensity of the collected amount of light; and

correlating the intensity of the collected light with the concentration of the analyte in the sample; and

the concentration of total protein was determined by:

generating an image of the processed sample by a camera;

measuring a number of pixels associated with protein deposits in the image;

correlating the number of pixels to a standard curve to obtain the concentration of total protein in the sample.

6. A method of measuring the concentration of total protein in a sample, the method comprising:

contacting the sample with a protein precipitation reagent to obtain a treated sample comprising a protein precipitate;

generating an image of the processed sample by a camera;

measuring a number of pixels associated with protein deposits in the image;

correlating the number of pixels to a standard curve to obtain the concentration of total protein in the sample.

7. The method of any one of claims 1-5, wherein the analyte is selected from the group consisting of calcium ions, potassium ions, chloride ions, sodium ions, glucose, lactate, creatinine, creatine, urea, uric acid, ethanol, albumin, alkaline phosphatase, cholesterol, pyruvate, beta-hydroxybutyrate, alanine aminotransferase, aspartate aminotransferase, and cetyl cholinesterase.

8. The method of any one of claims 1-5, wherein the analyte is creatinine, glucose, albumin, or alkaline phosphatase.

9. The method of any one of claims 1-5, 7, or 8, wherein the colorimetric reagent is 2,4, 6-trinitrophenol and a base; or the colorimetric reagent is 3, 5-dinitrobenzoic acid methyl ester, 3, 5-dinitrobenzoic acid or 3, 5-dinitrobenzoyl chloride, and alkali or alkaline buffer solution; or the colorimetric reagent comprises 3, 5-dinitrobenzoyl chloride; or the colorimetric reagent is a reagent system comprising copper ions, a hydroperoxide, and an oxidizable dye.

10. The method of any one of claims 1-5, 7, or 8, wherein the colorimetric reagent is one or more of glucose oxidase, hexokinase, basic copper tartrate, basic ferricyanide, and horseradish peroxidase.

11. The method of any one of claims 1-5, 7, or 8, wherein the colorimetric reagent is bromocresol green.

12. The method of any one of claims 1-5, 7, or 8, wherein the colorimetric reagent is a reagent system comprising 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitrotetrazolium Blue (NBT).

13. The method of any one of claims 1 or 2, wherein the colorimetric reagent comprises catechol violet, benzethonium chloride, or catechol red.

14. The method of any one of claims 3-12, wherein the protein precipitation reagent is one or more water-miscible solvents (such as alcohols, e.g. isopropanol, methanol or ethanol, ketones, e.g. acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, tetrahydrofuran); or the protein precipitation reagent is a salt (such as ammonium sulfate or a salt comprising a polyvalent metal ion); or the protein precipitation reagent is trichloroacetic acid or trichloroacetic acid and acetone; or the protein precipitating agent is a polymer (e.g., a non-ionic hydrophilic polymer such as polyethylene glycol and dextran).

15. The method of any one of claims 3-12, wherein the protein precipitation reagent is an aqueous surfactant.

16. The method of claim 15, wherein the aqueous surfactant is benzalkonium chloride or benzethonium chloride.

17. The method of any one of claims 1-16, wherein the sample is a urine sample.

18. The method of any one of claims 3-17, further comprising generating an image of the processed sample using a predetermined amount of light collected from the processed sample.

19. The method of claim 18, wherein the processed sample is imaged from a slide containing the processed sample.

20. The method of claim 18 or 19, wherein the image is a black and white image.

21. The method of any one of claims 18-20, further comprising displaying an image of the processed sample on a user interface.

22. The method of any one of claims 3-12 or 14-21, wherein the predetermined amount of light is sufficient to measure the amount of protein precipitation from an image of the processed sample.

23. The method of any one of claims 1-22, wherein the predetermined amount of light can be obtained by independently performing one, two, or three empirical experiments using a standard sample having a known concentration of analyte or total protein.

24. The method of any one of claims 1-23, wherein the predetermined amount of light is constant.

25. The method of any one of claims 1-24, wherein the predetermined amount of light is selected by a user of the camera.

26. The method of any one of claims 1-25, wherein the camera comprises a filter.

27. The method of claim 25, wherein the filter is configured to pass wavelengths corresponding to a range of wavelengths of a color produced by a reaction between the colorimetric reagent and the sample.

28. The method of any of claims 1, 3, or 7-27, wherein the camera comprises an auto-exposure function, and wherein measuring a period of time that an aperture is open comprises using the auto-exposure function to determine the period of time.

29. The method of claim 28, wherein the determining is based on at least one of the aperture size and the predetermined amount of light.

30. The method of any of claims 1, 3, or 7-27, wherein the camera comprises a sensor, and wherein opening an aperture for a period of time sufficient for the predetermined amount of light to be collected from the sample comprises:

opening the aperture;

detecting, by the sensor, an amount of light collected by the camera;

determining whether the amount of light collected by the camera is substantially equal to the predetermined amount of light;

closing the aperture after determining that the predetermined amount of light has been collected by the camera.

31. The method of any of claims 1, 3, or 7-27, wherein the camera comprises a sensor, and wherein opening an aperture for a period of time sufficient to collect the predetermined amount of light from the sample comprises:

opening an aperture for a period of time to collect an amount of light from the processed sample;

closing the aperture;

measuring, by the sensor, an amount of light collected from the processed sample;

determining whether the amount of light collected from the treated sample is substantially equal to the predetermined amount of light;

opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the second period of time is different from the first period of time; and

correlating the second period of time that the aperture is open with the predetermined amount of light to obtain a concentration of analyte or total protein in the sample.

32. The method of any of claims 2,4, or 7-27, wherein the camera comprises an auto-exposure function, and wherein measuring the size of the aperture comprises sizing the aperture using the auto-exposure function.

33. The method of claim 32, wherein the determining is based on at least one of the period of time and the predetermined amount of light.

34. The method of any of claims 2,4, or 7-27, wherein the camera includes a sensor, and wherein opening the aperture for a period of time comprises:

opening the aperture to collect an amount of light from the sample, wherein the aperture comprises a first aperture size;

closing the aperture;

measuring, by the sensor, an amount of light collected from the processed sample;

determining whether the amount of light collected from the sample is substantially equal to the predetermined amount of light;

opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the aperture has a second aperture size, wherein the second aperture size is different from the first aperture size; and

correlating a second aperture size of the aperture with the predetermined amount of light to obtain a concentration of analyte or total protein in the sample.

35. The method of any one of claims 1-34, wherein the analyte is creatinine.

36. The method of claim 35, further comprising determining a protein to creatinine ratio for the sample based on at least the concentration of creatinine in the sample and the concentration of total protein in the sample.

37. The method of any one of claims 1-36, further comprising performing a health assay based at least on the concentration of analyte, total protein, or a combination thereof in the sample.

Technical Field

The present disclosure generally relates to methods for measuring analyte and/or total protein in a biological sample. More specifically, the present disclosure relates to methods of using one or more colorimetric reagents, alone or in combination with protein precipitation reagents, to measure analytes and/or total proteins.

Background

To screen for a variety of health conditions, it is often necessary to analyze the amount of one or more analytes in a biological sample, such as red or white blood cells, calcium ions, potassium ions, chloride ions, sodium ions, glucose, lactate, creatinine, creatine, urea, uric acid, ethanol, albumin, alkaline phosphatase, cholesterol, pyruvate, beta-hydroxybutyrate, alanine aminotransferase, aspartate aminotransferase, acetylcholinesterase, and the like. In addition to measuring a particular analyte, it may also be helpful to measure the amount of total protein in a sample. For example, measuring total protein may be helpful in diagnosing kidney disease or acute kidney injury. Analysis of the concentration of specific analytes and total proteins in a biological sample typically requires that the sample be sent to a laboratory for analysis. This can lead to increased sample handling efforts, extended turnaround times for laboratory results, special transportation requirements, and increased storage times, which in turn can lead to bacterial contamination and/or bacterial overgrowth in the sample, changes in key protein levels, degradation of castings and cells, dissolution or formation of crystals, changes in sample pH, increased odor of the sample, and other deleterious effects on the sample.

Disclosure of Invention

Accordingly, the present inventors have identified a need for point-of-care assays that can effectively, accurately, and cost-effectively measure analyte concentrations, total protein content, or combinations thereof in biological and other samples.

