# Patent

## Title
Backscattering interferometric methods

## Abstract

Disclosed are improved optical detection methods comprising multiplexed interferometric detection systems and methods for determining a characteristic property of a sample, together with various applications of the disclosed techniques.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/645,660, filed May 11, 2012, which is hereby incorporated by reference.


BACKGROUND
1. Technical Field
The present disclosure related to analysis methods, and specifically to backscattering interferometric analysis methods and devices for use therewith.
2. Technical Background
Capillary-based analysis schemes, biochemical analysis, basic research in the biological sciences such as localized pH determinations in tissues and studies in protein folding, detection and study of microorganisms, and the miniaturization of instrumentation down to the size of a chip all require small volume detection. With the advent of lasers, light sources possessing unique properties including high spatial coherence, monochromaticity and high photon flux, unparalleled sensitivity and selectivity in chemical analysis has become possible; these technologies, however, can be both expensive and difficult to implement. In contrast, refractive index (RI) detection has been successfully applied to several small volume analytical separation schemes. For various reasons, RI detection represents an attractive alternative to fluorescence and absorbance: it is relatively simple, it can be used with a wide range of buffer systems, and it is universal, theoretically allowing detection of any solute, making it particularly applicable to solutes with poor absorption or fluorescence properties.
Recently developed methods utilizing refractive indices have been useful for measurements in aqueous systems, for example, to monitor biological interactions, but can be subject to noise from alignment and environmental issues. Accordingly, there is a need in the art for methods, systems, and apparatuses that can provide refractive index related measurements having improved sensitivity and reduced noise.
SUMMARY
As embodied and broadly described herein, the invention, in one aspect, relates to an interferometric detection system comprising a light beam that impinges one or more discrete zones along a channel.
In one aspect, the invention relates to a method for determining a characteristic property of a sample, comprising the steps of providing an apparatus adapted for performing light scattering interferometry, the apparatus comprising a substrate, a channel formed in the substrate capable of receiving a sample to be analyzed, a light source for generating a light beam, a photodetector for receiving scattered light and generating a plurality of intensity signals; and at least one signal analyzer capable of receiving the intensity signals and determining therefrom one or more characteristic properties of the sample; and interrogating the sample using light scattering interferometry.
It will be apparent to those skilled in the art that various devices may be used to carry out the systems, methods, apparatuses, or computer program products of the present invention, including cell phones, personal digital assistants, wireless communication devices, personal computers, or dedicated hardware devices designed specifically to carry out aspects of the present invention. While aspects of the present invention may be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class, including systems, apparatuses, methods, and computer program products.
Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method, system, or computer program product claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.



BRIEF DESCRIPTION OF THE FIGURES
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.
FIG. 1 is a schematic block diagram of an interferometric detection system.
FIG. 2 illustrates an exemplary fringe pattern that can be obtained from BSI analysis, in accordance with various aspects of the present disclosure.
FIG. 3 illustrates an exemplary configuration for a BSI system employing two sensors, in accordance with various aspects of the present disclosure.
FIG. 4 illustrates: (A) the signal output of individual cameras and their summation as the chip's position changes relative to the beam's position; and (B) the short term noise from individual camera and their summation as the chip's position changes relative to the beam's position, in accordance with various aspects of the present disclosure.
FIG. 5 illustrates an enhanced signal for an induced refractive index change using two cameras, in accordance with various aspects of the present invention.
FIG. 6 illustrates exemplary BSI configurations using: (A) a single large camera; (B) two cameras synced and connected to the same computer; and (C) two mirrors and an absorber, in accordance with various aspects of the present disclosure.
FIG. 7 illustrates an exemplary BSI configuration wherein an absorber is employed to block the centroid and undesired central fringes of a signal, and flat mirrors are used to focus the desired two fringes on one small sensor, in accordance with various aspects of the present disclosure.
FIG. 8 illustrates: (A) an exemplary alignment of mirrors such that fringes impinge the camera at the same angle; (B) differing widths of the base of two fringes falling on the camera at different angles; (C) similar widths of the base of two fringes falling on the camera at equal angles after adjustment of the mirrors, in accordance with various aspects of the present disclosure.
FIG. 9 illustrates: (A) fringes of varying intensity that occur when the beam does not impinge the center of the channel; and (B) fringes of equivalent intensity obtained after (C) the chip is moved horizontally with respect to the beam, in accordance with various aspects of the present disclosure.
FIG. 10 illustrates an exemplary configuration for a BSI system capable of monitoring sister fringes, in accordance with various aspects of the present disclosure.
FIG. 11 illustrates: (A) the short-term noise of fringes from the left and right sides of a signal and their sum, wherein the noise is in phase with a similar magnitude and wherein the level of noise spikes is reduced in the summed signal; and (B) individual signals showing increased noise due to induced mechanical vibrations, and the summed signal showing no significant increase in short-term noise, in accordance with various aspects of the present disclosure.
FIG. 12 illustrates calibration curves of glycerol solutions comparing single fringe and dual fringe detection methodologies, in accordance with various aspects of the present disclosure.
FIG. 13 illustrates a comparison of the signal to noise ratio observed for single and dual fringe detection methodologies, before and after the chip has been removed from the holder and replaced into the system, in accordance with various aspects of the present disclosure.
FIG. 14 illustrates a fringe pattern and windows W1, W2, W3, and W4, selected for sister fringe BSI, in accordance with various aspects of the present disclosure.
FIG. 15 illustrates the phase shift of signals over time upon mechanical vibrations of the optic bench, in accordance with various aspects of the present disclosure.
FIG. 16 illustrates the phase shift of signals over time upon mechanical displacement of the chip with respect to the beam, in accordance with various aspects of the present disclosure.
FIG. 17 illustrates the phase shift of signals over time upon mechanical vibration of different parts of a BSI instrument, in accordance with various aspects of the present disclosure.
FIG. 18 illustrates the phase shift of signals over time from mechanical instability of different parts of a BSI instrument, in accordance with various aspects of the present disclosure.



Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
DETAILED DESCRIPTION
The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.
Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which may need to be independently confirmed.
DEFINITIONS
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a substrate,” “a polymer,” or “a sample” includes mixtures of two or more such substrates, polymers, or samples, and the like.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
As used herein, the term “polymer” refers to a relatively high molecular weight organic compound, natural or synthetic (e.g., polyethylene, rubber, cellulose), whose structure can be represented by a repeated small unit, the monomer (e.g., ethane, isoprene, β-glucose). Synthetic polymers are typically formed by addition or condensation polymerization of monomers.
As used herein, the term “copolymer” refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and without limitation, a copolymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer.
As used herein, the term “bioassay” refers to a procedure for determining the concentration, purity, and/or biological activity of a substance.
As used herein, the term “chemical event” refers to a change in a physical or chemical property of an analyte in a sample that can be detected by the disclosed systems and methods. For example, a change in refractive index (RI), solute concentration and/or temperature can be a chemical event. As a further example, a biochemical binding or association (e.g., DNA hybridization) between two chemical or biological species can be a chemical event. As a further example, a disassociation of a complex or molecule can also be detected as an RI change. As a further example, a change in temperature, concentration, and association/dissociation can be observed as a function of time. As a further example, bioassays can be performed and can be used to observe a chemical event.
Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the methods of the invention.
It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
Back Scattering Interferometry
Rapid monitoring and detection of ultra small volume samples is in great demand. One analytical approach, Back-Scattering Interferometry (BSI), derives from the observation that coherent light impinging on a cylindrically shaped capillary produces a highly modulated interference pattern. Typically, BSI analyzes reflections from a capillary tube filled with a liquid of which one wants to measure the refractive index. The technique has been shown capable of measuring changes in refractive index of liquids on the order of 10−9. The BSI technique is a simple and universal method of detecting refractive index changes in small volumes of liquid and can be applied to monitor changes in concentrations of solutes, flow rates and temperature, all conducted in nanoliter volumes.
The BSI technique is based on interference of laser light after it is reflected from different regions in a capillary or like sample container. Suitable methods and apparatus are described in U.S. Pat. No. 5,325,170 and WO-A-01/14858, which are hereby incorporated by reference. The reflected or back scattered light is viewed across a range of angles with respect to the laser light path. The reflections generate an interference pattern that moves in relation to such angles upon changing refractive index of the sample. The small angle interference pattern traditionally considered has a repetition frequency in the refractive index space that limits the ability to measure refractive index to refractive index changes causing one such repetition. In one aspect, such refractive index changes are typically on the order of three decades. In another aspect, such changes are on the order of many decades. In another aspect, the fringes can move over many decades up to, for example, the point where the refractive index of the fluid and the channel are matched.
BSI methods direct a coherent light beam along a light path to impinge on a first light transmissive material and pass there through, to pass through a sample which is to be the subject of the measurement, and to impinge on a further light transmissive material, the sample being located between the first and further materials, detecting reflected light over a range of angles with respect to the light path, the reflected light including reflections from interfaces between different substances including interfaces between the first material and the sample and between the sample and the further material which interfere to produce an interference pattern comprising alternating lighter and darker fringes spatially separated according to their angular position with respect to the light path, and conducting an analysis of the interference pattern to determine there from the refractive index, wherein the analysis comprises observation of a parameter of the interference pattern which is quantitatively related to sample refractive index dependent variations in the intensity of reflections of light which has passed through the sample.
The analysis comprises one or both of: (a) the observation of the angle with respect to the light path at which there is an abrupt change in the intensity of the lighter fringes, or (b) the observation of the position of these fringes of a low frequency component of the variation of intensity between the lighter and darker fringes. The first of these (a), relies upon the dependency of the angle at which total internal reflection occurs at an interface between the sample and the further material on the refractive index of the sample. The second (b), relies upon the dependency of the intensity of reflections from that interface on the refractive index as given by the Fresnel coefficients. The rectangular chips also have a single competent from diffraction at the corners.
The first material and the further material are usually composed of the same substance and may be opposite side walls of a container within which the sample is held or conducted. For instance, the sample may be contained in, e.g. flowed through, a capillary dimensioned flow channel such as a capillary tube. The side wall of the capillary tube nearer the light source is then the “first material” and the opposite side wall is the “further material.” The cross-sectional depth of the channel is limited only by the coherence length of the light and its breadth is limited only by the width of the light beam. Preferably, the depth of the channel is from 1 to 10 um, but it may be from 1 to 20 um or up to 50 um or more, e.g. up to 1 mm or more. However, sizes of up to 5 mm or 10 mm or more are possible. Suitably, the breadth of the channel is from 0.5 to 2 times its depth, e.g., equal to its depth.
Typically, at least one the interfaces involving the sample at which light is reflected is curved in a plane containing the light path, the curved interface being convex in the direction facing the incoming light if it is the interface between the first material and the sample and being concave in the direction facing the incoming light if it is the interface between the sample and the further material. The sample is typically a liquid, and can be flowing or stationary. However, the sample can also be a solid or a gas in various aspects of the present invention. The first and/or further materials will normally be solid but in principle can be liquid, e.g., can be formed by a sheathing flow of guidance liquid(s) in a microfluidic device, with the sample being a sheathed flow of liquid between such guidance flows. The sample may also be contained in a flow channel of appropriate dimensions in substrate such as a microfluidic chip. The method may therefore be employed to obtain a read out of the result of a reaction conducted on a “lab on a chip” type of device.
In contrast to conventional BSI techniques, as illustrated in FIG. 1, the present invention provides systems, apparatuses, and methods for the analysis of samples, solvents, and systems using two fringes to improved sensitivity and reduce noise. In one aspect, the techniques and apparatus of the present disclosure can provide improved detection limits as much as 10 fold over conventional BSI techniques.
Intermolecular interactions are fundamental in all aspects of chemistry and biochemistry. In organic chemistry, intermolecular interactions have become increasingly more important as scientists explore and optimize synthesis, molecular recognition, and organocatalysis reactions and methods. Thus, the ability to monitor and analyze molecular interaction can be of great use in developing new compositions and methods. For example, many synthetic reactions utilize substrate-catalyst interactions to drive reactions to completion and obtain enantiomerically enriched products. Traditionally, these interactions have been studied using such methods as isotope incorporation, NMR, UV-Vis spectroscopy, fluorescence, and calorimetry. While these techniques can provide molecular interaction information, they often require high concentrations, large volume samples, and complicated methods. With respect to organic catalysis, significant research has been expended to design and develop new catalysts and catalyst systems.
Conventional BSI analysis techniques can be used to study interactions in both aqueous and non-aqueous solvents, such as, for example, metal-ligand interactions. In another aspect, BSI can be an efficient method to study small molecule interactions, especially when compared to other free-solution techniques such as calorimetry. The methods and techniques described herein can be performed for any aqueous or non-aqueous system and/or analyte species.
