DESC:Field of the invention
[0001] The present disclosure relates to an apparatus and method for performing molecular biological type diagnostic analyses in multiple formats. More particularly, the present disclosure relates to a molecular diagnostic technique based on principles of light diffraction using a device having a patterned substrate designed to enhance interferometric contrast.

Description of the Related Art
[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Sensors for detecting and measuring absolute or relative values of physical quantities such as chemical or biochemical concentration, magnetic or electric field strengths, pressure, strain, temperature, and pH, for example, in an environment to which the sensor is exposed, are well known in the art. Prior art sensors include direct reading sensors, which include, for example, a mercury thermometer or Bourdon pressure gauge, and sensors that employ transducers for converting an input signal or stimulus into an output signal of a different type. Thus, an infrared pyrometer converts infrared radiation into a useful electrical output signal readable by an electrical meter.
[0004] Prior art sensors also include optic sensors, which provide measured values directly or by means of transducers. A simple color comparison pH test apparatus is an example of optic sensor read directly whereas a camera light intensity metering system is optic sensor employing a transducer. The most sensitive optic sensor is of the interferometric type, which employs an interferometer to provide information about a condition sensed. An interferometer is an instrument that splits light from an input source into two or more light beams. The light beams are caused to travel through different paths with different effective optical path lengths so that an interference fringe pattern is produced when the beams are recombined. An analysis of the light and dark bands of the interference pattern provides a sensitive measure of the difference in
effective path length of the different optical paths.
[0005] A particular group of optic sensors that has experienced significant technical development in recent years includes integrated optic sensors. Integrated optic sensors are monolithic structures characterized by the integration of various optical components into a single optic waveguide construction An integrated optic sensor is typically a thin-film device comprising a waveguide constructed on a single substrate, which generally provides other optical elements or components to diffract, refract, reflect, or combine different beam portions propagating in the waveguide. Integrated optic technology is particularly useful in providing optical elements heretofore associated with interferometric sensors employing separate and discrete optical components. The prior art now includes integrated optic sensors that incorporate a variety of components including lenses, sensing fields, and filters on a single substrate.
[0006] The change in the refractive index changes the effective path length through the channel region, thereby changing the phase of the light beam as it emerges from the channel waveguide. Alternatively, if the channel waveguide is not directly sensitive to a particular environment, it may be coated with a material that is reactive to the environment, or to a component thereof, causing a change in the refractive index of the channel waveguide. An optical output beam from the sensor can therefore be used for measuring the relative or absolute value of the condition of the environment.
[0007] An optical biosensor technique that has gained increasing importance over the last decade is the surface plasmon resonance (SPR) technique. This technique involves measurement of light reflected into a narrow range of angles from a front side of a very thin metal film producing changes in an evanescent wave that penetrates the metal film. Ligands and analytes are located in the region of the evanescent wave on the backside of the metal film. Binding and disassociation actions between the ligands and the analytes can be measured by monitoring reflected light in real time. These SPR sensors are typically very expensive, as a result of which, the technique is impractical for many applications. Another optical biosensor, known as a resonant mirror system, relies on changes in a penetrating evanescent wave, which is similar to SPR and, like it, binding reactions between receptors and analytes in a region extremely close to the back side of a special mirror (referred to as a resonant mirror) can be analyzed by examining light reflected when a laser beam directed at the mirror is repeatedly swept through an arc of specific angles. Like SPR sensors, resonant mirror systems are expensive and impractical for many applications.
[0008] U.S. Patent No. 6,248,539 discloses techniques for making porous silicon and an optical resonance technique that utilizes a very thin porous silicon layer within which binding reactions between ligands and analytes take place. The association and disassociation of molecular interactions affects the index of refraction within the thin porous silicon layer. Light reflected from the thin film produces interference patterns that can be monitored with a CCD detector array. The extent of binding can be determined from change in the spectral pattern.
[0009] In similar arts, reflectance change on thin film surface due to interferometric effect from surface adsorption has been fabricated by growing a silicon or silicon dioxide layer of a desired thickness on optionally silicon substrates. Molecular receptors are immobilized on this sensor in a microarray pattern using a variety of techniques such as contact printing or piezoelectric inkjet printing. Receptor microarrays are of the order of 100 mm in diameter and cover the sensor surface. Reflectance signal is read out using a scanning laser beam and the difference in reflectance signal between a given receptor spot and the bare oxide surface indicates the level of surface adsorption. Such methods have their own limitations and disadvantages such as the fact that laser scanning on micro array pattern prevents measurement of molecular binding kinetics, an important parameter for certain applications such as drug screening. Furthermore, silicon being opaque in the visible region, necessitates reflection mode read-out leading to larger device footprint compared to one with transmission mode read-out. Also, even if real-time detection is possible, optical beam needs to traverse a fluid cell containing the sample, wherein thermal currents and mass transport within the fluid cell create slow drift that pollute molecular signal.
[0010] Kinetic binding measurements involve measurement of rates of association (molecular binding) and disassociation, wherein analyte molecules are introduced to ligand molecules producing binding and disassociation interactions between the analyte molecules and the ligand molecules. Association occurs at a characteristic rate [A][B]kon that depends on the strength of the binding interaction kon and the ligand topologies, as well as the concentrations [A] and [B] of the analyte molecules A and ligand molecules B, respectively. Binding events are usually followed by a disassociation event, occurring at a characteristic rate [A][B]koff that also depends on the strength of the binding interaction. Measurements of rate constants kon and kOff for specific molecular interactions are important for understanding detailed structures and functions of protein molecules. In addition to the optical biosensors discussed above, scientists
perform kinetic binding measurements using other separations methods on solid surfaces combined with expensive detection methods (such as capillary liquid chromatography/mass spectrometry) or solution-phase assays. These methods suffer from disadvantages of cost, the need for expertise, imprecision and other factors.
[0011] There is therefore a need in the art for an apparatus and a method thereof that provides highly sensitive and accurate analysis/detection of analytes along with efficiently making molecular binding measurements. It would be very advantageous to provide a method and apparatus of simultaneously assaying for multiple analytes using diffraction of light as implemented in present invention.
[0012] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0013] The 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 with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0014] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can 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.

