Patent Publication Number: US-2021190696-A1

Title: Dual-sensor detection of reflectance signals for thin-film based assays

Description:
PRIORITY CLAIM 
     This application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 62/950,833, filed on Dec. 19, 2019, the entire disclosure of which is hereby incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present disclosure relates generally to a thin-film element and corresponding device for analyzing a fluid sample, and more specifically to a device for measuring reflectance signals from both sides of a thin-film element. 
     BACKGROUND 
     Conventional thin-film based assays are performed by measuring reflectance signals from one surface of a thin-film element using a single sensor. In some cases, the single sensor may sequentially analyze multiple analytes from the same surface of the thin-film element. However, when multiple analytes are analyzed from the same surface, it is possible that one analyte can interfere with another. For example, in the case of an HbA1c assay, methemoglobin (the analyte for hemoglobin) can complicate the measurement of Fructosyl valine-histidine (fVH) (the analyte for HbA1c). 
     SUMMARY 
     The present disclosure is directed to a thin-film element that enables analytes to be analyzed on separate surfaces, and to a corresponding device that is configured to measure reflectance signals from the separate surfaces of the thin-film element to perform the analysis. In a general example embodiment, a device for analyzing a fluid sample includes a thin-film element comprising a first layer for processing the fluid sample to generate a first component and a second component. The thin-film element also includes a second layer configured to be impermeable to the first component to allow the first component to be retained by the first layer and permeable to the second component to allow the second component to pass through the second layer. The second layer includes a first reflective surface and a second reflective surface. The thin-film element further includes a third layer configured to retain the second component. The device additionally includes a first sensor positioned towards the first layer. The first reflective surface of the second layer is configured to generate a first optical signal by reflecting a first light modulated by the first component, where the first sensor is configured to receive the first optical signal. The device further includes a second sensor positioned towards the third layer, where the second reflective surface of the second layer is configured to generate a second optical signal by reflecting a second light modulated by the second component. The second sensor is configured to receive the second optical signal. 
     In another embodiment, the device includes a first light source positioned towards the first layer, where light from the first light source is modulated by the first component to generate the first optical signal. The device also includes a second light source positioned towards the third layer, where light from the second light source is modulated by the second component to generate the second optical signal. 
     In another embodiment, the device includes a first optical filter configured to filter the first optical signal before the first optical signal is received by the first sensor, and a second optical filter configured to filter the second optical signal before the second optical signal is received by the second sensor. In some embodiments, the optical filter can be located between a light source and a thin film element. In other embodiments, the optical filter can be located between a thin film element and an optical sensor. In still other embodiments, an optical filter can be located both between a light source and a thin film element and between a thin film element and an optical sensor. 
     In another embodiment, the first sensor comprises at least one of a photo multiplier tube, a contact-image sensor, a photodiode, and an image capturing sensor matrix, and the second sensor comprises at least one of a photo multiplier tube, a contact-image sensor, a photodiode, and an image capturing sensor matrix. 
     In another embodiment, the second layer comprises a gelatin and an optical masking material that provides the first reflective surface and the second reflective surface of the second layer. 
     In another embodiment, the optical masking material comprises TiO 2 . 
     In another embodiment, the first sensor generates a first electrical signal in response to the first optical signal and the second sensor generates a second electrical signal in response to the second optical signal. The device further includes a processor in communication with the first sensor and the second sensor to receive the first electrical signal and the second electrical signal. The processor is configured to determine or generate a ratio between the first component and the second component based on the first electrical signal and the second electrical signal. 
     In some embodiments, the sample comprises multiple components, for example, a first component and a second component. A first and second component are provided for illustration purposes only, three or more components may also be included in a sample. In some embodiments, the different components can include some property difference, such as but not limited to, molecular weight, size, molecular complexity, charge, van der Waals forces, hydrophobicity, hydrophilicity, and the like. In one embodiment, the difference can be molecular weight. In such an embodiment, the components can be calcium and albumin, which have very different molecular weights. The first layer can include at least one reagent for processing the fluid sample to generate the first component and the second component. In some embodiments, the at least one reagent is a compound that can generate or separate components based on a property difference. 
