Grating based sensor combining label-free binding detection and fluoresnce amplification and readout system for sensor

Abstract

A grating-based sensor is disclosed that has a grating structure constructed and designed for both evanescent resonance (ER) fluorescence detection and label-free detection applications. One and two-dimensional gratings are also disclosed, including gratings characterized by unit cells with central posts, central holes, and two-level, two-dimensional gratings. A readout system for such sensors is also disclosed. Various applications for the biosensors are described, including cell-based assays for assessing the effect of drug compounds, proteins, peptides and other materials on cell function. A biosensor embodiment optimized for a luminescent response at two different wavelengths is also described. Such luminescent response could be produced by fluorescence (either native or from an attached fluorophore), phosphorescence, chemi-luminescence, or other luminescence technology. Two different luminescence technologies could be combined on the same biosensor chip.

1 . A method of conducting a cell-based assay of a sample comprising one or more cells, the method comprising the steps of:
 (a) providing a grating-based biosensor substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for both 1) optical interrogation of the sensor with light in an evanescent resonance (ER) detection mode, and 2) optical interrogation of the sensor with light in a label-free detection mode;   (b) applying the sample to the biosensor;   (c) using the biosensor to measure at least one of following: (a) cell attachment of cells in the sample to the surface of the grating structure, (b) adhesion of cells in the sample to the sensor or to an extracellular matrix coating the sensor, (c) adhesion of cells in the sample to other cells, (d) morphological change of cells in the sample, (e) chemotaxis of cells in the sample, (f) protein exocytosis from the cell into the vicinity of the sensor, (g) ion flux in and out of the cell in the vicinity of the sensor, (h) cell migration, (i) cell flattening or rounding, (j) cell growth or differentiation, (k) cell death, (l) cytoskeletal rearrangement of cells in the sample, and (m) rearrangement, reorganization or changed expression of proteins inside or outside of the cells in the sample;   
     and
 (d) using the biosensor to measure the effect of at least one of the following on cell function of cells in the sample: (a) a drug compound candidate, (b) a protein, (c) a peptide or modified peptide, (d) an antibody or fragment thereof, (e) DNA including modified DNA (known in the art for eliciting biological effects), (f) RNAi including modified RNAi, (g) RNA (including modified RNA), (h) a chemokine, (i) a virus, (j) other cells, including bacteria or other organisms, (k) engineered binding domains (e.g., Affibodies, DARPins, Adnectins), (l) sugars or modified sugars, (m) ions, (n) lipids and modified lipids, (o) metals, (p) inorganic solvents, and (q) organic solvents 
 
   
   
       2 . The method of  claim 1 , wherein the cell function comprises a cell surface protein function or expression. 
   
   
       3 . The method of  claim 1 , wherein the cell function comprises cell viability or a change in cell viability. 
   
   
       4 . The method of  claim 1 , wherein the cell function comprises an internal cell function or expression. 
   
   
       5 . The method of  claim 1 , wherein the cell function comprises a membrane or membrane-bound cell function or expression. 
   
   
       6 . The method of  claim 1 , wherein the cell function comprises G Protein Coupled Receptors (GPCR) signaling. 
   
   
       7 . The method of  claim 1 , wherein the cell function comprises a cardiotoxic response to an ion-channel targeting drug. 
   
   
       8 . The method of  claim 1 , wherein the drug compound candidate comprises an ion-channel targeting drug. 
   
   
       9 . The method of  claim 1 , wherein the grating-based biosensor substrate is constructed as a two-dimensional periodic grating. 
   
   
       10 . The method of  claim 1 , wherein the biosensor is incorporated into a microplate. 
   
   
       11 . The method of  claim 1 , wherein the biosensor is incorporated into a mounting device having separated sample locations. 
   
   
       12 . The method of  claim 1 , wherein the biosensor is incorporated into a microscope slide. 
   
   
       13 . The method of  claim 11 , wherein the mounting device comprises a cartridge. 
   
   
       14 . The method of  claim 1 , wherein the measurements of part c) are made in the label-free mode. 
   
   
       15 . The method of  claim 1 , wherein the measurements of part d) are made in the ER detection mode. 
   
   
       16 . A grating-based biosensor comprising a substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for optical interrogation of the sensor with excitation light at least two discrete wavelengths in an evanescent resonance (ER) detection mode to thereby produce a luminescence response from a sample placed on the biosensor at least two discrete emission wavelengths. 
   
   
       17 . The biosensor of  claim 16 , wherein the luminescence response comprises a fluorescence response, such fluorescence being either native fluorescence or fluorescence produced by a fluorescent material attached to the sample. 
   
   
       18 . The biosensor of  claim 16 , wherein the luminescence response comprises a phosphorescent response. 
   
   
       19 . The biosensor of  claim 16 , wherein the fluorescence response is produced by a dye which is bound to a sample placed on the biosensor. 
   
   
       20 . The biosensor of  claim 16 , wherein a first emission wavelength is in the near infrared portion of the spectrum and wherein a second emission wavelength is in the visible portion of the spectrum. 
   