OBJECTS OF THE INVENTION
[0015] It is an object of the present disclosure to provide highly sensitive and accurate analysis/detection of analytes along with efficiently making molecular binding measurements.
[0016] It is an object of the present disclosure to provide a method and apparatus of simultaneously assaying for multiple analytes using diffraction of light.
[0017] It is an object of the present disclosure to provide an apparatus for molecular diagnostics and biomolecular sensing configured to measure various biologically relevant events such as target-receptor binding events.

SUMMARY
[0018] The present disclosure relates to an apparatus for molecular diagnostics and biomolecular sensing, and more particularly to a differential interferometric sensor configured to measure various biologically relevant events such as target-receptor binding events. In one aspect, sensor of the present disclosure comprises a plurality of micro-diffractive elements, also referred to as micro-structures hereinafter, patterned onto an optically transparent substrate so as to form a diffraction grating and providing at least two diffracted orders that respond differentially to homogenous molecular adsorption on the diffraction grating. According to one embodiment, optically transparent substrate is glass slide. In an implementation, difference signal generated from two diffracted orders can be configured as a direct indicator of the adsorbed thickness, thereby eliminating the requirement of printing receptions onto sensor surface, and also eliminating requirement of laser scanning as differential measurement of molecular binding kinetics can directly be done based on the difference signal.
[0019] In another aspect, sensor of the present disclosure can be patterned with a one-dimensional and/or two-dimensional diffraction grating with a depth of around 1/8th wavelength in reflection mode read-out and 1/4th wavelength in transmission mode. In an aspect, lateral spacing of the micro-diffractive elements can range from .4 to 10microns, while depth of the elements can range from 50 to 500nm. Sensor of the present disclosure can also be configured so as to have parameter space with probe beam larger than sensor surface, which facilitates effectiveness of detection in the assay and enhances signal output.
[0020] In another aspect, sensor of the present disclosure can be configured to include a light source, a means for supporting the patterned substrate, and a light detector positioned to collect diffracted light, wherein the light detector is operatively connected to appropriate data collecting devices. In another aspect, read-out beam from a visible light/laser source can be incident at an oblique angle such that zero and first order diffracted beams respond to molecular adsorption differentially, i.e. with opposite signs. Fluid cell fluctuations can be configured to affect both orders equally, enabling two diffraction orders to be used to cancel out fluid cell fluctuations. The light detector may be a single element photodiode or a multi-element CCD or CMOS camera.
[0021] In another aspect, diffracted patterns can enable measurement of real-time binding kinetics of molecules with common mode noise suppression in a sample used. Sample can be selected from any industry related to biological, chemical, automobile or any other industry in which the quality of liquid sample may need to be estimated. Biological applications of the present disclosure can include any area of bio-chips for genomics and proteomics, diagnostic chips, a combination thereof, among other like applications. In another aspect, patterns on substrate can be chosen in such a way as to have large diffracted signal intensity at desirable locations, such as away from regions of high noise such as that emanating from scattering of the main beam.
[0022] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[0023] These and other features, aspects and advantages of the invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:
[0024] FIG. 1 illustrates the schematic apparatus with patterned substrate.
[0025] FIG. 2 illustrates an exemplary diffraction pattern responding at different diffracted orders at respective refractive index.
[0026] FIG. 3 illustrates exemplary diffracted patterns at respective refractive index.

