Patent Application
20100167412
1. Field of the Invention
The present invention relates generally to sensors used in analysis of samples, and in particular relates to methods that allow integration of sample handling, reagent addition, and spectrophotometric measurement into an integrated handheld sensor system.
2. Description of Related Art
Sensor methods for quantification of volatile and nonvolatile compounds in fluids are known in the art. Typically, quantification of these parameters is performed using dedicated sensor systems that are specifically designed for this purpose. These sensor systems operate using a variety of principles including electrochemical, optical, acoustic, and magnetic. For example, sensor systems are used to conduct optical inspection of biological, chemical, and biochemical samples. A variety of spectroscopic sensors operating with colorimetric liquid and solid reagents have been developed. In fact, spectrophotometric indicators in analytical chemistry have become the reagents of choice in many commercially available optical sensors and probes.
Optical sensors possess a number of advantages over other sensor types, the most important being their wide range of transduction principles: optical sensors can respond to analytes for which other sensors are not available. Also, with optical sensors it is possible to perform not only “direct” analyte detection, in which the spectroscopic features of the analyte are measured, but also “indirect” analyte determination, in which a sensing reagent is employed. Upon interaction with the analyte species, such a reagent undergoes a change in its optical property, e.g. elastic or inelastic scattering, absorption, luminescence intensity, luminescence lifetime or polarization state. Significantly, this sort of indirect detection combines chemical selectivity with that offered by the spectroscopic measurement and can often overcome otherwise troublesome interference effects.
Because spectrophotometric indicators were originally developed for aqueous applications, their immobilization into a solid support is a key issue for their application in optical sensing. Polymeric materials for reagent-based optical sensors are often complex multicomponent formulations. The key formulation ingredients include a chemically sensitive reagent (indicator), a polymer matrix, auxiliary minor additives, and a common solvent or solvent mixture. In the past, it has been difficult to predict the best formulation of the sensor material to yield a certain desired functionality.
It is known that a variety of chemical substances absorb light in proportion to the concentration of the substance present in the sample. Furthermore, the light transmitted through such a substance has an absorption spectrum characterized by the light absorbing properties of the substance and the properties of any other medium through which the light travels. Such absorption spectrum can be prismatically revealed for analysis. By discounting the portion of the absorption spectrum attributable to intensity losses and other absorbers, the spectrum of the chemical substance can be isolated and its identity and concentration determined. The discounting, or “referencing,” is done by determining the absorption spectrum of the light source and any spectrophotometric components in the absence of the chemical substance. Referencing is usually done close in time and space to the measurement of the absorbance of the chemical substance to minimize error.
It is well known that portable, battery-powered devices for determining the concentrations of chemical substances are commercially available. Examples include portable photometers provided by Hach Company (Loveland, Colo., USA) and portable reflectometers by Merck (Whitehouse Station, N.J., USA). A detailed review of photometric and reflectometric systems is given in Comprehensive Analytical Chemistry, Chemical Test Methods of Analysis, (Y. A. Zolotov et al., Elsevier, N.Y. (2002)), and in a review paper given in Review of Scientific Instruments, (Kostov, Y. and Rao, G., Vol. 71, 4361, (2000)). The adoption of these systems makes chemical analysis outside of a laboratory possible.
Other methods utilizing test strips have been widely attempted for semi-quantitative analysis for a large number of analytes. Here, quantitative results can be obtained with disposable optical sensor elements, read by a photometer. In most instances, only a single analyte is determined by an optical sensor element. Since transmission absorbance is measured, it is difficult to produce disposable optical sensor elements for calibration free tests.
Disposable chemical sensors are well known in the art. For example, U.S. Pat. No. 5,830,134 describes a sensor system for detecting physico-chemical parameters designed to compensate for numerous perturbing factors, such as those resulting from the use of partially disposable monitoring units, thus eliminating the need for calibration steps.
