Abstract:
A system and method of quantitating the concentration of a molecule of interest in one embodiment includes establishing a plurality of test environments at a plurality of test sites, each of the plurality of test environments associated with one of a plurality of response curves, each of the plurality of response curves different from the other of the plurality of response curves, storing a combined response curve resulting from a summation of the plurality of response curves, exposing the plurality of test sites to a sample having a concentration of a molecule of interest, obtaining a plurality of quantitation signals, each of the plurality of quantitation signals associated with one of the plurality of test sites, associating a summation of the plurality of quantitation signals with the stored combined response curve, and generating a signal related to the concentration of the molecule of interest based upon the association.

Description:
FIELD 
       [0001]    This invention relates to diagnostic tests and more specifically to affinity based diagnostic tests. 
       BACKGROUND 
       [0002]    Diagnostic tests that can be performed at the point of care of an individual, such as at the bedside of a patient, at a care provider location, or at the home of the patient, are becoming increasingly popular. Diagnostic tests include tests directed to identifying biomarkers such as Nucleic Acid, protein, and small molecules. Many of the diagnostic testing devices incorporate affinity based sensors which are considered to be the state-of-the-art in detection of biomarkers. 
         [0003]    Affinity based biosensors function according to a “key-lock” principal in which a molecule with very high association factor to the biomarker of interest is used for detection. For example, a pregnancy test kit may incorporate a monoclonal antibody specific to a β-subunit of hCG (βhCG). The antibody is conjugated with a tag, e.g., gold, latex, or a fluorophore, which is used for detection. If the targeted molecule binds with the conjugated antibody, the tagged key-lock pair will be detectable such as by a visible test line. 
         [0004]    ELISA plates and microarrays (e.g., Nucleic Acid, peptide, and protein) incorporate a similar principal.  FIG. 1  depicts an ELISA assay  10  wherein antibodies  12  are immobilized on a substrate  14 . The substrate  14  may be positioned within a well (not shown). A blocker  16  is provided to cover the surface of the substrate around the antibody  12 . In a typical ELISA assay, a sample  18  is then added to the well in which the primary antibody  12  is immobilized. Next, the sample is incubated for some time. During incubation, the blocker  16  prevents the molecules of interest in the sample from binding to the surface of the substrate  14  in order to avoid false binding. During incubation, some of the molecules of interest  18  become bound with some of the antibodies  12  as depicted in  FIG. 2 . After incubation, the remaining sample is washed to remove the unbound molecules  18 . 
         [0005]    Subsequently, a secondary antibody  20  with a bound label  22  is added to the well, incubated, and washed resulting in the configuration of  FIG. 3 . As depicted in  FIG. 3 , the labeled secondary antibodies  20  are bound to the molecules of interest  18  that are in turn bound to the antibodies  12 . Accordingly, the number of labels  22  bound by the antibodies  20  to the molecule of interest  18  is proportional to the concentration of the target molecule. Depending on the type of label used, the number of labels can be finally detected using colorimetry, amperometry, magnetometry, voltammetry, luminescence, or fluorescence detection. Other label-free antibody processes such as surface plasmon resonance may alternatively be used. 
         [0006]    The quality of the diagnostic tests discussed above can be assessed using Limit of Detection (LoD) and Limit of Quantitation (LoQ) analysis. LoD, which is the minimum concentration of target molecules needed in order to create a large enough quantity of complexes that can be recognized by the available means of detection, is affected by the mutual affinity between a probe molecule and a target molecule, the density of probe molecules, incubation conditions, and the level of background and noise in the detection mechanism. As reported by Burtis, et al., “Tietz Textbook of Clinical Chemistry and Molecular Diagnostics,”  Elsevier Saunders,  2005, the LoD for a particular assay is generally considered to be the concentration of target molecules that produces a signal 2-3 times greater than the average background signal. 
         [0007]    The LoQ metric includes both an upper limit and a lower limit. The upper and lower limits as defined by Burtis, et al. “Tietz Textbook of Clinical Chemistry and Molecular Diagnostics,”  Elsevier Saunders,  2005, are the highest and lowest concentrations, respectively, that can be quantitatively measured within an acceptable total error for the particular assay. Generally, the acceptable error for a particular assay in the United States is defined by the Food and Drug Administration based upon the standard deviation of so-called “gold standard tests”. 