DETAILED DESCRIPTION OF THE INVENTION
[0027] In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present disclosure. It will be apparent, however, to one skilled in the art that embodiments of the present disclosure may be practiced without some of these specific details.
[0028] Those of ordinary skilled in the art will realize that the following detailed description of the exemplary embodiment(s) is illustrative only and is not intended to be in any way limiting. Other embodiments will readily suggest themselves to such skilled persons having the benefit of this disclosure. Reference will now be made in detail to implementations of the exemplary embodiment(s) as illustrated in the accompanying drawings. The same reference indicators will be used throughout the drawings and the following detailed description to refer to the same or like parts.
[0029] One should appreciate that the disclosed techniques provide many advantageous technical effects including configuring and processing various feeds to determine behaviour, interaction, management, and response of users with respect to feeds and implement outcome in enhancing overall user experience while delivering feed content and allied parameters/attributes thereof.
[0030] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0031] Photolithography, also termed optical lithography or UV lithography, is a process used in micro fabrication to pattern parts of a thin film or the bulk of a substrate. It uses light to transfer ageometric pattern from a photo-mask to a light-sensitive chemical "photo-resist", or simply "resist," on the substrate. A series of chemical treatments then either engraves the exposure pattern into, or enables deposition of a new material in the desired pattern upon, the material underneath the photo resist.
[0032] In an aspect, sensor substrates can have surface topographical features that are much larger in scale than analyte specific receptors. It will be understood that sensor elements may be constructed with molecular scale topographical features, wherein, for instance, sensor elements can include templated grooves having size of a large molecule and having a complementary geometry and interaction to the analytes in the sample. In other words, sample specific receptors may comprise molecular to microscopic size indentations that would provide complementary shape and interaction to specific proteins or cells, which can be the analytes being estimated or tested for.
[0033] In another aspect, "grating region" is a region composed of grating structures and plurality of holes provided in the grating. "Center of gravity of a grating region in a plane" means the point of intersection of a line passing through the center of gravity of the grating and perpendicular to the plane. "Center of gravity of a grating" is the center of gravity of the grating region when thickness of the grating is uniform in the grating region. Diffractive optic elements manipulate light by using principles of diffraction, wherein portions of wavefront can be retarded selectively using microstructure pattern on a substrate such as glass, but other materials can be used as well. Diffraction permits a more versatile and powerful means of steering light. Microstructure pattern may be surface patterns formed on surface of the substrate, or bulk patterns formed through bulk of the material. Diffractive optic substrate may have complicated patterns, but their versatility in manipulating light can be useful in current objective of optimizing properties of the diffracted light.
[0034] The present disclosure relates to an apparatus for molecular diagnostics and biomolecular sensing, and more particularly to a differential reflectance based sensor configured to measure various biologically relevant events such as target-receptor binding events. In one aspect, sensor of the present disclosure comprises a plurality of micro-diffractive elements, also referred to as micro-structures hereinafter, patterned onto an optically transparent substrate so as to form a diffraction grating and providing at least two diffracted orders that respond differentially to homogenous molecular adsorption on the diffraction grating. According to one embodiment, optically transparent substrate is glass slide. In an implementation, difference signal generated from two diffracted orders can be configured as a direct indicator of the adsorbed thickness, thereby eliminating the requirement of printing receptions onto sensor surface and also elimination requirement of laser scanning as differential measurement of molecular binding kinetics can directly be done based on the difference signal.
[0035] In another aspect, sensor of the present disclosure can be patterned with a one-dimensional and/or two-dimensional diffraction grating with a depth of around 1/8th wavelength in reflection mode read-out and 1/4th wavelength in transmission mode. In an aspect, lateral spacing of the micro-diffractive elements can range from 5 to 10?m, while depth of the element can range from 50 to 200nm. Sensor of the present disclosure can also be configured so as to have parameter space with probe beam larger than sensor surface, which facilitates effectiveness of detection in the assay and enhances signal output. According to one embodiment, pattern of the instant disclosure has a non-resonant grating. In another aspect, detection of analytes by the proposed sensor can be based on intensity shifts.
[0036] In another aspect, sensor of the present disclosure can be configured to include a light source, a means for supporting the patterned substrate, and a light detector positioned to collect diffracted light, wherein the light detector is operatively connected to appropriate data collecting devices. In another aspect, read-out beam from a visible light/laser source can be incident at an oblique angle such that zero and first order diffracted beams respond to molecular adsorption differentially, i.e. with opposite signs. Fluid cell fluctuations can be configured to affect both orders equally, enabling two diffraction orders to be used to cancel out fluid cell fluctuations.