Another U.S. Pat. No. 5,156,972 discloses a chemical sensor based on light absorption, light emission, light scattering, light polarization, and electrochemically and piezoelectrically measured parameters. Scatter controlled emission for optical taggants and chemical sensors have been disclosed in U.S. Pat. No. 6,528,318. Sensor arrays that use reference and indicator sensors are known and described in U.S. Pat. No. 4,225,410. Here, a sensor can be individually calibrated, such that each analysis can be read directly.
U.S. Pat. No. 5,738,992 discloses a method that utilizes a reference material to correct fluorescence waveguide sensor measurements. U.S. Pat. No. 5,631,170 teaches a referencing method for fluorescence waveguide sensors by labeling the waveguide with a reference reagent.
Two-wavelength, or dual-beam, methods are known in spectrophotmetric analysis. In “Referencing Systems for Evanescent Wave Sensors,” (Stewart, G. et al., Proc. Of SPIE, 1314, 262 (1990)), a two-wavelength method is proposed to compensate for the effect of contamination on the sensor surface. U.S. Pat. No. 4,760,250 describes an optoelectronics system for measuring environmental properties in which feedback-controlled light sources are used to minimize problems associated with the light source stability and component aging. A similar feedback-controlled two-wavelength method is described in U.S. Pat. No. 3,799,672. A dual-beam reflectance spectrophotometer is described in “Optical Fiber Sensor for Detection of Hydrogen Cyanide in Air,” (Jawad, S. M. and Alder, J. F., Anal. Chim. Acta 259, 246 (1991)). In Jawad and Alder's method, two LED's are alternately energized. The ratio of outputs at the two wavelengths is used to reduce errors caused by the background absorption of the sensor element for hydrogen cyanide detection. These two-wavelength methods are effective to minimize errors caused by optical and mechanical component aging and long-term stability problems of light sources. However, errors associated with variations in the effective optical path length of disposable test elements have not been solved.
A disposable sensor system comprising a discardable or disposable measuring device and further comprising one or more sensors is disclosed in U.S. Pat. No. 5,114,859. Furthermore, analysis of multiple analytes is done with microfabricated sensors as described in U.S. Pat. No. 6,007,775.
Many standard methods for determining the concentration of a chemical and biological substance in a liquid sample involve multiple steps. A sample usually requires a pretreatment such as filtering and dilution. The treated sample needs to be transferred to a measurement chamber such as a cuvette. An analytical reagent is added to the sample in the cuvette by a single or multiple aliquots. Mixing the reagent with the sample thoroughly is essential for many applications. Finally, optical properties of the sample-reagent mixture are measured by bench-top apparatus and converted to a concentration unit by an embedded microprocessor.
A multi-step analytical procedure is time consuming. In addition, more steps usually lead to more operational errors, such as sample contamination. Thus, any simplification of conventional analytical procedures is desirable.
By analyzing relationships among the sample, operator, and sensor apparatus, one may recognize that an ideal sensor device may be like a temperature probe. To determinate the concentration of chemical and biological substances, a combined-electrode approach is probably the only approach that closely resembles a sensor for the measurement of physical properties. Unfortunately, reliable electrodes for analyzing a majority of chemical and biological species are not available. On the other hand, many reliable methods based on absorbance and fluorescence measurements have been developed. In addition, inexpensive optical and electronic component are widely available.
U.S. Pat. No. 5,844,686 discloses a hand apparatus comprising a pipetting means, an integrated photometer, and disposable pipette tip. The hand apparatus requires seals at both the distal and proximal openings of the tip. For carrying multiple reagents, a partition wall inside the tip is required. Presumably, the reagent or reagents are in liquid or solid powder format since seals at the openings are required. To bring the reagent to mix with the sample, one has to break at least one seal. For multiple reagent situations, one has to break a seal and a partition wall. In addition, U.S. Pat. No. 5,844,686 requires an optical path for absorbance measurements, such that the optical path of the photometer is directly across the wall of the pipette tip. The hand apparatus allows a sample to be withdrawn into the pipette tip and evaluated photometrically by the photometers integrated into the pipetting electronic means. The apparatus disclosed in U.S. Pat. No. 5,844,686 provides an optical reference path by means of attenuated total reflection element that is permanently connected to the pipette part of the apparatus. The function of the optical reference path is not defined in U.S. Pat. No. 5,844,686.