         [0008]    The range of concentrations between the lower LoQ and the upper LoQ for a particular assay is the dynamic range of the assay. The lower LoQ is related to the LoD which, as discussed above, is related to the affinity probe molecule density on the substrate. Thus, a high affinity probe molecule density is incorporated in many assays so as to increase the sensitivity of the particular assay. The upper LoQ, however, is typically a function of the maximum density or concentration of probe molecules that can be achieved in a particular assay. 
         [0009]    Given a particular concentration of affinity probe molecules, the normalized signal obtained from a particular assay does not vary linearly with the concentration of the analyte of interest. By way of example,  FIG. 4  depicts a response curve (RC). A “response curve” as that term is used herein is a curve showing the relationship between various concentrations of a molecule of interest in a plurality of samples and a plurality of quantitation signals obtained from the plurality of samples for a particular assay. In  FIG. 4 , a typical signal response versus target concentration for a particular assay is depicted as a response curve  30 . Within the range of signals of  FIG. 4 , a lower LoQ is defined by the line  32  at about 10 −12  mol/l while the upper LoQ is defined by the line  34  at about 10 −10  mol/l. Between the lower LoQ  32  and the upper LoQ  34 , the response curve  30  is shaped like an ogee. The dynamic range of the assay which produces the response curve  30  of  FIG. 4  is about two orders of magnitude. The dynamic range of a typical assay is generally between 1 to 3 orders of magnitude. 
         [0010]    The dynamic range of a particular assay limits the usefulness of the assay in applications wherein the expected variation in tested samples is several orders of magnitude. By way of example, detectable proteins in human serum range in abundance from grams to tenths of picograms per milliliter. Furthermore, serum protein abundance can change by as much as 10,000-fold on stimulation. As reported by S. F. Kingsmore, “Multiplexed protein measurement: technologies and applications of protein and antibody arrays,”  Nature Reviews Drug Discovery,  No. 4, pp. 310-320, 2006, many of the proteins that are most frequently measured, such as acute phase reactant pentraxins or chemokines, also exhibit large variations. Accordingly, the expression range for many biomolecules can span 6 orders of magnitude or more and a convenient assay should have an equal dynamic range while the dynamic range of a typical assay is generally between 1 to 3 orders of magnitude. 
         [0011]    In multiplexed assays and microarrays the issue of narrowly limited dynamic range is especially relevant as detection of multiple biomarkers is critical for improving diagnostics. Microarrays, however, fall short of achieving the levels of quantification that established optimized tests, e.g., ELISA, can realize, even in conjunction with other time consuming lab techniques, e.g., serial dilution. Accordingly, while microarrays are heavily used and are experiencing an exponential growth, such usage is primarily directed to detection of molecules of interest rather than in quantification of molecules of interest. The use of multiplexed assays and microarrays even as detection mechanisms, however, is limited. For example, even healthy individuals carry markers which, when present in greater concentrations, can indicate the presence of a disease. 
         [0012]    In response to limited upper LoQs in earlier assays, some manufacturers have developed substrates which allow for higher densities of the immobilized probe molecule and thus higher dynamic range. Such substrates generally include a three dimensional structure which, while providing a greater dynamic range, may also increase the assay variation. Some of such approaches result in the binding kinetics and stability of probe molecules and may even cause reduction in dynamic range. 
         [0013]    Another approach to increasing the dynamic range of assays is to perform a series of dilutions on a sample such that one of the diluted samples provides a target concentration that is within the dynamic detection range of an assay. Such an approach has been reported by Patrick Domnanich, et al. “Protein microarray for the analysis of human melanoma biomarkers,”  Sensors and Actuators B: Chemical, Vol.  “In Press, Corrected Proof”, 2008. This simple yet effective approach has the disadvantage of requiring a considerable number of additional steps (including multiple dilutions and multiple runs of the actual assay) which add to the test time and increases costs and operating expenses. 