     In another embodiment, the sample comprises a human or animal blood sample, the first layer includes at least one reagent for processing the fluid sample to generate the first component and the second component, the at least one reagent comprising a lysing agent, a denaturing agent, and a protease for processing the blood sample to provide Hb and a peptide derived from HbA1c, where the first component of the blood sample comprises the Hb and the second component of the blood sample comprises the peptide (e.g., glycopeptide) derived from HbA1c. 
     In another embodiment, the third layer comprises at least one reagent configured to process the second component to generate a third component of the sample, and the second light is modulated by the third component. 
     In another embodiment, the thin-film element is moveable between the first sensor and the second sensor in a direction substantially perpendicular to a direction defined from the first sensor to the second sensor, such that a plurality of the first optical signals are generated by the first reflective surface and received by the first sensor and a plurality of the second optical signals are generated by the second reflective surface and received by the second sensor upon the movement of the thin-film slide. 
     In another embodiment, the first layer is a top layer, the second layer is a middle layer, the third layer is a bottom layer, the first sensor is a top sensor, and the second sensor is a bottom sensor. 
     In a general example embodiment, a method of analyzing a fluid sample includes moving a thin-film element between a first sensor and a second sensor in a direction substantially perpendicular to a vertical direction defined from the first sensor to the second sensor. The thin-film element comprises a first layer for processing the fluid sample to generate a first component and a second component, and a second layer configured to be impermeable to the first component to allow the first component to be retained by the first layer and permeable to the second component to allow the second component to pass through the second layer, where the second layer comprises a first reflective surface and a second reflective surface. The thin-film element also includes a third layer configured to retain the second component. The method also includes simultaneously generating a first optical signal by reflecting a first light modulated by the first component off of the first reflective surface and generating a second optical signal by reflecting a second light modulated by the second component off of the second reflective surface, and simultaneously receiving the first optical signal by the first sensor and receiving the second optical signal by the second sensor. 
     Additional features and advantages are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Also, any particular embodiment does not have to have all of the advantages listed herein, and it is expressly contemplated to claim individual advantageous embodiments separately. Moreover, it should be noted that the language used in the specification has been selected principally for readability and instructional purposes, and not to limit the scope of the inventive subject matter. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the present disclosure will now be explained in further detail by way of example only with reference to the accompanying figures, in which: 
         FIG. 1  is a side view of an example embodiment of a thin-film element. according to the present disclosure; 
         FIG. 2  is an exploded perspective view of the thin-film element of  FIG. 1 ; 
         FIG. 3  is a side view of an example embodiment of a device and thin-film element. according to the present disclosure; and 
         FIG. 4  is a flow chart showing an example embodiment of a method of using the device and thin-film element of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure relates to methods and apparatuses for analyzing a fluid sample, for example a human or animal sample, on a thin-film element. Fluid samples can be blood or a blood component or other liquids. Fluids can include, but are not limited to blood, urine, saliva, cerebral spinal fluid, bile, sweat, seminal fluid, plasma, serum, vaginal fluid, tears, vitreous fluid, or the like. In one embodiment, the fluid sample is a human or animal blood sample. 
       FIGS. 1 and 2  illustrate an example embodiment of a thin-film element  10 , according to the present disclosure.  FIG. 3  illustrates an example embodiment of an analysis device  100  according to the present disclosure which is configured to analyze a fluid sample dispensed on the thin-film element  10  shown in  FIGS. 1 and 2 . 
     As illustrated in  FIGS. 1 and 2 , the thin-film element  10  includes a plurality of layers. In the illustrated embodiment, the thin-film element  10  includes a first or top layer  20 , a second or middle layer  30 , and a third or bottom layer  40 . Those of ordinary skill in the art will recognize that additional layers can be added to the thin-film element  10 , or the existing layers can be divided into additional layers, without changing the function of thin-film element  10  as described herein. 
     In some embodiments, the thin-film element  10  can be formed on a support layer  42 . Support layer  42  can be a transparent material such as, but not limited to polyester or other transparent plastic material. In some embodiments, this support material can remain on the element during use. 
