WO2002005698A2

WO2002005698A2 - Method for extracting fluorophores from biological samples

Info

Publication number: WO2002005698A2
Application number: PCT/US2001/021981
Authority: WO
Inventors: Omanand Koul; Victor S. Sapirstein; Yongwu Yang
Original assignee: Seroptix, Inc.
Priority date: 2000-07-13
Filing date: 2001-07-12
Publication date: 2002-01-24
Also published as: AU2001273404A1; WO2002005698A3

Abstract

The present invention relates to a method for preparing a biological sample for detection of a diseased or infected state, such as, for example, HIV, Hepatitis A, Hepatitis B, Hepatitis C, or a combinatioln thereof. The method provides a supernatant fluid having a detectable amountof free compounds wherein the presence of the free compounds may be used to determine the presence of the diseased or infected state.

Description

METHOD FOR EXTRACTING FLUOROPHORES FROM BIOLOGICAL SAMPLES

FIELD OF THE INVENTION The present invention relates to the detection of the presence of a diseased or infected state and more particularly to a sample preparation technique to enhance such detection in a sample.

BACKGROUND ART Spectroscopy has been used to determine the chemical and physical composition of matter. For example, quantitative or qualitative measurement of the absorption, emission, or scattering of radiation upon irradiating a sample with radiation allows the abundance and identity of a sample's constituents to be determined. (Hereinafter, absorption, emission, and/or scattering of radiation of any wavelength or bandwidth are referred to collectively as spectroscopic signals unless otherwise stated.) These determinations are based on the fact that the sample constituents give rise to unique spectroscopic signals or fingerprints, which can generally be represented as a spectrum or . plot of the absolute or relative intensity of the spectroscopic signal at various wavelengths. In this regard, biological samples in a diseased or infected state comprise compounds that are indicative of the diseased or infectious state. Therefore, the presence, e.g., abundance of the compounds can be used to discriminate a diseased or infected sample from a disease-free or non-infected sample. For example, U.S. Patent Application Nos. 09/224,141 filed December 31, 1998 and 09/436,207 filed November 8, 1999, which are hereby incorporated by reference in their entireties, describe the identification of fluorescence wavelength ranges wherein the fluorescence spectrum of a sample will yield information that may be used to detect and identify the presence of infection or disease.

Analysis of spectra, however, is often complicated by the fact that the spectroscopic signals of different sample constituents overlap. Such spectral overlap is particularly important in biological samples, which, in addition to the compound of interest, often include a complex matrix of concomitant species. Morever, when the spectral overlap is intrinsic to the shape and characteristics of the spectroscopic signals being measured, even high resolution spectroscopic instruments are insufficient to completely discriminate the desired spectroscopic signals from the spurious spectroscopic signals of the concomitant species. Problems caused by spectral overlap are further aggravated when the quantity of the compounds of interest is low, such as at early stages of a disease or infection. Thus, conventional analyzers require that the disease or infection progress for a period of time before detection. Obviously, such a progression time undesirably delays the onset of treatment for an infected individual.

To overcome these obstacles, the 09/224,141 and 09/436,207 patent applications disclose preparing a biological sample, such as plasma, by passing the sample over adsorbants such as C-M Affi Gel Blue or activated charcoal to reduce spurious signals and improve the ability to discriminate a diseased or infected state from and a non-diseased ^• or non-infectious state. The present invention relates to a different method of preparing a biological sample prior to detection of a diseased state.

SUMMARY OF THE INVENTION

The present invention relates to a method for preparing a biological sample for detection of a diseased or infected state, such as, for example, HTV, Hepatitis A, Hepatitis B, Hepatitis C, or a combination thereof. The method comprises combining the biological sample with a fluid to obtain a first mixture and heating the first mixture to a temperature and for a time sufficient to obtain a second mixture comprising a supernatant having a detectable amount of free compounds and wherein the presence or abundance of the free compounds may be used to determine the presence of the diseased or infected state. During the method of the present invention, free compounds are preferably dissociated from proteins to enhance a spectroscopic property of the free compounds such as, for example, a fluorescence intensity or a fluorescence yield.

