Optical reader system and method for monitoring and correcting lateral and angular misalignments of label independent biosensors

An optical reader system and method are described herein that can detect a lateral and/or angular misalignment of one or more biosensors so that the biosensors can be properly re-located after being removed from and then reinserted into the optical reader system. In one embodiment, the biosensors are incorporated within the wells of a microplate.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an optical reader system and method for detecting a lateral and/or angular misalignment of one or more biosensors so that the biosensors can be properly re-located after being removed from and then reinserted into the optical reader system. In one embodiment, the biosensors are incorporated within the wells of a microplate.

2. Description of Related Art

A major challenge today is to design an optical reader system that can properly re-locate a label independent detection (LID) microplate after it is removed and then reinserted back into the optical reader system. In particular, what is needed is an optical reader system that can detect and correct a lateral and/or angular misalignment of a re-positioned LID microplate. This need and other needs are addressed by the optical reader system and method of the present invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention includes an optical reader system and method that uses one or more fiducial markings (e.g., position sensors) on a LID microplate to monitor and correct if needed any lateral and/or angular misalignment of the microplate. In one embodiment, the method includes the steps of: (a) placing the microplate onto a translation stage; (b) using one or more fiducial marking(s) on the microplate to determine a first position of the microplate; (c) removing the microplate from the translation stage; (d) re-inserting the microplate back onto the translation stage; (e) using the fiducial marking(s) on the microplate to determine a second position of the microplate; (f) comparing the first position and the second position of the microplate; and (g) if there is a difference between the two positions, then addressing the lateral and/or angular misalignment of the microplate by: (1) moving the translation stage so that the microplate is located at or substantially near to the first position; or (2) not moving the microplate but instead adjusting via software a measured reading (e.g., resonance wavelength) based upon the known position error and a known translation sensitivity. Likewise, steps (a)-(g) could be accomplished by using a stationary holder for the microplate and instead the optical beams can be moved that interrogate the stationary microplate. In another embodiment, the optical reader system can be used to monitor and correct a lateral and/or angular misalignment of a biosensor (which has a fiducial marking) that is not incorporated within a microplate.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring toFIGS. 1-11, there are disclosed several diagrams and graphs which are used to help describe the optical reader system100and method1100of the present invention. As discussed below, the optical reader system100is capable of performing two functions: (1) detecting a biological substance124(or a biomolecular binding event) on a biosensor102; and (2) detecting and correcting any lateral and/or angular misalignment of the biosensor102which is caused by the removal and subsequent reinsertion of the biosensor102into the optical reader system100. Prior to discussing the second function, a brief description is provided about how the optical reader system100can detect a biological substance124on the biosensor102.

As shown inFIG. 1, the optical reader system100is used to interrogate a biosensor102(e.g., resonant waveguide grating (RWG) biosensor102, a surface plasmon resonance (SPR) biosensor102) to determine if a biological substance124is present on the biosensor102. The optical reader system100includes a light source106(e.g., lamp, laser, diode) that outputs an optical beam104which is scanned across the biosensor102. Typically, the biosensor102is moved so the optical beam104can be scanned across the biosensor102. Alternatively, the optical beam104itself may be scanned with a mirror, galvanometer, electro-optic or acousto-optic scanner or other suitable adjustable optical element, across a stationary biosensor102. While the optical beam104is scanned across the biosensor102, a detector108(e.g., spectrometer, CCD camera or other optical detector) collects an optical beam112which is reflected from the biosensor102. A processor110(e.g., DSP110, computer110) then processes the collected optical beam112to obtain and record raw spectral data114which is a function of a position (and possibly time) on the biosensor102. Thereafter, the processor110analyzes the raw spectral data114to create a spatial map of resonant wavelength (peak position) data which indicates if a biological substance124is present on the biosensor102.

