Portable microarray assembly

Methods and systems are provided for imaging a microarray of a microarray assembly. The microarray assembly may be configured as a protein microarray and may be used to visualize protein expression levels in sample for disease detection and/or diagnosis. In one example, the microarray assembly may comprise a laser pointed in a first direction, a camera positioned parallel to and vertically below the laser, a first dichroic mirror vertically aligned with the laser for reflecting light emitted from the laser, a second mirror vertically aligned with the camera and horizontally aligned with the first dichroic mirror, and a chip coated in a nitrocellulose film and including an array of wells containing one or more biomolecules.

FIELD

The present description relates generally to systems and methods for imaging and analyzing biomolecule microarrays.

Microarray systems may be used to detect and quantify biomolecules such as antibodies, antigens, oligonucleotides, and RNA for a variety of clinical purposes such as understanding gene expression, gene regulation, protein production, protein modification, etc. Biomolecules may be adhered to microarray spots or wells formed on a glass slide or chip, and a fluorescent label, such as an organic dye, may be bound to a target subset of the biomolecules. Light from a laser may be directed to the biomolecules, and those biomolecules tagged with the fluorescent label may emit light in response to excitation from the laser. In some examples, light emitted from the luminescent labels may be gathered by a lens system and an image of the illuminated biomolecules may be captured. The image may then be analyzed to quantify expressions levels of the target biomolecules. Specifically, the intensity of different wavelengths of light may be compared to determine the relative intensity of emissions at wavelengths associated with the fluorescent tags. Because the fluorescent tags are bound to the target biomolecules, biomolecule expression levels or concentrations in the samples being tested, may be inferred based on intensity levels of light produced by the fluorescent labels.

In one example, microarrays may be configured to measure protein levels. Specifically, protein microarrays may be configured to measure antibody levels in a sample (e.g., lysate, serological, blood, or synthetic samples). As such, protein microarrays may be used to identify and/or diagnose infectious diseases. Proteins (e.g., antigens) may be affixed to a nitrocellulose film coating on the chip. When incubated with patient samples (e.g., whole blood, serum, plasma, saliva) containing antibodies specific to the printed antigens, the antibodies may bind to the antigens on the array. Probe molecules tagged with the fluorescent label may then bind to the antibodies, illuminating the antibodies when exposed to the laser beam. A clinical diagnosis may be made based on antibody expression levels as inferred from the luminescence, and therefore concentration, of the fluorescent labels.

However, the inventors herein have recognized potential issues with current microarray systems. As one example, image acquisition in most microarray systems is accomplished by a laser scanner included in the microarray system. Laser scanners may be necessary to deliver images at high enough resolution qualities to make accurate biomolecule identification and quantification determinations. However, laser scanners are often very large, and as a result current microarray system are stationary and not transportable. Further, many microarray systems may include multiple laser scanners for wavelength multiplexing, increasing the size, weight, and expense of such microarray systems. Thus, samples including the biomolecules must be taken to the microarray and scanner for analysis. As such, it may take a significant amount of time to render a diagnosis due to the delay associated with transporting the sample from a patient to the microarray scanner.

In another example, diagnosis determination may be difficult due to the low sensitivity of the protein microarrays. Specifically, organic dyes used to fluorescently label the biomolecules may have relatively broad emission and absorption bands, resulting in increased amounts of background noise in the biomolecule images. Additionally, light emitted from the organic dyes may be of low intensity. Further, the dyes may be prone to optical fading (photo bleaching) after exposure to intense excitation light. As such, image resolution may be low, and differentiation and quantification of the biomolecules may be challenging, resulting in reduced accuracy of disease diagnoses in protein microarrays.

In one example, the issues described above may be addressed by a microarray assembly comprising a laser emitting in a first direction, a camera positioned parallel to and vertically below the laser, a first dichroic mirror horizontally aligned with the laser for reflecting light emitted from the laser, a second mirror horizontally aligned with the camera and vertically aligned with the first dichroic mirror, and a chip coated in a nitrocellulose film and including an array of wells containing one or more biomolecules. In this way, the size of the microarray may be reduced by utilizing a camera instead of a laser scanner for acquiring an image of the wells. The size may be further reduced by positioning the camera and laser in parallel with one another, thereby increasing the portability of the microarray.

In some examples, the assembly may further include a cuvette for housing the chip, the cuvette including one or more of an aperture and groove for receiving the chip. The cuvette may additionally include an optically clear window integrally forming a front wall of the cuvette. The front wall of the cuvette may be pointed towards the first dichroic mirror for receiving a light beam produced from the laser. By including the cuvette for the chip, instances of contamination may be reduced.

In other examples, the biomolecules may be tagged with a fluorescent label, where the fluorescent label may comprise Quantum Nanocrystal fluorescent-nanoparticles (QNC). By using QNC to label the biomolecules as opposed to organic dyes, the sensitivity and resolution of the images captured by the microarray may be increased, allowing for improved accuracy in the diagnosis of infectious diseases.

DETAILED DESCRIPTION

The following description relates to systems and methods for imaging and analyzing biomolecule microarrays. A biomolecule microarray assembly, such as the biomolecule microarray assembly shown inFIGS. 1-6, may be used to detect and estimate an amount of biomolecules, such as proteins, in a sample of lysate, blood, serum, tissue, cell cultures, etc. The biomolecule microarray assembly may include several components, as shown inFIGS. 1 and 6, such as a camera, laser, and dichroic mirrors for imaging the biomolecules.FIG. 8shows an example method for imaging the biomolecules with the biomolecule microarray assembly. Biomolecules from the sample may be loaded onto a nitrocellulose coated microarray chip, an example of which is shown inFIGS. 4 and 7. The biomolecules may be labelled with one or more fluorescent tags to visualize biomolecule expression levels during imaging of the chip. In some examples, the microarray chip may be inserted into a chip cover or cuvette, such as the cuvette13shown inFIGS. 4 and 7, for preventing contamination of the sample.

Once the chip is inserted in the microarray assembly, light from a laser may be reflected via a first dichroic mirror onto the chip. In response to excitation from the laser beam, the fluorescent tags may emit light back towards the first dichroic mirror. The emitted light may then propagate through the first dichroic mirror and on to a second mirror, where the emitted light may be reflected to a camera for image acquisition. By including the second mirror in series with the first dichroic mirror, the camera and laser may be positioned parallel to one another, thereby reducing the size of the microarray assembly. As such, the portability of the microarray assembly may be increased relative to microarray assemblies with only one dichroic mirror.

Turning now toFIG. 1, it shows a schematic of a biomolecule analysis system100. The biomolecule analysis system100may include a biomolecule microarray assembly10and a computer122. In particular,FIG. 1shows a two-dimensional schematic diagram showing components of the biomolecule analysis system100and how they may be electrically coupled to one another. As such, the actual sizes and relative positions of the components of the biomolecule microarray assembly10may be different than shown inFIG. 1.FIGS. 2-6, described further below, include three-dimensional schematics of the biomolecule microarray assembly10, showing the relative sizes and positions of the components within the assembly10. As such, the function of components of the biomolecule microarray assembly10may be described with reference toFIG. 1, while the positioning of each component within the assembly10may be described with reference toFIGS. 2-6.

In a preferred embodiment, the biomolecule microarray assembly10may be configured as a protein microarray for quantifying protein levels in one or more of lysate, whole blood, plasma, serum, saliva, CSF, or synthetic sample. As such, the biomolecule microarray assembly10may be used in the diagnosis and/or detection of infectious diseases. Specifically, antibody and/or antigen expression levels in a blood sample from a patient may be analyzed using the biomolecule microarray assembly10to identify a disease afflicting the patient. However, it should be appreciated that in other embodiments, the biomolecule microarray assembly10may be configured to image and analyze biomolecules other than proteins such as: DNA, cDNA, mRNA, siRNA, peptides, carbohydrates, lipids, whole cells, etc. Said another way, the biomolecule microarray assembly10may be any one of a protein, DNA, RNA, peptide, tissue, antibody, carbohydrate, lipid, or other microarray reader.

The biomolecule microarray assembly10may also be referred to herein as biomolecule microarray assay imager10. The microarray assembly10may include a microarray chip12, a portion or all of which may be coated with a nitrocellulose film14, the nitrocellulose film14including a plurality of binding locations or wells16onto which a sample may be loaded. Thus, the binding locations16may be arranged on the nitrocellulose film14to form an array15. The array15of wells16may chemically bind sample biomolecules of the sample to the chip12. In the description herein, the wells16may also be referred to as spots16and/or binding locations16. A sample may first be loaded onto the film14prior to insertion of the chip12into the microarray assembly10for imaging. The sample may in some examples comprise one or more of: blood, mucus, tears, sera, aqueous humor, or other bodily fluids that include antigens and/or antibodies of interest for infectious or non-infectious disease diagnosis. However, in other examples, the sample may include one or more of a tissue culture, cell culture, skin, hair, bone marrow, brain or other organs, feces or urine, and cerebral spinal fluid, for gene expression level quantification. Thus, the sample may include a plurality of biomolecules, of which only a subset may be desired for analysis. As such, target biomolecules, which comprise the subset of sample biomolecules desired for analysis, may be tagged with a fluorescent label. The fluorescent label may be any suitable fluorescent tag which may bind to the target biomolecules and emit light of a different wavelength than a light source in response to excitation from the light source, such as a laser. The array15of binding locations16may be referred to as an assay once the biomolecules from the sample have been chemically bound to the binding location16, and have been tagged with the fluorescent label(s).