One aspect of the present disclosure provides a method of measuring the concentration of an analyte or total protein in a sample. The method includes contacting a sample with a colorimetric reagent to obtain a treated sample. The method further comprises focusing a camera comprising an aperture on the processed sample; opening the aperture for a period of time sufficient to collect a predetermined amount of light from the sample; and measuring a time period during which the aperture is open. The time period during which the aperture is open is then correlated to the concentration of analyte or total protein in the sample. In certain embodiments, the method measures the concentration of the analyte. In certain embodiments, the method measures the concentration of total protein.

In another aspect of the method of the present disclosure for measuring the concentration of an analyte or total protein in a sample, the method comprises contacting the sample with a colorimetric reagent to obtain a treated sample. The method further comprises focusing a camera comprising an aperture on the processed sample; opening an aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample; and the size of the aperture is measured. The size of the aperture is then correlated to the concentration of analyte or total protein in the sample. In certain embodiments, the method measures the concentration of the analyte. In certain embodiments, the method measures the concentration of total protein.

One aspect of the present disclosure provides a method of measuring the total protein concentration in a sample. The method includes contacting the sample with a protein precipitation reagent to obtain a treated sample including a protein precipitate. The method also includes generating an image of the processed sample by the camera and measuring a number of pixels associated with protein deposits in the image. The number of pixels was then correlated to a standard curve to obtain the concentration of total protein in the sample.

Another aspect of the present disclosure provides a method of measuring the concentration of an analyte and total protein in a sample. The method includes contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate. The method further includes determining a concentration of an analyte in the treated sample by focusing a camera including an aperture on the treated sample; opening the aperture for a period of time sufficient to collect a predetermined amount of light from the processed sample; measuring a time period during which the aperture is open; the time period during which the aperture is open is correlated to the concentration of the analyte in the sample. The method further comprises determining the concentration of total protein by generating an image of the processed sample by a camera; measuring a number of pixels associated with protein deposits in the image; the number of pixels was correlated to the standard curve to obtain the concentration of total protein in the sample.

In another aspect of the method of the present disclosure for measuring the concentration of an analyte and total protein in a sample, the method comprises contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate. The method further includes determining a concentration of an analyte in the treated sample by focusing a camera including an aperture on the treated sample; opening an aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample; measuring the size of the aperture; the size of the aperture is correlated to the concentration of the analyte in the sample. The method further comprises determining the concentration of total protein by generating an image of the processed sample by a camera; measuring a number of pixels associated with protein deposits in the image; the number of pixels was correlated to the standard curve to obtain the concentration of total protein in the sample.

In another aspect of the method of the present disclosure for measuring the concentration of an analyte and total protein in a sample, the method comprises contacting the sample with a colorimetric reagent and a protein precipitation reagent to obtain a treated sample comprising a protein precipitate. The method further includes determining the concentration of the analyte in the treated sample by focusing the camera on the treated sample; opening an aperture to collect light from the sample; measuring the intensity of the collected amount of light; the intensity of the collected light is correlated to the concentration of the analyte in the sample. The method further comprises determining the concentration of total protein by generating an image of the processed sample by a camera; measuring the number of pixels associated with protein deposits in the image; the number of pixels was correlated to the standard curve to obtain the concentration of total protein in the sample.

In certain embodiments of the methods of the present disclosure, the analyte may be selected from the group consisting of calcium ions, potassium ions, chloride ions, sodium ions, glucose, lactate, creatinine, creatine, urea, uric acid, ethanol, albumin, alkaline phosphatase, cholesterol, pyruvate, beta-hydroxybutyrate, alanine aminotransferase, aspartate aminotransferase and acetylcholinesterase. In certain embodiments of the methods of the present disclosure, the analyte may be creatinine, glucose, albumin, or alkaline phosphatase.

In certain embodiments of the methods of the present disclosure, the colorimetric reagent can be 2,4, 6-trinitrophenol and a base. In certain embodiments of the methods of the present disclosure, the colorimetric reagent is methyl 3, 5-dinitrobenzoate, 3, 5-dinitrobenzoic acid, or 3, 5-dinitrobenzoyl chloride, and a base or alkaline buffer. In certain embodiments of the methods of the present disclosure, the colorimetric reagent is a reagent system comprising copper ions, a hydroperoxide, and an oxidizable dye. In certain embodiments of the methods of the present invention, the colorimetric reagent comprises 3, 5-dinitrobenzoyl chloride. In certain embodiments of the methods of the present disclosure, the colorimetric reagent may be one or more of glucose oxidase, hexokinase, alkaline copper tartrate, alkaline ferricyanide, and horseradish peroxidase. In certain embodiments of the methods of the present disclosure, the colorimetric reagent may be bromocresol green. In certain embodiments of the methods of the present disclosure, the colorimetric reagent may be a reagent system comprising 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitrotetrazolium Blue (NBT). In certain embodiments of the methods of the present disclosure, the colorimetric reagent may comprise catechol violet, benzethonium chloride, or catechol red. In certain embodiments of the methods of the present disclosure, the colorimetric reagent may comprise catechol violet.

In certain embodiments of the methods of the present disclosure, the colorimetric reagents may comprise one or more reagents suitable for immunoassays. For example, the colorimetric reagents may comprise one or more analyte-specific antibodies and/or enzymes suitable for use in an enzyme-linked immunosorbent assay (ELISA) or enzyme-Enhanced Immunoassay (EIMT) technique.

In certain embodiments of the methods of the present disclosure, the protein precipitation reagent may be one or more water-miscible solvents (such as alcohols, e.g., isopropanol, methanol, or ethanol, ketones, e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, or cyclohexanone, tetrahydrofuran); or the protein precipitation reagent is a salt (such as ammonium sulfate or a salt comprising a polyvalent metal ion); or the protein precipitation reagent is trichloroacetic acid or trichloroacetic acid and acetone; or the protein precipitating agent is a polymer (e.g., a non-ionic hydrophilic polymer such as polyethylene glycol and dextran). In certain embodiments of the methods of the present disclosure, the protein precipitation reagent may be an aqueous surfactant. In certain embodiments of the methods of the present disclosure, the aqueous surfactant may be benzalkonium chloride or benzethonium chloride.

In certain embodiments of the methods of the present disclosure, the sample may be a urine sample.

In certain embodiments, the methods of the present disclosure may further comprise generating an image of the processed sample using a predetermined amount of light collected from the processed sample. In certain embodiments of the methods of the present disclosure, the treated sample can be imaged from a slide containing the treated sample. In certain embodiments of the methods of the present disclosure, the image may be a black and white image.

In certain embodiments, the methods of the present disclosure may further comprise displaying an image of the processed sample on a user interface. In certain embodiments of the methods of the present disclosure, the predetermined amount of light may be sufficient to measure the amount of protein precipitation from the image of the processed sample. In certain embodiments of the methods of the present disclosure, the predetermined amount of light may be constant. In certain embodiments of the method of the present disclosure, the predetermined amount of light may be selected by a user of the camera. In certain embodiments of the methods of the present disclosure, the camera may include a filter. In certain embodiments of the methods of the present disclosure, the optical filter may be configured to pass wavelengths corresponding to a wavelength range of a color produced by a reaction between the colorimetric reagent and the sample. In certain embodiments of the methods of the present disclosure, the camera may include an auto-exposure function, and wherein measuring the period of time that the aperture is open comprises using the auto-exposure function to determine the period of time. In certain embodiments of the methods of the present disclosure, the determining may be based on at least one of an aperture size and a predetermined amount of light.

In certain embodiments of the methods of the present disclosure, the camera may comprise a sensor, and wherein opening the aperture for a period of time sufficient for a predetermined amount of light to be collected from the sample comprises: opening the aperture; detecting an amount of light collected by the camera through the sensor; determining whether the amount of light collected by the camera is substantially equal to a predetermined amount of light; after the metering camera has collected a predetermined amount of light, the aperture is closed.

In certain embodiments of the methods of the present disclosure, the camera may comprise a sensor, and wherein opening the aperture for a period of time sufficient to collect a predetermined amount of light from the sample comprises: opening the aperture for a period of time to collect an amount of light from the treated sample; closing the aperture; measuring, by a sensor, an amount of light collected from the processed sample; determining whether the amount of light collected from the treated sample is substantially equal to a predetermined amount of light; opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the second period of time is different from the first period of time; the second time period that the aperture is open is correlated to a predetermined amount of light to obtain the concentration of the analyte or total protein in the sample.