In particular, the biosensor102makes use of changes in the refractive index at the sensor surface126that affect the waveguide coupling properties of the emitted optical beam104and the detected optical beam112to enable label-free detection of the biological substance124(e.g., cell, molecule, protein, drug, chemical compound, nucleic acid, peptide, carbohydrate) on the superstrate103(sensing region) of the biosensor102. The biological substance124may be located within a bulk fluid that is deposited on the superstrate103(sensing region) of the biosensor102and it is the presence of this biological substance124that alters the index of refraction at the surface126of the biosensor102. Thus, to detect the biological substance124, the biosensor102needs to be at least probed with an optical beam104and then a reflected optical beam112received at the detector108is analyzed to determine if there are any changes (˜1 part per million) in the refractive index caused by the presence of the biological substance124. In one embodiment, the top surface126may be coated with biochemical compounds (not shown) that only allow surface attachment of specific complementary biological substances124which enables a biosensor102to be created that is both highly sensitive and highly specific. In this way, the optical reader system100and biosensor102may be used to detect a wide variety of biological substances124. And, if multiple biosensors102are arranged in array like in a microplate126then they may be used to enable high throughput drug or chemical screening studies. For a more detailed discussion about the detection of a biological substance124(or a biomolecular binding event) using the scanning optical reader system100, reference is made to the aforementioned U.S. patent application Ser. No. 11/027,547.

It is well known that when an optical beam104is used to interrogate a biosensor102, then the resonance wavelength often has an undesirable dependence upon the exact spatial location at which the optical beam104strikes the biosensor102. The undesirable variation of the resonance wavelength is often caused by the non-homogeneity of the biosensor102which can be attributable to variations in the thickness of the waveguide and/or to variations in the grating period (for example) . In fact, a typical variation in the resonance wavelength can be as high as 3 pm per micron. Thus, if one desires to remove and replace the biosensor102from the optical reader100during the course of an experiment, the biosensor102needs to be repositioned to a high accuracy to prevent wavelength shifts induced by translation from overwhelming those wavelength shifts from biochemical binding. The impact, in terms of wavelength shift Δλ of such a translation sensitivity upon the measurement is thus

Δλ=ⅆλⅆx·Δ⁢⁢x.
Here Δλ/dx is the translation sensitivity (pm/μm) and Δx is the displacement (μm) of the biosensor102between measurements. This formula makes apparent two ways of reducing the impact of translation: 1) reduce the translation sensitivity, αλ/dx, by careful design of the biosensor102and/or the optical reader system100; or 2) reduce the amount of displacement αx that occurs between measurements.

To reduce the translation sensitivity, the scanning optical reader system100can be used to average these spatial fluctuations in the resonance wavelength. This has been shown to decrease the translational sensitivity by an order of magnitude to around 0.3 pm per micron.FIG. 2is a graph that shows the typical shape of the resonance wavelength (spectral shift) that can be obtained when scanning one 3 mm long biosensor102in one direction with a 100 μm diameter optical beam. It should be appreciated that the use of a “larger” optical beam104can help even more by further averaging down high spatial frequency variations. Although, a resonance wavelength translation sensitivity of 0.3 pm per micron works well in many applications, such a sensitivity can still be of great concern for systems attempting to detect small biomolecular binding events. Such small binding events can require resonant wavelength measurement accuracies of better then 0.05 pm. To address this problem one can minimize the translation induced wavelength error by ensuring that the biosensors102are properly positioned within the optical reader system100. This is done by the second function of the optical reader system100.

A detailed discussion is provided next about three different ways the optical reader system100can make sure that the biosensors102are properly positioned therein. Basically, the optical reader system100can detect and correct a lateral and/or an angular misalignment of the biosensor(s)102(microplate126) by using anyone or a combination of three different types of fiducial markings which can be located on either the biosensor102or the microplate126. The first type of fiducial marking is the edge of the measurement diffraction grating on the biosensor102(seeFIGS. 3-5and6A-6B). The second type of fiducial marking is a non-responding line602,602aand602blocated on the measurement diffraction grating of the biosensor102(seeFIGS. 6C-6D). And, the third type of fiducial marking is a fiducial diffraction grating702(position sensor702) that is separate from the measurement diffraction grating on the biosensor102(seeFIGS. 7A and 7B) . In yet another embodiment, the fiducial marking can be a coating (local metallic, dielectric coating) that is applied to a biosensor102or microplate126. This coating would have a sufficient reflectivity contrast so it could be detected by the optical reader system100.