A need exists for a cost-effective and time-saving handheld sensor system that provides a platform for development of easy-to-use, portable, and inexpensive sensors for a variety of applications. In addition, a need exists for a system that simplifies conventional spectrophotometric methods for chemical or biological analysis.
The present invention relates to a method that allows integration of sample handling, reagent addition, and optical measurement into an integrated handheld sensor system. With this system, analytical procedures based on wet chemistry absorbance, fluorescence, and other spectrophotometric measurements are simplified. A disposable reagent-carrying pipette tip provides means for sample pipetting and reagent addition, and defines an optical space for the optical measurement. Signal normalization based on an internal reference reagent or indicator and/or a second wavelength measurement can effectively reduce sensor errors caused by variations in the disposable pipette tip and its optical alignment with respect to the optical components of the handheld sensor system disclosed. The handheld sensor system disclosed in this invention provides a platform for development of an easy-to-use, portable, and inexpensive sensors for a variety of applications ranging from laboratory and field analysis to medical diagnosis and household testing.
A sensor system for determining the concentration of chemical and biological analytes is disclosed that is comprised of a disposable reagent-carrying pipette tip, a liquid handling unit to which the pipette tip can be detachably mounted, the liquid handling unit capable of withdrawing liquid into the pipette tip, at least one light source that is capable of emitting at least two colors of light, at least one photodetector, the detector capable of generating an electronic signal response indicative of light passed through or generated from the interior space of the pipette tip, and an electronic circuit means for processing, storing and transmitting the electronic signal response and controlling the light source.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and benefits obtained by its uses, reference is made to the accompanying drawings and descriptive matter. The accompanying drawings are intended to show examples of the many forms of the invention. The drawings are not intended as showing the limits of all of the ways the invention can be made and used. Changes to and substitutions of the various components of the invention can of course be made. The invention resides as well in sub-combinations and sub-systems of the elements described, and in methods of using them.
a is a disposable reagent-carrying pipette tip in which the reagent is dispersed in a porous plug which is located at the lower part of the pipette tip in accordance with one embodiment of the present invention;
b is a disposable reagent-carrying pipette tip in which a solid reagent is placed in a gap created by two porous plugs in accordance with an embodiment of the present invention;
c is a disposable reagent-carrying pipette tip in which a polymer film containing a reagent is coated on the interior surface of the pipette tip in accordance with an embodiment of the present invention;
a is a pipette tip with a light pipe molded onto the inside wall of the pipette tip in accordance with an embodiment of the present invention;
b is a pipette tip with a light pipe molded onto the outside wall of the pipette tip in accordance with an embodiment of the present invention;
c is a pipette tip that has a metallized exterior surface in accordance with an embodiment of the present invention;
a is a light source and detection arrangement where the light source is installed inside the liquid handling unit in accordance with an embodiment of the present invention;
b is a light source and detection arrangement where the light source is installed outside the liquid handling unit in accordance with an embodiment of the present invention;
While the present invention has been described with references to preferred embodiments, various changes or substitutions may be made on these embodiments by those ordinarily skilled in the art pertinent to the present invention with out departing from the technical scope of the present invention. Therefore, the technical scope of the present invention encompasses not only those embodiments described above, but also all that fall within the scope of the appended claims.
Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about”, is not limited to the precise value specified. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Range limitations may be combined and/or interchanged, and such ranges are identified and include all the sub-ranges included herein unless context or language indicates otherwise. Other than in the operating examples or where otherwise indicated, all numbers or expressions referring to quantities of ingredients, reaction conditions and the like, used in the specification and the claims, are to be understood as modified in all instances by the term “about”.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such process, method article or apparatus.