A preferred embodiment of the present method further comprises irradiating the supernatant with radiation, measuring quantitatively or qualitatively a fluorescence emission or absorbance of the free compounds, and determining the presence of a diseased state by analyzing the fluorescence emission or absorbance. The fluorescence emission or the absorbance is preferably compared to a signal characteristic of a non-diseased state.

In a preferred embodiment of the invention, the supernatant is irradiated with radiation that has at least one wavelength of from about 270 to 750 nm. The fluorescence emission is preferably detected over at least a portion of the range from about 400 to 850 nm.

In one embodiment, the biological sample comprises plasma. In a preferred embodiment, the first mixture comprises from about 4 to 8% by volume plasma, 0.9 to 1.5% by volume buffer, 0.3 to 0.9% by volume acid, and 88 to 96% by volume organic liquid. Preferably the buffer is a phosphate buffer having pH of from about 3 to about 5. The acid is preferably trifluoroacetic acid. The organic liquid is preferably acetonitrile. The first mixture may further comprise water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fluorescence spectra of an untreated Hepatitis C negative plasma sample and an untreated Hepatitis C positive plasma sample acquired with an excitation wavelength of 355 nm; and

FIG. 2 shows fluorescence spectra of the samples from FIG. 1, which have been treated according to the method of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, a method is provided for preparing a biological sample for detection of the presence of a diseased or infected state In particular, the method is suited for preparing biological samples for spectroscopic determination of a diseased or infectious state comprising, for example, HF/, Hepatitis A, Hepatitis B, or Hepatitis C. The spectroscopic determination generally involves measuring the presence or abundance of compounds that are associated with the diseased or infected state to discriminate a diseased or infected sample from a disease-free or non-infected sample. For example, as described below, the compounds may be fluorescent or may exhibit an absorbance, which can be detected by spectroscopic techniques. In untreated samples, however, spectroscopic signals from the compounds may be overwhelmed by background or spurious signals from concomitants in the biological sample.

A variety of techniques can be used to interpret a spectroscopic signal, such as to ascertain the presence or abundance of a compound in a sample. Additionally, spectroscopic information can be used to provide information regarding the environment of a compound, such as, for example, whether or not the compound is bound to a protein. Such techniques include but are not limited to, for example, determining the absolute or relative amplitude of a spectrum at one or more predetermined wavelengths or determining the absolute or relative area underneath all of or a portion of a spectrum. The wavelength corresponding to the peak or minimum spectroscopic signal and/or the relative shift in either of these values can also be used in this regard. In a time dependent measurement, the emission lifetime can be used to help identify a compound. Fourier techniques, wherein spectroscopic signals are interpreted as a function of their frequency content, can often be used in situations where spectroscopic overlap complicates time or wavelength based analyses. Multivariate techniques, such as, for example, neural nets or principal components analysis, wherein information from more than one wavelength or spectrum is utilized in a comprehensive analysis, can also be used to interpret overlapped spectra. Any of the above mentioned techniques or similar techniques that provide information regarding the presence, abundance, or environment of a compound within a sample are applicable to the presently recited method. The present method greatly enhances the ability of a spectroscopic technique to discriminate biological samples infected with a disease or pathogen (e.g., HTV viruses) from samples not infected with such diseases or pathogens. For example, the method of the ■ invention preferably provides a supernatant liquid, comprising compounds, which have been dissociated from proteins in the biological sample, as described below. The dissociated compounds in the supernatant liquid may exhibit enhanced spectroscopic properties such as, for example, a fluorescence yield and or intensity compared to compounds associated with the protein molecules, as found in an untreated sample. The term "compounds" as used herein includes, but is not limited to, molecules of any size or complexity, fragments of molecules, such as, for example, a DNA fragment or a denatured protein, or complexes comprising more than one chemically and/or physically associated molecule. The terms "fluorophores" or "fluorescent compounds" refer to compounds which emit detectable fluorescence upon irradiation with a suitable light source.