         [0014]    Recently, other solutions have been proposed to increase the dynamic range of an assay. One such solution for a point of care device disclosed in U.S. Patent Application Publication No. 2006/0019404 A1 includes the use of two strips with different dynamic ranges. Different dynamic ranges can be achieved by using different antibodies and, by combining the results, a wider dynamic range for the assay can be achieved. Other solutions reported by N. Ohmura, et al., “Combinational Use of Antibody Affinities in an Immunoassay for Extension of Dynamic Range and Detection of Multiple Analytes,”  Analytical Chemistry,  Vol. 75, No. 1, pp. 104-110, 2003, and A. P. Drabovich, et al., “Smart Aptamers Facilitate Multi-Probe Affinity Analysis of Proteins with Ultra-Wide Dynamic Range of Measured Concentrations,”  Journal of the American Chemical Society,  Vol. 129, No. 23, pp. 7260-7260, 2007, include the use of two or more probe molecules, either antibodies or aptamers, with different affinities for the analyte in order to extend the dynamic range. Such approaches require an overlap between the dynamic ranges of the different probe molecules used and are thus limited by the availability of multiple antibodies or aptamers having the desired affinities. 
         [0015]    A need exists for a device and method of performing a quantization assay incorporating low cost antibodies. A further need exists for low cost assays including multiplexed assays, protein arrays, lateral flow devices, sandwich assays, competitive assays, or bead based arrays which provide accurate quantization results and a method of using such arrays. Methods and devices which provide relatively linear results would be a further benefit. 
       SUMMARY 
       [0016]    In accordance with one embodiment, a system and method of quantitating the concentration of a molecule of interest includes establishing a plurality of test environments at a plurality of test sites, each of the plurality of test environments associated with one of a plurality of response curves, each of the plurality of response curves different from the other of the plurality of response curves, storing a combined response curve resulting from a summation of the plurality of response curves, exposing the plurality of test sites to a sample having a concentration of a molecule of interest, obtaining a plurality of quantitation signals, each of the plurality of quantitation signals associated with one of the plurality of test sites, associating a summation of the plurality of quantitation signals with the stored combined response curve, and generating a signal related to the concentration of the molecule of interest based upon the association. 
         [0017]    In accordance with another embodiment, a method of quantitating the concentration of a molecule of interest includes establishing a first test environment at a first test site, establishing a second test environment at a second test site such that a response curve for the second site is different from a response curve of the first site, storing a combined response curve resulting from a summation of the response curve for the second site and the response curve of the first site, exposing the first test site and the second test site to a sample having a concentration of a molecule of interest, obtaining a first quantitation signal from the first test site, obtaining a second quantitation signal from the second test site, combining the first quantitation signal and the second quantitation signal, associating the combined first quantitation signal and second quantitation signal with the stored response curve, and generating a signal related to the concentration of the molecule of interest based upon the association. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]      FIG. 1  depicts a schematic of a prior art test site within an ELISA array with an antibody and blockers formed on a substrate as a sample is added to the test site; 
           [0019]      FIG. 2  depicts the test site of  FIG. 1  with a molecule of interest bound to some of the antibodies of  FIG. 1  after the test site has been incubated and washed; 
           [0020]      FIG. 3  depicts the test site of  FIG. 2  after a labeled secondary antibody has been added and the test site has again been incubated and washed so that the labeled secondary is bound to the bound molecules of interest; 
           [0021]      FIG. 4  depicts a response curve for a typical assay showing the normalized output signal of a system as a function of the concentration of an analyte of interest in a tested sample; 
           [0022]      FIG. 5  depicts a multi-site biosensor system configured to expose a single sample to different environmental conditions allowing determination of the concentration of a molecule of interest in a sample over an extended range of concentrations; 
           [0023]      FIG. 6  depicts a platform for providing a number of different test sites in the form of a microarray; 
           [0024]      FIG. 7  depicts a procedure that can be used to establish different test environments at various test sites on a platform so as to expose a sample to multiple test environments; 
           [0025]      FIG. 8  depicts a combined response curve showing the normalized output signal of a system as a function of the concentration of an analyte of interest in a tested sample when the response curves for two sites with different test environments are summed; 
           [0026]      FIG. 9  depicts a combined response curve showing the normalized output signal of a system as a function of the concentration of an analyte of interest in a tested sample when the response curves for two sites with low affinity modulation are summed; and 
           [0027]      FIG. 10  depicts a combined response curve showing the normalized output signal of a system as a function of the concentration of an analyte of interest in a tested sample when the response curves for two sites with high affinity modulation are summed. 