In one aspect, the present disclosure provides methods and configurations that can detect position shifts and/or intensity changes in the fringes of a BSI signal, and can significantly reduce alignment constraints while enhancing the signal to noise ratio of the resulting data. In one aspect, the inventive approaches described herein utilize two fringes, one on each side of the interference pattern. In another aspect, the inventive approaches described herein utilize a plurality of fringes on each side of the interference pattern. In yet another aspect, the inventive approach utilizes one or more windows, each comprising a plurality of fringes, on each side of the interference pattern. In still another aspect, the inventive approach utilizes a Fourier Transform (FT) analysis of one or more windows positioned on each side of the interference pattern. In another aspect, this technique can simplify instrument alignment and reduce and/or eliminate sensitivity to vibration and alignment. In another aspect, the present techniques comprise use of both sides of the fringes of a signal, as compared to a conventional BSI technique which utilizes the fringes on a single side of the signal. In such an aspect, the limit of quantification (LOQ) and limit of detection (LOD) can be significantly improved over conventional BSI techniques.
In one aspect, use of the techniques described herein can provide improved detection limits of 10 fold or more, for example, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, or more, as compared to conventional BSI techniques.
Channel
The channel of the present invention can, in various aspects, be formed from a substrate such as a piece of silica or other suitable optically transmissive material. In one aspect, the material of composition of the substrate has a different index of refraction than that of the sample to be analyzed. In another aspect, as refractive index can vary significantly with temperature, the substrate can optionally be mounted and/or connected to a temperature control device. In yet another aspect, the substrate can be tilted, for example, about 7°, such that scattered light from channel can be directed to a detector.
In one aspect, the channel has a generally semi-circular cross-sectional shape. A unique multi-pass optical configuration is inherently created by the channel characteristics, and is based on the interaction of the unfocused laser beam and the curved surface of the channel that allows interferometric measurements in small volumes at high sensitivity. Alternatively, the channel can have a substantially circular or generally rectangular cross-sectional shape. In one aspect, the substrate and channel together comprise a capillary tube. In a further aspect, the substrate and channel together comprise a microfluidic device, for example, a silica substrate, or a polymeric substrate [e.g., polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA)], and an etched channel formed in the substrate for reception of a sample, the channel having a cross sectional shape. In one aspect, the cross sectional shape of a channel is semi-circular. In another aspect, the cross sectional shape of a channel is square, rectangular, or elliptical. In other aspects, the cross sectional shape of a channel can comprise any shape suitable for use in a BSI technique. In another aspect, a substrate can comprise one or multiple channels of the same or varying dimensions. In various aspects, the channel can have a radius of from about 5 to about 250 micrometers, for example, about 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, or 250 micrometers. In still other aspects, the channel can have a radius of up to about 1 millimeter or larger, such as, for example, 0.5 millimeters, 0.75 millimeters, 1 millimeter, 1.25 millimeters, 1.5 millimeters, 1.75 millimeters, 2 millimeters, or more.
In one aspect, a microfluidic channel, if present, can hold and/or transport the same or varying samples, and a mixing zone. The design of a mixing zone can allow at least initial mixing of, for example, one or more binding pair species. In another aspect, the at least initially mixed sample can then optionally be subjected to a stop-flow analysis, provided that the reaction and/or interaction between the binding pair species continues or is not complete at the time of analysis. The specific design of a microfluidic channel, mixing zone, and the conditions of mixing can vary, depending on such factors as, for example, the concentration, response, and volume of a sample and/or species, and one of skill in the art, in possession of this disclosure, could readily determine an appropriate design.
In one aspect, a channel comprises a single zone along its length for analysis. In another aspect, a channel can be divided into multiple discrete zones along the length of the channel, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zones. If a channel is divided into zones, any individual zone can have dimensions, such as, for example, length, the same as or different from any other zones along the same channel. In one aspect, at least two zones have the same length. In another aspect, all of the zones along a channel have the same or substantially the same length. In various aspects, each zone can have a length along the channel of from about 1 to about 1,000 micrometers, for example, about 1, 2, 3, 5, 8, 10, 20, 40, 80, 100, 200, 400, 800, or 1,000 micrometers. In other aspects, each zone can have a length of less than about 1 micrometer or greater than about 1,000 micrometer, and the present disclosure is not intended to be limited to any particular zone dimension. In one aspect, at least one zone can be used as a reference and/or experimental control. In yet another aspect, each measurement zone can be positioned adjacent to a reference zone, such that the channel comprises alternating measurement and reference zones. It should be noted that the zones along a channel do not need to be specifically marked or delineated, only that the system be capable of addressing and detecting scattered light from each zone.
In another aspect, any one or more zones in a channel can be separated from any other zones by a junction, such as, for example, a union, coupling, tee, injection port, mixing port, or a combination thereof. For example, one or more zones in the flow path of a sample can be positioned upstream of an injection port where, for example, an analyte can be introduced. In such an aspect, one or more zones can also be positioned downstream of the injection port.
In yet another aspect, a channel can be divided into two, three, or more regions, wherein each region is separated from other regions by a separator. In one aspect, a separator can prevent a fluid in one region of a channel from contacting and/or mixing with a fluid from another region of the channel. In another aspect, any combination of regions or all of the regions can be positioned such that they will be impinged with at least a portion of the light beam. In such an aspect, multiple regions of a single channel can be used to conduct multiple analyses of the same of different type in a single instrumental setup. In one aspect, a channel has two regions, wherein a separator is positioned in the channel between the two regions, and wherein each of the regions are at least partially in an area of the channel where the light beam is incident.
In one aspect, if multiple regions are present, each region can have an input and an output port. In one aspect, the input and/or output ports can be configured so as not to interfere with the generation of scattered light, such as, for example, backscattered light, and the resulting measurements. It should be noted that other geometric designs and configurations can be utilized, and the present invention is not intended to be limited to the specific exemplary configurations disclosed herein. Thus, in one aspect, a single channel can allow for analysis of multiple samples simultaneously in the same physical environment.
In one aspect, a separator, if present, comprises a material that does not adversely affect detection in each of the separated regions, such as, for example, by creating spurious light reflections and refractions. In one aspect, a separator is optically transparent. In another aspect, a separator does not reflect light from the light source. In such an aspect, a separator can have a flat black, non-reflective surface. In yet another aspect, the separator can have the same or substantially the same index of refraction as the channel. In yet another aspect, a separator can be thin, such as, for example, less than about 2 μm, less than about 1 μm, less than about 0.75 μm.
Any one or more individual zones along a channel, or any portion of a channel can optionally comprise a marker compound positioned within the path of the channel. In one aspect, a marker compound can be positioned on the interior surface of a capillary such that a sample, when introduced into the channel, can contact and/or interact with the marker compound.
A marker compound, if present, can comprise any compound capable of reacting or interacting with a sample or an analyte species of interest. In one aspect, a marker compound can comprise a chromophore. In another aspect, a marker compound can comprise a ligand that can interact with a species of interest to provide a detectable change in refractive index.
Light Source