In certain embodiments of the methods of the present disclosure, the camera may include an auto-exposure function, and wherein measuring the size of the aperture comprises using the auto-exposure function to determine the size of the aperture. In certain embodiments of the methods of the present disclosure, the determining may be based on at least one of a time period and a predetermined amount of light.

In certain embodiments of the methods of the present disclosure, the camera may include a sensor, and wherein opening the aperture for a period of time comprises: opening an aperture to collect an amount of light from a sample, wherein the aperture comprises a first aperture size; closing the aperture; measuring, by a sensor, an amount of light collected from the processed sample; determining whether the amount of light collected from the sample is substantially equal to a predetermined amount of light; opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the aperture has a second aperture size, wherein the second aperture size is different from the first aperture size; correlating the second aperture size of the aperture with a predetermined amount of light to obtain a concentration of the analyte or total protein in the sample. In certain embodiments, the methods of the present disclosure may further comprise determining the protein of the sample based on at least the concentration of creatinine in the sample and the concentration of total protein in the sample: ratio of creatinine.

In certain embodiments, the methods of the present disclosure may further comprise performing a health assay based at least on the concentration of the analyte and/or total protein in the sample.

The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

Drawings

Fig. 1 is a flow chart of a method according to one embodiment of the present disclosure.

Fig. 2 is a partial schematic view of a camera opening an aperture to collect light from a sample according to an embodiment of the present disclosure.

Fig. 3A is a photograph of six sample aliquots prepared according to the methods of the present disclosure. The colorimetric techniques used in sample preparation are noted on sample aliquots. In the figure Pro ═ total protein, Cre ═ creatinine.

Fig. 3B is a series of experimental images generated by a camera according to the method of the present disclosure. The shutter speed corresponding to each image is displayed above the image.

Fig. 4A shows photographs of a series of prepared samples according to the methods of the present disclosure.

Fig. 4B is a correlation curve showing the relationship between the amount of creatinine in an experimental sample and the shutter speed measured according to the method of the present disclosure.

Fig. 5 is a flow chart of a method according to an embodiment of the present disclosure.

Fig. 6A is a series of experimental images generated by a camera according to the methods of the present disclosure and showing the amount of precipitated protein in the prepared samples.

Fig. 6B is a correlation curve showing the relationship between the amount of protein in the experimental sample and the protein precipitation measured according to the method of the present disclosure.

Fig. 7 is a diagram illustrating a camera and a prepared sample according to an embodiment of the present disclosure.

Fig. 8A is a correlation curve showing the relationship between the amount of glucose in the experimental sample and the period of time required to collect a predetermined amount of light from the sample.

Fig. 8B is a correlation curve showing the relationship between the amount of albumin in the experimental sample and the time period required to collect a predetermined amount of light from the sample.

Fig. 8C is a correlation curve showing the relationship between the amount of alkaline phosphatase in the experimental sample and the period of time required to collect a predetermined amount of light from the sample.

Figure 9 provides images of samples with different creatinine concentrations treated according to the methods of the present disclosure.

Fig. 10 is a graph showing the relationship between the amount of protein in an experimental sample and the shutter speed measured according to the method of the present disclosure.

Fig. 11A is a graph of determining the amount of protein in a sample using a multivariate quadratic equation, according to an embodiment of the disclosure. FIG. 11B is a graph of the amount of protein in a sample versus sliding window standard deviation image characteristics across multiple devices. FIG. 11C is a plot of the amount of protein in a sample versus the average contour region image characteristics across multiple devices. Fig. 11D is a linear plot of a multiple quadratic regression using two image features on a single device, according to an embodiment of the present disclosure.

Fig. 12A is a graph of determining the amount of protein in a sample using an artificial neural network, according to an embodiment of the present disclosure. Fig. 12B is a structural diagram of an artificial neural network. x1 is image feature 1, x2 is image feature 2, and xn is image feature n. Figure 12C is a graph of the amount of protein in a sample versus histogram sliding window standard deviation image features across multiple devices. Fig. 12D is a linear graph of neural network training using image features on multiple devices, according to an embodiment of the present disclosure.

Detailed Description

In general, the disclosed materials, methods, and devices provide improvements for measuring analyte and/or total protein concentrations in biological samples. In particular, in certain embodiments of the present disclosure, analytes and/or total proteins in a biological sample are measured using a single sample, a single image, and/or a single assay that eliminates multiple testing methods, extensive sample handling, transportation, and storage. The measurement can be carried out in the clinic in time, and the change of the sample can be reduced compared with the traditional method. For example, samples analyzed according to the methods of the present disclosure may have less bacterial contamination and/or bacterial overgrowth, minimal or no change in key protein levels, less casting and cellular degradation, less crystal dissolution or formation, and minimal or no change in pH, appearance, and/or odor, as compared to samples analyzed according to conventional methods.

As used herein, the term "biological sample" generally refers to a sample of tissue or fluid from a human or animal, including but not limited to whole blood, plasma, serum, spinal fluid such as cerebrospinal fluid; lymph, abdominal fluid (ascites), samples of the external skin, respiratory, intestinal and genitourinary tracts, tears, saliva, urine, blood cells, tumors, organs, tissues and in vitro cell culture components.

As used herein, the term "total protein" refers to all proteins in a sample, including protein fragments of any size.

In various aspects of the methods of the present disclosure, the sample volume is between about 10 μ Ι _ to about 500 μ Ι _; for example, from about 10 μ L to about 300 μ L, or from about 10 μ L to about 200 μ L, or from about 10 μ L to about 100 μ L, or from about 50 μ L to about 300 μ L, or from about 50 μ L to about 200 μ L, or from about 100 μ L to about 500 μ L, or from about 100 μ L to about 300 μ L, or from about 100 μ L to about 200 μ L. In some aspects, the methods of the present disclosure require less sample volume than traditional methods for measuring analytes, total proteins, and combinations thereof.

The methods of the present disclosure include determining total protein by contacting a biological sample with a colorimetric reagent to produce a treated sample for determination of an analyte and/or a colorimetric or protein precipitation reagent. Depending on the concentration of analyte and/or total protein in the sample, the reaction between the colorimetric reagent and the analyte and/or total protein may cause the solution to change color to different degrees. The camera may be used to capture an image of the processed sample by collecting an amount of light from the sample through an aperture of the camera, where the amount of light collected is indicative of the color of the sample.

In certain embodiments, the camera collects (or a sensor associated with the camera collects) a predetermined amount of light regardless of the color of the sample. The predetermined amount of light collected may be determined experimentally, for example by one, two, three or more empirical experiments, to obtain one or more values of the predetermined amount of light correlated to the concentration of analyte or total protein in the sample. The predetermined amount of light should not be greater than the maximum amount of light acceptable by the camera or less than the minimum amount of light acceptable by the camera.

The amount of light collected by the camera may depend on the exposure time, aperture size, and other characteristics of the camera. For example, using a PL-D725MU-T USB 3.0 camera (PixelLINK) with a CMOS light sensor and an objective lens with a magnification of 10x and a digital aperture of 0.28, a predetermined amount of light can be collected from a sample containing albumin at a concentration of 1mg/mL for an exposure time of about 170ms to about 180 ms. This time represents the amount of time that the camera's aperture is open to collect light onto the sensor of the other light collecting means. Therefore, the predetermined light amount may be defined by the exposure time or shutter speed of the camera, rather than a value directly representing the light amount (e.g., photons).

In another example, a PL-D725MU-T USB 3.0 camera (PixelLINK) with a CMOS light sensor and an objective lens with a magnification of 10x and a digital aperture of 0.28 was used to collect a predetermined amount of light from a sample containing glucose at a concentration of 1mM at an exposure time of about 130ms to about 140 ms. In another example, a predetermined amount of light can be collected from a sample containing alkaline phosphatase at a concentration of 1. mu.g/mL using the PL-D725MU-T USB 3.0 camera described above at an exposure time of about 135ms to about 145 ms.