Preferably, the method comprises the steps of: (a) obtaining a biological sample;

(b) combining the sample with a buffer to obtain a first mixture;

(c) combining the first mixture with a volume of acid to form a second mixture;

(d) combining the second mixture with a volume of an organic liquid; (e) heating the mixture at a temperature and for a period of time sufficient to obtain a third mixture comprising a supernatant having a free compound therein and partially soluble solids or insoluble solids; (f) isolating the supernatant from the solids; and (g) measuring a fluorescence or absorbance of the free compound to determine the presence of a diseased or infectious state. It should be noted that the abovementioned steps are shown individually for convenience only. Thus, one of ordinary skill in art will understand, for example, that two or more individual steps may be combined or even performed in a different order to that shown above. The biological specimen or sample used in the present invention may be any biological material such as plasma, serum, blood particles, blood, bodily fluid, or tissue. The term "specimen" is often taken to mean a biological material that has been subjected to essentially no processing, whereas, the term "sample" is taken to mean a product that results from even a basic level of processing a specimen. For example, plasma is a sample that results after removing cellular material from a blood specimen. However, because the method of the present invention may be applied to a substantially unprocessed specimen such as tissue or to a partially processed sample such as plasma, the terms specimen and sample are used interchangeably hereinafter. Biological samples suitable for use in the present invention may be obtained from an individual, animal, or other organism using methods that are known in the art.

Plasma or other blood fluids are particularly convenient samples to use for identifying a diseased or infectious state of an individual. Human plasma, for example, contains as many as 100 to 125 proteins, many of which, such as albumin and lipoproteins, serve as carriers for smaller metabolically important compounds, as well as their metabolites. Additionally, as discussed below, at least some of the compounds associated with the proteins emit fluorescence or have an absorbance, which is useful for identifying a diseased or infected state. For example, Applicants of the present invention discovered that features of the fluorescence spectrum of plasma between about 380 nm and about 600 nm are a function of the presence or absence of a diseased state such as, for example, HIV. Treating a sample according to the method of the present invention accentuates differences in these features, thereby improving the ability to discriminate between a spectrum obtained from an infected sample and from a non-infected sample.

The biological sample is combined with a buffer to form a first mixture. Any buffer composition that provides adequate buffering capacity at a desired pH may be used in the present method. Preferably, however, the buffer is a phosphate buffer, such as, for example, an aqueous solution comprising potassium dihydrogenphosphate, anhydrous monosodium phosphate, trisodium phosphate dodecahydrate, or any combination thereof. The buffer may also comprise organic liquids. When the biological sample is a liquid such as for example, plasma, the combining step can be achieved by mixing the buffer and plasma to obtain a substantially homogeneous solution. If a biological sample comprising substantial solids such as a tissue sample is used, the combining step may further require homogenizing the buffer and tissue, as by blending, digestion, or other methods known in the art. It may also be useful to chemically lyse cells in the biological sample. The buffer should maintain the pH of the sample within an appropriate range for dissociating the compounds carried by or otherwise associated with the proteins in the sample. However, the pH should not be so high or low as to inhibit or perturb the fluorescent properties of the dissociated molecules. Preferably, the pH of the buffer is between about 2.5 and 5.5, such as, for example, between about 3.5 and 4.5, and most preferably about 4. Compounds released from the proteins at about pH 4 include, for example, pterins. One of ordinary skill in the art understands how to prepare a buffer according to the desired pH and buffer capacity.

Relative to the amount of plasma or other biological sample used, the buffer should be present in an amount sufficient to provide adequate buffering capacity. Preferably, the buffer solution comprises from about 0.6 to 1.3 molar buffer, for example, between about 0.9 and 1.1 molar and most preferably around 1 molar buffer. To avoid overly diluting the biological sample and compounds therein, the volume of buffer solution is preferably from about 10 to 50% as large as the volume of biological sample, preferably from about 15 to 25% as large. The exact volume and buffer concentration, however, can be determined by one of ordinary skill in the art and depends, for example, on the particular biological sample chosen for analysis, the diseased or infectious state in question, and the amount of acid used, as described below.