     First layer  20  of the thin-film element  10  is configured to initially receive a fluid sample  80  for analysis. Fluid sample  80  may be a blood sample including serum, plasma, or whole blood. In an embodiment, first layer  20  may include one or more reagent  50  that processes fluid sample  80  to produce analytes, such that the one or more reagent  50  generates a first component  60  and a second component  70  from the fluid sample when the fluid sample is placed on the first layer  20 . In an embodiment, first layer  20  may be transparent or partially opaque so as to not affect reflectance of an optical signal modulated by the fluid sample  80 , as explained in more detail below. In some embodiments, the composition of the first layer may be 30 μm polymeric beads supported by a water soluble polymer. 
     In an example embodiment, the one or more reagent  50  can include a lysing agent, a denaturing agent, and/or a protease for processing the fluid sample, for example, to generate hemoglobin (Hb) and a peptide derived from HbA1c. In this example, the first component  60  of the blood sample may include the Hb and the second component  70  of the blood sample may include the peptide derived from HbA1c. The one or more reagent  50  in first layer  20  may lyse the red blood cells, denature the hemoglobin, and release fVH (HbA1c analyte) from the hemoglobin molecules as a result of proteolysis. As described in further detail below, the denatured hemoglobin molecules may then be retained in first layer  20  due to the composition of second layer  30 . Denatured modified methemoglobin has characteristically strong light absorption at ˜540 nm, which enables detection by a sensor detecting light reflecting from first layer  20 . 
     In an embodiment, the one or more reagent  50  can include a glucose oxidant such as glucose oxidase. 
     Second layer  30  of thin-film element  10  is configured to be impermeable to the first component  60  (e.g., Hb) of the fluid sample, and permeable to the second component  70  (e.g., the peptide derived from HbA1c) of the fluid sample. In this manner, the second layer  30  enables the second component  70  to pass therethrough to third layer  40 , while the first component is retained in first layer  20 . In an embodiment, the permeability/impermeability through second layer  30  may be based on molecule size and/or molecular weight. In other embodiments, the permeability/impermeability through second layer  30  may be based on ion exchange, ion transport, barrier layers, and the like. 
     Second layer  30  may include at least one reflective surface so that the first component  60  retained by first layer  20  may be analyzed separately from the second component  70  retained by third layer  40 , and vice versa, as explained in more detail below. For example, the at least one reflective surface may include a first or top reflective surface  34  and a second or bottom reflective surface  36 , which enable the first component  60  and the second component  70  to be analyzed from opposite sides of the thin-film element  10 . In an embodiment, second layer  30  may be formed of a gelatin, and may include an optical masking material such as TiO 2  that creates the at least one reflective surface including top reflective surface  34  and/or the bottom reflective surface  36 . 
     In other embodiments, the second layer can be formed of BaSO 4 . This barium layer can include a reflective material such as TiO 2  that creates the at least one reflective surface. 
     Third layer  40  of thin-film element  10  is configured to retain the second component  70  (e.g., the peptide derived from HbA1c) of the fluid sample once it has passed from first layer  20  through second layer  30 . In an embodiment, third layer  40  may be transparent, partially opaque so as not to affect reflectance of an optical signal modulated by the second component  70 , as explained in more detail below. In some embodiments, the third layer  40  can be formed of a material such as, but not limited to, gelatin, synthetic polymers, and the like. 
     Third layer  40  may include one or more second reagents  90  configured to process the second component  70  to generate a third component  95 . The second reagents  90  can be the same or different than the first reagent. In one embodiment, they are different. In an embodiment, third layer  40  may include one or more second reagent  90  that processes the second component  70  into a chromogen, e.g., third component  95 , once the second component  70  is received by third layer  40 . For example, with the HbA1c example, where the second component  70  includes fVH, the one or more second reagent  90  in third layer  40  can process the fVH (e.g., Oxidase→H 2 O 2 →HRP→Blue Leuco Dye cascade), where the result can be detected by a sensor measuring reflectance. 