In the first way, the optical reader system100scans a biosensor102and uses the resulting raw spectral data114to create a spatial map of reflected power that enables one to precisely locate the edge of the gratings in the biosensor102.FIG. 3is a graph that shows the typical shape to the power evolution of the resonance wavelength when the optical reader system100scans a square biosensor102. To determine the edges of the biosensor102, various edge detection algorithms can be used. As an example,FIG. 4is a graph that shows the result of applying a derivative filter on the power profile shown inFIG. 3. By detecting the centroids of the positive and negative peaks of the differentiated signal, one can accurately determine the position of the biosensor102. Again, in this case the fiducial marking is the edge of the measurement diffraction grating on the biosensor102.

To estimate the repeatability of this type of position measurement, a square biosensor102was scanned 500 hundred times. The position of the detected edge then was measured with respect to the position encoder data on a translation stage128which supported the biosensor102(or microplate126) (seeFIG. 1). Results of this test are shown inFIG. 5. The typical standard deviation of the measurements is in the range of 0.25 microns, which is in fact very close to the resolution of the encoder on the translation stage128(seeFIG. 1).

This and other types of position measurements are important, because when a microplate126which contains an array of biosensors102is removed from and then reinserted into the optical reader system100, one essentially loses track of the absolute translational position of the microplate126. However, upon reinsertion, when the optical beam104is scanned across the microplate126, by detecting the location where the edges of the grating(s) occur on the biosensor(s)102, one can “recalibrate” the translation stage128(e.g., linear stages128) so it can very precisely move the microplate126back to the same position the microplate126was in before it was removed from the optical reader system100. For a more detailed discussion about how the optical reader system100can detect the edges of a measurement diffraction grating in a biosensor102, reference is made to the aforementioned U.S. patent application Ser. No. 11/027,547.

Additionally, one may use various edge detection concepts to monitor the two dimensional (2D) lateral position of the microplate126(seeFIGS. 6A-6D) . In one such edge detection concept, a square biosensor102is scanned in both an x direction and a y direction to determine the lateral 2D position of the microplate126(seeFIG. 6A). In another edge detection technique, one can scan a triangular biosensor102where the x-position is given by the position of the first edge detection and the y-position is given by the distance measured between the two edges detections (seeFIG. 6B).

In yet another other edge detection concept, one can use the second type of fiducial marking(s)602which are non-responding line(s)602located on the biosensor102to monitor the lateral 2D position of the microplate126(seeFIGS. 6C-6D) . In one example, the biosensor102has a design as shown inFIG. 6Cwhere a non-responding line602was made diagonally across the biosensor102. This diagonal non-responding line602enables one to estimate both the x and y positions of the biosensor102with a single 1-dimensional beam scan. In particular, when using such a diagonal non-responding line602the rising edge of a power vs. position trace is used to determine the x-position and the difference between the rising and falling edges is used to determine the y-position. In yet another example shown inFIG. 6D, the biosensor102has two off-center non-responding lines602aand602bthat are set at the edge of the biosensor102which allows one to also use the center portion of the grating to detect a biological substance124(or a biomolecular binding event) . An advantage of the last example is that one can put fiducial markings602aand602bon all of the biosensors102which allows one to obtain more data that can be averaged to improve the re-positioning accuracy. However, the drawback of this example is that a complete measurement requires two scanning steps, one scanning step for the position measurement and one scanning step for the biochemical measurement itself. It should be noted that non-responding lines602,602aand602bcan be generated by having some areas without a diffraction grating or without a waveguide.