In some examples the fluorescent label may include Quantum Nanocrystal fluorescent-nanoparticles (QNC). QNCs are inorganic spheroidal particles that exhibit electronic quantum states confined to concentric layers of the materials that comprise the particle. QNCs may be composed from II/VI and II/V semiconductor alloys that are surface-functionalized to enhance bonding to bio-molecules such as biotin, proteins, and streptavidin. QNCs may have greater fluorescence brightness, optical stability and narrower emissions bands than organic dyes. However, in other examples, the fluorescent label may include other fluorophores or organic dyes such as food dye, ruthenium-based fluorescent dye, ethidium bromide, fluorescein, or green fluorescent protein (GFP). It should also be appreciated that multiple types of QNCs may be used for the fluorescent label, where each type of QNC may reflect a different wavelength of light. Thus, multiple types of QNCs that reflect different wavelengths of light may be utilized in the same assay15.

Thus, the chip12may be removably coupled to the microarray assembly via a door (e.g., door214shown inFIGS. 2-5). The chip12may be relatively planar and may include a flat surface upon which the thin porous nitrocellulose film14is attached. The sample may be deposited onto the array15of target binding locations16included on the nitrocellulose film14. The target binding locations16may each be configured to bind to one or more sample biomolecules in the sample. Thus, the target binding locations16may be configured to bind to one or more proteins, in examples where the microarray assembly10is a protein microarray. However, in other examples, the target binding locations16may be configured to bind to one or more oligonucleotides in examples where the microarray assembly10is a DNA microarray. The nitrocellulose film14may be sized to approximately a 12 mm by 12 mm square. However, in other examples the film14may be sized to be larger or smaller than 12 mm by 12 mm. Further, in other examples, the film14, may be shaped differently than a square, such as circular, triangular, rectangular, etc. Thus, the chip12may be coated in a nitrocellulose film, which may be spotted with a plurality of target binding locations to form an array.

The target binding locations16may be approximately circular, and the diameter of the binding locations16may be in a range between 100-1000 microns. Further, the target binding locations16may be relatively evenly spaced from one another, where the spacing between target binding locations16may be in a range between 200 and 2000 microns. In some examples, the spacing of the target binding locations16may be based on the size of the target binding locations16, where the spacing between target binding locations16may be approximately twice the diameter of the target binding locations16. However, in other examples, the spacing may be greater or less than two times the diameter of the target binding locations16. The film14may be configured to include approximately 1600 target binding locations16. However, in other examples, the film14may include more or less than 1600 target binding locations16.

In addition to the porous nitrocellulose, the binding locations16may include one or more binding molecules to which the biomolecules in the sample may bind. Thus, the binding molecules of the binding locations16may affix/immobilize the biomolecules to the target locations16on the chip12. In examples where the microarray assembly10is configured as a protein microarray, the target binding locations16may comprise purified antigens, to which antibodies in the sample may bind. Thus, antigens may be spotted onto the nitrocellulose film14, at the target binding locations16. However, it should be appreciated that in other examples, the target binding locations16may comprise molecules other than antigens for binding to biomolecules of the sample other than antibodies. For examples, the target binding locations16may comprise antibodies, to which antigens in the sample may bind. In still further examples, the target binding locations16may comprise oligonucleotides or cDNA for binding to DNA or RNA in the sample. Alternatively, the target binding locations16may comprise only porous nitrocellulose, and may not include antigens or other binding molecules for binding to the sample biomolecules. In such examples, proteins in the sample may bind directly to the porous nitrocellulose at the target binding locations16.

Additionally or alternatively, a reactive mixture of probe molecules including any of the suitable fluorescent labels, may be introduced to the sample biomolecules, once the sample biomolecules have adhered to the binding locations16on the chip12. Specifically, the probe molecules may be configured to include QNC for fluorescently labelling the biomolecules in the spots16. The probe molecules may be configured to selectively bind to the target biomolecules. In this way, only the target biomolecules may be tagged with the fluorescent labels. In examples where the sample includes antibodies and the microarray assembly10is configured as a protein microarray, probe molecules labeled with QNC, may be configured to selectively bind to only target antibodies in the sample. Said another way, the probe molecules labeled with QNC may be conjugated to the target antibodies in the sample. Thus, the probe molecules may be introduced to the binding locations16after the target antibodies have affixed to the binding locations16for fluorescently labeling the target antibodies.

To enable the mixing and binding of the biomolecules from the sample with the nitrocellulose film14, an assay cover17may positioned over the nitrocellulose film14. In some examples, the assay cover17may fully enclose the chip12. However, in other examples, the assay cover17may only enclose the nitrocellulose film14and binding locations16. One or more of the sample, a blocking solution, washing solution, and/or the reactive mixture may be mixed with the nitrocellulose film14in the assay cover17. Further, the assay cover17may fluidically seal the nitrocellulose film14from the environment. Thus, the assay cover17may function as a container, holding various aqueous mixtures which may aid in binding the sample biomolecules to the binding locations16, inhibiting binding of and/or removing sample biomolecules that are not the target biomolecules, and fluorescently tagging the target biomolecules. Put more simply, the assay cover17may permit reactions between the nitrocellulose film14and various aqueous mixtures during development of the assay.

Further, the assay cover17may serve to prevent contamination of the sample and film14. By enclosing the film14in the assay cover17, an amount of foreign materials such as dust, dirt, bacteria, enzymes, fungi, viruses, etc., that accumulate on the film14may be reduced. Specifically, the assay cover17may fluidically seal the film14from the outside environment prior to insertion of the chip12into the assembly10.

Once the target biomolecules have been fluorescently tagged and chemically bound to the nitrocellulose film14, the assay cover17may be removed from the chip12, and the film14may be dried by flowing dry air over the surface of the chip12. After the biomolecules in the sample have been bound to the nitrocellulose film14and fluorescently labeled, the chip12may be inserted into a cuvette13.

The chip12may be inserted into the cuvette13, and the cuvette13may fully enclose the chip12prior to insertion of the chip12in the microarray assembly10. However, in other examples, the cuvette13may be inserted into the assembly10prior to insertion of the chip12into the cuvette13, and the chip12may therefore be inserted into the cuvette13after the cuvette13has been inserted into the assembly10. The structure of the cuvette13is described in greater detail below with reference toFIGS. 4 and 7. Cuvette13may serve a variety of purposes.

The cuvette13may fully enclose and/or hold the chip12, and center the chip12within the assembly10. Thus, the cuvette13may serve as a holder for the chip12, which retains the chip12in a fixed position within the assembly10during imaging of the binding locations16. In some examples, the assembly10may include one or more mechanical stabilizers41which may interface with external faces of the cuvette13, for retaining the cuvette13in a fixed position within the assembly10.

In some examples, the assay cover17may not be used to facilitate mixing of the sample and reactive mixture with the chip12. In such examples, the cuvette13may provide the same above-mentioned functions as the assay cover17. Thus, the cuvette13may in some examples function as a container, holding both the chip12, and various aqueous mixtures which may aid in binding the sample biomolecules to the binding locations16, inhibiting binding of and/or removing sample biomolecules that are not the target biomolecules, and fluorescently tagging the target biomolecules. Said another way, the cuvette13may permit reactions between the nitrocellulose film14and various aqueous mixtures during development of the assay.

Further, the cuvette13may serve to prevent contamination of the chip12. By fully enclosing the chip12in the cuvette13, an amount of foreign materials such as dust, dirt, bacteria, enzymes, fungi, viruses, etc., that accumulate on the chip12may be reduced. Specifically, as shown below with reference toFIGS. 4 and 7, the cuvette13may fluidically seal the chip12from the outside environment, once the chip12is inserted into the cuvette13.

The chip12may be inserted into the microarray assembly10for imaging of the binding location16. Specifically, in some examples where the cuvette13is not included in the assembly10, the chip12may be inserted directly into the microarray assembly10. However, in other examples where the cuvette13is included in the microarray assembly10, the cuvette13including the chip12may be inserted into the microarray assembly10. The cuvette13may include an authentication device which may be either mechanical or electronic, which may engage with the microarray assembly10when the cuvette13is inserted. The authentication device may ensure that the array15is imaged only when the cuvette13is inserted in the microarray assembly10.

Once the chip12is inserted into the microarray assembly10, the array15may be imaged by the microarray assembly10to visualize biomolecule expression levels. Imaging of the array15, may include exciting the fluorescent labels via a laser18, and capturing the light emitted from the fluorescent labels with a camera30. The laser18may be a violet diode laser, and may emit a light beam in a range of wavelengths between 400 and 450 nanometers. Light emitted from the laser18may travel in a first direction towards a first dichroic mirror22, as shown by light propagation arrow19ainFIG. 1. Before reaching the first dichroic mirror22, light from the laser18may first pass through a diffusing element20positioned between the first dichroic mirror20and the laser18. The diffusing element20may be positioned approximately perpendicular to the direction of light propagation from the laser18to increase uniformity of the beam profile of the light from the laser18. Thus, by including the diffusing element20the uniformity of light intensity across the surface area of the array15may be increased. Said another way, the intensity of light received by each binding location16may be relatively the same after passing the laser light through the diffusing element.