The present invention relates to a method that allows integration of sample handling, reagent addition, and optical measurement into an integrated handheld sensor system. This invention discloses a method of integrating the four most important components found in conventional analytical system—fluidic device, reagent, optical and electronic components—into a compact, handheld sensor apparatus. With this system, analytical procedures based on wet chemistry absorbance, fluorescence, and other spectrophotometric measurements are simplified. A disposable reagent-carrying pipette tip provides a means for sample pipetting and reagent addition, and defines a body of a material for the optical measurement. Signal normalization based on an internal reference reagent and/or a second wavelength measurement can effectively reduce sensor errors caused by variations in the disposable pipette tip and its optical alignment with respect to the optical components of the handheld sensor system disclosed. The handheld sensor system disclosed in this invention provides a platform for development of an easy-to-use, portable, and inexpensive sensors for a variety of applications ranging from laboratory and field analysis to medical diagnosis and household testing.
The present invention pertains to a method and apparatus for determining the concentrations of chemical substances (analytes) by utilizing their reactive properties with certain chemical reagents; for example, the analyte-reagent reaction producing a product that has a visible absorption spectrum different from the reagent and analyte themselves. In operation, the present invention measures the reagent-containing test element response to specific analytes through a change in light absorbance, luminescence, light scattering, or other light-based response. The analytes described in this invention are chemical species, but this invention can also be envisioned to include biological systems where bioanalyte interactions stimulate similar test element response. As an example, such biological systems could be immobilized enzymes that stimulate light response proportional to an analytes concentration, for example, luciferase response to adenosine triphosphatase (ATP).
Materials utilized as analyte-specific reagents incorporate dyes and reagents known in the art as indicators. As used herein, analyte-specific reagents are indicators that exhibit calorimetric, photochromic, thermochromic, fluorescent, elastic scattering, inelastic scattering, polarization, or any other optical property useful for detecting physical properties and chemical species. Analyte-specific reagents include organic and inorganic dyes and pigments, nanocrystals, nanoparticles, quantum dots, organic fluorophores, inorganic fluorophores and similar materials.
A sensor system for determining a concentration of chemical and biological analytes is disclosed, which is comprised of a disposable reagent-carrying pipette tip; a liquid handling unit to which the pipette tip can be detachably mounted, the liquid handling unit capable of withdrawing liquid into the pipette tip; at least one light source that is capable of emitting two colors of light; at least one photodetector, the detector capable of generating an electronic signal response indicative of light passed through or generated from the interior space of the pipette tip; and an electronic circuit means for processing, storing, and transmitting the electronic signal response and controlling the light source. In one embodiment, the reagent contains a reference indicator and responsive indicator that reacts with the analyte to produce a spectrophotometric change. In an alternate embodiment, the reference indicator is negligibly responsive to the analyte and its spectrophotmetric characteristics is substantially different from that of the responsive indicator.
a,
1
b, and 1c demonstrate a disposable reagent-carrying pipette tip 12 in accordance with embodiments of the present invention. The system disclosed in the present invention introduces a reagent into the pipette tip 12 by means of polymer coating and/or dissolution through a porous plug 14. The reagent 16 is needed to react with analytes in order to produce a color product. This system is simpler than prior art systems in which in order to bring the reagent to mix with the sample, one has to break at least one seal or a seal and a partition wall.