Referring now to the third type of fiducial marking, the optical reader system100in this case scans a fiducial diffraction grating702(position sensor702) which is preferably located on a microplate126(seeFIGS. 7A and 7B). As shown inFIGS. 7A and 7B, the optical reader system100can interrogate the fiducial diffraction gratings702which are relatively close to the biosensor102. Then, in real time measure the position of the microplate126aand126band if needed make the translation corrections before the interrogation beam104reaches the biosensor102. This allows continuous scanning with real time position correction.

As can be seen in the exemplary microplates126aand126bshown inFIGS. 7A and 7B, one can put a fiducial diffraction grating702and a biosensor102in each measurement well704a(seeFIG. 7A). Or, one can put a fiducial diffraction grating702at the beginning and at the end of the microplate plate126b(seeFIG. 7B). In the last case, the fiducial diffraction gratings702are located outside the measurement wells704band will be in contact with air or with the glue that holds together the microplate126. The design of these particular fiducial diffraction gratings702in terms of a grating period should be optimized to generate a resonance wavelength close to the one that would be generated if the fiducial diffraction gratings702were in contact with the aqueous buffer solutions likely to be used in the wells. This is because the global spectral range of the optical reader system100is limited by the spectral width of the light source106and detector108, and it is important to keep the resonance within the operational band of this source/detector system100.

An advantage of having multiple fiducial diffraction gratings702across the microplate126aand126bis that one can average the data and obtain a better measurement accuracy. Another advantage of having multiple fiducial diffraction gratings702on a microplate126aand126bis that it allows one to monitor thermal dilatations of the microplate126aand126b. To measure thermal dilations of the microplate126aand126bone can optically scan the microplate126aand126band record the locations of the fiducial diffraction gratings702(F1, F2, F3. . . )(or other types of fiducial markings) . Then, after some time and possibly a temperature change, one may rescan the microplate126aand126band again record the locations of the same fiducial diffraction gratings702(F1, F2, F3. . . )(or other types of fiducial markings). If the microplate126aand126bhas grown or shrunk due to temperature change, then the relative locations of the fiducial diffraction gratings702(or other types of fiducial markings) will have changed (i.e., Δ21=F2−F1will have changed, and Δ31=F3−F1will have changed . . . )

In an alternative embodiment, the fiducial diffraction gratings702and the measurement diffraction gratings can have different resonance wavelengths. To have different resonance wavelengths, the fiducial diffraction gratings702and the measurement diffraction gratings can be made with different grating periods. Or, they can be made with waveguides that have different thicknesses. In this embodiment, the resonance wavelengths can be detected by measuring the evolution of the power of the two peaks corresponding to the different gratings areas. Then, the edge detection can be made based on the relative power of both peaks.

In yet another embodiment, the fiducial gratings702can include features that are perpendicular to the scanning direction of the optical beam and other features that are at a certain angle such as 45 degrees with respect to the scanning direction. In this way, one can determine misalignments in both directions.

A discussion is provided next about several other uses of the fiducial diffraction gratings702(position sensors702) in addition to their use in helping with the repositioning of the biosensor102or microplate126. When the fiducial diffraction gratings702are not in contact with the liquid that is measured in the wells of the microplate126, then those fiducials are completely isolated and their resonance wavelength is affected only by disturbing external effects such as temperature variations or angular misalignments. As a result, one can use the fiducial diffraction gratings702to monitor those external effects as follows:

1. Angular Monitoring—FIG. 8shows the typical wavelength shift that can be measured as a function of the incidence angle when interrogating biosensors102at normal incidence with single mode fibers. As can be seen, the angular sensitivity is in the range of 10 pm/mRd which can make the angle monitoring very critical. One way that this angular variation can be monitored is to interrogate the fiducial diffraction gratings702by using a multimode fiber instead of a single mode fiber for the light injection. Indeed, as shown onFIG. 9, when this configuration was tested we obtained angular sensitivities in the range of 432 pm/mRd which is an order of magnitude greater than the sensitivity that obtained with the single mode fibers. So, by comparing the resonance wavelength measured with a multimode fiber and the one measured with the single mode fiber, one can better deduce any angular misalignment of the microplate126after reinserting it into the reader100by using the multimode fiber.