Upon reaching the first dichroic mirror22, light from the laser18may be reflected by approximately 90 degrees, and may propagate toward the chip12in a second direction as shown by the light propagation arrow19binFIG. 1. Thus, the first dichroic mirror22may be configured to reflect light from the laser, while allowing other wavelengths of light to pass through. As such, the first dichroic mirror22may only reflect light of wavelengths between a first range of 400 and 450 nm. Said another way, the first dichroic mirror22may be selectively transparent to light of wavelengths longer than 450 nm. Thus, only light with a wavelength greater than 450 nm may pass through the first dichroic mirror22without being reflected. Further, the first dichroic mirror22may be positioned at approximately a 45 degree angle with respect to the laser18, so that light emitted from the laser18is deflected approximately 90 degrees towards the chip12. More specifically, a first surface21of the first dichroic mirror22which faces the laser18, may be orientated at approximately a 45 degree angle with respect to the first direction of light propagation. Thus, the first dichroic mirror22and second mirror24may be orientated to that their first surfaces21and25, respectively are orientated at a 45 degree angle with respect to a light source end602of the laser18from which a laser beam is emitted. Said another way, the laser18may be pointed at the first dichroic mirror22so that light from the laser18may reach the mirror22at an incident angle of approximately 45 degrees. However, in other examples, the mirror22may be angled with respect to the laser18at an angle greater or less than 45 degrees. Further, the first dichroic mirror22and second mirror24may be orientated to that their first surfaces21and25, respectively, are orientated at a 45 degree angle with respect to the lens31of the camera30. Additionally or alternatively, the first dichroic mirror22and second mirror24may be orientated to that their first surfaces21and25, respectively, are orientated at a 45 degree angle with respect to the front wall406and optically clear window608of the cuvette13.

Light from the laser18may reach the chip12, and excite the fluorescent labels (e.g., QNC). Said another way, when exposed to light from the laser18, the fluorescent labels chemically bound to the target biomolecules may emit light back towards the first dichroic mirror22in a third direction which may be approximately opposite the second direction as shown by fluorescent emission light arrows23a. Light emitted from the laser is shown by the solid arrows19aand19b, while light emitted from the array15is shown by dashed arrows23aand23b. Emission light from the fluorescent labels of the array15may be of a different wavelength than the light from the laser18. Specifically, the wavelength of the emission light may be greater than the wavelength of the light emitted from the laser18. In some examples the wavelength of the emission light from the array15may be in a range between 500 and 900 nm.

As described above, the first dichroic mirror22may only reflect light emitted from the laser18. Thus, since the light emitted from the fluorescent labels of the array15may be greater than that from the laser18, the light emitted from the fluorescent labels may pass relatively unobstructed through the first dichroic mirror22and on to a second mirror24. Thus, as shown inFIG. 1, the array15, first dichroic mirror22, and second mirror24may be relatively aligned with one another along a straight line, with the first dichroic mirror22being positioned between the second mirror24and the array15. However, light emitted from the array15may be reflected off a first surface25of the second mirror22. Thus, the second mirror24and first dichroic mirror22may reflect different wavelengths of light. Specifically, the second mirror24may reflect a second range of wavelengths of light that is greater than the first range of wavelengths reflected by the first dichroic mirror22. In some examples, the second mirror24may reflect substantially all wavelengths of light. Thus, the first dichroic mirror22may be configured to reflect electromagnetic waves only with wavelengths up to 450 nm, whereas the second mirror24may be configured to reflect electromagnetic waves up to 1000 nm. Additionally, in some examples, the second mirror24may not reflect light with wavelengths less than 450 nm. In this way, light from the laser18may be reflected by the first dichroic mirror22, and light emitted from the array15may pass through the first dichroic mirror22without being reflected. However, the second mirror22may be configured to reflect light emitted from the array15, and as such, upon reaching the first surface25of the second mirror22, light emitted from the array15may be reflected approximately 90 degrees towards the camera30.

Thus, the second mirror24may be orientated at approximately a 45 degree angle with respect to the array15, in the same or similar orientation as the first dichroic mirror22. Thus, the second mirror24and the first dichroic mirror22may be orientated substantially parallel to one another. In this way, incident light from the array15may be reflected off the first surface25of the second mirror24in a fourth direction, substantially parallel and opposite the first direction as shown by fluorescent emission light arrows23b. Thus light reflected off the second mirror24may propagate substantially parallel to and opposite the direction of propagation of light emitted from the laser18.

Camera30may therefore be aligned approximately parallel to the laser18. Said another way, the camera30and laser18may be positioned on the same side of the first dichroic mirror22and the second mirror24. By including the second mirror24, and positioning the camera30parallel to the laser18, the compactness of the microarray assembly may be increased, and therefore the size of the microarray assembly10may be reduced. In this way, the laser18may emit light in a first direction towards the first dichroic mirror22, and the camera18may receive light emitted from the array15in a fourth direction, the fourth direction opposite the first direction. The camera30may therefore by orientated so that a lens31of the camera30faces the second mirror24. Specifically, the lens31may face the first surface25of the second mirror24at approximately a 45 degree angle.

Thus, the camera30may be orientated parallel with respect to the laser18so that the lens31faces the same direction as the source of light from the laser18. In some examples, the camera30and laser18may be positioned adjacent to one another such that no additional components separate the laser18and camera30. However, in other examples the camera30and laser18may be spaced away from one another. In this way, the parallel arrangement of the laser18and camera30may be referred to herein as an optically folded configuration or arrangement since the light emitted by the laser18, and the light received by the camera30propagate in approximately parallel but opposite directions. Said another way, the camera30, laser18, first dichroic mirror22, and second mirror24may positioned in an optically folded arrangement, so that light emitted from the laser propagates in a parallel and opposite direction to light reflected towards the camera from the second mirror.

Before reaching the camera30, light emitted from the array15and reflected off the second mirror24, may pass through one or more filters. In one example, a bandpass filter26may be positioned between the camera30and the second mirror24. Additionally or alternatively a longpass filter28may be positioned between the camera30and the second mirror24. In examples where both the longpass filter28and the bandpass filter26are included in the assembly10, the bandpass filter26may be positioned more proximate the second mirror24than the longpass filter28. Together, the longpass filter28and the bandpass filter26may only be optically clear to a desired range of wavelengths of electromagnetic waves. In this way, only light in the wavelength range of that emitted from the fluorescent labels may pass through the filters28and26en route to the camera30. As one example, only light with a wavelength of less than 800 nm may pass through the filters28and26. Thus, by including the filters26and28, background noise (e.g., light emitted from the array15not from the fluorescent labels) may be significantly reduced as compared to microarray assemblies not including the filters.

Additionally, in some examples, the filters may be selectable. Thus, the assembly10may be adaptable to multi-color multiplexing detection of several fluorescent labels by providing multiple filters that may be selectable to filter different wavelengths of light based on the desired fluorescent label. As such, the camera30may be configured to independently detect two or more signals from multiple fluorescent labels of different colors.

Although in the preferred embodiment of the microarray assembly10described above, second mirror24is included in the assembly10, it should also be appreciated that in other embodiments, the microarray assembly10may not include second mirror24. In such embodiments where second mirror24is not included in the assembly10, the camera30may be positioned so that the lens31directly faces the array15. Said another way, the lens31may be positioned perpendicular to the direction of light propagation from the array15, so that light emitted from the array15may pass through the lens31and be captured in an image by the camera30. Thus, the camera30may be positioned perpendicular to the laser18, so that light emitted from the array15may be received and imaged by the camera30, after the light passes through the first dichroic mirror22. Therefore, in examples where mirror24is not included in the assembly, light emitted by the array15may pass in a relatively straight line to the lens31of the camera, and may not be reflected by any mirror en route to the camera30. Further, the filters26and28may be positioned between the first dichroic mirror22and the lens31of the camera30in examples where the camera30is positioned perpendicular to the laser18and second mirror24is not included in the assembly10. Thus, the filters26and28, lens31, and camera30, may be aligned antiparallel to emission light arrows23afor capturing light emitted from the array15. Said another way, array15, first dichroic mirror22, filters26and28, lens31, and camera30may all be aligned with one another in a substantially straight line perpendicular to the first direction shown by light propagation arrows19a.

In this way, the lens31may gather light produced from the fluorescent labels, and the camera30may capture an image of the array15. The camera30may be a digital camera, configured to acquire digital images of the array15and the spots16positioned therein. Specifically, light emitted from the array15in response to excitation from the laser18may be captured by the camera30. The resolution of the image of the array15may depend on the distance of the camera30from the array15, the focal length of the lens31, the magnification of the camera30, and the surface area of the array15. In some examples, the camera30may be a 5 megapixel camera. However, in another example, the camera30may be a 3 megapixel camera. In still further examples, the megapixel of the camera30may be a range between 2 and 7 megapixels. The focal length of the lens31may be one of 30, 25, 17.5, or 12 mm. However, in other examples the focal length of the lens31may be a range between 5 and 30 mm. A width of the array15may in some examples be 25 mm. However, in other examples, the array15may have a width of 12.5 mm. In still further examples, the width of the array may be approximately 6.25 mm. Further, the width of the array15may be a range between 5 and 30 mm. The optical path length between the array15and the camera30may be approximately 120 mm. However, in other examples, the optical path length between the array15and the camera30may be greater or less than 120 mm. Thus, the resolution of the images captured by the camera30may be in a range between 2 and 10 microns. Specifically, by reducing the size of the array15to a width of approximately 12.5 mm, and configuring the camera30with a magnification of 0.45 and lens focal length of 25 mm, the resolution of the image may be improved to 4.9 microns.

