This invention relates to methods of determining the presence and concentration of analytes in a test sample.  More specifically, the present invention relates to methods for non-invasively determining the analyte concentrations in human or animal subjects.  Most specifically, the present invention relates to a non-invasive methods for the determination of blood glucose concentration in a human patient. 
The analysis of samples and the determination of the presence or concentration of chemical species contained therein is a common and important process in chemistry and biology.  Particularly important is the analysis of biological fluids, such as blood, urine, or saliva, to determine the concentration of various constituents.  Also of great importance is the measurement of the concentration of various chemical constituents embedded within biological materials, such as tissue.  Chemical analysis of blood, urine, and other biological fluids is crucial to the diagnosis, management, treatment, and care of a wide variety of diseases and medical conditions.  In the case of diabetes, monitoring of blood glucose levels several times a day is necessary to the efficient management of this disease in many patients.  Analysis of various blood components is of importance in both the diagnosis and treatment of diseases of the circulatory system.  For example, the level of various types of cholesterol in the blood has a strong correlation with the onset of heart disease.  Urine analysis provides valuable information relating to kidney function and kidney disease.  The concentration of alcohol in the blood is known to be related to a subject""s physical response time and coordination and can provide information related to, for example, the individual""s fitness to drive a motor vehicle. 
Additionally, there are many instances where it is desirable to measure the local concentration of chemical constituents in tissue, either in-vivo or in-vitro.  For example, in stroke victims it is important to monitor the degree of brain edema or the concentration of various metabolic chemical constituents in the brain that serve as indicators of brain function.  Such indicators include fatty acid compounds, water, blood, lactates, and certain proteins and lipids.  Other specific examples may include the monitoring of tissue oxygenation or tissue blood perfusion as a means to of gauging the metabolic function of a human or animal subject. 
Moreover, in many applications, a xe2x80x9creal-timexe2x80x9d measurement of chemical concentration in biological fluids is important.  Current invasive methods require that a sample of fluid be removed from a subject and then analyzed in one or more chemical tests.  The tests can be expensive and require skilled technicians to remove and analyze the samples.  Furthermore, the analysis of samples may have an undesirably long turn-around time.  Additionally, the tests are usually made in centralized clinical laboratories with a resulting complexity of sample tracking and quality control.  These circumstances create additional problems related to the potential change in the chemical composition of the fluid between extraction and analysis and, even more detrimentally, the possibility of a sample being confused with the samples of other patients. 
It is also advantageous to analyze the chemical nature of sample materials without physically extracting a sample from the subject.  For example, it is advantageous to examine the chemical makeup of human blood without taking a blood sample.  In addition to time and cost considerations such invasive testing causes skin trauma, pain, and generates blood waste. 
For all of the foregoing reasons methods of xe2x80x9cnon-invasivexe2x80x9d testing have long been considered an attractive alternative to invasive testing.  However, prior non-invasive testing methods have suffered from a number of practical drawbacks.  The present invention is a method of analytical and quantitative testing for the presence of chemical species in a test sample.  The method is non-invasive and has wide utility, being easily applicable to the non-invasive measurement of humans, animals, plants, or even packaged materials.  Being highly versatile the method is broadly applicable to both in-vivo and in-vitro samples. 
1.  Brief Description of Related Art
The concept of non-invasive testing is not unknown in the art.  What has been elusive is the ability of quickly, easily, cheaply and accurately conducting measurements. 
Certain infrared (IR) detection techniques are known and have been used to detect the presence of chemical constituents in the blood.  Specific examples include the IR detection of oxygen saturation, nitrous oxide concentration, carbon dioxide concentration, or measurement of oxidative metabolism, and blood glucose levels.  The goal of these inventions is the determination of human blood chemistry.  A typical present technology projects light into the body while measuring the light after it passes through the body.  Comparing the input beam with an exit beam allows a rough determination of blood chemistry.  Unfortunately, these techniques suffer from a number of inadequacies, most especially, tissue interference, lack of specificity, and limited accuracy.  A number of prior art patents describing such techniques are set forth below. 
Kaiser describes, in Swiss Patent No.  612,271, a technique for using an IR laser as a radiation source to measure glucose concentrations in a measuring cell.  This technique uses venous blood passed through extra-corporeal cuvettes at high blood flow rates.  This has the undesirable effect of heating the blood and requiring that the blood be removed from the patient""s body.  Kaiser does not describe a non-invasive technique for measuring glucose concentration. 
March, in U.S. Pat.  No.  3,958,560, describes a xe2x80x9cnoninvasivexe2x80x9d automatic glucose sensor system which projects polarized IR light into the cornea of the eye.  A sensor detects the rotation of this polarized IR light as it passes between the eyelid and the cornea.  The rotation of polarized light is correlated to glucose concentration.  Although this technique does not require the withdrawal of blood, and is thus, xe2x80x9cnoninvasivexe2x80x9d the device may cause considerable discomfort to the patient due to the need to place it on the patient""s eye.  Furthermore, March does not use an induced temperature gradient or absorbance spectroscopy as does the present invention.  As a result, the present invention involves no physical discomfort and is more accurate. 