In one aspect, the light source generates an easy to align optical beam that is incident on the etched channel for generating scattered light. In another aspect, the light source generates an optical beam that is collimated, such as, for example, the light emitted from a HeNe laser. In another aspect, the light source generates an optical beam that is not well collimated and disperses in, for example, a Gaussian profile, such as that generated by a diode laser. In another aspect, at least a portion of the light beam is incident on the channel such that the intensity of the light on any one or more zones is the same or substantially the same. In another aspect, the portion of the light beam incident on the channel can have a non-Gaussian profile, such as, for example, a plateau (e.g., top-hat). The portion of the light beam in the wings of the Gaussian intensity profile can be incident upon other portions of the channel or can be directed elsewhere. In one aspect, variations in light intensity across the channel can result in measurement errors. In still another aspect, if portions of a light beam having varying intensity are incident upon multiple zones or portions of a channel, a calibration can be performed wherein the expected intensity of light, resulting interaction, and scattering is determined for correlation of future measurements.
The light source can comprise any suitable equipment and/or means for generating light, provided that the frequency and intensity of the generated light are sufficient to interact with a sample and/or a marker compound and provide elongated fringe patterns as described herein. Light sources, such as HeNe lasers and diode lasers, are commercially available and one of skill in the art could readily select an appropriate light source for use with the systems and methods of the present invention. In one aspect, a light source can comprise a single laser. In another aspect, a light source can comprise two or more lasers, each generating a beam that can impinge one or more zones of a channel. In another aspect, if two or more lasers are present, any individual laser can be the same as or different from any other laser. For example, two individual lasers can be utilized, each producing a light beam having different properties, such as, for example, wavelength, such that different interactions can be determined in each zone along a channel.
As with any interferometric technique for micro-chemical analysis, it can be advantageous, in various aspects, for the light source to have monochromaticity and a high photon flux. If warranted, the intensity of a light source, such as a laser, can be reduced using neutral density filters.
The systems and methods of the present invention can optionally comprise an optical element that can focus, disperse, split, and/or raster a light beam. In one aspect, an optical element, if present, can at least partially focus a light beam onto a portion of the channel. In various aspects, such an optical element can facilitate contact of the light beam with one ore more zones along a channel. In one aspect, a light source, such as a diode laser, generates a light beam having a Gaussian profile, and an optical element is not necessary or present. In another aspect, a light source, such as a diode laser, can be used together with an optical focusing element. In another aspect, a light source, such as a HeNe laser, generates a collimated light beam and an optical element can be present to spread the light beam and facilitate contact of the light beam with at least two zones along the channel. Such a light beam configuration can allow for multiple measurements or sample and reference measurements to be made simultaneously or substantially simultaneously within the same channel.
In various aspects, an optical element, if present, can comprise a dispersing element, such as a cylindrical lens, capable of dispersing the light beam in at least one direction; a beam splitting element capable of splitting a well collimated light beam into two or more individual beams, each of which can be incident upon a separate zone on the same channel; a rastering element capable of rastering a light beam across one or more zones of a channel; or a combination thereof.
In yet other aspects, one or more additional optical components can be present, such as, for example, a mirror, a neutral density filter, or a combination thereof, so as to direct the light beam and/or the scattered light in a desired direction or to adjust one or more properties of a light beam.
Impingement
At the channel, the light beam should have a profile such that the light beam impinges the channel in the area of interest. In one aspect, the light beam impinges the channel over a single zone or portion of the channel. In another aspect, the light beam impinges the channel over multiple, for example, 2, 3, 4, or more zones. In one aspect, the intensity of the light beam is uniform or substantially uniform across each of the discrete zones of interest along the channel. In another aspect, the light beam is dimensioned so as to fill or slightly overfill the channel or at least that portion of the channel to be interrogated. In another aspect, the light beam is aligned such that it impinges the channel perpendicular to the central axis of the channel. In yet another aspect, the width of the light beam is parallel to the longitudinal axis of the channel.
In another aspect, the alignment of the light beam exhibits no or substantially no tilt or skew with respect to the channel. In one aspect, the alignment of the light beam and the capillary can be such that the resulting fringe patterns are positioned above or below the incoming beam. In one aspect, misalignment or poor alignment of the light beam with the channel can result in distorted and/or skewed interference fringes due to the uneven distribution of light beam energy at the point of impingement with the channel.
Other than alignment of the light beam with respect to the channel, the position and orientation of the light source, optional optical element, channel, and detector, can vary according to a particular experimental design, provided that scattered light can be generated by reflective and refractive interactions of the light beam with the substrate/channel interface, sample, and optional marker compounds. One of skill in the art in possession of this disclosure could readily determine an appropriate arrangement of the light source, optional optical element, channel, and detector.
When incident upon a channel having a sample therein, and optionally one or more marker compounds positioned in zones along the channel, the incident light can scatter and comprise elongated interference fringes due to reflective and refractive interaction with the sample, channel walls, and marker compounds, if present. These elongated fringe patterns can comprise a plurality of light bands whose positions shift as the refractive index of the sample is varied, either through compositional changes or through temperature changes, for example. In one aspect, the scattered light comprises backscattered light. In another aspect, the optical elements and channel can be positioned so as to result in side scattering of the light beam. In yet another aspect, the optical elements and channel can be positioned to measure light passed through a channel, such as, for example, when using a fluorescent analyte or probe species, or an absorbing species.
Alignment and stability of the laser beam in a BSI analysis can significantly affect the noise of the resulting signal. Pointing instability, which can be about 0.03 mrad, can be a one of the major sources of noise in BSI. As the laser beam moves (e.g., wanders) on the microchannel during data acquisition, the distribution of energy found in each fringe changes. Moreover, as the beam wanders, the position of the fringes can shift, producing noise and limiting the measurement. Other environmental factors, such as, for example, changes in temperature, mechanical vibration, and mechanical movement of parts relative to the rest of the instrument, can exacerbate pointing instabilities.
In one aspect, and while not wishing to be bound by theory, the total energy in the fringe pattern must be conserved. For example, the total light out must be constant if the total light energy in is a constant. Thus, sister fringes are complementary in function due to conservation of energy and changes in a fringe due to alignment or vibration issues should manifest in an opposite manner in sister fringes (i.e., fringes on opposing sides of the centroid). FIG. 2 illustrates numbered sister fringes located on opposing sides of the centroid in a BSI signal. For example, the fringe numbered 5 on the left side of the centroid is a sister fringe to the fringe numbered 5 on the right side of the centroid.
In another aspect, a laser beam can be translated across a channel/capillary to mimic laser pointing instability, mechanical vibrations or misalignment. In such an aspect, one side of the fringe pattern (relative to the fringe pattern centroid) will dim while the other side will brighten. These changes can be problematic when the analysis utilizes a fringe position sensing method, as the method depends on the ability to integrate the properties of the fringe (intensity, shape, etc.) to determine the position from the center of mass. In another aspect, inaccurate alignment of the laser beam can create changes in the distribution of fringe intensity, along with position shifts of fringes in the signal leaving the chip.
Detector
A detector detects the scattered light and converts it into intensity signals that vary as the positions of the light bands in the elongated fringe patterns shift, and can thus be employed to determine the refractive index (RI), or an RI related characteristic property, of the sample. Exemplary properties that can be detected and/or quantified using the inventive techniques can comprise, without limitation, changes in conformation, structure, charge level, level of hydration, or a combination thereof. In other aspects, the progress of one or more chemical reactions can be monitored, such as, for example, that can occur in an aqueous or a non-aqueous solvent.