A reagent or reagents 16 may be immobilized in the pipette tip 12 in several ways, as shown in
In one embodiment the disposable reagent-carrying pipette tip 12 demonstrated in
When the pipette tip 12 is manufactured by an injection molding process, other features may be incorporated into the sensor system in order to improve system performance, as shown in
In one embodiment, the pipette tip 12 has a metallized exterior surface 20. As shown in
The sensor system is comprised of a liquid handling unit 30 to which the pipette tip 12 may be detachably mounted, the liquid handling unit 30 capable of withdrawing liquid into the pipette tip 12. In one embodiment of the present invention, the liquid handling unit 30 may be a motorized pipette controlled with a microprocessor. The microprocessor and its auxiliary circuit may be used to control the light and read the output of the photodiode. In an alternate embodiment, the liquid handling unit 30 may be a manually operated pipette, in which necessary electronics may be built into the pipette for spectrophotometic measurements. In both embodiments, synchronization between liquid sample withdrawal, spectrophotometric measurement, and discharging the liquid from the pipette tip 12 when the measurement is completed is necessary.
The sensor system is also comprised of at least one light source 40, which can be any means that is capable of emitting light energy. Many light sources 40 may be selected for this application, such as multi-color LEDs, diode lasers, or miniature light bulbs. For the purpose of signal normalization, the light source 40 should be capable of emitting two colors of light. This can be achieved by using a multi-color LED or multiple LEDs and other light sources.
The sensor system further comprises at least one photodetector 50 or light detector, which can be any means that is capable of detecting light energy and converting the energy to electrical output signals that are indicative of the test elements response to the target analyte or analytes. It is understood that many commercially available photodetectors 50 or light detectors could be used to achieve the desired performance, such as photodiode, micromachined photo multiplier tube, or photocell, and are well known in the art. For absorbance measurement, miniature photodiodes and phototransistors may be used. For chemiluminescence and fluorescence measurements, photomultiplier tube (PMT) may be used. If a white light is used as the light source, a color sensor may be selected. Similarly, if a single wavelength light source is used, a detector that covers a wide range of spectrum is suitable. In one embodiment, the detector 50 is comprised of photodiodes, phototransistors, photomultiplier tubes (PMT), color sensors, and detectors that cover a wide range of the spectrum. Other light sources 50 known in the art may be used.
The light source 40 and detector 50 can be arranged in several ways, as shown in the figures. In one embodiment, both the light source 40 and detector 50 are installed inside the liquid handling unit 30, and there is no clearly defined optical path length, as shown in
In another embodiment, both the light source 40 and detector 50 are installed in a device 52 detachable from the liquid handling unit 30. This configuration is demonstrated in
In the arrangement in which the light source 40 and detector 50 are installed in a device detachable from the liquid handling unit 30, an ultrasonic wave generator may be embedded in the sensor system. The ultrasonic wave can help sample-reagent mixing. Because of the intimate contact of the pipette tip 12 with the wall of the chamber, a thin-film heating/cooling element and temperature sensor can be fixed on the interior wall of the chamber for temperature measurement and control. The ability to control sample temperature allows the system to measure samples with different initial temperatures and allows for either a standardization of measurement temperature to possibly an elevated temperature from the ambient and/or an increased tip temperature to accelerate the reagent-sample reaction.
The light source 40 may be installed inside the liquid handling unit 30 in such a way that provides illumination to the light pipe 18 described in
The sensor system is also comprised of an electronic circuit means 60, as shown in
The invention also provides methods for quantitating the concentration of an analyte by measuring an optical property of a sample or a change resulted in by the sample-reagent reaction. A method for determining analyte concentration of a chemical and biological substance is disclosed, which is comprised of providing a reagent-carrying disposable pipette tip; mounting the pipette tip to a liquid handling unit measuring at least two initial spectrophotometric parameters before a liquid sample is drawn into the liquid handling unit; drawing the liquid sample into the pipette tip; measuring two response spectrophotometric parameters at a give time or multiple times; calculating a normalized parameter using initial parameters and response parameters; and converting the normalized parameter to a concentration of analyte. The spectrophotometric parameters are absorbance, fluorescence, and other spectrophotometric measurements.