[0037] In another aspect, diffracted patterns can enable measurement of real-time binding kinetics of molecules with common mode noise suppression in a sample used. Sample can be selected from any industry related to biological, chemical, automobile or any other industry in which the quality of liquid sample may need to be estimated. Biological applications of the present disclosure can include any area of bio-chips for genomics and proteomics, diagnostic chips, a combination thereof, among other like applications. In another aspect, patterns on substrate can be chosen in such a way as to have large diffracted signal intensity at desirable locations, such as away from regions of high noise such as that emanating from scattering of the main beam.
[0038] Figure 1 illustrates a schematic representation of an apparatus of the present invention. Figure 1 depicts a substrate that can have a micro-structure pattern grated on it, which can show a property of differential diffraction pattern in one or two dimensions for a required sample. In an embodiment, microstructure pattern can have a substrate such as a glass slide, wherein a grating is provided onto the substrate. The grating provided onto the substrate surface can comprise a plurality of pitches, which can be arranged in a first direction, a second direction, or a plurality of directions depending upon the requirement.
[0039] In another embodiment, microstructure pattern can be etched to an optimal depth on a substrate, which can result into a diffraction pattern with different diffracted orders responding differentially to a molecular surface adsorption. Resulting diffracted orders can enable measurement of real time binding kinetics of molecules with common mode noise suppression in a sample.
[0040] The substrate can be transparent, opaque and can be selected from a group containing glass, fiber, plastic or in combination. Complex patterns on a flat surface may be created by a variety of means and photolithography is one of the preferable means employed in the present invention. Photolithography can be employed in many ways: light can be used to initiate a reaction to activate or deactivate surface species, and by the appropriate application of masks, micro structure patterns can be formed. The deposited material is either held to the substrate by physical adsorption, or can be covalently bound to the surface or a surface layer deposited on the substrate for the specific purpose of binding to the pattern recognition species.
[0041] In another aspect of the invention, lateral scale of the features can range from .4-10 microns and depth or pitch of the micro features on a glass slide can range from 50-500nm. In implementation, light from a source, which can be a laser diode, can illuminate the apparatus at oblique incidence generating a 2D diffraction pattern, where different diffracted orders respond differently to refractive indexes (bulk as in Figure. 2, or surface as in Figure. 3). Ratio of intensities from the two different orders can eliminate common mode ratio, as shown in Figure 2. Refractive index sensitivity can range from 2x10-5 RIU (in transmission mode) to 3x10-6 RIU (reflection mode). In an embodiment, assay can be performed by contacting patterned substrate with the sample or analyte containing medium. Solid substrate upon which the pattern of low recognition molecule is laid may be transparent, partially transparent, or reflecting at the wavelength of the incident illumination. In the case of a transparent substrate, analyte specific receptors may be patterned on surface of the substrate. Once the recognition element that is capable of specific binding (e.g., protein, oligonucleotide, antibody, etc.) is laid out on the surface in a preselected pattern, the medium to be assayed can be ctacted with the substrate, allowing analytes present in the medium to bind to their complementary recognition element.
[0042] When a particular analyte is present in the sample, subsequent binding event between analyte and its complementary recognition element can be accompanied by a change in local thickness of the layer on the substrate and/or in the local index of refraction. Both the change in thickness and in index of refraction can alter optical properties at interface between substrate and medium in regions where binding has taken place. In another embodiment, patterned substrate itself can produce an observable diffraction image but the binding events alter the intensities of the diffracted signal.
[0043] In an embodiment, assay method can include an extension for detection of multiple analytes in a sample medium and can involve producing multiple microstructured patterns of recognition elements within the same substrate. Pattern for each type of recognition element may either be distinct from that of others or may be the same, but simply located in different regions of the substrate. Non-limiting examples of simple distinct patterns can include different geometric elements (lines, circles, etc.), same geometric elements but arranged with different periodicities, same geometric elements with the same periodicity but rotated with respect to each other, provided the patterns do not have rotational symmetry. Patterns may also be a mixture of any of the above. In case of multiple patterns of multiple recognition elements, it is desirable to have regions of high intensity such that at least one region is distinct to each pattern employed. In other words, diffraction patterns may overlap but at each pattern can have at least one unique spot.
[0044] In another embodiment of the invention, an assay may also be performed in situ by placing the substrate into a chamber into which a sample can be introduced. In applications in which moisture may be problematic, substrate may be placed in a cell that is partially evacuated in order to reduce moisture. This is advantageous where it is desirable to reduce signal strength that may arise due to water condensation. However, in cases where analytes, but not their partner receptors, are favoured by water, presence of water condensation (also called ‘condensation figures’) can be utilized to enhance diffracted signal. In another embodiment of the invention, assay may also be performed in situ by placing substrate into a chamber into which a medium can be introduced. An analyte in a sample can be selected from any industry such as chemical, biological or automobile industry depending upon application area. It should be obvious to a person skilled in the art that above mentioned application is not restricted and can be extended to others area also.
