Method for detecting the presence of a target analyte in a test spot

Abstract

An apparatus and method for imaging metallic nanoparticles is provided. Preferably, the invention provides for an apparatus and method for detection of gold colloid particles and for accurate reporting to the operator. The apparatus includes a substrate holder for holding the substrate, a processor and memory device, an imaging module, an illumination module, a power module, an input module, and an output module. The apparatus may have a stationary substrate holder and imaging module which are proximate to one another. The apparatus provided for a compact sized system without the need for complex motorized devices to move the camera across the substrate. Further, the apparatus and method provide for automatic detection of the spots/wells on the substrate, automatic quantification of the spots on the substrate, and automatic interpretation of the spots based on decision statistics.

1 . Apparatus for detecting metallic nanoparticles on a substrate, with or without chemical signal amplification of the metallic nanoparticles, the apparatus comprising in combination:
 a substrate holder for holding the substrate, the substrate holder being stationary;   a processor;   a memory device in communication with the processor;   an imaging module in communication with the processor, the imaging module being stationary;   an illumination module for illuminating the substrate;   a power module;   an input module for receiving operator instructions; and   an output module for outputting whether metallic nanoparticles have been detected,   wherein the imaging module and the substrate holder are proximate to one another, and   wherein the processor controls the imaging module, illumination module input module and output module.   
     
     
         2 . The apparatus of  claim 1 , wherein the imaging module and substrate holder are greater than 30 mm from one another and less than 356 mm from one another. 
     
     
         3 . The apparatus of  claim 2 , wherein the imaging module comprises a photosensor and wherein the photosensor is less than 70 mm from the substrate holder. 
     
     
         4 . The apparatus of  claim 1 , wherein the processor, memory device, imaging module, substrate holder, illumination module, output module, input module, power module are contained within one housing. 
     
     
         5 . The apparatus of  claim 1 , wherein the nanoparticles have been amplified with chemical signal amplification. 
     
     
         6 . The apparatus of  claim 1 , wherein a type of lighting from the illumination module is selected from the group consisting of side-lighting, back-lighting and front-lighting. 
     
     
         7 . The apparatus of  claim 1 , wherein the memory device includes compensation module, the processor accessing the compensation module to compensate for distortion in an image acquired by the imaging module. 
     
     
         8 . The apparatus of  claim 7 , wherein the compensation module compensates for grayscale distortion. 
     
     
         9 . The apparatus of  claim 7 , wherein the compensation module compensates for spatial distortion. 
     
     
         10 . The apparatus of  claim 1 , wherein the memory device includes a program configured to perform the steps of:
 acquiring multiple images by the imaging module of a substrate in the substrate holder, the substrate having at least one test spot containing a test sample and at least another spot that is a control or a second test spot, the multiple images being taken at different exposures; and   determining, based on the multiple images of the spots, the presence of metallic nanoparticle complexes in the one test spot as an indication of presence of one or more of target analytes.   
     
     
         11 . The apparatus of  claim 10 , wherein the step of determining the presence of said metallic nanoparticle complexes in the spot containing the test sample comprises:
 performing regression analysis on portions in the multiple images containing the one test spot and the control or second test spot to generate functions of exposure time versus intensity for each of the spots;   selecting an exposure time;   determining intensity for the one test spot and the control or second test spot for the selected exposure time based on the functions generated;   determining whether the one test spot containing the test sample contains metallic nanoparticle complexes based on comparing the intensity of the one test spot with the intensity of the control or second test spot at the selected exposure time.   
     
     
         12 . The apparatus of  claim 11 , wherein the selected exposure time is an optimal exposure time. 
     
     
         13 . The apparatus of  claim 1 , wherein the memory device includes a program configured to perform the steps of:
 automatically detecting spots on substrate in the substrate holder, the substrate having a plurality of wells; and   automatically determining the wells based on the automatic detection of at least a portion of the spots.   
     
     
         14 . The apparatus of  claim 13 , wherein the step of automatically determining the wells comprises:
 automatically determining spacing between at least some of the detected spots;   automatically determining spots which are located within at least one well based on the spacing.   
     