     In other embodiments, many different analytes can be detected with an appropriate reagent. In some embodiments, bound/free analytes can be used. Analytes can include, but are not limited to glucose, blood urea nitrogen (BUN), creatinine, sodium, lithium, calcium, magnesium, unconjugated bilirubin, conjugated bilirubin, unconjugated delta bilirubin, and the like. 
     In some embodiments, a reagent may not be required. 
     As illustrated in  FIG. 3 , the device  100  is configured to accept the thin-film element  10  and analyze the thin-film element  10  from opposite sides. In the illustrated embodiment, the device  100  includes a first or upper assembly  110  and a second or lower assembly  120 . First assembly  110  may include a first light source  112 , a first optical filter  114  and a first sensor  116  configured to be used to analyze the first component  60  retained by first layer  20  of the thin-film element  10 . The lower assembly  120  may include a second light source  122 , a second optical filter  124 , and a second sensor  126  configured to be used to analyze the second component  70  retained by third layer  40  of the thin-film element  10 .  FIG. 3  illustrates that first light source  112  illuminates at 45 degrees and first sensor  116  reads at 45 degrees (total oriented at 90 degrees relative to one another). In other words, the light beam reflects and is detected at 90 degrees. A similar configuration is illustrated for second light source  122  and second sensor  126 . However, in some embodiments, to avoid specular reflections, the light source can illuminate at 45 degrees and the sensor can read the signal at 90 degrees relative to the sample. 
     Though  FIG. 3  shows the thin-film element  10  as set within the device  100 , it should be understood that the thin-film element  10  is moveable between first assembly  110  and second assembly  120 , for example, in a direction substantial perpendicular to a vertical direction defined from first assembly  110  to second assembly  120 . When positioned as shown in  FIG. 3 , a plurality of first optical signals  140  may be generated when a first light  135  from first light source  112  reflects off of first reflective surface  34  and is modulated by first component  60 , while a plurality of second optical signals  150  may be generated when a second light  145  from second light source  122  reflects off of second reflective surface  36  and is modulated by second component  70 . 
     In the illustrated embodiment, first light source  112  is provided above first layer  20  and is configured to project a first light  135  onto first layer  20  so that the first light  135  may be modulated by the first component  60  of the fluid sample retained by first layer  20 . First light source  112  may include, for example, one or more light-emitting diode (“LED”) lights or another type of lighting structure understood to those of ordinary skill in the art. Those of ordinary skill in the art will recognize that other configurations for first light source  112  are possible, for example, by placing thin-film element  10  in a non-horizontal configuration with first light source  110  to the side, or by placing first light source  112  in another location and using a reflector above first layer  20  to guide first light  135  towards first layer  20 . 
     In the illustrated embodiment, second light source  122  is provided below third layer  40  and is configured to project a second light  145  onto third layer  40  so that the second light  145  may be modulated by the second component  70  of the fluid sample retained by third layer  40 . Second light source  122  may include, for example, one or more LED lights or another type of lighting structure understood to those of ordinary skill in the art. Those of ordinary skill in the art will recognize that other configurations for second light source  122  are possible, for example, by placing thin-film element  10  in a non-horizontal configuration with second light source  122  to the side, or by placing second light source  122  in another location and using a reflector below third layer  40  to guide second light  145  towards third layer  40 . 
     In an alternative embodiment, a single light source can be used in place of first light source  112  and second light source  122 . For example, the single light source could project light towards both sides of thin-film element  10 , with reflectors being used to direct the first light  135  towards first layer  20  and the second light  145  towards third layer  40 . 
     In the illustrated embodiment, first optical filter  114  is provided above first layer  20  and configured to filter a first optical signal  140  generated by the first light  135  being modulated by first component  60  and reflected off of first reflective surface  34 , before the first optical signal  140  is received by first sensor  116 . In an embodiment, first optical filter  114  may be a band pass filter of an appropriate wavelength for the assay being run. Those of ordinary skill in the art will recognize that other configurations for first optical filter  114  are possible, for example, by placing the thin-film element  10  in a non-horizontal configuration with first optical filter  114  to the side. Other types of optical filters can include, but are not limited to absorptive filters and dichroic filters. In some embodiments, a diffraction grating or a monochromater can also be used to select a particular wavelength of light. 