A significant source of error in a system using a disposable element is caused by variations from one disposable element to another, such as variations in geometric parameters of the disposable elements or variations in alignment of the disposable element with respect to the pipette. The error caused by these variations can be eliminated by signal normalization. Several signal normalization methods may be used. For example, as shown in the Examples below, absorbance values at one wavelength may be used as the main signal and absorbance at another wavelength may be used as a reference signal. The main absorbance value may be normalized by calculating the difference in the signals or the ratio or the combination of both.
There are many ways to select the reference wavelength at which the reference signal is measured. The reference wavelength could be substantially different from the main wavelength at which the main signal is measured. The reference wavelength could be any wavelength at which the reagent by itself exhibits some spectral features. For instance, if the reagent is a dye, then the reference wavelength could be the main absorption peak while the main wavelength could be the main absorption peak of the reagent-analyte reaction product. If the reagent has no spectral features that can be measured, a reference reagent can be added into the reagent composition. For example in a calorimetric measurement, if the reagent is colorless, a dye can be added to the reagent composition as the reference reagent. In this case, the main absorption peak of the reference dye could be chosen as the reference wavelength.
In one embodiment, the reagent contains a reference indicator. One of the spectrophotometric parameters is measured from a reference indicator and the other spectrophotometric parameter is measured from a response indicator. The indicators reacts with the analyte to produce a spectrophotometric change. The second parameter is a measure of analytical information. In another embodiment, the reagent does not contain a reference indicator and the first parameter is measured from the reference wavelength. The reference indicator is negligibly responsive to the analyte and its spectrophotmetric characteristics is substantially different from that of the responsive indicator. A normalized parameter or signal is calculated from the main signal and the reference signal, as further described in the Examples below. In one embodiment, the normalized parameter is calculated according to the difference between the first and second parameters, the ratio of the first and second parameters, or a combination of the difference and the ratio.
The invention is illustrated in the following non-limiting examples, which are provided for the purpose of representation, and are not to be construed as limiting the scope of the invention. All parts and percentages in the examples are by weight unless indicated otherwise.
As shown in
The pipette tip 12 containing chlorine sensitive reagent film was first loaded onto the pipette 10. The computer turns the green (525 nm) and red lights (630 nm) sequentially, and takes respective readings (Go and Ro) from the photodiode while the green and red lights are turned on. Then, chlorine standard solutions were drawn into the pipette tip. The solution in the pipette tip 12 was flushed out and back into the tip by injecting to a 5 ml disposable polyethylene beaker and aspirating back to the pipette tip. This process was repeated three times to accelerate mixing and release of the reagent immobilized from within the polymer film and mix well with the sample. Finally, The DC voltage output from the photodiode (G and R) was recorded while the green and red lights were turned on. Absorbance, calculated as log(R0/R) or log(G0/G), is shown as a function of chlorine concentration in
The results obtained using a second pipette that has a light-to-voltage sensor (TSL 257R from Taos Inc. (Plano, Tex., USA)) are also shown in
A method to immobilize a chlorine sensitive reagent N,N-diethylphenylenediamine (DPD) in a porous polymeric plug 14 inserted inside the pipette tip 12 was demonstrated, using the configuration as shown in
The pipette tip containing the chlorine sensitive reagent was first loaded onto the pipette. The computer turns the green (525 nm) and red lights (630 nm) sequentially, and took respective readings (Go and Ro) from the photodiode while the green and red lights were turned on. Then, chlorine standard solutions were aspirated into the pipette tip. While the sample flowed through the porous plug, reagent dissolution took place. No other forced mixing was required. Finally, the DC voltage output from the photodiodes (G and R) were recorded while the green and red lights were turned on.
Photodiode output G and R are shown as a function of chlorine concentration in
A light source and light detector configuration was used, as shown in
While the present invention has been described with references to preferred embodiments, various changes or substitutions may be made on these embodiments by those ordinarily skilled in the art pertinent to the present invention without departing from the technical scope of the present invention. Therefore, the technical scope of the present invention encompasses not only those embodiments described above, but all that fall within the scope of the appended claims.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated processes. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. These other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