     
         15 . The apparatus of  claim 13 , wherein the step of automatically determining the wells comprises:
 automatically determining patterns for at least a portion of the spots detected; and   automatically comparing the patterns with predetermined patterns for wells.   
     
     
         16 . In a substrate having a plurality of spots containing specific binding complements to one or more target analytes, at least one of the spots is a test spot for metallic nanoparticles complexed thereto in the presence of one or more target analytes, another spot is a control spot or a second test spot for metallic nanoparticles, with or without signal amplification, complexed thereto in the presence of a second or more target analytes, a method for detecting the presence or absence of the one or more of the target analytes in the test spot, the method comprising the steps of:
 acquiring multiple images of the test spot and the control or second test spot, the multiple images being taken at different exposures; and   determining presence of said metallic nanoparticle complexes in the test spot as an indication of the presence of one or more of the target analytes based on the acquired multiple images of the spots.   
     
     
         17 . The method of  claim 16 ,
 wherein the control spot is selected from the group consisting of metallic nanoparticle conjugated directly to the substrate via a nucleic capture strand, metallic nanoparticles printed directly on the substrate, and a positive result of metallic nanoparticles complexed to a known analyte placed in a separate well.   
     
     
         18 . The method of  claim 16 ,
 wherein the test sample is a nucleic acid from a wildtype nucleic acid sequence, and   wherein the comparison sample is a nucleic acid from a mutant nucleic acid sequence that is related to the wildtype nucleic acid sequence   
     
     
         19 . The method of  claim 16 ,
 wherein the substrate includes a plurality of wells, at least one of the wells containing the test and comparison spots;   further comprising the step of determining an optimal exposure time for the well; and   wherein the images acquired are taken at the optimal exposure time and at least one exposure time which is less than the optimal exposure time.   
     
     
         20 . The method of  claim 19 , wherein the step of determining an optimal exposure time comprises determining an exposure time which results in a predetermined saturation of the image acquired. 
     
     
         21 . The method of  claim 16 , wherein the step of determining the presence of said metallic nanoparticle complexes in the spot containing the test sample comprises:
 performing regression analysis on the portions in the multiple images containing the test and comparison spots to generate functions of exposure time versus intensity for each of the spots;   selecting an optimal exposure time;   determining intensity for the test and control spots for the optimal exposure time based on the functions generated;   determining whether the test spot containing the test sample contains metallic nanoparticle complexes based on comparing the intensity of the test spot with the intensity of the comparison spot at the optimal exposure time.   
     
     
         22 . The method of  claim 21 , wherein the image acquired results in pixels assigned for the comparison and test spots, the pixels having pixel values
 wherein the step of performing a regression analysis comprises performing a regression analysis on the pixel values in the comparison and test spots.   
     
     
         23 . The method of  claim 22 , wherein the step of selecting an optimal exposure time comprises determining an exposure time which results in a predetermined saturation of a portion of the image acquired which contains the test and comparison spots. 
     
     
         24 . The method of  claim 23 , wherein the step of determining intensity for the test and comparison spots for the optimal exposure time based on the functions generated comprises interpolating or extrapolated the functions generated. 
     
     
         25 . The method of  claim 24 , wherein the step of comparing the intensity of the test spot with the intensity of the control spot at the optimal exposure time comprises performing statistical analyses on the intensity of the comparison and test spots to determine if the intensity of the test spot is similar or dissimilar to the comparison spot. 
     
     
         26 . The method of  claim 25 , wherein the step of performing statistical analyses comprises performing differences between means testing. 
     
     
         27 . In a substrate having a plurality of spots containing specific binding complements to one or more target analytes, at least one of the spots is a test spot for metallic nanoparticles, with or without signal amplification, complexed thereto in the presence of one or more target analytes, another spot is a control spot or a second test spot for metallic nanoparticles complexed thereto in the presence of a second or more target analytes, an automatic method of detecting the plurality of spots comprising the steps of:
 acquiring at least one image of the plurality of spots composed of metallic nanoparticles on the surface of the substrate;   compensating for at least one type of distortion in the acquired image; and   automatically determining locations of at least some of the plurality of spots composed of metallic nanoparticles based on the compensated acquired image.   
     