     In the illustrated embodiment, second optical filter  124  is provided below third layer  40  and configured to filter a second optical signal  150  generated by the second light  145  being modulated by second component  70  and reflected off of second reflective surface  36 , before the second optical signal  150  is received by second sensor  126 . In an embodiment, second optical filter  124  may be a band pass filter of an appropriate wavelength for the assay being run. Those of ordinary skill in the art will recognize that other configurations for second optical filter  124  are possible, for example, by placing the thin-film element  10  in a non-horizontal configuration with second optical filter  124  to the side. 
     In the illustrated embodiment, first sensor  116  is provided above first layer  20  and is configured to receive the first optical signal  140  after the first optical signal  140  has been generated by the first light  135  being modulated by first component  60  and reflected off of first reflective surface  34 , and after the first optical signal  140  passes through first optical filter  114 . In an embodiment, first sensor  116  may include at least one of a photo multiplier tube, a contact-image sensor, a photodiode, and an image capturing sensor matrix. Those of ordinary skill in the art will recognize that other configurations for first sensor  116  are possible, for example, by placing thin-film element  10  in a non-horizontal configuration with first sensor  116  to the side. 
     In the illustrated embodiment, second sensor  126  is provided beneath third layer  40  and is configured to receive the second optical signal  150  after the second optical signal  150  has been generated by the second light  145  being modulated by second component  70  and reflected off of second reflective surface  36 , and after the second optical signal  150  passes through second optical filter  124 . In an embodiment, second sensor  126  may include at least one of a photo multiplier tube, a contact-image sensor, a photodiode, and an image capturing sensor matrix. Those of ordinary skill in the art will recognize that other configurations for second sensor  126  are possible, for example, by placing thin-film element  10  in a non-horizontal configuration with second sensor  126  to the side. 
     The device  100  may further include or be placed in communication with a processor  180 , which may control the elements of first assembly  110  and second assembly  120  individually or as a whole, sending signals to first assembly  110  and second assembly  120  and receiving signals therefrom. In an embodiment, processor  180  may receive a first electrical signal generated by first sensor  116  in response to first sensor  116  sensing the first optical signal  140 , and may receive a second electrical signal generated by second sensor  126  in response to second sensor  126  sensing the second optical signal  150 . The first electrical signal and the second electrical signal may indicate, for example, measured intensities of the first optical signal  140  and second optical signal  150 , respectively. 
     In some embodiments, multiple measurements can be made over time to calculate a rate of reaction. In some embodiments, these multiple measurements over time can include at least an early blank reading and then a final reading. A response can be calculated by subtracting the early blank from the final reading. 
     Processor  180  may then process the signals, for example, by generating a ratio between the concentration of first component  60  and the concentration of second component  70  of the fluid sample based on the first electrical signal and the second electrical signal. In some embodiments, image processing can be used to obtain a result if the sensor is included in or part of an imaging reflectometer. 
     In other embodiments, processor  180  may then process the signals, for example, by calculating concentrations of two different analytes or sample components based on the first electrical signal and the second electrical signal. 
     Other types of algorithms can be used for calculation. Other algorithms can include, but are not limited to, a product of two measurements divided by a constant to yield a risk score or measuring the amount of interferent for the analyte of choice and an algorithm that eliminates bias. 
       FIG. 4  illustrates a method of performing an assay using the thin-film element  10  with the device  100  of  FIGS. 1 to 3 , according to an example embodiment of the present disclosure. Those of ordinary skill in the art will recognize that certain steps may be omitted from or added to those shown in  FIG. 4  without departing from the spirit and scope of the present disclosure. 
     At step  200 , a fluid sample is dispensed on first layer  20  of thin-film element  10 . The fluid sample may be, for example, a human or animal blood sample including serum, plasma, or whole blood. The fluid sample may be added to first layer  20  before thin-film element  10  is inserted into device  100 , or the fluid sample may be added with thin-film element  10  already positioned between first assembly  110  and second assembly  120  of device  100 . 