       
    
    
     DESCRIPTION 
       [0028]    For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and described in the following written specification. It is understood that no limitation to the scope of the invention is thereby intended. It is further understood that the present invention includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the invention as would normally occur to one skilled in the art to which this invention pertains. 
         [0029]    Referring to  FIG. 5 , there is depicted a representation of a multisite biosensor system generally designated  100 . The biosensor system  100  includes an I/O device  102 , a processing circuit  104  and a memory  106 . The I/O device  102  may include a user interface, graphical user interface, keyboards, pointing devices, remote and/or local communication links, displays, and other devices that allow externally generated information to be provided to the biosensor system  100 , and that allow internal information of the biosensor system  100  to be communicated externally. 
         [0030]    The processing circuit  104  may suitably be a general purpose computer processing circuit such as a microprocessor and its associated circuitry. The processing circuit  104  is operable to carry out the operations attributed to it herein. 
         [0031]    Within the memory  106  are various program instructions  108 . The program instructions  108 , some of which are described more fully below, are executable by the processing circuit  104  and/or any other components as appropriate. Affinity databases  110  and response curve (RC) databases  112  are also located within the memory  106 . 
         [0032]    The biosensor system  100  further includes environment control equipment  114 , environment detector suite  116 , and a label reader  118 . The label reader  118  may be included in a single device along with the other components of the system  100 . 
         [0033]    Alternatively, one or more of the components of the system  100  may be provided as a separate device which may be remotely located from the other components of the system  100 . 
         [0034]    The environment control equipment  114  is configured to establish and maintain environmental conditions, in this example, within a microarray  120  depicted in  FIG. 6 . Various methods may be used to form the microarray platform  120 . By way of example, U.S. Pat. No. 5,807,522 discloses a method for forming microarrays. The microarray platform  120  includes a number of different subarrays  122 . The subarrays  122  include a number of test sites  124 . The number of subarrays  122  as well as the number of test sites  124  within each of the subarrays  122  may be varied within the scope of the invention. The microarray  120  further includes two calibration arrays  126  and  128 . In some embodiments, the processor  104  may also be positioned on the microarray  120 . 
         [0035]    As reported by D. R. Jackola et al., “Entropy-favored human antibody binding reactions with a non-infectious antigen,”  Molecular Immunology,  Vol. 45, No. 5, pp. 1494-1500, 2008, temperature changes the binding efficiencies of some molecules. Accordingly, in the embodiment of  FIG. 5 , the environment control equipment  114  is operable to establish a temperature profile within the microarray platform  120 . The precise temperature within each of the test sites  124  and the calibration arrays  126  and  128  may be detected by the detector suite  116 . 
         [0036]    The test sites  124  and the calibration arrays  126  and  128  are prepared with a capturing agent effective for capturing a biomolecule of interest. Further details regarding the biosensor system  100  are provided with reference to the procedure  130  of  FIG. 7 . The processor  104  executes the program instructions  108  to execute at least some of the procedure  130  of  FIG. 7 . In different embodiments, the procedure  130  may be modified to include more or fewer steps depending upon the specific criterion. 
         [0037]    At block  132 , a molecule of interest is identified and then a probe molecule with an affinity for the molecule of interest, such as an antibody, a synthetic peptide, aptamer, etc., is identified (block  134 ). A binding efficiency coefficient for the molecule of interest (a) with the identified antibody is then identified for at least two different environmental conditions (block  136 ) and stored in one of the affinity databases  110  (block  138 ). 
         [0038]    The response curves associated with the at least two binding efficiency coefficients are then identified (block  140 ) and stored in the RC databases  112  (block  142 ). At block  144 , the microarray platform  120  is prepared by depositing the desired amount of the selected capturing agent in each of the test sites  124  and the calibration sites  126  and  128 . In alternative embodiments, a subset of the test sites  124  and the calibration site  126  may be prepared with a first capturing agent while another subset of the test sites  124  and the calibration test site  128  may be prepared with a second capturing agent so as to allow two separate tests to be conducted within a single microarray platform  120 . Additional configurations within a single microarray platform  120  may also be used. By way of example, each of the test sites within one of the subarrays  122  may be prepared with the same capturing agent while each of the subarrays  122  includes a different capturing agent. 