By collecting a predetermined amount (i.e., discrete) of light from each sample, the camera can produce an image or images having substantially the same exposure or brightness regardless of the color of the sample. Thus, the camera settings (e.g., shutter speed and/or aperture size) may be varied between images to achieve a predetermined exposure level in each image of the processed sample. These camera settings may be measured to indicate an optical property of the treated sample (e.g., the colorimetric intensity of the treated sample). For example, if the aperture size is held constant, the shutter speed required to achieve the desired exposure level may vary depending on the colorimetric intensity of the processed sample. Shutter speed can then be measured and correlated to the concentration of analyte and/or total protein in the sample. Conversely, if the shutter speed is held constant, the aperture size required to reach a predetermined exposure level can be measured and correlated to the concentration of analyte and/or total protein in the sample. By generating an image with a predetermined exposure level, the camera may be configured to produce a clinically useful image of the sample, regardless of the colorimetric intensity of the processed sample. In certain embodiments, the method can be used to measure the concentration of an analyte and/or the concentration of total protein, wherein the concentration of the analyte is determined using a first colorimetric reagent and the concentration of total protein can be determined by a second colorimetric reagent.

Accordingly, the methods of the present disclosure include contacting a sample with a colorimetric reagent to obtain a treated sample; focusing a camera comprising an aperture on the processed sample; opening the aperture for a period of time sufficient to collect a predetermined amount of light from the sample; measuring a time period; and correlating the time period to the concentration of analyte or total protein in the sample. Another method of the present disclosure includes contacting a sample with a colorimetric reagent to obtain a treated sample; focusing a camera comprising an aperture on the processed sample; opening an aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample within a predetermined time; measuring the size of the aperture; the size of the aperture is correlated to the concentration of analyte or total protein in the sample. The time period or aperture size can be correlated to the concentration of analyte or total protein by comparing the time or aperture size to a standard curve. The standard curve may represent the amount of time or the size of the camera aperture for a range of concentrations of analyte or total protein in the sample.

In addition to, or as an alternative to, measuring the concentration of the analyte or protein from the color intensity of the sample, the methods of the present disclosure further include measuring the concentration of total protein in the sample by contacting the sample with a protein precipitation reagent to obtain a treated sample comprising a protein precipitate. Protein precipitation may be visible after reaction with the protein precipitation reagent. The total protein concentration can be measured by generating an image of the treated sample with a camera and analyzing the image. Image analysis may include, for example, measuring a plurality of pixels associated with a protein deposit (e.g., with a protein deposit pellet or an image region composed of protein deposits). The number of pixels can then be compared to a standard curve reflecting the correlation between pixels and protein concentration to obtain the concentration of total protein in the sample. The test method can be performed faster than traditional tests, allowing for greater dynamic range, lower assay costs, and/or using less reagents and materials. In some embodiments of the present disclosure, the total protein is measured using a camera image, while the other analyte is not measured.

The methods of the present disclosure may be used to measure the concentration of a variety of analytes, including but not limited to one or more of the following compounds that may be present in a biological sample: calcium ions, potassium ions, chloride ions, sodium ions, glucose, lactate, creatinine, creatine, urea, uric acid, ethanol, albumin, alkaline phosphatase, cholesterol, pyruvic acid, beta-hydroxybutyrate, alanine aminotransferase, aspartate aminotransferase, and acetylcholinesterase. In some embodiments, the analyte may be selected from one or more of creatinine, glucose, albumin, and alkaline phosphatase.

As described above, the methods of the present disclosure include contacting a sample with a colorimetric reagent to obtain a treated sample as represented by step 101 of method 100 in the flowchart of fig. 1. The colorimetric reagent may be selected based on the desired application and the type of analyte to be measured. For example, colorimetric reagents for measuring creatinine include, but are not limited to, one or more of the following: (1)2,4, 6-trinitrophenol and an alkali buffer, (2) methyl 3, 5-dinitrobenzoate, 3, 5-dinitrobenzoic acid or 3, 5-dinitrobenzoyl chloride, and an alkali or alkali buffer, and (3) a reagent system comprising cupric ions, hydroperoxide, and an oxidizable dye. For example, colorimetric reagents for measuring glucose include, but are not limited to, one or more of glucose oxidase, hexokinase, basic copper tartrate, basic ferricyanide, and horseradish peroxidase. For example, colorimetric reagents for measuring albumin include, but are not limited to, bromocresol green. For example, colorimetric reagents for measuring alkaline phosphatase include, but are not limited to, the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitrotetrazolium Blue (NBT) systems. For example, colorimetric reagents for measuring total protein include, but are not limited to, catechol violet, benzethonium chloride, and pyrogallol red. In certain embodiments of the methods of the present disclosure, the colorimetric reagents may comprise one or more reagents suitable for immunoassays. For example, the colorimetric reagents may comprise one or more analyte-specific antibodies and/or enzymes suitable for use in an enzyme-linked immunosorbent assay (ELISA) or enzyme-Enhanced Immunoassay (EIMT) technique.

In certain embodiments of the present disclosure, the colorimetric reagent may comprise two or more components (e.g., an alkali or alkaline buffer other than 2,4, 6-trinitrophenol). In certain embodiments, these components may be added to the sample separately and sequentially. For example, the sample may be contacted first with the base and then with 2,4, 6-trinitrophenol. In certain embodiments, the components of the colorimetric reagent may be contacted with the sample simultaneously. For example, 3, 5-dinitrobenzoic acid or 3, 5-dinitrobenzoyl chloride may be dissolved in an alkaline buffer prior to contacting the sample.

The colorimetric reagents used in the methods of the present disclosure may be added in specific volumes based on the desired application and the type of analyte to be measured. For example, the volume of colorimetric reagent is between about 1 μ L to about 500 μ L; for example; for example, from about 1 μ L to about 300 μ L, or from about 1 μ L to about 200 μ L, or from about 1 μ L to about 100 μ L, or from about 1 μ L to about 50 μ L, or from about 50 μ L to about 300 μ L, or from about 50 μ L to about 200 μ L, or from about 100 μ L to about 500 μ L, or from about 100 μ L to about 300 μ L, or from about 100 μ L to about 200 μ L.

Protein precipitation reagents for use in the methods of the present disclosure can be selected based on the desired application and also based on the type of analyte to be measured in the sample that is also being analyzed to obtain the protein concentration (e.g., protein precipitation reagents can be selected so as to reduce interference between the reagents and the analyte and colorimetric reagent reactions). For example, protein precipitation reagents include, but are not limited to, one or more of, water-miscible solvents (such as alcohols, e.g., isopropanol, methanol, or ethanol, ketones, e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, or cyclohexanone, tetrahydrofuran), salts (such as ammonium sulfate or salts comprising polyvalent metal ions), trichloroacetic acid (alone or in combination with acetone), polymers (e.g., nonionic hydrophilic polymers, such as polyethylene glycol and dextran), aqueous surfactants, benzalkonium chloride, benzethonium chloride, and any combination thereof.

The protein precipitation reagent may be added in a specific volume based on the desired application. For example, in certain embodiments of the methods of the present disclosure, the volume of protein precipitation reagent is between about 1 μ Ι _ to about 500 μ Ι _; for example, from about 1 μ L to about 300 μ L, or from about 1 μ L to about 200 μ L, or from about 1 μ L to about 100 μ L, or from about 1 μ L to about 50 μ L, or from about 50 μ L to about 300 μ L, or from about 50 μ L to about 200 μ L, or from about 100 μ L to about 500 μ L, or from about 100 μ L to about 300 μ L, or from about 100 μ L to about 200 μ L.

As described above, the method of the present disclosure includes focusing a camera including an aperture on a processed sample, as shown in step 102 of method 100 shown in fig. 1. The camera may be any device having an aperture that, when opened, can collect light from the processed sample (i.e., take an image of the sample). The camera may include various imaging systems. In some embodimentsIn this regard, the camera may include a desktop imaging platform configured to image a biological sample suspended in a cassette or slide. In particular embodiments, the camera may be associated with a urine or blood deposit analyzer, e.g., sedvuetm DX^TMUrine sediment analyzers (IDEXX Laboratories, inc., Westbrook, ME). In such embodiments, the treated sample may be pipetted or otherwise dispensed directly into a cartridge disposed in the inlet end of the system. In other embodiments, the processed sample may be imaged from a slide comprising the processed sample, and the method may include inserting the slide, the cartridge, or another container comprising the processed sample into an inlet end of the imaging system.