An organic acid, preferably trifluoroacetic acid, is added to the first mixture to obtain a second mixture. In this regard, a spectrophotometric grade of acid is well suited for use in the present invention. For example, spectrophotometric grade trifluoroacetic acid has minimal absorbance at ultraviolet and visible wavelengths greater than about 270 nm and has minimal fluorescent impurities. Generally, the first mixture and acid may be combined by mixing, although further homogenization or digestion may also be desirable in order to achieve a substantially homogeneous mixture.

The acid is preferably added to the first mixture in the form of a liquid comprising more than about 60% by volume acid, for example, more than about 90%, or most preferably, more than about 99% acid by volume. When the biological sample is plasma, the volume of organic acid added to the first mixture is preferably from about 20% to 75% as large as the volume of buffer used, preferably from about 40 to 60% as large. The amount of acid should be sufficient to separate the fluorescent compounds from their associated proteins. Although some of the bound compounds may be dissociated from the proteins immediately upon adding the acid, a substantial portion are dissociated during the subsequent heating step, which is described below. The exact amount and strength of acid, however, can be determined by one of ordinary skill in the art and depends, for example, on the buffer used, the particular biological sample chosen for analysis and the diseased or infectious state in question. An organic liquid is then added to the second mixture to obtain a third mixture. Although a variety of organic liquids may be used, it is important that detectable amounts or concentrations of compounds dissociated from the proteins are soluble in the chosen liquid. Acetonitrile is a particularly useful liquid in the method of the present invention. Spectrophotometric grades of acetonitrile, which have minimal fluorescent and absorbing impurities, are also suited for use in the present method.

The volume of the organic liquid should not be so large as to overly dilute compounds extracted from the biological sample. For example, when the biological sample is plasma, the volume of organic liquid, such as, for example, acetonitrile, is preferably about 5 to 25 times as large as the volume of plasma, more preferably about 12 to 18 times as large. The exact amount of liquid, however, can be determined by one of ordinary skill in the art and depends, for example, on the particular biological sample chosen for analysis and the diseased or infectious state in question.

Subsequently, the third mixture is heated to a temperature and for a period of time sufficient to dissociate compounds from the proteins or other biological molecules in biological sample. For example, the mixture may be heated to from about 65 to 115 °C, preferably to from about 90 to 110 °C. Generally, the mixture is heated for more than about one-half hour, preferably more than about 1 hour. Following such a heating step, a substantially clear supernatant is formed over solids, which are partially or wholly insoluble in the supernatant. At least in part, the solids comprise proteins which have been precipitated from the third mixture. Compounds, previously associated with the proteins or other biological molecules within the biological sample are substantially dissociated from the proteins and extracted into the supernatant liquid. The supernatant and solids can be separated using techniques known in the art, such as filtration, centrifugation, selective adsorption, or combination thereof.

Applicants have previously discovered that reduced nicotinamide adenine dinucleotide (NADH), thiochrome, holo-lipoproteins, and holo-albumin contribute to the fluorescence spectrum of human plasma. The oxidized form of nicotinamide adenine dinucleotide (NAD) exhibits no fluorescence in the 380-600 nm wavelength range. It has been documented, however, that the presence of HTV viruses results in a reduction of niacin and NADH through the effects of interferon gamma's induction of the catabolism of tryptophan, the precursor to niacin. Thus, Applicants believe that at least a portion of the fluorescent compounds extracted into the supernatant phase comprise NADH, which has been dissociated from proteins in the sample and is indicative of the presence of HTV. The supernatant obtained from the heating step is better suited to spectroscopic analysis than untreated biological samples such as plasma. Removing large molecules such as proteins from the sample improves the spectroscopic quality of the sample by reducing spurious scattering and fluorescence signals associated with these molecules. Additionally, dissociating the compounds from the proteins may increase the fluorescence yield of these compounds, thereby, improving the ability to discriminate between a sample infected with a disease and a sample free of disease. It is known that interactions, such as, for example, energy transfer between bound compounds can inhibit the observed fluorescence yield compared to the free compounds. Samples prepared according to the present invention may be analyzed using spectroscopic techniques that are understood in the art. A particularly powerful approach for analyzing such samples is described in U.S. Patent Application Nos. 09/224,141 filed December 31, 1998 and 09/436,207 filed November 8, 1999, which are incorporated herein by reference in their entireties. Briefly, these applications describe instrumentation and algorithms suitable for discriminating a diseased or infected sample from a disease-free or infection free sample.