Hutchinson, in U.S. Pat.  No.  5,009,230, describes a glucose monitor which uses polarized IR light to non-invasively detect glucose concentration in a person""s blood stream.  The method requires an external IR source, which is passed through a portion of the human body.  However, the accuracy of measurement is limited by the wavelengths of the polarized light beam (940-1000 nm) being used.  Unlike the present invention, Hutchinson relies on detected changes in the polarization of the incident light beam.  Furthermore, Hutchinson does not use an induced temperature gradient as does the present invention. 
Similar limitations are found in Dahne, et al.,  in U.S. Pat.  No.  4,655,225, which describes a similar spectrophotometric technique.  Dahne uses a directional external IR radiation source to emit a beam.  Reflected and transmitted light from the beam is used to determine the glucose concentration.  Dahne differs from other techniques in using radiation at wavelengths between 1000-2500 nm.  Unlike Dahne, the present invention is not confined to using wavelengths between 1000-2500 nm.  Dahne also does not use an induced temperature gradient as does the present invention. 
Mendelson, et al.,  in U.S. Pat.  No.  5,137,023, uses a different concept known as pulsatile photoplethysmography to detect blood analyte concentration.  The instrument of Mendelson is based on the principles of light transmission and reflection photoplethysmography, whereby analyte measurements are made by analyzing either the differences or ratios between two different IR radiation sources that are transmitted through an appendage or reflected from tissue surface before or after blood volume change occurs in response to systolic and diastolic phases of the cardiac cycle.  Once again, the technique requires the use of external IR sources and is susceptible to interference from body tissue and other blood compounds. 
Rosenthal, et al.,  in U.S. Pat.  No.  5,028,787, discloses a non-invasive blood glucose monitor which also uses IR energy in the near IR range (600-1100 nm) to measure glucose.  As with the above-mentioned devices, these wavelengths suffer from poor analyte absorption which results in poor resolution and insufficient specificity. 
Cho, et al.,  in PCT No.  PCT/DE95/00864, discloses a blood glucose monitor which uses heat flux generated in a patients fingertip to measure metabolic rate.  Indirectly, this approximation of metabolic rate is used to measure approximate glucose concentration. 
Major steps forward are embodied in the glucose measuring techniques disclosed in the patents to Braig, et al.,  U.S. Pat.  No 5,313,941 (""941).  However, the ""941 patent requires an independent external IR source to determine blood analyte concentration. 
Optiscan, Inc of Alameda, Calif. has expanded the concept of gradient absorbance spectroscopy and demonstrated the utility of non-invasively measuring differential absorbance to determine blood glucose concentration in human subjects in U.S. patent application Ser.  No.  08/816,723 and 08/820,378, both of which are hereby incorporated by reference. 
2.  Scientific Background of the Invention
An understanding of the present invention requires an understanding of the concepts of transmission spectroscopy and gradient spectroscopy. 
Basic transmission spectroscopy identifies analytes (an analyte is defined as a chemical species sought to be identified by the present invention) by comparing a light beam passed through a test sample to a reference beam not passed through the sample.  Typically, transmission spectroscopy requires the test sample be removed from its native environment to a sample holder for analysis.  The absorbance spectrum of the sample is examined.  At specific wavelengths (known as analyte absorbance peaks) the light from the beams are compared.  By using Beer""s Law and comparing the sample beam with the reference beam in selected absorbance regions the absorbance of a sample may be measured and a determination of analyte concentration may be made.  This is known as classical transmission cell spectroscopy.  Strictly speaking, this method is unsuitable for non-invasive measurement.  Significant problems being the need for extracting samples and the inability to accurately determine the pathlength of the beams used to analyze in-vivo samples.  Progress has been made in overcoming these limitations as shown in the patents to Braig, et al.,  in Pat.  No. ""s ""941, ""847, and ""672 and in the patent application Ser.  Nos.  08/816,723 and 08/820,378.  These patents and patent applications have laid the groundwork for the novel advances embodied in the present invention and are hereby incorporated by reference. 
An understanding of the radiation emission characteristics of matter are also needed.  All objects at a temperature greater than 0xc2x0 K emit electromagnetic radiation in the form of photons.  Ideal blackbody radiators (objects having an emissivity coefficient em=1.0) radiate energy according to the Stefan-Boltzmann Law and Planck""s Equation (i.e. radiation output increases with increasing temperature).  Additionally, many non-blackbody objects demonstrate near-blackbody radiation characteristics.  For example, the human body""s spectral radiation characteristics are very similar to that of a blackbody radiator and may be described as a xe2x80x9cgraybodyxe2x80x9d distribution (for example, having an em of about 0.9).  These radiative characteristics provide known sources of IR radiation which may be used to non-invasively analyze the constituents of a test sample. 