In certain other embodiments, the camera may be a digital camera, a camera phone, or another portable device, and focusing the camera on the processed sample may include positioning the camera to face the sample. Where a portable device is used, a stand, tripod, armature or other support may be configured to position the camera in a particular position relative to the processed sample to image the sample (i.e. collect a predetermined amount of light from the sample). Focusing such a camera may include manually controlling the focusing via a user interface of the camera such that the processed sample is within a focus range of the camera. Additionally or alternatively, focusing the camera may include focusing on the processed sample using an autofocus function of the camera. Such an auto-focus function of the camera may use a sensor to detect the distance between the processed sample and the camera and adjust the focus of the camera so that the processed sample is within the focus range of the camera.

As described above, the method of the present disclosure includes opening the aperture for a period of time sufficient to collect a predetermined amount of light from the sample, as shown in step 103 of method 100 in fig. 1. Fig. 2 further illustrates a simplified schematic diagram of an example camera 210, the example camera 210 featuring an aperture 220 and a light sensor 230 for receiving light 260 from a sample 290. When the aperture 220 is open, light 260 from the sample 290 may enter the camera 210, contacting the light sensor 230. The optical contact sensor may be used to analyze visual characteristics of the sample 290, to generate an image of the sample, or to perform some other type of processing.

The aperture 220 may include any opening through which the camera 210 may collect light 260 over a period of time to determine the color or light intensity of the sample, or to generate an image of the prepared sample 290. For example, the aperture 220 may include a shutter of the camera 210 that exposes the light sensor 230 to light 360 from the sample 290 for a period of time (i.e., a time corresponding to the shutter speed). The size of the aperture 220 may be fixed, for example set by the manufacturer of the camera 210, such that it is substantially the same size each time the aperture is opened. For example, the digital aperture 220 may be in the range of about 0.025 to about 0.5, or about 0.03 to about 0.5, or about 0.025 to about 0.25, or about 0.03 to about 0.5, or about 0.05 to about 0.25, or about 0.05 to about 0.5, or about 0.1 to about 0.25, or about 0.1 to about 0.5.

In embodiments of the present disclosure where the aperture size is constant, the amount of light 260 collected by the camera 210 through the aperture 220 may depend primarily on the period of time that the aperture is open. In alternative embodiments, the size of the aperture 220 may be adjustable to allow a higher or lower flow rate of light 260 into the camera 210 via the aperture, and opening the aperture may include selecting or determining the size of the aperture. The aperture size may be manually selected by a user of the camera 210, for example, through a user interface of the camera. Alternatively, the aperture size may be determined by the camera 210 through an automatic exposure ("auto exposure") function of the camera.

Opening the aperture 220 for a period of time sufficient to collect a predetermined amount of light 260 may allow the camera to take an image of the sample. The method may include using an amount of light collected from the processed sample (such as a predetermined amount) to generate an image of the prepared sample 290. The image generated by the camera 210 may include a full-color image, a black and white image, or any other image suitable for measuring analytes and/or proteins in a processed sample. Alternatively, the image may be digitally generated by a light sensor 230 such as a Charge Coupled Device (CCD), Active Pixel Sensor (APS), Complementary Metal Oxide Semiconductor (CMOS), Foveon X3 sensor, or other sensor. In such embodiments, the method may include detecting the collected light 260 at the light sensor 230 and/or converting the incident light into an electrical signal. In some examples, such a light sensor 230 may detect incident light 260 and record it as a series of pixels, representing discrete points on the imaged prepared sample. Such pixels may be selectively sensitive to light of a particular wavelength or range of wavelengths. For example, the pixels may be subdivided into pixels that selectively collect light of wavelengths corresponding to green, red, and blue, respectively. Alternatively, such pixels may be sensitive to all collected light in the wavelength range to which the sensor is sensitive (i.e., produce a black and white image). The light sensor 230 may measure the wavelength and/or amount of light collected by each pixel of the camera 210 and store it in an array of values in the camera's memory or data storage device. In a particular example, each pixel may include a digital value corresponding to an amount of light 260 collected at the pixel, an average intensity, brightness, coloration, or another characteristic of the incident light. The method may further include displaying an image of the processed sample on a user interface in communication with the camera. Additionally or alternatively, the image and/or associated information may be sent to a remote computing device, such as a server, a cell phone, a computer, or another external device.

As described herein, the predetermined amount of light represents the amount of light collected by the camera to produce an image having a predetermined brightness or exposure. The predetermined amount of light may be specified according to the amount of light incident on a photosensor of the camera, a voltage generated by converting incident light into an electrical signal, the brightness or optical properties of the generated image, or by some other means. The predetermined amount of light 260 collected by the camera 210 may be constant such that each time the aperture is opened 220 an image of uniform exposure and/or brightness is produced. The predetermined amount of light 260 may be affected by various elements of the camera or imaging system. For example, the predetermined amount of light may be associated with a lens, an aperture, a light sensor, or other elements of a camera. The predetermined amount of light 260 can be selected based on the amount of light required to produce a clinically useful image of the processed sample 290.

In embodiments in which the amount of protein precipitate is measured, the predetermined amount of light 260 may be sufficient to measure the amount of protein precipitate from an image of the processed sample (e.g., by visually inspecting the image of the processed sample), by counting the number of pixels associated with the protein precipitate (e.g., a protein precipitate pellet or an image area containing the protein precipitate), and/or by processing the image using a program, algorithm, or some other method. In another embodiment, the predetermined amount of light 260 may be determined from a desired exposure level, brightness, contrast, or intensity in the image generated by the camera 210 based on a brightness histogram of the image of the processed sample 290.

The predetermined amount of light 260 may be selected by a user of the camera 210 through a user interface of the camera 210, and the method may include inputting the predetermined amount of light. The predetermined amount of light 260 may be selected or adjusted by a user to ensure sharpness of the image and/or to facilitate post-processing of the image. Additionally or alternatively, an automatic exposure ("autoexposure") function of the camera 210 may be used to determine an optimal or clinically useful exposure level. Such an automatic exposure function may use an algorithm that uses information about the prepared sample 290 and/or settings of the camera 210 to determine the amount of light 260 that the camera should collect to produce a clinically useful image. For example, the predetermined amount of light 260 may be determined based on an average brightness of the prepared sample 290, a brightness histogram of the prepared sample, a recognition and/or balance of high and low light for the prepared sample, or some other method.

For a given sample, the amount of light 260 that camera 210 collects through aperture 220 (i.e., the exposure level of the image produced by the camera) is typically affected by two camera settings: the size of the aperture 220 and the period of time the aperture is open. Depending on the optical characteristics of the processed sample 290, a larger or smaller aperture size and/or a longer or shorter opening time of the aperture 220 may be required to collect a predetermined amount of light from the sample. For example, if one sample is darker than another, a larger aperture size and/or longer shutter speed will be required to collect the same amount of light 260 compared to a brighter sample.

In one embodiment, the method of the present disclosure includes measuring a period of time that the aperture is open in order to collect a predetermined amount of light, as shown in step 104 of method 100 in fig. 1. In some embodiments, the various camera settings may be determined using the auto-exposure function of the camera prior to generating an image of the processed sample. In a similar manner as the auto-exposure function can use the optical properties of the sample to determine the optimum exposure level, the auto-exposure function can also be used to determine the appropriate camera settings to achieve the predetermined exposure level. In some embodiments, the auto-exposure function uses a light sensor of the camera to evaluate the optical properties of the processed sample and determines the appropriate shutter speed of the camera before opening the aperture. The auto exposure function may also use information about a predetermined light amount and/or aperture size when determining a time period during which the aperture should be opened.

In another embodiment of the present disclosure, the camera may include a sensor that measures the collected light in real time, such that the camera may actively respond by closing the aperture when a predetermined amount of light has been collected. Accordingly, such embodiments may include: (i) opening the aperture; (ii) detecting an amount of light collected by the camera through the sensor; (iii) determining whether the amount of light collected by the camera is substantially equal to a predetermined amount of light; (iv) the aperture is closed after the determination camera has collected a predetermined amount of light. In addition, the sensor may be sensitive or selectively sensitive to any wavelength range suitable for a particular application. Such a sensor may continuously measure the amount of light entering the aperture, or may measure the amount of light collected by the aperture at a plurality of discrete points in time after the aperture is opened, in order to intermittently measure the amount of light collected. When the sensor determines that a predetermined amount of light has been collected by the aperture, the sensor may be configured to close the aperture via a controller, switch, mechanical component, or other element of the camera.