     At step  202 , the fluid sample dispensed on first layer  20  of thin-film element  10  reacts with the one or more reagent  50  of first layer  20  to create the first component  60  and the second component  70 . The reaction may take place before or after thin-film element  10  is inserted into device  100  and/or positioned between first assembly  110  and second assembly  120  of device  100 . 
     In other embodiments, a first component and a second component may already exist in the fluid sample and no reaction to produce them may be required. In one embodiment, a first component may be glucose and the second component may be albumin, both in a blood sample not requiring a reaction. 
     At step  204 , first component  60  is retained by first layer  20  because second layer  30  is impermeable to first component  60 , while second component  70  migrates through second layer  30  to third layer  40  because second layer  30  is permeable to second component  70 . As with step  202 , the migration of second component  70  through second layer  30  to third layer  40  may take place before or after thin-film element  10  is inserted into device  100  and/or positioned between first assembly  110  and second assembly  120  of device  100 . 
     At step  206 , second component  70  is retained by third layer  40 . In an embodiment, third layer  40  may include one or more reagent  80  to react with second component  70  once second component  70  migrates through second layer  30 . For example, the one or more reagent  80  may generate a third component from second component  70  that will act to modulate second optical signal  150 . As with steps  202  and  204 , step  206  may take place before or after thin-film element  10  is inserted into device  100  and/or positioned between first assembly  110  and second assembly  120  of device  100 . 
     At step  208 , if thin-film element  10  is not already positioned between first assembly  110  and second assembly  120  of device  100 , thin-film element  10  may be manually or automatically positioned between first assembly  110  and second assembly  120  of device  100 . In an embodiment, thin-film element  10  is moveable between first assembly  110  and second assembly  120  in a direction substantial perpendicular to a vertical direction defined from first assembly  110  to second assembly  120 . Those of ordinary skill in the art will understand that different insertions directions and/or configurations are possible. 
     At step  210 , processor  180  initiates an analysis procedure by activating first assembly  110  and second assembly  120 . In  FIG. 4 , processor  180  is shown to control first assembly  110  and second assembly  120  simultaneously and independently, though those of ordinary skill in the art will recognize that first assembly  110  and second assembly  120  can also be controlled sequentially or together with a single control structure that activates both assemblies using a single signal. 
     At step  212 , processor  180  causes first light source  112  to project the first light  135  towards first layer  20 , while at step  214 , processor  180  causes second light source  122  to project the second light  145  towards third layer  40 . The first light  135  and second light  145  may be, for example, light signals generated by one or more LED&#39;s. In the embodiment illustrated in  FIG. 3 , the first light  135  and second light  145  are projected at an angle of 45° to facilitate accurate measurements, but those of ordinary skill in the art will recognize that other configurations may be possible as describe herein. 
     In other embodiments, fluorescence and/or luminescence can be used. 
     At step  216 , the first optical signal  140  is generated as the first light  135  reflects off of second layer  30  and is modulated by the first component  60  retained by the first layer  20 , while at step  218 , the second optical signal  150  is generated as the second light  145  reflects off of second layer  30  and is modulated by the second component  70  retained by third layer  40 . In an embodiment, the first optical signal  140  is reflected by the first reflecting surface  34  of second layer  30 , while the second optical signal  150  is reflected by the second reflecting surface  36  of second layer  30 . It should further be understood that modulation by the first component  60  includes modulation by additional components generated from the first component, and modulation by the second component includes modulation by additional components generated from the second component. For example, as explained above, one or more reagent  80  contained by third layer  40  may generate a third component from the second component, which then modulates the second optical signal  150 . 
     At step  220 , the first optical signal  140  is filtered by first optical filter  114 , while at step  222 , the second optical signal  150  is filtered by the second optical filter  124 . As will be understood by those of ordinary skill in the art, the first optical filter  114  and the second optical filter  124  should each have an appropriate wavelength for the assay being run. In an embodiment, the first optical filter  114  and the second optical filter  124  may be automatically adjusted by processor  180  to an appropriate wavelength, or manually adjusted by a user based on the assay being run. 