The detector can, in various aspects, comprise any suitable image sensing device, such as, for example, a bi-cell sensor, a linear or area array CCD or CMOS camera and laser beam analyzer assembly, a photodetector assembly, an avalanche photodiode, or other suitable photodetection device. In one aspect, the detector is a photodetector. In another aspect, the detector is an array photodetector capable of detecting multiple interference fringe patterns. In yet another aspect, a detector can comprise multiple individual detectors to detect interference fringe patterns produced by the interaction of the light beam with the sample, channel wall, and optional marker compounds. In one aspect, the scattered light incident upon the detector comprises interference fringe patterns. In another aspect, the scattered light incident upon the detector comprises elongated interference fringe patterns that correspond to the discrete zones along the length of the channel. The specific position of the detector can vary depending upon the arrangement of other elements. In one aspect, the detector can be positioned at an approximately 45° angle to the channel.
The intensity signals from the detector can then be directed to a signal analyzer for fringe pattern analysis and determination of the RI or RI related characteristic property of the sample and/or reference in each zone of the channel. The signal analyzer can be a computer or a dedicated electrical circuit. In one aspect, the signal analyzer includes the programming or circuitry necessary to determine from the intensity signals, the RI or other characteristic property of the sample in each discrete zone of interest. In another aspect, the signal analyzer is capable of detecting positional shifts and/or intensity changes in interference fringe patterns and correlating those positional shifts and/or intensity changes with a change in the refractive index of at least a portion of the sample. In another aspect, the signal analyzer is capable of detecting positional shifts and/or intensity changes in interference fringe patterns and correlating those positional shifts and/or intensity changes with a change in the refractive index occurring in a portion of the channel. In yet another aspect, the signal analyzer is capable of comparing data received from a detector and determining the refractive index and/or a characteristic property of the sample in any zone or portion of the channel.
In one aspect, an additional camera or detection device can be utilized to monitor sister fringes, for example, located on opposite sides of the centroid. FIG. 3 illustrates an exemplary configuration wherein separate cameras are utilized to monitor respective sister fringes on opposing sides of the centroid. If a perturbation occurs in the laser beam, it can result in a shift, for example, of distance +x, in the fringes on one side of the fringe pattern. At the same time, a compensatory change occurs on the opposite side of the fringe pattern, having the same magnitude, but in an opposite direction (i.e., −x), as illustrated in FIG. 4A. This effect can be experimentally observed by moving the channel horizontally with respect to the beam position.
When both sides of the fringe pattern are monitored, the sum of the opposing signals can also be monitored. In one aspect, the summed signal can be a point-by-point addition of the phase output of each fringe, plotted, for example, versus time. In one aspect, such an approach results in reduced noise due to the fact that changes in a fringe, for example, due to alignment or vibration, on one side of the centroid are compensated by changes in the respective sister fringe on the opposing side of the centroid. In another aspect, short-term noise in both sides of the fringe pattern (i.e., in sister fringes) is in phase, but in the opposite direction. Thus, such short-term noise, or a significant portion thereof, can be filtered out using conventional electronics and/or software, as illustrated in FIG. 4B.
In another aspect, one or more windows on each side of the interference pattern can be monitored. In one aspect, the Fourier Transform of one or more windows can be monitored. In such an aspect, the specific position and/or size of a given window can be selected by the user or by a computer so as to optimize instrument output. In one aspect, a window can be positioned so as to comprise fringes that contain useful information for a given sample or analysis. FIG. 5 illustrates an enhanced signal for an induced refractive index change using two cameras. It should be noted that various mathematical algorithms can be applied to one or more signals or a range of signals on each side of the interference pattern, and that one of skill in the art, in possession of this disclosure, could readily select an appropriate signal or range of signals and mathematical algorithm for a given experiment and/or analyte.
In one aspect, as described above, two cameras can be used to monitor opposing sides of the BSI signal. In another aspect, such an approach can require separate computers to control each camera. In another aspect, a single large sensor can be utilized in lieu of separate sensors (e.g., cameras), as illustrated in FIG. 6A. In such an aspect, both sides of the fringe pattern can be monitored by the same sensor simultaneously. Such an aspect can also be beneficial in that the single sensor has the same orientation with respect to both sets of fringes being monitored. In another aspect, separate cameras can be used to monitor opposing fringe patterns, but the cameras can be connected to the same computer and synced together such that they collect data at the same time, as illustrated in FIG. 6B. In yet another aspect, mirrors and an absorber can be used in the optical path, as illustrated in FIG. 6C, wherein the absorber can block the centroid and any undesired central fringes such that each of the two mirrors can reflect the desired fringes to the sensor(s). In yet other aspects, a single or multiple mirrors of varying shape or design can be employed to achieve the same result. For example, one or two a D-shaped mirrors can be used to direct fringes to a detection device. In another aspect, an exemplary BSI configuration can comprise an absorber employed to block the centroid and undesired central fringes of a signal, and flat mirrors to focus the desired two fringes on one small sensor, as illustrated in FIG. 7.
In an aspect where separate mirrors are used to reflect portions of the BSI signal, the position and alignment of each of the mirrors can affect the resulting data and reduction in noise. In one aspect, the angle of each fringe with respect to the sensor can be adjusted. For example, it can be desirable for the optical paths of each fringe to have an identical path and angle with respect to the horizontal axis, as illustrated in FIG. 8A. In such an aspect, the mirrors can be aligned such that the two fringes are parallel. The sensor can then be positioned such that it is centered between the two fringes. The mirrors can then be adjusted again such that each of the two fringes falls onto the sensor with the same angle. In this aspect, alignment of the mirrors and thus, the angle of the beams impinging the sensor, can be confirmed by comparing the base of each fringe. Fringes having differing widths at their base are not aligned at the same angle, as illustrated in FIG. 8B. Fringes having the same widths at their base are aligned at the same angle, as illustrated in FIG. 8C.
The intensity of each of the fringes can also be used to determine if the beam is aligned with the microfluidic channel. If the two fringes exhibit varying intensities, as illustrated in FIG. 9A, the beam is not aligned with the center of the microfluidic channel. In such a case, the chip can be moved with respect to the beam (or vice versa) until the fringes exhibit the same intensity, as illustrated in FIGS. 9B-C.
In yet another aspect, the shape of each of the fringes can be used to evaluate alignment of the instrument. In this aspect, the point of impingement of the beam against the channel can be adjusted until the two fringes exhibit similar shapes. If the shape of the two fringes is similar or identical, it can suggest that the channel is symmetrical at the point of impingement.
Thus, in various aspects, fringes on opposing sides of the centroid can be used to optimize and/or align the beam, chip, sensor, and any mirrors and/or absorbers that can optionally be present. In one aspect, such a technique can be used to obtain fringes having at least one of similar base widths, similar intensities, similar shapes, or a combination thereof. In another aspect, such a techniques can be used to obtain fringes having similar base widths, similar intensities, and similar shapes.
In one aspect, when using the techniques described herein to align a BSI configuration, the short-term noise present in each of the two monitored fringes is in phase or mostly in phase, having a similar magnitude, but in opposing directions. In such an aspect, this short-term noise can be filtered out, as illustrated in FIG. 11A. Even when a perturbation, such as, for example, a mechanical vibration, is induced, the intensity of the noise can be reduced through the techniques described herein. FIG. 11B illustrates an example wherein the instrument bench as intentionally jarred to induce a mechanical vibration. While a significant amount of noise is present in each of the individual fringes as a result of the vibration, the summed signal shows a significant reduction in the noise compared to the individual signals.