In yet another embodiment, the aperture may be opened multiple times (i.e., multiple images generated). This may be particularly desirable in situations where the camera lacks an auto-exposure function or is unable to determine the time period or aperture size required to collect a predetermined amount of light during the first opening of the aperture. In certain embodiments, the camera may open its aperture to take a preliminary first image of the sample, and then use information about the first image to adjust the camera settings for a second or subsequent image. In such embodiments, opening the aperture may comprise: opening the aperture for a period of time to collect an amount of light from the treated sample; measuring the amount of light collected from the treated sample; determining whether the amount of light collected from the treated sample is substantially equal to a predetermined amount of light; opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the second period of time is different from the first period of time; the second time period that the aperture is open is correlated to a predetermined amount of light to obtain the concentration of the analyte or total protein in the sample. The second time period may be determined based on an analysis of the amount of light collected from the sample during the first opening of the aperture. For example, the camera may use an algorithm that correlates a period of time that the aperture is open and an amount of light collected from the processed sample, and the method may further include determining a second period of time based on the first period of time and the first amount of light collected that will allow the camera to collect a predetermined amount of light from the sample.

In some embodiments, the camera may use an iterative approach to collect a predetermined amount of light. In this case, the aperture may be opened a plurality of times, with each successive time period being different from the previous time period. The time period for which the aperture is open may be iteratively increased or decreased by a predetermined amount of time (i.e., an iterative "step"). Additionally or alternatively, an algorithm may be used to determine the magnitude and/or direction of the iterative change over the time period that the aperture is open. This may allow the camera to capture successive images with progressively predetermined amounts of light.

Additionally or alternatively, the camera may open the aperture multiple times for different periods of time to capture images over a range of exposure levels. The multiple images produced by the camera may be post-processed to determine which image was produced with a predetermined amount of light. In such embodiments, measuring the period of time that the aperture is open may include measuring the brightness, color, or other characteristics of the plurality of images, and selecting the image from the plurality of images that is closest to the predetermined exposure level (e.g., the image produced via collecting a predetermined amount of light). The time period that the aperture associated with the image is open can then be used to correlate shutter speed with the concentration of analyte or total protein in the sample.

The measured time period can be used to quantify the concentration of analyte or total protein in the treated sample. In some implementations, the measured time period can be displayed on a display or user interface of the camera. In other embodiments, the measured time period and/or shutter speed may be transmitted to a computing device, memory, remote computer, server, or other system for further analysis, processing, or correlation, for example.

In some embodiments, the methods of the present disclosure further comprise correlating the period of time that the aperture is open to a predetermined amount of light to obtain the concentration of the analyte or total protein in the sample. One such embodiment is shown in step 105 of method 100 in fig. 1. As previously described with respect to step 101, the sample may be contacted with a colorimetric reagent to obtain a treated sample that may exhibit different degrees of staining depending on the concentration of the analyte of interest or total protein present in the sample. For example, three samples are shown, containing the colorimetric reagent without creatinine, and three treated samples, containing the colorimetric reagent and 2000mg/dL of protein and 1000mg/dL of creatinine. Using the methods of the present disclosure, the concentration of creatinine or total protein in the treated sample can be estimated by measuring the coloration, brightness, chroma, intensity, opacity, or other optical properties of the sample, for example, by taking an image of the treated sample.

In certain embodiments of the present disclosure, if the optical characteristics of the images generated are predetermined (e.g., a uniform amount of light is collected to generate each image), measuring the various camera settings that generate such images may allow a user to estimate the concentration of analyte or total protein in the sample. For example, instead of using the same camera settings to take differently colored images (where the coloration of the image is indicative of the concentration of the analyte or total protein in the sample), the same image may be taken from different samples, and the camera settings used to achieve the same image may represent the concentration of the analyte or total protein. In one example, the image exposure (i.e., the amount of light collected from the sample) may be held constant while the shutter speed is measured. Measuring the time period that the aperture is open (as reflected by the shutter speed) can be used to determine the concentration of analyte or total protein in the treated sample.

Fig. 3B shows six images generated by collecting predetermined amounts of light from the six samples shown in fig. 3A. Each image representing the sample of fig. 3A is located in a corresponding position in fig. 3B (i.e., the top left image in fig. 3B corresponds to the top left sample in fig. 3A, and so on). As shown, the six images produced by the camera appear to have substantially the same color, brightness, and/or exposure despite the differences in staining observed in the sample. This is because the camera opens the aperture for different periods of time (568, 436, 252, 782, 1224 and 598 milliseconds as shown) for each image in order to produce the same color and/or exposed image regardless of the original sample color.

Those skilled in the art will recognize that the period of time that the aperture is open will vary depending on the characteristics of the camera and the processed sample to be imaged. For example, in certain embodiments of the methods of the present disclosure, the time period may range from about 0.1ms to about 10 seconds, e.g., between about 0.1ms to about 5s, or about 0.1ms to about 1s, about 1ms to about 10s, or about 1ms to about 5s, or about 10ms to about 10s, or about 10ms to about 5s, or about 100ms to about 10s, or about 100ms to about 1 s.

Figure 4A shows a sample image generated by the camera for another series of treated samples including a colorimetric reagent and a known amount of creatinine ranging from 0 to 2000 mg/dL. Like the images in FIG. 3B, these images are taken by collecting a predetermined amount of light from the sample, thus producing images of substantially the same color and/or exposure. As is evident from the images, the samples containing more analyte (creatinine in this case) are darker and therefore the aperture needs to be opened for a longer period of time to collect the predetermined amount of light. As shown in fig. 4B, the shutter speed measured by the camera at which the camera collects a predetermined amount of light from the sample can be correlated to the amount of creatinine in the sample, which represents a standard curve reflecting the relationship between the amount of analyte in the sample and the shutter speed.

In some embodiments, correlating the period of time that the aperture is open to a predetermined amount of light may include performing a regression analysis. In such embodiments, preliminary tests may be conducted and an experimental relationship between known concentrations of analyte and shutter speed, or total protein and shutter speed, may be plotted. The regression line can be used to estimate a regression function related to the two variables so that subsequent experimental data can be interpreted based on the regression function. In such embodiments, correlating the period of time that the aperture is open to a predetermined amount of light to obtain the concentration of the analyte or total protein in the sample may comprise using a regression function to estimate the concentration of the analyte or total protein in the sample.

In other embodiments, the memory of the computing device, controller, processor, server, or other computing unit associated with the camera may include data correlating shutter speed to a predetermined amount of light to obtain a concentration of the analyte or total protein (e.g., a stored regression function). In such embodiments, correlating the period of time that the aperture is open to the predetermined amount of light may comprise determining, by the computing device, a concentration of the analyte or total protein. Such determination may be made based on at least one of the type of analyte, the type of colorimetric reagent, and/or the measured period of time that the aperture is open. Such determination may also be made based on protein content, the type of colorimetric reagent, and/or the measured time period during which the aperture is open. After determining the concentration of the analyte or total protein, the amount may be displayed on a display, or a user interface of a camera or a device associated with the camera. In other embodiments, the concentration of the analyte or total protein may be transmitted to a computing device, processor, memory of a camera, memory of a computing device, or a remote computer, server, or other system for further analysis, processing, or diagnosis.

As explained above with reference to the method 100 represented in fig. 1, in some embodiments, the size of the aperture of the camera may be held constant, and opening the aperture may include opening the aperture for a period of time sufficient to collect a predetermined amount of light. Measuring the concentration of the analyte or total protein in the prepared sample may then include measuring the time period that the aperture is open to collect a predetermined amount of light. The measured time period can then be correlated with a predetermined amount of light to obtain the concentration of analyte total protein in the sample.

In another embodiment, the period of time that the aperture is open (i.e., the shutter speed) may be held constant. In such embodiments, the size of the aperture may be varied for each image, and the size of the aperture may be measured to determine the concentration of analyte or total protein in the sample. For example, the time period may be in the range of about 0.1ms to about 10 seconds, such as between about 0.1ms to about 5s, or between about 0.1ms to about 1s, or between about 1ms to about 10s, or between about 1ms to about 5s, or between about 10ms to about 10s, or between about 10ms to about 5s, or between about 100ms to about 10s, or between about 100ms to about 1 s. In these embodiments, the predetermined amount of light is when the digital aperture is between, for example, about 0.01 to about 0.99, about 0.055 to 0.9, or about 0.1 to 0.5.