Images captured by the camera30, may in some examples be transferred to a computer122for analyzation thereof. Computer122may be any computing device configured to access a network including but not limited to a server, personal computer, laptop, a smartphone, a tablet, and the like. In some examples, the microarray assembly10may be electrically coupled to the computer122via a wired connection such as a USB port.

Computer122may include a logic subsystem123and a data-holding subsystem124. Computer122may additionally include a display subsystem125, communication subsystem126, and/or other components not shown inFIG. 1. For example, computer122may also optionally include user input devices such as keyboards, mice, game controllers, cameras, microphones, and/or touch screens.

Logic subsystem123may include one or more physical devices configured to execute one or more instructions. For example, logic subsystem123may be configured to execute one or more instructions that are part of one or more applications, services, programs, routines, libraries, objects, components, data structures, or other logical constructs. Such instructions may be implemented to perform a task, implement a data type, transform the state of one or more devices, or otherwise arrive at a desired result.

Logic subsystem123may include one or more processors that are configured to execute software instructions. Additionally or alternatively, the logic subsystem123may include one or more hardware or firmware logic machines configured to execute hardware or firmware instructions. Processors of the logic subsystem123may be single or multi-core, and the programs executed thereon may be configured for parallel or distributed processing. The logic subsystem123may optionally include individual components that are distributed throughout two or more devices, which may be remotely located and/or configured for coordinated processing. One or more aspects of the logic subsystem123may be virtualized and executed by remotely accessible networking computing devices configured in a cloud computing configuration.

Data-holding subsystem124may include one or more physical, non-transitory devices configured to hold data and/or instructions executable by the logic subsystem123to implement the herein described methods and processes. When such methods and processes are implemented, the state of data-holding subsystem124may be transformed (for example, to hold different data). For example, the data holding subsystem124may be configured to store images from the camera30. The images may be modified, and/or analyzed based on instructions stored in the logic sub system123.

Data-holding subsystem124may include removable media and/or built-in devices. Data-holding subsystem124may include optical memory (for example, CD, DVD, HD-DVD, Blu-Ray Disc, etc.), and/or magnetic memory devices (for example, hard drive disk, floppy disk drive, tape drive, MRAM, etc.), and the like. Data-holding subsystem124may include devices with one or more of the following characteristics: volatile, nonvolatile, dynamic, static, read/write, read-only, random access, sequential access, location addressable, file addressable, and content addressable. In some embodiments, logic subsystem123and data-holding subsystem124may be integrated into one or more common devices, such as an application-specific integrated circuit or a system on a chip. Together, the logic subsystem123and data-holding subsystem124may be configured to store images captured from the camera30, and analyze the images to determine biomolecule expression levels in each of the target binding location16of the array15.

Thus, the logic subsystem123may include one or more algorithms for processing and analyzing data received from the camera30. Thus, in order to compare the intensity of different wavelengths of light emitted from the different binding locations16, the logic subsystem123may include one or more algorithms or software for image analyzation. One or more of a combination of different algorithms for data normalization and statistical techniques, such as artificial neural networks, multivariate statistics and tree algorithms may be stored in the logic subsystem123for analyzing the images captured by the camera30to detect for and/or quantify biomolecule expression levels in the sample based on relative intensities of different wavelengths of light. In this way, in examples where the microarray assembly10is configured as a protein microarray, light received from the binding locations16and captured by the camera30may be compared and analyzed to detect for biomarkers of infectious diseases. However, it should be appreciated that the images may be analyzed to detect for and/or quantify gene expression levels, oligonucleotides, antibodies, antigens, etc.

When included, display subsystem125may be used to present a visual representation of data held by data-holding subsystem124. As the herein described methods and processes change the data held by the data-holding subsystem124, and thus transform the state of the data-holding subsystem124, the state of display subsystem125may likewise be transformed to visually represent changes in the underlying data. For example, the images captured by the camera30may be displayed to a user via the display subsystem125. Further, modifications to and/or analysis of the images may be displayed to the user via the display subsystem125. Display subsystem125may include one or more display devices utilizing virtually any type of technology. Such display devices may be combined with logic subsystem123and/or data-holding subsystem124in a shared enclosure, or such display devices may be peripheral display devices.

When included, communication subsystem126may be configured to communicatively couple user device122with one or more other computing devices, such as controller34of the microarray assembly10. Thus, in some examples, the microarray assembly10may be electrically coupled to the computer122by a wired connection as shown by the dotted line inFIG. 1. However, in other examples, the microarray assembly10may be wirelessly coupled to the computer122via any suitable wireless connection for data transfer such as Wifi or Bluetooth. Thus images taken from the camera30may be transferred to the computer122via either a wired electrical connection and/or a wireless connection. However, in other examples, the microarray assembly10may include a memory chip33which may be removably coupled to assembly10for storing images captured by the camera10. The memory chip33may be any suitable memory storage device such as a USB memory stick, SD card, micro-SD card, etc. Images may be transferred from the memory chip33onto the computer122by removing the memory chip33from the microarray assembly, and inserting the memory chip33into the computer122.

Communication subsystem126may include wired and/or wireless communication devices compatible with one or more different communication protocols. As non-limiting examples, communication subsystem126may be configured for communication via a wireless telephone network, a wireless local area network, a wired local area network, a wireless wide area network, a wired wide area network, etc. In some embodiments, communication subsystem126may allow computer122to send and/or receive messages to and/or from other devices, controller34, via a network such as the public Internet.

Although images from the camera30may be transferred to the computer122for analysis, it should be appreciated that in other embodiments, the microarray assembly10may perform image analysis on its own. For example, the controller34of the microarray assembly10may include computer readable instructions for analysis of the images captured by the camera30. Thus, the controller34may be configured and may function the same or similar as the logic subsystem123and data-holding subsystem124of the computer122described above. Said another way, logic subsystem123and data-holding subsystem124may be included in the controller34of the microarray assembly10for processing and analyzation of the images captured by the camera30. In such examples, the microarray assembly10may not be electrically coupled to the computer122. Further, in examples where image analysis is performed by the microarray assembly10, the microarray assembly may further include a display (shown below with reference toFIG. 3) such as display subsystem125.

Controller34may be configured to operate one or more of the laser18and camera30for capturing images of the array15. In some examples, the controller34may be configured to receive input from a user via either the computer122or a display screen or button pad located on the microarray assembly10. Thus, a user may send signals to the controller34via either the computer122or through a display screen or button pad located on the microarray assembly10. Based on signals received from the user, the controller34may send signals to the laser18for emitting light, and/or to the camera30for capturing an image of the array15.

Power to the controller34, laser18, and camera30may be provided by the computer122in examples where the microarray assembly10is coupled to the computer122via a wired connection. However, in other examples, the microarray assembly10may include its own battery32for powering the various components of the microarray assembly10. The battery32may be any suitable battery such as lithium-ion, lead-acid, solid polymer electrolyte, molten salt, etc. The battery32may be a rechargeable battery, and may be charged by a wired electrical connection to computer122, or other electrical source such as an electrical socket. The battery32may provide electrical power to the laser18, where the current of said electrical power may be any current level within a range of currents between 1-2 amps. In still further examples, the microarray assembly10may include its own power cord for receiving power from an electrical socket.

Although in the example ofFIG. 1, microarray assembly10is described as being capable of imaging and/or analyzing microarray assays, it should also be appreciated that other types of assays may be imaged and/or analyzed using the microarray assembly10. For example, the microarray assembly10may be configured to image and/or analyze colorimetric assays. In such examples, the microarray assembly10may include a white light source in place of laser18. Thus, a source of white light may be used to illuminate the array15instead of the laser18. Further, the first dichroic mirror22may be replaced with a beam splitter capable of reflecting only a portion of the white light incident on it from the white light source to the array15. The orientation and position of the white light source within the assembly10may be the same or similar to the laser18. Similarly, the orientation and position of the beam splitter may be the same or similar to first dichroic mirror22. In this way, white light from the white light source may be reflected onto the array15by the beam splitter. Based on the chromophores of the biomolecules in the assay, the biomolecules may absorb certain wavelengths of the white light, while reflecting others.

The colors reflected by the chromophores may determine the wavelengths of light captured in the image of the array by the camera30. The colors captured by the camera may also be adjusted by various filters, such as filters26and28. For example, one or more filters may be utilized to exclude certain wavelengths of light reflected from the array, and may only allow a desired range of wavelengths to pass through to the camera30for image acquisition. En route to the filters, at least a portion of the light reflected from the array15may pass through the beam splitter, and on to the second mirror24, since the beam splitter may only reflect a portion of incident light, and may allow the remaining portion of incident light not reflected, to pass there-through. In this way, by allowing light to be both reflected and transmitted, the beam splitter may serve to reflect at least a portion of the white light emitted by the white light source to the array15, for illuminating the biomolecules in the assay. Additionally, the beam splitter may serve to allow at least a portion of the light reflected by the array15, to pass through the beam splitter and on to the filters and camera30for imaging of the array15.