Once a desirable signal is obtained, for example, by summing the respective fringes, a signal analyzer can be used to determine one or more characteristic properties of the sample. In another aspect, the signal analyzer can utilize a mathematical algorithm to interpret positional shifts in the interference fringe patterns incident on a detector or the summed signal. In another aspect, known mathematical algorithms and/or signal analysis software, such as, for example, deconvolution algorithms, can be utilized to interpret positional shifts occurring from a multiplexed scattering interferometric analysis. In other aspects, a signal analyzer can be used to interpret and/or compare the Fourier Transform of one or more windows on each side of the interference pattern.
The detector can be employed for any application that requires interferometric measurements; however, the detector can be particularly useful for making universal solute quantification, temperature and flow rate measurements. In these applications, the detector provides ultra-high sensitivity due to the multi-pass optical configuration of the channel. In the temperature measuring aspect, a signal analyzer receives the signals generated by the photodetector and analyzes them using the principle that the refractive index of the sample varies proportionally to its temperature. In this manner, the signal analyzer can calculate temperature changes in the sample from positional shifts in the detected interference fringe patterns. In one aspect, the ability to detect interference fringe patterns from interactions occurring along a channel can provide real-time reference and/or comparative measurements without the problem of changing conditions between measurements. In one aspect, a signal analyzer, such as a computer or an electrical circuit, can thus be employed to analyze the photodetector signals, and determine the characteristic property of the sample.
In one aspect, the systems and methods of the present invention can be used to obtain multiple measurements simultaneously or substantially simultaneously from discrete zones along the length of a channel. In such an aspect, each zone can provide a unique measurement and/or reference. For example, a series of reactive species can be used as marker compounds, positioned in zones along the channel, each separated by a reference zone. In another aspect, temporal detection can be used to measure changes in a sample over time as the sample flows through the channel, for example, with a flow injection analysis system.
In another aspect, two or more samples, blanks, and/or references can be positioned in the channel such that they are separated by, for example, an air bubble. In another aspect, each of a plurality of samples and/or reference species can exhibit a polarity and/or refractive index the same as or different from any other samples and/or reference species. In one aspect, a pipette can be used to place a portion of a reference compound into the channel. Upon removal of the pipette, an air bubble can be inserted between the portion of the reference compound in the channel and a portion of a sample compound, thereby separating the reference and sample compounds and allowing for detection of each in a flowing stream within the channel. In another aspect, each sample and/or reference compound can be separated by a substance other than air, such as, for example, water, oil, or other solvent having a polarity such that the sample and/or reference compounds are not miscible therewith.
In one aspect, the sample is a fluid. In another aspect, the sample is a liquid, which can be a substantially pure liquid, a solution, or a mixture. In a further aspect, the sample can further comprise one or more analytes. In one aspect, a sample can be introduced into the channel via an injection port at, for example, one end of the channel.
As the light beam impinges one or more discrete regions of a channel, the resulting interference fringe patterns can move with a change in refractive index. The ability to analyze multiple discrete zones simultaneously can provide high spatial resolution and can provide measurement techniques with an integrated reference.
Interferometric Detection System
In one aspect, the invention relates to an interferometric detection system and method that can be used, for example, for detection of refractive index changes in picoliter sized volumes for chip-scale analyses. Conventional backscattering interferometry, as illustrated in FIG. 1, utilizes interference fringes generated by backscattered light to detect refractive index changes in a sample. The backscatter detection technique is generally disclosed in U.S. Pat. No. 5,325,170 to Bornhop, and U.S. Patent Publication No. US2009/0103091 to Bornhop, both of which are hereby incorporated by reference. With reference to FIG. 1, a conventional backscattering interferometric detection system 10 comprises a laser 12 that produces a light beam 14. The light beam can be directed through one or more neutral density filters 16 to reduce the intensity of the light beam, before being reflected on a mirror 18 and directed to impinge an etched channel 22 on a chip 20. The chip can also be positioned on a temperature controlled support block 23 and/or an X-Y translation stage 24. After various reflective and refractive interactions with the channel and sample, the scattered light can be directed to a detector 25, and the intensity signals generated by the detector interpreted by a computer based signal analyzer 28. As described above, multiple detection devices, for example, cameras, can be used such that each detection device monitors an opposing fringe pattern. In addition, mirrors and/or absorbers can optionally be used to control and direct the optical signal from the microfluidic channel to the one or more detection devices.
In another aspect, the inventive interferometric detection system and methods are capable of measuring multiple signals, for example, along a length of a capillary channel, simultaneously or substantially simultaneously. In one aspect and while not wishing to be bound by theory, the refractive index changes that can be measured by the multiplexed interferometric detection systems and methods of the present disclosure can arise from molecular dipole alterations associated with conformational changes of sample-ligand interaction as well as density fluctuations.
The detection system has numerous applications, including the observation and quantification of molecular interactions, molecular concentrations, ligand-metal interactions, non-aqueous electrochemical reactions, ultra micro calorimetry, flow rate sensing, and temperature sensing.
In one aspect, the detection systems and methods described herein can be useful as a bench-top molecular interaction photometer. In another aspect, the detection systems and methods described herein can be useful for performing bench-top or on-site analysis.
In another aspect, the interferometric detection system comprises a substrate, a channel formed in the substrate for reception of a sample to be analyzed, a light source for generating a light beam, directing the light beam onto the substrate such that the light beam is incident on at least a portion of the channel and thereby generate scattered light through reflective and refractive interaction of the light beam with a substrate/channel interface and the sample, the scattered light comprising interference fringe patterns, a photodetector for receiving scattered light from each of the two or more discrete zones and generating a plurality of intensity signals, and at least one signal analyzer for receiving the intensity signals and determining therefrom one or more characteristic properties of the sample along the length of the channel.
In one aspect, the present invention provides a method for determining a characteristic property of a sample comprising the steps of providing an apparatus adapted for performing light scattering interferometry, the apparatus comprising a substrate, a channel formed in the substrate capable of receiving a sample to be analyzed, a light source for generating a light beam, a photodetector for receiving scattered light and generating a plurality of intensity signals, and at least one signal analyzer capable of receiving the signals from sister fringes in the resulting signal and determining therefrom one or more characteristic properties of the sample, and interrogating the sample using light scattering interferometry.
Detection of Chemical Events
The disclosed systems and methods can be used in connection with the detection and determination of a wide variety of characteristic properties of a sample. For example, the invention can be used to determine absolute or relative refractive index (RI) of a sample, for example a fluid either flowing or static. The disclosed systems and methods can also be used in connection with detection and determination of chemical events, for example the formation of hydrogen bonds in an organic reaction.
In one aspect, the disclosed systems and methods can be used in connection with a step of performing a chromatographic separation or a separation of the sample prior to the determining the characteristic property step.
Analytical Detection Events
In one aspect, the invention also finds use as a detector for other chip-scale analytical schemes including chromatographic separations and FIA. In another aspect, it is possible to detect catalyst species and their interaction with chemical reactants. In another aspect, the interferometric techniques can be used to quantify environmental analytes. In yet another aspect, micro-thermometry can be performed, wherein the device has the capability of measuring small temperature changes (in the 10−3° C. range) allowing for calorimetry and fundamental chemical binding studies to be performed in picoliter volumes.