         [0039]    Once the microarray platform  120  is prepared, a sample is introduced into the selected set of test sites  124  (block  146 ). Prior to introduction, the sample may first be prepared by processes such as filtration and amplification, including the addition of reagents and other control preprocessing. A control substance is also introduced to the calibration sites  126  and  128  (block  148 ). 
         [0040]    If not already established, the environment within each of the selected set of test sites  124  is controlled to establish, within each of the selected set of test sites  124 , a different test environment (block  150 ). In this example, the environmental condition that is controlled is temperature. Accordingly, a thermal profile is established across the microarray platform  120 . Depending upon the particular embodiment, this may be accomplished by providing a unique heater/cooler for each of the selected set of test sites  124  or subarrays  122 . In other embodiments, heat is applied at one end of the microarray platform  120  and a heat sink is thermally attached to the opposite end of the microarray platform  120  to establish a thermal gradient across the microarray platform  120 . The environment at the calibration sites  126  and  128  is also controlled (block  152 ). 
         [0041]    The sample and control substances are then incubated at the established test/calibration environment for a predetermined time (block  154 ). During the incubation, the actual test environment within each of the selected set of test sites  124  is monitored by the environment detector suite  116  and data indicative of the established test environment is provided to the processing circuit  104  (block  156 ). Additionally, the actual calibration environment within each of the calibration sites  126  and  128  is monitored by the environment detector suite  116  and data indicative of the established calibration environment is provided to the processing circuit  104  (block  158 ). Bonding activity at the calibration sites  126  and  128  is also monitored and data indicative of the activity is provided to the processing circuit  104 . The bonding activity at the calibration sites  126  and  128  may be analyzed by the processing circuit  104  to modify control of the test site environment (block  160 ). In other embodiments, the calibration sites  126  and  128  are used to reconstruct the actual environment to correct for errors, e.g., at block  172 . 
         [0042]    When the sample has been sufficiently incubated, the test sites  124  are washed (block  162 ) and a labeled secondary antibody is introduced into the selected set of test sites  124  (block  164 ) and incubated (block  166 ). The selected set of test sites  124  are then washed (block  168 ) and the labels remaining in the test sites  124  are detected by the label reader  118  (block  170 ). Based upon the signals associated with the number of labels remaining in the selected set of test sites  124 , the concentration of the molecule of interest within the sample is calculated by the processing circuit  104  (block  172 ). 
         [0043]    Calculation of the concentration of the molecule of interest is possible because the test environments for the test sites  124  are controlled to provide a combined response curve having a combined minimum LoQ that is at or below the minimum concentration which is desired to be quantitated and a combined maximum LoQ that is at or above the maximum concentration which is desired to be quantitated. Additionally, the test environments for the test sites  124  are controlled such that the ogee of the response curve associated with a particular test site  124  overlaps the ogee of the response curve of at least one other test site  124  throughout the concentration range of interest. The foregoing may be understood with reference to  FIG. 8 . 
         [0044]      FIG. 8  depicts a combined response curve  180  that extends substantially linearly throughout a concentration range of interest that is bounded by a minimum concentration  182  and a maximum concentration  184 . The concentration range of interest is larger than the dynamic range of any single test site  124  for a given test environment. 
         [0045]    By way of example, the response curve  186  is associated with a first test site  124 . The response curve  186  has a minimum LoQ  188  and a maximum LoQ  190 . The minimum LoQ  188  is greater than the minimum concentration  182  while the maximum LoQ  190  is much less than the maximum concentration  184 . Accordingly, the test site  124  associated with the response curve  186  can be used to provide reliable quantitation of samples with a concentration slightly above the minimum concentration  182  of the concentration range of interest and much less than the maximum concentration  184 . Accordingly, the test site  124  associated with the responses curve  186  cannot be used to provide reliable quantitation of concentrations throughout the concentration range of interest. 
         [0046]    With continued reference to  FIG. 8 , the response curve  192  has a minimum LoQ  194  and a maximum LoQ  196 . The minimum LoQ  194  is less than the maximum LoQ  190  for the response curve  186 , while the maximum LoQ  196  is greater than the maximum concentration  184 . Accordingly, the test site  124  associated with the response curve  192  can be used to provide reliable quantitation of samples with a concentration at the maximum LoQ  190  for the response curve  186  and up to concentrations that are at the maximum concentration  184  of the concentration range of interest. 