     
         28 . The method of  claim 27 , the step of acquiring being performed by an image module which is less than or equal to 356 mm distance from the surface of the substrate. 
     
     
         29 . The method of  claim 28 , wherein the image acquired by the image module includes all or substantially all of the surface of the substrate. 
     
     
         30 . The method of  claim 28 , wherein the image module is a photosensor. 
     
     
         31 . The method of  claim 30 , wherein the photosensor is stationary. 
     
     
         32 . The method of  claim 27 , wherein the at least one image is acquired using an image device; and
 wherein the step of acquiring at least one image comprises acquiring the image without moving the image device and the substrate relative to one another.   
     
     
         33 . The method of  claim 27 , wherein the step of acquiring at least one image comprises acquiring a plurality of images to obtain an optimal image. 
     
     
         34 . The method of  claim 27 , wherein the step of correcting at least one type of distortion comprises correction of grayscale distortion. 
     
     
         35 . The method of  claim 34 , wherein the at least one image is acquired using an image device with a field of view; and
 wherein the correction of grayscale distortion comprises applying a compensation model for brightness across the field of view for the image device.   
     
     
         36 . The method of  claim 35 , wherein the compensation model is derived by acquiring images using a consistent light source at different brightness values and by using a calibrated set of filters to generate curves for the images acquired at the different brightness values. 
     
     
         37 . The method of  claim 27 , wherein the step of correcting at least one type of distortion comprises correction of spatial distortion. 
     
     
         38 . The method of  claim 37 , wherein the correction of spatial distortion comprises:
 generating a plurality of points distorted by the spatial distortion;   generating a plurality of points undistorted by spatial distortion;   generating a model based on the plurality of distorted and undistorted points; and   applying the model to the image acquired.   
     
     
         39 . The method of  claim 27 , further comprising the step of performing adaptive thresholding on at least a portion of the image acquired. 
     
     
         40 . In a substrate having a plurality of wells, the wells containing at least two spots containing specific binding complements to one or more target analytes, at least one of the spots including a test spot having metallic nanoparticles, with or without signal amplification, complexed thereto in the presence of one or more target analytes, an automatic method of detecting the plurality of wells comprising the steps of:
 automatically detecting at least a portion of the spots on the substrate;   automatically determining the wells based on the automatic detection of at least a portion of the spots.   
     
     
         41 . The method of  claim 40 , wherein the step of automatically determining the wells comprises:
 determining spacing between at least some of the detected spots; and   automatically determining spots which are located within at least one well based on the spacing.   
     
     
         42 . The method of  claim 40 , wherein the step of automatically determining the wells comprises:
 determining patterns for at least a portion of the spots detected; and   comparing the patterns with predetermined patterns for wells.   
     
     
         43 . The method of  claim 40 , wherein the substrate includes a legend identifying a configuration of at least one of the wells, and
 wherein the step of automatically determining the wells comprises reading the legend and determining a well configuration based on the read legend.   
     
     
         44 . The method of  claim 43 , wherein the legend is a bar code. 
     
     
         45 . The method of  claim 43 , wherein the legend is a grouping of spots. 
     
     
         46 . The method of  claim 40 , wherein the step of automatically determining the wells comprises:
 determining spacing between the spots on the substrate; and   determining configuration of the plurality of wells based on the spacing between the spots on the substrate.   
     
     
         47 . The method of  claim 46 , wherein the step of automatically determining the wells further comprises the step of determining spots on the substrate which are in a straight line with one another, and
 wherein the step of determining spacing comprises determining, for the spots in a straight line, the spacing for spots adjacent to one another.   
     
     
         48 . The method of  claim 47 , wherein the step of determining the spacing between the spots comprises:
 determining centroids for the spots on the substrate; and   calculating the spacing by determining distances between the centroids of the spots adjacent to one another.   
     
     
         49 . The method of  claim 46 , wherein the step of determining configuration of the plurality of wells comprises comparing the spacing determined with predetermined spacing. 
     
     
         50 . The method of  claim 49 , wherein the step of determining a configuration of wells comprises:
 comparing patterns in the determined spacing with predetermined patterns of spacing for known configurations of wells; and   assigning configurations for the plurality of wells based on the step of comparing the patterns.