Turning now toFIGS. 2-6they show schematics of the biomolecule microarray assembly10which may be used to image and visualize the presence and/or amount of biomolecules in a sample collected on the microarray chip12.FIGS. 2-6show three-dimensional schematics of the microarray assembly10. Further, the relative sizes and positions of the components included in the microarray assembly10are shown inFIGS. 2-6. Thus,FIGS. 2-6may be drawn approximately to scale. However, it should be appreciated that in other examples, the relative sizing and positioning of components in the microarray assembly10may be different than depicted inFIGS. 2-6. SinceFIGS. 2-6all show schematics of the microarray assembly10, theFIGS. 2-6may be described together in the description herein. Thus, components of the microarray assembly10introduced in the description of any one ofFIGS. 2-6, may not be reintroduced or described again. Further components of the microarray assembly10already introduced inFIG. 1may not be discussed again in the description ofFIGS. 2-6herein.

The microarray assembly10, as shown inFIGS. 2-6, may comprise six or more walls, defining a housing201of the microarray assembly10. Each of the walls may include an interior face (proximate to interior components of the microarray assembly10) and an exterior (or outside) face, the exterior face visible to a user. Specifically, the microarray assembly10may comprise a front wall202with a front exterior face opposite from a back wall204. Further, the assembly10may comprise a first side wall206opposite from a second side wall208, and a top wall (e.g., top face or surface)110opposite from a bottom wall (e.g., bottom face or surface)212. The top wall210may comprise multiple components such as a door214for receiving the microarray chip12containing the sample to be analyzed. Further, in some examples as shown inFIG. 3the top wall110may include a display screen302and/or a button pad304for receiving input from a user and/or for providing feedback to the user.

Focusing now onFIG. 2, it shows an exterior perspective view200of a first embodiment of the microarray assembly10shown above inFIG. 1. Specifically, the perspective view200and shown inFIG. 2, may be an axonometric projection of the microarray assembly10, showing the assembly10as viewed from a skew direction in order to reveal more than one side of the assembly10. Further, it should be appreciated that all perspective views of the assembly10shown herein (e.g., perspective views300,400, and500shown below with reference toFIGS. 3, 4, and 5, respectively) may also be axonometric projections of the microarray assembly10. In the embodiment shown inFIG. 2, the microarray assembly10may be configured as an image capturing device only. Thus, the microarray assembly10as shown inFIG. 1may be configured to capture images of the array15(not shown inFIG. 2), and may not be configured to analyze the images.

The microarray assembly10may be shaped as a rectangular prism as shown inFIG. 1. However, it should be appreciated that other shapes and/or sizes of the microarray assembly10are possible. The microarray assembly10may include a door214positioned on the top wall110for receiving the chip12and/or cuvette13(not shown inFIG. 2). Further, the microarray assembly10may include a USB port216and/or a power switch218on the front wall202. However, it should be appreciated that one or more of the USB port216and power switch218could be located at other positions on the microarray assembly10such as on any of the other walls204,206,208,210, and212. The USB port216may be configured to electrically couple the microarray assembly10to a computer (e.g., computer122shown inFIG. 1) for transferring images captured by the camera (not shown inFIG. 2) to the computer for analysis thereof. Additionally, the microarray assembly10may draw power from the computer or a wall socket through the USB port216. Thus, the battery32may be charged through a wired electrical connection provided by the USB port216to any suitable power source such as the computer, electrical socket, vehicle power outlet, etc. Further, the power switch218may be adjusted by a user to power on and power off the microarray assembly10. The power switch218may be any suitable digital or mechanical two-way switch.

Moving on toFIG. 3, it shows an exterior perspective view300of a second embodiment of the microarray assembly10including a display screen302and/or button pad304. Specifically, in the embodiment shown inFIG. 3, the microarray assembly10may be configured to both capture and analyze images taken of the array15(not shown inFIG. 3). Thus, in the example shown inFIG. 3, the microarray assembly10may be configured to display results from an analysis of the array15(shown above inFIG. 1) via the display screen, where the results may include identification and/or detection of a disease, antibody expression levels, biomolecule expression levels, gene quantification, etc. As such, the microarray assembly10in the example ofFIG. 3may provide an infectious disease diagnosis.

In order to receive input from a user, and display results from the image analysis to the user, the microarray assembly10may include the display screen302and/or button pad304. As shown in the example ofFIG. 3, the display screen302and/or button pad304may be positioned on the top wall210of the microarray assembly10. However, it should be appreciated that in other examples, the display screen302and/or button pad304may be positioned elsewhere on the assembly10, such as any of the other walls202,204,206,208, and212. The display screen302may be any suitable display screen such as an LCD, CRT, flat panel display, plasma, etc. In some examples, the display screen302may additionally comprise a touch display. Further, the button pad304may be included with a plurality of buttons306. The button306may allow a user to toggle through options displayed on the display screen302. Thus, the button pad304may allow the user to interact with and manipulate the display screen302.

Results from analysis of an image captured of the array15may be displayed on the display screen302. As described above, the results from the analysis of an image of the array15may include a quantification of biomolecule expression levels. Thus, a concentration and/or amount of one or more antibodies, proteins, genes, etc. may be presented on the display screen302. Since an infectious disease diagnosis may be inferred from antibody expression levels, an infectious disease diagnosis may additionally be presented on the display screen302.

Thus, in the embodiment of the microarray assembly10shown inFIG. 3, the assembly10may be configured as a “one step” analyzer that upon input from the user via the display screen302and/or button pad304, may both image the array15, and analyze the the image by employing a method, such as the example method provided below with reference toFIG. 8. Specifically, a controller of the microarray assembly10(e.g., controller34shown inFIG. 1) may include computer readable instructions for executing a method, such as the method described below with reference toFIG. 8, to image the array15and/or analyze an image of the array15, to provide an indication of biomolecule expression levels in the array15.

In other words, in some examples, the microarray assembly10may be configured to perform both image acquisition and image analysis. By conducting both the image acquisition of the array15, and analysis thereof, the microarray assembly10in the embodiment shown in FIG.3, may determine concentrations and/or amounts of the target biomolecules (e.g., protein concentrations) in the array15. These results may be displayed to the user via the display screen302. Further, evidence of disease may be inferred and displayed to the user via the display screen302based on the concentrations of the target biomolecules. In this way, infectious disease diagnosis and identification may be performed by the microarray assembly10in certain embodiments.

Turning now toFIG. 4, it shows an external, exploded, perspective view400of a third embodiment of the microarray assembly10including the chip12and cuvette13. Specifically,FIG. 4, shows the chip12and cuvette13detached from the microarray assembly10prior to insertion of the chip12and cuvette13into the assembly10.

The cuvette13may comprise six or more walls, for enclosing the chip12, when the chip12is inserted into the cuvette13. Each of the walls may include an interior face (proximate to interior components of the microarray assembly10) and an exterior (or outside) face, the exterior face visible to a user. Specifically, the cuvette13may comprise a front wall406with a front exterior face opposite from a back wall408. Further, the cuvette13may comprise a first side wall410opposite from a second side wall412, and a top wall (e.g., top face or surface)416opposite from a bottom wall (e.g., bottom face or surface)414. The top wall416may include an aperture418for receiving the chip12. Thus, the chip12may be inserted into the cuvette13through the aperture418. The walls406,408,410,412,414, and416of the cuvette13may be fluidically sealed along their edges, so that gasses, liquids, and/or solids may only enter or exit the cuvette13through the aperture418. Specifically, the aperture418may include a mating groove420which may be configured to receive the chip12. The groove420may be centered on the cuvette13, for holding and retaining the chip12. The edges of the chip12, may fit into the groove420, for inhibiting movement of the chip12relative to the cuvette13.

In this way, the cuvette13may hold and retain the chip12at approximately the center of the optical beam produced by the laser18(shown above inFIG. 1). Further, the cuvette13may hold the chip12perpendicular to the optical beam produced by the laser18when inserted into the microarray assembly10. The cuvette13may be constructed from any suitable material or combination of materials such as plastic, glass, acrylic, and stainless steel.

The door214of the microarray assembly may be adjustable between an open position to allow one or more of the chip12and cuvette13to be inserted into the assembly10, and a closed position. Door214may be adjusted to a closed position during imaging of the chip12. Specifically, when the door214is closed, the door214may be in sealing contact with the housing201of the assembly10, so that substantially no light may escape from within the assembly10to outside the assembly10. Thus, the door214may optically seal the interior and exterior of the assembly10. In this way, laser light may not escape from the assembly10, and light from exterior the assembly10may not pass into the assembly10. As such, background noise may be reduced when imaging the array15.

Further, the door214may include an electronic interlock which may enable the laser to be powered on, only when the door214of the microarray assembly is closed. In one example, as shown inFIG. 4, the electronic interlock may include a conductive rubber seal404positioned on an interior surface of the door214. Further the interlock may include one or more metallic (e.g., stainless steel) pins402positioned on the assembly10. When the door214is open, as shown inFIG. 4, the rubber seal404and pins402may not be in contact with one another, and as such power to the laser18may be inhibited. However, when the door214is closed, the rubber seal404may physically contact all of the pins402, forming a complete series circuit. As such, power to the laser18may be enabled when the door214is closed, and the circuit of the electronic interlock is closed.

Turning now toFIG. 5, it shows an external, exploded, perspective view500of the microarray assembly10including the chip12and cuvette13, where the cuvette13has been inserted into the assembly10through the door214. Specifically,FIG. 5, shows only the chip12detached from the microarray assembly10. Thus, as shown inFIG. 5, the cuvette13may be inserted into the assembly10prior to insertion of the chip12into the cuvette13and assembly10. However, in other examples, it should be appreciated that the chip12may be inserted into the cuvette13, prior to insertion of the cuvette13into the assembly10.