Acquisition of a fluorescence sample from the presently prepared samples comprises irradiating the sample with radiation having a sufficient energy for inducing a fluorescence emission from a predetermined compound, e.g., molecule. Preferably, the sample is irradiated with at least one wavelength of from about 270 to 750 nm, more preferably from about 270 to 400 nm. Lasers such as, for example, Nd:YAG lasers that emit a fundamental or harmonic line in the ultraviolet are ideal excitation sources. The resulting fluorescence emission may be measured quantitatively or qualitatively using a light sensitive detector. A particularly useful fluorescence instrument comprises a dispersion element and a charge coupled device detector (CCD) for measuring the intensity of the emitted fluorescence as a function of the wavelength of the emitted light. Preferably, the detected fluorescence comprises radiation with at least one wavelength of from about 400 to 850 nm, more preferably of from about 400 to 600 nm. Such an instrument provides a powerful means of discriminating partially overlapped spectra obtained from complex samples comprising multiple fluorescent compounds.

Algorithms based on characteristics of the observed spectra, such as, for example, the total fluorescence, the wavelength of peak intensity, the shift in the wavelength of peak intensity, or the peak intensity, and fluorescence lifetime may be used to further discriminate partially overlapped spectra. A particularly powerful class of algorithms comprise multidimensional or multivariate techniques, which simultaneously utilize multiple parameters from one or more spectra. Combining multiple parameters into a single analysis or algorithm increases the predictive accuracy of the technique. Related techniques such as neural network algorithms can be "taught" to discriminate a diseased or infectious sample from disease free or infection free samples. Each of these techniques is suitable for use with the sample preparation method of the present invention.

Prior to performing a spectroscopic analysis, the supernatant of the present invention may be treated to a chromatographic separation, such as, for example, liquid chromatography or electrophoresis, in order to further enhance the difference between a diseased or disease-free sample. In particular, microfabricated devices such as those described in U.S. Patent No. 5,858,195 to Ramsey discloses systems and methods for performing fluid manipulations in channels etched in a microfabricated substrate. Within such devices, electrokinetic pumping allows rapid mixing, reaction, and separation of minute quantities of samples, reagents, and products. The method of the present invention may be adapted for use on such devices and, following formation of the supernatant, spectroscopic detection of the compounds may be accomplished with or without a separation step.

The following non-limiting example further discloses the method of the present invention.

Example 1

A sample of plasma from a Hepatitis C infected individual and a control sample from an individual free of Hepatitis C were obtained. A fluorescence instrument comprising an ultra-violet excitation source and a multiwavelength detector was used to obtain a Hepatitis C fluorescence spectrum 1 and a control fluorescence spectrum 2 from the Hepatitis C infected sample and Hepatitis C free control sample, respectively. Spectra 1,2 were obtained prior to treatment with the method of the present invention. The spectra are shown in Figure 1. Although Hepatitis C spectrum 1 exhibits a somewhat larger intensity than control spectrum 2, the spectra are not substantially resolved and it is difficult to discriminate between the infected and non-infected samples on the basis of these spectra. Subsequently, a portion of each plasma sample was treated separately according to the following steps:

A) A 0.5 ml volume of plasma was combined with mixing with a 0.1 ml volume of 1 M phosphate buffer of pH 4 to obtain a first mixture. B) A 0.05 ml volume of trifluoroacetic acid was added with mixing to the first mixture to obtain a second mixture. C) A 7.5 ml volume of acetonitrile was added with mixing to the second mixture to obtain a third mixture.