Measurement of End-Point Values
In one aspect, multiplexed BSI can measure end-point values of phase for the reaction between molecule A and molecule B as a function of the concentration of molecule B to determine the binding affinity of the complex and/or to quantitatively determine the concentration of the A-B product at reaction equilibrium. End-point concentration bioassays can be used in both research and clinical diagnostic applications.
Determination of Kinetic Parameters
In a further aspect, BSI can determine kinetic parameters. That is, the interferometric detection technique described herein can be used to monitor various kinetic parameters, such as, for example, binding affinities, of a chemical and/or biochemical analyte species. The use of BSI for the determination of a kinetic parameter can provide one or more advantages over traditional techniques, for example, free-solution measurements of label-free species, high throughput, small sample volume, high sensitivity, and broad dynamic range. A BSI technique can be performed on a free-solution species, a surface immobilized species, or a combination thereof. In one aspect, the species of interest is a free-solution species, wherein at least a portion of the species of interest is not bound or otherwise immobilized. In another aspect, at least a portion of the species of interest is surface immobilized.
In one aspect, a BSI technique can be used to analyze and/or quantify one or more molecular interactions, such as, for example, a dissociation constant for one or more binding pair species.
The sensitivity of a multiplexed BSI technique can allow analysis and/or determination of at least one kinetic parameter to be performed on a small volume sample. The volume of a sample comprising at least one species of interest can, in various aspects, be less than about 1 nL, for example, about 900, 850, 800, 700, 600, 500, 400, 350, 300, 250, or 200 pL; less than about 600 pL, for example, about 580, 550, 500, 450, 400, 350, 300, 250, or 200 pL; or less than about 400 pL, for example, about 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 280, 250, 230, or 200 pL. In one aspect, the sample volume is about 500 pL. In another aspect, the sample volume is about 350 pL. The sample volume can also be greater than or less than the volumes described above, depending on the concentration of a species of interest and the design of a particular BSI apparatus. A species that can be analyzed via BSI can be present in neat form, in diluted form, such as, for example, in a dilute solution, or any other form suitable for analysis by a BSI technique. The concentration of a species of interest can likewise vary depending upon, for example, the design of a particular BSI apparatus, the volume of sample in the optical path, the intensity of a response of a specific species to the radiation used in the experiment. In various aspects, the species can be present at a concentration of from about 1 pM to greater than 100 mM.
The concentration of one or more analyte species in a sample can be determined with a BSI technique by, for example, monitoring the refractive index of a sample solution comprising an analyte species. A property, such as, for example, refractive index, can be measured in real-time and the kinetics of an interaction between analyte species determined therefrom. Other experimental conditions, such as, for example, temperature and pH, can optionally be controlled during analysis. The number of real-time data points acquired for determination of a kinetic parameter can vary based on, for example, the acquisition rate and the desired precision of a resulting kinetic parameter. The length of time of a specific experiment should be sufficient to allow acquisition of at least the minimal number of data points to calculate and/or determine a kinetic parameter. In one aspect, an experiment can be performed in about 60 seconds.
An apparent binding affinity between binding pair species can subsequently be extracted from the acquired data using conventional kinetics models and/or calculations. In one aspect, a model assumes first order kinetics (a single mode binding) and the observed rate (kobs) can be plotted versus the concentration of one of the species. A desired kinetic parameter, such as, for example, KD, can be determined by, for example, a least squares analysis of the relationship plotted above. A suitable fitting model can be selected based on the particular experimental condition such that a rate approximation can be determined at the end of the analysis. One of skill in the art can readily select an appropriate model or calculation to determine a particular kinetic parameter from data obtained via BSI analysis.
In one aspect, BSI can be utilized to measure a free-solution molecular interaction. In another aspect, BSI can be used to measure both a free solution property and a immobilized interaction within the same channel. In a further aspect, BSI can measure label-free molecular interactions.
BSI can be used in any market where measuring macromolecular interactions is desired. In one aspect, a BSI technique, as described herein can be combined with various electrochemical studies. In the food industry, as well as in biodefense applications, a rapid methodology that can assay for the presence of toxins, xenobiotics, allergens, additives, or biowarfare agents whether chemicals, viruses, or cellular pathogens such as certain bacteria can be useful as evidenced by a large number of such items for which no easy to use tests are readily available today.
In other aspects, BSI using a dual fringe detection method as described herein can detect a horizontal displacement of the beam and channel of less than about 100 nm.
In various aspects, use of the sister fringe approaches described herein can improve BSI signals affected by mechanical instability and vibrations, beam pointing and intensity instability, chip displacement, optical component displacement with respect to other components, and optic bench vibrations. In other aspects, the use of such complementary fringes can provide one or more of the following advantages: 1) reduce and/or eliminate the effect of optic bench vibrations, 2) provide compensation for signal perturbations resulting from mechanical displacement of the chip with respect to the beam, 3) reduce and/or eliminate the effect of laser beam pointing instability, 4) reduce and/or eliminate the effect of mechanical vibration of one or more parts of an instrument with respect to one or more other parts of the instrument, and/or 5) provide compensation for noise resulting from noise due to mechanical instability and/or non-equal expansion or contraction of instrument optical and mounting components. In other aspects, the design of a BSI instrument can be simplified over conventional analytical instruments in that the use of sister fringes can allow for orthogonal arrangement of components.
EXPERIMENTAL
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
In a first example, signal to noise studies were performed using glycerol calibration solutions to evaluate the effect of the multiple fringe monitoring approach on instrument performance. FIG. 5 illustrates that as the refractive index (RI) of a sample in the channel is changed, the two fringes monitored by separate sensors have the same direction and similar magnitudes, suggesting that summing the two signals can enhance the overall signal by a factor of two while maintaining noise reduction.
Calibrations curves of glycerol solutions were performed to compare the limit of quantification (LOQ) and S/N of the standard one fringe detection method with the LOQ of the sister fringe detection method. The LOQ typically obtained for a conventional BSI configuration wherein a single fringe pattern is monitored, is 500-700 μM glycerol. In contrast, the LOQ obtained using a dual fringe detection method, as described herein, ranged from 60 μM to 90 μM glycerol, as illustrated in FIG. 12, representing an approximate 5-10 fold improvement in LOQ. Similarly, an improvement in the signal to noise ratio of a factor of two was observed, increasing from about 50 with a single fringe detection method to nearly 100 with a dual fringe detection method, as illustrated in FIG. 13. This improvement in signal to noise ratio remains even after the chip is removed from the holder and replaced into the system with no realignment of the mirrors or camera.
FIG. 14 illustrates an interference pattern with fringes disposed on each side of the pattern, and wherein windows (e.g., W1, W2, W3, W4) are positioned over various portions of the interference pattern. The zero intensity portion of FIG. 14 located in the center of the plot corresponds to a region where there is a space between the mirror that directs the backscatter pattern. In such an approach, windows W1 and W4 are sisters, W2 and W3 are sisters, etc. In one aspect, the use of a single mirror system could simplify and/or improve and simplify instrument alignment. In another aspect, a camera, such as, for example, a 2D camera having a large sensor, could assist in aligning a system or minimizing steps to do the same. In using a sister fringe technique wherein multiple windows are monitored, further improvements in detection limits (LOD) can be realized, as described below in Table 1, for example by a factor of 2.45. A reduction in long term drift by improving the limit of quantification (LOQ) by a factor of 1.4 was also realized.