In this way, the cuvette13may be removably coupled to the assembly10. As shown above inFIG. 4, the cuvette13may be decoupled from the assembly10, and as shown inFIG. 5, the cuvette may be inserted through the door214and recoupled to the assembly10. The cuvette13therefore, may be inserted into the assembly10, and then the chip12may be inserted into the cuvette13. In this way, the cuvette13may retain and position the chip12within the assembly10. Cutting plane502defines a cross section of the assembly10shown below inFIG. 6.

Moving on toFIG. 6, it shows a cross-sectional view600of the microarray assembly10taken along cutting plane502shown above inFIG. 5. Thus internal components of the microarray assembly10are exposed inFIG. 6. As such,FIG. 6, shows the relative positioning and orientation of the different components of the microarray assembly10with respect to one another. For example,FIG. 6shows how the camera30and laser18, may be positioned substantially parallel to one another. In this way, the camera30may be orientated so that the lens31points in the same direction as a light source end602of the laser18from which a laser beam is emitted from the laser18.

Further, an axis system605is included inFIG. 6. Axis system605includes a horizontal axis604and a vertical axis606. The axis system605may be used to describe the relative positioning of components of the microarray assembly10. Components described as vertically above, may be positioned vertically above relative to the vertical axis606. Thus, a first component described as vertically above a second component, may be positioned at a greater positive vertical point along axis606than the second component. Similarly components described as to the “right” or left” may be positioned to the right or left along the horizontal axis604.

The laser18may be positioned vertically above the camera30. However, the first dichroic mirror22may be positioned horizontally in line with the laser18. Further, the second mirror24may be positioned horizontally in line with the camera30. The first dichroic mirror22, second mirror, and array15may be positioned vertically in line with one another. Thus, the first dichroic mirror22, second mirror24, and array15, may be aligned along a line parallel to the vertical axis606. Further, the first dichroic mirror22, second mirror24, and cuvette13including the array15, may be horizontally offset from the laser18and the camera30.

The cuvette13may include an optically clear window608positioned on the front wall406of the cuvette13. The cuvette13may be orientated approximately perpendicularly to the laser18, so that the front wall406including the window608faces a direction perpendicular to the direction of propagation of light emitted from the laser18before it reaches the first dichroic mirror22. Said another way, the laser may be orientated so that the light source end602points towards the first dichroic mirror22in a first direction. The window608and wall406may face the first dichroic mirror22in a second direction, the second direction perpendicular to the first direction. The window608may allow light to pass relatively unobstructed through the cuvette13. In this way, the laser beam reflected off the first dichroic mirror may pass through the window608to the array15(not shown inFIG. 6). Further, light emitted from the fluorescent labels of the array15may propagate out from the cuvette13through the window608back towards the first dichroic mirror22and the second mirror24. As such, the cuvette13may be orientated in the assembly10, so that the window608faces the first dichroic mirror22and the second mirror24. The first dichroic mirror and second mirror24may be orientated at approximately a 45 degree angle with respect to the front wall406and window608of the cuvette13. Further, the cuvette13, may be orientated approximately perpendicular to the laser18and camera30, so that light propagating from the array15out through the window608, may travel in a direction perpendicular to the direction of light emitted from the laser18before it reaches the first dichroic mirror22.

As shown inFIG. 6, the cuvette13may be positioned so that it is centered with and horizontally aligned with the first dichroic mirror. Further since the array15and chip12, may be centered within the cuvette13, the cuvette13may center the array15and chip12on a light beam produced by the laser18and reflected off the first dichroic mirror22. In this way, the optically clear window and array15may be centered on the light beam produced by the laser18. Further, the optically clear window608may be sized to permit relatively uniform light intensity dispersion across the surface area of the array15.

Thus, the first dichroic mirror22and the second mirror24may be positioned at approximately a 45 degree angle relative to the laser18, camera30, and cuvette13including the chip12and array15. Further, the cuvette13may be positioned perpendicular to the laser18and the camera30so that light passing in and out of the cuvette13may travel in a direction substantially perpendicular to the light passing between the laser18and the first dichroic mirror22and between the second mirror24and the camera30. As such, light from the laser18may be reflected approximately 90 degrees towards the cuvette13including the array15. Light emitted from the fluorescent labels of the array15may pass through the window608and first dichroic mirror22en route to the second mirror24, where the light may be reflected approximately 90 degrees towards the camera30by the second mirror24. Thus, light reflected from the second mirror24may travel opposite the direction of light emitted from the laser18towards the first dichroic mirror22. In this way, the laser18and camera30may be orientated parallel to one another so that the light source end602of the laser18from which light propagates faces the same direction as the lens31of the camera30.

Continuing toFIG. 7, it shows a perspective view700of the cuvette13and chip12prior to insertion of the chip12into the cuvette13. In some examples, the cuvette13may be constructed from a suitable plastic material. However, in other examples, the cuvette13may be constructed from a plastic and glass. Specifically the optically clear window608, which may be positioned on wall406may be comprised of glass, while the rest of the cuvette13may be constructed from plastic. In some examples, the optically clear window608may form all or a portion of wall406, and as such may be integrally formed within the cuvette13. However, in other examples, the window608may be formed separately from the cuvette13and may be attached to the cuvette13. The glass that comprises the window608may be constructed from any suitable materials such as optically-clear glass, fused quartz, Aclar, and Topaz. Additionally or alternatively, other materials such as polystyrene and acrylic may be used in constructing the window608.

All of the walls406,408,410,412,414, and416, may be fluidically sealed at their edges, to prevent gasses, liquids, or solids from entering the cuvette13other than through the aperture418. As shown inFIG. 7the groove420for accepting and retaining the chip12, may be centered between walls front wall406and back wall408. Further the aperture418and groove420may be centered between the side walls412and410. In this way, the chip12may be centered along a central axis of the cuvette13. The chip12may be removably coupled to the cuvette13via the groove420. Thus the chip12may be inserted and removed from the cuvette13for cleaning and/or sterilization.

The nitrocellulose film14containing the array15(not shown inFIG. 7) may be positioned near a bottom of the chip12, so that when the chip12is inserted into the cuvette13, the film14is positioned more proximate the bottom wall414than the top wall416of the cuvette13. The window608may be sized to permit light entering the cuvette13to reach the entire surface area of the film14. As such, the size of the window608may be constructed based on the size of the film14and array15.

FIG. 8shows a flow chart of a method800for imaging an array (e.g., array15shown inFIG. 1) and analyzing images taken of the array to determine biomolecule expression levels in the array of a microarray assembly (e.g., microarray assembly10shown inFIGS. 1-6). The array may comprise a plurality of binding locations (e.g., binding locations16shown inFIG. 1) for binding biomolecules of a sample to the array. The array may be positioned on a nitrocellulose film (e.g., nitrocellulose film14shown inFIGS. 1 and 7), the nitrocellulose film forming a coating on a microarray chip (e.g., microarray chip12shown inFIGS. 1, and 4-7). Once biomolecules from the sample have been bound to the binding locations, the chip may be inserted into a chip cover (e.g., cuvette13shown inFIGS. 1, and 4-7) before being inserted into the microarray assembly. Once secured in the assembly, a laser (e.g., laser18shown inFIGS. 1 and 6) may be powered on to excite fluorescent labels chemically bound to a portion of the biomolecules on the binding locations. Specifically, a controller (e.g., controller34shown inFIGS. 1 and 6) may be in communication with the laser for adjusting operation of the laser. In response to excitation light from the laser, the fluorescent labels may emit light which may be captured in an image by a camera (e.g., camera30shown inFIGS. 1 and 6). The controller may send signals to the camera for capturing images of the array. In this way, the controller34may include computer readable instructions for executing a method such as method800. As such, method800may be executed by the controller34based on input from a user via one or more of a computer (e.g., computer122shown inFIG. 1), display on the microarray assembly (e.g., display302shown inFIG. 3), or button pad on the microarray assembly (e.g., button pad304shown inFIG. 3).

In some examples, where the microarray assembly is configured as an image capturing device only, such as in the embodiment described above with reference toFIG. 2, the image acquired by the microarray assembly may be analyzed to determine biomolecule expression levels by a source external to the microarray assembly such as the computer. However, in other examples, where the microarray assembly is configured as both an image capturing and analysis device, such as in the embodiment described above with reference toFIG. 3, the controller of the microarray assembly may include computer readable instructions executable to both image the array, and analyze the image to determine biomolecule levels in the array.

Method800begins at802which comprises receiving user input. As described above, user input may be received by the controller from a touch display or button pad included on the microarray assembly. However, in other examples the user input may be received from a computer in communication with the controller of the microarray assembly via either a wired or wireless connection. The user input may include commands for one or more of: powering on or off the laser, taking a picture with the camera, and selecting one or more filters (e.g., filter26and28shown inFIGS. 1 and 6) for filtering pictures captured by the camera.

Method800may then continue to804which comprises determining if the chip is inserted into the microarray assembly. In one example, it may be determined that the chip is inserted if a door (e.g., door214shown inFIGS. 2-5) through which the chip is inserted into the assembly is closed. The position of the door may be determined based on a voltage and/or current output from a circuit included on an interior surface of the door. Thus, the current in the circuit may change depending on the position of the door. Specifically the current in the circuit may increase when the door is in a closed position relative to when the door is in an open position.