[0045] In an implementation of the proposed assay method, an analyte-specific recognition element can be bound to surface of substrate, and when light is incident on analytes bound to said analyte-specific receptors, a pre-selected number of diffraction spots in pre-selected positions spaced from said beam of light indicative of the presence of analytes bound to said analyte-specific receptors can be found. After sufficient time substrate can be illuminated with sufficient laser beam light and then diffracted light can be detected from the substrate and diffracted light for the presence of different order diffraction spots can be analysed on the basis of intensity shift. The intensity shift of the signal reflects the position of pattern having an negative effect on the detector. Diffraction spots can represent binding of analyte to said specific receptors on the substrate’s surface.
[0046] In another embodiment, area of technology of the proposed biosensor can include, but is not restricted to, biological industry such as bio-chips for genomics and proteomics, diagnostic chips, or automobile industry such as fuels where quality of fuel is to be estimated, or chemistry such as chemicals where estimation is needed at molecular level or any other industry where liquid or semi-solid or paste like sample can be used to estimate its quality.
[0047] A light source may produce monochromatic beam, wherein light can have a wavelength in the range from ultraviolet to infrared, but preferably a coherent and collimated light beam, such as would come from a laser (e.g. diode, He—Ne, Nd:YVO4, Argon-ion).
[0048] In an aspect, transparent substrate of the present disclosure and resulting differential read-out of the proposed sensor can be attached on top of an inexpensive CMOS camera such as mobile phones to measure molecular binding at sensor surface.
[0049] Figure 2 illustrates an exemplary graph showing differential response of (0,0) and (0,1) diffracted orders to bulk refractometry. Ratio signal (curve B) may be able to bring out step-wise injection of fluids of different refractive indices in presence of signal drifts as shown by the curves A and C. For quantification of low intensity signals, a sensitive CCD array detector or a PMT may be used. For further signal enhancement, lock-in detection as well as amplification schemes known to those skilled in the art may be employed. Image, or a part thereof, obtained as an electronic signal from the detector can be stored on a computer and an image analysis software can then be used to identify patterns on the substrate that give rise to observed diffraction image, thus identifying analytes that are present in the sample. A code may be written on the substrate itself that can identify which analyte-specific receptors are present. Presence of signals at specific locations relative to a standard encoded location within the diffraction image can correspond to presence of specific analytes. Quantification of signals at defined locations enables quantification of amount of different analytes.
[0050] Figure 3 illustrates an exemplary graph showing differential response of (0,0) and (0,1) diffracted orders to surface adsorption. Detected signal is illustrated and can be analyzed through the generated curves A, B, and C.
[0051] In another embodiment, micro-structured patterned layer itself may be invisible to source for several reasons, including that the layer of recognition elements is very thin and its refractive index is closely matched to that of the substrate. If the layer of recognition elements is not very thin with regards to the original substrate, an inert material can be added such that this inert material covers rest of the substrate and reduces effective thickness of the patterned layer. In this case, refractive index of patterned layer and of inert material should be closely matched. Another reason that the patterned layer may not be visible to the source is that the layer of recognition elements is very thin and refractive indices of the substrate, the thin layer, and of the medium are very similar.
[0052] In another aspect, the present disclosure can include an interferometric method for determining presence or absence of a target analyte in a sample. The method can include using a laser beam having a wavelength ? to probe at least a portion of a substrate. Portion of the substrate having a reflecting surface, here in this case, can be a microstructure, which can be exposed to the sample. Reflecting surface can include at least a first region having a layer of recognition molecules specific to target analyte, and a second region that does not include a layer of recognition molecules specific to the target analyte. The method can further comprise measuring a time dependent intensity in a photodetector of a substantially reflected diffraction signal of the probe beam while probing the first region and the second region. In one aspect of the disclosure, time dependence arises from relative motion of incident laser beam with respect to the substrate.
[0053] In another aspect, there can be a scale free or label free quadrature interferometric step-detection method of determining the presence or absence of a target analyte in a sample. The substrate can have a spatially patterned structure of receptor molecules specific to the target analyte. The method further can include detecting intensity change in a far-field diffraction pattern.
[0054] It is understood that the specific order or hierarchy of steps in the methodology disclosed is an illustration of exemplary approaches. Based upon design preferences, it is understood that the specific order or hierarchy of steps in the processes may be rearranged. Some of the steps may be performed simultaneously.
[0055] There may be many other ways to implement the invention. Various functions and elements described herein may be portioned differently from those shown without departing from the spirit and scope of the invention. Various modifications of these embodiments will readily apparent to those skilled in the art in view of present disclosure, and generic method defined herein may be applied to other embodiments.
[0056] All structural and functional equivalen ts to the elements of the various embodiments of the invention described throughout the disclosure that are known or later come to be known to those ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the invention.
[0057] The above description and drawings are only illustrative of preferred embodiments which achieve the objects, features and advantages of the present invention, and it is not intended that present invention be limited thereto.