     At step  224 , first sensor  116  generates a first electrical signal in response to first sensor  116  sensing the first optical signal  140  after passing through first optical filter  114 , while at step  226 , second sensor  126  generates a second electrical signal in response to second sensor  126  sensing the second optical signal  150  after passing through second optical filter  124 . In the embodiment illustrated in  FIG. 3 , the first optical signal  140  and second optical signal  150  are received by first sensor  116  and second sensor  126  at an angle of 45° to facilitate accurate measurements, but those of ordinary skill in the art will recognize that other configurations may be possible. The first electrical signal and the second electrical signal are then relayed to processor  180  for further processing. The first electrical signal and the second electrical signal may indicate, for example, measured intensities of the respective first optical signal  140  and second optical signal  150 , which may indicate respective concentrations of the first component  60  and second component  70 . 
     In some embodiments, other types of optical outputs can be utilized. These can include, but are not limited to surface plasmon resonance (SPR) diffraction by ring resonators, output by a waveguide, output by an interferometer, or output by a photonic detector. 
     At step  228 , processor  180  receives the first electrical signal from first sensor  116  and the second electrical signal from second sensor  126  and performs an analysis using the two signals. In an embodiment, the analysis includes a comparison of a concentration of the first component  60  based on the first electrical signal and a concentration of the second component  70  based on the second electrical signal, for example, by calculating a ratio between the concentrations and making a determination based on the numerical value of the ratio. 
     It is contemplated that an advantageous use of thin-film element  10  and/or device  100  could be in the performance of an HbA1c assay, where methemoglobin, which is the analyte for hemoglobin, complicates the measurement of Fructosyl valine-histidine (fVH), the analyte for HbA1c. The use of separate layers (e.g., first layer  20  and third layer  40 ) for measurement of each analyte, has several advantages, for example, reduced assay time, increased reliability, and reduced cost. 
     In the example of an HbA1c assay, a fluid sample (e.g. whole blood) can be dispensed on first layer  20 . The one or more reagent contained by first layer  20  may then lyse the red blood cells, denature hemoglobin, and release fVH (HbA1c analyte) from the hemoglobin molecules as a result of proteolysis. The denatured hemoglobin molecules (e.g., the first component) are retained by first layer  20  due to the gelatin in the second layer  30 . In this case, detergent modified methemoglobin has characteristically strong light absorption at about 540 nm, which allows detection by first sensor  116  by measuring reflectance. The first electrical signal generated by the first sensor  112  can therefore indicate the concentration of hemoglobin (Hb) in the blood sample. 
     Fructosyl valine-histidine (fVH) (e.g., the second component) is small enough that it can pass through the gelatin in the second layer, so it passes through second layer  30  to third layer  40  where it is retained. At third layer  40 , the fVH can be processed (e.g., Oxidase→H 2 O 2 →HRP→Blue Leuco Dye cascade), where the result can be detected by second sensor  126  by measuring reflectance at about 670 nm. The second electrical signal generated by the second sensor  126  can then indicate the concentration of HbA1c in the blood sample. 
     Once the processor  180  receives the first electrical signal and the second electrical signal, the processor may determine the result of the assay by calculating the ratio of HbA1c:Hb. Determination of the ratio of HbA1c:Hb in this way is advantageous over systems that measure both concentrations on the same side of a thin-film element, for example, because it is possible for one analyte to interfere with the other. 
     In other embodiments, a slide without reagents can be used. Therein, components of the sample such as but not limited to unconjugated/conjugated bilirubin can be used to transport a component that can modulate an optical signal. Unconjugated bilirubin is typically bound to albumin. In some embodiments, this unconjugated bilirubin can be retained in the top layer because the albumin is impermeable to the second layer. In contrast, conjugated bilirubin can make its way through the second layer into the third layer because it is not bound to albumin. Then, direct measurements by reflectance can be performed and the processor can provide a result. 
     It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims. 
     Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. 
     The terms “a” and “an” and “the” and similar referents used in the context of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure. 
     The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” 
     Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. 
     Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects those of ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. 
     Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein. 
     Further, it is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.