D) The third mixture was heated at 100 °C for two hours to obtain a fourth mixture comprising a clear supernatant and partially or wholly insoluble solids. E) The clear supernatant was separated from the solids.

Subsequently, the clear supernatant obtained from the Hepatitis C plasma and the control plasma were subjected to fluorescence analysis as described above to obtain a treated Hepatitis C spectrum 3 and a treated control spectrum 4, respectively, as shown in Figure 2. The results clearly demonstrate that the method of the present invention dramatically increases the differences between fluorescence spectra obtained from diseased or infected samples and disease free or infection free samples. For example, a peak amplitude and area of treated Hepatitis C spectrum 3 are both significantly greater than a peak amplitude and area of treated control spectrum 4. Moreover, the treated Hepatitis C spectrum exhibits a red shift (shift to longer wavelengths) compared to the treated control spectrum. These distinctions between the treated spectra are significantly greater than the differences between the spectra of the untreated plasma samples. This indicates that the treating the samples according to the present invention provides the ability to more selectively distinguish between positive and negative samples. Additionally, the present invention provides a more sensitive detection of

HTV infection and other diseases, particularly at the early stage of the infection when the infection is undetectable by conventional methods.

While various descriptions of the present invention are described above, it should be understood that the various features can be used singly or in any combination thereof. Therefore, this invention is not to be limited to only the specifically preferred embodiments depicted herein.

Further, it should be understood that variations and modifications within the spirit and scope of the invention may occur to those skilled in the art to which the invention pertains. Accordingly, all expedient modifications readily attainable by one versed in the art from the disclosure set forth herein that are within the scope and spirit of the present invention are to be included as further embodiments of the present invention. The scope of the present invention is accordingly defined as set forth in the appended claims.

Claims

THE CLAIMSWhat is claimed is:

1. A method for preparing a biological sample for detection of a diseased or infected state, said method comprising: combining the biological sample with a fluid to obtain a first mixture; and heating the first mixture to a temperature and for a time sufficient to obtain a second mixture comprising a supernatant having a detectable amount of free compounds and wherein the presence or abundance of the free compounds may be used to determine the presence of the diseased or infected state.

2. The method of claim 1 wherein said diseased or infected state comprises at least one of HIV, Hepatitis A, Hepatitis B, Hepatitis C, or a combination thereof. ^'

3. The method of claim 1 wherein the free molecules are dissociated from proteins to enhance a spectroscopic property of the free molecules.

4. The method of claim 3 wherein the spectroscopic property is a fluorescence yield.

5. The method of claim 1 further comprising the steps of: irradiating the supernatant with radiation; measuring quantitatively or qualitatively a fluorescence emission or absorbance of the free molecules; and determining the presence of a diseased state by analyzing said fluorescence emission or absorbance.

6. The method of claim 5 wherein the fluorescence emission or the absorbance is compared to a signal characteristic of a non-diseased state.

7. The method of claim 5 wherein the radiation has at least one wavelength of from about 270 to 750 nm.

8. The method of claim 5 wherein the fluorescence emission is detected over at least a portion of the range from about 400 to 850 nm.

9. The method of claim 1 wherein said first mixture is heated to a temperature of from about 65 to about 115 °C.

10. The method of claim 1 wherein the biological sample comprises plasma.

11. The method of claim 10 wherein the first mixture comprises from about 4 to 8% by volume plasma, 0.9 to 1.5% by volume buffer, 0.3 to 0.9% by volume acid, and 88 to 96% by volume organic liquid.

12. The method of claim 11 wherein the buffer is a phosphate buffer having pH of from about 3 to about 5.

13. The method of claim 11 wherein the acid is trifluoroacetic acid.

14. The method of claim 11 wherein the organic liquid is acetonitrile.

15. The method of claim 11 wherein the first mixture further comprises water.