TABLE 1







Improvement in Detection Limit Using Windows














Sum
W1
W2
W3
W4
Average

















LOQ
0.252127
0.380926
0.332905
0.386532
0.340597
0.36024


LOD
0.312128
0.870859
0.72956
0.672167
0.785504
0.764523









In another example, mechanical vibrations in the optic bench were made by pushing on the optic bench with heavy weights to increase noise and generate spikes in the resulting signal. In such an instance, if a user leans on the optic bench, the signal baseline can be displaced or offset. Such mechanical vibrations can be at least partially compensated using the sister fringe approaches described herein, as illustrated in FIG. 15, and can reduce instrumentation cost by reducing or eliminating the need for optic benches and/or vibration isolators.
FIG. 16 illustrates signals from an experiment wherein the chip was mechanically displaced with respect to the beam. In this experiment, buffer was injected into the channel and the chip was displaced in 1 μm steps from right and left of the center. In this experiment, the introduction of sample causes a large offset in the signal due to displacement in the chip's position with respect to the incident beam. In one aspect, this displacement results because the channel sits on a heat-insulated chip holder connected to the optic bench via plastic screws and translational stages. When two similar sister regions are selected for analysis, this type of noise (e.g., from displacement due to sample introduction) can be reduced or eliminated.
In another aspect, displacement of the beam with respect to the channel can present a significant problem due to the pointing instability of the laser beam. Pointing drift can be significant with, for example, a He—Ne laser, and can have a significant impact on the signal to noise ratio.
FIG. 15 illustrates the signal from an experiment wherein a portion of a BSI instrument is subjected to mechanical vibration. Such a vibration of different parts of the instrument can result in the same signal changes as optic bench vibrations (i.e., large spikes in the signal and potentially a small offset). In this experiment, different parts of the instrument (e.g., laser source, mirror, chip holder, chip, and camera) were lightly tapped. The use of sister fringes effectively filters the vibrational noise from mechanical vibration of one or more portions of an instrument.
FIG. 18 similarly illustrates the signal from an experiment wherein the different portions of a BSI instrument were subjected to mechanical instability. In this experiment, buffer was injected into the channel and then the chip holder and round mirror were pushed and/or perturbed. The pushing motion results in a mechanical displacement from the default position, causing errors in the signal (e.g., a baseline offset). The use of sister fringes compensates for the offset and reduces the noise in the resulting signal.
In another aspect, the use of sister fringes in BSI can advantageously illuminate the center of the channel, whereas side illumination can lead to a loss in signal magnitude. In conventional BSI, it can be difficult to determine if the beam is hitting the center of the channel when interrogating only one side of the interference pattern. In contrast, a sister fringe approach can assist with alignment and signal optimization. In addition, construction of a BSI instrument can be simplified, as sister fringes can allow the orthogonality of components to be determined. In contrast, alignment in a conventional BSI instrument is difficult and it can be challenging to determine if an incoming beam and reflecting centroids are co-planar.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

## Claims

1. A method for determining a characteristic property of a sample comprising the steps of:
(a) providing a substrate having a channel formed therein for reception of a sample to be analyzed;
(b) introducing a sample to be analyzed into the channel;
(c) directing a light beam from a light source onto the substrate such that the light beam is incident on at least a portion of the sample to generate scattered light through reflective and refractive interaction of the light beam with a substrate/channel interface, and the sample, wherein the scattered light comprising interference fringe patterns including a plurality of spaced light bands whose positions change in position or intensity in response to changes in the refractive index of the sample;
(d) detecting positional shifts and/or intensity changes in fringe patterns positioned on opposing sides of a centroid; and
(e) determining the characteristic property of the sample from the positional shifts of the fringe patterns.

2. The method of claim 1, wherein interrogating comprises detecting backscattered light on the photodetector, and wherein the scattered light comprises a plurality of interference fringe patterns.

3. The method of claim 1, wherein the light source comprises a HeNe laser or a diode laser.

4. The method of claim 1, wherein at least one of the channel, light beam, detector, or a combination thereof is aligned so as to produce sister fringes having at least one of a similar base width, similar intensity, similar shape, or a combination thereof.

5. The method of claim 1, wherein at least one of the channel, light beam, detector, or a combination thereof is aligned so as to produce sister fringes having a similar base width, similar intensity, and similar shape.

6. The method of claim 1, wherein the sample is non-aqueous.

7. An interferometric detection system comprising:
(a) a substrate;
(b) a channel formed in the substrate for reception of a sample to be analyzed;
(c) a light source for generating a light beam, the light source being positioned to direct the light beam onto the substrate such that the light beam is incident on at least a portion of the channel, to thereby generate scattered light through reflective and refractive interaction of the light beam with a substrate/channel interface and the sample, the scattered light comprising interference fringe patterns including a plurality of spaced light bands whose positions shift in response to changes in the refractive index of the sample;
(d) a first photodetector for receiving scattered light from a fringe pattern disposed on one side of a centroid, and generating a plurality of intensity signals;
(e) a second photodetector for receiving scattered light from a fringe pattern disposed on the opposing side of the centroid, and generating a plurality of intensity signals; and
(f) at least one signal analyzer for receiving the intensity signals and determining therefrom one or more characteristic properties of the sample at one or more of the discrete zones along the length of the channel.

8. The interferometric detection system of claim 7, wherein the scattered light comprises backscattered light.

9. The interferometric detection system of claim 7, wherein the substrate and channel together comprise a microfluidic device.

10. The interferometric detection system of claim 7, wherein the light source comprises a laser or a diode.

11. An apparatus adapted for light scattering interferometry, the apparatus comprising:
(a) a substrate;
(b) a channel formed in the substrate capable of receiving a sample to be analyzed;
(c) a light source for generating a light beam capable of being directed onto the substrate such that the light beam is incident on the channel and thereby generates scattered light comprising interference fringe patterns;
(d) a photodetector for receiving scattered light from two sister fringe patterns disposed on opposing sides of a centroid and generating a plurality of intensity signals;
(e) two mirrors and/or lenses adapted and positioned to direct the scattered light from two sister fringe patterns disposed on opposing sides of the centroid onto the photodetector; and
(f) at least one signal analyzer capable of receiving the intensity signals and determining therefrom one or more characteristic properties of the sample.

12. The apparatus of claim 11, further comprising an absorber positioned between the channel and the photodetector to block the centroid.

13. The apparatus of claim 11, wherein the light source comprises a HeNe laser or a diode laser.

14. A method for determining a characteristic property of a sample comprising the steps of:
(a) providing a substrate having a channel formed therein for reception of a sample to be analyzed;
(b) introducing a sample to be analyzed into the channel;
(c) directing a light beam from a light source onto the substrate such that the light beam is incident on at least a portion of the sample to generate scattered light through reflective and refractive interaction of the light beam with a substrate/channel interface, and the sample, wherein the scattered light comprising interference fringe patterns including a plurality of spaced light bands whose positions change in position or intensity in response to changes in the refractive index of the sample;
(d) detecting positional shifts and/or intensity changes in fringe patterns positioned on opposing sides of a centroid; and
(e) determining the characteristic property of the sample from the positional shifts of the fringe patterns, wherein determining comprises performing a calculation with two sister fringes positioned on opposing sides of the centroid.

15. The method of claim 14, wherein interrogating comprises detecting backscattered light on the photodetector, and wherein the scattered light comprises a plurality of interference fringe patterns.

16. The method of claim 14, wherein the light source comprises a HeNe laser or a diode laser.

17. The method of claim 14, wherein at least one of the channel, light beam, detector, or a combination thereof is aligned so as to produce sister fringes having at least one of a similar base width, similar intensity, similar shape, or a combination thereof.

18. The method of claim 14, wherein at least one of the channel, light beam, detector, or a combination thereof is aligned so as to produce sister fringes having a similar base width, similar intensity, and similar shape.

19. The method of claim 14, wherein the sample is non-aqueous.