ADVANTAGES OF THE INVENTION
[0058] It is an object of the present disclosure to provide highly sensitive and accurate analysis/detection of analytes along with efficiently making molecular binding measurements.
[0059] It is an object of the present disclosure to provide a method and apparatus of simultaneously assaying for multiple analytes using diffraction of light.
[0060] It is an object of the present disclosure to provide an apparatus for molecular diagnostics and biomolecular sensing configured to measure various biologically relevant events such as target-receptor binding events.
,CLAIMS:1. A sensor comprising:
a plurality of micro-diffractive elements patterned onto an optically transparent substrate to form a diffraction grating having multiple pitches arranged in one or more directions, wherein said patterning provides at least two diffracted orders that respond differentially to homogenous molecular adsorption on said diffraction grating.

2. The sensor of claim 1, wherein said optically transparent substrate is glass slide.

3. The sensor of claim 1, wherein said substrate is selected from one or a combination of glass, fiber, plastic, or a combination thereof.

4. The sensor of claim 1, wherein said patterning is performed by means of photolithography.

5. The sensor of claim 1, wherein difference signal generated from said at least two diffracted orders is configured as an indicator of adsorbed thickness.

6. The sensor of claim 1, wherein said plurality of micro-diffractive elements have a lateral spacing ranging from 0.4 to 10 microns.

7. The sensor of claim 1, wherein said plurality of micro-diffractive elements have a pitch ranging from 50 to 500 nm.

8. The sensor of claim 1, wherein parameter space with probe beam is larger than surface of said sensor.

9. The sensor of claim 1, wherein the sensor further comprises a light source configured to generate a read-out beam, a means for supporting said optically transparent substrate, and a light detector, wherein said light detector is configured to collect diffracted light.

10. The sensor of claim 9, wherein said read-out beam is incident at an oblique angle such that zero and first order diffracted beams respond to molecular adsorption differently.