Furthermore, an analysis of radiation behavior shows that, in objects at a constant and uniform temperature, photons emitted from the interior of the object are reabsorbed within 10-20 xcexcm of the point of origin.  Thus, an external radiation detector cannot detect radiation emitted from deeper than 20 xcexcm inside an object.  Under these conditions, only an object""s surface emission spectrum is detectable by a detector.  This poses a significant problem for non-invasive measurement techniques seeking to analyze chemical characteristics present deeper within an object. 
The field of gradient spectroscopy was developed, in part, in an attempt to overcome the photon reabsorption problem.  The Optiscan patent applications 08/816,723 and 08/820,378 disclose a thermal gradient, induced by a single temperature event and a measurement of differences in signal magnitude to non-invasively measure human test samples.  The Optiscan applications use a temperature gradient induced by a single temperature event to non-invasively determine the glucose concentration in a human test subject by analyzing differences in signal magnitude at selected wavelengths. 
In the absence of a temperature gradient, emitted photons are reabsorbed by a test sample, after they travel only a short distance.  By inducing a temperature gradient in a test sample, photons travel greater distances before reabsorption by the sample.  This allows radiation emitted from deep inside the sample to reach the surface where it can be detected by a radiation detector.  Additionally, the larger the gradient, the greater the detector signal is, improving the signal-to-noise ratio.  The present invention utilizes this phenomenon to analyze the chemical composition of the sample without the need to pass an externally applied light beam completely through the sample. 
Briefly, in the context of the present invention, a temperature gradient exists where the temperature of a material varies according to some arbitrary function, usually related to depth or time or both.  For example, if some material is at an initial temperature (e.g., 37xc2x0 C.) and a surface of the material is cooled to some lower temperature (e.g., 10xc2x0 C.) a gradient is induced in the material with the cooled surface being at approximately 10xc2x0 C. and the deeper (and as yet unaffected) regions being at approximately 37xc2x0 C. A temperature gradient exists between the two extremes. 
The present invention integrates all of the above-mentioned concepts to provide a method of analyzing the constituents of a test sample. 
Accordingly, an object of the present invention is to provide a new and improved method for detecting and quantifying various chemical analytes present within a test sample.  In particular, an object of the present invention is to determine the absolute or relative concentration of chemical species contained in a test sample medium.  Another object of the present invention to provide a non-invasive method of quantifying various chemical analytes within biological media. 
A specific object of the invention is to provide a new and improved method for measuring the concentration in human, animal, and plant subjects of chemical species, such as glucose, insulin, water, carbon dioxide, alcohol, blood oxygen, cholesterol, bilirubin, ketones, fatty acids, lipoproteins, albumin, urea, creatinine, cytochrome, various proteins and chromophores, microcalcifications, and hormones, white blood cells, red blood cells, hemoglobin, oxygenated hemoglobin, carboxyhemoglobin, organic molecules, inorganic molecules, and inorganic molecules, such as phosphorus or various drugs and pharmaceuticals in blood, urine, saliva, or other body fluids.  A further object of the invention is to make such measurements non-invasively, quickly, easily, and with extreme accuracy. 
The present invention describes a method for quantitatively determining the chemical composition of a test sample.  Test samples may be chosen from a broad range of in-vivo or in-vitro samples.  The present method uses a radiation detector, a data processing means, and a means for inducing a periodic thermal gradient in the test sample.  The method generally comprises the steps of providing a test sample, inducing a thermal gradient in the sample, using the detector for measuring analytical signals from the sample at one or more predetermined wavelengths.  Simultaneously, one or more reference signals are measured at reference wavelengths.  The analytical and reference signals are compared to determine a parameter.  The parameter may be phase difference, signal amplitude difference, or frequency difference.  The parameter information is correlated with empirically determined analyte concentration information by the data processing means, thereby determining the analyte concentration of the sample.  This information is transmitted as an electrical signal for further processing. 
A particularly useful parameter is a measurement of the phase difference (or phase delay) between said analytical and one or more of said reference signals.  The magnitude of the phase difference is correlated with data stored in a data processing means to determine analyte concentration. 
The accuracy of the method is substantially enhanced by inducing a periodically modulated temperature gradient in the sample, measuring the reference and analytical signals, continuously monitoring the parameters between the reference and analytical signals, and then integrating the parameter information over a test period.  Correlation of this information with empirically determined analyte concentration information allows the analyte concentration of the sample to be determined and transmitted as an electrical signal for further processing.  Alternatively, when using a periodically modulated temperature gradient and phase difference information, the phase may be monitored at reference and analytical phase signal xe2x80x9czero crossingsxe2x80x9d to determine phase delay and thereby determine analyte concentration. 
Additionally, the present method may be used to monitor analyte concentration at varying depths inside a test sample.  This is accomplished by introducing two or more periodic temperature gradients in a sample at two or more driving frequencies.  The resulting signals are processed to extract phase information and determine analyte concentration at varying depths within a sample, This has particular usefulness in analyzing analyte concentrations in test samples having non-uniform properties. 
Finally, the method of the present invention may be used to non-invasively determine the blood glucose concentration of human and animal test subjects. 
Other features of the invention are disclosed or made apparent in the section entitled xe2x80x9cDetailed Description of the Inventionxe2x80x9d.