   
       21 . The biosensor of  claim 16 , wherein a first emission wavelength corresponds to the emission wavelength of a first dye associated with a sample placed on the biosensor and wherein a second emission wavelength corresponds to the emission wavelength of a second dye associated with the sample. 
   
   
       22 . The biosensor of  claim 20 , wherein the first dye comprises Cyanine-5 and the second dye comprises Cyanine-3. 
   
   
       23 . The biosensor of  claim 20 , wherein the grating structure comprises a two-dimensional periodic grating structure, the periodic grating structure is periodic in first and second dimensions, and wherein the first and second dimensions are mutually orthogonal, and wherein the periodic grating structure in the first dimension is optimized for optical interrogation of the biosensor to produce fluorescence from a first dye and wherein the periodic grating structure in the second dimension is optimized for optical interrogation of the biosensor to produce fluorescence from a second dye, the first and second dies emitting fluorescence at different wavelengths. 
   
   
       24 . A grating-based biosensor comprising a substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for optical interrogation of the sensor with light in an evanescent resonance (ER) detection mode to produce a luminescence response from a sample placed on the biosensor at two discrete wavelengths, and wherein the luminescence response at the two discrete wavelengths is produced from two different types of luminescence. 
   
   
       25 . The biosensor of  claim 24 , wherein one of the two types of luminescence comprises fluorescence; such fluorescence being either native fluorescence or fluorescence produced by a fluorescent material attached to the sample. 
   
   
       26 . The biosensor of  claim 24 , wherein one of the two types of luminescence comprises phosphorescence. 
   
   
       27 . The biosensor of  claim 24 , wherein one of the two types of luminescence comprises chemi-luminescence. 
   
   
       28 . The biosensor of  claim 24 , wherein one of the two types of luminescence comprises electro-luminescence. 
   
   
       29 . The biosensor of  claim 24 , wherein the luminescence response at least one of the two discrete wavelengths is produced by a dye which is bound to the sample. 
   
   
       30 . The biosensor of  claim 24 , wherein the grating structure comprises a two-dimensional periodic grating structure, the periodic grating structure is periodic in first and second dimensions, and wherein the first and second dimensions are mutually orthogonal. 
   
   
       31 . A method of conducting a cell-based assay of a sample comprising one or more cells, the method comprising the steps of:
 providing a grating-based biosensor substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for both 1) optical interrogation of the sensor with light in an evanescent resonance (ER) detection mode, and 2) optical interrogation of the sensor with light in a label-free detection mode;   applying a sample to the cell,   using the biosensor to measure cell attachment of cells in the sample to the surface of the grating structure, and   using the biosensor to measure the effect of a drug compound candidate on cell function of the cells in the sample.   
   
   
       32 . The method of  claim 31 , wherein the measurement of cell attachment is made in the label-free detection mode. 
   
   
       33 . The method of  claim 31 , wherein the measurement of effect of the drug compound candidate is made in the ER detection mode. 
   
   
       34 . The method of  claim 31 , wherein the cell function comprises a cell surface protein function or expression. 
   
   
       35 . The method of  claim 31 , wherein the cell function comprises cell viability or a change in cell viability. 
   
   
       36 . The method of  claim 31 , wherein the biosensor is incorporated into a microplate. 
   
   
       37 . The method of  claim 31 , wherein the drug compound candidate comprises an ion-channel targeting drug. 
   
   
       38 . The method of  claim 31 , wherein the cell function comprises a cardiotoxic response to an ion-channel targeting drug. 
   
   
       39 . The method of  claim 31 , wherein the grating-based biosensor substrate is constructed as a two-dimensional periodic grating. 
   
   
       40 . The method of  claim 1  or  claim 31 , wherein the biosensor is used for measuring the effect of a drug candidate on ligand gated ion channels. 
   
   
       41 . The method of  claim 1  or  claim 31 , wherein the cells are endogenous target expressing cells. 
   
   
       42 . The method of  claim 1  or  claim 31  wherein the biosensor is used in either the ER or label-free mode to measure the addition of extracellular matrix components as a soluble material or as a sensor coating amendment to the biosensor. 
   
   
       43 . A method of conducting a cell-based assay of a sample comprising one or more cells, the method comprising the steps of:
 (a) providing a grating-based biosensor substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for both 1) optical interrogation of the sensor with light in an evanescent resonance (ER) detection mode, and 2) optical interrogation of the sensor with light in a label-free detection mode;   (b) applying the sample to the biosensor; and   (c) monitoring interactions of at least two different cell types wherein one cell type is measured in the label-free and another cell type monitored in the ER mode.   
   
   
       44 . The method of  claim 43 , wherein cell-based assay comprises a cell penetration experiment wherein a first layer of cells is placed on the sensor and wherein a second layer of cells of another cell type are placed top of the first layer of cells, the experiment measuring the ability of cells of the second layer to pass through the first layer of cells.