         [0047]    Thus, quantitation of samples within the concentration range of interest, which extends from the minimum concentration  182  to the maximum concentration  184 , is possible by summing the signals obtained from the test sites  124  associated with the response curves  186  and  192 . The concentration associated with the combined signal is obtained using the response curve  180 , which is a summation of the response curves  186  and  192 . Because the response curve  180  is a summation of the response curves  186  and  192 , the minimum LoQ  198  for the response curve  180  will be a concentration slightly less than the minimum LoQ for the most sensitive response curve (response curve  186 ), while the maximum LoQ  200  for the response curve  180  will be a concentration slightly greater than the maximum LoQ for the least sensitive response curve (response curve  192 ). 
         [0048]    Therefore, by carefully controlling the environment at each of a plurality of test sites  124 , a combined output signal may be obtained which has a relatively linear relationship to the concentration of a molecule of interest. Moreover, while only two response curves were needed to fully cover the concentration range of interest in  FIG. 8 , the test environment at additional sites may be further controlled to provide response curves that are higher than the response curve  192  (to the right as depicted in  FIG. 8 ), or lower than the response curve  186  (to the left as depicted in  FIG. 8 ) if increased dynamic range is desired. 
         [0049]    An important consideration in selecting the environmental factor(s) controlled at block  150  of  FIG. 7  is to select two or more test environments that result in response curves that overlap in the desired manner. An “overlap” is a range of concentrations which are within the dynamic range of at least two response curves. Linearity of a combined response curve can be optimized by controlling the test environment at different test sites  124  to provide an overlap as depicted in  FIG. 8 . 
         [0050]    As the overlap is increased, the combined response curve increases in slope. By way of example,  FIG. 9  depicts a response curve  210  and a response curve  212 . The affinity at the test site associated with the response curve  212  is only slightly less than the affinity at the test site associated with the response curve  210 . This low modulation of affinity results in a combined response curve  214  that exhibits an increased slope as compared to the response curve  180 . The dynamic range of the combined response curve  214 , however, is much less than the dynamic range of the combined response curve  180 . Accordingly, decreasing the overlap allows for a greater dynamic range for a fixed number of test site environments. 
         [0051]    As the overlap is decreased, however, the slope of the resulting combined response curve is reduced. By way of example,  FIG. 10  depicts a response curve  220  and a response curve  222 . The affinity at the test site associated with the response curve  222  is significantly less than the affinity at the test site associated with the response curve  220 . This high modulation of affinity results in a combined response curve  224  that exhibits a decreased slope as compared to the response curve  180 . The dynamic range of the combined response curve  224 , moreover, is greater than the dynamic range of the combined response curve  180 . The combined response curve  224 , however, also has a flattened region  226 . In the flattened region  226 , the potential error in the quantitation of analyte concentration is increased. Providing another test site with an affinity greater than the affinity of the test site associated with the response curve  222  and less than the affinity of the test site associated with the response curve  220 , however, can eliminate the flattened region  226 . 
         [0052]    In the foregoing example, the temperature within each of the test sites  124  was controlled to provide different binding efficiencies. In embodiments incorporating CMOS technology, a temperature gradient within the chip can be established using on-chip resistors such as polysilicon resistors or integrated Peltier elements. Low power on-chip temperature sensors can be used to accurately measure the temperature within each of the test sites. A multisite biosensor can thus be implemented on a printed circuit board, glass, plastic substrate, or on a CMOS chip with gold, glass, epoxy, polymer, or gel coating, or even in a well plate such as a 96 well plate. If desired, control, readout, and also sensing for the control can be provided in the printed circuit board or CMOS chip. CMOS technology allows multiple sensing sites to be fabricated in close proximity. This assists in maintaining uniformity of non-controlled environmental factors amongst the test sites. The chip can be part of a system using stand alone microfluidics or a capillary principle or may be used with a separately provided device. The signal estimation and the assay data can be hard coded on the CMOS chip if desired. 