In another example, the method at804may comprise determining if the chip is inserted based on a state of a mechanical or electrical switch of the assembly. In some examples a bottom of the chip and/or chip cover may interface with the switch of the assembly, to transform the state of the switch. Thus, the state of the switch may change depending on whether the chip and/or cover is inserted into the assembly.

If it is determined that the door is open and/or that the chip is not inserted into the assembly, then method800may proceed to806which comprises not powering on the laser. In this way, the laser may only be powered on when the chip is inserted into the assembly, and the door of the assembly is closed to prevent light from escaping or entering the assembly during imaging of the array. Method800then returns.

However, if it is determined that the door is closed and/or that the chip is inserted into the assembly, then method800may continue to808which comprises turning on the laser for a duration and directing light emitted from the laser in a first direction towards a first dichroic mirror (e.g., first dichroic mirror22shown inFIGS. 1 and 6). The first direction may be a direction away from the laser and towards the first dichroic mirror (e.g., first direction shown by light propagation arrow19ainFIG. 1). In some examples, the duration may be an amount of time. The duration may be a pre-set value stored in the memory of the controller. However, in other examples the duration may be adjustable based on input from the user. Thus, the user may adjust the amount of time that the laser is powered on. In still further examples, the duration may be based on the configuration of the assembly, such as the wavelength of light produced by the laser, intensity of the light beam produced by the laser, distance between the laser and the array, distance of the laser to a diffusing element (e.g., diffusing element20shown inFIGS. 1 and 6), type or wavelength of the fluorophore used in the probe molecules to fluorescently tag the target biomolecules, resolution and/or sensitivity of the camera, concentration or amount of biomolecules expressed or fluorescently tagged on the microarray chip, etc. Light emitted from the laser may propagate in a first direction towards the first dichroic mirror orientated at approximately a 45 degree angle to the direction of light propagation from the laser.

Method800then continues to810which comprises reflecting the light emitted from the laser towards the chip off the first dichroic and exciting the fluorescent labels in the array. Thus, in some examples, the method at810may comprise reflecting light approximately 90 degrees off the first dichroic mirror towards the array. Thus, the light traveling in the first direction may be reflected approximately 90 degrees so that after reflection it propagates in a second direction towards the array, the second direction perpendicular the first direction. For example, the second direction may be the same or similar to second direction shown above with reference toFIG. 1by light propagation arrows19b.

After exciting the fluorescent labels at810, method800may then proceed to812, which comprises reflecting light emitted from the array, specifically the fluorescent labels, off a second mirror (e.g., second mirror24shown inFIGS. 1 and 6) in a fourth direction (e.g., fourth direction shown by light propagation arrow23binFIG. 1) towards the camera. Light emitted from the array may be emitted in a third direction, the third direction being parallel but opposite the second direction and perpendicular to the first and fourth directions. For example, the third direction may be the same or similar to the third direction shown inFIG. 1by emission light arrows23a. In some examples the fourth direction may be opposite the first direction of light emitted from the laser towards the first dichroic mirror. Said another way, the fourth direction may be 180 degrees opposite the first direction. Thus, the method at810may include reflecting the light emitted from the array approximately 90 degrees towards the camera.

Light reflected off the second mirror may then be captured at814. Thus, the method at814may comprise capturing an image of the array with the camera. Specifically, the controller may send signals to the camera for capturing an image of the array. In some examples, the camera may capture more than one image. Camera settings may in some example be pre-set, or in other examples, may be adjustable based on user input. The camera settings may include: a number of images to capture, filters, exposure duration, focal length of a lens (e.g., lens31shown inFIGS. 1 and 6), f-stop setting of the lens, camera gain, frame transfer speed, timing of the image acquisition relative to powering on of the laser, etc.

Additionally, digital information corresponding to the image may be acquired at814. Specifically information corresponding to a location of each of a number of spots (e.g., spots16shown inFIG. 1) of the array may be acquired at814. For example, a spot-by-spot delimited list of each spot's column and row placement in the assay, its name, ID, and its block location if the microarray is divided into individual print blocks may be acquired at814. Further, the method800at814may comprise identifying images of the array that are rotated, inverted, and/or reversed. For example, the method800at814may comprise determining an angle of rotation of the image of the assay, which may be any angle between 0 and 360 degrees, and adjusting the orientation of the image so that it aligns with pre-set orientation conditions. Thus, at814, the image acquired may be one or more of rotated and/or flipped (e.g., reflected across an axis) so that the orientation of the image acquired at814matches a pre-set orientation. Said another, during each successive iterations of method800(e.g., during multiple image acquisitions), each of the images of the one or more assays may be adjusted so that their orientation relative to one another is approximately the same.

One or more of the images taken at814may then be stored at816. In some examples the images may be stored in non-transitory memory of the assembly such as on the controller and/or a storage device (e.g., memory chip33shown inFIG. 1). Additionally or alternatively, the method at816may comprise transferring the images taken at814to the computer for storage therein. In still further examples, the images may be stored in non-transitory memory of any suitable device for storing digital images such as a memory chip or card, flash drive, etc. Specifically information corresponding to the location of each of the spots of the array may be stored at816. For example, the spot-by-spot delimited list of each spot's column and row placement in the assay, its name, ID, and its block location if the microarray is divided into individual print blocks may be stored at816.

Thus, after storing the images at816, the image capturing of the array may be complete. Said another way, the steps in method800up to and/or including816may be executed to capture and store an image of the array. As described above, in examples where the microarray assembly is configured as an image capturing device only, method800may return after storing the images at816. Thus, in some examples, method800may return after storing the images at816.

However, in still further examples, where the microarray assembly is configured as an image capturing device only, one or more images stored at816may be transferred to a source external to the microarray assembly, such as the computer for analysis to determine biomolecule levels in the array. The images may be transferred via a direct electrical connection between the microarray assembly and the external source, and/or may be transferred via the storage device. For example, a memory chip (e.g., memory chip33shown inFIG. 1) may be removed from the microarray assembly, and inserted into the computer or other external source, and the images contained on the memory chip may be uploaded to the external source. In yet further examples, the images may be transferred via a wireless connection such as Bluetooth, Wifi, or other electromagnetic wave frequency suitable for wirelessly transmitting data packets containing image data.

In examples, where the microarray assembly is configured as both an image capturing device and an image analysis device, such as in the embodiment described above with reference toFIG. 3, the controller of the microarray assembly may perform the analysis of the one or more images stored at816to determine biomolecule levels in the array.

Thus, in some examples, method800may continue from816to optional steps818and820which may be executed to perform the analysis of the one or more images stored at816. It should be appreciated that in examples where the microarray assembly is configured as an image capturing device only,818and820may be executed by a source external to the microarray assembly (e.g., computer122shown inFIG. 1). Specifically computer readable instructions may be stored non-transitory memory (e.g., data-holding subsystem124shown inFIG. 1) of the external source, where the instructions may be executable by a controller (e.g., logic subsystem123shown inFIG. 1) of the external source to perform a method such as818and820of method800.

However, in other examples, where the microarray assembly is configured as both an image capturing and image analysis device,818and820may be executed by the controller of the microarray assembly.

Thus, method800may continue to optional step818which comprises determining the spot locations after storing the images at816. Specifically, the spot location analysis performed at818may comprise identifying the location of each of the spots on the assay, including the biomolecules. The biomolecule expression levels at each spot may then be subsequently determined based on a wavelength analysis at820at described below. The spot location determination process may begin by acquiring the spot-by-spot delimited list of each spot's column and row placement in the assay, its name, ID, and its block location if the microarray is divided into individual print blocks. Information about the spot may be stored during image acquisition at814and image storage at816as described above. Based on an origin, a series of field points where each spot is expected to be located, may be determined.

Identification of the origin of the array (e.g., array15shown inFIG. 1) can be accomplished using an image recognition method. In the first method, a set of fiducial spots are printed onto a first row of binding locations (e.g., binding locations16shown inFIG. 1) of the array to serve as an image-recognition and alignment feature. The fiducial spots may be a pattern of spots separated by blanks, dilution series, or any other features that fluoresce. An image may be created of a first row of spots and that portion of the image may be saved as a recognition template. This template can be re-used for any microarray that has been printed with the same pattern of fiducials appearing in the first row. With this template as a guide, the corresponding pattern in each image the may be located and used in that subset of the image to identify the spot located at the origin, which may be the first row and column in the array. Once the origin is identified, the projected field points for all spots are calculated based on their coordinates derived from the image information stored at816. If no recognition spots have been printed on the microarray, the first row of spots can serve as a surrogate recognition template for its own microarray image.

Subsequently, a series of measurements may be performed to identify the actual location of each spot. Using the calculated field points, a centroid of intensity located in a region surrounding each field point may be located. The centroid may be equivalent to the first moment of the spot region, defined as:

Where lpis the pixel intensity at the pixel p,XpandYpare the distance vectors to the pixel p from an arbitrary reference location, and N is the total number of pixels in the region. Once the centroid is located, the field point is re-centered on the spot centroid. With this new location as an origin, the method may include locating inflection points in the gradient of intensity along vertical and horizontal lines through the region. These points may define the lines of steepest descent in the gradient of the signal, points that are directly related to the spot boundaries. At these inflection points, the gradient will be maximized and the second derivative of the intensity with respect to the radial coordinate will be zero:

Averaging the two inflection point locations along each axis provides the coordinates of the actual spot center. With this point as the new center, the method may then include searching for a circle of signal whose intensity is bounded by adjustable lower and upper limits relative to the local background. These limits define the sensitivity of the location algorithm relative to the background. A single sensitivity setting may be used for all images produced by a particular combination of spot diameter, print pitch, and assay protocol. The method may include keeping a running inventory of the calculated spot center and the actual spot centers during the microarray spot location routine. From the differences of these sets, adjustments for possible image rotation and drifts during the printing may be made. As such, the need to rotate or stretch a “grid” over the microarray to account for deviations in spot locations from their projected centers may be reduced or eliminated.