Fig. 5 shows a flow diagram of such a method 500 for measuring the concentration of an analyte and/or the total protein concentration in a sample.

As described above, steps 501 and 502 of method 500 may include similar steps as steps 101 and 102 of method 100. In some embodiments, contacting the sample with the colorimetric reagent (101, 501) and focusing by the camera (102, 502) can be performed substantially the same as in method 100. The method 500 may further include opening the aperture for a discrete period of time, wherein the aperture size is related to the concentration of the analyte or total protein in the sample. For example, step 503 includes opening an aperture for a predetermined period of time, wherein the aperture is of a size sufficient to collect a predetermined amount of light from the sample. Then, as shown in step 504, an aperture size that allows the camera to collect a predetermined amount of light during the period of time that the aperture is open may be measured.

As previously described with respect to shutter speed, an optimal aperture size (i.e., an aperture size that allows the camera to collect a predetermined amount of light over a predetermined period of time) may be determined by the auto-exposure function. In such embodiments, opening the aperture for a period of time may include using an auto-exposure function to open the aperture such that the size of the aperture is sufficient to collect a predetermined amount of light from the sample. Thus, measuring the size of the aperture may include using an auto-exposure function to determine the size of the aperture. As previously mentioned, such determination may be based on at least one of the optical properties and/or time period of the treated sample and the predetermined amount of light.

Additionally or alternatively, methods involving measuring aperture size may include taking a plurality of images to collect a predetermined amount of light. In some embodiments, the camera may include a sensor, and the method may include: (i) opening an aperture to collect an amount of light from a sample, wherein the aperture comprises a first aperture size; (ii) closing the aperture; (iii) measuring the amount of light collected by the sensor from the processed sample; (iv) determining whether the amount of light collected from the sample is substantially equal to a predetermined amount of light; (v) opening the aperture for a second period of time if the amount of light received from the sample is not substantially equal to the predetermined amount of light, wherein the aperture has a second aperture size, wherein the second aperture size is different from the first aperture size; (vi) correlating the second aperture size of the aperture to the concentration of the analyte or total protein in the sample. Such a method may use the first opening of the aperture to guide the second or subsequent aperture opening, using an algorithm, an iterative method, any of the methods described with respect to shutter speed, or some other method.

The method 500 may include correlating the size of the aperture with the concentration of analyte or total protein in the sample, as shown in step 505. Correlating the size of the aperture with the amount of analyte or total protein may include comparing the measured aperture size to a standard curve to estimate the amount of analyte or total protein. Such a standard curve can be generated, imaging a series of samples containing known amounts of analyte or total protein, and plotting the measured aperture size for each sample relative to the known concentration. As previously mentioned, regression lines can be used to estimate regression functions related to two variables, so that subsequent experimental data can be interpreted based on regression functions related to, for example, measured aperture size and analyte or total protein concentration.

In some embodiments, methods 100 and 500 can include determining the concentration of analyte and total protein in a sample. The amount of total protein in the sample can be estimated by analyzing an image of the sample, for example, an image generated when a camera collects light to measure analyte concentration. The method may include generating an image of the processed sample by the camera (steps 106 and 506); measuring the number of pixels associated with protein deposits in the image (steps 107 and 507); the number of pixels is correlated to the standard curve to obtain the concentration of total protein in the sample (steps 108 and 508).

In one embodiment, measuring the amount of protein precipitation comprises performing a visual analysis of an image of the processed sample generated by the camera. For example, when the aperture of the camera is opened to collect the previously measured amount of light, an image of the processed sample may be generated during a previous step of the method. As previously described, an image may be generated by collecting light and recording it as an image using the camera's light sensor.

The image of protein deposits may include precipitated protein globules, which may appear in the image in varying amounts depending on the concentration of protein in the sample. FIG. 6A shows experimental images of treated samples containing 0-1000mg/dL protein. As seen in fig. 6A, the protein may be visible to the naked eye and appear in the form of discontinuities in an otherwise uniformly exposed or colored image. In some embodiments, an image of a protein precipitate, such as the image seen in fig. 6A, may be the same image used to measure analyte concentration (i.e., the image produced when a predetermined amount of light is collected from the sample). Additionally or alternatively, a separate image may be generated for protein quantification, and the method may include opening an aperture to collect a second amount of light from the processed sample and/or generating an image of the processed sample.

The method may include processing the image of the protein precipitate. In some embodiments, the image of the protein precipitate may be post-processed to increase the clarity of the image, increase contrast, increase focus, or facilitate quantification of the protein. For example, the camera may be coupled to or in communication with a computing device that includes a controller, processor, memory, or other component. Post-processing may include using an image processing program on the computing device or executing instructions stored in a memory of the computing device. Additionally or alternatively, aspects of the image may be manually adjusted by a user of the camera through interaction with a user interface of the camera or an associated computing device.

In some cases, visual analysis may be used to quantify the amount of protein precipitation in a sample. In one embodiment, the method may include visually analyzing the image generated by the camera to measure the concentration of protein precipitate in the sample. For example, a user of the method may view the image and count precipitated protein globules, estimate the region of the image containing protein precipitates, or perform some other visual analysis.

In some embodiments, algorithms, programs, or software may be used to quantify protein precipitation in the treated sample. In some implementations, a computing device coupled to or in communication with the camera executes instructions (e.g., instructions stored in a memory of the computing device) in order to measure an amount of protein precipitation in the processed sample. Such instructions may be executed by a processor of a computing device to automate a portion of the measurements described above with respect to the naked eye technique. For example, the instructions may be configured to measure the amount of protein precipitate in the processed sample by measuring or determining the number of pixels associated with the protein precipitate (e.g., corresponding to a protein precipitate pellet or an image region containing the protein precipitate). Additionally or alternatively, the instructions may cause the computing device to measure the amount of protein precipitate in the processed sample by measuring an area of an image of the processed sample that includes the protein precipitate. Measuring the region including the protein deposit may include determining a number of pixels of the image associated with the protein deposit. Distinguishing the protein precipitate from the background of the image may include analyzing a value associated with the pixel. For example, each pixel in the image may include a value corresponding to the amount of light collected, intensity, brightness, coloration, gray scale, or other optical characteristic of the pixel (e.g., a pixel in an 8-bit image may be represented by a number between 0 and 255, where 0 corresponds to black and 0 corresponds to white). In a particular example, the threshold may be set such that pixels having a value above the threshold are considered to comprise protein deposits, while pixels having a value below the threshold are considered to be the background. In this case, determining the number of pixels associated with protein precipitation may include determining the number of pixels above or below a certain threshold.

Additionally or alternatively, precipitated proteins may be measured by determining the homogeneity of the resulting image. Images that are more heterogeneous (i.e., less homogeneous) may represent a greater amount of precipitated protein, while homogeneous images may represent a lesser amount of protein. In such an example, homogeneity of the image may be determined by analyzing a numerical value associated with each pixel in the image (e.g., a value associated with the collected light, as described above). In a particular example, determining homogeneity of an image may include calculating a standard deviation of values associated with each pixel in the image, where a higher standard deviation may be the result of a greater amount of precipitated protein (or vice versa).

Fig. 6B shows a graph of the concentration of protein in the sample versus the measured amount of protein precipitation in the image of the treated sample. As shown in fig. 6B, the "response" is related to the homogeneity of the image, as determined by the methods previously described. The amount of protein precipitate in the treated sample is determined using image analysis software that calculates the homogeneity (i.e., response) of the image and correlates the homogeneity to the amount of protein in the imaged sample. In some cases, correlating the amount of protein precipitate in the treated sample to the concentration of protein in the sample can include performing a regression analysis. In this case, preliminary tests can be performed and an experimental relationship between a known concentration of protein and the precipitate in the sample image can be plotted. The regression line can be used to estimate a regression function related to the two variables so that subsequent experimental data can be interpreted based on the regression function. In such embodiments, correlating the amount of protein precipitate in the treated sample to the amount of protein in the sample may comprise using a regression function to estimate the amount of protein in the sample.