         [0053]    Temperature is not the only environmental factor which can be used to establish different test environments. The electric field in which incubation is conducted has been shown to modify the binding efficiencies of molecules as reported by J. J. Gooding et al., “Electrochemical modulation of antigen-antibody binding, ”  Biosensors and Bioelectronics,  Vol. 20, No. 2, pp. 260-268, 2004, M. L. O&#39;Grady, et al., “Dynamic Control of Protein-Protein Interactions,”  Langmuir,  Vol. 24 (1), pp. 316-322, 2008, R. J. Heaton et al., “Electrostatic surface plasmon resonance: direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches,”  Proceedings of National Academy of Science,  Vol. 98, No. 7, pp. 3701-3704, 2001, and  I. Wong et al.,  “Dynamic control of biomolecular activity using electrical interfaces,”  Soft Matter,  Vol. 3, No. 3, pp 267-274, 2007. 
         [0054]    In some embodiments, the electric field within each of the test sites may be controlled to be different from the electric field within each of the other of the test sites. The electric field can be used to modify, e.g., local concentrations, pH, etc. The underlying mechanisms include electrolysis and ion attraction. The use of an AC signal may further provide local mixing by creating movement of ions within the sample, e.g., through electrohydrodaynamic effects. Many voltage ranges which provide the foregoing effects are CMOS compatible. Accordingly, a CMOS chip with surface electrodes may be used. Depending upon the desired effect, one, two or more electrodes may be provided at a particular test site and either exposed or isolated from the test sample. Another fabrication method incorporates glass slides with electrodes (e.g., ITO, gold) or plastic or paper membrane with printed electrodes (e.g., carbon, gold, silver). 
         [0055]    Another approach to controlling the test environment is through the use of magnetic beads and a controlled magnetic field. The magnetic beads are functionalized with molecules that have high affinity against a range of biomolecules, e.g., antibody, epoxy, Nucleic Acid, or aptamers. As the magnetic beads are moved across the sensor sites, different molecules will bind to them at different rates, thereby scraping the surfaces of the test sites and “cleaning” the test sites. Controlling the magnetic field within the test sites affects the extent of “cleaning” that occurs at each test site, thereby modifying the binding efficiency of different molecules depending upon the strength of the bonds formed between the molecules and the antibodies. An external magnet may be used in various embodiments to bias the magnetic field to the desired values since CMOS integrated coils can only generate moderate magnetic fields. 
         [0056]    Another approach is to modulate the affinities using different pressure at different test sites while incubating/washing the spots since pressure can be used to modify affinities as reported by M. L. Yarmush, et al., “Immunoadsorption: strategies for antigen elution and production of reusable adsorbents,”  Biotechnology Progress,  Vol. 8, No. 3, pp. 168-178, 1992. Pressure modification can be achieved in a fluidic setup. 
         [0057]    The type of sensor or sensors incorporated into the label reader  118  will vary depending upon the particular label used. Various embodiments may thus use luminescence, fluorescence, colorimetric, electrochemical, impedance, and magnetic sensors. The sensors should be configured to allow isolation of the signal produced by a selected one or more test sites. Likewise, the sensors incorporated into the environment detector suite  116  may include IR sensors, and Hall sensors. AMR sensors or GMR sensors may be provided to monitor the density of magnetic beads on a test site surface. ISFETs or CMOS based charge detection circuits may be used in electrochemical embodiments. 
         [0058]    The concentration of a molecule of interest can thus be obtained over a wide range of concentrations even if the range of concentrations exceeds the dynamic range that can be realized with a single test site. Moreover, the procedure  130  can be used in a variety of test site platforms including 96-well plates, plates with fewer or additional wells, microarray platforms, printed circuit board platforms, CMOS chip platforms, multiplexed assays, protein arrays, lateral flow devices, sandwich assays, competitive assays, bead based arrays or other appropriate platforms. The procedure  130  may further be used for the detection of a variety of molecules of interest as well as different types of molecules in addition to antibodies. By way of example, the procedure  130  may also be used for the detection of Nucleic Acid, protein, or small molecules. 
         [0059]    While the invention has been illustrated and described in detail in the drawings and foregoing description, the same should be considered as illustrative and not restrictive in character. It is understood that only the preferred embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the invention are desired to be protected.