2. Temperature Gradient Monitoring—FIG. 10is a graph that shows the temperature sensitivity of an interrogated fiducial diffraction grating702when the temperature cools by 21° C. As can be calculated from the wavelength changes of the curves shown, the sensitivity coefficients are −10 pm/° C. for TE mode and −26 pm/° C. for the TM mode. Therefore, temperature changes of as small as 0.01° C. can perturb the measured resonance wavelengths by 0.26 pm, which is of significance for small biomolecular binding events. Additionally, even if in-well referencing is used (see U.S. patent application Ser. No. 11/027,509 entitled “Method for Creating a Reference Region and a Sample Region on a Biosensor and the Resulting Biosensor and U.S. patent application Ser. No. 11/027,547 entitled “Spatially Scanned Optical Reader System and Method for Using Same”) temperature gradients, and in particular changes in temperature gradients, inside wells may still be large enough to induce resonant wavelength shifts of concern. Assuming that the fiducial diffraction gratings702are in contact with glue, then the temperature variation is the major parameter that makes the resonance wavelength fluctuate over time. With this knowledge one can then use the wavelength fluctuations measured across the fiducial diffraction gratings702on the microplate126aand126bto deduce the temperature gradient fluctuations and check that they are under acceptable levels.

From the foregoing, it can be readily appreciated by those skilled in the art that the present invention also includes a method1100for monitoring and correcting if needed any lateral and/or angular misalignment of the microplate126. As shown in the flowchart ofFIG. 11, the method1100includes the steps of: (a) place microplate (with one or more fiducial markings) on holder which in one embodiment is the translation stage128and in another embodiment is a stationary holder (not shown) (step1102); (b) using one or more fiducial markings on the microplate126to determine a first position of the microplate126(step1104); (c) removing the microplate126from the holder (step1106); (d) reinserting the microplate126back onto the holder (step1108); (e) using the fiducial marking(s) on the microplate126to determine a second position of the microplate128(step1110); (f) comparing the first position and the second position of the microplate126(step1112); (g) if there is a difference between the two positions, then addressing the lateral and/or angular misalignment of the microplate126(step1114) by: (1) moving the translation stage128so that the microplate126is located at or substantially near to the first position (step1114a); or moving the optical beams104so that the microplate which is on the stationary holder appears to be in the first position (step1114b) ; or (3) not moving the microplate126or the optical beams104but instead adjusting via software a measured interrogation reading (e.g., resonance wavelength) based upon the known position error and a known translation sensitivity (step1114c); and (h) if there is no difference (or no substantial difference) between the two positions, then interrogate the microplate126while it is in the second position (step1116).

It should be noted that the term angular misalignment as used above is the skew that is caused by the microplate126being rotated in the Z axis if the X&Y are the lateral axis. Alternatively, it should be noted that an angular misalignment can also be caused if one performs a “skewed” scan across the microplate126where one simultaneously moves the X&Y motion stages in a coordinated skewed motion.

It should also be noted that in most of the drawings herein, were made based on the assumption that the sensor is spectrally interrogated. This means that the sensor is interrogated at a fixed incidence angle with a broad spectral source and that the wavelength is detected in the reflected beam. The source is then a broad spectral source and the detector is a wavelength sensitive detector such as a spectrometer. However, it should be appreciated that the principle of the present invention can also be extended to an angular interrogation approach where the biosensor is interrogated with monochromatic light and then a resonant angle is detected in the reflected beam.

Furthermore, it should be noted that there are configurations of the present invention that do not need to use scanning to position, re-position and/or interrogate the biosensor102. One such non-scanning system involves the use of a vision system. The vision system would create an image of the biosensor(s)102, the optical beams104, and/or the fiducials on a position sensitive detector (e.g., CCD camera). And, this vision system could make use of the fiducials by looking at the position of the fiducials imaged on the CCD camera and then make the appropriate adjustments.