After determining the location of each of the spots at818, method800may then proceed to820, which comprises performing a wavelength analysis to determine the presence and/or levels of biomolecules at each spot. In some examples, only one wavelength of emitted light may be used for analysis at820. Specifically, fluorescent tags of only one wavelength of light may be used to tag the target biomolecules. Thus, based on the emission intensities of the wavelength of light used to tag the target biomolecules, one or more of an amount, concentration, and/or level of the target biomolecules at each spot may be inferred. However, in other examples, the analysis at820may include multiplexing and analysis of two or more wavelengths of light, corresponding to two or more target biomolecules fluorescently tagged using different but simultaneously-excited labels. Put more simply, in examples where two fluorescent labels are used that have different wavelength emissions spectra, quantification of the intensity of each wavelength corresponding to the two or more labels may be used to determine biomolecule expression levels. In some examples, the light emitted by the different fluorescent tags may be sufficiently separated in wavelength, to discern the different tags. However, in some examples, various statistical algorithms, or data visualization software programs may be used to analyze the relative intensities of different wavelengths of electromagnetic waves emitted by the array and captured by the camera. In some examples, the analysis may include determining the presence and/or levels of antibodies. As such, the analysis at820may be used to diagnose an infectious disease. However, in other examples, the analysis may include determining the presence and/or levels of antigens. In still further examples, the analysis may include determining the presence and/or levels of one or more of DNA, RNA, peptides, etc. Thus, the method800at820may include generating results from the analysis, where the results may comprise an estimation of biomolecule levels/concentration in the array. Further the results may include an infectious disease diagnosis based on antibody concentrations/expression levels.

In some examples, method800may return after determining the concentration and/or levels of biomolecules in the array. However, in some examples, the method800may continue from820to822which comprises displaying the results via a display screen (e.g., display screen302shown inFIG. 3). Thus, estimations of biomolecule expression levels, (e.g., antibody, protein, antigen, DNA, and/or gene concentrations) derived from the analysis at820may be presented to via the display screen at822. In this way, the method800at822may comprise displaying concentrations of one or more of antibodies, proteins, DNA, RNA, etc., and may additionally including displaying an infectious disease diagnosis. Method800then returns.

In this way, a microarray assembly may comprise a laser emitting in a first direction, a camera positioned parallel to and vertically below the laser, a first dichroic mirror horizontally aligned with the laser for reflecting light emitted from the laser, a second mirror horizontally aligned with the camera and vertically aligned with the first dichroic mirror, and a chip coated in a nitrocellulose film and including an array of wells containing one or more biomolecules. The camera mat further comprise a lens, and the camera may be orientated so that the lens is pointed in the first direction for capturing light reflected off the second mirror. In some examples, the assembly may further comprise a cover for housing the chip, the cover including one or more of an aperture and groove for receiving the chip. The cover may further include an optically clear window, the optically clear window integrally forming a front wall of the cover, where the front wall of the cover may be pointed towards the first dichroic mirror in a second direction, the second direction perpendicular to the first direction. The optically clear window described above may be sized to allow uniform light dispersal from the laser across a surface area of the array of wells containing the one or more biomolecules. The chip may be centered along a central axis of the cover, and the cover may center the array and optically clear window on a light beam produced by the laser. In some examples, the first dichroic mirror and the second mirror may be orientated at a 45 degree angle with respect to the lens of the camera and a light source end of the laser, from which a laser beam is emitted. Additionally or alternatively, first dichroic mirror and the second mirror may be orientated at a 45 degree angle with respect to the front wall of the cover. The microarray assembly may further comprise a door, adjustable between a closed position and an open position for receiving one or more of the chip and cover, where in the closed position, the door may optically and fluidically seal an interior and an exterior of the assembly. In some examples, the door may include an electrical circuit for monitoring the position of the door, where the voltage and/or current in the circuit may depend on the position of the door. In such a circuit, the current in the circuit may increase when the door is closed relative to when the door is open. Structurally, the cover may include 6 walls fully enclosing the chip, where the walls may be fluidically sealed at their edges, so that fluid communication between the interior and exterior of the cover is provided by the aperture only. The microarray assembly may be configured as a protein array and as such the one or more biomolecules may include one or more antibodies.

In another representation, a system for quantifying biomolecule expression levels may comprise a housing, a laser pointed in a first direction, a camera positioned parallel to and vertically below the laser, a first dichroic mirror horizontally aligned with the laser and orientated at a 45 degree angle with respect to a light source end of the laser for reflecting light emitted from the laser, a second mirror horizontally aligned with the camera and vertically aligned and parallel with the first dichroic mirror, a chip coated in a nitrocellulose film and including an array of wells containing one or more biomolecules, and a controller with computer readable instructions for capturing an image of light emitted from the array in response to excitation from the laser. In some examples, the one or more biomolecules may be tagged with a fluorescent label. The fluorescent label may comprise Quantum Nanocrystal fluorescent-nanoparticles. The system further of any of the above embodiments may further include a display screen for presenting images of the array captured by the camera to a user. An optical path length between the array and the camera may be approximately 120 mm.

In yet another representation, a method for imaging an array of a microarray assembly may comprise inserting the array into the microarray assembly, verifying the array is inserted in the microarray assembly, powering on a laser for duration and directing a laser beam produced by the laser in a first direction towards first dichroic mirror en route to the array, reflecting light emitted from the array in response to excitation from the laser off a second mirror in a second direction, the second direction opposite the first direction, and operating a camera to capture light reflected off the second mirror. The method may further comprise selecting one or more filters positioned between the camera and the second mirror for allowing only a desired range of wavelengths of light to reach the camera. Any of the above embodiments of the method may additionally or alternatively comprise identifying and locating each of a plurality of spots positioned on the array, where the spots may comprise one or more biomolecules chemically bound to the array. In any of the above embodiments of the method, the laser, camera, first dichroic mirror, and second mirror may be positioned in an optically folded arrangement, so that light emitted from the laser propagates in a parallel and opposite direction to light received by the camera.

In this way, a microarray assembly may include a laser for exciting fluorescent labels tagged to target biomolecules in a sample. Specifically, the fluorescent labels may include Quantum Nanocrystals fluorescent-nanoparticles (QNC). A laser beam produced by the laser may be reflected off a first dichroic mirror and directed towards an array to which the target biomolecules may be chemically bound. Laser light may reach the array via an optically clear window in a cover which houses a chip containing the array. When excited by the laser light, the fluorescent labels may emit light, which may propagate out from the cover through the optically clear window, back towards the first dichroic mirror. However, the first dichroic mirror may be optically transparent to light in the wavelength range emitted by the fluorescent labels, and as such light emitted from the fluorescent labels may pass through the first dichroic mirror and on to a second mirror. The second mirror may be configured to reflect substantially all wavelengths of light, and as such, light emitted from the fluorescent labels may be reflected towards a camera positioned in parallel with the laser. In this way, a lens of the camera used to gather light emitted from the fluorescent labels, and a light source end of the laser from which laser beam is emitted may face the same direction.

A first technical effect of increasing the compactness and therefore reducing the size of the microarray assembly is achieved by including both the first dichroic mirror and second mirror in series with one another so that the camera and laser may be positioned parallel to one another. By reducing the size of the microarray assembly, the portability of the assembly may be increased. In this way, the microarray assembly of the present invention may be transported and/or carried by a user.

Further, a second technical effect of increasing the resolution of images captured by the camera of the microarray assembly is achieved by utilizing QNC as the fluorescent labels of the biomolecules. By increasing the resolution of the images of the array, the accuracy of detections and/or quantifications of biomolecules present on the array may be improved. As such, the accuracy of infectious disease determinations may be increased.

Note that the example control and estimation routines included herein can be used with various microarray assembly configurations. The control methods and routines disclosed herein may be stored as executable instructions in non-transitory memory and may be carried out by the control system including the controller in combination with the various sensors, actuators, and other microarray assembly hardware. The specific routines described herein may represent one or more of any number of processing strategies such as event-driven, interrupt-driven, multi-tasking, multi-threading, and the like. As such, various actions, operations, and/or functions illustrated may be performed in the sequence illustrated, in parallel, or in some cases omitted. Likewise, the order of processing is not necessarily required to achieve the features and advantages of the example embodiments described herein, but is provided for ease of illustration and description. One or more of the illustrated actions, operations and/or functions may be repeatedly performed depending on the particular strategy being used. Further, the described actions, operations and/or functions may graphically represent code to be programmed into non-transitory memory of the computer readable storage medium in the microarray assembly system, where the described actions are carried out by executing the instructions in a system including the various microarray assembly hardware components in combination with the electronic controller.

It will be appreciated that the configurations and routines disclosed herein are exemplary in nature, and that these specific embodiments are not to be considered in a limiting sense, because numerous variations are possible. For example, the above technology can be applied to various microarray types. The subject matter of the present disclosure includes all novel and non-obvious combinations and sub-combinations of the various systems and configurations, and other features, functions, and/or properties disclosed herein.