In some embodiments of the present disclosure, the amount of protein precipitation in the treated sample is correlated to the amount of protein in the sample using the multivariate quadratic equation provided in fig. 11A. Multiple images of protein precipitates are acquired from different locations within the sample in step 1. The image is then processed in step 2 by a number of image processing pipelines that extract protein precipitation features in the image. In some embodiments, the image processing pipeline may include sensor noise calibration, contrast and edge enhancement, edge and contour detection, contour region, fourier transform, and/or standard deviation. In step 3, the features extracted by the image processing pipeline are correlated with proteins using a multivariate quadratic equation, as follows:

wherein x₁Is image feature 1; x is the number of₂Is image feature 2; x is the number of_nIs the image feature n.

FIG. 11B shows a plot of the concentration of protein in samples on multiple devices versus sliding window standard deviation image characteristics. FIG. 11C is a graph showing the relationship between the concentration of protein in the sample on multiple devices and the mean profile area image characteristics. FIG. 11D shows a linear plot of a single device after performing multiple quadratic regression on two image features.

In some embodiments of the present disclosure, using an artificial neural network as provided in fig. 12A, a meridian will be approachedThe amount of protein precipitate in the sample is correlated to the amount of protein in the sample. Multiple images of protein precipitates are acquired from different locations within the sample in step 1. The image is then processed in step 2 by a number of image processing pipelines that extract features of protein deposits in the image. In certain embodiments, the image processing pipeline may include a sensor noise calibration, contrast enhancement, and/or fourier transform and/or a histogram of standard deviation. In step 3, the features extracted by the image processing pipeline are associated with proteins using an artificial neural network. The structure of the artificial neural network is provided in FIG. 12B (where x₁Is an image feature 1, x₂Is the image feature 2, x_nIs the image feature n). FIG. 12C is a graph showing the relationship between the concentration of protein in samples on multiple devices and the histogram sliding window standard deviation image features; and figure 12D shows a linear plot of a number of devices after training the neural network on the image features.

In other cases, a computing device, controller, processor, server, or other computing unit associated with the camera may include data that correlates the concentration of protein precipitates in the processed sample to the amount of protein in the sample. In such embodiments, correlating the amount of protein precipitate in the image to the amount of protein in the sample may comprise determining the amount of protein by the computing device. Such determination may be made based on at least one of the number of pixels associated with the counted protein deposits in the image, the measured region including the deposited protein in the image. After determining the concentration of the protein, methods 100 and 500 may include displaying the concentration on a display or user interface of the camera or a device associated with the camera. In other embodiments, the concentration of the protein may be transmitted to a computing device, processor, memory of a camera, or remote computer, to a server or other system for further analysis, processing, or diagnosis, for example.

In some embodiments, methods 100 and 500 further include determining a health condition of the user based at least on the determined concentration of the analyte and/or protein in the sample. In some cases, a computing device (e.g., a computer, server, processor, or controller) in communication with the camera may use the determined concentration of the analyte and/or protein to determine a health condition, diagnose a disease, express a risk factor, or provide some other information about the patient's health. In one embodiment, the relationship (e.g., ratio) of the amount of protein and analyte may be indicative of a health condition, and determining the health condition may comprise determining whether the relationship of analyte and creatinine is above or below a threshold level. In a particular embodiment, the analyte may be creatinine and methods 100 and 500 may include determining the urine protein: creatinine ratio ("UPC ratio"). In this case, diagnosing a health condition (e.g., proteinuria) may include determining whether the UPC ratio is above or below a threshold. Other health conditions and analyses are envisioned by one of ordinary skill in the art.

The example methods 100 and 500 shown in fig. 1 and 6 are intended as illustrative, non-limiting examples. The steps described herein may be performed sequentially or in parallel. Further, various steps may be performed in a different order than described herein, and some steps may be omitted, skipped, and/or repeated. Additional or alternative elements of methods and additional or alternative components of systems are contemplated as will be apparent to those skilled in the art.

Fig. 7 is a schematic diagram of a system 700, such as the system used in either of method 100 or method 500, according to an example embodiment of the present disclosure. System 700 may include a camera 710, a multi-well sample plate 720, a computing device 730 (including a processor 740 and a memory 750), and a server 760. Perforated plate 720 may include a plurality of holes 722.

Camera 710 may include one or more light sensors, such as Charge Coupled Devices (CCDs), Active Pixel Sensors (APS), Complementary Metal Oxide Semiconductors (CMOS), FOVEONA sensor or other sensor. As understood by one of ordinary skill in the art, for a given applicationSuch a light sensor may be sensitive to any wavelength range of interest. In one embodiment, the light sensor may be sensitive to a range of wavelengths corresponding to the visual spectrum or a portion of the visual spectrum (i.e., wavelengths between about 190nm and 700 nm). Additionally or alternatively, the light sensor may be sensitive or selectively sensitive to wavelengths in the infrared and/or ultraviolet range. In one embodiment, the light sensor may be selectively sensitive to a range of wavelengths corresponding to wavelengths reflected, emitted, or transmitted from the prepared sample, e.g., wavelengths corresponding to a color of the prepared sample after reaction with a colorimetric reagent, wavelengths corresponding to an emission spectrum of a fluorophore, or other ranges of wavelengths. Such a light sensor may also be a black/white light sensor, so that all incident light in the range to which the sensor is sensitive is detected and recorded, independently of the wavelength.

The camera 710 may also include an aperture 712 and one or more optical filters 714. The optical filter may pass only light within a particular wavelength range to the aperture 712. Such filters 714 may be configured to selectively pass or block light of a particular wavelength or wavelength range such that the camera 710 collects only light of the particular wavelength or wavelength range. For example, the range of wavelengths passed by the filter 714 may be related to the reflection, emission, or transmission spectra of the processed sample. The wavelength range may correspond to the color of the colorimetric reagent or a product produced by a reaction between the sample and the colorimetric reagent. In this manner, the camera 710 may record only desired wavelengths, thereby reducing noise or unnecessary image content. Additional or alternative uses of the optical filter 714 are also contemplated.

In another exemplary embodiment, the analyte or total protein may be targeted by one or more fluorophores in the reagent. The fluorophore may emit light in a first specific wavelength range when excited by radiation in a second wavelength range. Thus, in such embodiments, the system 700 may additionally include an excitation source (e.g., a laser) that emits light in the second wavelength range to excite the fluorophore. In this case, the filter may be configured to block all wavelengths outside the emission spectrum of the processed sample.

As shown, camera 710 is communicatively coupled to computing device 730. In various embodiments, such communicative coupling may be achieved using WiFi, through bluetooth, or via a wired interface (e.g., a USB cable). Alternatively, in some implementations, the camera 710 may be coupled to the computing device 730 through a wired connection, such as an ethernet interface. In some implementations, the camera 710 may be a camera attached to or integrated in a mobile computing device (e.g., a cell phone). The mobile computing device may access the public internet to transmit images (e.g., candidate images or target images of biological cells) to the computing device 730. In some implementations, the camera 710 can additionally or alternatively be communicatively coupled to the server 760. For example, in some embodiments, camera 710 may transmit an image to server 760, server 760 may perform image processing or analysis (e.g., post-processing of the image, determination of the concentration of analyte or total protein in the sample), and server 760 may then transmit the resulting information to computing device 730.

As shown, computing device 730 includes a processor 740 and a memory 750. Memory 750 includes instructions 752 stored thereon. Memory 750 may include volatile memory (e.g., RAM) and/or non-volatile memory (e.g., hard disk drive). Memory 750 may also be communicatively coupled internally to processor 740. The processor 740 may be configured to execute instructions 752 stored in the memory 750 (e.g., to perform various computing tasks). Additionally or alternatively, memory 750 may store images (e.g., images recorded by camera 710) and information related to the images. Memory 750 may further store information related to the measured concentration of an analyte or protein in a sample.

The instructions 752 stored in the memory 750 of the computing device may include instructions related to performing the methods described herein. For example, the instructions 725 may instruct the camera to focus on the processed sample, open and/or close the aperture 712, generate an image of the sample (e.g., a sample located within the well 722 of the multi-well plate), measure a period of time that the aperture 712 is open, and/or measure a size of the aperture. Such instructions 725 may also cause the computing device 730 to correlate the determined time period and/or aperture size to the concentration of analyte or total protein in the sample. Additionally, the instructions 725 may relate to processing of images produced by the camera 710. For example, the instructions 725 may cause the computing device 730 to process an image of the sample, measure the concentration of protein precipitates in the processed sample, and/or correlate the concentration of protein precipitates in the processed sample with the concentration of protein in the sample.